418 0ilS and Fats

David Firestone and Mattn Peter Yuraw●

z,Cttrer帥 俺
us,F00J and prIJg Ad何碑おfrari19,

41.1.01 41.1.03
AOAC Omcta,Method 031.11 AOAC Omcla‖ Method 984.20
C)ils and Fats MoLture 3■ (D:33 and Fa皓
Preparndon tt Test Sampte Ka"F3scheF MethOd
Procedure First Actlon 1984
F3再R Adttn 1381 Final Adon 1988

rsc/rcmc,f― _AcAC mor

Utt clear sedimntetee liquid― ple diFeCdy after inverting the
container several血 .rliquid smple∞ n述 ぉ 貿慮 ment,reLase
ali sedment fron wal13 0f COntaittr and dstribute unifomy ドα appliCable tt alkdine or oxidzed dis and fats.)
阻
t h r o u g h o u t d l e c i l f o r輸 抽蔵 o n o f 航 s 価, a n d v o l 調 e m a t t e F .
F o r d e m 述皿 ぃ h w h i c h F e S u l t t n i g h t にbcet痢 ed by possible
A,APParatws anJ Reagm体
・ S
P I e S C I I C e o f H 2 0 g●" 1 0 d i n e v t t u e j , 的 m p l e a s f ollowtt Addan―
h y d r o u s N a 2 S 0 4 i n 甲Ⅳd o n o f l - 2 g p e r 1 0 g s m p l e , a n d h o l d h o拓 arr F泣 れを″r″r4rわ"徳 s抑 冴み_Manual or automated,
oven at 50° C,Sdr vlgorously and fllter to obtain clear nlmte,PFe― with stttr.
W S O l i d ― p l e f o r m t t O n o f m t t s m e a n d v o l a t i l e m a t t e ●) r 絶 ガ F 持がた, r F a g α 止―べ 術 l i z e d , w i d l H 2 0 e q u i v a l e n t
aS diFeCd in 92t12律β41.1.磁),For oher deteminations,Inelt
CaS mg H20/mLreagent.Attilablecomly orprepm asf
t e s t s u l e i n d F y i n g O v e n a t a t e m p e r a m a t l e a s t 1C0 °
abovemp.If
lowst Dissolve 133 g 12 in 425 ■ lL dry pyridine in dry
血 脚田 部 I d i r d y . r m i d o r c O n t a i n s 申 山 距 L 蝕 t e r t e s t s a n ‐
P l e i n s d e o v e n ` F o r di ― m t i c n s w t t c h t t g h t b e a r e c t e d glass,stopperod
by dle bottie. Add 425 mL dry ethylene glycol
wsence ofmols8● 距 . g " i t t n e v a l u e ja,t陀p o r t i o n osftにs m p l e mGl10Elethyle鴎 、CooltoK4°Cinicebahandbubblein 102-105g
i n t t t i n g o v e nC ,actF5 01° C0 °a b o v e m p , a d d i n g l - 2 g m y d r O uS 0s 2 ' M i X W d l a n d l e t s t a n d 1 2 h . R e a g e n t i s r e a s o n,abbulty s t a b
N%S04 per iO g sanple,Mix test smple thorou抑呼 and fllter h restandardize for each s9mes of detettnations.Scandardize&遠ly
arying OVen.Tc ttaFd醐距i赴け,keep dもandfatsincool place and
widlsodiulntaHInte・
2H20・ l mgsodiuntamte・ 2H20‐ 0｀
lS66mg
Protect fFOn ttght and血
H20・AStematively,standardize widl weighed H20 1■medlyl alcc‐

Refennce:JAOACa,429(1981). holasfo1lowtt TransferaccwatelywttaH10unt(50 mg)H20的

timtiOn vessel and ttrate to el∝
M皿 週由c end Point・
Calculaに C=
ng H20/d reagenほ
41.1.02
0&ガ を,■_2 Medloxyethancl― pyttdne
Fおcttr rcagttr Jil″
AOAC O御 ctal Method 026.12
Mb3sture and Voindl● Mater CⅢ l).
in 0383 and Fa低 (d)挽 “ 姥 MJツタ
"_,Anhydrous CHC13 mettl dcchol(1+1
Vatuum Oven― od or 2+1).
F3Rtt Actlon 1926
日nat Atton a De― rnarOn

Weigh,to nearest O,01g,5-25 g prepared test sample,contain‐

sOm鵬 叫 れ rne_,wgende監 4は 判 巨Cat nOt崎確 lt孟
. ing玉 100 mg H20,into titration vessel,dissolve in anhydrous
wm sonencun曲 ぬ oFOumy曲 胡 戴 ve田 諭 航 d述 組 .
CHC13 medlyl alcchol.Titrate with undiluted or diluted(lⅢ l)
Karl FischeF reagent to electronetric end Pcint・
CaHy cut blank
Wagh5士 住2gpFepndtestsmpleintoAlmoismedshca5cm
test using same alnounts ofrengent,thiuent,and solvents.Subtract
diameter and 2 cm tt widl dght―
flt dぃ ver COVer.Dry to con,
blank titer.
stantwelghtin vacuumovenatunifomtempemme 2い 25°C above
b P O f H 2 0 ■的 由 略 フにs s u r e , w h i c h s h o u l d 1b0c0≦n l m H g
(13.3 kPaj.Coolinefflcientdesiccator30min andwetht COnstant 戦α % = 辮
weight is amined"“en su韓 韓iVe l h dryl■ 8 pe的赴 軌。wな m‐
t i o n a l l o s s 0o5f%凱, R e p o ■
坊l o s s h w e i g h t a s 時
m dasn■
d vola‐
dle matter. 仰θ を「Pyridinebfree Karl FischerFeagents are avttlable frOm iabt
oratory reagett suppliers.)
Refemncest五減 駒 ど.Cttn,1軌 1347(1926).
泌9AC 14 247(1931);lS,560K1932). ReFerence:JAOAC 67,299(1984j.

0 2002 AOAC tNTERNATtONAL

AOAC OFFiCiAL METHODS OF ANttYSS(2000)
01LS AND FAT
Chapter 41,p.3

T a b i e 9 8 5 . 1 9 D(egnr3m1Oり
守f H20 at dmbrent 41.1.07
temperature3(°C) AOAC Offtc,a:Method o21.08
Tempera‐ Tempera‐ index of Refmct:on
ture of Oits and Fats
３ 範 勢 賛 ３ 研 範 ３ 範 ４ ４ 範 招 福 範 好 組
２

15 0.99913 0,99505 40 0。 98852 Fir3t Actton 1921

16 0。
99897 0.99473 50 0。 98807 Final Action

17 0.99880 0.99440 51 0.98762 A C e w a r D r r a t r O n s

18 0。
99862 0.99406 52 0.98715 Detemine index ofrefraction e)witt any standard instrulnent,
６

19 0.99843 0。99371 53 0.98669 reading oils at20°

or 25°
C and fats at40°
C .PlaceinsmmentsO that
20 0.99823 0,99336 54 0.98621
み 総 品 に セ ぐ 艦
21 0.99802 0,99299 55 0,98573
perature仰 .2° C)H20 hOugh prisms.Approximate teElperature
９

22 0。
99780 0。
99262 56 0,98525
corections ofbuけrOrefractometerreadingsmybemadettf01low_
23 0.99757 0.99225 57 0.98475 ing fomula:
︲ ２

24 0.99733 0.99186 58 0.98425

25 0.99707 0,99147 59 0.98375 R=R'+【 (r'― め
26 0。
99681 0.90107 60 0.9鶴 24
whereR=reading reducedtO standardtemperature,R'=re記 且ng Ob_
27 0,99654 0.99066 61 0。 98272 tainedattemperature■ T=standard temperature,and【 =0.55
28 0.99626 0.99024 62 0.98220 for fats and O.58 for olls.
29 0,99597 0.98982 63 0.98167 R e a d i n g s o f i n s m m e ndtttt ctdtyt cgainv eb■e r e d u c e
30 0.99568 0。
98940 64 0.98113 standard temperature by subsd価はng factor O.000365 foF O.55 and
31 0。
99537 O.000385 for O.58 in fomula As temperature rises,■ falls。 lnsm‐
m e n t u s e d m y b e s t a n d a r d i z Ce ,d t hw ei ot rl e tH i2 c0 a la ,t O f2
H20 at ttttemperame ttng l.3330.Any corectton found shOuld
be made on alirettngs.Index ofrefracton vaFieS With density and
I f v o l u ro 距
ftt is measured attemperamre r c 1 0 sin
e salne
t o ■ direction.

"ytt R3PaCtOmerer
ら Kg/mLj=DT'(g/mLj+0,00068(r― り
Tochargeinsmment,OpendOubに pFiSmby means of韓礎w head
and Place few drops smple on pFiSm Or,if prefered,open"sIIIS
where correctoncoettcient O,00M8is aPproximttofaCmalcOef‐ slightly by tuming scttw head and pour few dropsに
立smple intO
icient for sample is known,it should be usedj. 血 nel‐ shape aperture between pnsms.C10se pris耐鼠皿 ly by dgiぃ
ening screw head.Letinsmment stand few min before reading,sO
( b ) C d C u l a t e s p e c i f l c g r a v i t y ( s p e C i n c g r a v i t y T j o f s a m p l e 筑
m temperamre Oftest sample and ins― ent will be sane.Clean
temperamre ras fol10w主
PriSEISbetween readingsby wiping oil o賊
widl soRcloth,then with
COtton pad moisにned wih solvent g"trichloroettlene,toluene,
e・
Specttc gravityT=DwaI,Or ortttroleurnetherj,and崎 的 .
MethodofmeasurementisbaseduponObservationOfpositionof
where DT=weight per unit volume(g/mL)ofsample at rand b o r d e r l i n e o f t o t。
anl irne nreoecnlは
述t o f a c e s o f n i n t g i a s s p r i
』■0ィ=density of H20 at r(frOm Table 985.19). BFing tMs border line ink)Fleld of vision of telescope by dng rO伍
double prismby means ofalidade h folbwing manner Hold se
l f s a m p l e a n d H 2 0 a r e W e i g h e d i n s a m efltty
p y andmove
c n o m ealidade
t e r , abackwardor
t s a m e forwarduntil fleldofvision is
砲mperatu峰 (0, divided into light and dark Portion.Line dividing these po
`拍田
Ю よI line,"and,as arule,will not be sharp line but 虹 band ofc。
spedflc gravityT=7_p1/Hi。 Colors are eliコ ninated by rotating screw head of cOmpensator until
ShW,C010rless line is obdttd.AdiustbOrderline sotttitfalison
of substance d缶
POint Of intersectton of cross hairs.Read″ 沌cdy on
絶坪 α拘う″り,一DifFerence between results of minations
2 deに
scale of sector,esdmating 4dl decimal place.Take≧
3 re調ほngs,ap‐
done simultaneously or m quick succession by same analyst should
PrOtthng inに rsecdon alternately from one neld 。
t。
ther,and aver―
nct exceed 2 units of4は
decimal place.
age.Range of readings should be却 .0002.Check correctness of
insmmentasin A,or widl quartz plate that accompames it,using
Referenctt Sran筋だ M2れ て 泳 ル r ttβ Aゅ s着ザ θお ,F何島 切 ″
monobromonaphthdene,K420tC=1.6587),and make necessly cor―
Der:ッ″ど 、
セs,6th Ed.,1979,Pergamon Press,New
recton in reading.
York,NY.
Taylor,J.K.■ 彪α施 を切 A,aryrたα′晩 醜おょ a gyzerg3日 utyroreractOnerer
り,I.M.
Kolthoff&P.J.Elving(Eds),1967,hterscた nce Pub‐ Place 2 or 3 droPs flitered tesl smple on surface oflower pns血
lishers,New York,NY,Part I,Sec.D‐ 4,Vol.7, Close prisrs and触 的ust mirOr until it gives sharpest reading.If
Ch.81,“ Measurement of Densiけ and speciflc Grav‐ reading isindistnctaFterrunningconstanttemperature H20hOugh
ity,"pp 4561-4610. insmment fOr someよ me,test saH耳〕 le is unevenly distributed on

◎ 2000 AOAC tNTERNATiONAL

01LS AND FAT AOAC OFF,C8AL METHODS OF ANALYSiS(2000)
Chapter 41,p 2

41.1.04 41.1.06
AOAC Ottcial MethOd 920.242★ AOAC Ofncia‖ Method 98S.19
specnc cravitt tAppagentl of OitS (Apparentl weight per untt votumo
Pycnometer Method and specttc cravitt of Fatt and o3,3
FiFSt Action 1920 Pycnomet hthod
Final Ac8o■ Ftrst Act,on 1985
Repeated Fi『st Action 198S Finat Acdon 1994
Surp,us 1934
′
SO―AO広 C日 ●めoJ
6α″rわ″i sでをAppenttx B,safety notes on chrom拒 延id and sul‐
A , Pn 「
c r P r e
駈 c acd.
WeightofgivenvoluHle ofliqddfat atdesired temperatuFe iS de‐
A, StanJaRttz脅 宙o口ofP/cPcmerer teminedinpycnometeFpreViouslycalibratedatsametettratureゃ
Care的 1ly ciean pycnometer(ca 50 mL capacity,臨 mble Class B , A p p a F B r u S
lnc,,No.15123‐ 50,or equivaent)by ttling with chromic acid (a)わ "例 材 ユ コ 働 50nLcttdty,斡 d仰 配 宙 hcapandaleト
cleamng solu歓 〕
n and letting standseveFal h.Ewty pycIIclttterand noneter gradmed in ol° c(臨 mble Class hc.No.15123‐ 50,or
i n s e t h o r o u g h i y w i d l L Ol;l虚W i t l r e c e n d y b o i l e d H 2 0 p r e V i o uequlvdenty.caremlly
sly clean pycnOmterby n■
i ng wihch叩 直c acid
d鯛 直ng sddon andletting stand several hon.Enw PycnOWter
c o o l e d t o c a C2,0a° n d p l a c e i n c o n s t a n t t e n l p e m m e b a t h Ca.t 2 5 °
and由nse horounly宙 h H20・
ARer 30 min,adiust H20 1eVel to rOper pOlnt on Pycnclneter and
●)脇 er ba統.屯 onstanc temperature,held at temperature
s t o p p e , r e r l o v e f r o m b a t h , wり
i pwei凸
th clean cloth oF tOWel,and °
い C ) a t W t t C h d e t e m i n a t i o n iっ
s. t o b e m a d e く
w e i g h . E m p t y p y c n o m e t e r , n n s e s e vコ
e区r将w
a li直
thalooholandthen
a carrbrB」On crPycnOmetr
ether,letdry completely,relnoveed胸
『vapor,and weをh.醜 輸mまne
F i l l p y c n o m e t edrI,Foe,C覗
ently ttled H20 preVioudy coo
weightofcontttnedH20at25°C by subtrndtt weight Pycnometer
io ca 5。C below constanttenperam badl,(b),Carefttly ttsert and
fr o m its weight when illed wih H 2 0 ・
sett hemometer avoidlng any ttr bubbles,and Place in collst
B, Der― fnatrO, temperatuFe bath.After l h,attuSt H20 1eVel to prorr point
Fili ciean,dry pycnometer widltest sample rViCuSlypycnOmeter,stopper,read
Cooled to temperature(りt00.1°C.Renove
PyCnCHleter fronbah wtte dry wm ciean clodl,let cool if neces―
ca 20°
C,Piace in constanttenperame bath 30C,翻 mh じ uSt
at 25°sary,and wdgh的 はl ng.Empty pycnometeF,nnSe several unes
O n l e v e l t O p r o p e r p o i n t o n P y c n o m e t e rwith
, a alcchol
n d s tanddleneher,let
o p p e F . R e m dry
o v ecompletely,remcve
frOm ether vapor,
bath,wipe dry,and weigh as in A.Subttact wdght empけ FeplaCe themometer and caP,and weigh to O.l mg.
pycnometeF frOm its weight when illed with oil md dhttde differ‐ Vdime(mLj ofpycnometer a temperature ri yT
ence by waght H20 at25°C,asdeteminedinA.卸 面 entiS Specinc
C(appareno. yT=〔 G7■ ,伊ゃお毛Qが lⅢ 。(T一Dl
【
gravity at 25/25°

Ifspedac gravity at 20720 直 isd r判

where W and W=weight c)ofpycnometerempty and fdled with
Prowdas above andasin
A but subtract 5°
C from each tempeFature specifl乱
0丁 言denSity(grnLj ofH20 attemperature
rr20,reSpectively;れ
くり,frOmTable 985.1,;and a=mean coefrlcientOfcubic exPansion
of pycnomter e=0・ooOo10 foF bOrOSillcaに
glass,0.000025 for
soda line giass).
41.1.05 ' Der― ragFOn
AOAC OfRcta3 Method 020.213 メ″ a r 的0 乱姥″
( o O : な a n 2 r a r s“ 陥二 Weigh Kto O,l Hlg)
Spec18c enVtt Of olL empty, cllan pycncmeter wi`h cap and thermometer. Fill
Tmpedure Co― tlon pycnoneter w血 峰si sample wtth is tt tempeFtture below ttt of
ftrst Actron 1920 脇r:θ
constant temperanR bah and proceed as under Carゎ ″ゲ
F輸臨:ActSon ″θ"を控 え
,メ
Repea8ed First ActBon 1985
∽河″宅rrar″″.―Meit fat attemperattre ca
●)Fars prir at粛
10 above its mel曲
lg"nt miX Well,and let stand at elevated tem,
脱崎 aiF bubbles.Proceed as in(a).
lF spccnc gravity of ollis de腕ぱ惑at odler dlan standard tem‐ perame to eli闘
確 5°C mttbecdculated
at 2占
perature,approximate specirlcgraviけ E CarcFrwOns
as follow賞 (O CalCulate waghtper unit volume DもOf Sample attempera―
m r e r i n g / m L a、s f o l 蕊

C=C'+0,00064(r-25° C)
外 tg/mL淳
学 A
whcre C=sPeclflc gravゅ at 25/25° C,α =spcclnc graviけ at
r/25°C,T=temperature at which speciflc wvlけWas detettned, where W and,7=welght(g)。 f py●
nOneteF empty and fliled wih
and O.00064=nean coFreCdOn for C.l° test smples VT=volume ofpycnchetcr(InLh attemperature

0 2000 AOAC tNTERNAT10NAL

 0 にS A N D F A T AOAC OFFC,AL METHODS OF ANALYSS(2000)
Chapter 41,p.4

Tabie 921.08 Buwrorerractometer rB8dino3 and tndtces 41.1.08

of refractton
AOAC Otttc`at Method o20,156
!ndex of index of Meiting P。:nt Of Fate and Faty Actds
W31ey Method
40.0 1.4524 60.0 1.4659 Ftrst Actlon 1920
40.5 1.4527 60.5 1.4662 Finat Actlon
41.0 1.4531 61.0 1.4665 A 用 田卸 !
41.5 1.4534 61.5 1.4668 A ぢ∽ 力θr _ w " 夕r " 加 “″. ―
べp e c i n c g r a v i t y s h O u l d b e s a m e a s u l a t
42.0 1.4538 62.0 1.4672 o f t t t o b e e x a m i n e d . P r e P a r e b y S e p m t e b o l l i n g

牌艦督 蹴総辞総 紺lil程

42.5
附蹴総鉛淵￨
1.4541 62.5 1.4675
43.0 1 . 4 6 4 5 63.0 1.4678
43.5 1.4548 63.5 1.4631
艦 撤督翻 撒 蹴 1撤鉛離 艦
44.0
44.5
1,4552
1.4555
64.0
64.5
1.4685
1.4688
mre unnt fOr use.
45.0 ユ Det― rnarOn
1.4558 65.0 1.4691
45.5 1.4562 05.5 1.4694
46.0 1.4565 鰐 艦 襴 蝸 鮒 甘縦 盤 総 総 鰍
86.0 1.4697
1-1.5 cmdianeter and weighca200mg.Removediskswhensolli
46.5 1.4569 66.5 1.4700
狙d let Stand 2-3 h to obtain no―
lコ対,・
47.0 1.4572 67.0 1.4704
47.5
48.0
1.4576
1.4579
67.5
68.0
1.4707
1.4710
げ鞘繊隅欄鮒艦 端翻総胡艦
48.5 1.45鶴 08.5 1,4713 鮒唱&鞘温盤 鷲 樹 品淵冊盤
49.0
淵 謎 艦 鮒艦 路盟 品
1.4586 69.0 194717
49.5 1.4590 艦鮎
Plt and remove disks.
69.5 1,4720
50.0 1.4593 70.0 1.4723
50.5 1.4596 70.5 1.4726 Place 30 x3.5-3.8cmに st tube,containing dcchOl―
H20 miXture,
51.0 1.4600
inta1135x10cmbeakercOn面 胡ngた eandH20,andleaveundmix‐
71,0 1.4729
51.5 1.4603 71.5 1.4732
艦 淵 艦 騨 紺 批 盤 艦 鷲品 i 棚 樹
52.0 1.4607 72.0 1.4735 itsown.Loweraccurate dlemomece,dlatcanbeゃ adtoO.1°C,into
52.5 1,4610 72.5 1,4738 testtube undibulb isjust abovedisk.To secure even temperatw in
53.0 1.4613 73.0 1.4741 dl paFtS 9falCOh01■
H20 miXture around disk,shr gently widl dler_
53.5 1.4616 73.5 1.4744 nometer.Slowly heat H20 in beaker,constantly sdrring widl air
54.0 1.4619 74.0 1.4747 s的にam or odler suitable device.
54.5 1.4623 74.5 1.4750 When temperature ofalcohol―H20 miXture dsesto ca6°C below
55.0 1.4626 75,0
mp of的 ,disk ofttbegins to shrivel and gradually rolis up intoト
1.4753
55.5 1.4629 75.5 1,4756
やgularmss.Lower曲 げHlometerundi fat脚配icle is even withcen‐
terofbulb.Rctate themoneterbulbgendy andsO regulateheatthat
56.0 1.4633 76.0 1.4759
ca10minisFequiredforlast2° CincFeaSeintempenture.Assoon as
56.5 1.4636 76.5 1.4762 如 massbecottssPheFiCa,配 adttmometer.msis wiに y mp.At
57.0 1.4639 ″.0 1`4765 his point,temperawe ofbath mustbe=1.5° C above mp ofsample.
57.5 1,4642 77.5 1.4768 Conduct 2 additional demin倒 はOns exacdy as above.Second and
58.0 1.4646 78.0 1.4771 third results should agree closely.
53.5 1.4649 78.5 1.474 Ifedge ofdisk tOuches side ofmbe,make new dem菰 natiOn.
59.0 1.4652 79.0 1.477

41.1,09
AOAC OffBcial MethOd 920.157
Meiting Polnt of Fats and Faty Actds
prlsm surfaces.As,is greatly affected by temperature,use care to Caplitary Tube Method
keep ttmperature constant.Caremlly adiuStinsmment,using stan‐ First Action 1920
dard fluid supplied with it,convertinsmmentreading tO,fromTa‐ Ftna!Acticn
ble 921.08.
Draw ca 10 mm melted and nltend fatinto dlin― wall capi■ tty albe,
Referencett Lewkowitsch,勧 卸 比沼r確 統″ ,。
gメa″″A″ rysおげ 1_n sealendofmbewitlfatin sMll aaHle.Do notbum tt Ho
θ'生Faな 伽 ″W佐確s,6h Ed.,1,312(1921). t l l b e s c o n t a i t t的
n g邦 fc aa t 1 o6 判
めi n r e t i g e d o年r1■0 C°. A ト
ユSθa C陸 閉.あa26,512(1907). 触れtllbetotturatetlemometer卸組mtoo.2° C,so ttlowerend
泌 OAC 48,128(1965). is even就h bottom ofHg bulb.Suspendin 600 nLbter&述 ffllled

0 2000 AOAC,NTERNATiONAL
AOAC OFttCtAL METHODS OF ANALYStS(2000) OILS AND FAT
Chapter 41,p.5

With H20SO tlat t l e m o m e t e r i s i I I I n e r s e d c a 3 0Cm l n , S t a r t i n g 8-10°

below mp o f s a . m p l e , a ps pc l ya s h et ■
o incFeaSe b暉
a t lc a t t m p e 劇
O . 5C°/min,agitating H20 h batl by small sttam ofair or witt slow
st的.Take as mp temperam at which substance becoIIles mnspart
m p.l)eRteepmoerlt宜
e n t . M a g n i t t i n g g l a s s i s u s t t t o d e t e c t c o鴫 av‐
erage of3 deteminations tshOuld agree C).wihin O.5°
References:J吹OAC 69,247(1986).
E/SDA β″ぇCれを閉.,ガ と,13(IV),P,448.
晩 wkowitscL C乃 2秘た】 打″
cttragy ωttA″ 研 体ザ
θ協島r物なatt w竹彪s,6th Ed.,1,325(1921).
Wiley,P所
″″r2S att P″cr確ゲAgた″ rar
″″
A″rysな,2nd Ed.,3,309(1906r10.

コ”＞ｕョ ｕゴＬ〓“り
41.1.10
AOAC Offictat Method 042.18
Ttter Tost for Oil● and Fat8

ｕ＞０ ● く
Ftrst Actlon 1942
Finat Actbn 。
〓０ こ   ｕｎ ＯＦ Ｊ“＞ｕヨ  は可 Ｌ宝津

A SParFcarfOng rOr 7rerl随 !的 Omerm

ryP2.―Etched sにmP glass.
駒 滅 ― Mercuy.
Rangをα"″s″ た「―お江inus 2 to+68°
あ宮httsわ ChO.2° C.
-385-390 mm. WiOこ‐MouTH BoTTLE
駒 勉"鶴 抗
S,c確.―Consmcted of suitable dlemometer ttbng of eidler
pet Dねmete,plain tontけpe:6r7mm;diaEle‐
plain or lens frontけ
ter,lens frontけ
pe:CrosssectionofstemmustbesuchdlatitwHlPass
through 8 mm Fing gage but not enter 5 mm slot gage.
助 肱 <oming no― l or equa,suitable th―omtric glass.
晩 ngtl,15-25-;di劉 陀帆 5。 5mmtonotgreaterthanhatofstem.
Dな 艇 e r r 9 加 防 比 脇 げ う″め 修 - 2 °C 肥 た - 5 0 4 6 0 m l n . FiOure 942.13-TRer et:r:ng。 車 mbiy.
°
Dお 1硬 cゎ 密 C 秘 = 注 P o 脇 r O P ザ r 陸 閉 θ秘` , 2 ぇ- 2 0 - 3 5 m m .
陸柳 一 BetWeen highest gradu江 lon
ザ 競 昭 冴 弛 け
and exPansiOn chanber,10 mm. a A P P a r a r r s
物 なわ″抗a 刺姥ぇ_ 」T o p e m i t h e a t i n g 8t5o°
Cとe S p a c e お
ove
Shring ttter assembly,as shown 圧
in Fkュ
●942.18,consisting of
Hg to be evacuated or illed wih N2 0r Oher suitable gas.
2 L beakeL 450 mL wide‐
noudlbottle odght 190Hlm,id of neck,
T9P"″なん―<31ass nng. 38的 ,dにr test tube(25x100コ岡o,and Sdtter K2-3 HIn Oneo■
C″ 』比α r:θ
".― All lines,rlgures,and letters to be clear‐
cttt and
end bentin fom oflooP,19 mln dimetery.
distinct.Eachdegree marktobe longerthanreHlai面 ng lines.Gradu‐
ations to be numbered at O and at each muldPleC.of2° a Derrmrnatran
五″切確rsわた-45 mm. Heat l10 g glycerol―
KOH soludon(25 g KOH in 125 g glycerol)
施 注地 . _ 6 仏O A C n t e r T e 亜, " s e r i a l N o . d, 狙H l a n u f a c t u r e r ' s to 150°
C in 800 mL beaker and add 50 mL oil or melted fat,previ―
name or rademark mustbe etched on stem.Words・ ■5 111m imner‐ ously Flitered ifnecesstt to remove foreign substances.(Alhough
' m u s t a l s o b e e t c h e d o n s t e m , a s w e l l a s l i n e e x t e 岨
s l o ポ n g a r o u n d saponincatiOn Often takes Place dmostimmediⅢ ly,continue he■‐
stem 45 mln above bottom of bulb. ing and frequeni 五stヤ
ng 15 HttL Do not heat>150° C.)
,― E「or at any point on scale rnust 0,2°
scar2 2rr。 be≦C. When saponlflcationお
conplete,usudymttperecdyhO‐
S勉″』αだi“だo″.―Thermometer must be standardized at ice mogenecus solu血 ,cool slighdy and add 200-300 mL H20・A fter
pointandatintervals ofca20° C,forcondは on of45 mmimmerslon, complete soludon of tle soap,add with stiring 50 mL dilute H2S04
andforaveragestemtemperatuofemergentHgcolumof25° C. (16 mL H2S04in 70 nL H2Cめ ・He■ S01uはon,with frequent stirring
Cas夕.―Themometer must be supplied h suttable case on which and addidon of H20 if necessary,until layer of山SiSfaw樋COm‐
appears markings“ AOAC Titer Test,"宅セ to+68°C in O.2° C." pletely melted and dear.SimOn offaqueous acid layer,add H20 t
( M θ確= F o r i n t e r p r e t i n g d l e s e s p e c i n c a t i O n s , f o l l o w i n g d e nfaW
d d O naCidS,boi1
s 2-3航
n,and agan siphm ofFaquecusiayeL Repe江
apply:) treamnt widl H20 utt Wash H20 iS neud to mhyloran
Totallengh is over―au lengtl offlnished insmment.Dialneter is move fatty acids sc as not to include H20,and ilter whil
that measured with Fing gage or HllcrometeL Lengは l ofbulb is dis‐ 血 o u n t t P a p e r ・H e a t t o 1 3 Cげ o n h o t p l a t e t o r e m os v eo ―
f
tance from boし 的m to beginning of enalnel backing.Top of her‐ H 2 0 a n d p o u r f a t t y a c i d smibnettoO dhけ
eightof57 mm from boト
mometeris toP of flnished insmment. tOHL r H20 1S Presentin fatty acids,血 ヒre鼠1に,and theat.
ASTM 36C titerに stthemoneteris satisfactory(FisherScientnc Fill H20 batl and ttuStt0 20° C for a1l oils and fats with ttters
Co.or Scientiflc Products,Inc.). ≧35°C,and to 15-20° C below titer for oils and fats wih titers

0 2000 AOAC tNTERNAT10NAL

 01LS AND FAT AOAC OFFICtAL M日 間ODS OF ANttYSS(2000)
Chapter 41,p.6

く35°C。(H20 1eVelshould be l cm above smple Lvel.)Place tesc 鴨 bb 965.32 Wetth崎 】 的 etyL町 °n acccrdha的
tube containing fatty acids in apparatus,Hgure貿
脇18.Insert her‐
mometerto immerslon mark and equttstantfrom sides ofmbe.sur
expected hyd的
灘 期 耐
(/
HydrOXH Value Webht.o
vertically with sdFingrOdatrate Of 100complete urand‐
doWnmo‐
0 - 2 0 1 0 ±0 。
1
tions/min,starting agitation while tenperamre 10°
is≧C above titer
20t50 5
point.(surershOuldmovethroughvedcaldistanceofca3.8cm.If
preFered,stiring may be perfomed mechmically.)Continue sur‐ 50-100 3
ringuntiltemperatureremains constant30Sorbeginstorisein<30s 100-200 2
interval.Immediately discondnue stiEing and observe rise in tem―
Perature`RepOrt asはterhghest pointreachedby hemometer.Du‐
plicate deteEHnations should no― lly agree within O.2°
C.
agent blank.Place nasks on steam bath under smdard taper reflux
Reference: 泌 OAC 25,72気 1942). condenserandheat l h(Do not utt hotPlate or mantに。 )Add10 mL
H20 mugh condenser and heataddidond 10 min.晩 t ttasks cOol
withcondenserattached.Add25 mL″ ,buけl acch。1,cahalftOugh
41日1●11
condensert relnove condenser,and use rest to wash down sides Of
AOAC O甘 1● lal Method 921,09★
aask.Add l mLphenolphthaleinand dtate to faintJnk endPOint
Acetyt Value of Otis and Fats wid1 0.5M alcoholic KOH.
問rst Acdon 1921
F3na!Actlon Add 10 1nL Py占 dine,neutralized to menolphthaein,t。 に立
Surplus 1965 WdOn fOr acidity dete航 nation.Swid gently to mix,add i nL
phenolphthalein,and atrate to f血碇 pink end lttint wih O.5M alco‐
S222601■ 2&017,10th Ed. holic KOH.

Hydroxyl value=lB Ⅲ (7Xy/の 一樹 X n01述 ty x56.1′

W
41.1.12
AOAC Official Method 965.32
where y=mL KOH for acidity ttration,β i mL KOH forreagent
HydrOxy:va,ue of 01t8 and FaL
bl‐ ,C=g sg田,le foracidけ timdOn,s=回 L KOHforacetylated
A ∝守t a t l o n M e t h o d
salnple,W=g smple used for acetyla飾 .
Firet Actacn 1965
Fina:Action 1989 Rettrence既挽 R34,335《 1901).
ユ閉抗 C陸 夙 1日L627(193o.
AOCS― ACAC科 陶rnOr 五比i臨 8。C梅恥 Ad_』 財.17,31鴻 (1945).
AOCS Method Cd 13-60 KRevised 1993).
(HydrOXylvdueis numberofmg KOH equivdenttc hydroxylcon‐
tent of l g oil or的
,Applicable to fatty oils and derivttves such as 泌 似 C48,131,175(1965).
fatけ』COh01s,mono‐ md dglycerides,and hydroxystearic acid.)

A 用 “抑 a g 41.1.13
AOAC O付 :oiat Method 020.158★
e)ル ″ :冴
′″
2.―Reagent grade,redistiled筑
114-115°
C.
lodin●Ab60rption Number of Oits and Fate
(b)Accrを回げりどガ】夕.―ACS,純 sh.
Hanu3 MChOd
,J″ ″ キ ,c冴 た 伽 ゎ ガ ガ 閉β ″ 住部 .― MiX 3 volumes reagent
(C),メ
Flret Adttn 1920
(a)With l VOlume(b)justbefore use. Ftnal ActBon
(d)″‐β″ゥraた。角θt.へ eagent grade.Neutralize wid1 0.5M Surplu●1997
K O H t o f na ki n pt hがe n o l P h t h a l e i n e n d P o i n t .
(c)Arcoた o!た ″。″ss,閉 ゎだ的航能 SraP3Ja芝 初 加 :θ L-0。 5M. はu reports should speciw methOd used.)
(ShOuld be対 .5M so that blank titers do not require ref11ling of A R 即 研t
50 mL buret,)
rraれ,s :。″j“g sθど
″rfθ _Dissolve 13.2 g pure 12 in l L
″.―
ユ Den9‖"fnarfOrr

“務″魚"=挽 C Appendix B,safetynotes onplpets and acedc anhyと 景

粧艦独
盟淵缶
品継拙洲盤盤盤斜艦温
価 de,) o3mLj.TttLmaybediSSolvedbyhettng,butttluttnshouldbe
According to expected hydroxyi value,weigh fo1lowing alnounts cold when Br2iS added,Hanus L s01utton my also be prepar
ofsample into 250 mL standard taper glass‐ stoppered Erlenmeyers fo1lowH Measure 825 nL CH3C00H ttd dissolve 13.615g12 in it
to nearest Fng fOr acetylation: widlaidゃfheat.Cool,anddtate25 mLwihO。 lM Nar2S203,路 27
W e i g h 9 . 0 - 1 1 . O g t e s t p o r t i o n i n t o a n c h e r n a s k f o r t t i(stt
d t y APpendiX均
deter ・Measure anodler Portion of200 mL CH3C00H
mination。 (For fatty acids such as hydroxystettic acid,take and add 3 mL Br2・ T05 mLofdlissolution add 10mL 15%KI solu―
O.9-1.lg.) t i o n , a n d t t r a t e w i t h h e O . l M C aN la C2 uS l2 a0 t3 e・ v o l u m e B r 2 S 0 1 い
Pipe1 5.O nL reagent(o intOnaskcontaining aceけL飲 加 smple.
はon required to double halogen content of remaining 800 m
For samples with O-20 hydroxyl value,add additiona1 5 mL solution as follow革
pyndine.Mix thorougtty by gende swining.HPet5 mL reagent(o X=2ktt whereX=mL Br2SOluよonrequlred;β ョ800 xhiosulfate
(and 5 mL pyttdine,ifused in acetylation)intO ancther aask fOr re_ e q u i v a l e n t o f ol n ,m aL n d1 2 C S iO l tu hはi c s u l f a t e e q u i

0 2000 AOAC iNTERNA刊 ONAL

AOAC OFFICrAL METHODS OF ANALYStS(2000) 01LSAND FAT
Chapter 41,p.7

l mL Br2 S01utlon.If necessary,reduceコ

nixed solution to proper Table 920。150 Te8teamp!ow● l口
h低
concentration by diluよ
o■with CH3C00H・
kx】
ine vatue g Samplo・ Accuracv.ma
a DeFemrnarrOn 3 10.58-8.46 ±0 . 5
Weigh ca O.5000g的,or O.2500 g oil(0,100m.2000 g of olls 1 0 3.17-2.54 0.2
thathave highabsorbentpoweo,int0 mL 5∞giass‐stoppeFed flask 20 1.59-1.27 0.2
orbottle anddissolvein 10 mLCHC13。
Wittpipet add25 mLHanus 40 0,79-0.63 0.2
12 SOludon,draidng pipet deflnite time,andletstand30 minindark, 8 0
0.40-0.32 0。
2
shaking occasionally。 cess hme.欧
(For accurate results use exact L
120 0.26-0.21 0,1
s h o u l d bme%≧
Of amount added.)
160 0.20-0.16 0.1
A d d 1 0 m L 1 5 % K I s o l u d o L s h a k e d l o F O u g h l y , a n d a200 dd loO mL 0.1640.13 0.1
魚eshly boiled and cooledH20,WaShing down my tee 12 0n StOpper. a For100 and 15096 exceSs,res陣 は市 け.
T i t t a t t 1 2 W i h S t a nldMa rNdaO2。
S203,adding ttrdually,wmcOn_
stant鏡 通 ng,undl yellow solution tllrns amOst colorless.Add few
d r o P s s l c h i n d iocr■
9(miX Ca l g soluble stath witl enough cold
H 2 0 t O m a k e d, la id nd p1 a0 s0 にn L b o t h n g H 2 0 , a n d b 駒 o ir la r aヵ
cr ao g 御
l切成弱
t t n. ―P i p e t 2 0 m L W i i s s 0 1 u t t o n i n t o 5 0 0
while sdIIing)and COndnue dmtion indi blue entirely disappears. ErlenmeyeF COntttning 150 mLrecendy bOiled and cooled
Toward end ofttration,stoPper botde and shake vigorously,so that
15 nL 15%KI soluは o n.Tirate immediately with lM O。Na2S203・
a n y L r e m a l n l n g i n s o l u t i o n mh yC bH eC tちa k e n u p b y t t s o l u t i o n .

Conduct 2 blank deteminations along wldl deteminな

lon on 距 1=2X
smple.NumberofmLO。 lM Na2S203required by blank伊 33-2X
) minuS
mL used in detemination(9 giVes Na2S203 eqttvdent of L ab‐
sorbed by he fat or oil.Calculate%by weight ofSOrbed(12
12お where X=mLO.1〃 Na2S203requiFed fOr 12 COntent andB=mL re‐
number,Hanus methodj. 0。1.
quired for total halogen content.yCi ratio must be l.10±
ュ De― rnagOn
(β―S)X〃 X1269
12 number=
g smple
Use sample weight calculated as 2gexlttcted lodine value,or
w h e r e 〃i s n o l a rOifけ
N a 2 S 2 0 3 lSu。
は
on. from Table 920.lS9. Ⅲ

Rタッいをai Marchゴ 998

wagh m e l t e d 俺r
a ne d 側t e s t w r t i O ,n 的 i, n5 t∞on L d e 狙
stopperednaskcontaining20mLCC14・
glass‐ WithPipet,add25 mL
41.1.14 L SOlutiOn,draining pipet dendte dme.Excess of 12 ShOuld be
AOAC O鷲 :● lat Method 920.159 50L607)of amount added,that is,100-150留
b of amount absorbed.
,odine Aboo中 tiOn Number
Swirl,andletbotdestandindark30min at25° と5°C.Letoils witt 12
of Ot:s and Fats
values>150(linSeed and Perillうstand l h.
Wi,3 MethOd
同rst Actlon 1920 Add 20 mL 15%KI soluは on and 100 mL recendy bolled and
Final Actlon cooled H20・Titratethe Lwid10・lM Na2S203,942.27G22A.1.13),
A,Peagenrs added gradually,shaking consmdy undl yellow solution tums al‐
mostcolorless.(Vigorous nagnetic sttHng is convenient.)Add few
WttSあ れ確 Wれ ガθ私一 〈r)Dissolve 13 g resubttlttd 12 in l L
drcPs starch indcator,(mix ca l g soluble starch wih enough cold
CH3C00H,and Pass in dFied(mOugh H2S04)C12 und1 0riginal
H20 tO make thin pasに,add 100 mL bolll昭 H20,andboilca l min
Na2S203 dtration of solutton is doubltt
not quiに
charac胡胡c
while stiFring)and cOnよ
nue dtration until blue endR31y disappears.
colorchmge atendPolntindicatesProperamountofCL・
COnvttent
Toward end ofreacはon,stopper bottle and shake vi30rously sO that
method is to reserve somle oforiginal 12 S01udon,add sHght excess of
C L t O b u l k o f s o l u t i o n , a n d b r i n g t o d e s i r e dany d12remaininginsolutioninCC14maybetakenupby
t e r b y r e a d d i d o n s o f r e ― KIsoludon.
served Poltion.)Or:o)Dissolve 16.5 gIClin l L CH3C00H・ Conduct2 deteminationsonblanksinsame HlannerastestportiOn,
but wihout fate Slight variβ
ttons in temperame appreciably affectti―
Store in amberbotlle sealedwith paraffm undiready foruse.Wtts
soludons are sensitive to temperamre,molsmre,and light.sk)Ie in
terofLsolutiOn,as CH3C00Hhas highccefflcient ofexPansiOn.Itis

dark at<30°C.Detemine ycl ratic as fo1lows: e s s ed■

江, t h e r e f o r e ,t血b l a n k s a n d d e t e r t t n a t i o n s o n s m p l e b e

乃ガれ2 cθ "r例!.一‐Pipet 5 mL Wtts s01ution inlo 500 mL made at salne time.Na2S203 equiValent of L abSOrbed by smp
Some」 taken i mL standard Na2S203駅
Erlenmeyer contthing 150 mL samrated c12 H20 and 岱s 】utton requlred by blank●
)一nL
beads.Shake,heatto bpl and boil ttiskly 10 min,Cool,add 30 mL 旺思"n deteminai側o.calculate%by weightofL absOrbed as h
H2S04(1+49)and 15 mL 15%KI solution,and d t r a t e n158B
切 i m m e dG22
i a t 41.1.13)and
ely 19po点
as L Ilumber,Wぃ
met監赴
with O.lM Na2S203・ R e f e r e n c e : 泌θA C 4 8 , 1 2 7 ( 1 9 6 5 ) .

0 2000 AOAC iNTERNAT10NAL

01LS AND FAT A O A C O F F I C t t t M t t O D S O F A N t t Y S S ( 2
Chapter 41,p.8

41.1.15 c)A的 ,統r ttraに

夕._Accurate to封
.0001g.
AOAC Otticia:Method 993.20
(DF::確府._Asttess,coarse grade tthaman No.541 is sdt‐
kxヨine Va,uo of Fats and Oi18
able).
W i i S ( C y c ot xo aいn e r A c e t l C A c i d Sotveno Method
Firet Actlon 1903 Qめ 】し'α
jrο
y2れ
.―Capable ofEttntaining 100°
c wihin±1.5°
C.
Fina3 Action 1998
a用 倒町切 rg
′
UPA朗 OCS― AOAC ntnor e),θ ttsれ筋ゎ冴姥 rてりSθ
′ ″「-15%.Dissolve 15 g KI in
所。
100 mL H20。
(Applicable to de碗 nation ofbdtt vAue for fats and tts which
do not contain cottugated dOuble bonds.) ●)辞 葛S'a″:確 sοれrjο ■.く ア)Dissolve 13 g resublimed lin l L
acetic acid,and pass in dried(hough H2S04j Cl unhl α ttinal
働″
ガο″: Wtts SOlution causes severe bums,vapors can cause Na2S203 dmtiOn Of s01田怠 on is not quite dOubled。
(Characteristた
lung and eye damage.Use ofttme hood is recom‐ colorchange atendPcintindicatesproper amountofcl.Convttdent
mended.挽 をAppendix B,Laboratory Safety for proce‐ medlodis to reserve some oforiginal l solution,add slight excess of
dures on safe handling of acids and organic solven低 Clto bulkofsch敵 加,andbttng todesired ttterby re畑 飲温s ofre‐
(CyC10hexane). served portion.)Or:像)Dissolve 16.5 g IClin l L acetic acid
Detemine rycl血 as fo1lows:
挽2Table993.20A fordleresults ofdleinterlabomory study sup‐
pOFting acceptance ofthe method. (r)】冴 挽 ctte荘 .―Pipet 5 mL Wtts s01ution into 500 mL
Erlenmeyer nask∞ ndning 150 mL saturated Ci■ H20 and Some
A PrrncrPre
skly 10 mine Cool,
glass beads,Shake,heat tt bolling,and boil b五
Fat or oll is mixed with iodine monochioride solution to add30mLH2S04 SOlution(1+49)and15 mL 15%KI solution,and
halogenatedouble bondsin fatoroユ.Excess iodinelnonochloFide iS dtrate l―ediately with O.lM Na2S203・
reduced k〕free lodine in Presence ofPomsslum lodide,and ttelo‐
rarれaragg秘∽確 ″■― Pipet 20 mL Wtts sOlution into
d i n e i s m e a s u r e d b y t i t r a t i o n w i t h s o d i u n t h t t d f a t e t t i n g 口)拘
starchas
500 mL Erlemeyer nask containing 150 mL recendy bttled and
indicator.
cooled H20 and 15 mL 15%KI sol配 側 .Timtei_ediately widl
Iodine valuo(IV),Calculated as cg iodine absorbed Per g ofsam‐
O。 lM Na2S203・
ple(%10dineabsorbeo,lSameasureofunsaturationoffatsandoils.
a APPararws
距 l=2X
(a)OJttS‐ Sり pc″ ″滋 筋 夕FaJる 。-500田 L 3,-2X
-10m mL,forpreparing
(b)crass_srcPP2湖 湾 胸 ″ ′た"ぃ な 。
standard solutions. w h e r e X = m L O , l M N a 2 S 2 0 3 r e q u i r e d f o r l c o n= tm eL n tr e a‐n d β
(0路 れ脇力佐 』ゆ 例髭府・ 一ゼ )25 nL,forWtts and 15%Potas‐ oisnct l.10±0,1,addlor
quiredfOrtotalhalogencontent.Ifycl.蕊
sium iodide(KI)soludOns.o)2 nL,for starch soludon。(3)50 mL, Ci to oorttct ratio.
fOr H20・ s t m z e d w i i s t t l u t t n m a y b e oebrtcaiianle d f r
a)Rq″θ suppliers(Speci,wihout Carbon ttmchlcFide).
″2,Ptrr.-20mL,witlnllingnasLforcycloheXane.

Tabte 993.20A:nteriaboratory study recu3に for det― inatlon ofiodine value by Wl,3 mmOdu● ino c8rbOn tetrachiorBde 30iVent
or cyclchexanHcetlc acld(lⅢ l)803vmt

Mean Vatue RSDn% RSDRI%

Sample CTCa CHXう CTC CHX CTC CHX CTC CHX CTC CHX
SunfBowor 133.6 132.9 1.4 t7 3.4 2.3 1.1 i3 2.6 1.7
Renned palm 5 3 . 1 0.1 0.2 0.3 0.4 0.2 0.3 0.5 0.7
Cnttde nsh 109.1 108.5 0.7 0.5 1.7 1 . 1 0.6 0.5 1.6 1.0
Tung 164.5 163.1 2.0 ■4 3.1 2.5 1.2 0.9 1.9 1.5
Ta‖ow(beeD 47.2 46.9 0.2 0.2 0.5 0.4 0.5 0.5 1.0 0。 3
Crude paim 52.5 52.6 0.3 0。
4 0.4 0。
5 0.5 0,8 0.8 1.0
used fryhg 37.7 37.7 0.1 0.2 0。
2 0.3 0.4 0.5 0.4 0,9
Paim kemel 18.2 18.3 0.01 0.01 0.03 0.04 0.1 0,1 0.2 0.2
Olive 82.3 82,2 0.2 0.5 0.6 0.8 0.3 0。
6 0。
7 0.9
HS30宅 1 102.6 102,3 0.5 0.8 1.8 1.9 0.5 0.8 1.7 1.8
HSBO‐ 2 74.7 74.8 0.5 0.4 ■0 0.6 0.6 0.5 1.3 0,8
HFOど 73.0 72.8 0.4 0.4 0.7 0.6 0.6 0_6 0.9

: 討 叫
◎ 2000 AOAC tNTERNAT10NAL
AOAC OFFICiAL METHODS OFANALYSiS(2000) 0 ' L S A N D F A T
Chapter 41,p.o

Table 993.20B Test 3ampte weighほ P r e p a r e a t lke adsett e2m ibnlaat■

i o n s t o r u n w i t h

Value Test samote.o Accuracv`mo group.

Add 15 mL cyclohexane―

０
５
3 10.53-8.46
acedc acid solvent,Cい
),to each test
samPに and swin to ensure htt itis cottletely dissolved,

０
２
10 3.17-2.54
Dispense 25 mL Wtts sOludOn hto nask cont

０
２
20 1.59-1.27
s t o p p e r f l a s k , a n d s w i n拭
t。x.IElmedately sethmer for l.Oor2.Oh,

０
２
40 0.79LO,63
dependingonlodinevalueofsamplecVく 150,1.Oh;IV≧ 150,2.0め

０
２
80 0.40-0.32 and store nasks in dark at 25°
±5°C for duration ofreacton.
120 0.2640.21 0.2
Remove flasks from dark,add 20 mL KIsolution,C(b),and mix.
160 0.20-0.16 0.2 Add 150mLH20 and gradually ttrate wtthO.lM standardNa2S203
200 0。1640.13 0.2 solution,D,widl constant and vigorous shaking or mechanical stir‐
ring.Continue tittng until yellow color has almost disappeared.
Addl-2 mL starch hdcatorsolution tt flasks and condnue ti観遭ng
Storeinmberbottle sealedwithParflnundireadyforuse.Wtts until blue colorhasjust disappeared.o傷″ Ifreactton is not的田遠‐
solutions are sensidve to temperamre,曲 mre,and ttght.Store in natedby addidon ofKI and H20 W綱 ほn3min past■ O or2.O h reac‐
dark atく30°C, don dme,sttle must be discarded.The― ple must beよ mted
(C)Sθ′ め た Sttrch sθ ′″fθ ″.―Mix paste of l g starch with small within30minofrenctionte劇 両nation;ifnct,theanalysisisinvalid。 )
a l n o u n t c d d H 2 0 ' W h i l e s t i F r i n g , a d d 2T0e0S tm Lfboorl l i n g H 2 0 ・
見 98rCWratrOn
sensidvity,place 5 mL starch sol耐 on in 100 mL H20 and add
O . 0 5 m L O . l M l o d i n e s o l ub tl iu oe n ;c 的
olor Produced must be ('一 ざ)XM X1269
Iodine value(IV)=
d i s c h a r g e d b y O . 0l5M msLo dOi。u n t h i o s u l f a t.e仰θ
s ″:
olu血 vvt offat or oil
1%starch solution,comlnercialy avttlabに ,is suitable.)
(OPし rass,I協 ど'勧 "a惚 確 rttcr2θ 7)・Finely grind and dry to
where B=dmtiOn Ofblank(mL);S i tiraton Oftestsmple(mLj:
constant weighe ca(め
llo。
befOre using in D. M i m o l a r oi fけ N a 2 S 2 0 3 S O l u t t n .
( O S 材 : 閉 統加 ″r r a 々
仰 α2 S 2 0 J5 ・
F 2 の W r ″r i"θ. 判 , l M . S t a n ‐
R e f e r e n c e s″
i &PA″
P P 1 6 陸れ 6 2 , 2 3 3 9 ( 1 9 9 0 ) .
皿 ze as in】
D.
一〈め 的 施 hわriC確″ t職 ・ ―モOnCenmted,sp gr ユAOACれ ,77,67気 199o.
(o Ac施 。
l.19。(2)Acgr,caご J″rc2rr4θ 2)・Glacial.(3)S″ rrHガ
c ac泌 Rをッお泌 Ma"oた rp97
'「 COncenmted.
rrr2Sθ
O Cy挽 れ拠陀,一脚確 f Eratic results may resuk ifcydchex‐
ane is ola i.e.,contains oxidzable;髭
matt研
ど0).] 41.1.15A
一Mix cyclchexane,o, AOAC Officiat Method 994.02
い)CyCわ 確期 軸 放 actt s蕊蕊 、
and acedc acid,oo),lⅢl(V′
V).Ven,absence ofoxkttzable mat‐ Lead in Edibl● 01,s and Fats
terinsolventbyshaking 10mLsolventwih l mLsat―daqueous DiR斑真Graphlte Fumace
K2Cr207S01udon and l mLH2S04,OC)'Nogreencolorshouldap‐ Atomlc ALcrptton SpecmphotOmarBc Method
Ftrst Actlon 1904
Pear・
a srangarararrOn Orscrrrm mfag研 ねre sOrrfOn rJPACrAOcS― ACAC胸 袴moJ
Accurately weigh O.16-0,22 g dhea rlnely ground K2Cr207, OoFexEAFopte」 AOAC M的 怜or
C(d),的 nearest O.0001 ginto 500 mLflask,dissolvein 25 mLH20,
K A p p l i c a b l e t o d e t e m i n a t i o n o f t r a c e a m o u1n8tμ
巴床g ] O f i e a d h s障
add5 mL HCl,Co(ア ),and 20 nL KI solution,C● ),and rOtate to a l l け
pes ofqttde or reined edible cils and fats.)
mix.m stand 5血 n.Add 100 mL H20・ Titrate wih s(述 um
thiosulfate solution,C(o,shaking contittuouSly until yellow color
ざ夕をTableめ、02A forttt resul〔 s ofthe interlaboratory study sup‐
h a s a r m o s r d i s a p p e a r e d . A d d l - 2 mほ
Lc asttoarrscohliu配
tion,C(C),
porting acceptance ofthe method.
andcontinue adding thiosulfate soludon slowly unは
lbluecolorjust
dsappears` 6αt t fれrοS ″ A p p e n d i x B , s a f e t y n o t e s o n d l e s a f
organic solvents and the safe handling of special chem‐
N a 2 S 2 0 3 S d u t i o n mけ
o ,l M菰- 2 0 . 3 9 4 x w t K 2 C r 2 0 , ical hazards for cyclohexane.DisPcse Of Waste sol‐
vents in approprlate manner compadble with
島 DeremJnarrOn applicable environmental rules and regulations
Meittest sample,ifnot already liquid(dO not exceed sttm口
le melい A . P r f r r p r e
ing point by>10°C).PasSに stsmple mough dOuble layer ofttiter
Oil or fatis vaponzed in graphite mmace,with or without plat―
p a p e r t O r e m o v e a n y s o l i d i m p u n d e s a n d t r afom,connected c e s o f H 2to0 (atottc
n i t rabsorptbn(AA)spectrophotoneter.Pb
atiOn
may be perfomed in dr oven,ca 100° C,but should be completed
concenmtionisdeteFttnedfromabsorptionmeasuredat283.3nm.
w i t h i n 5 H 3u 0n s±) . T e S t S a m p l e m u s t b e a b s o l u控=
t eAllyl d F y . o v θ
」a S S W a F e m u t st d ub te eお, c l 価d e 如 c o m p l e t e)l y d r y ・ a APPararug
Let altered test sample cool to 68-71°
C.Immediately weigh (a)A勧 旋 力 sθrprjttrAA,w2c竹 ″ ο`韓 ″ .―Equipped wi血
amount of test sample indicated in Tお
le 993.20B into clean,的 `や
a k h e i g h t " m o d e a n d c po rn it ni tn eu ro ,u os r" “
mode and pen
500 mL nask,Ba). corder(fu11‐ . 2s);eleCtrOde‐
SCale response in費 less discharge lamp,

0 2000 AOAC tNTERNATtONAL

01LS AND FAT AOAC O肝 OAL METHODS oF ANALYSts(2000)
Chapter 41,p.lo

Tabte 994.02A3nteHaboratory study resulte ror dittd graphite fumace absorption spectttphOtOmetrlc de-3natbn oftend
edibl●o‖8 and fat3
r
Leve8 1 Leve1 2 Leve1 3

No.ofiabs 14 16 16 14 16 14
Mean 0.083 0.086 0.046 0.057 0.018 0,024
S, 0.005 0.006 0.003 0.004 0,002 0.002
SR 0.007 0.017 o.olo o.oo5 o.oo5 o,oo4
RSDr,% 6.1 7.3 5.9 6.7 11.3 10.3
RSDR,% 9.0 19.3 22.6 8.5 30.2 14.6
ra 0.014 0.018 0.008 o.oll o.oo6 o.oo7
Rb o.o21 0.046 o.o29 o.o13 o.o15 o.olo

No.ofiabs 15 16 16 15 15 16
Mean o,o84 0.090 0.052 o.o58 o.o24 o,o26
S, 0,004 0.003 0,004 o.oo2 o.ool o,ool
SR 0.018 0.019 O.012 o.o13 o.oo7 o.oo8
RSDⅢ % 5.3 3.7 7.0 3.5 4.9 5.0
RSDR,% 21.1 20.8 22.3 22.3 29.7 26,9
ra 0.012 0,009 0.olo O,006 0.oo3 o.oo4
RD O.o50 0.053 0.o32 0.o37 o.o20 o.o22
a r‐
2.8 x sr
DR=2.8 x sR・

C o l l a b o仰e
砲 ttdy data wee 臨
s t1 aけ
aはt t t y z e d u s i nけ
g t oe us 出
t s a s " e n hA ユ
S S c t t A n t t C r l7飢
3,95311990).

orPbhollow cadlodelamp;deutenum,zeeman9 oreqdvalentback‐ e)Er2crガ

く c●りβ JCap封】e of ttntttning 60±
″.― 2°
C.
ground co「 ection. (0'θ rrres._20 HL.Polyedlylene,or polypropylene‐cappedi
( b ) C r a P れ たタル 閉 a c β a r 。
開乾を, . ―P l a c e d i n A A s p e c t r o ‐
m e t a l f (r Ce le e。a n h O r O u g h l y w i t h w a m t t t r i nc S ea c i d ( H N 0 3 )
photOmeter,e〉equipped wih control lttit for temperame prO_
w i d l d1ilseは d H 2 0 , a n d d r y i n d r y i n gC . o) v e n a t c a 8 0 。
gra― ng.
― No― (9開 CrCP″r.― モ apable ofaccurately delivering (10ぃ
20●
C)C“ ″ 確 勉院 ・ l(叩 COatedj.
for Vttan spectrophotome砲
ぅ.
(d)Prarr9秘 .―Pyrolytic,cottbined with graphite tube,c),Or
(h)P,α =,S・
pyrOlydcally coated gmpttte tubet Atomization offwall orottplat‐
fomcanbe used Precislonandsensidviけ offplatfomis2x higher a魚 昭寧加西
dlan off wall. (う の Ci。
れ2滋″ .―Analyttcal pde,
(b)ル 例 筋 .二Wen deined,conta拭 ng 2%phosPhOrus.
●)筋 α,泣 ,閉 叫 み 先 → a2%(I」 v)L∝ ihin sOlutiOn h cyclchex‐
Tabl●994.02B Pttgram8 fOr graphite ttmace atomiョ
野ustng
maxlmum po_heating and gas 3bp ane.Dissolve2glecithin,(b),andddutein 100mLcydohex
(d)Jrat w脇 筋 ,一Reflned oll Kany edible),Witl Pb concen‐
Gas low, t r a t i o nl ≦μ
Step ° C Ramp ttmeo s Hold d「 le,s mL7min 8湾q チS t o r e i n m e■
de‐
e polyettlene botde.
( 0 ル ″ s 腕 材 胡 ″r わ
な「くr ) s ,たs 従ο筋あ - 1 0 理 峰 .
― ― ― U n ∞a t e d t u b e , o f r w a ‖
Dllute organOmetallic standard conOStan,5000 Elag,avttlable
２ 範
。

1 100 ・10
300 from Condnenta1 0il Company,Ponca City,OK 74602,USA,or
2 650 60 300 equivAenけwih Oil blank,(d).Prepare and store in polyedlylene or
3 1900 o 5 0 polypropylene― capped botdes,■o.υ )S,碗 だ 切 ☆:昭 sθ :“_
4_____― ―-2700_ 1 3 50 `と船 .一Diluにstock soludon,律 xr),Widlblank solution,(d),tO Pb
Pyro‐coated ttbet wm platrOrrn
concentrations(Mg):0.020:0。 050;0.100.Prepare daily in poly―
edlylene or polypropyleneccapped bodes,B(D.
２ 範
。

1 200 10 300
2 650
O Arg例 .―Puri呼≧99・ 99%.
60 300
５ ３

3 1700 0
a PrwaratrOnOrTst白 朔呼 た,Brank3研 五jo,angStangarrs
0
4 2700 1 50 Placetestsamples,blanksolution,Cc),and standardworking so―
l u t i o n の,
, C (i on くo V e n 2 a°
C t, a 6n 0d ±a l l o w t o e q u i l i b r a

◎ 2000 AOAC,NTERNATiONAL
 AOAC OFF,CIAL METHODS OF ANALYStS(2000)
0!LS AND FAT
Chapter 41,p.11

Table 994.02C Mod‖ toattone for Vartan 3peCtrophotOmeter

陥陥陥陥陥る泊尚
３
。 ． 。． 。． 。 ． 。
20.0 Normal
２

３
100 40.0 Nomal
３

３
550 60.0 Normal
４

３
550 40.0 Norrnal
５

０
550

． ０・ ０・
1.0 Nomal
６

０
2500 1.0 Nomal

０
2500 5.0 Normat

1 200 10.0 3.0 Nomal No

2 200 20,0 3.0 Nomal No
3 650 60,0 3.0 Nomat No
4 650 40.0 3.0 Normat No
5 650 1.0 0.0 Normal No
6 2000 0.7 0,0 Nomal Yes
7 2000 5.0 0.0 Nomat Yes
8 2000 Nomal No
陀 協 Use 10 rtL forlnlttn3

Rernove tom oven and shake vigorously.Weigh together 5.00ga c a t w r a F m 3

test portion and 5.00gequllibratedmatrix modiflerP Cc),in 20 mL ( r ) M e a s u r e p e a k h e i g h t o n c h t t r e c o r d e r t F e a d i n g o f d i s p l a y

襴灘艦艦騨鮒総捕鞘
‐
bottle,B(o,andmix horougtty.Proceed similarly withblank sol■
tion and standard working soluは
ons.
二 P均 脚 陶 fron orAPParatug
Perfom mib瑚
υ)Draw calibrationcurve as functionofAof3 standardwoFking
ng Steps before commencing tts切 :
solutions,C(e)o),(COHにCted for blank solution),aganst dleir Pb
(F)SWitCh On AA sPectOphotometeF and deuteriulnbackgFOund
concentrations.Autocali岐
ぶ 。ncanbe appliedwhen udng sOphis止‐
corrector.Attust iamp curent,slit widh,and ampincatiOna Set
cated equipment.
wavdength at 283.3nm.
o Rudめ concenmtiOnOftttpoHionnromcalimionarve,o.
(2)OptimiZe posidOn of graphite Fumace atomizeri set required
o Report resultt as mean ofFeSults of2 single deteminttons
prOgramoncontrol unit律 2 Table既 o2B forprogFan fOrgraph‐
Hけ
'取
り'are lneL
江 r m
障
ite fumace ato血zer,andTable卿 402Cformttiflcationstobeused
with Varian spectrOphotometerj.Place platfom(if aVAlable)in
,枇
監甜艦群艦縛
gr響 阻te tube.Pretreat tube and/or platFom by cycling through the 比 System StJranfrrw
tealperature prowm atleast lx. O亀 脚 勉 朗 み ― Direrence between resultt of 2 detemina‐
(ヨ)PretreatpipetdPbeforeeachiniectiOni npetanddlendiscard はo n s P e r f o m e d o n s a n e t e s t p o r t i oonL, buyn dsearm es aolpnee問
20 1tL cyc10hexane. condiはons should nOtexceedrepembility value calcutated mmdata
見 D e r e m r n a r r O n showninTable班 働 ,atrcalculatingpFed飾 aS hnctiOnOfde‐
腕 ned IIlean vduet
lniect 20 Lμaliquots of blank soluは
on tor 10「止 for Varian
血 わ″り・ ―Difference between results in 2 different
spectrophotometeo,standard working sOlutions,and test portiOns, (b)比 脚
D,intographite ttmace.Betweeneachanalysis,initiate temperature
progran and record absorption,A.
撫盤給器 か 鮮
1紺端 臣
鯛鮒逆
ing precision as function oFdetemined mean value.
References:A″αi C陸秘.54,67A(198分 .
A二 助 2crrCSC.1,3∝198o.
Tabre 994.02D Repeaね billty and reproduclb13ity value3 P″
″幼 ″ C 陸れ 6 3 , 1 1 8 3 ( 1 9 9 1 ) .
ユ AOACI″ r.72,34(1989);77,951,1537(1994).
Product _ RepeataЫ li町
,r Reproducb‖ lty4 R
Rをッ活夕ど=March 2000
Edible ol! 0.19x ma 0.30x m
ⅢAdopted as a Codcx Reference Medlod tType H)for atOmic absOrption
Cocoa butter______坐 lsX m o.68x m
a Corresponding mean concentratton vatue spectrophotOmtt oflead h sPecined animlor mixed attml and
t a b 拒的 P r o d u C 低
・

0 2000 AOAC tNTERNATtONAL

01LS AND FAT AOAC OFFICIAL METHODS OF ANttYSS(2000)
Chapter 41,p.12

41.1.16 41.1.18
AOAC Official Method 965.33 AOAC Offic:al Method 920.160
Peroxide Va:uo of 0118 and Fat8 Sapon‖loBtion Number
TRratlon Method Of 01ls and Fats
(Koettstorter Numbeゅ
First Action 1965 Titrimdttc Method
Final Action 1969 First Acuon 1920
Final Action
AOCSrAOAC MernOd
A . 町
向 町!
(Hθ!夕 i COnduct analysis in diffuse daylight oF in artiflcial light
shielded from direct light source.) A J c θれο" c P θr a s s f ″ れメガr θ
ヌ: ″
夕 s θ! ″
,どθ" [ ゴA θA C 1 9 ,
A.,“ ュ打f3 427(1936)].――(F)Reflux l.2 L alcoho1 30 min in distiHing nask
with 10 g KOH and 6 g granulated Al(or Al foil).Distil and collect
(a)Ac夕 rJc acJ″―cんJθr句しrtt Sθ″″rj。■.――MiX 3 volumes
l L after discarding rlrst 50 mL.Dissolve 40 g KOH in this l L
CH3C00H With 2 volumes CHC13,USP・
alcchol,keeplng temperatureく 15°C while dssolvmg alkali.Keep
め 乃 raSす 筋 腕 : ルs 】″! わら 財 ″″J 班 ― 助 s s o l v e e x c e s s K I
soludon in glass‐stoppered botde.Or,(2)Crush 40 g KOH in
in freshly bolled H20・
Excess solid must remain.Store in dark.Test
185 Hlmmortar.Add45 ggranulatedCaOand grind m二 xhretopow‐
daily by adding O.5 mLto 30 mL CH3COOHtCHC13,0);hen add
der.From l L alcchol add 100 mL to mortar and transfer to nask,
2 drops l%starch solutlon,tniX Ca l g soluble starch with enough
insing mortar widl several more poH3ons.Add remainder of
C 0 1 d H 2 0 m m a k e ,t ah di dn 1p 0a 0s にn L b o l l i n g H 2 0 , a n d b o i l c a
alcohol to flask,shake mょxmreと 5 mdnl and invertbeakeroverneck
ing).IFsolutiontums blue,requiing>l drcP O.lM
l min while sti「
Of aask.Repeat shaking several times dudng day.Next morning
Na2S203 tO diScharge color,prepare fresh solution.
niter s。
ludon into clean,dry,giass,stoPpered bOttle.
C)助 沈″ ″れ39Sttesra滋 ″Wれ ,:θ な.判 .l and O.01M.Pre‐
2 7 G ″
P a r e a n d s t a n d a r d i z e .a1s. 1i3n)既
A .For O,01M,dilute
ュ DH_rnarOn
O.lM widl freshly bolled ttd cooled H20。
E DetermrnarfOn AccuFately weighca5 gniteredOil into250t300mLErlenmeyer.
P i p e t 5 0 m L a l c c h o l i c K O H s o l u t i of nl a is nk め
,山
なd n g p l p e t d e f l ‐
(■)河 αrs ttθ体.二weigh 5.00±0.05 g testportion into 250 mL
stoppered Erlenmeytt Add 30 mL CH3C0011tCHC13,a), nite time.Connect nask widl ttcondenserandboil undlfatis oom‐
glass―
and swirl to nssolve,Add O.5 nL saturated KI solutionP(b),範 m Pietely saponlfled(ca 30 minj.Cool,and dmte wih o.5M HCl,
Mohr pipet,lec stand widloccasional shaking l min,and add 30 mL936.lSGをgA.1,06j,直ngphenolphhale血.Conductblank佐使用対‐
H20・S10wlyは廿ate with O,lM Na2S203 WithVigorous shaking undi 面 m a l o n g w 血 t h a t o n ― p l e , u s i n g s a l n e p l p e t f o r n e a s u r i n g
yellow is almost gone.Add ca O.5 mLl%starch soluは on,and con‐ KOH solution and draining salne time.
unue tiratiOn,shaking vigorously torelease all 12 frOmCHC131ayer,
until blueJust disappears.If範 .5 mLO。lM Na2S203iS uSed,repeat
de断 航 nation with,01M Na2S203・ Calculate saponincatiOn number
(mg KOH required to saponl守 l g fatj=28.05伊 ―D/g Oil
Conduct blank detemination ttly KmuSt be幻 .l mLO。 lM
Na2S203)・Subtract from test portion dtration.
w h e r e =β
nLO.5M HCirequiredbyblankandS=mLO.5M HClre‐
P e r o x i d e v 1a l iu e q( u航i v a l e n t p1 el r oo xr if da xJt 〃
kj g=x 。
ざ
q 直 配 f o r s m p l e .
1000/g sample,where S=mL Na2S203(blank COrrecteo and材 =
molariけNttS203 S。 lution.
O Margarれ 2.―Meltfatbyheatingwihconstant銅ringon hot 41,1.19
plate atlow heat,or heatin air oven at 60t70° C.(Avoid excessive AOAC Ofric`a:Method 920.161★
heat and long exposure■40°C.)When completely melted,hold in Sotub:e Acid● in OI:3 and Fats
w a m p l a c e u n t l a q u e o u s p o H i c n a n d mhoasvte osfe tsdoeld赴 . FinBt Actlon 1920
D e c a n t o i l i n t o c l e a n b e a k e r a n d ufglhl tWehra ttmtaЮ
n No.4,or Finat Actlon
equivalent paper.Do not rcheat unless necessary to obtain clear丘 1- SuFpru3 1965
trate.Proceed as in a)・

Reference臣
ユ A れ θ】C 確 統 肋 c . 2 島3 4 5 ( 1 9 4 9 ) . S2226船 0,10th Bd.
AOCS Medlod Cd 8-53.
JAθAC48,175(1965).
41.1.20
AOAC Offtcia:Method 920,162★
41.1.17
insolublo Acids
AOAC Offに ,a:Method 940.27★
hner Numbeゆin oils and Fats
(H●
Thiocyanogen Number
Ftret Acticn 1920
of 01ls and Fats
Final Action
Firet Action 1940 SuFptuS 1965
Su■olus 1965

Sce 26.026r26.027,10th Edt S夕β26n31,10dl Bd.

◎ 2000 AOAC iNTERNAT10NAL

 AOAC OFFiCiAL MttMoDS OF ANALYStS(2000)
0'LS AND FAT
Chapter 41,p.13

41.1.21
AOAC Officla!Method 940.28
Faw Actd3(Free)
in Crude and Refined oi:s
Tltratlon Method
First Action t940
F3nat Action

HttrOnar cOrOnseed PrOrrcrg nsccrarOPAOAC Mb船 oビ

(a)=″ cr″冴βοJFs.― ―Weigh 7.05 g well‐ mixed ollinto 250 mL

flask or4 oz bottle.Add 50 mL alcchol,prevlously neutralizedby
adding2 1nLphenolphthaleinsolutionandenougho.lMNaOHto
produce faint permanen:pink.Titrate with O。 25M NaOH,936.16
●22A.1.12),witl Vigorous shaking undl peFmanent faint Jnk
a p p e a r s a n d p e r s ils tmsiと
n.Reporl as percent free fatty acids ex‐
pressed as oleic acidi mL O.25M NaOH usedin hは ■い。n coHe‐
sponds tO this percent.
(b)あ 瑠 防″ θ体.―TO ca 50 mL acoholin clean,dry 150 mL
aask,addfewdrops oftheolland2 mLPhenolphmlein.Place nask
in H20 at60-65°C until wann,and add enoughO.lMNaOH to pro‐
duce faint pemanent pink.Weigh 56.4 g ollinto the neutralized a‐
cohol and titrate with O。
lM NaOH,9托 16(ュ ,c Appendix A),
occasbndly waming and vigorously shtting mixmre unは l same
faint peHnanenc Pink appears in supemate alcoh01,MuldPly mL
O。lM NaOH byO.05 andreport as percent free fatty acids exPressed
as oleic acid.
Free fatty acids my aso be expressed in ttms of acid vdue
(mg KOH necesstt to neutralize l g oll)。

Acid value=percent fFee fatty acids(as 01eic)xl.99

41.1.22
AOAC Offtcla:Method 972.28
F a t t t A c t d s riont aOけi l S a n d F a t s
Hexane Di● t[1!ation
F10ure 972.2卜 Ltquidaltqu:d extractor.
Fittt Actlon 1972
Final ActBon 1985

■OACrAOい Mb防 何 a Paggm的

A . P r r n c r p r e 50%.Dissdve 50g
0 1 l o r t t i s sn ae pd o 胡
and,after acidflcation,fatty acids and 瓦齢 盤陥昭解盟陥「
u n s a p o n i n a bmに
atter are removed by condnuous extraction witl
hexane.欧 trachca cfsecond saPonirled tt Or fat wid10ut acidiflca‐ 認 濫群盛灘 塩 協鞘 撒 側諸韻描
tionyields unsaponirlable matter.Fmy acids are deteEninedby dif‐ ,TX 79008ゆ‐
68,USA,″
hexane has been t
ference. 融苦
ユ A p p a r a t w s (C)肘 rrog2″ ∽ ″ り g夕″的減cnO″ 姥 肋閉 .

(O Li7″ 泌村均″芝 筋 確Craぇ司 s2夕ngure 972.28.With magnedc a PrePararran Or Test sampre

stirring disk, cOndenser, and refluxing flask (available as
No.4112A‐ E,Tc―
Shoot,Tx 773ぃ
y Howe Scienti範 ,PO BOx 7246,Cut and
,USA).A ttmilar liquid―
liquid extractor is avAl‐
胡酬 靴糾納 斜 1撚密輔晶 艦鮎
able from Konに s,Vinetand,NJ 08360,USA.
縦 撤盤路艦鱗鞘盟鞘靴胡隠盟齢
:艦路脚 wam
(b)Caれ う:″ガθ″ み冴 ″陶 r控ぇ― Sargeni_welch
盤締ifnecessary
ag″ ガご ざ
=こ

婆 瑠 胎 8洋 智
卜00° '研 ewvde証 ,的 四 宙徒 mag確 伍C鋭 Hng筑
H20,and 縄オ ・ ペ to d
( C ) 打夕a r れど 碑α″r r 2 。
ァθれをr 姥 確 乃 r 均 伽 所 宅 角a 抑 確 . E T c r a r y F aA ■
c r J g J an ng ど
aponfrrabre ttarer
″ゎぇ― Sargent‐
(d)Rο,ary gyaPο Welch Noo S‐31210‐06,or Acidify freshly saponiied sample by slowiy adding 6M HCl ca
equivalen4 which releases vacuum with N2 0r He. 5 mLjtoreacdonvessel.Test withmethyl orange indicat

0 2000 AOAC tNTERNATtONAL

0 1 L S A N D F A T AOAC OFttC3AL MttODS OFANttYttS(2000)
Chapter 41,p.14

hatsoluはonisstrongly acid`Fillinnertubeofextractorwidlhexane, 41.1.23

immediately insert into position in reacdon vessel to prevent AOAC Otticial Method 925.41
backnOw Of aqueous solution,and attach condenser.Add 100 mL AcidBげ 。latiiel 3inand O‖Fats
hexanetoround‐bottomaask,addNo.20Sだ boilingchporinverted (RetcheH卜Meittt and Polenske Va,uo8)
mp tube,attach to side am,and begln renuxing Witl magnetic胡 回 Tltrimetric Method
rotttng at minimum speed.While checking to see thtt enulttons do Firat Actlon 1925
notise hreactionv弱 鴻1,gradutty incMserotationtoca5001Pmmd Fina3純 tion
降flux rate to 200-250 dropJmin.輸 血 距 refluxing with stiIIing ん 用“ 抑 的
2.Oh.Cool round‐bottom aask and ilter ttlu血,if cloudy,trough
(う Sθ ″:″“わだ的ガ宮g sο れ!わ払―く1■ 1).Prepare as h 936.16
2-3 clnpaperon ttueddsk.Remove solventeiheron steambtthwitl
Gec Appendix A).
streamofN20rinrowevattmめ 単 現 )町1噌Vacuumcmtiously l面n,
O C 々 呼 r 0 4 - S O J a! わhw ―
J“Add 20 mL l+l NaOH solution
t l e n f u l l v a c u u m w i t l n aC s kb a it mh m rm si et di iO nd 82 げ
0min.
to 180 mL pure glycerol.
Cool,IeleasevacuumwihN2,dry Outersuば 猛eofround‐ bottomaask,
(O SiI商 R “ め擁 . ―G t t N o . 6 , C a r b o m n d u m C o .
and wdn・Repe狂80° assure conmnt
C vacuuln stepe rnecessary,的
崎 ghL Cac田旋 %tod faw ttplus unsapomflable mamrtcrude E De― rnatrOn
SubmtValue fOFunSaponiflable ttmtt F,to 6t油血 total
飽呼 減 dS)。 Accurately weigh 5■ 0.l g smple into dean,dry 300 mL
mty acids. round‐bottom aask.Takeく 5 g ttEwに wih certain fats or denva_
dves,e.g.,距 tylated monoglycendes,ha require more alkali for
見 tJngaponWabre Marer
complete sapomflcation tttm available widl prescribed anount
Starting withfreshly saponifledsample,D,extract(wid10ut acidi‐
gttCer01_soda solution,A(b).Add diluent oil wm ReicherteMeissl
Flc述oo aSinE.Coolround‐ bottomnask and transferhexane sch‐vaue範 .2,e.g,cottonseedoil,to sampleaask toprovidetota of5 g
don to 250 mL separator,dnsing gendy with 10 mL hexane. sample plus diluentoil.Add 20 mL glycero14oda soludon and heat
DeteHnine unsaponiflable matter as in,33.08(受241.1.39), witl swining over flane or ireproofboard‐ covered hot plate until
Shake vigorously,...''but use hexane in‐
ParagFaph l,beginning“ c o m p l e t e l y s a p o n i f l e d , a s s h o w nt ubrye mbよ
ecoming perfecdy
stead ofether. dear.NooilshouldremainonsurfmtwallsofflaskaFeWet'y SOlu‐
don.晩 t nask c001to ca lCЮ °C(caS minj and dssolve contents h
Reference:ムサ θAC SS,84駅1972).
135± l mL ttendy政 減ed H20 Witl minimun loss ofH20 VapOr.

rЦtt「

A〕
〔 t B ,

Figure 925.41-Apparatus for determining ReicheH卜 Meissi and Polenske valu● ●:(A)wlth rubberstoppers and(B)Wlth
giass iointS.

◎ 2000 AOAC iNTERNAT,ONAL

 AOAC OFFIC,AL MFMODS OF ANALYSiS(2000)
0,LS AND FAT
Chapter 41,p.15

(135 mL H20 1S COnveniently measured frOm 125 mL Erlenmeyer

淵器
∫網協総猛
亀 軋°
m make l
prevloudy c倒 的ratedめ dehver 134.6± 1.O g H20■ 25°C.)Add
6mLH2S04(1+4)如 d15piecesoFSiCt Disは1,usingapparatus witl
鱗撒「
呂
dimenslons given inFigure 92541.(AdapteFmaybeusedfordisdlト
ing in的110 mL volumetric flask.)Rest nask on JeCe Offlreproof
S u P P O r t w i t t crehnoに
leF cmdiaIIleter,and regulate name sc as tO
collect l 10mLdistillatein30±
2min(measurehme frompassage of
f l r s t d r o p 1 ol fa dt ie s は
f r o m c o n d e n s e r t t r,elceetitviinngg dniass‐
ゅ
t i l l a占
tpe 山
i n t o r e c e i v i n g m fp le aF sa kt u ar te にo f C c. a 2 0 °
When l10 mL has disは1led,substitute 25 1nL graduate forreceiv‐ 鰐鮒 艦 鮒鵠 さ
equivalent).
鞘 靴 識 ヰ f株
ing flask,remove flalne,and disconnect distill菰 on head 8om con‐
densero Mix withOut violent shaking, mnerse nask contaidng
出s は1 l a t e a l m o s t c o m p l Ce t He 2l 0y 1i 5nn航
, 1f 5l °
iter hough dry お風
だ〔鞘路冊濫警療職 推欲盟甜盟
9 cm aloderately retentive paper(S&S589 White Ribbon is
総ほ 襴
satisfactory),and titrate 100 mL nitrate with O.lM NaOH,
936.16(s夕 をA.1.12),using Phen01phthalein.Sclution shOuld 認錯縄 辮 酬 :縦
Slore in refFigerator.
remain Pink 2-3 inin.
O rbrtts,`切 り ″ro鏡 S。′
″r:ο
″.―Approximately O.o5M.Di―
R e i c h e Mr et i‐s s i v a l u e = 1 . l x lute 60 mL iscPrOPanol―KOH s。 lution,(e),With 440 mL
( t i t r a t i o n o f tsiamltnipOlne ― O f b i t t d o X 5 / g s m p l e isopropanol and 500 mL nethyl alcohol.store in amber Or“
Life―
time Red"Pyrex bottle.
Remove remainder ofsoluble acids from insoluble acids on ilter
0 冨 り 湖 冴″ 胡 閉 仇 ― D i s s o l v e 3 0 0 m g t t m o l b l u e i n 2 5 m L
by washing widl血●e15 mL pOrtiOns 15° C H20,eaCh Prevlous, O.05mcoholたKOFI solutioL o,and add 75 mL isopropanol.
ぃ s t t h o u t t c o n d e n,s2研
5 mL graduate,and l10 nL recdvhg
C Preparatran OrFattyAcrJ 3orutrOn
nask.Dissolveinsoluble acidsbypassing tree 15 鵡ons mL脚 neu‐
鯛 alcohol through niter paPert each PortiOn having pFeV10usly (め ざq″ ″"a″ jο れ― Tmsfer O.5-0.7 g wel卜 mixed,meited fat
ito 20 x 150 mm test tube with lip, and add 5 mL
passed hOugh condenser9 25 mL graduate,and l10 mL receiving
flask.Tirate cOmbined alcchollc washings lM wihO。
NaOH,using isopropanol― KOH solution,(o,and boiling chip.Place tube tO
phenolphmlein. d e p h o f 5 c m i n b o n i n g H 2 0 b a t h 2 0 m i n t o,saanpdoenviafpyo的
‐
rate iscPropanol,leaving solid soap.sttpper,and analyze within
Polenske value=KtiratiOn Of smple― はtration ofblank) 48h.
x5′gsmple
●)晩 姥秘 協 !′ ο″ゲ ?″ 御rリ ザ ac材 ″ ガ″どわ れ ryzを々 埼
scaP.一Add 5 mL isoprcPanol―
KOH solution,(e),t。10 mL H20
仰 θセf Uniess these directions are fo1lowed in every1,repro‐
det拭
and2 drops ttmol blue soludon,(D,in smali beaker or aask.Add
血 dble results cannot be obtained.)
H2S040や 1)drOpヤ ise,withconstantstiring,ulは
linidalbluecolor
References:JttθAC 13,255(1930)i38,319(1955); tums to yellow,Orange,and Flnally red.This volume H2S04,mea‐
39,355(1956);40,50,(1957);68,249(1985).
灘 灘 盟 総 齢 瑠 艦 靴 艦 鮒 テ 鞘
acid soludon,use ofmore H2S04tO hydrolyze scap isindicaに ditop
41.1.24 ofchromatographic column should be yellow.)
AOAC Otticia:Method 9弱 .04☆
(C)靭 胸 い な ゲ 5o4Pa″ ″ oFracrjθ″ ゲ raり αC施 卜 ■ dd to
Butyrl●A●ld soaP,whiLcoohngtubeincoldH20,numberofdttPs H2S04(2+1)
(M。1。
Per cenり
in Fat
Column ChmrnatOgmphlc Method
F3rst Actlo■1950 辮 継 縄 脳 憚 襴 継 盟 韓 灘
Final Act3on
t a i n e d . A d d 1 0 m L h e xbauntey―
l a c c h O ll ust。
ion to tube and hOr―
Surptus 1994
oughly mix widl giass rod.Aqueous phase should cling to whi
A.ApPararws precipitate of K2S04,al10wing easy decantation of solvent soludon
Chttθ″昭rogttβ力:Cr″bこ、 一千Fuse15cmsecdonof3811modtubing of fatty acids.This soludon of acids is ready for c
to20cmof22 mmttbing,wttdlinmmisfusedto 5cmof7 mmtub― a PrPararrOn Or cnrOmaruraPnfc Corum何
ing,with dttp tip.
Overlay 35 gPIttngHlaterial widlhexane―butyl alcch01 solution
E Feagents in mortar.Mix witt pestle to fom slury.Place smali glass wOol
phg loOsdy in constricttdendofc01umn and gently tamp into place
(a)S''cfご αご:法――Mallinckrodt Specialty Chemical Co.,
with glass rod,Place ttnger over constricted end ofcoluHm and add
No.2847.Heatin shallow Pan or evaporating dish 18 h at 175°
C. hexane‐butyl alcohol soluton unは l reservoir is half RHl.using tea‐
Store in desiccator or dghtly sealed cOntAner.
spoOn,underlay prepared slury beneath solvent.Jiggle spoon up
(b)β rcttοご白鉛0′grec″―grycθ′J。′″rJο″.__E)issolve 700 mg and down along side ofreservoirp and iet flocculent siury setele tO
bromocresol green in 700 mL ethylene glyc。
l by warlning on bottom of colu醐 .
steam bath,c001,and add ca200 mL H20・Prepare O,lM NH40H After adding all packing matehal,remove flnger and let s01vent
by diluting ca 6,6 mL NH40H t01 L wilh H20・
Add 40 nL ofthis flow out andPacking llatedal settie.Apply 5-101b(34.5-68.9 kPつ

◎ 2000 AOAC tNTERNATiONAL

OILS AND FAT AOAC OFFIC,AL MttMODS OF ANALYSIS(2000)
Chapter 41,p.16

air pressure to top ofcoluHln to speed up flow ofsolvent41.1.26

and facili‐

ｒ
囲e unifompacking ofslury.ReLasepressuttjustbeforelastpor・ AOAC Offioial Method 957.13

ヽ
“
onofsdventHnksintocoluI皿
.IfCOluHlnlooksunifom,packed, Ac:d3(POtyun3aturated)
itis ready for use,ifnot,add more hexane‐butyl alcchol soludon to in Olis and Fats
reservoir and again apply pressure as before.Prepared coluEln spectOphctOmetrlc Method
should have now rate Ofca 3.5コ正ノmin wihout use ofpressure.If Ftrst ActBon 1957
鼠ow rate is<3 mWmin,addmorebromocresolgreen― glycolsOlution Ftnal Action
to pacttng material,and FemiX.Ifflow mte is》4 mmin,add mOre
H2S103 tO paCking natenal,and ttmix.Hexane― butyl alcohol solu‐ AOCS― AOAC MetnaJ
t i o n , r e c o v e r e d d u F i n g p r e p a r a t i o n o f c o l uA l .m ,Pm ar y r bn e c u rs eP d r s eu b s e ‐
quendy to prepare cher cdumns or for chFOmatOgraphy. NatuconiugatedcOnstituentsaredettminedbymeasunaguV
E C h r o m a t o g r a p n y o r F a r / A c r a absorption at specified wavelengths in Purified solvent.
Decant卸 町e n t s o l u d o n o f a t t a c i d S O n t oP ta oc pk ば Nonconiugated POlyunsaturated constituents are partially cOniu‐
edCOlulmand
i m 賦戴 a t e l y s t a r t c o l l e c t n g e l u a t e i n 2 5 0 m L E rglaetm ee dy b ty
t Ah se tstoionngga,isCn0 1KSO0H1―
ution,andabsoFpt10nsofcot
faWaCidSOlutioncompletely setlesimpacking,washdow■ 施 他 of gatedcOnsthentsareredete航ned.The%ooniugateddiene,dene,
mservolr wm血 磯 5 mL pOHions Lx_‐ butyl alcchol ttuttL ttt tetrae距,and pentaene acids are o,latedfrom
c抽 predeteminedaby
each w翻 略 sink into PckittbefoFe reming申抗 ,Yellowbmd simultanecus equ江 lons.
shouldalwaysbeobservedatwtttopOfCOluHm;dlisbandcontains昨 Method is applicable to detemination ofpolyunsat
a n i C a c i d s e 2 S 0 4 a
海 n
) d
a n a
d C i
W d
i l s u
n l
o 免
t H D v e . 能 noic曲的ugh pentaenoic,in aIBlmal and vegetable fats contttdng
鴫
Ifproduct contains butterfat,second disは nct ye1low band due to only natural or cttisomeFS,OnlyaE10untsofprefomedcOniu‐
sma■
butyric acid appears and slowly IIllgrates down coluHln,breaking g a t e d m a t e r i a l , a n d o n l y s m i l a n o u n t t O f P i g m
away from top band.Long‐ chain fatty樋田s,X36,paSS rapidly tion may undergo considerable change during the alkali
t h r o u g h cloml ■a n d d o n o t f o m y e l b w b a n d s . E l u a t e v o l u lisonedzation.Medlod
tt be‐ is not applicable,or is applicable only witl
tween eluは on oflasttrws oflong‐ chttnacids and「 lrsttraces ofbu‐ speclflc Precautions,to hydrogemted oils or odler tts containing
tyAcacidwillbe20430mL.Whenloweredgeofyellowbuけ HC acid rrarts isOmers of unsaturated fatty acids,to ish oils or sittlar fats
zone is l cm from lower end of chromato野 containing acids more hghly unsatumted tt pentaenoic,to crude
翌古cCOlum nge
mtiOn∞ 陀軟x Frsthctioncontainslongtchainlids ttdisu調 cils or smples containing pigments whose absottdon undettoes
け
changes during alkali isomeFiZation,or Ⅲ fats and Oils containing
100±10 HL.Next 120 mL hction contains htyric acid.
large anounts ofprefomed conJugated fatty acds.
ぇ r r t r a t r O n O r F a t y 宮F
A 酎r a c r r a g
a A p P a r a t w 3
Add l dropttmol胡 距 胡 utt fo昭 曲 Ю nLeluatbeingdmted.
Titrate each taction tt fntpemarent awearance ofpupletblue end (a)rsο,″協 jθ ″aPPararA.ぺ ce Figure 9野 .13A.)(r)c伽 _
″ 防 れ - 1 8 0 ± 0 . 5 C°. C a p a c i t y s u t t c i e n t t t i m ‐
磁 だ 切 η t t a r″
POlnte using ca O.05M KOH,o,diSPensed tom 50nL bwtfor ist
mtion and tom 5 nL h劇 merse25 x250mlnPyrextesttubestodepthof l14Hun(4.5 in.).F
捉仰 duated inは01 mL forSecOnd ttB
飲鴻.EndpOlntforeachmdiOnis鈴 ばpbutお subiecttOfadingbecam badlliquid use Fisher Scientiflc Co.No.B‐ 219 badl wax or DC 550
OfC02 abSOrption.TheC02efttdisrgligible rtimionぉ collducted
mpldly witlline事 闘 価 .rnece職 叩 ,d胸 的 Rmybecariedouth
C02‐tCe amosphere whichtendsめ make血 述 m values mom repro‐
山rble.(PasS air mugh 20m aqueOus KOH的 1胡愉 .位m mugh
H20,and ana■ y into timion n容 に)
Blank corecticns are not requttd.Alkali added to ttE101 blue
soludon takes care ofblank,
ExPresS butyAc は a tc r通a t i o n a s % o f s u m o f d l e 2 品眺st i 位, c a l c u ‐
latedtoneares10.1%.E泣リヮたr Long‐ chainacidtimtion‐26.lmL; lmm
〔
OJ‖ 口
Fy
buけHC acid dtration=2.83 nLisum=28,93.Mble%buけ riC acid=
2.83x100/28.9=9.8.

Referencett Jttθ
AC 39,212(1956);召 附,531(1957).
︱︱ Ｉ Ｆ

CAS‐107‐
92-6(butyFiC aCi●

41.1.25 出AHOHET[R
AOAC O甘 :●
lal Method 922.11★
Faty Acids Gaturated and unsaturated)
in 0113 and Fats
Lead SalいEther Method
First Action 1922
同nal Action 1964
surprus 1965
Figure 057.13Arconstanttemperature bath and
挽 夕28.039,1lth Ed. acceseoriec.

◎ 2000 AOAC,NTERNAT10NAL
AOAC OFFICtAL M口 MODS OF ANALYSiS(2000) 0'LS AND FAT
Chapter 41,p.17

い・
︱
Ｎ
いＮ

・
〓︱
EttD ViEW

BOTTOH
Figure 957.13ロ トーRight,distr`bution head●.Left,8nanSfold.A:l dimension3 are in gnm.

l a c e b a t h i n i n As bu ol xa wねi t h i n s u l a t e d aux on steamb批 3 L Removefrom sに畑 lbathiFeplaCe reflux mbe

F l u i d , D o w C o l n i n g C, oP 中
cover having holes for stiH● r and cork supports fOr test tubes. 胡 hdisuningmP,75° comecting tllbe,and髄 瞳 止Place nask in
(2)確 sr″仇れ― Pyrex,1lpped,25x250 Hlm. H20 bah Or執 減 能 陀ating mande and disは 1,collecting disは
1late h
c)Dttrib″ :町力御な.一Toittesttubes snugly.Tubing incenter2 L Erlenmeytt store h glasttstoppered botde.Absoluに
」 dcchol ls
ofheadhasboth endsopen and2 small holes 25 and38 Hlm,respec‐ sぶsfactoFy if OfcomPmble puFiけ (aS ObtainedorpuriFledj,
dvely,from botton.(Sβg ngure ps7.13B,right.) ω rs。 。cr竹 (22,イ ‐筋 B的 中を閉腕2).―NIST cttned grade
“)M物 切 ら 広 二 Wit1 10outle低 ,卸 戯lcomecLdめ 50mmlongcap‐ 研 spectralgrade(闘 HiPS66Co.,Speci』 け ChemiCJS,Po BOx968,
i l l l y t t l b e , l I I l m b o r e , C a″F p u ni ug sw e9 d5 o7 u. d1,e鍋t s .
leFt.)い Borger,TX 79008‐ 0968,USA).Hexane or cydohexane is satisfac‐
(う 「irrag切加脇 加 2姥ぇ 一 ConSmctfrOm 6 mm od tdbing bent tory ifA requirements are met,Purify as follows Placeca9 cnglass
in shape ofU‐ mbe,heightca380m吼 widthca30m.Hll Hlanom‐ wool above stopcock atlower endof80 x4.5 cm fllter mbe.Add ca
eter ca half full witt H20 COntaining l drop methyl orange and 30 cm sihca gel(Grade 12,28t200 mesL Fisher No.S‐ 157,cr
l drOpH2S04・TOadiustnowofN2,attachca90cnrubberhttng to e q u l v a l.ePnOめ
urisooctane slowly into mbe,f11ling
o n e o f N 2 0 u t i t t s o n d s d b u d n g h e a d . F i l l 1 0 0 m L g r a d u a tCOFk e w iSttpper
tl covered widl Al foil loosely in toP of tube and let
H20 and invertin cOntttnerofH20・InSert end ofmbbertubing un‐ isooctane iltermugh ttlica gel,collecting h 2
dergraduate.TumonN2 Supply andmeasurerate ofdisPlacementof new silicagel as onen as necessatt toyieldis00ctane confOming to
H20hgraduateo Rateofnow shouldbe50-100mmin.Mark level A ll航t.CheckA ofl cn layerofthe isooctane agttnst H20 thrOugh
ofliquid h manOmeter at tlis flow rate. range ofwavdengdls usedinanalysis.A coml翠 edwittH20 Setat0

●)物 ガ ″ 筋 '切 確 え一coveFing mge of 220-360 nn wih mustbe幻 .070 at all wavelenghs,and dle resultantA versus wave―
wavelengdlscale readable to O.lnm.BechanModel DU‐ 7側O is sat6 length curve EluSt be smooth,Otherwise refllter and recheck A.
istttory.AdiustH21mpwimnocellinbeamsometerbalancesatlow‐ (C)Pθ rassれ閉 町 att.ttc_gゥ ∽ rs。筋rゎた._6.6%KOH for
est posttL wavdengh osually翌 11回ぃ .Shl Widlhs are航位 劇for 25 min i30medZationo Weigh ca 750 g edlylene glyc01 inめ lL
absorption measurewnts at 262,268,and 274 nln wheFe,at flnal bal‐ round‐bottom Pyrex 8ask.Close wih hollow stopper containing
ancing,コ苛uSments EluSt be O.8rO,9E聞砿 short outlettube and inlettubeゃ aching to bottomofflask.Connect
sθrPr,ο, cをどJs.―一 i n l e t t t bf er e te o N 02 2 ‐
S 0u .p 0p 1l %y 0( 2く) a n d b u b b l e N 2 trO
(c)Aう QuartZ, matched pairs in lengths
liquidduring a1lstages ofprepltt to exclude ali ttF andtO agitate
l.000士 位は万On H偽 如 fmedwitlH200riSoom■ e,mustmatchwithin
001 A unt liquid slighdy.Place in di bath at 1004150°
Ciraise bah tempera―
mtt t0 19ooc and hold 10 min to dry glycol.Remove badl and let
a Feagents badltemperature drop to 120°
C.Slowiy and carefully add 60 g 857o
(a)M夕 !肋れ班 αttοれ惚.―Check A ofl cmlayerofmethyl alco― KOH,keeping schは on under N2・Remm to 01l bathireheat bath to
hol agttnst H20 at220■mand山 的 ugh range ofwavelengtほ used in 190°C and hold atthis temperature 10 min.Remove nЮm bath,and
analysis.A at220 nm mustbe<0.4 and curve should be smooth incool.Renove hoHow stopper md close wih sold stopper.Store h
range 262-322 nm.Otherwise puttfy as follows,and recheck A. refrigerator at caC 4°
K40°D under N2・
Place 2 L methyl alcohol frOm new drum or glass bOttlesinto Check
3 L KOHcontentby adding 10.00g KOH― glycoisdution toca
double‐
neckstandardtaperdisは
1 ling flask,add10gKOH and25gZn 90 mL medlyl alcchol,neutralized witl lM HCito PhenOl
dust`Stopper one oudet and Place FeauX Condenser血
oher,and re― e n d P o i n t . T i t t a t e w i t h s t a n d a r d i z eld PliMn kHjCuls tu ad宜
isap―

0 2000 AOAC tNTERNATtONAL

0にS AND FAT AOAC OFFiCIAL MttMODS OFANALYSlS(2000)
Chapter 41,p.18

pears.%KOH=mL xmolぶ ty x5.61/weight solutiont lf%KOH islution is required,make siHilar dilutions ofblank,Ifblanks dO not
n o t 6 . 5 - 6 . 6 , d r y s o m e g l y c o l b y h e a t i1n9g0 °
Cu nadse Fa bNo2v■
e , check,repeat tests,increasing aow of N2・
and ttustt0 6.6%KOH.
(C)Fθ ″歿,力C明 “8"eap的 “ぉ研“頑 ed由 ,2ア%【 0■ r5加 あ
(d)Pθ ttSS'″ ″ ぃ とr餌 免姥―g,cθ どsο′ ″rFο″。 一‐ 21%KOH for K O 確ア 協 ! 航 ―P r o c c e d a s i n o , e x C e p t u s e 8 0 m g s m p l e a n
15 minisomenzation.Prepare as h(c),eXCeptuse 210 g 85%KOH.
21± 0.1%KOH― glycol solu敵 加,and isomettze exacdy 15 min.
T的 阻に,and打 じ
ustt0 21± 0.1%KOH,if necessa呼 .
見 sprrrOpnOran4貯 わ 白四Jrng3
(O Nirragtt gth―Pretpu点
ned grade,範.01%02・
1fpolyunsattlrated fatty acidconttituentis known tObe absen,or
a PrePararran Or ttst sanPre
itsabsenceisconfrmedduringanalysis(nOmaximundetectedatits
Melt sample carefully on stealnbah,sdrhoroughly,and niter if
a n a y d c a l w a v e l e n g,的
no SpectrcPhOtometeric reading is required
not dear.
i n r e g i o n o f istoswお onandnoα attlttregionneedbettludedin
E DerermrnarrOn equations.Forexmple,cottonseedoilis known tO cOntain no pOly‐
(■le 6.6%KOH method is Prefered when samples cOnttn only
unsaturated constituents nore highly unsatunted than dienoic
linoleic and linolenic acidsi dle21%KOH medlodispreferedwhen O i n O l d C 抵組) . H e t t e , h a n a l y S s oおd
f山l , m e t t u r e m e n t s a r‐
e崎
smples contttn linoleic,linolenic,and arachidonic acids.When quiredOnly江 233 nm,andequttbnttcalculatelholeicお dcOntent
requlres a ontt a tliS Wavelengul.
pentaenoic acids are present,21%KOH Inethod must be used.)
0 ) 乃 r C 弱 略 a 材 ″ ウ郷 α触″冴 a c 施. ―I n めi n L P y r e x CorrectioB forbackground absorption isused ody when measur‐
Cup tdiameteF 14 m,height 10 mmj weigh enough 対on的 test脚 ing very smll mces Of fatty acids.When fatty acid is
JveAreadingofと 0。 2tca200mg).DroPcuPinto75 mLisooctanein m o r e t t t r a c e , b a c k g r o u n d c c r r e d O n s如 da rteh enicrt r e q u i
150 mL beaker,and rotate beaker to dissolve smple,w― ing if u s e m a y l e a d t o e r r o n e.oWuhse nr eα
soufl 低
any polyunsatumted
necessary. Cool to room temperature, transfer to 100 mL consdtuent証悔risttxttzation,atits analyは cal wavelengh,is>1.α
g l a ssst‐O p p e r e d v o l u n e t r i c f l a s k , d i l u t e t o v o l u m e now ibackground
t h s o l v ecorrection
n t , a n d should be mde.No background cOrec‐
血 x t h o r o u g h l y , M e a s u r e A i n u v F e g i O n a g a i n s t n ations t c h eare
d toc e be
n cHlade
o n ‐ater isomenzation with 21%KOH.
t a i n t t lg v es n。t , d i l u t i n g s o l u t i o n ( a n d / O r u s i n g o d a働
l e r 確wrarOng
cell lengtt if
necessary so tlatobservedA is O.2-0.8).Alsc take Rttxttngs onboth
s i d e s o f s P e t t n e d w a v d e n g h s t o d e t e m i n e t h a t H l(o i m u m ル:ウ
a xAbso,〃 is 乃 p r∽ ‐ α'夕 C船 :"″閉h― calculate a for
r e s可電
e n t . C o m p o n e n t i s c o n s i d e r e d a b s e n t i f m a x i n u l n i s n o t f o u n d hdetemination,Eくa),uSing subscripts,
each wavelengh recorded in
characteristic reg10n and no fmercalculadons are mde 体re‐ in曲 233,268,315,346,to designate each individual a.
gion.Measure at:Dienoic,233 nmi面 enoic,262,268,274 nm; 為 岬 町 ,a=Aん 4や 脇 A=ob― edabscFbanC,う =clllengdl
tetraenoic,308,315,322 nmi pen的耐 c,346 nm. h u 工与組 C = g t e s t p o H i o n t t d i l u t i o n u s―e dm efnotr.A ―
(b)Fθr″θ″cθ ″7θ
可叫ra姥 り″″sa初″'2″αctas,こ び79【θれ I n f o 1 l o w i邪
n g, s eu qb us 漁
c F i p t s 2 , 3 , 4 ! a n d 5 r e f e r
25材れなθ た―weigh loO mg(的
腕2れ密r,。 n earest O,5 mgjtestpor‐
triene,temene,and pentaene oonstituents,I● speCtively.
don into l mL Pyrex glass cup.Weigh ll.0± 0.l g ofthe 6.6%
KOH一 glycol solution into 25 x 250 mm PyrexにSt tube,Conduct Absorptivity at 233 ntt corrected 的甲forお 的n
と2blankdettnationswith testportion.Covermbe widldistribuト b y a c i d o r e s t e F g2 r= Oa u2 p3 s3 = α 勾
ing headandcomectto acap111叩 tube on malllfold,Adiustf10WOf
where a。= 0.07 for esters and O.03 for scaps and fatty acids,
N2tOpemit斉 Ot100mLN2tOpaSstroughtubrmin.晩 t N2SWeep
through tube l min toremove d,hen imEletteto depthofl14m Absorptivity at 268 nm coFeCttd for background absorption=
inbah at180± 1° C.Check temperature frequently and standardize a3=2.81e68 泌 “2α+a27'〕
themometer at frequentintervals.
After20Httnremovedistribuは ngheadanddroP l mLc■ pcontain‐ Absorpt市 i t y a t 3 1 5 n m ceoc「 t e d f o r bgarよound absorption
ing weighed sample into tube,note exact tirne,and reJace head. = a 4 ‐ 2 . 5 E a 3 1 5班 %十“ a322)]
Dropclean i nLcupintoblanks.Keeping distFibuhnghead inplace,
A b s o r p t i v i t y a t 3 4J6= α3 n l4n6= α
renovetube fttmbath,swirl vigorously few s,andremm tObathiaF‐
ter l min,examne soluは on.Ifclear9 return tobatliifnot clearDindi‐
α確どac泳 .―r quantides within bttkets of a34 0r α
c a t i n g i n c o m p l e t e s a p o n i f l c a t i o n , s w i d t u b e 2 -(D6研
3 d m e s昭 a n d r e m m t O
are O or negative,no characttsdc absorption mlaxim are present
bath.At i min intervals,repeat swirling until saPoniflcaは onis comb
and coresPonding consdtuent is reported as absent.As prefomed
plete.Keep bath temperature at 180± 1° C.
constituents are usually present in small amounts,bⅢ kground ab‐
Exacdy 25 min afterdropping testportion into tube,remove from
sorpticn corecはons are usually requlred.Ifiarge amounts of pre‐
bah,wlpe clean,and place in 3 L beakerto cool,condnuing to pass
fomed constiments are present,dlis method is not applttable.
N20Ver Soludon.ColdH20bahmay alsobeused.After cooling,re‐
HoweverP RO baCkgroundcorecdons aretobe appliedto readingsin
movehead,andwashlowertubttg with 20 nL騨 点ned mehylalco‐
pentaenoic region,346 nm.
hol,collecting washings intesttube.Wash宙 thmethyl dcoholfrom
beaker;do■ot use wash botde. COniugated diene,%=C2=0・ 91%
Insert glass strringrod 30cmlong witlcwvedend atbottominto
testtube and move cup up and down to mix solution.Transfer solu‐ C o n J u g a t e d t r i e n e0,・ 4% 7= aC 33 ‐
宜on to 100 m正̀glass_stOppered volumeHc flask,dilute to volume
ConJugated tetraene,%=C4=0・ 45α4
with pudied methyl alcchol,and mix thoroughly.Measure A asin
( a ) , u S h g K O Hg ―
l y c o l b l a n k a s r e f e r e n c e . I f d u t i o n o f s m p l e s c ‐ COniugated pentaene,%=C5=0・ 39宅

◎ 2000 AOAC iNTERNAT10NAL

AOAC OFFtCIAL METHODS OFANALYSiS(2000) 01LS AND FAT
Chapter 41,p.19

(c)Attα 〃 !ツ'rf2ざ
ル ″″ ″ 9ZJZgar″ cθなr加御 句 こび%拓 θ■ Linolenic acid,%=y=1.102,3 0'88a'4 0・ 02aを
25"j″ お0閉昭rirarわ み一 calculateattbreaChWavelengtlindetemi‐
′ Ar劉 】由徹Ddc acid,%=Z‐ '4 1・
naはon,(b).α =A/bc. 1.65α 8位 告

Absorptivity at 233 nm corrected for coniugated diene acids Pentaenoic acids,%=P=1.982's

'233 α
ち=α
originally present=α 2 ・
°3 c)s御
? 脇 ∽ 純 力= あどβ2 材α夕確 a c i な q ′“″筋 θW , c 肱 あ た, 昭: カ
A b s o r p t i v i t y a t 2 6■8c印
o r e c t e d f o r b a c k g r o i S°
°rpはon and 律 た″ね冴 as切 %C20 50%C22 paraFzο た αc施 ,_
forunttstroyedcottugateddettdお
α'3=4.03レ'268 /ta'2α
+αを 一α3
7'1十 Linoleic acia 79=X=1.o9α
'2 0・57a'3 0・ 26a'4 0・ 03α告

A b s o w t i v ia け
t 3 1 5 n m c odr rfeocrにb a c k g r o u n d a b s o T P t i o n a n d ′
L i n o l e n i c a c-i0t.%8=8ya='14.+1090・
a5を
α
for undestroyed cOniugated ene=tetra
2'4=2.06Ea'315 /Kaち a4 無 賀価d° dc acid,%=Z=1.65ど4 1・ ′
67α
随Ⅲ2'322)〕 5
′
(d)「 ο″Cθ″
ブ“garをどac,どs,5.5%【 θ rr,25碑 :, Pentaenoic acids,%=P=1.45α 5
,sθtteriz筋 ほ ― 〈r)wi!肋 “!bact抑 例 泌 co椛 一
c的 な 。
c)め 朋 Cθ閉?。 S'わ れ 一

Bm血 ぬ d,%=X
= 1 . 0 8 6…a1七 t t u g a t e d P 0 1 y u n s a t u r a t e d Ca 4d +d Cs 5, % = C 2 + C
2 6ヰ0
. 3 2 4 K a 'おθ 8 .α
4 昭
3 b α )
3 崎 T C t a l C °
ttt nOnconJugated polyunsaturated acids,%=X+y+Z→ P
Bnolenic acid,%=r=1.980げ 268 2268) 4.92惣 を15 23崎) T°

Ar抵
肛
血ぶcお
こ%=Z=469tah5
p 名
鴻 批警 !老
局増餅(腎が4珊岳
赳fヽ
路斜
あ
(2)降 ■防 地 ″ 腕 ∽rrgα 一
t t α″ 昭 ″ ″ど。 Saturated acids,%=79 total fatty acid
まC a t t% dc 十
o n i u g a t e d% n ao cn ic do ゃ
nJugated aci
hnoleic a c i t % = X = 1 3. 20a48' 36+ a0 ・
4'0 2
メ4 ■ (%前
H瑞 '4 盤 ‐
枇繊甜盟か 艦鞘品キ
諸縄穏
鑑品統れ臣登i49勿
'2'4 Reference臣 XC
ンLて 42,354(1959).
江edlod Cd 7-58.
(oAう prPrルfrをsル ″″θ″ οげ町 西2″Cθな!J勉 秘 ■ 2r%斉 θ■ AOCS B40,487(1957):4名
'for each waveLngth 233,268,
r 5 ″れ なa 胸″乾a 此加. < a l c u l a t e α C A S5 ‐
06t32‐
1(araChidodc acid)
315,and争拓nm。(If nO mximum is found,report comPonent as O 60‐
CAS‐ 33‐3 oinoleiC acio
without her measurement or caculation.) CAS-463‐40‐ l cinOlenic acid)
112‐80‐1(deiC acio
=α'23 3 %
Absorptivity at 233 nm=a七 CAS‐

' 2 6 8 a 2 6 8 4 1 . 1 . 2 7
A b s o r p t i v i t y a 't 3 =2 α
6 8 n H l = α

☆
樹
緊柵 P縛謎
艦]生
盤盟￨;l繁 [;:5 a31S 6 α 346 A風 革
"経
PrBParatBon of mthyl Esters
t Action 196S
(F)「 θ,cθn」
i″garをど ac,どs,2ゴ %【 θ″, r5閉 :れ ,sθttβr‐ Fl時 Finat Actlon 1984
jttri9PL― ric medlod will not
<SpectroPhotomeに 的にndate
d間 be―

"mm
tween acids witt same number of double bonds but different chain ,acn,何 lt ut sα 1nce
t t um re pr確 9 6 5orsur′け万●AcrJ

齢 鴨路鞘: 能
kllown,assunle that tlese Pentaene acids alChain lengh
re present inis un‐
equa1 41.1.28
amounts,and apply third set of equatlons.) AOAC Omcta:Method 960.33
7res c例 勉 i円筋 g C20″ Zraを″β αc拡 ― Fa8け
(r)sa初 Acids in Oi:s and Fats
'3 0・ 口 ratl°
n J Methw Este碍
Bnoに た 延d,%=X=1`09a'2 0・ 5花 2位 竹ゃ0.00勿 '5 堵
3oron Trinuorsde Method
Linoledc aciこ%=y=1.102'3 0・ 88a′ on 1969
4+0・312's First Ac宙

財S
i崎 c淵 開蹴協 。
r
Arachidottc acid,%=Z=1.65が 4

P e n t a e n o i c a c i d s , % = P = 1 .′
51 4 α A.角 ;ncipぬ

8C22″ ″勉御 2 aC拡 ―

(2)挽 印 icS∽ ″勉:4:■ Glycendes and PhosphOlipids are saponiaed,and fatty acids are
liberated andestentted in presence ofBF3 CI』yst for further analy‐
Linoleic ac畑 '4 0・
,%=X=1.09a'2 0・ 57a'3 0・2(筋 12att siS by IR,96534E(sα 2 41.1.36),or CC,9G22F(影 を41.1.29).

◎ 2000 AOAC iNTERNATtONAL

 0にS AND FAT AOAC OFF,C:AL METHODS OF ANALYSiS(2002)
Chapter 41,p.20

Mcthod is applicable to common animl and vegetable Oils and Tablo 969.33 DeterFnination offlask size and amount of
fats,and fatty acids,UnsaponiFlables arenotremoved,andifpresent 6 r e a g e n t f r o m a p p r o x i m a t e t e s t s a m p l e s
in iarge arnounts,may interFere with subsequent analyses.
0.5M NaOH BF3 Reagent
Method is not suitable forpreparation ofmethyl esters oFfatty
靭
ac‐ Flask,mL mL mL

５
０
ids containing mttOr anOuntt of epOxy,hydroperoxy,aldehyde,

５
100-230
ketone,cyclopropyl,and cyclopropenyl groups,and coniugated

５
０

７
250-500
Polyunsaturated and aceけ leniC COmpounds becausc of partial or

０
０

９
complete deslluction of these groups. 500-750

０
０

２
750-1000
B,Appararぃ
(a)魚″Crゎ ″メaSる。-50 and 125 mL aasks witl ouに
r Standard
taperjoints
(b)COn滋 ぇ髭,一Water‐ cooled,redux,with 20-30 cm jacket solution isstiliteJd.Add additiOnal saturated lution to NaCis。
ncat
and standard taper innerjoint. heptane solution into neck of nask.Transfer ca l mL upper heptane

a Reagents

(a)助 ″″ rr"″οガ2修 昭4ga″,-125 g BF3/L medlyl acoh。 1.

盤器 沌
品,替i絣紺詳
盤拙軸胤淵 糾准
centation of 5-10夕らfor GCt
Avalable cOIIlmercially or pre脚 時 as follows:Weigh2 L flask con‐ To recover d.ry esに応,transttr aqueous and hepme phases tO
tAning l L methyl alcohol.Coolin ice badl and widl dask sは ll in
250 mL separatoR tttract win tw。 50 mL pOrtiOns petroleum ether
bath,bubble BF3 frOm Cylinder trough giass tube hto methyl ac伊
op 30-60° C)or hexane.Wash combined exmcts with 20 mL Poト
h o l u n t i l 1 2 5 g is so お
rbed.Work in hood.BF3 muSt be nowing 的 n S H 2 0 u n t i fl r ea ec i td O‐ m e d l y l r e d i n d i c a 町
t odrr.ODursy O v e r 価
t h r o u g h g i a s s t u b e b e f o r e i t i s p l a c eldi tiins arnedm ouvneは
d f r o m Na2S04,Fllに
, and evaporate solvent under strealn of N2 0n Stealn
methyl alcohol to prevent hquid from being drawn into cylinder
batl.Ifsmple isく500 mg,reduce vOltunes of solvent and H20・
valve system.Gas should not flow so fast dlat white fumes emerge
M o r e v o llぶ
e esters may be lostifevaporation is Prolonged
frOm attk.Reagentお stabに2 years.(Ca″ r肋 :RemoveBLvapO職 streamofN2 iS t00 vigorous.For IR spectroscopy,te航 nate evapo‐
wih effectve fume removal devtte.Avo超 contact with Sttn,eyes, ration as soon as solventis removed.ForCC,mehodis apPlicable to
and resplratOry ttact.)
鮎 け a c i d S W 8i t Ch ≧a t o m s , i f s o l v e n t i s n o t c O m p l e t e l y r e
●)筋 例物湖 たs曲 秘 ル 競 w協 北及 -0・ 5M.Dissolve 2 g
(b)乃 rrarけ ac,お.―Add fatty acid to aask,hen add lu‐
BF3S。
N a O H i n 1 0 0 m L m e h y l a l c o h o l c0o.n5t%aHi2n0i・ng≦
鴨鴨 t e p r e ― はon,and condnuc as in(a)With 2 min boiling under reflux.
cipimにOfNa2C03 fOming onlong smding may beignored.
(C)″?ra″夕._Pw,as deに rttned by Cc.Iffatty acids cOnttn―Referenctt Attl働 秘 .38,51(1966).
ing≧
20C atoms are absentinfator oil,hexane maybesubsdmted. IUPAC 2.301,7h配 .
夕r り! ″″S p rr ″
i 9判
み .1%in a)%alcohol. ユA れ. θ: ! C t t F L S O C . 4 S , 1 0 3 ( 1 9 6 8 ) .
(d)財
ユAttC S8,396(1975);62,709(1979).
(e)Mr″ g夕み―モ Ontaining tt mg bふ g.
ユ 働 比筋脇″ogた 247,63(1982).
Check newbatchesofreagen低 ,PartiCularly BF3,bypreParingand
chromatographing nedttl esters of puFe Oleic acid.If extraneous
R2νおedi】拓arch r997
C22 regiOn witt BF3),reieCt reagent.
peaks appear(in Cが 中A d o P t e d a s a C o d e x R e f e r e n c e M e d l o d c y p e I D f O r a c
, Prepararo″ ron thauttde oflin01eate(in the fom ofgiycerides)in SPecial foods,
(6α″rfθ rざ 22 Appendix B,safety notes on disは1lation and petro―
leum ether.)
41.1.28A
(WOrk in h00d.Wash all glassware imlnedately after use.If
AOAC Ofnctal Method 996.06
fatty acids contAning>2 double bonds are present,reinove atr
f r o m m e t h y l a l c c h o l a n d f l a s k b y p a ls os fi Nn 2g fi en W 田
s正
Fat tTotat,saturattd,and unsaturated)
nt‐
rea■
utes.Methyl esters should be analy2ed aS Soon as Possible.If nec― in Foods
essary,heptane solution may be kept under N2 in refrigerator.For Hydrolyttc Extraction Cas chromatographic Rttethod
Fi英st Actaon 1996
pr010nged storage,sealin ampule and stOre in freezer or add equiv_
Revised 2001
alent of O.0057o2,6‐di‐ 姥rr― butyl‐4-medlyIPhenol(BHT).For IR
analysis,solventremovalmustbeascompleteasPossibleiforCC, (Applicable to dete■航nation of fatin foods`)
5-10%solution is suitable.)
Precise weighing is notrequired Testsample size needbe known 6α″rわ″f BOron tttfluoride may be fatal ifinhaled.
only to deteHnine size offlask and amounts ofreagents,according to
Table 969.33. S夕2 Tables 996.06A―C forthe results ofthe inに
rlabOratory study
supporting acccPtance ofthe metlod.
Sample ca 350 mg is preferred for CC.
(a)Fθ ″Jttrs α ″″ θjrs.―_Add salnple to flask and then add A . 所n c i J e
methanolic NaOH sol賦 On and bOiling chp.Attach condenser,加 d F a t a n d t t t y a c i d s a r e e x t t a c t e d m m f o o d b y
r e n u x u n t i l f a t g 1 0 b u l e s d i s a p p e a r ( u s u a l l y 5 - 1 0(aCidiC
m i n ) hydrolysis
. A d d B F 3fOr
S 0 most
1 u ‐prOducts,alkaline hydtrolysis for dairy
tion froHi bulb or auto■ latic Pipet ttugh condenser and continuc products,and cOmbinatlon for cheese).Pyrogallic add is added to
b o i l i n g 2 m i n . A d d 2 - 5 m L h e p t a n e t h r o u g h minimize c o n d oxidative
e n s e r degradation
a n d b O iofl fatty acids during analysis.
l■un longer.Remove heat,then condenser,and add ca 15 mL satu‐ Tttglycende,航 undecanoin(Cilか mal standard.Fat
,iS added as inに
rated NaCi soluton.Stopper nask and shake vigorously 15 s while
is extmcted into cher,tlen mehylated to fatty actt Hlehyl esters

◎ 2002 AOAC iNTERNAT10NAL

 AOAC OFttC,AL METHODS OF ANALYStS(2002)
0,LS AND FAT
Chapter 41,p.21

Table 996.06A intertaboratory study resurts for deteHninaJon oftota3 fatin food by hydr。 ,yuc extracti。_aS Chromatography

No`oftabs
SR
R° RSDr,% RSDR,% excluded
Wheaい based cereal 1.96 0.208 0.260 0.582 0.728 10.6 13,3
peanut butter 46.3 086 2.37 2.41 664 1.86 512 2′
10
Fish sticks 11.2 0.354 0.541 0,991 1.51 3.14 4.80 2′
10
Parrnesan cheese 26.5 0.540 4.17 1.51 117 204 158
Chocolate cake(baked) 13.3 0.929 1.95 2.60 5.46 7.00 14.7
Fruit snack 3.92 0.087 0.146 0.244 0.409 2.22 3.74 1710
Ground beef 21.9 1.11 182 3 1 1 5.10 5.06 8.32 1′
10
Yogurt _____一 一一―― ― 1.46 131 0.367 0.622
・ Blind dupHcates 8.98 152 __
め 「= 2 . 8 x s r

38品
危括脊培
Rher cochttn
st o,Gttbbsに

( F A M E s ) N n g B t t h m e t h a n o l . F A M E s a r e q u a n d (D確
t t v d昭
yαmれ
″ eた
伊″ 防::￨た
gg秘 ガ否.
sured by caplllatt gas chromatography(CCj agよ nst Cilo mtemal
(D BaSttrs.―Aluminum and PlastiC・
standard.Tot』fat is caculated as sum ofindividual fatty acids ex―
(h)ざれα修″″α々r baれ.一MAnt対 ぷng 70-80°C
pressed as tltiglyceride equivalents.Samted and mOnounsamated
fats are c』
culated as sum ofrespectve faw aCidS.Monounsawated (1)革e碗 院 挽.―Supporting common glassware.
fat includes only cお
fOm. 一With nitrogen strealn supply,maintaining 40士
C)脇 姥″院れ。
3.Appattrtrs 5°
C.
a αあ S 陸 え D e s i t t d b r M o i o n 拭併 C e n t d 価
ge
( a ) C a s 説拘z 3 a r a g r a P h r EC qo u―i 碑
胡 with hydrogen aalne
b逮 糾
lonization detector,capill切 rCOlumn,splitmodeiniector,OVen tem‐
perature prograttng sufflcientto implement ahold‐ raE町>hOld se‐ O)均 θ″れた,″θわr attcR c2″均駒g2.―Opdonali mttntttning
600 x g.
quence.Operating cOnditionsi tempenture(。 C>inieCtOr,225;
detector,285i initialに mp,loO(h01d4m拘 悦 ― P,3°9mini nnal (m)C″ ツjりごて 加昭Ctめ″θッ御.―Mantaining 100± 2°C.
telnp 240;hold 15■ tn;ctter gas,陀liurn;flow rate,0.75 mWmin; (ゆ 路 ″傷 材協βえ
linear velodty,18 cJtts「it ratiO,200,1.
(O CaSaゅ crsゎれ勉院S.-25 mm,porosity“ A,"extra coarse,
(b)Capj脇 ヮ ∽ 脇 み一 駐 P観 減ng the FAME Pair of adiaCent 175 μm.
PeakS OfCは3andC201andtheFAMEtrioofadiaCentpeaksofC22;:,
(p)恥 惚 ″脇れ ッ 協な.―Aboutll nL.
C2は3,and C20“With a resoluはon Ofl.O or greater.SP2560 100 m x
(q)Pれ を
れo!たcJο
sをどraP caPs._Withpolyvinylliner,to ntvials.
O.25 111m with O.20 11m rllm is suitable.
秘たrメ蕊 , (r)駒 作 ′
拓′′
FcO″ sマ
βra.一TO nt vials,
(C)Mり 。
(d)説 oppごrs.―Synthetic rubber or cork. C Reagenrs
(e)〃 ヴθ″ :″律″′ 可略 夕院 S姥■ (a)ル r9gα】たac拡

T a b l e 9 9 6 . 0 6 B i n t e H a b o r a t o r y s t u d y r e s u t t t f o r d eo tn e mo if n as 宙
aturated tttin food 町
s t hu yt d格r o 3 y t t c e x t r a c u o 障
gas chromatography

No ofiabs
% Sr SR
P RSDr,% RSDR,%
RC
Whea卜 based cereal 0493 0.0391 0.0522 0109 0146 7.92 10.6 1′
10
Peanut butter 8.72 0.257 1.31 0.720 5.07 2.95 20。7 1′
10
Fish sticks 300 0.223 0.572 0624 160 7.44 191 1′
10
Parmesan cheese 174 0.311 2.46 0871 6.89 1.79 141
Chocolate cake(baked) 356 0171 0.304 0.479 0.851 4.81 8.55
Fruit snack 1,27 0.0242 0.0362 0.0678 0,101 1,90 2.33 2′
10
G「ound beef 998 0.636 1.39 1,78 3.89 6.38 139 1′
10
0.986 1 0.170 0.476 2.1 17.2
a Blind dupHcates
°
「= 2 8 x sr

i猛1龍
脊Ъhe「
cochttn o「
Orubbsほ
式

◎ 2002 AOAC,NTERNATiONAL
0'LS AND FAT AOAC OFFlC,AL METHODS OF ANALYSiS(2002)
Chapter 41,p 22

T a b l e 9 9 6 . 0 6 C i n t e H a b o r a t o r y s t u d y r e s u t t s f o ro nd eotfe mmionnao宙
u n s a t u r a t e d f a t i n f c o d町s ht yu d偽r O l y t t c e x t roaト
c“
gas chromatography

No,ofiabs
S型邸plla X,% Sr SR μ RC RSD「 ,% RSDR,% exctuded
Whea卜 based cereat 0280 0.0320 00560 0.0896 0,157 1 1 . 4 20.0
Peanut butter 22.3 0.411 1 1 1 115 3 . 1 1 1.84 4,96 2′
10
Fish sticks 133 0.165 0.313 0462 0.876 902 171
ParFneSan cheese 6.43 0.271 1.09 0.759 305 420 17.0 1
Chocolate cake(baked) 3`79 0.413 1.27 1.16 356 10.9 33.5
Frul snack 1.03 0,0453 0.734 0.127 206 417 67,7 2/10
Ground beef 8.38 0.930 1.96 2.60 5.49 105 22.0
Yogurt 0,345 0.0222 0.0542 00621 0152 6.42 15.1 1 ′
1 0
8 Blind duphcates
b r=2 8x sr.

38品
穐島Ъ
予培Rherhran
C∝Grubbs
o「 test

(b),ノ ″Cれど ο″ ,c acたと-12M and 8,3M.To make 8.3M HCl, Ctュ。 ―tridecanolc methyl esに 「,Cl.。‐ tetradecanoic methyl ester,
add250 mL 12M HCito l10 mL H20・ MiX well.Store atroom tem‐ C性 1‐ 9‐ にradecen01c methyl estr,CttЮ ― pentadecanoic methyl es‐
perature(20r25° C)。 ter,Clil‐ 10-pentadecencic methyl esに r ,Cit。‐ hexadecanoic metlyl
j″
(c)A"初 ″ れわ泌え冴滋 .-58%(w/w)` ester, C16:1‐ 9‐hexadecenoic methyl ester, C17:0‐ heptadecanoic
rり!ど
(d)Dj夕 え一Pudty aPpropnate for tt extraction.
れ夕 m e t h y l e s t e r , C 1 7 : 1 ‐1 0 ‐h e p t a d e c e n o l c m e t h y l e s t e r ,
一Attdrous. C阻 :げ OCtadecanoic methyl esttr,Cl&r9-OCtadecenoic medlyl esに ,
(e)施 rつた″″ でrttA‐
C18:2‐ 9 , 1 2 ‐o c t a d e c a d i e n c i c methyl ester,
(DDれ a″
θ′.-95%(v/v).
C l & 3 9 ' 1 2 , 1∝
5‐t a d e c a t r i e n d c t t h y l re,s軌
に。 ―ettOSanott me的 1
(g)拘 れ`れター Nanograde
ester,C_1‐8‐ eicosenoic methyl ester,C202‐11,14-eicosadienoic
(h)働 ′
θ万"秘 . methyl ester,C20:3 11,14,17‐eicosatrienoic methyl ester,and
(1)駒】f″
閉S″rrar2.一
Anhydrous. C22びdOCOsttmttc IIlettl ester.Prepare hdi宙 dud FAME standard so6
C)』ο″れrr"″θ″材夕″aga″ r,-7%BF3(W/W)in methanol,made 1utons as follows:Break toP ofglass vial open and carefully mnsfer
from conlmercially avttlable 14%BF3S01斌 On.Preparein the hood. contents to 3Hdran glass vialち Wash oFiginal vial with hexane to en‐
(k)Dセ rり′赫 でr― P夕初 rを 切 筋 2,れ 洵 ″.-1+1(V′ V). sure compleに mnsfer and add washings todran 3‐ giass vial.Add
(1)rr′ょり ご ι″ ′
ガタ j″ r2閉 αr sra″rrar″sθど ″riθ ″.―Cll:。 ‐triun‐ l.OmLCiloFAMEstandardsolution,(2),dlute tO total volume ofca
d e c a n o i n ; 5 . 0 0 m g / m L i n C H C 1 3 ・A c c u r a t e l y w e i g h 2 . 5 0 g 3.O mL wih hexane.Individual FAME standard solutions are stable
Cll,0‐ triundecanoin into 500 mL volumetric aask`Addca400 mL u p t o l w e e k w h e n s t o r e d i n r e f r i g e Cr )a .t o r ( 2 - 8 °
CHC13 and mix until dissolved Dlluteto volume with CHC13・ In‐
a E沼 ″,c"on o′ Far
vert flask at ieast 10 additional times.Triglyceride internal stan‐
dard solution is stable up to l mOnth、 vhen stored in refrigerator nnely ghnd andhomogenizeに st盟 叩 leS】 OrtO exttacは onof施 .
C). [ 肘θt t f W i t h m a t A x e s o f u n k n o w n c O m p o s i t i o n , i t
(2-8°
‐ s a りt o a n a l y z e t e s t p o r t i o n w i t h o u t a d d t i o n o f i n t e m a l s t a n d a r d t o
(m)Fα rゥαご′ ″"2,カメ′2sr2rs rFA〃 どs,sra″ガα,″sθ J″
ensure against interferenceso Should interfettng peak be found,the
riθ ″ j.―(ゴ )〃i逆a FAMEs sttzJa″sθ 筋と わ″ .―Reference mixture
areaofCH inに malstandard peak must be corectedbefoゃ perfom‐
contお ning series of F小
MEs,including Cl&1ごなand rrans(avAlable
mじ calculations.Use 2.OmLcmolofom insteadofintemalstmdard
as GLC‐85 from Nu Chek Prep,Elysian,MN 56028,USA,orequiv―
solution.]
alent).To prepareコ mxed FAMEs standard soludon,break toP of
(a)乃 。 dS 2斉Cr泌 残 筋 "″ 。血 ごな a胸″cれを2sを.一Accurately
glass vial,open,and carefully transfer contents to dramgiass
3‐ vial.
Wash ongind vi』 with hexane to ensure complete transfer and add weigh ground and homogenized test portion (containing ca
washngsto 3-dran giass vial.Diluに to ca 3 mL witt hexane, 100-200 mg fat)intO labeled Mttonnier flask,Force matttal into
″ガα′ゼ sοJ″ rわれ.<11:0-Undecanoic methyl nask as far as possible,Add ca lm mg pyrOgallic
(2)CH.FAME s惚
esterin hexane Use only in preparation ofindividual FAME stan‐ 2 . O l j n L t h g l y c e H d e i n t e m a l s t a n d a r d s o l u tAidodn ,aCf(e1w)b。
oiト
dard solutions,(3)To prepare Cllo FAME standard solution,break ing granules tO flask.Add 2.O mL ethanol and HHx well until entire
tesl Portion is in solution.Add 10.O mL8,3M HCl and EniX Well.
lop ofglass vial open and carefully transfercontents to 50 rnL volu‐
methc flask.Wash odginal vial with hexane to ensure complete Place flask into basketin shaking water bath at 70-80° C setat mod‐
transfer and add washings to 50 mL volumetric flask.Dilute to vol‐ erate agitation speedゃ Maintain 40 ndn.Mix contents of flask on
ume with hexane Cll.FAME standard solution is stable up to Vortex IIllXer eVery 10■
lin to incorporate particulates adhehng to
l weck when stored at O°C sides of flask into solution.After digestion,remove nask from bath
(3)れ どル泌″αどFA〃 Esra"滋 材 wr″!ゎ,s.―Standard soludons of a n d a 1 l o w t o c o 0 1 t o r o o m t e m p e r a t u rCe)(.2A0dtd2 5e°n o u g h e t h a ‐
each of fo1lowing FAMEsi C4:0‐ tetranoic methyl ester, ■ol to Flli bottom reservoir of flask and lnix gendy.
C6:0'hexanoic methyl ester, C8:0‐ OCtanOic methyl ester, (b)Dα "抑 勉 `Cな.―Accurately weigh ground and homoge‐
Cl。:。‐
decanoic mcthyl cster, C12:0‐dOdecanoic methyl ester, nized test portion (containing ca 100-200 mg fat)intO labeled

◎ 2002 AOAC!NTERNAT10NAL
AOAC OFFiCIAL METH00S OF ANttYSIS(2002)
0'LS AND FAT
Chapter 41,p.23

T a b r e 9 9 6 . 0 6 D R e t e n d o n H m e O f f a t y a c i d s a n d m e t h y l e s tMOiOnnieraask.Force
tr matettalinto nask as faF aS pOssible.Add ca
FAME〕
〔 100 mg pyrogallic Ⅲ ld,C(a),and 2.00 mL thglycedde inttmal
Relattve retentton standard solutlo■,C(1).Add a Few bolling granules to nask.Add
tinles
Fatundd Retention tirne nlin rin ll・
n ini ミ
↑
rtヽ
2.O mL ethanol and mix well until entire test portiOn is in solution.
4:O Butyric 10.49 Add 4.O mL H20 and mix well.Add 2.O mL NH40H,C(C),and mix
0.46
6:O Capro海 12.36 0.54 well.Piace nask inco basketin shaking water bath at 70-80°
C set at
8:O Caprylic 1569 0.68 moderate agitation speed Maintan lo mint Mix cOntentsofnaskon
10:O Caprに 20.39 0.89
11:O Undecanoic 22,99 1.00 首艦 F 熊i 協 品i 1獄 艦 祐 盟 糾 謎 話転 盟
12:O Lauric 2558 1.11 bath and add a Few droPs OF phenolPhdlalein.Keep solution ba
13:O Tridecanoic 28.15 1.22 n ゆW i t h
(が addition Of alnmonium hydroxi
14:O Myristic 30`65 1.33 to Flll bottom reservoir of flask and mix
・ gendン
14:l Myristoleic 32.63 1.42
14:l tra19s‐
Myristelaidic 32.01 139 (C)Ch夕 2S2.一Accurately weigh grOund and hOmOgenized test
15:O PentadecanOic 33,04 1 . 4 4 portion(containing ca 100-200 mg fat)intO labeled MoiOnnier
15:l PentadecenOic 3498 1.52 flask.Force maに 占al into flask as far as Possible.Add ca loD ng
16:O PalmiSc 35.41 1 . 5 4
pyrogallic acid,C(a),and 2.00 mL tnglycende intemalstandard so―
16:1″■,s‐Palmitelattic 36.39 1.58
lution,C(1).Add a few bOiling gmnulesto flask.Add2.O mLedlanol
16:l Palmitoteic 36.88 1.60
and mix weli until entireに
st pOrtion is in schは
on.Add4.O mL H20
17:O Margaric 37.54 1.63
加d miX Well.Add 2.O mL NH40H,C(C),and mix well.Place nask
171l Margaroteic 38。92 1.69
18:O Steattc 39.78 1.73
into basketin shaking waにr bath at 70r80°C set at moderaに agita―
18:l rrans 6‐P etroseLnic 40.50 1.76 tion speed.Maintain 20 min.Mix contents ofnask on vortex mxer
18:l rra,stElaidに 40.61 1.77 every 10 min to incorporate pttniculates adheing to sides of nask
18:1,竹 打s ll=Vaccenic 40.72 1.77 into solution.Add 10.O mL 12M HCl and place nask into b。 1ling
18:l Petroselenic 40.90 1.78 stealnbatlttdmaindn 20 min.Mix flaskcontents every 10血 n us‐
18:1 01eic 40.99 1,78 i n g V o r t e xはe
田 r.RemovenaskfrOmsteambadlandallowtOc001to
181l Vacoenic 41.18 1.79 r 0 0 m に m p e r a t u r e ( 2 0 - 2 5C。
め. A d d e n o u g h e d l a n o l t o n a s k t o n l l
18:1 0dadeceno俺 41.54 1.81 bottom reservoir and Ettx gendy.
18:2″口ns‐Linoletaidic 41.69 1,81
18:2″口,se卜Linolelaidic 42.11 1.83 A d d 2 5 m L d e t h y l e t h e r t o M t t o n n i e r n a s k f r o m )( ,a 0) 「
,(●
C).
18:2″コ,s12‐Linolelaidic 42.53 1.85 Stopper aask and place in centtfuge basket.Place basketin wr
48:2 Linoleic 42.87 1.86 action shaker,secuttng flask in shaker with rubber mbing.Shake
20:O Arachidic 43.75 4.90 flask 5 min.Rinse stopper into flask with diedhyl ether― petroleum
18:3g‐Linotenic 44.25 1.92
etheHmxture,C(k).Add25 mLperoleumether,stopperflask,and
20:l Eicosenic cis 5 44.42 1.93
shake 5 min.Centrifuge flask(inbasket)5 min at600 x g。 (河θttf lf
20:l Eicosenic rrans ll 44.45 1.93
20:l Eicosenic ds8 centrifuge is notavallable,a1low contentsto set atleast i h until up‐
44.67 1 . 9 4
20:l Eico韓 niC JS ll 44.82 1.95 per layer is clear.)Rinse stoPperinto nask with dichyl eher― pe‐
20:l Eicosenic cis 13 44.99 1 . 9 6
lroleum ether mixture.Decanteher(tOp)layerinto 150 mL beaker
18:3 Linotenic 45.02 1 . 9 6
and carefully rinse lip of flask into beaker ttith diethyl
18:2 Linoteirconiugated 46.35 1.97 ether―petroleuEl ether mixturec Slowly evaporate ether on aln
sに
18:2 Linoleictoniugated 45。40 1.97 bath,using nitrogen stream to aid in evaporation.Residue reman_
21:O Heneicosanoic 45.69 1.99 ing in beaker contains extracted fat.
1 8 : 2 L i n o t e i ―o n l u g a t e d 46.18 2.01
18:4 0dadedめ traenoic 46.39 2.02 二 何ettyraro何
20:2 Eicosadienoic 46.65 2.03
Dissolve extracted fat residue in 32■
mL chlorofbnn and 2-3 mL
22:O Behenic 47.46 2,06
20:39‐Eicosatrienoic diethyl ether.Transfer mixture to 3 draln glass vial and then evapo‐
47.94 2.09
22:l Cetoleic 48.27 2.10
rate to dryness in 40°C water bath under nitrogen stream.Add
22:l Erudc 48.50 2 . 1 1 2.O mL7%BF3 reagent,Cc),and l.O mL toluene,C(g).Se
20:3 Eicosatrienoic 48.68 2.12 with screwcap tOp coneaining Teflon/silicone sepmmo Heat vial in
20:4 Arachidonic 48.94 2.13 oven 45 min at 100°C.Centiy shake vial ca cvery 10コ 直n.(肘θ々f
23:O Tttcosanoic 49.22 2.14 Evaporation of liquid froin viais indicaに
S inadequate seals:if tlis
22:2 Docosadieno,c 50.17 2.18 occurs,discard solution and repeat dle entire procedure。
)Allow vial
2410 Lignoceric 5079 2,21 to cool to rOom temperature(20-25° C).Add 5.O mL H20,1・ O mL
20:5 Eicosapentaenoic 50.96 2.22 hexane,and ca l.O g Na2S04,C(1),Cap via and shake l前 n.Allow
24:l Nervonic 51,92 2.26 layers to separate and then carefully transfer toplayerlo anothervial
22:3 Docosatnenoic 51.98 2.26 contAning ca l.O g Na2S04。 (Hθセf TOp layer contttns FAMEs in―
22:4 Docosatetraenoioc 62.28 2.27
cluding FAME oftnglyCeride intemal standard solution。 )
22:5 Docosapentaenoic 54.75 2.38
oic 55.82 2_43
IttectFAMEs ontoGC column ortraIIsfertoautosamplervial for
GC analysis.

0 2002 AOAC iNTERNAT!ONAL

 0に S AND FAT
AOAC OFF,CIAL METHODS OF ANALYSts(2002)
Chapler 41,p.24

Tabte 996.06E Factors ltc)fOr conversion of FAMEs to FAME standard solutions and 2 μ
L ofmixed FAMEs standard solu‐
trigiyceride equivalents
tionl Use ttxed FAMEs standard sOlution to optimize chrOmato‐
皿 a卓 ― _ F A F と つた 町ecdng any test sdutions.ARer dl
graphc ttsponseあ
4:O Butyttc 0.8627 chromatog〔Nc cOnditiOns have been optimized,1対 ect test solぃ
0.9868
6:O Caproic
tions from E.
08923 0.9897
8:O Caprylic 0.9114 0.9915 a carcura何 。何s 、
10:O Capric 09247 0.9928 Total fatis the sum of fatty acids frOm ali sources,exPressed as
ll10 Undecanoic
triglycerides.ExPressing lneasured fatty acids as triglycerides re―
09300 0.9933
12:O Lauric 09346 0,9937

樹矩 r4補
quires mathematical equivalent of cOndensing each fatty acid

13:O Tridecanoic
14:O Myristic
0,9386
0.9421
0.9941 濫 曲札器 推 品端 母
iち私
描灘
0.9945 groups and l methine group are added k)every 3 fatty acids.
t4:l Tetradecenenoic 0.9417 0。
9944 Calculate retention times for each FAME in individual FAMEs

蹴 盟品総擦
粗濃:樹蟹盈盟艦漁 盟
15:O Pentadecanoic 0.9453 0.9948
15:l Pentadecenoic 09449 09947
to identify FAMEs in mixed FAMEs standard solution.Use addi―
16:O Palmkic 0.9481 0.9950 donal FAME solu伍 ons(frOm the same supplier)when necessatt for
16:l Hexadecenoic 0,9477 0.9950 complete FAME identiけ vedflCation。
17:O Margaric 0.9507 0.9953 (a)Calculaに r e S p O n s e F a c t o r (rReFa)c的
h faty acid Fas followsi
17:4 MarOaroleic
18:O Steattc
0.9503
0.9530
0.9952
0.9955
昨端予粋
18:1 0ctadecenoic 0.9527 0.9955
t8:2 0ctadecdieoic 0.9524 0.9954 where Psf=peak area ofindividual fatty acid in mixed
18:3 Linolenic 0,9520 0.9954
=peakareaofCti:。fatけaCldin mよedFAMEs
dardsolutioni Pscll:。
18:4 0ctadectetraenoic 0,9517 0,9954
standard solutloni町を11。こ Weigh〔Of intemal standard in mixed
FAMEs standard sOlution;and Wl=weight ofindividual FAME in
20:O Arachidic 0.9570 0,9959
航 xed FAMEs standard solution.
20:l Eicosenic 0.9568 0,9959 0わ セ=Peaks Of known idendty withOwn k■rel■ ive reに n don
20:2Eに oSadienoic 0.9弱 5 0.9958 dmes are tisted in Table 996.t16D.When peaks ofunknown identity
20:3 Eicosatrienoic 09562 0.9958 are observed during dle chromatOgraPhic run,attempt to ttmti母 (
20:4 Arachidonic 0`9560
such peaks using MS,FTIR,etc.Peaks of unkown idendty should
0.9958
not be included in the summalon when quandfying fatin the test
20:5 Eicosapentaenoic 0.9557 0.9958
smple.)
21:O Heneicosanoic 0.9588 0.9961
●)Calculate arnount of individual(航
glyCerides)(7Tc)in tes:
22:O Behenic 0.9604 0.9962 sample as follows:
22:l Docosaenoic 0.9602 0.9962
Pt,xttc.1:。xl.0067
22:2 Docosadieno:c 0.9600 0。
9962 WFA〃 コ
Ptctlo xR,
22:3 Docosatrienoic 0.9598 0.9961
22:4 Docosatetraenoic 0。
9595 0.9961 WTCt=yFAMB XrTct
22:5 Docosapentaenoic 0.9593 0.9961
22:6 Docosahexaenoic 0.9590 0.9961 w h e r e n i = p c a k a r e a o f f a t t y a ci io dn ,; iM n牲 t =e Ws et l gp ho t■
11。
23:O Tricosano,c 09620 o f C l l n i n t e m a l s t a n d a F d s al d dp eO dr t ti oO に
n0,0g6i7上
=conver‐
0.9964
slon ofintemal standard from triglyceride to FAME;Prcll拘 =peak
24:O Lignoceric 0.9963 0.9965
area ofCilo inにmal standard in test pO止 10n;andrTci=conversion
241l Nervonic 0.9632 0.9965 factor for FAMEs to triglyceddes for individual fatty acids(sC2 Ta‐
・
FAl'S the conversion factor for cOnversion of FAMESs to oorresponding
ble 996.trE)`
faty acids
a撤 (HθrCf lf procedure is fO110wed exactiy,ytcHo should be
偽Ct° rゎ「∞ nttrSonげ FAMEs的 価g呼師 des ttr
品 潜 呂 群 :鷲 O・0103・ )
(C)CalCuiaに amount oftOtal fa:in test smple(sum Of ali fatty
acidsi expressed as triglycerides[including ctt and rrazs fOHns of
m o n o u n s a t u r a t)eads afcOi1d1s0〕
W s i
え OC Dere何 わagm
To阻 1ね t,%=(Σ WDTc/叱 s t 高。
P 。n )

R c l a t i v e rnetに
ion times(vs FAME of riglyceride intemal stan―
dard solution)and response factors ofindividual FAMEs can be ob―
whe r e峰
駈鎮 m P =にW e i g h t s to flpに
Ч o n止, g .
tained by GC analysis Ofindividual FAME scandard solutions and ( d ) C a l C u l a t e w e i g h t o) fa s e af cO h1 1 F0 aW tS t: y acid(叱
航 xed FAME standard solution,町 ect Ca 2 μ
L cach ofindividual 略 =眸 AMtt X鼻
対

⑥ 2002 AOAC tNTERNAT10NAと

AOAC OFFiCIAL METHODS OF ANttYS!S(2002)
01LS AND FAT
Chapter 41,p.24A

Where rFAi〓 COnversion factors for conversion of FAMEs to their lar liquid stationary phase(diethylene glycol polysuccinate,
COFeSpOnding fatty acids(S22 Table 996.06E). butanedol polysuccinate,ethyに ne glycol polyadiP■ e,etc.)or other
(e)CalCulate percent of saturated stFat
s劉inに
叩L(w/w;eX‐ liquid(e.g"CyanosnicOnes)meetng emdency andresolution spec‐
.)asfOl10W既
pressedas samratedfattyacids:sumofC40,C60C80,Cに 1品 c 配l o n s . N o n p o l a r p h a s e c a n b e u s e d t t c e r t a i n
C o n d i t o n c o l u m n w h i l e d s c o n n e c t e d f r o m d e t e cCt o r a t 1 8
S■ ur■ ed fat,%=(Σ sAurated呼 叱 車沖Rm)X100% with curent ofinei gas at 20-60 mminfor≧ 16 h and fOr addi‐
t i o n a 1 2 h a tC . 1C 9o 5n °
diは
o n i n g にm p e r a c u r e m a y v a r y w i t h s p e ‐
(O CalCulateamountofmonounsaturatedfatintestsample(W′ W; cnc liquid phasc.
expressed as sum of only cな form of monounsaturaにd fatty acids ガ″
(d)ずン g?,一Maximum volume 10 μ
L,graduated to 0 L.
1卜
:配i c,お
[ C 1 6 1 , !C ,i Cチ c.1,etc.1)aS fOllow鋭
(e)R夕 Cο材2え-0-2.5 or5.O mV range,<1.5 s response rate(time
Monounsaturated fat,%= for pen to Pass from O to 90%following momentary i
10070 signal),25 cm minimum paper width,and 25-100
(Σ urated,7'托
honouns■ 宜
"mn)X1007o speed,with attenuatorswitch to change range.Ifinte
Polyunsaturated fat,70= pOnse Wtth ttequate se雨 血 y ttd stts施 的リ
(Σ
p o l y u n s a t u叱取
r a碑血
t .e )d X呼1 0 0 % 摺 紺 :古路 鮮
Ifdlemal conductivity detectoris used,conditions rnust be rnodi_
( 肋姥r T e s t s a m J e s 高nc gO d h拭y d t t g e n a t e d Mf a ct o mw ‐
iH fled
y た as followsi ColuHln,2-4 m x4 Hlm id;support,grain size
plicated chromatograms due to large number of isomers formed・ 15〔H250 μ m(No.60-100);statiOn句 ′phase,15-25%;carier gas,
during hydrogenatお n process,One general indに
ation of hydroge‐ He Or H2;nO auxlll叩 gasitemperaturett c01uml 180t200° C,iniec‐
n狂lon is presence of Ct&:rraな peak(S).For hydrogenated的 tor 40-60°C above that ofcolumn;carrier gas flow,60-80 mWmin;
chromatograms,use the following guidelines to calculaに FAME reCOFder,0-l mV range;and amount sample ittecに d,0.5-2 μ L.
peak areasi rra″ s peaks elute"orto ,therefore,include
Cな all peaksCorrection factors must be used.
between Cl&l cis and 2CtCな Cl性 in Calculation of Ct&2 Peak area
“ aR密 印"ts
Often Cl&ir″ な Peak"COnttstsof broadsenesofpeaks EduetOpO‐
sitional isoners from hydrogenation];inClude a1l of these in (a)arrigrga一 Cl&1 N2,He,Ar饂 ed andcontainingくlomg 02/kg,
r 惚湾 p e a k a r e a ) (b)Orル rgascs.― H2,99・94%,free from organic impundes.Air
Or 02,free from organに impuntに s(セ ppm hydrocarbons equiva―
R e f e r e n c e : ユA θA C れ ■8 0 , 5 5 5 ( 1 9 9 7 ) ; 8 2 , 1 1 4 気
1999). lentto CH4)・
R タッ体αな M a r c h 2 0 0 2 (C)Rヴ カ切“2S=施 rrs.__Known mixtures of medlyl escers or
medlyl esにrs ofoil ofknown compositioh,pFeferably similar to that
o f m胡 筑』 t o b e t t a l y z e d .
41.1.29
,Opearrng c。 何JrdOnS
AOAC Ottcia:Method 963.22
Followingvanables areinvolvedinselectingappropnaに teSt Con‐
Methyl Ester3 0f Faty Acids in Otts and Fa低
ditionsi lengdl and id ofcoluFIln,temperature ofcoluHln,camer gas
Gas Chromatographic Method
flow,resolution required,size of sample,and tine of analysis.Size
Ftrst Actlon 1963
Finat Acuon 1984 ' of samPle should be such that linear respOnse of detector and
electrometer is obtained.In general,following condtiOns will elute
AOAC― ′ lJPAC MemoJ m e t h y i s t e a r a t e w i t h i n c a 21 05 0 0m ti hn e oa rt e≧d c a l p l a 夕
t eT sa _ G を
C"JOpted_AO広 c ttemar ble 963.22):
A.Prrncripre IfapParatus permits,lnJector shouldbe atca2tXl° C anddetector at
temperattre above tlat ofcoluEln.Flow ofH2 tO detector should be
Methyl esters offatty acids from animal and vegetable fats having
ca half that of磁 航 er gas(H2f10W may be equaltO camer gas flow
8-24 C atoms are separated and det卸
耐 nedby gas chromatography.
withN2and 2 mm(ld)C01uIIInた 回OWOf02Ca5-10timestlatOfH2・
Method is not applicable to ePcxy,oxidized,oF p01ymedzed fatty
acids,

3 . t A p p a r a r u s
Fo1lowing conditions are fbr use with aame ionization detectori
(a)Gas cれ pttrag″ βれ._With航 航mum dead sPace in iniec―
tion system,which is maintaned at 20-50° C higher dlan coluHm
temperamre.Mantain c01uIIln temperature wihin± 1°C to atleast
220°C.If progratnmed heated,dual coluHlns are recommended.
ヌ
r]陀rcθ
″rれ″
?sったCれ4ク
セrイr,″
.2〕
(b)COr″ ″■s._1_3 mx2-4 mm(lo giass or stainless steel(do
not use staniess steel with polyunsaturated components wi血
>3 double bonds).Use ShOrtcolumn whenlong‐ chAn(>20C atoms)
acids are present.
(C)PaCttg.― Acid‐washed and silanized diatomaceous earth,
with narrow range(25 μ m)grホ n s12e betWeen 125-250 μ m
(No.60-120),Average graln size is inversely related to id and di―
recdy related to column length.Coat with 5-20%p01yestertype po‐

◎ 2002 AOAC tNTERNATiONAL

0'LS AND FAT AOAC OFF'C:AL METHODS OFANALYStS(2002)
Chapter 41,p.24B

rc伽続確stt C切 姥rイr,p.2〕
rTc・

⑥ 2002 AOAC iNTERNATiONAL

 AOAC OFFCIAL METHODS OF ANALYStS(2000)
C)3LS AND FAT
Chapter 41,p.25

Tab!o983.22 Conditlons to● !ute meulyl stearate within C18,and C18 medlyl esters appear in order:stearate(18:の
,Oleate
ca 15 min at≧
2000 theoretica,ptates (18:1),lin01eate(18:2),and hnolenate(18:3).C20 Saturated ester
Concentration of Column ( a r a C M d i C , 2 0a:pop euasrusd 1け
b8 e: f3 orに
e, sb に
ut m a_y be砲
1側
'淵 ternperattre, versedon some collHnns,orpOsitions may change withcolumn use.
Cdumntt mm 撚私
Car盟 錯材 Oc
比 carcurarrOns

５ ０ ５
2 15-25 175
︲ ︲ 郷 Use method Ofnomalization,which assumes ali components of
3 20-40 180 test wiion are represented on chromatogram,sc hatsum Of areas
4 40-60 185 under peaks represents 100%Of consttuents(tOtal elutton)。
185 Ifinsmment is equipped with integrk)L use igures shOwn.If
■ot,use mangulation:Draw iines for each peak tangenttO sides and
i n t e s encgは b a s e l i n e . C a l c u l a t e a r e a o f r e s u l t‐i n g tri
E Perramance sPcrPcarrOn3
plying height Kccrrected for a■
y change in attenuation)by%base.
P e r f o m t t d y sxitsu roen 航o fl mS eに
a的
r a t e a n d1 m0 e1 的
eat Feo r a u t o m a t i c d l y a t t e n u a t e d p e a k , o b t a i n p e a k w i d d l b y d r a w i n g
in caequal proportions ctgⅢmethyl esters froncocoabutteoo Ad‐ tangentstoOutersidesofPeakKdleSemustbefullchartspan,and up
just sample size,coluIIln temperature,and carier gas now sc Per%Ofpeakmustbeusedjandintersecttgbaseline.Caculate
that area
me的 lSにarate peak is recorded ca 15 min after solvent peak,ca% by muttyttg height tcoreCted for atten
full scaleo Measure base widtt in mm of methyl stearate(ッ 1)and If signiflcant alnounts of cOmpOnents withく
12 C atoms are ab‐
methyl oleate(w2)betWeen points of intersechon widl baselines eof nt,calculate percent by weight of each component,express
t a n g e n t s d r a w n t o i on ni e pc oはi n t s o f c u r v e s . A l s o m e a s u r e r e mt e tn h‐y l e s t e r ,
はo n d i s t a n c e i n I I I m ( D f r O m s t a r t t o p e a k H l a x i m u n f o r m e t h y l
s t e a r a t e a n d d i s t a n c e i n m m b e t w e e n p e a k m a1 x i m u n f o r m e 的 q=q x100s ci
stearate and me的 1 01eate,ユC』culate the餌 胡cal plates,″ ●館‐
ciency),and res01ution,R: w h e r e q = a r e a o f p e a k c o m s p o n d i n g t o c oq m= p o n e n t i
suHl oF areas under dl peaks.
″=16(S/ッ 1)2 I n ∝的占n c a s e s , e . g . , i n P e s e n c e o fく
c 1o 2m p Co n ae tn ot ms s ,w i 血
large dfFerences
i n ,m ao nl de t pc ru el sa er n cw ee t ot fh 低s e c O
R=2//Kwiキ W2j
grouPs,cOrmtion factors must be used tt 慮 cOnvert庫
areas intO
Select condidons to 6btain,2000 and R≧
w e i g h t p e F e n t . D e t e m i tn ie oc no f―a c t o r s b y a n a l y t t n g k n o w n
1.25。in addiはon9
linolenicacid(183)mettlestershouldbeseparatedfromarachidic erence standards of rttdlyl esters of ccmposidOn
aCid00:OmdgadOlettacid(20:1)eSters.Columnswillshow sample grad‐under identical 脇宜 q干ng cOnditionsi For Feference standard,
ua1loss in R宙 th use;when value becomes≦ 1.25,replace. Percent by weight componenti=曳 xlo08 31
ぇ D e r e r m r n a r r O n
where■ =weight Of compOnenti in reference standard gi=
With apparams showing stable baseline,lnJect O,1-2Ⅲ 5-10%
heptane soluは tod weightOfali componentsinreference standard.Calculate f
on of methyl esters,96933A Gcc 41.1,28).If mce
ChFOmatOgram:
components are desired,smple Hlay be increased≦ 10x Pierce seP
tumofinletportandquicklydschargemedlyl esters.Widldrawnee‐ Percent(area/距 つOf COmponenti=q x10け Σq
dle and ncte on chart飢 臣出 peak due to airor solven与 mrking start
r e f e r e n c e p o i n t , A d i u s t m e t hryalmeosuに
n t s o m t t o r p e a k iost■ a ‐ from which calculate co「 factor for each component
ection
tenuated>8、preferably less.Change setting ofattenuatoras neces‐
sary tokeeppeaksonchartpaper.Markattenuatorsetttlg onc権 止. 氏 =(芯罪 3)X(Σ qゴq)
For deteminatiOn of acids《 312,10Wer cdumnに mperature is
Deteminecorectionfactorsrelativetopal血 dctttr16=1,SO脱
needed;for>C20,higher.Temperature programttng is useful in
such cases,e.g.,with acidsく C12,inieCt at 100° C and raise temperat て,
X・ = ――
ture 4-8° 9min to Opdmunは or prograln up to a nxed temperamre 【16
andcontinue atconstanttemperature until aucompOnents areeluted.
If apparatus does not use progrmmed heating,operate at 2 rlxed Then to calculate percent of each cOmponent(as methyl esters),
にmperamres between 100° and 195°C. multiply its areaby appropriatecorection factor,andsun corected
areas:
a rgenrrrrcatrOn
Analyze reFerence standardmixturesundersame operatingoondi‐ Percent by weight componenti=(rix c)x lo幌 (rixq)
t i o n s a s f o r 鵡O
t ens,tM脚
easure retention dstances(D fOr known
esters.Plotiog S as function of nuttber of C atoHぃ
of acids.Under In certain cases,e.g.,when all cOmponents are not eluted,use h‐
isotherlnal cOnditiOns,graphsOfsmightchainestersOfsanedegree temal standard,S,such as CぉorCw methyl ester,and detemine its
of unstturation should be straight lines,approximate,Parallel. COFreCtion factor.Then,
Identify peaks from test portion from these graphs,interpolating if
PeFCent by weight oFcomponenti as metlyl ester=
necessary. Avoid condidons which penmit“ Inasked peaksr'1.e.,
(Ws/W)X(IPrで OX(crcs)x loo
which are not suFflciently resolved.
Esters appearin order ofincreasing number ofC atOHls and ofin‐ w h e r e w s i n g i n t e m a l s t a n=dtaortda la nmdg″ t e s t p o H よ
on,and
creasing unsamratiOn for sarne number of C atoHlst C16iS ahead of subscnpt S refers to intemal standard compOnent.

0 2000 AOAC iNTERNAT10NAL

01LS AND FAT AOAC OFFICI札 METHODS OF ANALYSiS(2000)
Chapter 41,p.26

Reportresults to following s i g n i n c a n t f l g u r e s , wTiatbhl oo n e9 9 r1 l. 3

g 0u r ie nb te e‐H a b o r a t o r y e t u d y r e 3 u : t e f o r f a t y a c i d 8 i
yond decilnal pointin all cases:3 for>10%,2 for l-10%,and one for
く1%. 鞘韻 弘:名
品
紹 品R払
培
麒Pd
Faty acid sR RSDR,% Sr RSDr,%
,P「 ecfs′
研
Ftth dに,area pettentage
(a)R?夕 α勉うJ′ 均ん― TWO Single deteminations performed on 14:0 0.58-0.98 8.31-13.12 0.49 5,3
same day by salne operatorwith same apparatus on salne sample for 16:0 0.44-1.91 5.66-10.02 0.54 2.9
珂 OrCOmponents(>5%)should■ Ot differby>3%relative,wih an 16:1 0.55-2.59 6.80-26。73 0.51 4.3
absolute value of l%. 18:0 0.05-0.42 3.05-14.43 0`06 1.8
一」 18:1 0.23tO.68 1.92-6.71 0。 19 1.6
(b)Rη ″れをめ'Iゎ。 TWO Single deteminations perfomed in
18:2n-6 0.05tO,13 2.37-10.69 0.01 1.2
different iaboratories for maJor components should not differ by
18:3nt3 0.04-0.17 3.86-23.14 0.04 5.6
>10%relative,with an absolute value of 3%.
18:4n-3 0.07-0,24 2.43r6.30 0.04 1.5

２ 抑 卸 卸 卸 抑 如 如
。
ｏ ﹁ お い 巾 巾 伸 市
References:IUPAC 2.302,7th醜 . 0.02-0.17 7.78-34.25 0.01 5.5
】AttC 46,146(1963);50,21気 1967),57,336(1970); 0。15-0.45 4.46-18.13 0。 13 7.3
62,709(1979). 0.01-0.08 10.29-27,71 0,01 8.6
0.03-0.07 16.08-67.73 0.03 17.4
R2ν体を″=March r997 0.04-0.07 27.47-47,93 0.01 3,1
ⅢAdopにd as a Codex ReFerencc Medlod cypeII)fOracid ttdrolytts,bo‐ 0.07-0.19 8.86t43.83 0.02 2:5
0.06rO.15 4.91-13.77 0.04 3.5
rOn Hnuttde oflinoleate on the fOm Ofglycerides)in special foods.
0.43‐2.06 5.48-9.78 0.25 1.9
22:0 0.02-0.38 11.77-141.42 0.02 8.9
22:1 0.14-0.80 8.88-17.24 0。 11 7.9
41.1.30
22:4n-6 0,08-0.17 38.29‐ 03.55 0.06 26.3
AOAC Officta:Method 991.39
22:5nt6 0.04rO.08 13.14t50.60 0.03 18.6
FaHv Acids in Encapsutated Fish 01:o 22:5n-3 0,10-0.32 8.84-16.20 0.13 6.7
a n d F i s h O i l M e t h y : Ea sn td e rE st “ 22:6n-3 0.69-1,44 7.50-16.09 0。 29 3.7
Oa3 ChRDmatOgraphic Method 24:0 0.02-0.14 48。38-102.04 0.01 10.9
First Action 1991 24:1 0.07-0。73 41.22-78.73 0,03 7.4
Fina:Actlon 1995
s h o l l s , a b s o l u t e w e i g h t ( m g / g t e slto np)o 成

／︲ ヽ
__ 同

５
９
20:5n-3 2.98t31.10 5。38-19.75 7.17
AOCSrAttC m

ヽ
５
３
22:6n-3 2.60-13.27 4.24r12.60 3.68
能 夕Table 99139 for the results of dle intedabomtory study sup‐ Ethyt ester concentrate,area percentage
Porting acceptance ofthe nethod. 14:0 0.06 17.07
16:0 0.11 12.24
A P r r n c r p r e
16:1 0。10 30.96

Tese portiOns are weighed into Tenon_lined screwHcap giass 18:0 0.15 7.00
tubes that contain appropriate intemal standards.FatけacidS Ofoil 18:1 0.50 5.72
ぉ1 研 e t t l e s t e r s a I I I ‐18:2n-6
s m p l e s a r e d e F i V a t i z e d b m e t t l ;eHsltee的 0.06 7.93
18:電 賄■3 0,08 12.85
ples require no derivatizationt Prepared mettl esters are mlyzed
by CC instrument equipped with fused silica colunn coated wi血 18:4n-3 0.15 7.62
20:0 0.03 10.12
bondedPolyglycolliq岨 dphase,oxygen scrubberin cariergas line,
20:1 0。45 3.19
andflalne lonizadon detector,Medlod detemines areapercentages
20:2n-6 0.10 32.42
of 24 fatty acids and absolute weights tmgrg sample)Of EPA
20:3n-6 0。11 35,22
(all―Cお‐ 5,8,11,14,17-eicosapentaenoic acid or 20,5n-3)and DHA
20:3n-3 0。07 29.32
( a l l , C f S - 4 , 7 , 1 0 , 1d 3o ,c 1o 6s ,a 1h 9e ‐
x aoei■
c a c i d o r 32 )2 .: 6 n ‐ 2014n-6 0.09 7.37
a ApParar崎 204nt3 0.15 7.86
20:Sn-3 1.54 5.鶴
(a)CaS統 ″閉αr9graPれ― with aalne loniztton dete的 ,cap11‐ 22:0 0,07 73.94
lly coluIIln inieCtiOn system(sPlit mode preferred at sPlit ratio of
22:1 0450 4.68
l:50),and Suitable data processor.Ndef ln ishoils analysis,san‐ 22:4n-6 0。41 84.99
ples are usualy sumcienttO pemit operation in sPllt mOde.)Oper― 22:5n-6 0.14 43.08
ating conditions: temperatures― ‐lnJection po■250°Ci detector 22:Sn-3 0,36 8.52
temperature 270°C;oven progra― ed from 170 to 225° C at 22i6n-3 1.40 7.51
l°C / m i n t n o i n i t i a l o r i n a l h o l o . H e l i u m O r h y d r o2g4e:n0 c a r n e r g a 0.11
s 158.70
(99.99%pure,or bette,With Oxygen scrubber h line. 24:1 0.23 35.16

(b)CC oθ′ 卯 `―Fused silica9 30 m x O.25 mm(or O.32 mm) Ethyt ester concentrate,absolute weightて mq/g test portion)
coated witt bonded Polyglycol,based on Carbowax‐
20M(e・g., 20:5n・3 20140 9.15
Supeicowax‐10,Supelco,Inc.,Bellefonte, PA 16823,USA,or 22:6n,3 14.15 8.97
a SR and RSDRfornSh。
eqdvalent coluIIln that provides salne eludon pattem as that inus_ ‖s are ranges of va:ues obtained in the collaboratMe
study or 4 dlrerent Rsh o13.RSDR Values are etevated for analttes that
trated inFigure 99139 andbaselhe separation of21:5n‐
3,23:0,and rarety exoeed O.1-o.2拷 ot total analytes(20:0,2013n‐
6,22!0,22:4n‐ 6.
22:召
h‐6). 22:5n‐6,24:0,and 2411)

◎ 2000 AOAC iNTERNAT10NAL

 AOAC OFttC!札 M目 猟)DS OF ANttYttS(2000)
0,LS AND FAT
Chapter 41,p.27

０ ・Ｃ ””０ ”
卜 ・ｔ ｉ 口 ”
卜 ・ ︼”０ ”

？占 ”

” ・ＣＯ ”的田
” ｏこマ “０ ﹁
Ｃ
０ “マ ︼

０ ぃ０ 一

０ ”０ ↓

い０ ” ︺ ０ “”“
０ ・こ付 “０ 一

０ ・〓い ”田的
０●ＥＯ﹂００Ｅ

０
・〓 ” 嵐 雨
Ｏ Ｌ〓 ■ ０ ︼

” ・ｔ守 ”０付
Ｏ

”
ｏこ 守 ”０何

０ ・エ ロ ””的
”田
付
字品 “

 
０ ・ “ ぃ０付

０ ・ い ”的田
守石 “

︱一
〓

Ｃ
［］
］
罵

罵
硫 側

罫
協 思
捕試掛1品 mtlon tt menhattn tt fatyお
d methw eSteぉ
。
n印
o対bL ttsed
樹
培品嵩
盟艦呂
苫播温
景品辞
(c)a%Mttr,2切 ″ cra触″ ツαセ″腕 抗 _Maintained at l∞°
C. ume with isOoctane.駒醇 1・ O mL PortiOns into screw‐ cap glass
Dry heater bl∝
k may be used. m b e s a n d e v a P o n t e s o l v e n t h Sg te On Fd ee ms bm em S O if n N
c)crass rzba_16x125 mm.With leak‐ dght Teflontlined 能 ezer ifnot to be used imElediately.
screw caps.
O yiaな - 2 m L , w i t h s c r e w c a p o r c r i m p c a P K f O r a u t o s t t見w 最阿
。 jめ角
l e r柳 . q四 膚rOn arlJAnarysfg
O仇 一 御 虎 r`oュ"apPtts tO all encapsulated materids,in‐
( O A ″ け施 a ル α: 御c a ― A c c u r a t e t o 却
. G 的l g .
cluding nonesterifled faty acids,widl excePtton of
(9Dけ れ曲 8鶴 SO″
rcc )
(h)0ね SdWa″.二Volumetric aasks,25 and 100 mLi volume血 Accuntely weighca 25 alg enl mg)diintO glass ttbe contな ぃ
Pipets,l and 2 nL;Pasteur 障低.
p的 i n g m e t h y l e s t e r I S , E . A d d l .5 5M m Le Ot 。
hanolicNaOH,blanket
with nitrogen,caP,mix,and heat 5 min at 10o° c.Co01,add
aR餌 寧"ra
2 mL BFain methanol,C(a),blanket with N2,Cap tightly,mix,
(a)β οttθ “句 α″οガル .―BF3,12%in methanol.2nLmberglass
and heat 30 1nin at 100°C.Cool mixture to 30-40° C,add l mL
a n P o u l e s ( S u P e l c O , I n c . , C a t . N o3.032‐
0,or equivdent reagent,
isoocta■e,bla■ket with N2,CaP,and shake vigOrously for 30 s
sealed in amber giass mpoules for extended shelf hfe)。(C例 材;例f
while still warm.
BF3 in medlanol is a corrosive Mgent and mutt be handled with
care.思 ddeye andskincontactbyuseofprontiveshieldandrub‐
ber gloves.Use cnly h Propedy Ottndng fume h00d。 c 側 禾 脳 モ 辮 靴
) 鞘
isooctane layer separates from aquecus lower Phase, transfer
●)2Jfθ M力 り′御ゼ β佐メ 容伽 .―Reagents Of99Ⅲ %「 直け as
detemined byTLC and CC analyses m chekPreP,Inc.,Elysian, isooctane layer to a cLan glass tube,blanket with N2,and Cap.
鵬 ,Ci No.N‐ 23‐M Enethylester3 andCat.No.N‐ 23‐E Eethyles_ 欧m c t a q u e O u s l o w t t P h a s e a S e c o n d d E l e W i
ter3,Or equivalenり,oわ ″f On lMluest,the CharlestOn Laboratory, l mL isooctane.Combine isooctane exmcts and concenmte tO ca
Soudleast Fish済絶s cen玉 、IWadonal Marine Fisttedes Service,PO lmL hshamばdry N2・
Box 12607,Charleston,SC 29412,USA,will Prov間 にcapsules of InieCt l-2 1tL into CC system.
collaborative sttdy sampに よ[Steam― deodoFiZed mettaden oiJ fOr
(b)財 閉り′θre的 I夕sr2rs._Accurately weigh≦ 15 mg仰 .l mgj
u s e h o P t i m i t t n g G C e)q u i P m e n t ・
methyl or ethyl esterinto glass ttbe contthing aPProPnate ester IS,
(C)絶 宅 c“rg拓 所2c確 碑途 、 一 sOdum hydroxide,methanol,
E.Add l mLisooctane,blanketwithN2,Cap,andmよ thorougtty.
isooctane,and sodium chloride.(働 ″r′ ο″r s22物 財 歳 β,Safety
notes on scttum hydroxide,methanol,and isooctane.) InieCt l-2に into CC System.Ifpeak height of ls is幻 .5 th■of
EPA or DHA peaに repeat malysis using 2.O mLIS.
a PrParaFron o′ sOrurrOns
a carcwfatrOns
(a)Arcoれ θrたsθガれ"ゎ だ拘I:あ夕 .-0.5M.Dissolve 2.Og NaOH
in methanol and dihte to ltXl mL with methanol. G ) A 万 2 α P c にC 純 8 a 一 C a l c u l a t e a r e a p e r c e n t a g e s o f f a t t y a c i d
(b)Sο tt cれ 勉 ―一Saturated soludon.Dissolve 36 g NaClin methyl esters or ettl esters as follow試
100 mL H20・
Area percent fatty acidx=tAx/tAT―
As)]x100
二 PrepararFan orSranJarJs
Accurately wttgh ca25 mg仰 .l mg)of23:O methyl orethyl ester where Ax=area counts of methyl or ethyl ester X;AT=total area
intemal standard(IS)int0 25 mL volumedc nask,and dihte to vOlt counts for chromatogram;and AIs=area counts of IS.

0 2000 AOAC tNTERNAT10NAL

01LS AND FAT AOAC OFFICtAL METHODS OF ANALYSiS(2003)
Chapter 41,p.28

(b)確 をれ′げ EPA a″ ガD「 Aれ θ:rs._<hLulate EPA or DHA, C)OaS Cれ rθttarθ ,一

graPた ―
With name 10nization detector
mg/g oil,as follows: [963.22B タ 41.1.29)or equivalent system].Electrottc integrator
Gタ
iS Preferable.
Ax xi略XCF.x101J0
EPA or DHヽmg/gゴ aR顕 寧抑rs
All reagents are analytical reagent quality.

whereAx=arcacounts ofEPA orDHA;AIs=areacounts ofintemal (a)″ ‐HCXa″ タ

standard;CFx=theoredcal detector corection factor for EPA or (b)rb′ ″2″タ

-50ワ )aqueous solution(v/V).
(C)MCrあ α″οJ.―
DHA(0.99 fOr EPA,0.97 for DHA);WIs=Weight of IS added to
sample,mg;ys=sample weight,mg;and l.04 is factornecessary
(d)どto す 夕r.__Freshly distilled, free of Peroxides and
れン′夕rれ
.
express rcsult as mg fatty acid/g oil(rather than as resldue.
methyl esteつ
(C)M夕 り ″容″r s . 一
夕と
をカ ゲ E P A t t D r r A れ Calculate EPA οルをれ,― Toluene-2-hexaneK90+10,v/v).
o r (e)TLC冴 夕,Cttp:略 ざ
DHA,mg/g csters,as follow鋭 ( 0 肋 修挽ノ タr ″ ″沈″ど s θルr : ο
catt S筋 ″. 一-5 mg/mL.Dissolve
50 mg inethyl erucate in 3-4 mL hexane and diluteto 10 mL with
EPA or DHA,mg/g=箸 X1000 hexane.
維 輯 諮 ″.一-200g/L.Dissolve 24 g AgN03in
(g)S′ルタrがrratt sο れ,Jθ
H20 and dilute to 120 mL with H20・
where teHns are same asin(b),eXCept use l.08,factor necessary to (h)y夕 ′ れメr r2,racθsa″Oar夕 ,″r夕r″α′sra″ガar″ sο′ ″
e x P r e s s r e s u l t a s n g f a t t y a c i d / g o l l K r a t t r t h a n arわ
s ″e.-0.25
t h y l emg/mL.Dissolve
ster). 25 ng methyl tetracosanoate in
References:AOCS Offlcial Mettod Ce lb-89. 3-4 mL hexane and dilute to 100 nlL with hexane.
ユAθ AC五 円,7S,488(1992). ″, - 0 . 5 g 2 , 7 dをi c m O r O f l u o r e s c e i n 7 L . D i s ‐
( i ) 比C w r a y ″αg 夕
solve 50 mg 2,7を dchlorofluorescein in 100 mL 50% aqueous
methyl alcohol by warlnlng and stirring.
41.1.31
'旅 標 確 tron orFaけ AcrF撤功 〃 Esrers
AOAC Official Method 985.20
T a k e r 授n
e p t円a t i v e t e s t s m p l e o f c a t t m g o i l t t f a t a n d P r e P
Erucic Acid in 01ls and Fats
soludon offatty acid methyl esters in hexane(ca 2CH50 mg/mL)ac‐
Thin‐Layer and Gas Chromatographtc Method8
cording to 96933 G夕 241.1.28).
First Action 1985
Final Action 1992 HLayer〔労 的m節 兜 陣 口,y
二 恥 '何
確 ″rq″ararfθ″.― 一Place 60 g silica powder in
lUPAC-40AC胸 0」 (a)ELC P挽 ″
500 mL round‐ bottomユ ask with 120 mL AgN03 S01ution,(g),
(Applicabに to animal and vegetable olls and fats,including those and shake l inin to obtttn fully homogeneous slury sufficientfor
containing cetoleic acid[c体-11‐ docosenoic acid which occurs in 5 Platest Spread slurry in usualinanner to give layer thickness on
ishoilsl,and tO hydrogenated olls and fats which may contain both Plate Of Ca O.5 mm.Let plates Partially ttr dry(preferably ca
cな‐and rttzs‐ isomers of docosenoic acid.) 2°C
301止 n in dark),then fully dry and activate plates in 100±
oven for 2.5h.Use Plate aS S00n as Possible after activation。
(Ac―
A.Principre
C for l h haybe satisfactory ifPlates are notdark‐
tivation at l 10°
Constment fatty acids are converted to methyl esters,separated ened.)Score lines through ccadng 10 mm from sides and ttP of
layer thin‐
by low temperamre argentatiOn chromatography cLC),
eachPlate before use to reduceedge effects duing development.
and quanttated by gas chromatography. ‐s i n g a p p l i c a t o r , d e P o s i t
ザ れ加り ′2 S t t r s ,U一
●) A P P ′j c a r J″。
a Apparartrs 50 HLmethylestersolution,D,in narrow streak ca 50 mm long on
left of Plate,atleast 40 mm from side,and 10 Hlm froHl bottom.
(a)CttSttG″ .-50 nL beakers,100 and 500 mL round‐ bottom
L solution contttning equal volumes ofthe
Silnilarly apply 100 μ
flasks,and sma11,pointed―bottom glass tubes.
methyl ester soluton and melhyl erucate standard soluton,C(o,
もFor ethyl ether,equipped with N2
(b)Dな ガJJar,例 姥 ッ,cを oa rightside ofPlate.Take care when applying solutons because
streaコ
n,
coating is fragile.Apply 50 Lμmethyl erucate standard solution
(C)Cο ′ ″れな.―く31ass,ca 200 x 10 HIHlid,with glass wool or tO Plate as above,betwee■other 2 applicaticns to help idendfy
sintered― glass ilters),
giass ilter(Or use smali funnels with sintered‐ methyl erucate band after develoPment(Figure 985.20).PlaCe
with sihca gel packing. bottom edge ofplate in ethylether,C(d),undlether ascends to ca
″.Regulated at 100± 2°C.
(d)θ ツタ 5 mln above area of apPlicatiOn,concentrating methyl esters in
naHow band.
(e)比 CPた 7″S.一-20× 20cm"glass,to bc spread with silica gel G
(E,Merck)or equivalent. oDタ ッタ′θPれ 夕みrザ P′α′ 2S,一 Pour TLC developing solvent,
C(e),intO tank to ca 5 mm depth and Place tank,with lid,in
た一
(o ELC appitcaゎ 二
For depositing sample solution as narrow
band or streak. deep―freeze unit at-22° C,or as near to lemperature as Possi‐
ble。(In certain cases,it may be advantageous to line tank with
(g)TLC』 セツ冴。βれg勉 ″た With lid.
filter paper in contact with develoPing solvent.)After 2 h,
(h)TLC冴 rre乾 夕″れ,4<apable of maintaining dcvcloPing
タマP‐
place plate carefully in tank and let solvent ascend to ca/2tO%
tank and contents at-20 to-25° C. height of Plate.Then remove Plate,allowing solvent to drain
(1)yy脇 4p off,and evaporate residual solvent from plate with N2 Stream

◎ 2003 AOAC tNTERNAT10NAL

 AOAC OFFCIAL MmSOFANttYSS(2000)
O i t S A N D F A T
Chapter 41・p.29

一
国製
Satd FA Me esters Erucic acid,%= x100
rt x[(拓
/h)十(〃2/r2)]
transtisomers
鶴一
Methyt erucate t印
品粉鑑盛冊鮒路松1酬 〔縄瑠絆:納
 
Methy,cetoleate
一
Other monoenes 一
arett
に are他
前ned h%,〃
d ttculated
i価 are c』
一
的;辞
品
一
Dlenes and polyenes
材1 = 1 0 0 - r l
一
一 。 一
γ2 ‐ 1 0 0 r2

翻躙徽 は 翻
１ ２ ３

Sampl●
Methyl erucate
Sample+methyt erucate
r 2 = r―と
r3
同g u r e 9 8 5 。
2 0 - T y p t c a , t h i n ,‐a y e r c h r o m a t o g r a m s h o w ‐ where
ing sepaFatiOn of methyl oeters Of eruc:c acld,ceto3oic
a c i d , a n d t 1t 8n 0字m e r e o f ed no oc io c● a c i)dF.r●
actton ra=堅
designated methyt erucate w::t usun,,y contatn methyl
esters cf other nonoenolc ac,d3.

under hood.Replace plate in tank,and let solvent ascend tO top

OfPlate.Then remove place and,as before,dtt with N2 Stream.
Spray plate with spray reagent,C(1).VieW Plate underUV light

卜
and locate band containing methyl erucate derived from test
艦 躙 艦 ド
耕
縦品鞘 :r灘
綿替t納:撒貯協 `:獄
: れ P r C r 3 O」n
(middle sample;Figure 985.20). 一 Difference between results Of 2 tに
(a)R印 欧煎妨】り 。 teEttna‐
(d)Sη a″!ゎ″ゲ "2協 メ容″ J駒げjθ な.ヽ crape o鮮witt great tions PerfomedsimultaneouslyorinrapidsuccessiOnon same saln‐
c a r e t o a v o i d 1 0 s s e意
sn,ibnagn dn ectotnlヒe r u cvaetde fdroo由
m pに,by sane operator,under same conditions,and fOr erucic acid
test portion(Fraction l).Si航 larly remove layercontaining remttn‐
:言
盟
獄ま
」格
韻艦 ‖
竹辞
揖置
村よ
ヽま
』嵩
嵩裾
!告
品長
督樹
‖fず
獄
『指
将古
苫f監
d e r o f f a t t y a c i d s s tf r po Om rにt i O n l o c a t e d a b o v e a n d b e l o w m e t h y l
e r u c t t e b a n d i n t o s e p a r a t e b e a k e r ( F r a c t i o n 2 ) . A d d t o earnountく
a c h b e a5%,precls10n
ker deCFeaSeS.
l.O mL metlyl tetracosanoate inteml standard solutton,CQ),and ー
(b)R2P「鑓 わ:!:ケ Difference between resultt in 2 different
10 mL dhyl edler.Stir and mnsfer cOntents to separate columns or iabs on sane test Hlatedal fOr erucic acid present in amounts>5%
funneis,3(o,caCh COntaining ca l g silica gel.Extract mehyl esters should not exceed 2.2g/1oo g sample or 5%of deに mined value,

鮒盤拠rater.ForewicacH く
5%,wd_
wstt h ar
us唯 3研 four 10mLpoEtionsofewld比 想 oolect血 強 ぃ 1∞ nL

ナ 撒 棚 ェ 娯艦 References:P″″ APPi C確 開.54,2759(1982)iSi 302(184).

and evapoFate au sottent with N2 Stream under hood to concentrate
ユ As縦 .Pめ こと A脇 町sぉ18,77(198o.
methyl esにお 斑 botmm of tubes.Dissolve methyl esに 、 in ca
20-50 11L hexane. CAS‐112‐
86‐
7 cmcic ac10

見 Cas Cttromarurapn/

Sク
9 6 3F.G2`22B4―
1 . 1 . 2 9 ) , e x C e41.1.32
p l i tAOAC
t eO付c t,oiallMethod
- 2 975.30
1tL hexane
盤縄縄紺s鞘瑞陥瀞 艦劇機路‖ 繊￨ Docosenolo Acid in Oits and FatG

areas mm chromtOg皿 Gas Chromatographic Method

路拙盤 を 艦古 漁洗辞 F3RBt Action 1975

Finat Action 1984
A . P r r n c r p r e

1
mcic ackェ,an isomerofdocosenoic acid,is characteristic acidof

岱
『盤猛蹴器替齢 堵
鑑機脳 濫柵 rapeseed.01l or fat is converted to medlyl esters,which are deter‐
mined by GC,with methyl tetracosanoate as intemal standard.

◎ 2000 AOAC t卜 『ERNATtONAL

C),LS AND FAT AOAC OFFiC:AL METHODS OF ANttYSiS(2000)
Chapter 41.p.30

ユ A P P a r a r w s 41.1.33
OC密 統″秘西θ g門肱 二 Varian Model 1740tlα Packard
HeWlett‐ AOAC O宵 io,at Method 977.17 r
ねndemtorand3生 Q.9mlX
5700的能s,orequlvalent,witlaal.eiOd刻 Poiymers and Oxldation Products
l旭ho.2mゅ od stainless steel coluEm COntaining 15%dietlylene of Heated Vegetable 01:s
glycol succinate(DECS)on 30r100 1nesh Chromosorb W,acid Gas Chromatographi● wethOd fOr Non‐EluHon Materla18
washed,Operating condtionsi temperatures― lnJector 250°
C,de‐ Ftrst Action 1977
tector 290°C ,coluHln 190°C;He carier gas flow rate adiuSted to Final Action 1984
give retentiα ltime ofca 17 min for mehyltetracosanoate.Measure
Accurately weigh ca40mg Oiland 10 mg ttheptadecmdn intemal
peak areasby electronicintegrator,diskinttgrator,ordangultton.
(b)rraな否r夕 rttcar筋メ西た-125 mL conical iask with stan‐standard(Alltech‐ Apphed Science Laboratories,Inc.,No.21322 Eno
longer
dard taper 24/40 joint and neck elongated and constdcted to avお
lable])into 50 mL Erlemeyer.For ease of handling,
8-10 mm id. empty entre vial inにmal smdard supplied(100 mg)into tared
100 mL volumedc aask,weigh accmtely,dissolve triglycer
a Feagents hexa■e,anddiluteめVolume.Tc avoid volume changes,immediately
(6α″rわ″i S2をAppendix B,safety notes on distillation,sodium mnsfer lo mL aliquots to nine 50 mL Erlemeyers and caremlly
metal,hexane,magnesium,and methanol.) e v a p o r a t e s o l v e n t u n d e r N 2 S t r e a m . F l a s k s
(a)HC盟確.一つistil reagent grade hexane and dry‐over stored an町 at O° C if desixtt Weigh test portion dimtly intt nask after
drous Na2S04 befOre use. tempemure equlllbrium.Add4 nL O。 5M alcoholic NaOH solution
( b ) 財夕統御ο4 α″り″″匹. ―D i s d l w i t h M g t u m i n g s . and boilhg chip,and reflux 10 min witl H20 COndenser.Add5 mL
(C)S述 ″閉 加夕1祐え確 sθれ!筋 .―APproximately l%in anhy‐ BF3‐metlyl alcohol,Preparedash96933De)G空41.1,28),trough
drous methyl』 cohol.Clean ca l g Na metalin hexane,的 With nl_ condenserandboi12価n.Add2-5mLPetroleumedlerttP30-60° C)
ter paper,and dissolve in 100 mL anttdrous lnethyl acchol h and boil l min more.Cool mixttlre and mnsfert0 30 mL sepmorP
250 mL conical iask ntted with slllca gel drying mbe. washtt aask several dmes widl total of 10 mL petrolem edler.
C)MC的 こ`r解 ″勤 勉 ″ J加 筋 .-2 mymL hexane.Dis― Shake l航 n to extract memyl esters into petrolem edler.Drain
s o l v e 1 0 0 m g n e t t l e r u c a t e s i g E l a C h medlanolicKlowerjげ温韓inttsecondseparatoFandeXmctagainwidl
emical Co.,99.5%C22::;
N o . E 3 5 1 0 ) i n h e x a n e i n 5 0 m L v otlou mVeotlr‐ i10c nLpetroleumether.Combine
a a s k a n d d i l upemleun に eher extracts,and wash
ume with hexane. with5 mLPortiOns H20unは l wattungS are acid‐ ■ee whenに stedHおdl
mehylred.Dry widlttdrOusNa2S04,preferably by pouring s
(0〃 夕′ り `Cを 確cθs物 姥S々 脇滋材 sοれ,jθ ″.-2 ng/mLhexane.
D i s s o l v e 1 0 0 m g廿a Cm Oe Sh ay nl oにa t ce h _o Au Pl pにl i e d S c iPle
e n trOugh
ce narow glass colum containing Na2S04;WaSh colum
Labor菰抗es,Inc.,or Sigma Chettcal Co.,lignOcenc acid mehyl wid15mLttpetroleumeher.EvaporatesolventunderN2HAdlaidof (
esに対 997o C24;No.L l126 1replaCed by No.6766])in heXane in s t e a m b a t l . B e c a u s e o f s m a l i v dおu, tm tn s of fe er s に
dry esters b
50 mL volumettc flask and dilute to volulne witt hexane. l nLに st tube‐ shaped volumetric nask or cone‐ shaped vial to fac■ i‐
tate FeH10Val wih synnge foriniechOn Onto CC column,
E DerermrnarrOn
Analyze sample wih aame iOnization detector as in 963.22B― C
(a)降 igル″→ βθ″s2ヵCrOrrar価 的 [統 OS御餌宅 R22:1・ Pipet
(s夕 を41.1.29)。MeaSure peak areas of chromatogram by eidler
l,2,and3 mLmettylerucate standardsolutionhtoseparate 25 mL
planimeter or electronic integrator.
conical nasktt nttth3 mL Hlettltetracosanoate立
如dardtthtion
into each nask and hexane tomake 8.O mL total volume.ItteCt2
L μ
ofeach solution into gas chromatograPh.
Nonelution mtedal,%=
R22:1=(馬
私 1′
晩 0(P24/P221)

Where R22,1=Weight response factor for erucate relative to

tetracosancatei町 221=ng docosenoate(eruCate);町 24=mg where PA'=peak areaofintemal standardi W'alld W=weights in‐
t e t r a c o s a n o a t e ; P 2 4 = p e a tk r aa cr Oe sa aにn c a t e , a n d P 2 2 1 = p e a k at re emaa l
s t a n d a r d a減
n do n t9 e sr te 脚
sPechvely:and PA=total area o
docose■oate. chromatogram。
(b)D釘 使 ″οた αc′ な れ ヵ な α″ど0:な。 ― Accurately weigh ca
Reference: 拡 OAC S8,898(1975).
50 mg oil into dry transestedflcation nask,add 3 mL methyl
tetracosanoate standard sohtion and 10 mL NaOCH3 SOlution,and
renux l h.cool,add 5 mLhexane,swirl,add7 mLlM HCl,stopper, 41.1.34
AOAC Officia:Method 982.27
and shake vigorously l min.Add H20 until hexane reaches con―
Polar Components tn Frying Fats
s t t c t t d n e c k a n d t t he ec xt a2 nⅢe l a y e r h t o g a s c h r o m a t o g r a p h .
ChromatogFaphic Method
First Actlon 1982
Docosenoic acids,%=律
2ノP20(比 〃手
略D R221 X 100 Final Action 1984

“= n g
where略 Oil.
rt/PACrAOAC Mo:わ oど
References:JムθAC 57,1161(197o:58,488(1975). A Prrncrpre
CAS-112‐ 85-6(docoSenOic acio Metlod assesses dete貞oration of used frying fats,and is applica‐
CAS-112‐ 86t7(eruciC acio ble to all fats and oils.Polar components are those conlponents of

◎ 2000 AOAC tNTERNA刊 ONAL

 AOAC OFFЮ IAL METHODS OF ANALYSS(2000)
0 1 L S A N D F A T
Chapter 41,p.31

f a t s dmeiに nedby coluEln chromatography underconditions sped‐

fied, and include polar substances such as monoglycerides,

●●

・●
diglyceddes,free fatty acids thatoccurin unused fats,as well as po‐

・
lar transfomatioa products fomed duttng tthg Of foodsmffs

  ▲ワ
and/or dwing heating.Nonpolar components are mosdy unaltered
t r i g l y c e r i d e s . F r y i n g f a t s a r e s e p a r a t e d boyg rcaoPlhuym n c h r o m 江

ａ ｌ
on silica gelinto nOnpolar and Polarcomponents.Polarcomponents
are detemned indirectly by subtractng concenmtiOn OfnonPolar

ｌ Ａ Ｔ ，
components.QualiけOf Separation can be checked by thin layer
chromatograp町.
a Appararws
a ) 6 θ′
協 ″. ―G l a s s , 2 . l cm id x 45 cm,with Teaon stopcock
and ground‐glassjoint.
1 2 1 2 1 2
(b)TLCPrares._PrecoatedsllicagelⅢ id10utnuOrescenceindi‐ FRACTrON
catoめ,20x20 ctt layer thickness‐ 0.25m. FiOure 982.27-Ewa!uation of efnciency of fractionat:on
a F e a g m t e by TLC ceparatlon of po3ar and ROnpOiarfraction:Fracr
tio■l contains nonpolar componente,and Fract`on 2
(a)紀 S。だ比納は一式斑lLa gel ttL paHiCle size O.063tO。 200m
σO - 2 3 0 m e s h A め鋼 , M m k N o . 7 7 3 4 , o r e q u l v a l e n t , a d i u s t t O H● 20
ontaine p。lar oomponents.
contentof5%容 f o l l o w t t Dlr&yl主g e l コl h i n p o F C e l a i n d s h i nC 1 6 0 °
oveni cool in desiccator to room tempe― e,Attust H20COntentめ
5 % , e . g " w e i g h 1 5 2 g s d i c a g e l a n db o8t tgo mH 2undercolulm,open 0 h 5 0 0 mstoPcock,andL r o u n d ‐ let sample solutton ttdn to level
aask win ground_glass stopper and― iCally shake l h. o f s a n d l a y e r . E nl Ou nにP o l a r c o m p o n e n t s w i t h 1 5 0 m L p e t r o l e u n
(b)酎 叫唯 Sθ !燿江れ加″脇 ― Petrolem e臓 ぃ 40-m° ctler ehertedler(87Ⅲ 13)contボ 距d i n 2 5 0 m L d r o p p i n g f u n n e l . A d i u s t
K87キ 13). aOw rate sc hat150 nL passes dlough cOlum within 60-70 min.
O SCa―Sa″ a―Analy血劇 reagent grade;pttfled by acid and ARer elution,wash any substance adhering tooutlet ofcoluコ m into
calcined. r o u n db‐
otton nask widl petroleum edlertetler(87+13).
C)"ray reagttL対 o,bdOphosphOric acid,10%in dcohol, h same mlln研 ,elute polar components into second 250 mL
a P r e P a r a r勃
r a n体ゴ round‐botoHl flask with 150 mL ethere Discard silica gel.
Wamlserll‐liquidandsolidfats toteHlpemture slighdy abovemp
Remove solvent from each■ acはOn widl a rotary evaporatorand
and mix dlorougtty;avoid overhettng.Remove visible tEttHdes
by flltration;ifH20 iS presen,use hydrophobic fllter. 1 6 0 C° H 2 0 b a t h o r w i t h N 2 S t r e a n i n 2 5 0 m L a a s k o n
Avoidlosses due to foaning.Ifrotary evaPoratoris used,shortly
二 PrePararrOn Or oOrrrmn
fore end of ev響 げ狐 On,introduce N2 intt System.Co01 residue to
nll cOlulnn with ca 30 mL petroleum edlerteher(87+13).PlaCe mbienttemperature and introduce N2 intO aask.weigh aasks.
wadofcotton woolinbotttmofcolumn and realove airby pressing
with glass rod. a catwrarrOng
In 100 mL gtts beake,Prepare siutt of 25 g silica gel and ca
C a l a l a c P o l a r o o w O r m t stPt wa/sV )pWei―
h mula
80 mL petroleumether― etler(87+13)and pOur slu『y into colunn
hЮ ugh8 cmglassttmel.Rinsebeaker,funnel,andsides ofcolulnn
with same solvent.Open stopccck anddrain solventto 10cmabove Polar∞ =雫 規∞
mpone醇
豆l i c a g e l . L e v e l t t l i c a g e l b y t a p p i n g c o l u m n .
Add ca 4 g seatsand through fumelinto colunln.Drain solventto
where A=g nonPolar fracdon,ど =g smple in 20 nL aliquct
sand layer.
(ca1 9.RepOrt result to olle decimal place.
見 C "鋪
「 β3 如 y
比締紹 chromaragraphy 輸iayer
Enrrcrenc/
T o d価 e 俺 騨榊
n e p o l a r c o m p o n e n t t b y d i f f e t t n c e , o n l y n o n P o l 打
fracton is used.HoweverP if separation is controlled by TLC,botl
Dilute polar and nonpolar fraction(lⅢ 9)in CHC13'Apply 2 Ⅲ
polar and nonpolar fracdons are requlFed,Separadon may also be
c o n t r o l l e d b y c h e c k i n g r e c o v e r y o f s a m p l e , b u t f o rSPOtSusingcapillattdispensingPipet.Developplate
smples conttin‐ withpetroleum
ing substantial amounts ofpolar material,recovery may be incom― ether― edlertCH3C00H(70+30+2)in ta■ k lined with ilter paper
for ca 35 min(ca 17 cmp.Remove plate and let solvenl evaponte.
plete because smali amounts of highiy polar material,generally
l-2%,are not●1■ ted under condidons specifled.
Spray Piate Wit110%molybdophosPhoric acid.Afterevap
Accurately weigh 2.5主0.lg(的 0,001g)test portion into 50 mL
of alcohol, heat plate in 120-130° C drying oven. Fraction l
volumetric aask,and ttssolve in ca 20 mL petroleum ether― cher
( n O n p 0 1 a r j s h o u l d b e f r e e O f p o l a r Fs iu gb us rt ea 算
2n 2c 7e )s . G g を
艦色
督Ⅲ轍 酷陥補ξ
::協 鑑 Reference臣
炉移rr2 S修
″Attrrを
!ル 筋 80,106K1978).
20 HL sample aliquot to coluIIIn,witlout disturbing surface.
JAOAC 64,1329(1981)i6S,375(1982).
Dry tw。250 HL round‐
bottom flasks in 103±
2°C oven,cool to IUPAC 2.507,7th Ed.
room temperature,and accurately weigh to O.001g.Place one flask P″″ APPi Cれ を秘.貿 ,2Z陽(198分 .

◎ 2000 AOAC iNTERNATtONAL

01LS AND FAT AOAC OFFICtAL METjtODS OF ANALYSS(2000)
Chapter 41,p.32

41.1.35 steamofN2・
Pipetl mLalcoholicKOH,c),inmeachnask,aushwitl
AOAC Official Method 979.19 N2,and Store stoprred in dark 5 h or overIE"ヒ `
。す ら crs「Methy:ene interrupted Add20 mL l.OM borate buffer,(a),50 mL H20,andl mL O.51M
Po,yunsaturated Faty Acids in tDlis H C i t o e a c h , d i l u t e t o v 0 1 u n e w i t h H 2x0.,Bapnedt血
four 3 mL
Spectrophotometric Method aliquots Ofeach standard soludon into test tubes,incubate,and pro―
First Action 1979 ceed asin C,ガ形セ"句筋αガθ″,begianing“ To 2 mbes(blanks)...''.
Finat Action 1984
Plot average A at each level aganstg μ
i撤
thlinolein/mL.
A,Peagenお

(a)Pο ttSSれ″“うo「 はこう宅βをみ-1.OM,PH 9,0.Dissolve 61.9g >協号

枇脇鮒i船鞘総 粗瀧艦
H3B03and 25,O g KOH inca800 mL H20 bysは 対ng and heating. not differ by>2.2%.
Coolto room temperamre and adiusttC PH 9・O With l.OM HClor References:五AθAcも 0,895(1977);61,1419(1978).
l.OM KOH,as required.Dlluteto l L wih H20 and mix.
(b)D】 ″夕Pοrass,″ ″ みο″セ う域枠ぇ-0・ 2M,pH 9.o.Dllute
200 mL l.OM buffer,a),to l L with H20 and mよ . 41.1.35A
AOAC Ofmoial Method o94.15
(0と ゃ帆 材徳g sο !肋 西.一翌 )並 転 w:″!Jθ な一担Lssolve 10 ng
Totat crs and rrarPetoctadecenoic tsomers
lipoxidase,from soybean,50 000 units/mg(ICN Pha― ceudcals,
and Generat Faty Acid Composition
Inc.,Life Sciences Group,Nuttdonal Blochenicals Divisio■ ,or
ln Hydrogenated Vegetabte Oils and Antrnal Fats
equivalent)in 10 nLice‐ cold dilute borate bufferD(b).Refrigerated
Caplilary cas chttmatographiHn陥 画
solution is stable 30 days.マ )Wθ たれg sθ rrrわ″._Mix 2,O mL stock
Spectrophotometric Memod
solution with 8.O mL ice‐ cold dilute borate buffer,● ).r large Firet Action 1994
n u m b e r o f a n t t y s e sb eapFeer fⅢ
o m e d , d i5l,uOに
m L s t o c k s oolnu は Final ActBon 1938
to 25 mL with ice‐ cold diluにborate buffeL(め .(3)あ αctta″″
sθ:″ r:θ″.ギ ipet4mLworking ena′ me s01u敵)n,(2),to 10mLvolu‐ (Applicおle to脚鵡dly hydrogenated vegetable olls andにrestrid
mettc flask and hold 5 min in boiling H20・ animl tts containing汚 %rran fatty ac赴 .Methodis not appl拒a―
ble tottdrogenatedmarinecils andpartia■ y hydrogenatedish Oiis,
c)A'あ わJた 抑 放 碑 り 加滅姥 寛 力 FttL-35M.助 ssolve l.40g
KOIII h acoholand dlute to 50 mL tt alcohol.臨響 距 純 sh daily. whichcmttn large levelsofcな ‐and rra西‐ isoElerS OfC16,C18,C抑
and C22 Cittn lengths.)
ユ PreparatrOn Or sampre
挽βTable 994.15 for the results of thedaboratoryinに study sup―
Accurately weigh ca 100 mg vegetable oil alld ttallsfer wih
―
″hexane into 100 mLvolumetric aask,whichhasjustbeeniushed p o r t i n g a c c e p t a n c e o f t h e m e t h o d .
with N2・ Dilutt to volume widl tthexane or acetone,Pipet i nL di‐ A P r r n c r P r e
luted soluton in的100 mL volumetric aask and completely evapo‐ Total rrα“s isOmer content consists of r,α ,s fatty acids
rate solvent under N2・ t'万船 ‐ 。Ctadecencate(18:lr);H10no‐ r西α西‐ Octadecadienoate(18:2c,
a DetermrnatrOn o r r c , d e s c r i b e d a s; 1
! 8
廊
瑠 : ,rn船 ctadecadienoate(18:2,o;and
2 め _ O
mono‐ぃぃ ‐ octadeCatrienoate(18:3 ccr,crc,and rcc,described as
Add l mL alcchollc KOH,(d),S01utbn to solvent‐ free residue in l & 3 つ] , w h i C h o C c u r i n h y d r o g e n a t e d v e g e t a b l e o i l s a n d t e H e s d a l
volumetric flask.Heat gendy on stealla bath to dissolve.Flush with
animal fats. Tctal r,α ″s content is deteimined by infrared
N2and h01d stoppered,in dark,mini■ lurn of5 h to oveHllght.After speC廿oPhotomety(IR)using methyl elaidate as extemal standard.
saponiflcation is complete,add 20 mL l.OM borate buffer,(a),and VariorSiSOmers of18:2rr,18:2r,and 18:3r are resOlved;their weight
50 mL H20,and mtx.Addl mL O.5M HCl,dilute to volume wih
percentagesaredetermittdbygtt chromatography.BasedonIRde‐
H20,and mix,
temination,weight percentage of 18:lriscalculated from equ組 on.
Pipet 3コ nL saponifled solution into each offour 13 x 100 Elm test Difference betteen total methyl octadecenoate(1811,assum of】1
tubes.To2tdbes(blankS),addO.10mLinactivatedenzymesolution, 18:l peaksin CC)and Calculated 18:1,gives weight percentage of
(c)(3),and miX Well.To remaining duplicate tubes,add O.10 mLcな‐ octadecenoate(18:lo.
w o r k i n g e n z y m e s o l u n o n , ( c )M (i 2x ) 。
by shaking vigOrously 30 ユ s A P P a r a r t J s
i― ediately afteradding enzyme solution,andletali mbes standex―
cん え,″ ″rograPみrcc).‐ 二With name ionizadon detectoL
posed to air atroom temperature 30 min.Zero spectrophotOmeter (a)CaS at
2 3 4 n m w i t h b l a n k t u b e s a n d m e a s u r e A o caplllattcolumniniectiOnsystem(splitratio■
freacted oils. 10o.Operttng con―
dttons:inJection Port 225° Ci dettctor 250° C.TenpeFature pro―
For oil,calculate g polyunsaturated fatty acid(PUFA)as
gramiinita1 150° C,progralnrate l.0° (yttn,flna1 200° C,「 lnalhold
trilinoに in/100 g oil=WxDFxlド ,where witttrilim師 胡流nal 20 min.的 !ef Operator may change opettng conditions to obtain
soludon from standard curve,and DF=dilution fltor.
optimum settndon ofisomeric fatty acid mettl
a PrepararrOn Or sね 何Jara Curye hydrogencamergas(299。 99%puFity)withOXygenscrubberinline.
Weigh ltXlmg cれcむ‐ trilinolein,99%suCheCkPrep,POBox295, ●)CCcθ れ脇解.―-100mxO.25 mm fusedsilicacaplll町 coluIIln
Elydan,MN 56028-0295,USA,orequivalenty,transfer quantittdvely coated wih SP‐256tl(available from Supelco,Inc.,Bellefonte,PA
to 100mL volulnettc nask,anddluteめ VOlulne wi血″‐ hexane orace‐ 16823,USA)Or other suitable capillary column coated with
tone.Pipet 10 mL aliquctinめ10D mL volumetric flask and ttlute to cyano―alkylpolysiloxane(e,gⅢSP‐2340,CP SIL‐ 88)that prOvides
volume wih salne solvent.Pipet 2,4,こ 8,10,and 12 mL ttqmttinto sane elutton patteコ n as in Figure 994,lS,
s e p a r a t e 1 0 0 m L v o l u I I l e t t c a a s k t t a n d e v a p oガ rみa t.一Maximunvolume
e e a c h t t d10 r yμ ttss h
L,graduatedtoO.1卜 止.
O CCリ g夕

⑥ 2000 AOAC tNTERNATrONAL

AOAC OFRCttt METHODS OF ANALYSS(2000) 01LS AND FAT
Chapter 41,p.33

T a b l o 9 9 4 . 1 5 3 n t e H a b o r a t o r y s t u d y r e s u k s f o r g a s c h r o m a t o g r a p h i o r i n f r a t t d e t e r m i n a t l o n oonf roaft yP aarctitda ,c3oym p o 3 田

hydrogenated veOetabl● o‖3

16:0
A 10.3 0.3 2.4 0.03 0.25 0.08 0,7
B 9.1 ■4 2.8 0.13 0.25 0.36 0.7
Cl 9.7 1.1 3.5 0.11 0.34 0.31 0.95
02 9.7 1.2 4.0 0.21 0.39 0。
59 1,09
Cl and C2 9.7 ■0 4.0 11
0。 0.39 0.31 0.10
D 10.7 ■0 3.2 0.10 0。
34 0。
28 0.95
E 9.7 3.7 3.7 0.13 0.36 0。
36 1.01
R 10.8 ■3 2.6 0.02 0.28 0.06 0.78
18:0
A 3.8 0.8 ■9 0.03 0.07 0,08 0.20
B 7.0 1.6 3.3 0 . 1 1 0。
23 0.31 0.64
Cl 6.7 1.2 2.4 0.08 0.16 0.22 0.45
C2 6.7 ■2 3.9 0.08 26
0。 0.22 0.73
Ct and C2D 6.6 2.6 4.8 0.17 0.32 0.48 0.38
D 7.3 0.6 ■2 0,04 0.08 0.11 0.22
E 6.9 2.9 3.6 0.20 0.25 0.56 0.7
R 5.8 ■8 4.4 10
0。 0.25 0.28 0,7
Total satlarated faw acldS
A 14.9 0.6 2.5 0.09 0.37 0,25 1.04
B 17.2 0.7 1.6 0 , 1 1 0。
27 0。
31 0。
76
Cl 17.6 2.0 2.9 0。
35 0.51 0.98 1.43
C2 17.3 1.0 4.6 0。
17 0.79 0.48 2.21
Ct and C2め 17.6 1.1 2.3 0.19 0。
40 0.53 1.12
D 19.0 0.8 1.7 0。
15 0.32 0,42 0,90
E 17.5 2.8 3.4 0.50 0.60 1.4 1.68
R 17.4 1.4 3.4 23
0。 0.58 0.64 1 . 6 2
13:l rtsOrners
A 4.9 5.2 範.4 0.25 1 . 7 0.7 4.96
B 14.9 2.1 9.5 0.32 1.41 0,90 3.95
Cl 17.4 5.3 12.5 0。
91 2.18 2.55 6.10
02 17.5 4.2 10.3 0.73 1.81 2,04 5.07
01 and C2D 17.4 6.9 10.9 1 . 2 0 1.90 3.37 5.31
D 26.6 3.6 9.6 0。
96 2.55 2.69 7.14
E 32.6 1.9 7.3 0.61 2.53 1.71 7.08
R 19.4 4.3 9.7 0.83 1.87 2.32 5.24
18:l o isorlers
A 24.9 1.1 3.8 0,28 0,95 0.73 2.66
B 24.7 2.4 7.1 0.59 1.75 1.65 4.9
Cl 28.1 3.2 6.9 0.88 1.94 2.46 5.43
C2 28。
2 3.3 7.1 0.93 2.01 2.60 5.63
Cl and C2D 28.3 4.2 6.5 1.19 1.84 3.32 5,16
D 34.3 2.6 6.1 1,02 2.11 2.86 5,91
E 34.3 1,9 10.5 0.66 3,61 1.85 10.11
R 32.2 i9 6.5 0.61 2.10 1.71 5.88
18:2{nい
6)
Ａ

53.0 0.2 ■0 0,10 0.53 0.28 1448

Ｂ

41.5 1.0 1.4 0,40 0.59 1.12 1 . 6 5

③ 2000 AOAC iNTERNATtONAL

01LS AND FAT AOAC OFFCt札 MttODS OF ANALYSS(2000)
Chapter 41,p.34

Tabl● 004.lS (● 。ntrn口。り

Samplea X,% RSD"% RSDR,%

Cl 33.6 0.7 1.7 0.22 0.57 0.62 1 . 6 0
C2 33.5 0.6 1.1 0.19 0.38 0.53 1.06
Cl and C2 33.6 1.8 2.0 0.59 0,66 1.66 1.86
D 13.6 1.2 2.2 0.16 0.30 0。
45 0 . 8 4
E 1 1 , 2 2.0 2,3 0,23 0,26 0.64 0.73
R 26.5 0.8 2.5 0.22 0.66 0.62 1.85
18:3(何 ‐3)
A 0.9 7.3 9.6 0,07 0.09 0。
20 0,25
B 0.8 li8 20.2 0,09 0.50 0.25 1.4
Cl 0.8 5,6 8.5 0.05 0.07 0。
14 0.20
C2 0.8 5,2 8.7 0.04 0.07 0。
11 0.20
Cl and C2 0.8 7.7 19.9 0.06 0.16 0.18 0.46
D 0,9 2.5 8.6 0.02 0.08 0.06 0.22
E 0.8 6.8 17.5 0,05 0.14 0。
14 0.39
R 1,0 3.1 8.4 0.03 0.08 0.08 0.22
18:2r tsomers
A ND° NAど NA NA NA NA NA
B 0.1 40.2 92.1 0103 O,07 O.08 O.20
Ct 0.2 19.8 96.3 0,03 15
0。 0,08 0.42
C2 0.01 25.0 78.9 0.02 0,07 0.06 0。
20
Ci and C2め 0.2 79.3 101.7 12
0。 0.16 0。
34 0.43
D 0。
3 25.8 100.5 0.07 0.26 0.20 0.73
E 0.03 28.5 66.9 0.08 0.20 0。
22 0.56
R 0。
1 80.0 100.7 0.09 0。
12 0.25 0.34
18:2!isorners
A 0。
3 25,8 49.8 0.08 0.16 0。
22 0。
45
B 0.7 3 3 . 1 55,0 0。
24 0.39 0.67 1.09
Cl 1.5 17.2 34,5 0.25 0.51 0,7 1,43
C2 ■6 2.1 35.5 0.03 0.56 0.08 1.57
Ct and C2D 1.5 6.0 34.2 0,09 0.51 0.25 1.43
D 3.3 5.1 22.6 0.17 0.75 0.48 2.1
E 3.1 2.6 27.3 0,03 0.84 0.22 2.35
R 2.3 4.3 20.3 0.10 0.48 0.28 1 . 3 4

18:3risomers
A ND° NA」 NA NA NA NA NA
B 0.03 152.3 193.7 O.05 O.06 O.14 O.17
Cl 0.1 8 6 . 1 136.6 0.04 0.06 0,11 0.17
C2 0.04 75.0 141.6 0.03 0.06 0.08 0.17
Cl and C2 0.04 93.81 133.8 0,04 0.06 0.11 0.16
D 0.2 55.4 76.9 0.12 0.16 0.34 0.福
E 0.2 31.7 79.7 0.06 0.15 0.17 0。
42
R 0.1 0.0 150。4 0.00 0.08 0.00 0.22
a卜晩喜 Ы endS ous闘 tt ∝ va市
Jttextrattdttm2dttmttattnesoattR瞥
o前Eョ blends offat extracted from盟
付neS)diluted Httth unhydrogenated corn ;D― 冊鞘鑑営ヤ
肝‖占
‖鞘鞘協雛濫転脚 線縄眠桶e
(partia‖ 。
y hydrogenated soybean soiけ
b Blind duplicates.
° NDョ
notdetded(content ot total faty acids(0.01%).
“
NA i not apptcab:e

⑥ 2000 AOAC iNTERNATiONAL

AOAC OFF,CIAL MttMODS OF ANALYSiS(2000) 0,LS AND FAT
Chapter 41,p.35

“ 18 23
3 1 1 7 !
湾！

34
3o l
・
︵
翻Ａ

3038
°
ｒ

32
1371♀
40
30 0S TIME(MIN)40 45

Figure 994.1-18 region ofthe gaa chromatogram 的 o frt haec偽

t d m e t h y l e s toemr 3p竹
artinl:y hydrOgen soybean
o13,using 100 m x O.25 inm fused stiica cap‖:ary●o:umn coated wlth SP‐ 2500.

ω ccaα `初 れ筋 θれ,一Ittect l-2 HL mettles俺 職 (in hexane

稲樹脱群「
母セ 麒 】
響報総ず
岩と
or heptane soludon)1沌 m test sを
m口 le into GC.Compare retention
t i m e s o f t e s t s a t t l e w i t h t h o s e o f C C rnegf‐
erence
n e s s c e l i s O . 1 - 1 . O m m w i t h N a C l o r K B r w i n d o w s . A l l ure
i n s ttM,15).
trumnts
mustbecheckedforwavelength accuracy andphotometricscale ac‐
見 r F D e rr ― n a t r O n O r T O r a r! t r a n g C o n 怖
curacy according to Mnufacturer's insmcdOns.Chart paper inust
be linearin eitler wavelengdl or wave number and calibrated in ei‐ Perfom asin 994.14 Gca 41.1.36A).
dler t r a n s m i os rs i ao bn S, O■r b a n c e , A .
a caturarrOng
a Feagenrg
Calculate weight percentages of fatty acid medlyl este
CC確 ″″確 Sran協泌 .―Mixttre of cお ‐and rrans‐isomers of sumlng unity response factoribr each component:
known compositlon;available from Nu Chek PrePj lnc.,Elysian,
MN5駅 ル8,or Supelco,Inc.,Bellefonte,PA 16823,USA.
晩 ,%草 Px/PT x 10tl
,PreParatran Or Mbrnyr Egrers
where Px=GC area counts Ofspecinc methyl● ster peak;PT=total
Melt solid fats or free fatty acids at temperature≦10°C above
area countt of ali fatty acid methyl ester peaks in chromatogran.
melungPOintandmix.Ifcloudy,魚 11erhЮ ughniterpape■ Ifdiluted
sample is cloudy due to H20,add smali portion of航 caculate weight percentage of 18:lr isoHlers,Wl&ば
町 drous
NttS04tO melted smple,mix,and let settie before taking pOrtion
for methyla飲〕 n.Using ca400r500 mg fat,pFepare methyl esters as 71&1,%=博 、はs―(1.74 x W18あ ) 0・84(M比 82,十 Wi&3か
in 96933(影 を41.1.28).
where ttra■==tOtal rrans cOntent deに mined by IR;W18:2rr=tOtal
二 CC Anarysrs crFatt/Acfa ConPcsrtrOrT
weight percentage of江 1182″ isomer peaksin CCi 7182=tOtal
(a)CC PCつ rma″確 SP変 "∽ 筋 ″S・ 一 Iniect l-2ル methyl es‐ weight percentage of江 1 182risomer peaks in GCi路 歓争 =tOt州
ters from CC reference standards,C,into GC,Select GC conditions weight percentage ofa11 18:3risomer peaksin CC:1.74 and O.84=
to obtttn resolutionoflnethyl esters atleast equlvalentto thatinFig_
c o r r e c t i o n f a c t o r s f o r r r a n s , r r a n s rf ra at t ty fa ac ti td ys tt
ure 994.15. acids,respecは vely.

◎ 2000 AOAC iNTERNAT10NAL

(｀
OLS AND FAT AOAC OFFICiAL MgrHODS OFANALYSS(2000)
Chapter 41,p 38

Calculate weight percentage of 18:lc isomer,Wi8:ici 6α材!わみi Scc Appendx B,safety notes on handling Of special

W i & l c , % = &路1 - W i & 1 ′

chendcal hazards‐ ―carbon disulrlde.最 %Material r
Safety Data Sheets,or equivalent,for each lcagent.
DispOse of waste solvents according to applicable envi‐
w h e r e佑
抑1 = t O t a l weight percentage of all d l e 1 8 1 lr o ni mseonmtea rl rp ue laekss a n d r e g u l a t i o n s .
in CC.

Reference主 ユAθACあ ,7軌 783(1995). S22 Table 96S.34 for he results ofthe interlaboratory study sup―
ユ Cれ万0閉燿rag∴sc,28,633(1990). porting acceptance of dle method.
JAOCS67,804(1990)Ⅲ 69,95(1992). A , P r r n c r p r e
R?yな夕か Marchゴ タ99 ln most naturally occurring vegetable Fats and oils,unsalurated
constiments cOntain only isolatet ice.,non‐coniugated,double
bonds in cお con「lguEぶon.The cな bonds lnay be isomeFiZed tO
41.1.36 rra,s cOnFlguraticn duing extracton and Processing procedures,
AOAC Ofrlctat Method 965.34 due to oxidaはon,converslon during headng,and/or partial hy‐
1301ated rrans:somers drogenation,A村ml and marine Fats may∞nttnmeasmble anounts
in Margarines and Shortenings ofn― lyoccuringrran isomers.Isolatedr_bondsinlong― chAn
inttared Spectomerlc Method fatty 4cids,eslers and triglycerides inay be measured by
Firet Actton 1965 infrared spectrome的 げ.Absorption band with maximum at ca
Finat Actio■1982 966 cm 1(10.3 μ m),ariSing from C‐ H defomation about r昭,s
Rev18ed Ftret Action 1997 double bond,is exhibited in specm of ali compounds containing
isolated rra″ s group.This band is nct observed in spectraofcore‐
AOCStAOAC〕 brnc」
sponding cおand saturated compounds,Measuremeitofintensiけ
t o 徒o n o f i s o l a t t d r t t b m d s i n n a d mof
M e t h O d i S a p p l i c a b l働e 耐胡 l ttsお sorption band under analyncally Controlled conditions
研 Focessed longttn wids,esm,andtFiglyuins widlr万 船 levels is the basis for qumtitative method for deten口 Ination of isolated
! ″酒 C O n t e n t . F o r h i g h a c c u r a c y , c o m m o n i n t e r f e h n g a b s o r p t i o n s
挙. 磁. M e t l o d t t n o t atが航 t L c aObnlley owriお h specinc時
associaにd widl glycerol backbone cf triglycerides and carboxyl
c a u t i ofnastめ
s a n d t t s c o n t a i n i n g げc
l toen iqい
mtidtt tOVer5の
.g,■コじOiめ;tO materials containing mdiOnal groupoffatty acidsmustbeeliminatedbyconversionofthesesaEl‐
gated unsattmtion●
ntensity ofC‐
H defomation aboutr″
附doぃ JeSめ their metttl esters prior tO anttysis.
groups which m田 町戸
blebond[e.g"Castoroil containing五 cinoleicacidoritSgeometrical
isomer,Hcinelaidic acid(12‐hy仕oxy‐ 9‐octadecenoic aci●
];to
丘 APParattr3
`
nixed glycerides having long and short chain moieties(e.g,,
diacetosteariゅ
;Or in general,to any matenal contalttng consutu_ 総1臨群摘辞
!骨
麟離盤選
齢群総 品
辮絆讐書
艦鞘輝盟濫艦輪能
966 cm l band of C‐H deformation ofisolated rran double bond, 紳 齢 艦 結: : 憾 ぷ8 冊 艦 品 艦 鞘 ミ
For direct analysis of triglyceFideS use procedure desc減
脱 dh SPeCtrometers should employ TCS or DTCS detectors orinsure
965.35 ts22 41,1.61].) lineariけ.

Tab!e965。 34 interlaboratory sttdly resu!t3fOr determinatlon of fnn3 380mer3 by infrared 8pctrOmtrlc method・

Sample __ No,oflabs _Mean,% Sr sn RSD..% RSD。 .% ヤ R・

５

Metl」 20.23 0.49 1.30 2.40 6.42 1.37 3.64

６

Tg‐1・ 6.09 0.22 0.60 3.65 9.82 0.62 1.68

６

Tg‐2′ 1 . 3 3 0.18 0.44 13.86 32.78 0.50 1.23

５

Tg‐39 16.14 0.20 1.19 1.21 7,38 0.56 3.33

６

Tg‐4g 16.02 0。 38 1.07 2.範 6.69 1.09 3.00

６

Tg‐5角 30.91 0.46 1 . 8 6 1,49 6.03 1.29 5.21

T9‐6′ _上 4 _ .25 0.81 1.22 1.71 2.59 2.27 3_42
宮
Based on coWaborative study resuRs ofthe rnodi“ed method.
D r‐
2 0 x sr
・
R=2 8x sR・
」
Me,1‐ Methyl elaidate‐memyi stearate with 21.lSん rrans.
・
Tg‐1=Trielaidin in zero‐ trans peanut oll with 6.14,6材
ans.
′
Tge2‐ Canola o引
。 TO‐3 and Tg‐4=ShoRening
n T9‐
5ョ Margarine
r Tg,6=Par fry o‖

◎ 2000 AOAC tNTERNATfONAL

AOAC OFRC'AL METHODS OF ANALYSS(2000) 01LS AND FAT
Chapter 41,p.37

E c a r r D r a t r O n
(b)rnrra″ ″ ど竹″泌 M印 !れgα !な.―Equipped with NaCl or
KBR windows and flxed Pah lengtt ofl mm.For use in null‐type For each standard soluは
on,calculate the exact weight Of medlyl
insmments,餌 rs of Cells matched to wittn O.01 absorbance unitselaidate diluted in 10 mL solvent.Analyze each solution and deter‐
are required.In spliい
bean type lnsmments widl both cells illed IIllne baseline corected infrared absorbance at 966 cm l as de‐
with solvent,electronic balance of 2 beams to within these limits s c b五e d i n C a n d H .
should be attained.
(a)乃 r勉 酒 働 確 所 <メθ%.二 Using Flrst‐ order regresslon anal‐
0降 r″ rrricPaSな。一Class A;1は25,and 50 nL.
a)P,grs,_class A;1,3,4,5,7,and 9 mL. 曲 1脚 斜 魁 股 掛 誰 賭 見 辞弔 督 甘 料 鮒 朋 撚
●)D■ ″sめたn舛姥″rp″crs,― For 81111堪
of infrared cells. standard solution axiS)as
o‐ a funcは
o n of he gram Of met町 1
(DA″切 ∽′腕ran確.二wid1 60 g capaclけ ,Capable of weigh‐ elttdate/10 mL ofsohtion←
aXiS).
ing O,2±0.0001g.
(b)Fθ r,ぃ ∽ ″ただ >rθ%「 押拘ceed asin(a)uShg 10,30,50,
(DS'Ctt Wacrう αれ.―For melting solld fats. and 70%rratt smdard s01utions.
a Reagenほ Once cdib鵡 on data have been established,they shOuld be
:S″
(O Carb04″ Dry,reagent grade.Use with cade‐
" 』2 rCS2j・ heck e d p e d o d i c a l l y t o .e Fn os ru r te t gh he ei sr t v qa ul ai nd ti
quate vendlation. dve acc― y and precision,primary Or secondtty reference should
●)Pttα り S勉滋 魅 .―Medlyl elttA分',and methyl oleate; be mehylated and analyzed wih each group Of smples,then
メ"%puriけ (aVttlablefro■e.g.,NuCheckPreP,Inct,POBox295, che●ked toensure thatmeasuredrrar levelisin agreement with he
Elyttan,MN5〔 紐28,USA). known or established mean value.

a PreParagon 何
orraSrね
J 3orutrOng 見 PreparagOn or■ ぉtsanPLs
ω M夕rり:θra滋確 sra″ れ だ sθ′ 所 例 s「 (ゴ ― )a″ θg/mと。 Ac― 一 Melt solid fats on am sに
bath and mix before smplhg.Samples
curately weigh o,5± 0.01 g methylelなdaに Primary standard tothe 血 t a p p e t t c l o u d y d uetopresenceofH20ShOuldbettatedwidl
nearest O.0001 g into 25 nL volunetric flask.Dllute to voluHle 町 drOus sodium sulfate and iltered mough ilter paper before re‐
With CS2,StOpper,andmix well.口 )a00207筋 L― Pipet5.00 mL moving smple for maly鵡
O。020g/mL elaidate standard solution,(r),int。 50 mL volumetttc C o n v e ■0 . 5 - l g t t t o m e t t l e s t e r s a s d e s c F i b e d i2n 9 6 9 3 3 律
nask and dilute to volume widl StOPper CS2・ and mix well. 41.1.28).
●)Mを !めこθ脇確 S=血 rr sθ れ,ぁ .判 .020g/mL.Accmtely Accurately weighO.20±0.01 g preparedneat medlyl esに rstc he
weigh O.5±0.01 g Eletwi oleate primary standard to dle nearest nearestO.0001 ginto 10mL volumedc nask,Dilute to volme widl
O . 0 0 0 1 g i n t o 2 5 m L v o l u m e t r i c f l atsok .vDollluumに
e w i t h C S 2 , CS2,Sttpper,mdコ 田X Well.
sbpperP and mix well.
a rnfrang Der― rnatrOn
oゴ ,4筋 a7恥 rrans撒 比 協 な .二WeightodlettarestO,0001g
into 10 mLvolumetric nasks,amOuntofmethyloleateprimtt stan‐ Setupoperating conditions ofinfmedsPectrometeraccording to
dard l昭ゼ table below)to± 0.005g.npet specifled volume of
O,0020g/mL methyl elぶ date standard soludon,GXr),intO each 隅 貰 盤 品 深 斜 鑑 縄 盟 l品 齢 七:ド1鷲霊‖だ群経協置をど世
nask and dilute to volume with CS2・ StOpper and mix well. Ployed should be identical for smples and standards.的 =gi lnta_
red spectrometer should be checked to ensuFe dltt it is Opera
withindlemanufacturer's established specincations.Checks should
0.0020g/mL
Nominal% Methw elattate Mettyl oteate includesignal‐ to‐ noise,andphOtometricandwavelenghaccuracy。 )
sねndard solutlon.mL DHmarV standardB c (a)Sttrた と 筋 筋 施 r″ 閉確 れな.―Fillcleaninfraredhqddcell widl
１

1.00 0.198 CS2,remOve any Ar bubbles,stoprrP and place in spectrometer's

４

4.00 0。
192 cell holdeF.COuea single‐
bean specmm to be used as attfeFenCe
●aCkgrOuno.Renll clean ce■widlsmple solution and collectsh‐
７

7.00 0.186
bealn spec血血 OfSampleA R調 ぃ mple specmm against back‐
gle‐
ground and convertto absorbance.
(d)raョ α tt a材 ■
弧 、rraな Sr仰あ 材 sο肋 腕 .一Using (め 助 確 θr Jza′ ‐う夕御,tな れ切α Ⅲ… Fill clean infrared liquid
O.020g/mL methyl elaidate siandard solution,(a)(r),and ceu witt cs2,remOve any ar bubbles,stoppeF,and place in refeト
O.020g/mL medttA oleaに standard solution prepare set of calibra‐ ence beal■cell holder.Flll second nlatched cell widi sample solu―
tion,remove any airbubbles,stoppert andPlace in
tion solttons by pipetting vOlumes ofsolutions(speCiied in table
holder.Collect specmm and cOnvertto absorbance.
below)into 10 mL volumettc aasks.助 lute to volume witt CS2,
stopper,and mix we11. 比 carcurarrOng

淵 蹴 樹 幣 3だ縦 樹 ,総
0.020g/mL 0.0209/mL 縄 鑑 替 総 脳
Nominal% Methyl elaidate Methyl oleate absorbancebandat966cm 1● タタFigure965.34j.Pointsin specmm
rraRS standard solution.mL standard solution.mL between whichlineXris drawn will vary withpeak absorbance.For
１ 範 ５ ７
。

1.00 9.00 accurate resuits,baselines mustbedmwn dle same forbodlsmples

3.00 7.00
andstandards.Forttgh!声 αtt contなningsmples,basdine shouldbe
。 。

drawn to the nlinimum located at 985 cm 1.Occasionally,this ttni‐

5.00 5.00
mum may be absent and the pointto which baseline is drawn wil
7.00 3.00 need to be eshmated.

0 2000 AOAC tNTERNATtONAL

01LS AND FAT AOAC OFttCIAL METHODS OF ANttYSS(2000)
Chapter 41,p.38

7086t― 41.1.36A
AP
AOAC Off:o18:Method 994.14
,6
純旧獄樹榊鵠鞘鞘隅品嵩
まd Ftts
infrared spectrophotometric Method
4
Ftrst Action 1984
言 。 Finar Action 1998
.2
AB一 単 、 (Applicお le to detemination of total isolated[1.e.,nOncOttugated]
′raな content h fats and oils contaning>5%!″ な fatty acids.Not
0 appltabL to smples containing>5%coniugated unsamtiOn[e.g"
1000 950 900
tung olll,mterials mttning functiona g
Wamunberlm‐ 1〕
sorption ofC―
H defo― tion around博
鶴 bond le.g"castOr oil con‐
taining Hcinoleic or dcinelaidc acids〕
,Or any materials where
speclic groups may absorb close to 967 cm 1110.3
ml.) μ

a秘 ,Fθ
″f s2β Appendix B,safeけnCにS On the safe handling Of
s p e c i a l c位
h e a■l h a z a r d s ―
a r b o n d i s lu dl e「. D i s p O s e o f
carbon disulttde in an apFopnate nallner compttble
with environmental rules and regulations.
: お
・1 AP 挽 θ T a b l e 生1
9身 4 for dle results ofthe intedaboratory study
AB
Porting acceptance ofthe methOd.
.05
A P r r n c r P b
1000 g30 m lsolated rraな
dOuble bonds(predominmt,惚 湾 cOnnguration h
W― L t m ‐・〕 Partially hydrogenated fats)show absorption at ca 967 cm 1
(10。3 μHめ deriVing最 om C‐ H defo-lon about持 唖 bond lsO‐
F i g u F e 9 6 5 . 3 4 H n f r a r e d惑 rs a陣 c f m e t h y t e s t e r s
late"招 肘 contentisdetemittdbymeasurenentofintensity ofhis
containing 70 and 2%,コ"o.
absorption.Triglycendes Or fatty acids are convetd to methyl es―
ters before mking IR measurements.Totalisolated rran cOntentis
calculatedusingcalttationcurveofabsorptionversus,″ 湾 content
(
Detemine baseline corrected abscrbance of absorbance band ofcalibmtion s01udons.

松 濫1梱.梅
鎖脳総鮮灘路窮 ど
逮穏艦 ユ A P P a r a m
r a r r a平冴c r r aθゎ確
P れ姥r r R ) .D ―
o u b bl ee a‐l n R o r F o u r i e r
ofpeak maxinun willva,y with insmment and rraA level ofsam‐
ple analyzed.PositblofmaximumshouldbeestablishedseParately
for each insmment using 707o,raw standard solution.恥 s same 縦辮c 1絆
瀧ヽ蹴 胡憚 陥艦 様ま
品だ
posi41on should then be used at au concenmtiOns.)
ness cdis O.1-1.O mln with NaCl or KBr windows.Allinsmments
must be checked for accuracy ofwavelength and Photometric scale
Using Flgure 9tお
34,calculate Ac as follows: accuracy according to manuFacmrer's insmctiOns.chart paper
HlustbelinearineidlerwavelengdlorwavenuHlberandcalibratedin
e i t h e r t t n s m iosrs laobns,o■
rbance,A.
Ac=otP― AB)
a月 中 fg

Using cttibration data for the appropnate tt

rttω
rangeは 10%Or (ゆ 6αだつ″ど's“ Dry,ACS grade.
"閉g rcs2)。
●)確 的 !挽 協 確 Sracた ざθJzr助 .-20 mgrmL.Accurate呼
>10%),Calculate weight equivalent of methyl elaidate h smple as
follows: weigh ca 2000 mg nchyl elaidate tpurity刃 9%)tO the nearest
O。l mg hto 100 mL volumettc nask,dluteto volume wih CS2 SO‐
intercept lution,and Hux thoroughly.
Methyl elttdate,g= Ac―
(O MCttr dca郷 成 鋭 施 卜 刻 理 価 β wareas h● )us‐
i n g m e h y l d e a t e t p u r i t y p 9 9 %週
) h SO に
fmehyl daidate.
(d)例 ど う白何jθ
″Jθ!″』わ明じ.判 .8,1.6,4,8,12,16,and20 mg mdlyl
規 ∞ elaidagmLin 19.2,184,1612,8,4,andOmynLmethyloleatWS2
solution,時
s「湾tvely.Accurately add l,2,5,lα
15,2Q and 25 mL
methyl elaidate stock solution,(め,intO sepane 25 nL vol― tric
nasks using plpets.Dilute contents of flasks to volume wm mthyl
RepOrt results to the nearest O.1%.
oleate stock solution,c).VeriSWdghtperntagesofmethylelaidate
Reference革 泌 θAC 48,437(1965).
andmthy1 61eate by ttPlllatt GC analysis ash卿
41S G2241.1.35A).
Iweightpercentages ofmedlyl elaidatedfFerl沌
mexPected valuesby
AOCS Methods Cd 1461如 d Cd 1495.
汚 %KfOrsmplescm成 拭ngpercentmdlylelaidatejor混%tfOrsam‐
R夕νな夕な ル灯
αrch F998 10%mehyl elぶ dateboF>2%(foF― pleS containing
ples cOntainingく

⑥ 2000 AOAC,NTERNAT!ONAL
AOAC OFFiCiAと METHODS OF ANALYStS(2000) 0'LS AND FAT
Chapter 41,p.39

on ofisotated rrang unsaturated faty

Table 994.14 8nter,aboratory study resuR3 rOrinfrared determina‖ ac旧
8 3n parttatiy
hydrosenated vesetable oti3

Samolea x.%rra,s RSD,1% RSDR。 % S, SR r R

A 6.2 4.8 34.6 0,3 1.8 0.84 5.04
B 15.5 4.2 11.3 0.7 1.8 1.96 5.04
Ct 18.9 4.6 33,7 0。
9 2.2 2.52 7.06
C2 19.1 3.7 10.3 0,7 2.0 1.96 5.49
Cl and C2b 19.0 5.8 10.5 1.1 2.0 3,07 5.61
D 3 0 , 1 3.0 9,0 0,9 2.7 2.52 7.06
E 34,6 1.0 11.3 0,3 3.9 0.84 5.04
R 21.6 4.1 8.8 0,9 ■9 2.52 7.06

」 l 襴
緒 結 掛 描 甜 博艦紺艦蹴描隅鮎肥晋
erence sample(pa'tatty hydrogenated 30ybean 80iけ
穏i嫌
驚認群 艦側洛
堺齢隅 淵鍋品 群挫甫_
B‖ 1 6ateS
nd du伊

ons.PerFom
>107o ndhyl elai的 め,Prepare ttsh calibralion sol岨 at 967 cm 1.This can be Obtained by supenmposing 2 spectra to
ofCS2・
step E l―edately because ofhigh volatiliけ draw basd施 .)
Draw anothersmighthnePdLitottnateandpassinghough
, P r e p a r a r f O n O r T e s r 軸″ apex ofanalyttal band asin ngure994.14.KLine meets zerolineof
M e h s o l i d f a t s o r f r e e f a t t y a c i d s o n s t e a n b a h o F i n Ochat V e n aatt tpoint
e m ‐ z apeX at 4 and baseline tangent at X.)
For transnlssion spectunl,measuFe dStances XZ and yZ Calcu‐
p e r a t u r e 1C 0 °above meldng ttint.r melted fat is doudy,81ter
述
p r e p a r e m e t t l e s t e r s lateおsorption of cd厳 on solution,AF
山 o u g h n i t e r p a p e r e U s h g c a 4 0 0 - 5 0 0 m g,的
asin 9ゆ ル:Accurate results depend on ttmy
節 ●解 4 1 . 1 , 2 8 x 距 Ai i log tXZ/海 ら
of fatty acd medlyl esters.BefoFe R analysiS,remove excessive
uSing
levels of impurities[etg.,nonsaponiflable matter,polymers〕 To calculate absoFptiOn of calimtionlutioL s。 At,read Ax at X,
suitablecleantuppFOCedurete.g"SaponacatiOnfo1lowedbyextrac‐ andAY at比
don of nonsaPoninabL matter,hill‐layer or column chromato控
urately wdghca400-500mgundilutedfatty 1
acidme的 Ai=AY… Ax
phy].)A∝
esにrSurl)totlenearestO。 l mginto25 nLvolumetFiCaaSk.Dilute
ユo t n g n e t t l e l a i d a t e r m L c d i b r a t i o n s o l u t t l t t a b s c
tovolumewithCS2S01uticn.PerfomstepEimmediatelyttcauseof
sus coHeswnding Ai vttues as ordinate.Draw bett smight line
high volatility of CS2・
mugh 7 pOints Plotted.For better accuracy,detemine linear re‐
丘 rfPヵ
コ胡 DerermrnarrOn
Fill cell with CS2 SOlution,and mtthing cen widltest smple
fFOm D or cdibration solution ton C(0.USe hypodemic syinge
witlblunted needle,andcen upnght,lnJect tombottomsobubbles
pass up hough ceu.when using double‐beam lR,place cell wih
CS2inreference beam.Place cdi wihtett smple oFCaibratiOn so‐
lution in sample beamo Scan spectrum(T or A)fron 1050 to
︵
一〓●●﹄

選認酬 群雷
撒電性 辮灘 酬 胡瑞
●ュ︶ｏｏＣ８一

● ０宮 口０ と０ ●ａ ｔ

md store itin memoFy OfinstruHlent datahandling system.Scantest

smple or calibration solution in same range as that for reference.
R江loobtainedspecmmagttnstbackgFOundSpecmmtoobtainme
”■Ｅ”宮８岸

ror A,Measure spectra of calibraは on soluはcns in oFder Ofincreas―

lng concenmiOns.

見 carctrra〔
腕 s

For each specmm,draw basdine tangent to peak ttnina tta‐

c e n t t o 9 6 7 cFml gl唖
G9 ″
9 4 . l o o″:oIMtθ i s i m p o r t a n t t o d r a w
corect baseline because of Hleasurement of basehne corected ab‐ ,0●0 00●
sorption.Absorption minima mightslightly vtt between smples. Frequency,cm‐ ￨
Concentration andamounlofrra,sunsaturationmay ini■ enccPosi‐
tion of absottton ddm Bestresults are obtained when baseline
forsampleisdrawnexacdy asbaselinein specmmofOneofcを 出敬竹 F i g u r e 9 9 4 .J1R4 卜 spectra cf paRialty hydrogenate
tion standards having approximately same intensity of absorption c a n o 3 a o l l m e t h y : e %s ●o
t e r:su像
tion in CS2D・

0 2000 AOAC tNTERNATiONAL

0にS AND FAT AOAC OFFiCIAL METHODS OFANttYSls(2000)
Chapter 41,p.40

ltdatat(Nθ
gression equationto「 ttf Onceobtained,calibratloncurve is used,it must have linear resPonse With adequate sensidvity and
does not have to be repeated aslong asinsmmentsettings,parts,or satisfactory baseline correction.
cells have not been changed.)
a Feagearg
D e t e m i n e a b s o r p t i o n o f, tA e8 s' tu 唱
sS a山
s ma pm に
e procedure as
forcalibration solutions.Usingcalibrationcurveorlinearregresslon (a)6α rrJ2,ど as.二He(prefered for betterresolution and coluIIln
equaton,deterlmne aIIlount M2)。 f mg mehyl elaidate/mL test l i f e j o r N 2 d r i e dn ianngdζ
1 0c omngt ぶ
Ong.
sample soludon,D,coresPonding toA3・ CalCulate peFCentrratt un‐ ( b ) O r Lr夕g いc s , 一
H 2 , 9 99 ・
%free fromorganic impurides.Airor
s a t u r a t e d f a t t y a c i d c o n t e n t a ss t sm me pt lt α l e l a i d a02,free
t e i nfrom
に organic in,wittes(く 2 ppm hydrocarbons equivalent
tO CH4)・
%,″ 酒 unsaturated fatty acid content(as Elethyl eladate) 鶴 確 s仰 あ ras._Mixttre ofcお andrrarumehylesters
oR瞭
=100x財 ノ〃1
of known composition close to that of marga口 nes.

where〃 a PreParafrOn OrsarTPre

1=amount of fatty acid methyl esters in test sample solu‐
tion,Ing. lsolate fatby 118●夕
92仇 を33.6,07).Using ca 350 ng fat prepare
methyl ester by 96,。 3 3(s2241.1.28).To methyl esters in
Reference臣 ユ Cれ″初aragぇ 最克 28,633(19901.
stoppered
glass‐ flask,add volullle ofheptane to yield l-109ら
solu‐
JAθ6S 67,804(1990j;69,95(1992). ″
はono Swirlto dssolve.
ユA θA C れ , 7 軌 7 8 3 ( 1 9 9 5 ) .
互 OC OPrarrng cOndfJong
Rビッなタ
ガf拘脇rch r999
Carrier gas flow depends on carrier gas and packing density.
Following aow rates are recommended:He,15-20 mlymini N2'
41.1.37 8-10 mL/min.Flow ofH2tO detectorshouldbe halfthatofcarrier
AOAC Offtcial Method 985.21 gasi now ofo2ShOuldbe ca5-10 dmesthatofH2・ ItteCtOrshould
Totatrrans Faw Acid isomers b e 斑2 0 - 5 0 °
C higher ttan colulm temperamre and detector should
in Margarines be at 250-300° C.
Ca8 Chttmatographic Mcthod
見 6C Ph所 口研旧礎 う口鈍W化回敵閉じ
Ftrat Actlon 1985
Final Action 1992 Perfomanalysisofmixtureofmethylelaidateandmethyloに ate,
Adiust methyl ester amount,coluHBn temperamFe,and carrier gas
(Not apphcable to margttnes containing hydrogenated marine nOw so that nettl elaidate peak is recorded ca 30 min aneriniec_
oils.) Hon and方 ヲ full SCale.Measure base widths in mm of medlyl
A P r r r r p r e elaidate(,1)and Eletlyl oleate(w2)OetWeen Pcints ofinteFSeCは On
wldlbaseline oftangents drawn tO innecは 。n pOints ofcurves.Also
Methyl esters of ttty acids from margarlnes are separated and
measuredistanceinttbetweenpettFLaXimumformedlylelお date
measured by CC to detettnetotal,脇酒 unsamrat10n content(rrans
and metlyl oleate,比 Calculaにresolution,R:
isomers ofunsaturaにd18 C actts).Resultsby this method are com‐
parable to dlose obtained by IR medlo4 96534●θ夕41.1.3o.
R= 2y
ユ A p p a r a r t r s 路 ヤ晩

(a)CaS Cれ ″肥 rograPれ.二With flame bnization detector and

minimuln dead sPace in inJecはo■system.Maintain column tenper‐
ature within±1°at ca 220°
C.
_6.lm(20 ftj x 2 Hlm id giass or stainless sに
(D ccr″ れた。 el
tpOlyunsaturated components with>3 double bonds my decom―
pose in stainless steel columR).
(C)Pa枕 れg.― Acid‐washed md silanized diatomaceous e卸 帆 Analyzereference standardmixtures undeF Same Condidons as for
lα卜120 mesh,coated with 15%OV-275 stationary phase(15%
OV‐ 275o■ 100r120 mesh Chromosorb P AW‐ DMCS is available
fron Supelco).OV‐ 275 col― s are extremely sensiは veto 02,par‐
dcularly at operating temperature.Carier gas and alllines must be
free of02・COnditionnewcolumnsby purgingwidlc狐 】ergas over―
nigh at room temperamre and then gradually increasing tempera‐
ture(1-2° 9航 n)tO temperature 10-20° C higher than ope劇 tng
temperame but nO hgherthan recomended HElitS.Ifcolumn h岱
been lcmoved fFOninSmmentOF eXpOsed to air,purge coluEm wih
c2田ter gas for l-2 h before heating.
一 Maximum volume 10ぃ
O rSyringを 。 ,gFaduated的 0.1ル .
C)R2Cθ rダタ∴-0-2.5 or5.O mV range,く 1.O s response rate(dme
for pen to pass froln O to 90%following momenttty introducよon of
100%signal),25 om mininumpaperwid曲 けand25-100 cm/hpaper
sPeed;equipped withattenuatorswitch to change mge.Ifintegrator
Se19ct conditions to obtain R≧0.5.
a Det_rnarrOn
With apparatus showing stable baseline,lnJect O。1-2 μLl-10%
hepme s01uttn of nethyl esters.Change setting of attenuator as
necessary tt keep peaks On chart paper.
sample.Measure retentlon distances of known esに rs and identify
peaks from sampに by comparison witlretention distances ofpeaks
from standard mixttres.ngure 985.21 」
shows aけ _
ca ChrOmat。
graPhic separation of mehyl esters ofmarganne fatty acids.
胤 carcuratrOn3
Use mehod ofintemal standardization(■ omalization),whch
assumes all components of methyl esters are FepreSented on
chroHlatogram,so that sum of areas under peaks represents 100%
of constituents(tOtal elution).Obtttn total rran cOntent by addi‐
tion of ali rra,s isomers.
,PrarsrOn
(う Re陣 腕 】り .―TWO Sirue deteHninations perfomedon sanrle (
day W Sane Operator wm salne appmms on sane smple foFtOta
r 脇な c o n t e n t ( 1 0 - 3 0 7 o ) s h O u l d n o t d f f.e6r pbeyl対
centage umt.

◎ 2000 AOAC iNTERNATtONAL

AOAC OFF,C,Aと METHODS OF ANALYS,S(2002) 0,LS AND FAT
Chapter 41,p.41

ration du五 ngextractionandprocessing procedures,as aresuit ofox‐

idation,converslon during heating,and lttdal hydrogenation.Ani―
m a l a n d m a r i n e f a t s m na y m ec ao sn ut rぶa b l e a l n o u n t s o f n a t u r a l
occumng rran fとtty acid geomenc isomers.Isolated′ ratt double
b o n d s i n lcoMnng ‐t t t y a c i d s t, y的 a c d m e h y l e s t e r s ( F A M E ) ,
SOaPS,and triacyiglycerols may be measuredby infrare
troscoPy.A unique absorPtion band wih amaximumatca966cm 1
( 1 0 . 3 1 ,t 宜
IゅS i n g f r o m aH Cd―
e f o m a t i o n v i b r a t i o n a b″
oざ
″ ut a r負
double bond,is exhibited in the spectraofali compounds contぶ mng
an isolated rttα ″sgroupittsbandisnot6bservedinthespecraofthe
c o r e s p O n d i n g s a tuunrsaatmerda taendd fcaお
tty acids
ment of he inに nsity of tlis absorption band detemines total iso―
lated仰 筋ど f a t t y a c i disst ■
not necげ
e stsoコ c O n v e n f a t a n ds to i l に
samples to FAME before analysis,
This method is not aPplicable to fats and olls contalning>1%。f
COniugated unsaturation(e.g.,mng Oil),tO materials containing
funcはonal groups tlat modify dle intensity of dle HC―
deformation
vibration about tle r,節dOublebond(e.g.,Castoroil whichcontAns
hydroxy fatty acids),or,in general,to any matedals contaittng con‐
sdmentsthathavefunctionalgroupsthatttvedSetOspecincabsOrp‐
tion bands at,or sufrlciendy close to interfere widl,dle 966 cm 1
(10。3脚 m)band Ofthe C― H defomation vibration of tle isolated
rra2,dOuble bond.

a A p p a n f u s

弱 o閉 ボ fatt rrrR,sP2は 的確 統 一Resolぃ

(a)Fθ ″rigr F■
tion of 4 cm l in dle spectral range of 1050-900 cm he insm‐
1.■
ment data handling system should pemit conversion ofdle spectra
Time to absorbance,scale exPansiOn of the x and y axes,readout of
wavenumbers to the nearest i cm l and absorbance tO dle nearest
Figure 985.21-Typica:chromatogmphic sepam付 on of O.0001 AU,and integration ofthe area under the absorption band at
m e t h y t e e t e r s伯“oy 「a c i d s f r o m m a r g a t t n e s . c = c r S , t966
= cm 1.The F「 IR spectroEleter is equipped wldl a deuterated
rrans.Notshown are componen低 18:コL18:2ctや t and n g l y c i n e s u l f a t e ( D T C S ) d e t e c t o r f o r g
20:0,which e3ute bemen 18:lc and 18:2cc.
滞 だ脳 謄 温 古鮒 縦 鞘 ぷ 認 線
o f ≦0 . 0 0 0 5 A U . 陀
コ966cm l bandfora l%trielaidinKTE)standard
(b)Rマpr`ガ距:所!:ゥ .―TwO Single deteminaは ons perFomed in must yield a signal― to‐
noise ratio(S/N)of>10:1.
different laboratories for total rraな
content(10-30%)shOuld not (b)Ar筋 ″arを ど ゎね′Rヂ 2c筋 4 rATR,苺 こ″ど ご夕″.一With a
differ by>1.5 percentage unil. ZnSe(Orequivalent)cryStal.Thecapacity ofthehorizontal ATRcell
isca50 μL,andthe cell mustmintainaconstantに mperature of65土
References:ユ Aれ θ】Cたβ
tt Soc.54,279(1977). .
2°C.
JAttiC 68,弧 1985>69,247(198o.
(c)A,αウrた はngO.3±0,0001g.
α配材協,c2.-60gcapacity;wei」
(d)Dゆ ωatt p脇豆cP"2お ・― For transferFing 50 11L test
41.1.37A
portions to the ATR cell.
AOAC Ofnciat Method 2000.10
DeteHnination of Tctaitsolated tans unsaturated (e)S=“ "Wα 姥″うα統.一For Hleldng fats.
Faty Acids in Fats and Oils a Reagents
ATRfTlR Spectroscopy
Pri艇 ヮ sra取ね必 .一」 TE and宜 olein(TO)Witl p航 ty of>99%
First Action 2000
(avalぉ le from Nu‐Check Prep,Inc.,PO Box 295,Elysian,MN
56028).
(ThiS methOd is applicable to namal or prOcessed ons and fats
conSsは ng oflong,chAn fatty acids,esters,and triglyce拭 des wih ,Prepara″ o何0′rans carrb胸 何。打Sね ,JarJs
r″四 levels of≧5.0%.)
Weigh,to he nearest O.0001g,(0.3-メ )g To andメ =TE into a
Sを2 Table 2領対.10forresultsoftheinterlaboratorystudysupport‐ 10 mL beaker, whereメ equals O.0015, 0.0030, 0.0150, 0,0300,
ing the acceptance ofthe method. 0.0600,0.0900,0.1200,andO.1500g,to prepare O.5,1,5,10,20,30,
40,and 50%r万 御 s calibration standards,respectively.
A. Pガ ,cripre
ln most naturally occumng vegetable fats and olls,unsaturated i cattb角 ″o何
constituents contain only isolated double bonds in the c,s conttgura‐ For each rttαな standard,calculate the exact%r汚閉じ,exPressed as
tion.Thesectsdoublebondsmaybeisomenzedk)the rhaれ S COnflgu‐ TE,%of total fat.Analyze each standard and detem曲 に the inに‐

◎ 2002 AOAC tNTERNATiONAL

01LS AND FAT AOAC OFFiCIAL MEl情 ODS OFANALYS,S(2002)
Chapter 41,p.42

Table 2000.10. rra,s Lovei3 0ftrietaidin in cotd‐ pressed,bl● ached,degu口 wned 30ybean oila
b Addet% Mea吼
M M u にN α
1 0.8 o,8
% Ret t RS既
100 75
% RSD島
21
% HORRAT
`′
5,0
2 ■0 1.0 100 105 29 7.3
3 5.0 5,1 102 2.3 3.1 1.0
4 10.0 10.3 103 36 51 1.8
5 1540 15.6 104 22 3.3 1.2
6 20.0 20.6 103 4.5 50 2.0
7 ___ 400 40.1 100 3.5 3.5 1.5
PreaS30n
8 0.4 11 68
9 0.1 56 134
10 39.6 2.1 3.4 1.5
11 0,1 16 143
12 4.5 2.7 5,3 ■7
13 14.7 0.6 4.0 1.5
14 1 . 5 5,9 12.4 3.3
15 23.7 2.8 4.2 1.7
16 203 1 . 1 4.7 1.8
23.7
The nrst 7 mixtures(Nos l through 7)are‖ Sted in Tabte lぃ ,AOAC加 484,1147(2001)〕 in the same sequence but under dttrent namesithe的‖ owing
1 0 m l x t u r e s ( N o s a t h r O u g h 1 7S )t ae rd ei ‖
n T a b l e 2 A OμA C r n 4 8 4 , 1 1 4 8 ( 2 0 0 1 ) l i n t h e s a m e s e q u e n c e b u t u n d e r d i r e r e n t n a r r l e s
A c c u r a c / s t a n a」βs T s e v e n p a t r s o f b t t n d d u p : i cコr
a t ea cayαs t a n d a r d s ( M i X t u r e N o s . 1 - 7 ) c o n s i S t e d porfe scsoeldd,‐
bteached,degummed sOybean ol and
k n o w n a m o u n t s o r T E T h e s e s t a n d a r d s w e r e p r e p a r me ed t rg kr 海
a!萌l y . T e s r sぃa品 、 二Ten pairs of bttnd dupttde(unknOwn)test Samp3es(MiXbre Nos
8 - 1 7 ) c o n s i S t e d omfm∞
ercial vegetable 「
obnlse。
n d e t T h eu ie 『
nmes were・
r e s p e c t i v,e駅け
〕y b e a n oi出 r a p e s e e d io ‖h y d r o g e n a t e d s o y b e a: np a ol ‖
m kernel
ol,i a blend of soybean oit,cotonseed oil,and hydrogenated scybean olli a Palrn blend kemel
o「 oil and hydrogenated soybean oili 2 blend3 0f SOybean di and
h y d r o g e n a t e d sio yab ebalne nod‖ o f r a p e s e e d a n d h y d; ra on gd e ns au tn en d何 Oゃw se or y。b e a n o ‖

′
′′
′，︲︲・ヽヽ
、
、
grated area underthe absorption band a t fledmaterial
9 6 6 c n l forr惚
a s″s‐
dfo■
e sifledに
c r i bstsamples,and(3)an
ed h G appropri_
and H.
ate rra″J‐free lnaterial such as the refined,bleached source cil for
Using a rlrst_。rderregression analysis,det銅 ne dle slope andin‐
test samples.
tercept Ofthe line that best fits the plot of tle area of dle r比
材ばband
f o r a l i t hぇ
es rs″
tandards(y aXiS)aS a funcdon 府 O f(%xr 昭 Using adisposable Jpet,transfer(WihOut weighng)ca50 μ L of
axiS).
Once a calibration curve has bcen established,check it pedodcally the neat(undluted in any solvent)reference background matehal.
to ensure hatit has not sMfted, Place he reFerence background mate五 al on dle horizontal(faCe_up)
ZnSe sampling surface ofttt ATR cell.The test on pol■
mustcθ加_
見 Prepa臼すo"of resr sampres
pた セry cOver tle horizOntal sface of dle crystalt C。 llect and save
Melt sohd fats gently on steani badl andx.Treat
Hよ test samples t h e s i nb ge la em ‐s p e c m m t O b e u s e d a s b a c k g r o u n d . C l e a n he
thatappearcbudy because ofthepresence ofwaterwith ttdrOus tal by wiping Offthe test portiOn with a disposable soft lint,free or
Na2S04 untilthey are clear and dlen ilに r. low‐lint tissue paper.In general,to IIllnimize concalnlnation,apPly

6わ frareJ De絶口研す partOfthe nextに St pO■ l on,and tlen wiPe it offthe crystal before re‐
,atron
S e t IuRp otpheer aFt「
ing Parameters according to the a p p l y i n g t h e Lc a t e5 s0 t μ
lpoon■ f o r a n d y s i s .

播品糧 鴛 梢譜館:瀞 瑞着挑鮒ぼ器】 盟鷲総 httzontal ZnSe cryst』

.It must∽
々β姥セリcover the surface ofthe
Place c a L5(0w iμ
thOut weighing)ofthe neattest portiOn on t

盤縦i楯 鮮品 芦群先路 総嫌盟錯温樹濫 crystalo Collect and save the single―

t i o n . Rl■
o t h es に
bcani spectrum of the test w
t s a m p l e sbienagml es‐
pecmm againstthat ofthe

鞘樹鮎脳ざ 鰐継告 総批鮒盤出

t群
麟器艦 reference background,and convertto absorbance.Save the abso中
tion spectrum.Repeat for otherに
stsmples.

緒鷲!i跳 縦塩ゴ 撚 触縦 増嶺督甜総ぶ 路 比 carcuragons

盟艦 鑑濫撚訳盟 s艦 濫損 艦
fats,maintan2° the
C. ATR響齢
cell at 65± 偽 襴 ふ 萎 麒 鮮
Materials for measuring:he reference background single‐
beam late the linear r e g r e s s 1 0 n e q u a d o n亜 f op rl to ht e o af rt eh ae v s % 貯
spectrum are(r)To for calibration standards,(2)the unfOrti‐ r″閉
じcalibration standards.

◎ 2002 AOAC iNTERNAT10NAL

AOAC OFFICIAL METHODS OFANttYS崎 (2002)
0にS AND FAT
Chapter 41,p42A

Using the slope and intercept generated for rratt stttdards,cal‐

umnis heaにd at analyHcaltemperature(ca 130r135°C).AdiustCar_
culate dle%r″ 円s fOr test samples by substimting the value ofthe der giass now sO thatretention time for buけ
Hc"id iS Ca 5 min.)
intcgratcd area of the ,s
r招band in the fouowing equationi Peak taling,even aFter column is conditioned,can somedmes be
reduced or eliminated by inieCtiOn 2 μL ttmethylchlorosilane
rrans m益 班 ,%=響 何 MCS)OntO column`
a Reagて 加ts

Report results to dle nearest O.1%. (a)ルrtts筋

閉 りかoメ
f』2 sθ
r 24だ
例.―CaO.5M in edlanol.Dis‐
solve 4.5 g KOH in 100 mL etlanol.
References:の c朋 拘修伽 αtt R釘Om2滋 Pttcrた容 (1999) ‐
ω ο脇θ -5托 ぃrnaqueous solution.
wたoric ac広
5h Edt,Amedcan Oil C陀 口Ists'Soctety,Cham‐ 律)ル β24yric actt sra取 ね材 sθ防 わ″.-0.4 Elg/nL H20・ Dis‐
paign,IL,Cd 14d‐99. solve 400 mg 4‐
buty五c acid refeFenCe Standard in 100 mL H20 and
泌 θい 7 7 , 4 5 7 - 4 6 2 ( 2 0 0 0 ) . dilute 10 mLofttts soluは
onto 100 mL wih water.Soluよ onmustbe
ユAて弘 Cあ r.MⅢ l144(2001). freshly prepared.
‐
0 五 確 m a r s 胸滋 材 w : “, :Lο―対 . 2 5 m g ″v : 山能 抵」 乱 H 2 0 ・
Dissolve 250 mg″―valttcacdin 100nLH20anddlute 10mLoftlis
41.1.38 so皿 onめ 100 mL witt wtt Solution mustbe mhly prep孤 迅
AOAC Omclat Method 990.27
ButyrSc Acid in Fatt Containing Buttettt ,PrepanJ〔 加 orSね ″JaH curve
Cas Chromatographic Method Use graduated Pipets to transfer to indvidual test tubes O.2,0.5,
F↓RBt Acuon 199o 1.0,2.0,3.5,and 5.O mL podOns Ofbutyric acid smdard sOludon,
Fina:Acuon 1993 C(C).TC each test tube,add
pet 2.Obyがeric acidmal
mLvお inに
stttdard solution,C(d),and,Iespectively,4.8,4.5,4.0,3.0,1.5,and
rUPACtAOAC ttettoJ O mL H20・StOpper test tubes and gendy mix each solution.Solu‐
dons in test tubes contain,resPectiVely,0.08,0。
2,0.40.8,1.4,and
的 n of butyric acid content of milktt or
(Applicable to detemi田 2.O mgbuけ Hc aCid and O.5 mg valenc acid.
butterfat oF ttXtures of fats contalung milkfat or butterfat.)
Stabilize column ≧ 30 min at analytical temperature (ca
1 3 0 - 1 3 5C°
).USettcrosynngetoiniectCalltLaliquctsofPrepare
Resulいofthe interlaboratory study ngsuppo対
he acceptance ofthe
s t a n d a r d s o l u t i o n s i n t u m . M e a s u r e p e a kc ha en id g h t t f
method:
ゴ0 0 % β ″r r g t t r
valedc acids tonearestO.5mm.PlotratiosG旭 けricaCid7Valericacio
vs corespOmよ ngweightsofbuty=c acid.的 ref Adiustmplinerat_
s,‐0,104i sR=0。242:RS,=3.0%;RSDR=7.0%
C確伽 朝宅を
t e n u a t i o n s o t h a t p e a k h e i g h t f o r b u t y i c a c i d f o rHhCg haecsitdb u け
確うル】 うLc配効 比 α防 ″,5θ%所 rTar
sr=0.043;sR=0・161;RSDr=2.4%:RSDR=9・ 0% standard is ca 80%fuli scale.)
R研″ど確肋ツα加″あ比 5%あ明rr2rar E DeremrnarFon
=0.008t sR=0・ 024;RSDr=4.5%;RSDR=13.1%
s『 Accurately weigh ca loo mg test pOrtion into 50 mL beaker.
A , P r r n c r p r e Pipet3 nL ethanolic KOH solution into beaker and add a few glass
beads`Coverbeaker with watch glass,PlaCe on bolling water batl,
F a t i s s a p o n i n e d w i t h K O H s o l u nt et do n w ia tn ld d l e n i s a c 拙
and heat≧10 1nin or until fat globules are no longer visible on sur‐
H3P04 tO liberate fatty acids,Water‐insoluble and water6soluble
Face.Remove watch glass and continue heating until ethanol has
f a t t y a c i d s a r e 1s 観e瓶o
p an r. aB tu HeけCd ab cy i品d ri ts t nd ee dに a s
complecely evaPorated.Let beaker cool.Pipet 5.O mL H2()intO
t h e f r e e a c i d b y g a s c h r o m a mt ao lg r sa tp ah ny d au rs di .n g a n i n に
beaker,cover with watch giass,and gendy swirl beaker tO cOm‐
B.Appattttrs pletely dissolve soapt lf necessary,warm mixture gently to cOm‐
_lo mL with grounい
(a)確 Sr配 な 。 plete diss01ution,Pipet 5.O mL H3P04 S01ution into beaker and
giass stoppers.
gendy swiri to coagulate precipitated fatty acids.Filter solution
(b)P,2rs.traduaに 42-5 mL. through small,auted,rapid paper.Pipet 5.0 1nL iltrate into test
(C)材 軌 り 勅 呼 .-l μ L. tube and add by Jpet 2.O mL valedc acid intemal standard
(d)CaS Cれ ″"α!θ tion.StOpper testtube and mix contents.
grapれ.―With nalne lonization detector,
on‐coluEm or all glass iniectiOn systett and record駒 e F湖
, ize coluHHl鴻01血 ■analyticaltemperamKca 1304135° o.
-61ass,ca21n x3 mm id,魚 UsemicrosyringetoinieCtCa l「
止 品nalsoludonontocolumn.Measure
(e)6θ 肋 ″い 1led with 10-15%sta‐
tionly phase suitable for free fatty acid analysis on 80-llXl mesh 減c and V2山鹿 樋ids to neanst O.5m.
peak heights for buけ
acid,washed slla胡zed suppo■ 。(FFAP or SP‐ 1220 is suitable sta‐ Catty out 2 deteminations in rapld succession.
tionary phase;迅 idon ofl%H3P04 Can improve resoluは on ofbぃ 脚θ 姥r(F)Rinse syinge thoroughly with water between every
:yric acid and solvent Peaks.Chromosorb W is suitable support.) 2 andyses and at completion of analyses widl dhted soap s01ution
Condition and stabilize coluHln for use at ca 130-135°
to minimize coroslon due to H3P04・
(2)After senes of sample in‐
C.
jections,iniect One Or more butyAc acid/valattc acid standard solu―
OV例修f COndition column≧ 48 httca 180°
C.Ifon‐ columttecton tions and check calibration curve vs corespondng peak heig比
is not uset setitteCは On port at≧175° C.If butyric and valeric ttd ratios obtおned from standard sOludons.(3)CaprOiC and caprylic
peaks are notsepmted atbaseli確 ,resolu血 加of coluIIlns containing acids may eluにafter valeric acid and cause interfering peaks in sub―
a n H 3 P 0 4 S t t O n a r y p h a s e c a n b e i m p r o vL eq du ba yn t‐t e Csequent
t n g lanalyses.Be
μ sure that dlese acids have elutedttfore another
tities of2.5%weight/volume H3P04S01ution into column whle col‐ test solution is ittected.〕

◎ 2002 AOAC iNTERNAT:ONAL

0,LS AND FAT AOAC OFFiCiAと METHODS OF ANALYSiS(2000)
Chapter 41,p.42B

Tabl●990。
27 Repeatabinty tr)and mpttducibili守(R)vatueS 41,1.39
tPヨ 0。95)for buttHC aCtd deteHninatlon in fats AOAC Omciat Method 933.03
Far Residue tunsaponlf3ablo)
of 01ls and Fa低
Stattstica
Ether Ex“■ct3on Method
No ofiabs 11 10 11
FiRBt Act3on 1933
Mean value,%(w柿) 3.46 1.79 0.18 Finat Action
r vatue(2.3 x sr) 0.29 0.12 0.02
Accurately weigh 2-2.5 g fatinto sapo直
flcadon nask(200 mL
R vatue(2.8x sn) 068 0.45 007
a sr=0104,0043,and 0 008 and sR=° Erlenmeyer with standard taper24/40outerjointis recc―
ended).
242,0161,and O.024 brfats l,
2,and 3,resF8対Vely. Add 25 nL alcoholand l.5 mL KOH soludon(3+2).SapOnify by
° 1:100%butterrati 2iend
b‖o「cream and vegetable oiに
ontaining about boiling,withoccasional swirling,on steanbah30 min underreaux
50%m‖ k f a t 3 : r e n n e d t a l i o w t lc o n t a i n i n g 5 % ね air condenser.(No loss of alcohol should occur during
saponirlcatiOn.)TranSfer alcoholic scap sOludon while still wam to
250 mL separator,ushg total of50 mL 闘nSe H20・ saPonincation
,carcuraめ ,s aask with 50 mLether and add etherto seParatOr.Shake vigorously,
Fittpeakheight胡 oOfbuwiC acid/valeric掘 andreadtondi‐ and letlayers separate and dttfy.Drain bweFiayer and pour edler
bration cwve weight httC acid equivalentto height配 施.
that"よ layeF mOugh tOp intO second separator containhg 20 mL H20・
Rinse powing edge with鵡 ,adding rinsings tO second sepmtor.
C a l c u l a t e b u t y ,i %c w ea ic g遇h t / w e i g h t , i n s 2 m p l e a s f o l l o w 既
欧 tractsoaPsolutlonwtttwo50mLPoltionsetherinsame manner.

hい嗣影=甲 Make total oF 4 extractions for marine ctis or other oils of ttgh
unsaponiflable content.

Rotate combined edler extracts gently with the 20 mL H20

where Wb=weight,mg,ofbutyric acid read from calib両
lon cutte, (VigOrous shaking atthis stage my cause troublesome emulsions)。
and Ч 3=Weight,mg,oftest ttdOn. Let layers separate and drain aqueous layere Wash wih o江 tヤ H卜
Ifrepeatお ilityvalueoiSSaは sfactory,analresultis IIleanof2de‐ tiona1 20 nLPortiOns H20,Sttking vigorously.Then washedlerso‐
teminations.If Fepeatabdity requirements are nct曲IIleヒ 鯛配 re‐ l u t i c nm e 3s はw i t l a l t e r n a t e 2 0 m5 LM pa Oq ru te io ou ns s K cO aH O 。
sults and cttqy out another 2 deteminationstle o■fat. and H20,Shakhg vigorously eachはIle.If emulsion foms during
wasMng,drain as muchaqueous iayeras Possible,leaving emulsion
a Repettbi,町 anJReppJwcrbrrrw
in separator wih etherlayerj and Proceed widl next washing.Af
Rη 夕"力 】り .―When the mean ofduplicate deにminttons lies 岨 rd KOH treament,wash eher solution successively wid1 20 mL
between any 2 mean valuesin Tおle 990.27,dle difference between
POrtiOns H20 until washings are no longer alkaline to
results of 2 decminations caried out in rapid successbn by dle
phenolphtlalein.
same analyst,using the same apparatus,for analysis ofittnd鹸 正test
materia shouldnotbe greaterthanthe rvalue thatcoresPondS ttthe Transfereher solu血鴻to 250 mL町 騨丸 ∞お∽l beaker,五 nse sep‐
higher of tle 2 mean values. arator and i低 脚 ng edge宙 h eher,and add ttsings to main solu―
― When he means ofduplicate detettmtons, t i o n酌
、a p O r a t e m c a 5 m L afnedr― q u t t d t t v e l地 y,は 韓v e d
R?乃 れ c,う 】:り・
s m a l P O H i O n s e t h e r , t o 5 0 n L f a t f l a s k o r E r l e n m eけ
yer previoぃ
o b t A n e d i n 2 d i f f e r e n bt il ea sb o f嗣o r a n a l y s i s o f i d e n t i c a l t e s t m a ‐
仕ied and welghed witlsimilar nask as tare.EvapoFate edler.When
t e r i a l , l i e b e t w e e n a n y 2 n e a n v a l u 附2 e s i7n, tThaeb ldei勇
fference
nearly all cher has been removed,add 2-3 mL acetone,and wttle
between the mean resul低 obtainedby those laboratories shoulanot
heating on stealnorH20bah,completelyFemOVesolventingendeair
be greater than dle R vdue血江 cottspOnds to dle hgher of the
current.Dry at 100° C for 30 mh penods to constant welght.
2 mean values.
Dissoive contents of flask in 2 mL edler,add 10 mL neutralized
ReFerencett P″ ″ 約2β 16ル れ 58,1419(1986).
(phenOlphthaleiゅ alcoh01,andは m te wit10.lM alcoholic NaOH(or
ンヽこ次C72,8∝ 1989)。
KOH)。 (≦ 0.10 mL is usually required.)COrec〔 weight residue for
CAS‐ 107-92-6 outyriC acd) f r e e f a t t y a c i d p r e s e n t ( l m L O . laMc iaol.k d i = 0 . 0 2 8 2

r cο
rT2・ ″!れ″を
sθ,Cあ4βセ″イメ
,″.43)

◎ 2000 AOAC tNTERNAT10NAL

AOAC OFFiCttt METHODS OF ANALYSS(2000) OILS AND FAT
Chapter 41,p.43

C O F r e C t W e i g h t r e s i d u e f o r r e a g e n t nbeldabnyk coo脱
nducting H 2 0 b r O u g h t d o w n b y s w i n h g s e p a r a t o r . P o u ru pm e t rh oe に
rsolu‐
determnation siindarly but Orlltting fat. tionfromtopofseparatorintolipttdconicalbeaker.Rinse separator
with petroleum ether and add rinsings to beaker.Add few pieces of
References:A″ りsr S8,203(1933).
brokenporcelainorSiC mdevaporate almostallofsolventon steam
JAOAC 28,282(1945);29,248(1946j.
bath.Remove lasttraces of solventin curent of C02 0r Otherinert
gas while warmingbeaker.To avoidoxidttonofresidue,do notex‐
41.1.40 p o s e t o a l 1r l w wh ai ml .e s は
AOAC Officiat Method 943.04 Dissolve unsaponiflable matterin 5 mL petroleum edler and mns‐
Squalene in O::s and Fats fer to adsorption colum.く Eluate,which is caught h 250 mL
Tltrimetric Method glass,stoppered 12naSk,shouldemerge dropwise,cal mWnin butno
Ftrst Actlon 1943 fastei gende pressure ttng used if necessly.)When sOlution has
Final Action been nearly drawn into column,add ca 5 nL petroleum etttr‐pre宙
e beakert Cα
ousiy used to iぃ l dnw adding solvent,prevlously used
A.Peagents
to insebeaker,in 5r10 mL poHions,always keeping srace ofcol―
θ一 D i s s o l v e 6 0 g
( a ) 働 座 印 ' 確冴 ″θt t s ' 切￨ り 加 冴滋 W ! ″r : 払 uEm COVered witlliquid,until total of 50 mL has eluttd(rCOlum
KOH in40 nL H20・
witt Teflon stoPcOCk and top reservolr is used,Promd aS abov
●)D:r″ 2 Pθ!ωs:跡たり ″raI:ル Sθ!″ れ.― Dissolve 28 g KOH
r:θ
trough addidon of 5 mL rinse:dlen dnse beaker witt remaining
in H20 and dilute的 lL. 40HLpetroleume鹸 、addtoreservdL andletPasstOughcolumn.)
(C)PCttte跡 2虎 ぇ―ぺ kellysdveBい 63-7げ C),研 eい Vale配・
Add ttw Jeces OfbrOken porcel血 or SiC and remove most of
(oA筋 ″れ切 θメ滋 aJsο,う 2″ 480-2御 秘asれ .―Adsorption alu‐
solventon steambadl.Finally pass current ofC020r Cherinert gas
述 n a f o r c h r o m a t o g r a p h i c a n a y s i5s4,0F,iosrh eerq uAi‐
vttent.
through heated flask undHast traces oF solvent are expelled.Cool
Keep in tighay c10sed container,away fron lnoisture. 1l traCes of
residue to room temperature under inert attosphere.い
(C)わ r:沈,2S″Jra確う″秘:た s】″!われ.-0.lM.Dissolve 8 g Br2 solvent must be removed before detemination is condnued.)
in20 mLCH3C00H・ Prepare another solutionby gradually adding,
助s s d v e u n t t ss io dr ub ee d h Ю5 m L C H C L a n d a
with cooling,5.45 mL H2S04tO miXture of20 nL CH3COOH and
pyndine sulfatebromide solutton to pFOVide≧ 5 0 % e x c e s s ( 1 0 m L i s
8.15 mL pyridine.Mix 2 soludons,cool,and diluに t01 L with
usually adequate)。 Let mixtutt reHlaln in dark 5 min and dttn add
CH3C00H・
_o.o5M.Prepare tttly 5 m L 1 0 % K I s o l u t i o n , t o g e t t F W i t h 4 0 MmiLX Hd2l0o・ roughly,
0ヽ 鵡 閉"挽 わS″ rratts,a滋 〃 wr2,わ れ
wash down any free 12 0n StOpper,and ttrate with O.05M Na2S203・
by diluti増0.lM solution,942:27A.1.13). Ggβ
Toward end of timtion add starch indic斑oL(mix ca l g soluble
ユ APPararus starchwithenoughcoldH20tOmakethinPaste,add 100mLboiling
Attο 例 初 取 ― P r e p ‐f r e s h c o l u H l n f o r e a c h d e t e m i n a tH20
i o n and boll ca l mm while 「ing),Shake
sは vigorousiy,and con‐
?ガ
iHlmediately before use.Place small wadofcottn tt constrictedend hnue dtration to disappearance of blue.Conduct bla■ k de価 na‐
ofglass mbe,8 mm d and30cmlong.corCOnvenience,column may tion on pyddne sulfate bromide soluは on siH五 larly and calculate
have Teaon stOPcoCk in stem and top reservoir of浮 的 nL capacity.) m L O . 0 5 M N a 2 S 2 0 3 e q u l V d e n t t o a b s o r b e d h a l o g en.Blank detemi‐
Add alumina adsorbent in ca 10 small portions until col―is ca nation on all reagents used should show cally pracは no halogen con‐
10 cln high.Apply gende s岨 側 and tamp each匹 由 on dumina s u m p t l o n . l m L O . 0 5 M N a 2 S 2 0 31‐ ・
71 mg squalene.Report resuits
lightly wih aattened end ofheavy ttlass rod.Place出wad sE盟 ofcoト as lng squalenJ100 g sanPle.
ton on top Ofcol― and tt lightly.Wash coluHln witlca 15 mL
References:拡 θAC X,4男 )(1943j;28,282(1945);
petroleurn edle,remove sucd価 ,and keep toP of c01uHln covered 29,247(1946):48,127(1965).
witl shallow iayer ofpetroleum eher until ready for use.
νな夕体 M α「
R を 転 玉妨
a per_rnarrOn
able
(Cttrわ″,SタタAppendix B,safety notes on distllation,fla― CAS‐111-02-4(squalene)
solvents alld petroleum ether.)
Accurately weigh(±20 mg)ca 5 g sample into 125 mL
41.1.41
Erlenmeyer wih standard taperjoint,add 3 nL concentrated KOH
AOAC Ottictal Method 986.19
solution and 20 mL alcchol,and boll under air condenseF 30 inin,
Trigtycerides in Fats
shaking occasionally.Cool somewhi and wttle still warm,add
and Oiis
50 nlL petroleunl eher;HIx,and transfer k)separator.Rinse flask
Cas Chttmatographic Method
with 20 mL alcoholandthen wit140 mL H20,adding nnsings to so―
First Action 1986
lution in scParatOr.Shake vigorously,let separate completely,and
Finat Action 1992
slowly dHttn sOap soluはon.Pourpetroleun ether extracば rom toP of
separator into another separator ng20 condぶ mL H20・Repeat ex‐ rJPA朗 OAC筋 倒腕oJ
traction of soap solution wi血50 mL petroleum edler.Rotate com‐
Ao Prrncfpre
bined exttacts gently with the 20 mL H20 and,after letting iayers
separate,discard wash H20・ Repeat washing by shaking vigorously Triglyceride groups having sallle C nunber ar
w i h 2 0 n L H 2 0 na n d i sa cg aおr d l o w e r l a y e r a fgas
t e chromatography
r s e p a r a tofi solution
o n t of oil or fat,under tempera‐
Washpetroleumehersolution with 20 mLdihte KOH solution and 雌 ‐ p r O g r ae ―
d c m t t ,t a施n d t t t t t e d b y r e f e r e n c e
hen with 20 mL portions H20 undl wash liquid is alkali‐free,shak‐ triglyceride solution.Triglycerides having same C nmber are not de―
ing vigOrously each time.After Flnal washng,drain iast droPs of temttned individually.Contentis detemined by peak狙 胡 o.

◎ 2000 AOAC tNTERNAT10NAL

OtLS AND FAT AOAC OFttCIAL M日 耐ODS OF ANttYStS(2000)
Chapter 41,p.44

,APParar“ ●コ打どReagents ing same C number expressed as percentage relative to tOtal

(a)効 S勧 加 宅 raPれ二 With facilities for column
on‐ iniec‐ 面giycendes cOntent by fomula
tion,oven temperature progralming up to 350°
C,如 d,preferably,
electronic integration.All‐
giass systenl is preferable.
KATc松,x100
(b)CO'″ 秘″.―Glass,ca O.5rO.6 m long,andみ 4 mmid,船 led w h e r e A _ : = c o r e C にd p e a k a r e a o f t r i g l y c e r i d e s g r o u p : ; A T = t o t a l
with 3%(orleSS HlethylPolysi10Xaneonacidbwashedsilattzedsup‐
c o r e c dに p e a k a r e a golfy cte減 ddes groups contained in smple
Po止 (OV‐1 lS Suitable)。 C肛拭ergas aow 50 mmin,He is FeCOm‐
“T = Σ A N ) .
mended as ctter gas but N2 may be used with some loss in
助 fference between results of 2 deteEttnadons camed out On
r e s o ol nu .はC o n d i t i o n c o l u m p nC o rf o3tr6o≧ hu swei talt 3 5 0 °
salne day by sane analystushg salne apparatus forsame test nate―
c a n e r g a soユ
w r a t e o f c a 5血mn口
.降 tを=Equivalent results may
五狙and foF triglyceddes Present>107o should not exceed l%abso‐
bc Obtained witl use oFshort(≦ 6正めC01ulm.〕 luに.For tFiglyceHdes present at levels ofく
10%,difference should
(c)rrigryCタガガタ s.―Pudty 99%.Prepare standard sdution in not exceed O.5%absolute.Triglyceddes present 5%are atくdeter‐
CH3Cl(Or diiSOpropyl edler)c側 面 ning ca 10 mgrd each Of Hllned iess accurately.
triCapFin,tricaprylin,trilaurin,timyhsun,tripalmitin,andtistearin.
Referencα】
物″ 叩 ,C陸 れ SL 1515(1985).
a mrernfnatron or rrrgrycerfres CarrecrrOn FgctOrg
With 2 1tL microsyFinge,iniectCa l l止
triglycerides standard sot
41,1.42
l u t i o n w i t h i n i e c t i O n a n d d e t e c Ct o ar nt de m p e r a t u r e s s e t a t c a 3 7 5 °
AOAC Offtclat Method 955.34
initial oven temperamre ca 220°
C.I― ediately commence pro‐
Fats(Vegettb:o
grammng oven ttmperame tOincrease ttrate 5° C/mintbut
ofca年 in Butterrat
not>5°C)andCOninueanalysisuntiltemperaturereachesca350°
C.
Sterct Acetate Melting Point Method
Manttn this temperature until all ttglycttdes have eluted mm Firet畑 !on 1955
coluHln. Fina,Actlon
AssumethattriladriniscompletelyrecoveredfFOmC01umn and
calculate corection factor,五
,for each remaining ttglyceride fDHSC― AOAC Mernc」
from A A p p a r a t w s
(O SPgCね ′秘た円 炉陸 ぇぺ eC Figure 9SS34A.
五=(qノ Oθ Xは」Aか 二 Heavy R宙
(b)P脇 筋 ″秘 SPar2ra。 Fe,hammered aat,tO ca
where AL=peak area foF 3 Hlm wide x15 mm long,on
trilaurin:An=peak area for standard one end.Mountin《 hssecting needle
holder.
triglycendei cL=COnCent述 側 Kng/mLjoftFilauriniCh=COncen‐
破ldon(mg/mLj oF standard triglyceride f.
DeteminertOn】 21可ecは。ns Ofstandard soluは
cn.Plot graphof
average vdues forrfor each triglyceFide agttnst correswndng C
nunber.Corec故 鴻 factor>1.l is unsatisfactory.Decrease station‐
ary phase loading orincrease camer gas flow rate to achieve accept‐
able corretton factors.
Plot vaues of retention dme for Oach standard dglycende peak
agalnst coresponding C nunber.S宙 よght line should be 6btained
from which expected retention dmes for odler dgttcerides can be
detenEIned.

,PreparaJt加 ゴ Sampre sorutrOn

Warmに st sample as necessary to comPleに ly liquefy.Homoge―
nize liquid test sample by gendy shaking container. Prepare
5 0 m g / m L os no l ou fはt e s t s a m p l e i n C H 3 C i K O r diiSOpropyl etleo.
For exmple,transfer ca l.25 g hquid sample to 25 nL graduated
flask usingぶ
pet and diSsolve smple(while Still止
quid)in 2-3 mL
solvent.Dllute widl salne solvent,and mttx.Iftest sample is known
to contain signiflcant amounts ofmonot or diglycerides or free fatty
acids,remove hese according to 96S.3S(sを241.1.61)befOre pro‐
ceedingwithanalysisasdescibedfordetemnationoftriglycerides
co「ection factors.

丘 De拒 宮
研inatron
F`Oure 055,34A口 JGlats『 ntcro■ Rer for steroi acetate
l d e n t i f y e a c hよ辞b y u s i n g i d e n t i n c a t i O n g r a pmhi.nDee に peak precip的 ぶo3.A:Top poAlon offBRer,capac町 l mL口 B:
a r e a s o f e a c h g r o u p o f t r i g l y c e r i d e s . C a l c u l a t e c o r e c t e d p e aLowerk a r eportio■
as of■ 8ter,Ground suHttces between A and
b y u s i n g c o r e c t i o n f a c t o s d e t e m i n e d e i t h e r b y c d c u l a t i o n o r b y iBn ―
hold f3iter pad.A and B are heid together by sprlngs,
terpoltton from graph of corection factors obtained for standard D.C:Filter fiask.E:Wlre twisted around stopper to hold
triglycerides.Dekメ コinequantity ofeachgroupoferiglyceFideS hav‐icwer end of spr3ngs.

0 2000 AOAC iNTERNAT10NAL

AOAC OFFIC,AL METHODS OF ANALYSS(2000) 01LS AND FAT
Chapter 41,p.4S

除 もも
ｕ
/ ミ
グ 色々 デ ｀

W 込

Figure 955.34E卜 ● rystalline fom8 0f free sterolo.A:Cholesterol,B:Phytosterol.C:Mlxed cholestero卜 ●hytoStero:.

ュ Der― rnatrOn Repettrecrystallization and iltratlon third andfoun time;then dry

°
To 15 g flltered fatin 150 mL beaker add 4 g KOH dissolved in precipitate l h筑1∞ C.
4 mL H20・ Add 20 mL 957o alcchol,cover with watch glass,and
DeteminempofrecFystanizedmedsterolacetates(temperature
ring occasionally.
heat O.5 h on stean bath,sは
at which liquid flrst staHs torun,detemned when heated atrate of
A d d 6 0 n L H 2 0 , m i X , a n d p o u r i n t o 4 0 0 m L b拭enagk e r c o n 面
1 8 0 n L 9 5 % a l c o h o l . W a m t o c a 4C0 ° 0ぷ け r mpis辺 。
a n d a d d 4 0 m L l % d t t i t O n i nO.5-1,0° C higheF han dlat ofpure butter sini‐
in alcchol,(Heatmay benecesstt todiSS01vedigitoninぅ Strandlet larly trett vegetable tt is itticated.
stand ovemightin rettgerator. a M r c r O c w t a r 問
Fliter cold ttxture with strong suchon on rapidqualitttive ll cm
Disscive sterol acetate remalmng frommpdeteminationin 2 nL
paperin Buchner.WhealiquidhasPassedtroughPOur50mLH20
over paper witlout stopping suction.Swin occaslonally.Condnue alccholin 20 mL beaker and add 3 drops 40%aqueous KOH.He江
to apply strong suction(H20 nlters rather slowip until all H20 haS on steambath 5 min.Add 10 mL H20 and mnsferliquidto 125 mL
passed throughPaperk〕 washoutmostofsoaps.Pour50mLalcchol SepaFatOr.Add 25 mL edler and shake.1,t layers separate,then
over Paper and condnue suction untn a11liquid has passed drough, drain and discard aquecus iayen Wash edler widl dree 5 mL por‐
Finally wash paper with four 50 mL脚鵡ons e瞳 ,lethng eachpor‐ tiOnS H20 and evaporate edler to dryness in 50 mL beaker.
はon pass tough compleに ly before adding next,
Add 10 mL 70%alcohol to residue and heat to dissolveo Cool,
陀
Co Separate preciP的
Dry paper and precipitate 15 min at 100°
place drop ofclear solution on slide,and examine drop microscopi‐
from paper. Crush or crumble preciPitate, and Piace it in
cally at 100… 200x for typical crystals of phytosterol or
18x150mmttsttube.Add2nLaceticanhydFideandheatin 130° C
phyttsに rol mixture G″田じ 陀 955.34B).
rol,cholesに
glycerolbath 15 mdn。 (PdPitate shoulddissolve hca5 mini donot
usedirectheat,車ncespatteringmay occurandmatettalmaybelost) ,DrgrrOnrn白 "。yery―ProwJure
Coolto ca 70°C.
Co品 Ыne iltrates lttn dgitonide precipi田 直
ons and add enough
caremily add4nLalcoholandmix.Filterhotsolutionby gravity
cholesterol dissolved in alcohol的combine with all dgitonin pres―
t h r o u g h p l e d g e t o f c o t t o n i n m dncgrtou bnei tKeP拭
regitype),receiV‐
ent.晩 t航 xture stand 3 h or ovemight.Fnter off pttciJtate and
ing flltrate h20mLbeaker.Placebeakeronsmallhotplateandcare‐
wash with H20'alCchol,and edle,hen suck dry.Cmsh precipitate
fully bFing liquid to gende bDil.Add H20 drOp by drop tthisterol
and tampitiighdy into paperextracは onthinble,Suspend thimble in
acetateisjustabouttoprecipitatebutsturemainsinsoluは on atbp.
standard taper Erlenlneyer closed byflux I● condenser and contttn‐
晩t cool,stirring occaslonally witl Pt spanla,fOr 15-20 mdn or
i n g s m a l l a n o u n t確 .o Hf e黎a t x y l e n e t o b p a n d i e t t h i n
longer.niterOn smlldiskofpaperinmicroBtthnerca 15mmdi…
contents hang in hot vapors ofboiling xylene 16 h.
alneにつ。 Suck dry andseparate precipitate frompaper.Place precipib
tate in 5 mL beaker and heat wi血l nL 95%alcohol to dissolve Remove dttmble and dry at 100°C undl xylene has evaporated.
completely.Cool beaker by setting in Pett dish ofice‐ H20・When Remove digitonin residue,weigh and transfer tt beaker,Dissolve
thoroughly dhilled,mattal usually sets to semisolld crystalline r e s i d u e i n e n o u g h H 2 0 t O m a k e c a 2 % d i g況.
i t Ao dn di n c as %o l u 敵
slury. volulne alcchol and heat on stealn bath.Addl mLれ ‐a賦りl alcohol
Transfer slury to sPecial mttroniter,using Pt spatula,and apply Kreagentgrade),C001,and 81ter ordgitonin compoundon Btlchner
suction.As liquid is drawn ttЮ ugh rliter,cOmpact precipitate by ofsuitable size.Suck dry andtransferprecipitate to watch glass.Dry
tamping with nat end Of glass rod of suitable size.(PreCipitate can at 100°
C until ali amyl alcchol is volatilized.DigitOnin may dlen be
thenbe deanly and completely removed from paperin fomofsmall
pulverized and is ready for reuse.
button or tabld.)Redssolve precipitate in salne 5 mL beaker wi血
additttna1 l mL hot alcohol(or O.5 mL ifprecipitate is very sma11), R e f e F e n C e S !i み
. N財:
夕性D α: りJ . 9 , 2 6 1 ( 1 9 5 5 ) .
and aner chilling to recrystallize,fliter second hme on lnicrofllter. JAOAC 38,338(1955);41,268(1958).

0 2000 AOAC iNTERNATtONAL

01LS AND FAT AOAC OFFiCiAL METHODS OFANttYSIS(2000)
Chapter 41,p.46

41.1.43 are cbtaned On successive itteCは

Ons ofidendcal volumes of stan‐
AOAC Official Method 970.50
Fats ttegetable)in
dard mixture.
B u t t e(め
r PrgPara筋″
fat
`
ザ∽′ ― ・ 一 Pack glass column,1.8m(6 fty x4
Oas Chromatographlc Method m id,wih commercid l-3%stationtt Phase on 100-120 mesh
Firet Action 1970
Cas‐ChromQOrdssolve O.年 1.2 g pOlysi10xane in 200 mL toluene
Finat Action
Or CH2C12rt01uene(lⅢl).Heatto dssolve(polysiloxane dissolves
fDH30-AOAC Mり 防ar slowly in solventmixttre).(Ca″
だ。″r Slloxanes are toxic,Weardis‐
A,PreParatrOn Or sanPre Posable gloves and use effective fume removal device when han―
dling.)Add solution to 40 ChromQandletstand
g Gas‐ 10minwi血
Obtain fatfrombutterby,如 .117Gをを33.6■5).Weigh5-10gf11‐
occasional gende stirFing.DryinFOtaFyeVaporatorheldh50°Cbath
tered fat into 150 mL beaker and proceed as in,5534B G22
orheaton steambatl with occasional gende sは
41.142),beginning“ ….add4g KOH.… ''.(Sterol acetaに need not
胡 ng and renove re―
be recrystallized udess mp is also desired.) sidual solventin vacuum oven at 50°
C.
Carefully wash inside of colurm and small amount glass w001
a用 似 gtttrs
with 5%solution dc出 駅 斌 metり lsilane in toluene,inse widl
(a)CC Cθ ′ ″閉″ Pacたれg.―― (F)S,αガθ′ ″り ″れas夕.―― JXR,or mehyl alcohol undl insings are neutral to indicator Paper,and ttr
OV-1,or OV-101 dimethylpolysiloxane,or OV-17,or OV-22 dry.Plug coluコm exit with small plug of silanized glass wool and
methylphenylPolyslloxane.(2)S″ PPθrr.― -100-120 mesh t r o u g hh ‐
ole septum,and plug iniectOn side arm wih%hOle
Gas‐Chrom Q.CO― ercially prePared Packing of l or 3%station‐tum.Addcoatedpacking materialhoughiniectiOnport,using im‐
叩 phaSe available from AlletchLAPplied SCience LaboratoFieS, nel ttd Jtttt tubing and tapping colum very gently during
Inc.,or Supelco,Inc. 畑 tion.Add%paCking mateFid tt time,remove funnel,and apply
(b)剛 り αご夕惚″ 一 Distilled in glass(BurdiCk and Jackson Labo ca 5-10 psi(34.5-69 kPaj N2 tO itteCtiOn Portwhile tapping gen
oratones,Inc.,or equivdenめ. to settle packing.Pack to 2.5 cmbelow inieCは
On side arln and Plug
″ W!″われ-0.4ロ ト Wdgh 40.O mg wih sdanittd glass wool.
O ChO陀 眼 地滋丹
cholestanestandaFdKAlltechaAppliedSctteLaboratories,Inc.)into 0∽ 越曲 前 昭 ザ 御ど 切伍 ― Heat≧8hat260°C wmcast10psi
tO VOlullle witl ehyl acemte. (34.5-69 kPaj N2 nOWing hough colum Shut off PFeSSure,dse
100 mL volutttric aask and diluに
(d)9修 ′夕ざ1髄 れ々r■ ar s″
比協rtr sθ!″ゎ″.-0.2 Hg/μL.Dlluに にmPeratureto290°C,andconはnueheatingttL Reducettmperature
10.O mL standard solunon,(0,t020,O mL with ethyl acetate. め 260°C,劇 じust N2 t0 5r10ド
i,and heat放
阻組ona1 8-12h

(Opざ どゎざ wttj体_2.O Hg/μ

″″′acgratt sraFra″ L.Wdgh o ん r r o ,4初“―C h r o m a t o g r a p h o ‐
sc ia t O2的Sぃ
l俺a c e t a t e
standard
22.2ngp_sitosterolacetatestandard(ICNPhamaceuticas,Inc.,Life solution tt detemine retention tines and FeSOlut
Sci9岡 unn.Mittmumof1600theottdcalplatesisrequlredforp_sitosterol
es GrouP,Ca90%purejinto 10mLvolumetricflaskanddilute
acetate peak.
to volume wih ethyl acetate.Comlnedal pbsitosterolacetateismix‐
破 o f c m p e s t e D l a c e明tmaitneo rt eCaOrmlpioenre neth)はand
Theoredca Jates=α ガアx16
p_sitosterol acetate,De腕 ‐
純concentralionofβ
SitOSterol acetatein
h噌 2-3● standard solution,暁
standard tt chromatogゅ 腕 五 陀 whereと =Hmp_sitosterolacetatepeakfFOmtteCdOnpoint andJ=
距a OfCampesterol いand
ace!分
p_sitostrolpeaks
acebい by drawingElm triangulatedbase widthofp‐sitosterol acetate peak.
l i n e s t a n g e n t tso osfkpた
eak andintersectingbaseline.Detemine ar‐
e a s o f r e s u l t i n g t r i a n g l e s b y m u l t i p l y i nag DetrmrnarrOn
hettht by%base.
Pipet l.OmLcholestaneintemalstandardsolutioninto3 dramvial
C o n c e n t r a t t t p _ s i t o s t t=d器a c e t 捷 contalning sterol acetates,rotate vialto wash down sides,
的dssolve,Iniect2-3ぃ
Lsample soluはon and 2-3●standardmix‐
‐
ture,0,斑 leaSt in duplicate.Identitt
SitOSterol
β acetate peak in
where Q=ng ster01 acetate standard/mL,4=areap_sitosterol smple
ac‐ from re俺耐on dHle in standaFd miXture.rheight ofsample
=area campesterol acetate peak.
etate peak,4nd兵 peak is>60%fuli scale,add additona1 1.O nL intemalstandard so‐
(ゆ勧ο ″容ra4夕 _β_s統
。S確″!α確ねたs勉再協材 ″競′ ″に判 .2躍 lution to smple solution,andrechromatograph smple and standard
c h o l e s t a n e pa,nsdi tlo.s0t距
tい M i x e
rol acetatゴ q u a l v o l u r n solutions.
Hlixture es Measure peak heights of cholestane and
(O and(0. p_sitosterol acetate peaks in mm.
a ApParar“ 3 ‐
ng βSitOSterol acetate/100 g smple=
(a)Gtt cれ ,omarog砕 れ_Barber‐ Colman Co.Mode1 5側 , 何 /″ x)X(Cノ Ci)X(SJSi)X(Or2)x10o

Searle Analytc Series 4740,orequivalent,widlH2flamelo減zation

detector and l mV strip chartrecorder.Temperatures(°
C〉 c01umn, where■and亀=height(m)ChOLstane andtosterol p_立 acetate
220-260;flash heater and detector,240-270,flow ratett N2(ultta peaks,降 spectivew,h standard mixture;最 and島=heightぃo
_ stoster01
8 - 1 7 2 k P a j t o e l u t e pp_立
h i g h p u r i t y g r5 a dp es ji ,( 21 03 屯 i t o s t e acetate
r o l a c tand cholestane vely,in
peaks,respecは
smplα
qand q=μg β‐SitOSterol acetate and L,respecavely,
cholestane/μ
datein 16r20 min;H2'Ca 40r45 mW航 対 air,3∞ 屯 40 mWmin,
i n s t a n d a r d m i x t u r e ig oc lh =o μ
lestane/1tL in sample;and o=mg
Attustelec廿 Ometer sensiHvity so that 2.5円 ‐
事β sitOSterol acetate
smplerltL.
gives ca 50ワうdeflection(ltr9-lo_lo amp Full scale deflecdon
w i t h l m V r e c o) rR 住
e p e a t i l l i e C t i C n s u n t i l c o n s t a n t p e a k hReferences:J■OAC
eights S3,623(1970,S4,643(1971).

0 2000 AOAC iNTERNAT10NAL

AOAC OFttCiAL METHODS OF ANttYSiS(2000) 01LSAND FAT
Chapter 41,p.47

41.1.44
AOAC Offtctat Method 067.18
BetaoSito3terO:in Butter O:1
Cas Chromatographic Method
First Action l範7
Fina'Actlon 1980
( A p d i c a b l e t O s m p ln ei sn cg ot nt tm お
ter oil.)
″‐
hexane andlet now mOughpackingundl″ _hexane reaches tOp Of
Packing material.Use column immediately.Do notlet dry.
E PreparatrOn Or resr sanPre
Dissolve 900 mg butter oll in 3 mL″ ‐ hexane.Quantitatively
tansfer solution,uslng dispOsablepipet,to digitonin― Celite column
andletpassthroughcolumnuntil solutionhasenteredPacttng mate‐
g f r e e p _ s i t o s t e r o 1dal.Wash / 1 0 0 gsmple
b u t beakertwice
‐ with 2 mL″ ‐ hexane and add each
wash to column,rinsing sides oftube.Wash c01umn with flve 2 mL

A , P r r r T c r p r e
portions″‐ hexane.After all hexane has entered cdumn,wash wih
flve 2 mL portiOns benzene.When iast portion benzene reach
Free 3‐ p_OH ster01s are renoved from butter oil by complexing l cm above top ofPacking田 逮京al,removerubbeFtubing and wash
with digitonin,androls sに are then removed froHl digitonideaceliに itterandoutersufaceofcolumn dPthoroughly widlbenzene tore‐
column by eluton with dimethyl sulfoxide● MSO).(6α ″,こ 0■f move traces of fat.(Fお lure to wash colum sides and column dp
DMSOcanbeharlnful.Avoid skin contactbywearingheavyrubber wim sOlvent will resuitin Poor chromatograms due to interference
gloves.Use effectve fumeFemOVal device.)Butter dl has apparent fromtriglycerides.)Discardhexaneandbenzene.BegineludOn with
range ofO-l mgβ ― sitOSteroW100 g andice crealnhas apparentvalue 10 mL DMSO before benzene falls below top ofpacking material.
ofca4 mg/100 g Fat from emulsiflers. Collect DMSO eluate in 15 mL screw‐cap cenmfuge tube.
a用 “ 抑 rs Add3 mL″ ― hexane to eluate,shake,and cendfuge.Transfer up‐
perlayercontaining sにrolsto second screw‐capcen宜 血ge tube.Re‐
a)DJ″ cを ο t t α 0 “
S a r r a . ―
celite 545,cr equivatent.
‐ -2 μ pett extraction ofDMS0 1ayerin aぃ ttube with two 4 mL PortiOns
(b)βSfrθ s脅万 。 どsra″ ″α ″″sθ !″ rfθ z.一 g p_sitostero1/μ L
″‐ Lxane‐bewnc(lⅢ l),Caninly mnsfering upperlayerto second
CHC13・ P r e p a r e f r o m A l l Al pt pe lc i卜e d S d e n c ae b■o r a t o r i e s , I n c . ,
ttlbe each dme.Vigorous,shake pooledupperlayers ttid1 3 mL H20
standardo5%p_sitosterol,5%campesに rol)(■ 010ngeravailablo. andmHttge untilc臨 ほ Removeupperiayer andevaporate underN2
02‐ 脇第御 徐■助 SはI Pure grade over KOH.(a″ ″す例 f挽 2Aμ or虚lLIed air in 30 1五 L beaker on stean btt T― fer ttdue to
pendixB,safety noに sondistillation,potassiumhydroxide,naHlm体 O.5 dFanSCrewrcapvi』 wtttw00.8mLportionsCH03・ ARerevapo‐
ble solvents,and hexane.) ming sOlventwitlN2 0raltered airoversteam防 向 redissolve sterols
a APParatws in O.l mL CHC13fOr GC mlytts.
(a)Cω 説 "θttrag叫 放 ― Operating conddon革 にmperamに s 見 D e r _ f n a r f O n
C>COluHln225-245andtteCdOnPoFtandiamelonizationdecc‐
(°
珂 ect 2-8卜
Lexmctedsmpleandcdculatep_sitosterol by con―
tor265-285.AdiustN2Cを 口tergasnowca50r60正 Wmin)to6btain
vedng peak area to weight,using dお ly standard curve.
following retendon dmes(min〉 Ch01eSterol 16r18,cmpesterol
22-24,and β ‐
SitOstero1 28-30.Use l.8m(6 ftj x4 mm id colunn mg β ‐SitOSteroyl∞ g butter oil=
c o n t a n i n g 3 % J X R s i l i c o n e L 1o 2n 0 l m側e s h C aC sh ‐
rom Q and (燿frOm curve/1000)x(100/ロ ニ珂ected)X(1007g sample)
c o l l d i d o n c o l u m 2 4 h a tC2 5w0i°
t h 1 5 - 2 0 p s i4(-110338。 kPajN2・
( b ) 乃r r a r m t t ,c 一
をM O n i t o r p e r f o m a n c e o f g a s c h r o m a t o g r aIpdhe n d f y p e a k s f r o m b u t t e F O i l S m p l e s b y c o m p a r i n g h e i r r
by noは ng separationofcmpesterol and sitosterolexpressedas tion tiHles peakto retention times ofknown 30mpOunds.Relative reten‐
r e s o l u t i o n = 2 D / K C + 3 ) , W h e r e D = d i s t a n c don e bdmes e t arei
w e e cholesterol
n t h e 2l.0,campestero1
peak 1.4,p_sitosterol l.7.
Hlaximar C=campesterolpeakbasewidth,andB=p―
sitosterolpeakR e f e r e n工D
ce革aFり
Sci叡
ら1 7 弧1 9 6 7 ) .
base widh.Peak resolution should be≧
1.6.
スOAC 52,600(190)i53,535(197《
め.
01可 CCtお え″肋 叱 ″. 二W m 1 0 μ L H 航 l t o n m i c r o snガ
ge,
d r a w l a●i r i n t O b a r e l , i n s e r t n e e d l e i n tCAS‐ o l4 6‐
o s83‐ u 5(p_sittsterol)
don,and draw de‐
SiFed alnount into barel.Remove needle fronl solution and draw
l口しair intO barel,Notevolumecnscale and adiusttO desiredvol―
41.1,45
une,if necessary.
AOAC O荷 ictai Method 970.51
(O Pttβ α″rゎ″ゲ Jttα〃 C″ ″4-Prepare standard soludon
Fats(Animaけ in v9getable Fats
of2 μ g β ‐sitOStero1/11L CHC13・ [Detemine composidon ofstandard a n d O i t s ( D e t e r m i no at te is ot ne r co fゅ C ぃ
asin 970.50Bc)G2241.1,43).]Obtよ n standard curves
ly d嵐
cover‐ G a g c h r o m a t o g rM ae pt hh に
od
i n g r a n g e l -g1 0p _μ sitosterol,using<3 points.P10t area of First Action 1970
b‐s i t t s t t r o l pnesatk mag‐
Sg iぶ
βtOsterol. Final Action 1974
,PrepararfOn Or cOrun,
on of some vegetable olls contains small amounts Of
(SterOl fracは
Dissolve,with heを直ng,300 mg Jig肋 ,あ in 5 mL H20,add to cholesterol.Sterol fraction of palE1 0il may contain considerable
mortar and Pesde contatlling 10 g Celiに ,and mix thoroughly. amount ofcholesterol.)
( P a C k t t g m a t e r i d c a n b e k e p t s e v e F t t m O n t l s i f s Ct o iF ne d a t 5 °
n i n m i x t u r e t o 2 xA,PreParatrOn Or sampre
d g h t l y c l o s e d c o n t a i n e L ) T r a n S f e r 3 g, dcgeiltioに
12 cm!ube with ca 5■lmid outflow tube and small giass wool Padat SaPonify and extract unsaponinお le matter from 2.5± 0.01 g fat
bottom,and closed with short length of gum rubber tubing and asin Paragraph l and 2,933.08Gce41.1.39).Discard aqueous solu‐
pinchcock,Pack ttHdy,using tmping rod.(Flow rate of tightly 止ons.Transferether extttctto 250 mLbeaker and evaporate to dry―
packed coluHln is O.5-0.75 mLlmin.)Saturate column wih 5 mL ness on steaHl bath under N2・ Dissolve unsaponiflable IIlatter in

0 2000 AOAC iNTERNAT10NAL

0にS AND FAT AOAC OFFICIAL MgrHODS OF ANALYS:S(2000}
Chapter 41,p.48

4-5 mL CHC13,ranSfer t0 4 dran vial,and evaporate to dryness.

Rinsebeaker withtree 3 nLPortiOns CHC13,taking special care to
dssolve any matenal on sides of beaker.Transfer ttndngs to vial 猛 群 `
a n d e v a p o r a t e t o d r y n e s s u nS dt eO r e N 2s ・
mples in freezer. venttontreaches 17 cmstoP li時.EvapoFate S01vent and vttwplate
underlongwave LⅣ lightin darkened room.Mark off sterol band
lsolaWon of Ste的 13 by Thin‐ Layer Chromatography (Sarne R,o.2-0.3,as p_sitosterol stmdardj
sterol band as follows(dOnotremove p_sitosterol standardj:Scrape
a FeagerPrs ttJ APParatws
ofF sterol band wim square end of stainless steel spamla tFisher
(a)TLC Pね たな.―Prepare nЮm silica gel PF 254 Ⅲ366 or HF N o . 1 43‐
7 51‐
0,orequivalentjinto100mLbeakerandtransferw
254+366(Bdnkmann lnstruments,Inc.,or equivalenゅ asin C,or
20 mL CHC13t0 70-toP diameter funnel containing folded
u s e p r e c o a t e d P l a t e s na iv pa li al ta eb "l te p ra es c` o町a t e d widl ttica
12.5 cEldiameterfllterpaper(S&S588,orequivalentj.Extract ster‐
gel HF254+366,500 11mthick;No.2112,available as specialorder ols witt flve 10 mL pOrtions CHC13 andeVaporate combined fllme
'tpreCCated
u P O n r e q u e sfり
r o m A n a l t e c h , I n c . , Qo ur a“n t a c I ぶ to near ttness on steaElbtth under N2・ Transfer residue to 3 dran
with sllica gel PK5F with nuorescent indicator,500 μm ttck; Vial(SCrew‐ cap wihAHinerj withCHC13 andeVaporate to dryness
No.4851‐830)froHl Whattan,Inc. under N2・ (Altemat市 ely,remove sterol band with TLC plate
(b)働 J鍬力 秘 ・ ― Dittlled h glass(Burdick andJackson Labo‐ MaPert elute sterols from silica gel with 70 mL CHCi3[fOurteen
ratoHes,Inc"or equivalent). 5 m L p o l t i o n s l , a n d e v a p o r a t e s o l v e n t t o n e a r admrbyantels s o n s に
( C ) E r L y r ぇ_2Arn姥 h y d r o u s0 ,. ≦ 0 脇a l c o h o l ( F i s h e r S c tuen dn etri fN)2l ・
c
Co.Er138,or equivaleno.
Gag chromatography of Sterois
(d)PCr″ ルw切 2統夕ぇ― Distilled in giass,bp 30-60° C(Burdick
and Jackson Laboratones,Inc.,or equivalentj. i neagmts
Oβ ‐S'わ れだ wれrfθ
Sをpr sra″ ■ L.Weigh 30.O mg
.-3 rg/μ U s e r e a g e n t s幡労O B ( a ン( d ) ●2 2 4 1 . 1 . 4 3 ) a n d f O 1 1 0 W i n g
ldriCh
p_sitosterol standardい Chemical 2)into
Co.,Inc.,No.S340‐
O働 競 鹿 ″JS幼 滋 ″ sοれ,わな-1.2 1tg/卜 ・Weigh 60.O mg
10 mL volumettc flask and dilute to volume with etlyl acetate. cholesterol standard l代1ltech‐
APphed Sdence Laboratories,Inc.)
ngconl‐
CoHIInercialmaterialismixttreofcmpesterolKearliereluは into50mLvolumedcnaskanddlutetovolumewidlehylntate.
POnentj and sitosttrol.
p― ●)6肋 :asra確 て肋:β s々rors肋 ″材駒 ″.判 .2 11gcholettane
, c aPPararな。一Glass plates,
(D恥 れ リ ケ Cれ″7tarttraPれ and O.6 μg cholesterol′ ltL, Mix equal volumes cholestane,
20x20cm(ca 8 x 8in.):DesagarBrinkmann applicator;E間は航 ng 労硝 O B O G ″ 4 1 . 1 . 得
),and ChOlesterol standard sdutionst
syHnge,10 μL;dettCCating storage
board,spotting ttmplate;micro‐ 材秘前″ 陥-0.6距 choles‐
(O CttβJβ″r_p_s,rosttror s腕
“ 5‐
cabinet, Fisher 8‐ 6; storage rack, SGA Scientinc lnct,
terol and l.5卜 gpositosにrOW卜.MiX equal volunescholesterol and
C‐4116‐3:devdoping tank tThOmas―Mitchell tank■ ow unavAl‐
β‐SittSterol,■ o,Standard lons.
sol直
able);10ngwave 15 watt UV lamp(use with UV‐absorbing eye‐
C ) C t t r e sヴ
を α確! 姥
α 車血 ″ W r 2 rθ
ど″. - 0 . 6 r g /. 卜
Weigh
glasses)orChFOmatO‐Vue cabinetequlppedwithone ortwo 15 watt
lamps and,preferably,C‐ 50 1ongwave transllluminator(Ul‐ 30,O mg cholesteryl acetate standard(ICN Phamaceuticals,Inc.,
or equlvalents.
Vlolet Products,Inc.〉 LifeSciencesGroupjinto50nLvolmedc nask anddilutetovol‐
tra―
scraP2L―
(9恥 れ'92'PJarι Optionali ndapt fromsealinguEle tubeWih e的1抵etate.
β Figure
with fritted disk(Coming Glass Works 39580,30M).S夕 見 APparatus
財伍47★●夕
249.2.25). e)Cas ttR舛 確的graPな―‐ Barber‐C olman Cot Mode1 5は洵,
a PrePararrOn Or Prares Searle Analydc Sttes 4740,orequivalent,wittH2回 alne loniaion
detector and i nV strip clコR recorder.Temperatures― olumn,
Ahgn5 matching20 x20cnglassJatesOnmountingboard,and
lt。 220-250°C;detector and nash he轟 、240-27げ Ci flow rates:N2
justbeforecoating,wittplates withtissuedmpenedwihacch。
remove any dustor flngerPnntS・AdiustapplicatortodeliverO.5mm Kulmhigh PuFity grade),20t25 psi(138-172 kPab
terol in 8-12 min;H2,Ca 40-45 mmin;airp 300-340 mmin.
位tck layer.Weigh 45 g silica gel into 500 mL Erlenmeyer,add
ElectroEleter seぷ宜vity l x 10r9 amp full scale deflecdon witl
130 mL H20,Shake vigorously 25-30s,and Pour hto applttator.
l mV recorder.
I― ediately coat plates widl silicagd suspension and let plates rest
AdiustelectFOmetersensitiviけso tlat l・
5 μ
g cholesterol gives ca
undisturbed unはl gelled(0.5-1め .Dry cOated plates≧2 h atl10°C
50%dedecは on.RepeatiniectiOnsuntilconstantpeakheightsareob‐
and store i n 狙dnegs 血
cabinetuntiljust before use,
tained on success市e inielt'Ons of identical volumes of standard
ユ 輸 侑oとり研 a静 o何aturapny IIllXhre.
L i t t d e v e l o J n g c h a n b e r w i t h b l o t t t t p a p e r a n d a d d 1 0(b)Pr2Pa″
0 nL ′
rわ″ゲcθ ―S2を97050C(b)●
筋 ″・ θ を41,1.43).
eher―petroleun ether(1+1)tO Chamber.Cover chamber and 0 命 材" : θ 肌― H e 述 1 外弘 h u n d e r c o n d d o n s
″t r g ザc o :,″
equllibFate 2 h. s p e c i f l e d i5n0 C9(7o0G。
を241.1.43).
Draw line across plate 17 cm frombottom andca l cm fromeach (d)Pをrrar″ 御 ご夕.一ChrOmatograph ca 2 1tL choles‐
side.Spot10 μL β― sitOSterol standard solution,(0,at pOint 2 cm terol‐
β‐sitOSterol standard mixture to determine retention dmes and
from bottom edge and 3 cm from l side of plate. 1)issoive resolution of column.Minimum of 1600 heoretical plates is re‐
unsaponiflable matter,A,in200μLCHC13andSpotentiresamplein BアX16,wheFe L
quiredforcholesterolpeaki tleoFetiCaplatesioツ
10い POrtiOns on imaginary line 2 cmfrombottomedge ofplate so =cmcholesterolpeak frominieCdOnpoint,and,=cm trimgulated
that spotcenters are O.75 cm apart.Rinse vial with caL 100
CHC13
μ rol
base wdthofcholesterolpeak.h addition,selttadon ofcholesに
and spotボnse solution in equal portions on top oF sample spots. and cmpesに rol peaks,expressed as peak resolution, should be

◎ 2000 AOAC iNTERNATiONAと

AOAC OFFiCiAL METHODS OF ANALYSiS(2000} OILS AND FAT
Chapter 41,p.49

≧2 . 2 . P e a k r e s o l u′ (dβoⅢnD=,2Wつ h e r e D = d s t a n c e i n c 41.1.47 m be‐

tween cholesに rol andcampesterolPeakmaximum:3=tFiangulaに d AOAC Orioial Method 897.02
base width of cholesterol peak;and P=dangulated base width of 011(COttOnseed)in oits
c a m p e s t e r o l p e a k . D e t e m i n e p e a k r e s o l u t o甲n l eo n h as v狐i n g c a and Fats ヽ
equal amounts cholesterol and campesterol(ca equal peak areas); Hatphen Test
s a m p l e w e cd にS h O u l d g i v e p e a k2 5h-e5i0g%ho低 f chat width. Flrst Actlon 1897
Final Actlon
a DeremrnatrOn
Caガ JoJlf S夕をAppendix B,safety nctes on na― able solvents,
Pipet l.OmLcholestaneintemalstandardsolutioninto3dralnwial toxic solvents,and carbon disulide.
containing extracted sterols,rotate vialto wash down sides witl h‐
eCt 2 μ
temal standard solutton,and swirl to dissolve sterols.町 L MiX CS2 COntaining l%S in soludon widl equal volume amyl al―
cohol.Mix equtt voluHles ofthis reagent and sample under exami‐
sample atleastinduplicate.Repeatwith2 μLcholestane‐cholesterol
nation,and ttat h batt of boiling satmted NaCl solutton l-2h.
standard mixttre,ldendfy cholesterolpeakinsample fromitsreten‐
Presence ofaslitde as lワひcottonseedoil produces pronounced char―
tlon dme in standardmixture.Ifcholesterol peak heightin smple is
actenstic red or orange―
red solution.Depth Ofcoloris proPol■ Onal,
>60%full scale deflection,add additiona1 1.O mL cholestane inter‐
to certain extent,to alnount ofccttonseed oll present and compara‐
nA ttandardsd岨 研 的 smple andchromatographsmple and並 如‐
はv e t e s t s w i t h k n o w n m i x t u r e s o f c o t t o n s e e d o i l g i v e a p p r o
dard mixttre as above.Measure cholestane and cholesterol peak
amount.
heights h mm.
Different dus reac`with difFerent intensities.Oils that ha
Calculate ng cholesteFOV100 g smple,correcting for intemalheated to 200r210°C[Allen,“ Co― ercia1 0rganic Analysis,"5h
standd as fol10W革 Ed.,2,177(1924)]and hydrogenated oils J卸 此 SOn,“Vegetお に
Fats and Oils,"2nd Ed.(1943)]reaCt Wih greatly diminished inten‐
ng Cholestero17100 g= s i t y . H e a t i nn g a t1 0C2航5 r0 e°n d e r s c o t t o n s e e d o i l
crr,x)x(oノ q)Xいぜ s、)x(072)X100 givhg reacは on lAう s.工S伍 仇 加.れど.18,711(1899)].Fat of tti‐
mals fed on cottonseed meal or ohercottonseed Products may give
where rrland生=height(mmjcholestaneandcholesterolpeaks,re‐ positve reaction by this test.
and品=height(剛ゅ chOlesに
spectively,in standard mixture;最 rol
Reference革 ユPharFL Cれど れ 6dl ser.,島39は 1897)。
andcholestanepeaks,respectvely,insmplα
qandq=距 chOles‐ Aあ .A″αJysr 22,326(1897).
terol and cholestane/μ vely,in standard mixture;oi=μ
L,respecは g Allen,`℃oHmercia1 0rganic Analysis,"5dl Ed.,2,
L in samplei andmgoこ
cholestane/μ Smple/μ L. 177(1924).
比 cOnrrrmarOry Test 軌7'SrA RgP.,1如
Com.Agだ c.え (ID,P,143.
脇 筋々 178,372(195o.
Presence of cholesterol may be conflrlned by CC of sterol ace‐ C陸 れ Rθν.韓 497(1964j.
tates.AfterdeteminingcholesterolbyGC,evaporatesmpletodry‐
ness on sに am bath under N2・ C001and add 3 mL Pynditt and l mL CAS‐8は〕 1‐29‐4(cottOnseed oll)
acetic拭血ydFide.Capvial,swidonsteambatlundi sterolsdissolve,
a n d c o n d n u c h e a i血n gb a Ot nh sl にh . E v a p O r a t e , u t t n g N 2 S t r e a m D
41.1.48
until no odcrofpyridine is detected.Chrom江 ograph sterol acetates
AOAC O付 1● lat Method 974.19
and cholesteryl acetate standaFd onsS01uは
and compaFe retention
い peaks.
Faty A● ids(Cyctopropeno in OitS
tiines of sample and cholesに ryl acet分
Ha:phen Test
Firat Action 1974
References:JAθ AC S2,774,778(1969);54,643(1971):
Final Actton 1989
62,368(1979).
A . P r r n c f P r e
CAS‐ 57‐88-5 chDleSterol)
Oilis dissolved in ttxture ofbutanol and l%S inCS2,andheated
in presence oflightin 1losed
狙alphen"Jgments
tube.・ which are
41.1.46 formed are measured spectrophotometdcally,
AOAC Offioial Method 921.10 a APPararus
01,(Rosin)in ct:s and Fats
( a ) T 枕うタユー< 】u l t u r e t u b e s , 2 0 x 1 5 0 m m w i t h s c r e w c a p s a n d T e f ‐
Quatitative Tttt l o n l i n e F S , P y F e X N o . 9 8 2 5 , 市a
o r leeqnじ
i,matched to witttn O.01ム
Procedure u n i t s w h le ln e員d w i t h H 2 0 ・
(b)CttSra″ r″々β夕″r″
″ θ″うα協.―Maintained at l 10°
C.
P o l 述z e p u r e c i l , o F d e i n i t e d l u t i o n w i t t p e t r o l e w l e t l e r , i n
(C)韓 だCrttβ んθ的開夕rgぇ― COvering range 34(卜 600 nm,with
200 111m tube.Rosin oll has polarization in 200 Hlm tube of+30 to
voltage regulation systern.Bausch and Lomb Spectronic 20 is satis‐
+40°
S ,while most dis read between+l and…
1,C[Lewkowitsch,
“ factory if 75
O。in.tube adapteF iS enlarged slightly with sandpaper
Chemical Technology and Analysis ofOils,Fats and Waxes,''6th
wrapped around conttal igure.
Ed.,1,35は 1921)].
a FeagerPrs
CAS‐ 8u12‐16‐
2(rosin 011) (a)″ ‐
コ″rα .■Redistil if color foms in solvent blank.
″θ′

⑤ 2000 AOAC tNTERNATtONAL

0 1 L S A N D F A T AOAC OFttCiAL METHODS OF ANALYttS(2000j
Chapter 41,p.50

J/7r Sο
oD:!″ r2s″ -170 precipitate S in CS2・
!ガjoた
。 Prepare care to keep arachidic acid and wash solutions at dennite
ddly. ternperature in order to apply solubility coFreChOns given below.
(C)の 】 印〃OPttC Sra滋 之 空 ottonseed ol methyl esに rs con‐ D i s s o l v e a r a c h i d i c a c i d o n tltulに at le cr c hw oi lt ,h b o i l i
taining known amount cyclopropene FatけacidS・ evaporate to dryness in weighed dish,dry,and weigh.Tc his
weightaddO.0025g/10mLof90%alcohol usedin crystallization
ユ DerermrnarrOn
and washing,if conducted at 15° C;if conducted at 20° C,add
Accurately weigh ca200 mg ollinto screw cap tube.Pipet20 mL O.OM5g′ 10 mL.
butanol and 5 mL l%S in CS2intO tube,cap tightly,and mix.Place
MP of arachidic acid hus obtained is 71-72° C.Weight mchidic
in beaker contalning prcPylene glycol mlntalned at Cl10°in oll
acid x 20 i approximate weight peanut oil present.Arachdic acn
bah inhood.KecPhoOdlighton.Holdpropyleneglycollevd above
has characteristic appearance and may be配 にndfled undeF mlCrO‐
toP of tube contents.After 2.5 h cooltube to room temperature h
s c o p e . A s l i t t l e a s 5 tu1t0o%iplecaa■
nbe detectedby this Hlethod.
beaker containhg aowing tap H20`Wipe tube dry and cLan with
soft paper towel.Measure A witlin 4 h at ttHlum ca 547■ m R e f e r e n c e s閉 θ ガ. 7 3 , 1 3 3 0 t 1 8 7 1 ) .
: ,6 ″乃
ヮ
against blank prepared with com oil or oher cyclopropene fatty 晩 wkowits帆 働 卸 筋 rrn″ b8り 筋 A″ ry品 ヴ
acd‐free oil.IfsarnpleA .04,increase
is範 sample size andoil blank α h ttαtt a脇 ダWtts,6th Ed.,2,316(1922).
up to l g and repeat deterHlinatlon. 】 Determine amount of
CyC10pFOpene fatty acids from standaFd CurVe prepared from CAS‐8002‐ 03‐ 7 tpeallut dl)
cydopropene standard.

References:JAOAC SS,1288(1972);56,82(1973).
41.1.50
AOAC Otttcta,Method 037.11
41.1.49 in o:ive,
011(Peanuゅ
AOAC O付 tola:MettBod 871.01 Cottonseed,CoHl,and Soybean 01ls
0t'(Peanuゅin ol13 and Fats M O d i n e d B e i 3 t e r T e s t
Modited Renatt Test Fid Actton 1037
F3rst Actlon 1871 Fina'Actton
Fhal Action A.R昭 算"的
Weigh20gollinto Erlenmeyer.SapOnifywitlalcoholic KOHso8 oA′ ∽筋佐 脚吃耐町4協 ッ院扇た w胸 修仲-1.5M.崩 ssolve 10 g
lu飲加,既抑.lalA tsg241.1.18)ineutralizeexacdy widlCH3C00H(1+ 100mL
KOHhpurinedalcOhol,切n.lmtt G資41.1.18),anddluteめ
l ap nh d l w摘a s h i n t o 8 0 0 - 1 0 0 0
3 ) , u s i n g P h e m叫 mL awih「
a s k宜ned
o O nalcchOl.
taiぃ (
ing boiling斌 xhtt ofl∞ nL H20 and l卸 nL 20a cH3C00MPb (b)的 め 劇 肋 ric a位卜 も pecinc gFaViけ1・16.Dilute 83 mL
nandmc。 。
solution.Bdll航 e rslngnask
l脚対ぶ囲胡soapby i― Check
concentratedacid(speCiflCgravity l.19)to 100mLwidlH20・
h H 2 0 ' S W n i n g O C αd o n a l y t O C a u s e s c a p t o s t i c k t o s i n . A t r c owidl
o ト speclac gravity splndle.
ing,decantH20andexcesstCH3C00ぱ b SOlution,and wash Pb soap
(c)Aた がわトー70%.Diluに 700 nL alcohol to 950 mL widl
widlcold H20 and dcchol,900 by volum.Add 200 nL ett cork,
H20・ Check by speciflc graviけor refractive mttx ttd adiuSt if
and let stand until scap ddI逸 即斑ett hett on H20 bah uSing ttaux necessary.
c o n d e n s e r , a n d b o inl. Wciat5l航
dis,nostofsoapwillbettsolvet
w t t l a r d s , w h 敵di c hn的 m u c h s t e a m , p a r t o f s o a p w t t b e ュl eregr nundis_
solved.CooletFttluSmofsoapt0 1卜 1 7 C
° a n d l e t s t a n d u n t i l a l l 伸
Weigh O,92 3 0r measure i nL oilinto 125 mL Erlenmeyer witl
s o l u b l e s o a p s s e p a r.a t e t c a 1 2 め standard taper outerjoint.If oil is IIleasured,use short Mohr pipet
Filter on Buchner and thoroughly wash insoluble Pb soaps with
with fairly latte ttening at uP,drain to lower mark,hold until me‐
blePbsoapslntoseparatorwithjetoFedlerD
ethen Washethersinsol工 niscus stoPSittng mplrt,anddFain tOmark agaln.Add 5 mLalco‐
altematng wih HCl(1+3)at end OfOperation iflittle ofscap stcks
holic KOH solution,and heat S min on sに am bath,ushg air
tO paper.Add enough HCl(1ゃ 3)sc hattOtal volume ofacid is ca
condenser to avoid loss of alcchol.Sw± l once or twice during
200 mLandenoughethertt make itttotal volul配 150-200 mL,and
shake vigorously several Elinutes Letlayers separate,drain offacid saponiflcation.Add 50 mL 70%alcohol and O.8 mL ofdle HCl.
layer,and wash etler once with 100 mL HCl(1+3)and hen vttth Wamm to dissolve any precipitate that may fom.
s e v e r a l p o r t i o n s o f H 2 0 u n d l H 2 0 W t t hInsert
i n g thermometer
s a r e nand
o cool
l o with
n g econtinuous
r a c d 的 agitation so dlat
methyl orange.Iffewundecomposedlumps OfPb soapremain(indi‐ temPeramre Falls ca l°(ンmin.Observe turbidityに nperature or
cated by s01ld Pattdes remaining after third washing wih H20), clouding point,which is temperature at which derl占 te precipitate
break uP by runttng offalmost all H20 1ayer,adding ll性le HCl,and irst appears.dftemperatureofsoludonis aboveroomtemperatllre,
shtting;then condnue washing with H20 aS before. c o o l i n g m a y b e a c c o m p l oi rs h be yd oh cホc a s i o n a l y i _ e r s i n g
Distil eher from solution ofinsoluble fatty acids and dry latterin
C≦ b e l o w t l a t o f soonl.uDは
s o l u t i o n m H 2 0 b a t h o f t e m p e r a t u r5e° o
flask by adding little absolute alcchol and evaporating on stealn
not iIIIIIlerSe nask below level ofcontents,and agitate continuously
bath.Dissolvedry faty acidsby waming with 100mL90%alcchol
to prevent premature fomation ofturbidty by local cooling.Solu‐
b y v o l u m e t C o o l s l o w l y tCo, s1h5a°
king to aid crystallization.Let
s t a n d 3 0 航n a t 1 5C °
. はo n m a y b e a g i t a t e d b y s t i r i n g w i t h d l e r m o m e t t r or
In presence of peanut oll,crystals of aralhidic acid separate nask.observe tuttidity teHlperature by looking drough solution to‐
from solution.Fllter,wash precipitate twice with 10 mL alcchol, ward good light,ortoward dark background with good lightngcol由
90ワ)by volume,and then with alcohol,70%by volume,taking from one side.)

◎ 2000 AOAC tNTERNATiONAL

AOAC OFttCiAL METHODS OF ANttYSiS(20001 01LSAND FAT
Chapter 41,p.51

Ifturbidity appears before temperatureC(olive r

reaches011)。
9° Use deepestred colors producedas basis for companson,andbe‐
13° causeofshortpeFiOdOfstablemaximuminに
C(cottOnseet com,Orsoybeanoils)presence ofpeanutollis in‐ nsity donottest>3 olls
dicated.ConflHn by 871.01(S2241.1.49). at one time.Standards containing known amounts ofにa seed oilin
oliveollthatgivelitdeornoplnkwihhistestshouldberunsimulta―
αりsr 62,9“
Referencett A″ 1937). n e o u s l y w i t h s a w l e . P r e l i m i n t t y F 0 0 m t e m p e m t u er se it ne ds it ‐g 市
JAて,AC 28,293(1945),32,363(1949). c t t o n o f s c a n d a r d s t t b e u s e Hd 2 0h mi ec te h‐o d .
CAS‐ 8002‐03‐7(peanut Oil) RefeFenCeS:JAθAC 19,493(193o;2は 418(1937).

41.1.51 41.1.53
AOAC Otticia:Method 929.08 AOAC O付 lclat Method 893.01
Sa:ad 01:s(Refined,Winttrized) 0H(SeSamO in OiiS and Fats
Coid Test Mod3fted Vi:3avecchia Test
procedure First Actlon 1893
Ftrst Actlon 1929 Finat Actton

nl1 4 oz oil test sample bottle widl oil at C,coFk

25° dghtly,and
sea wittparaffln.Compleに ly submergebottlehbucketcontaining
inett Cracked ice and add H20 undlit dses to top ofbOtde.Keep
bucket ilにd solldly withice tt removhg any excess H20 and add‐
lngice whennecessary.After5.5 hremovebo■ leandexarnineoll.If
it is properly wintered,smple wili be brilliant,olear,and limpid. use the very dilute solution sPecifled`)
Reference: 」は似 C12,203(1929).
Add2 mL furRralto 100 mLalcchol.Thoroughly EtX O,l mLof
this solution with 10 mL HCl and 10 mL sample by shaking h test
tube 15 s.Let mixture stand 10 min,observe color,add 10 mL H20,
shake,and agAn 6bserve color.Ifcrimson color dsappears,sesalne
cil is absent.(As furfural gives violettint witlHCl,itis necesstt to
成 12,67(1893);13,69(1894).
References:ユ Sθa C確 醜.I閉
JAθAC島 441(1923).

CAS‐ 8008‐74-0(seSanle oll)

41.1.52
AOAC Omciai Method 036.12
0 ‖t T e a s t t d ) l n O i l V e o ‖ 41.1.54
Qualltatlve Color Test AOAC Otttcial Method 920。 163
Fir●t Actacn 1936 Fats(Fore:gn)Contatning Tristearin in Lard
Finat Actton Melt3ng Pcint Method
Ftrst Acticn 1920
For prelimintty qualitative test use following F00m temperatuFe Final Action 1989
method:Measure exactly O.8 mL ttedc tthydride,1.5 mL CHCi3,
A P r r r r P r e
and O,2 mL H2S04intO testtube(18x150-is convenienり ,MiX,
Presence ofbeef的 ,ta1lows,and similar fats,as well as hydroge‐
andcooltoroom temperature.Add7dropsofol tobetesteddirectly
to reagents,mix,and cool agaln.ぼ o measure test dl,use glass tdb‐ nated andinterestenfledPOrk ttinpork fats and lard,is cにdby deに
rso‐ detemining difFerence between mp ofcrystauized tFiglycerides and
ing,4 HInod,andca2 11mid;7 dropsshouldweighcaO.22gぅ
mpoffatty acidsderivedfromthesetriglycendes.This valueisiarge
lutionofollinreagentsiscloudyanermixingandcooling,addacedc
for pure Pork fats and smali for beeffats.
飼 呼d r i d e d r O p w i s e , s h a k i n g a f t e r e a c h a d d i d o n u n d i s o l u t i o n s u d ‐
denly dears.Appreciable deviations from these amounts,Particu‐ a mr_rnafrm
l a r l y i n H 2 S 0 4 , C a u S e d i s d n c t v a r i a t i o n s i n c o l o r i n t e Weigh
n s m e s e5 sg i melted
nce and flltered lard intostoppered
glass‐graduate
mixedreagentdeterioratesslowly,donotmlxinadvanceoftesting, and add 20 nL warm acetone.Mix well,taking care dlat solution is
ARer5 mln,add 10 mL absolute edler fron graduate and mix im‐ dearandhastempemmre>30° C.Letstand 16r18hatconstantに m‐
mediately by inverting once.Tea seed oll foms brown solution perature of 30° C.Fine mass of crystals occuPyhg≦ 3 mL should
c h a n g i n g t o i n t e n s e r e d w i td sl ri en dm ri en ao cr hs eo s. mコa x i m u dml e n b e f o u n d a t b o t t o n o f g r a d u a t e . S h o u l d v o l u m e o f c r y s
and then fades slowly within few min,01ive cil fo― initial green terially exceed 3 mL,take smaller amounl of lard(3-4g)for new
solutktton additionofetler.This colorfades slowly tobrown‐ gray, test.If crystals obtained from 5 g lard are insufflcient,increase
occasionally passing through faintpink stage.Botholive cil and tea weight iard and volume acetone pFOpOrtionately.
seed oil evenmally fade to pemanentlight brown,Mixtures of tea Decant supemate acetone solution ton crystallized glycerides.
seed oll and olive oil show characteristic tease9d oll colors propor‐ Add血 ●e5mL PortiOns wam(30-35° C)acetOne trom small wash
はo n a l i n i n t e n s i t y t o a m o u n t o f t e a s e e bottle,taking d o i l p r ecare s e nnotto
t . break up depositin washing,and decant frst
2 pO由
For approximate quantitative estimadons,drop oll into reagents Ons.Achvely agitate third Portion in graduate an
as descibed above and let remain at F00n tempeFature S min.In lnovemnt transfer crystals to smali fllter paper.Using wash
meantime,cool 10 nL portion absolutt ether inH20・ ice‐After wash crystals with 5 successive sm1l potlons of tle warln acetone,
5減 n,place test mbe cOntaidng oil and reagents H20in1 ice‐
min, andremove excess acetone by sucttn.Spreadout pap
addcoldetheroaking carethatnoH20falls intotesttube),and mix. breaking up any large lumps,and Ar dry atroon temperame.
H20 bath and let colors develop while
Retum tube tt ice― is im‐
i↓ Thoroughly commnute mass and dete航 ne mp of crystals in
mersed in ice―H20・ COlors develop slowly and reach maximum .163.
closed l Hlm tube,using apparatus ttHHlaF tO that ofFigure捌
within ca 5 min, HeatH20inbeakerrapidlytoca55° C andmintainthistemperamre

0 2000 AOAC tNTERNAT10NAL

0!LS AND FAT AOAC OFFIC,AL METHODS OFANALYSiS(2000)
chapter 41,p.52

acids ‐
ofβ おn,275.14.r smpleis impure,Elean MW
paldttdsに
should approach山
筑 offatty ttds mm ttsに
航 n,284.49.

R e f e r e n c e s i t / S D AぇAJ″
″′れが 五
閉広 Circ.132.
JAθAC 3,2ほ
2 (1920j:4195(1920j;19,417(193o:
59,323(1976).
れ arysr 65,623(1940j.

CAS‐555‐
43‐1(triSiearin)

41.1.55
AOAC O付 lolal Method 974.20
Detectton of Fish
and Marine Aninla1 01:s
prccedure 1974

(APplicable in Presence ofvegetable oils and in absence ofmetallic

salts.)

磁 ″jθ
″f ざ22 Appendix B,safety notes on bromine.

Dissolve 30 drops oilin 8 mL CHC13in 50 mL beaker and add

10mLWiis sOlution,9郷 .159A(彪 241.1.lo.Add from buret,widl
constant stiring,Br2 CHaC00H(1+3-4)to endpoint where solu‐
hon appears tobleachout,andthen addstightexcess.Mix,inmedi―
ately pour into flatも
otton test tube,and let stand l-3 h until clear
supemate is obtained.Measure height of騨町 lpltate with mm mle
and compare with standards pFepmd With known amounts of ish
oil,including O,Anount of insoluble bromides is PropOrtional to
amountofash01l up toca 15%and as little as l%ish oil can be de‐
悔cted by slight precipitate on standing ovemight.
●Rin0
Figure 920。16← AppaFatuS tbr deterHlining 8■
Reference革ユAれ 0"Cれ 卸 .Sて だ。 47,23く 1970>
Point.
泌 OAC S7,1005(197o.

until therlnometer∽ Cithen heat again

=ying mp tube registers 50° 41,1.56
C.Renove
and dse temperature ofouterbath ratler quickly to 67°
AOAC Ofrictat Method 945.102
bumer.MPisreachedwhen fusedsdbstancebecomespeFfeCdyclear
0‖ (Minerat)in Fats
and transparent.
FtFSt Actlon 1945
Whenmp ofglycerides obtttnedby this ElethOdisく 68.6° C,Pres‐
A.Orarrratrye rest
ence ofbeeffatorotherfat shouldbe suspected,and mpof≦ 63.2° C ―P r o c 」 ure
i s e v i d e n c e t h a t s m p l e i s n o t p u r e l a F d . C O n d u c t d e t e m i n a t Pi lo na wc iet hl m L o i l o r m e i t e d f a t h E r l e m e y e r ; a d d i n L K O H s
control sample ofpure lard. tion(3+2)and 25 nL alcohol.Boll under refluxお r condens句
Conflm presence of foreign fat by taking mp of fatty acids pre‐ shaking occasionally,undl saponiflcation is ccmplete ca 5 H遠 ゅ.
pared from glycerides.After detemittng mP,transfer crystallized Add25 mL H20 and mix.In presence of)幻 .5%mineral oil,disunct
glycendes to 50 mL敬 克監ぁ add25 mL caO.5M alcoholic KOH,and turbidty appears.
h e ■o n s t t a m b a t l u n t i l s a p o n l i c a t i o n i s c,oPmopulre に
solutioninto ユ
Owantratrye Mah何
seParator conttning 200 nL H20'aCid,,add 75 拓 shake,
mL e血 ― FrnarA●ffon
and iet stand.Drain aqueous acd layer and wash etler solution
(Ca″rわ″f品%Appendix B,safety■ otesondisullatiOn,flammable
≧3 dmes withH20・ Transferethersolutontocle‐ dry 50mLbeaker, solvents,and petroに um ehert)
evaporaに ether on steambath,and inally dry acids at X洵 °C.Let ac‐ Treatunsaponiflableresidue,933n8G2241,1.39),withH2S04 aS
ids r卸 減 n atroom temperame ca2h andtttemine mp,Ifmp ofgly― below.When very small anlounts ofmineral dl a
c e n d e s , p l u s t w i c e d i f f e r e n c e b e t w e e n m p o f g l y c e F iunsaponiflable
d e S a n d m p residue
of for test may be obtained as fo1lows:
鮎 t y a c i d s , i3 s°
C守, t l e l a r d i s r e g a r d e d a s a d u l t e m t e d . Saponify 100 g fat by refluxing under air condenser 2 h wih

ConcluslonsmaybeconflrIIledfmherbyprecisedek】 55 mL KOH solution(3Ⅲ 2)and 240 mL alcchol,wtth occasional

HunttnsOf
mean MW of separated fatty acids.Dissolve ands in cOlodess,shaking.Cool,add300mLpetroleumetheroP35-60° C),andtrans‐
r e d i s t l e do 施
hol,caremlly neutralizediElmettately fer
b e to
f o separator.Rinse
r e u s e , a n d nask wit1240mL alcohol andaddrinsingsto
dtrate with O.5‐ ●.2M KOH,using phenolphthalein.Mean MW=separator.Add 480 mL H20 and Shake vigorously.Letlayers sepa―
weightfatty acids x 1000/tmLxlnol述
ty KOItluse●
.IFsampleispure rate,血 in lowerlayer,and transferupper layer to anodler separator.
lard,meanMWoffattyacidsshouldcorespondcloselyttthatoffatty Repeat extraction of saponifled fat with 300 mL petroleun ether,

◎ 2000 AOAC iNTERNAT10NAL

AOAC OFFiCiAL MttMODS OF ANALYSiS(2000) OILS AND FAT
Chapter 41,p.53

a n d c o m b i n e e x t r a c t s . W a s h e x t r a c t t w i c e iwointSh H6200 ,n L Table

P o ■966.17 Repoatability and reproduoib‖ Ry waluo3
u s i n g g e n t l e a g i t a t i o n . R e p e a t w a s h imnLgO 。
w5iMh ω
KOH,fol‐ O/c Hydrocarbon content
lowed by vigoFOuS agitation wm successive m mL po鵡 ons H20 atlevel of
p o reaxに
u n t i l w a s h i n g s a r ef r ae le k. aE lv ia ― tractto smali volume and
Deterrninations
dry with anhydrous NttS04・
Two sin91e deterrninations in l 0.09 0.14 0.33
Filter petroleuEl ether solution trough small cotton plug int0
laboratory should dttr≦
Babcock milk‐test botde,989.04A(a)Gを233.2.27),add few small
Singie deterrninations in difrerent 0。10 0.14 0.40
p l e c e s b r o k e n nP ,o ar nc de l r拭e m o v e s o l v e n t t t h ae la It Ii n g oiaboratories
n sに
should differ≦
bath while passing curent of air mough bOttle.Cool,add 5 mL
H2S04,miX,andkeepbOtdeinboilingH20bath30min,shaking oc‐
caslonally.Remove bottle fFOmbath,cool,and nll with H2S04until
surfaceFiSeS Wellinttgraduatedneck.CentFifuge 5 min at 1200中 m Gende air strealn will aid solvent removal.Transfer to weighed
andreadvolume ofurreactedresidue.Ifenoughmineral ollis avAl‐ 250 mL Soxhlet aask,insing witt three 20 mL pOrtions petroleum
able,obtain density asin 95住 48(sでを40.1.Oo,using smali SPrengel ether.ReFnOVe relnaining solvent by evaporation on wam surface
tube.Weight mineral oll can be closely approximated by mul位 ply‐ w itlgende airstream,keeping solutiOnbelowbp.Cooltoroomtem―
ing volume by O.88.Refracdve index ofcolorless residue should perature
be or and welgh.
in desicc■
く1.500 at 20°
C. Repeat with additbnal he述
昭 periods of 20 min9 co01ing,and
Reference:泌 θAC 28,285(1945). Wei」 ng undi change in weightis 3.5 mg.

CAS-8012-95-l Kmineral oll) Conduct blank deteminaは on without test Portion.

Hydrocarbons,%=(S― D x100/C
41.1.57
AOAC Officia:Method 966.17 whereS=gsampleresidue,3=gblankresidue,and Ci gsample.
Hydrocarbons(Saturated)in Glycerides
a Prcrgrm
A:umina Column Chromatographic Method
Ftret Action 1966 Following 95%confldence limits my be exPected(磁 g Ta_
Fina3 Action 1970 ble 966.17):

AOCSrAO四 0角に納oJ Conim presence ofhydrocarbons or mineral oil tt

ofIR specmm Ofresidue with that ofPure hydroclbons or min
A.ApParartJs anど Reagents oil,USP.
( a ) C んr O 初αr 9 g r a piた . 一W i t h T e d o n s t o P c o c k , 2 5 - 3 5 m m
c ねb 夕
diameter and 400 mln long. Referencei JttAC49,71,232(196o,
●)A筋 ″れ柳 拭 能、 ― Activated alui拭na,AlccaP grade F‐20,
8い2m mesh(Aluminun Co.of America,1501 Alcoa Bldg,Pitts‐
41.1.58
burgh,PA 15219,USA,or equivalenり .Dry alumina≧4 h at200°C
AOAC Ottlctal Method 961.11☆
before using,Store in sealed botdes in desiccator.Alumina must
Chick Edema Factor(Dloxins):n CttS and Fats
h a v e a l o l s t unrte ocも%
foい
ntに
o rettn glycendes.
Bi0383ay Method
ユ DerrmrnarrOn Firet Action l弱 1
Fina:畑 ,on 1962
H工xed test podon into 125 mL
Weigh 10± 0.01 g melted,well‐
Surplu3 1974
Erlenmeyer,and dissolve in 50-100 nL petroleun ether,waming
gently,if necessary. Scを28.113r28.117,12th Ed`
Preparecolumnbytampingplugofglasswoolintobottomoftube
so dlatsone ofgiass wooliSin constncted Portion above stoPcock.
Hll tdbe ca%full with petroleun ether(nsher scientittc Co. 41.1.59
No.B‐ 139,or equivalent)and add 200 g alumina ttmugh powder
AOAC Otticial Method 968.23
funnel.Tap side oftube to aidin packing,Cover witt ca l cm anhy‐
Chick Edema Factor
drous Na2S04・Elute atrateof80-90 drops(3.0-3.5 nLj/min,using
(Dioxins)in(Dils and Fats
stopcock for control.
Gas Chttmatographic Method
When solventheaddropsto ca l cHl,add sanple solution.Let sol‐ Ftrst Action 1968
vent layer drop to ca l cm above aluIIllna, collecung elutte in Finat Action 1989
5(沌mLErlenmeyer.Rhsesampleflaskwith 50mLpetroleumeher
A . P r r n c r p r e
and add to column.Repeat 3 times,adding each rinse to column
wttn solvent headdrops,ocalcm,and wash dbwn sides ofcoluBm Fat,oll,fatty acid,or lipid is treated with H2S04 and eXtracted
on transfering.Continue adding Pctroleun etherto colum untilto‐ With pe位oleum ether.Extractis puFifled on A1203 C01umn,further
t a l o f 4 0 0 m L h a s p a s(sIendv etrhtreodu ぶ v o l u m e t r i c ntreated
a s k c withan H2S04,and examned by electron capture GC.Peaks
s e r v e a s c o n v e n t t n t r e s e r v o i r f o r f l n a l a d d i t i o n o f s o witt
l v e n tretention
.) dmes relative to alttin(Rか
be:ween 8 and45indcate
Evaporate eluate on steaFn bah to 50二
75 mL.Sti拭 ng rod placed presence of chick edema factors(heXa‐ , hepta‐ , and
in flaskwill help prevent superheating andcOnsequentbciling over. octachlorodibenzo,p―dioxins).

◎2000 AOAC iNTERNAT10NAL

01LS AND FAT AOAC OFFDAL METHODS OFANttYSIS(2000)
Chapter 41,p.54

島 Reagents anJAPParatws itteCt S口L(equiVttent to 50 mg oiginal toxic fat)intO gas

はh S e J l g l a s s w a r e w i t h a p p r o w a t e s o l v e n t s b e f o r echrom筑ograph,Resulting
use.Do not chromatogram should exhibit sttes of r
store solvents in polyethylene contalners,Solvents availablepeaks fromwtt Raca 8-45.Peak heights should be between 10 and 25%
Burdick and Jackson Laboratories,or equivalent。 ) 側 1岡de deflection.Peaks■
8-13 are dueめ
heXachlorodibenzoBP‐
di‐
閉 2 協夕∴― D i s t n e d i n g l a s s , b p C3 .0 - m °
( う P 夕! 乃! 2 ″ oxln isomers:2p併 逮s at 17-22,to ttE 2 hq脚独iSOHErs,and peak at
(b)酌 り ↓夕rttrル ra肋 瓶 岡 統 胸 脇 所θg均 ヵ ンー Ehrc%alCOrnlj 35-45,to octa‐is倒
田eL
or a b s o l u t e. 0e1h%earl(c幻o h o l ) . (b)P″ わだ″ り S″rrZr,c″ ″ c`em2P3 Dissolve 2.5 g test
(O HC滋 ″夕 ・ PatOn h 10 mL hexane in 500 mL glass‐ stcPpered Erlenmeyer.
(0/Sθ θC″ れ2.―Disは1led in glass. Add 10mLH2S04,StOpperP and shake 30 s.Add 125 nLpetroleun
(e)Attri4 sraz`ね ″ ざθ ど
ガ θ払 一■.05 μg/mL isooctane. e巡 、stopperP and shake vigorously ca l min.Let separate and de‐
放 冴2 肥 ″わw βθ s ' 昭
ガ 挽 肥 夕M 呼 ル. - 1 . 5 % . cantupperlayerinto 500 mL Erlenmeyer,avoiding transferoflower
0勧 racゎ 材
P r e p a r e f r o m c o n c e n t r a t e d r e f e r e n lc ae b lt eo x mi mc t t v iK _a layer`Repeat
Vホ extraction widl additional 125 nL portiOn petroleun
sionofPesticides andlndusdalChemicals,UoS.FoodandDrugAd‐ eher.Evaporate combined petroleun ether extracts lo S nL.
midstration,200 C S`,SW,Washington,DC 202敬 与USA)in USP o Ar蜘 商昭 統 の み _BefOre use,dry ali solvents町
CottOnseed or other vegetable ガわれ f Do not contact toxic
dl.(6α shaking with anhydrous Na2S04・ Transfer evapomted petroleum
fat.) etherextractto aluminacolumn,(め ,using tota1 0f lomLpetroleun
(g)Acriya,caα比協円 筋И.― →FisherSdendttc Co.No.A‐ 540i do not ether.晩tdraintojuttaboveLvelofNa2S04・ KecPing liquid level
substtute.Acuvate loog poldonsby heating4hat260° C.Transfer above Na2S04筑 all dmes,elutt wid1 100mLpetroleumether(tac_
vity of
withoutcooling to dry container and close dghdy.Check acは はon l),50 mL 5%ehyl ether in petroleun edler KfraCは Onり ,and
AL03by analysis oflow PositVe reference smple,o,eXalnlnlng 100 mL25%ethyletherinpetroleumether tfmCtiOn 3),tFlowntes
AL03 8aCtions 2 and 34 Wih Sufrlciently activated A1203,ChiCk of 8-9 mmin are satisfactory.)Discard fFaCtOns l and 2 and col‐
edema factor elutes predominandy or entirely h fraction 3 as indi‐lectfraction 3 in 125 nLErlenmeyer.Add several bolling chips a
cated by gas chromatograms.KChromatoBFanS Should show senes e V a p O F a t e t O c a 2 n L o n s t e血 a lTnr悦a n s f e r r e距s週wihsHlallpor‐
ofpeaks widl ttbetWeen ca 8 and45.) 伍 o n s p e t r o l e l m e h e1r0的 m L g i a s
s t
s o
‐ p p e r e d P duate andmer
(h)Ar″加'″ α ごんrottarθ g,α″ れ,c cθJ″加 ″ ―
.― TO dry tube, e v a p o r a t e t o 3 m L u n d e r N 2 ・
17(oO x250mm,ntted ttbottom with ccarse porosity mttedglass OA″ 苫わM2S″ ル riC ac″】2叫 み― Add 2 mL H2S04tO pe‐
dskandTenonstOpcOck(mbewitloutdiskbutwithglasswoolplug trolttnethersdttn,stopper,andshake宙 gorously 30品 臨 sepa‐
at bottom may aso be usedj,add redisは dried
lled petroleum ehe■ rate and decant upper layerin的10 mL bea監 ぁ avoidng transfer of

／ ′︲ ヽ 、
町drOus Nが04,until mbe is%mll.Transfer 15 g any H2S04・
before use wihお Add2 nLpetroleumetlertowduate,swirl vigorously,
AL03的 mbe h smll ttons,tapplngめsene.After last portion haslet separate,and decant upper layer into beaker.Add O.5 g solid
s e t d e d a n d t t b u b b l e Ss 面施
s t o Pf s no sl iv ne gn め
t a d dy ‐
5 g‐
NaHC03tObeakerandsは rcaO.5 min.Lttstand5minanddecantpe‐
drous Na2S04・ Drain excess pemlem etler until i isjust above suretroleum etleriayerinto 2 or4 dram v胡 .Wash NaHC03 Wld12 nL
face ofNa2S04・ petroleum ether and decant washings into vial.Evaporate solvent
(1)CaSご 力″ θttα!οgrαPんjc cο′ “閉″,一Glass, 1.8-2.7m Justto dryness at room temperame in vial under N2・
(6r9ftj X4 mm id,packed wid1 2.5%SE 52 or odler Polysiloxane (e)CaS Cれ ,θ graPれメ.――
ttarθ Dissolve residue in 250 μL
staは 組twashed and silanizedい
onatt phase on抵 atOmceOus eコ
氏 isooctane,stopper vial,and rotate to wet stts with■solvenは ject
120
wihnarowrangeo5 μゆgrainsizebetween 125-250,m(6い 5 卜L solution (equivalent to 50 ng test portion)intO gas
meSゆ.COat support with substrate as followtt DissolveC h2.5=slll‐
F O m a t O g r a p h , c ) . P e a k s owfi t8h-■
45 having same rettntion
C O n e g L t t r u b b e r i n 3 0 0 m L C H 2 C 1 2 t t O l ule)nWei(hl Ⅲ
heat,Add d m e a s p e a k s 8o-f4■
5 mm reference toxic fat(O indiCate pr
97.5gGas,ChromQandletstand10minwi血 occasionalgentlestiFe ence ofchck edem factor.
dng.Dry in rotttr evaporator held Cinbatt 50°Apply vacuum to
PerfOm Feagent blank他 的問血ぶOn Wh each set ofsmples.
chromatographic tube and add small amounts of ocated support
Smoodl baseline should be obtttned in8尊region■
.
whiに tapping mbe at packing level after each addition.F11lto widlh
2.5 cin on exit side ttd 7.5oncI■ entrmce side and f11l remaining Rettrences:泌 O AC毎 ,384(1963】 48,構3(1965〉
space withsilanizedglasswool.ConditioncoluIIlnatol湾 口由ngpres‐ 50,874,1338(1967);51,940K1968)i S3,628(1970);
SuFe 2-5 days at 250° C. は ,528(1981).
二 With 63Ni electron capture or 3H con―
G)働 S協 ″初 rOg切元 地 位″ 22tb,702(1968).
centric―
type electron capture detector.Operate instmmentin accor‐
R2ッなaオ Marchメ 9%
dance with instructions of manuFacturer. Ob能 意n stable baseline
beforeusetChooseoperatingvoltagethatwincausebetweenO.6 and
fuliscale denecdon for O.l ng aldrin oル standardalttin soludonj 41.1.60
at sensidvity setting of l x 10 9 amp full scale.Keepc01umn temper‐ AOAC O打 :●la:Method 942.19
ature at 200±
1°C and attust N2 f10W So that aldrin elutes h Synthetic Co:ors tn 01ls and Fats
l-1.5 min d)25-0.33血 L
.E6r8mm]/min chart speedj.Iniect 2 μ Spectrophotometnc MethOd
alttin standard soluton before each reference or test sample. First Action 1942
Final純 tlon
a DerermfHatrOn
α
(a)A″rys活 α r研た,拡―Dissolve 2.5 g referenceA
ザ 呼ヵ″″
P e a g e n t s
l . 5 t / o ctfoa対t i n 1 0 m L C C 1 4 i n 5 0 0 m L gs it ao sp sp ‐
eredErlenmeyer ″ス ニ Mix l L CH3COOH With200 mL HCland
o Ac″ sοttr:θ
and proceed asin(Dく
d).Dissolve residue Linisooctane
250 μ and 100 mL H20・

◎ 2000 AOAC iNTERNAT10NAL

AOAC OFFICi札 METHODS OF ANALYSIS(2000) 01LS AND FAT
Chapter 41,p.ss

(b)Ac材 ざ ″ヱーCautiously add 400 mL H2S04t0 100 mL

θttrfθ in acid extracts and as green residue on papers after flltration of
H20・When cool,add 900 mL CH3COOH and mix. troleunl ether solutions.
一
(C)Sο冴:閉 研 乃Ifル Sθル筋 払 Approximaに ly 25%.Dissolve
A θAC 25,726(1942)!M,235(1951).
References:立
250 g NaOH in H20 and dihteto l L.

a sePararrOn a何 どrdentrFcatrOn
41.1.61
(6α″rjο 力:SceAppendix B,safety notes ondistillation,aammable
AOAC Off`oiat Method 965.35
solvents,and petroleuHl ether.)
G:ycerides
Place125mLoiland250mLpetroleumetherineachof6separa―
in Monoglyceride Concentrates
tors.Shake contents of flrst 50 wi血
mL Soludon X and,as soon as
Cotumn Chromatographic Method
iayers separate,transferlowerlayertoflaskccntaining250ELH20・
Firet Actlon 1965
Mix,and i― ediately extFaCt this diluted acid solution by passing Final Action 1969
successively throughtwo500nLseparators,eachcontaining 75 mL
A . A P P a r3a , 日
Petr01eum ether.Shake vigorously,let layers separate,and discard
l o w e r a q u e o u s l a y e r . R e p e a t t h i s p r o c e d u r e w i t h e a c h o f o t h e r 勧初確rOgrapれ
ω 5 sep‐ た例肱―ReseFVO士 ,250 mL widl Tenon
a r a t o r s , u s i n g s a l n e p e r o l e u me xettrhaecr,tcoo lrocF‐ S frOm the di‐
stopcockattacheduroughstandardtaper ipdpinnerjointto19/22位
luted acid solutions.Combine dle 2 petroleum ether extracts,wash
colum■ ,19 GO X290 mm,with outer standard taper 19/22joht
with three 25 mL portions H20,and fliter.
top and witl ccarse mtted glass disk and inner standard t
Extract combined petroleun ether solution with two 25L 1■ pOr‐
19722jointatbottom.Bcttomjointconnectstoadaptercon ng of
dons Solution X.Treat each acid extract separately by nixing wih
150 mL H20 and配 ‐ extracttg quicktt by paSSing trough two outer standard taper 19/22 30int comected k・
to Teflo
250 mL separators,cach containing 50 mL Pe位oleum ether.CoHl‐ (Available from LuFeX SCientinc,from PFint No.580241‐ 12.)
unehesepetroleumedlersoluは ons,washacid‐ f ttewith 15mL騨 ― ( b ) M t t n ―P a t t e r sK oe nn ‐
eyTwin shell Blendett orequiV
はOnS H20'and evaporate to dryness on stealnbath.Do not he■ dish formixng adsorbent.は vAlable frottPatterson‐Ke■ey Co.,Div.of
a f t e r r e m o v a l o f s o l v e n l . R e s i d u e m aEyx tcroanctta lD■
& C Y e l l o w Harsco, 100 Burson St, PO Box 458, East Str6udsburg,
No.9 or No.10(fOrlnerly FDaC Yellow No.3 or No.4,resPcc‐PA 18301‐0458,USA.)
Hvely)and pOSSibly trace ofExtract D&C Orange No.4 Kforme的
E Prwaratron Jsrfrca cer
FD&C Orange No.2)if latter dye was oFittndly present in iarge
amounts.Identify color spectrophotomencally as in chapter on .No.S‐679,grade
Place ca 10 g silica gel KFlsher Scientinc C。
color addittves. 923,100-200 mesめ in tared weighing bottle and cap immdiady.
ShakeFlrstseparatorcontainingttlutedollwith25mLSoludonL Weigh to nearest mg and subtracttare wdght.Remove cap and t町
let separate 20 min,and transfer lower layer to flaskning 2 h at 200°
C.Remove from oven,cap immedately,and let cool
contお
30 min atroom temperature.Raise caP momenttly to equ
2 0 0 m L 2 5 % N a O H . M i x , a n d a d d 2 0 0 mCL0 0H12,0a・
nd remove
temal pressure wih atmosphereo Weigh,reheat 5 min at C, 200°
color from tts alkaine s01ution by passing successtte 100 mL Poト
cool,and reweigh.Repeat 5 min dryhg cyde unti1 2 consecudve
ough two 250 mL separators,each containing 75 mL petro‐
tions tど
weights agree within 10 mg.Calculate H20 COntent a
leum ether.Discard ex位 【ted alk:岨ne soluはon.Continue his
Solutton/treamentofollin other5 separators,andttnally combhe
the2petroleumetherexmcts.Washwith血 ●e50 mL portions H20 耐二 s解尋
品
and extract with two 20 mLPortiOns Solution ng tt
layers
leは sepa‐
rate 5航 n.Drain lowOr layers into flask containing 300 mL 25% Adiust H20 COntent ofonginal sllica gei to 5%as fo
NaOH,mix,mdadd300 mL H20・ C001,andremove color from tlis
d la地 g e l x ( 5 )- わ
H 2 0 t O b e a d d e d ig i gn d o 占 】5
alkaline solutton by passing successive 100 mL w胡ons trough
2 separators,each containing 75 mL petroleum ether.Combine pe― Weigh silica gel to be attusted in blender and add calculated
t r o l e u m e t h e r s o l u d o n s , w a s h f r e e f r o m a2l0k,atlria nwsiftehr耳t o a m o u n t H 2 0 t O g l v e i n a l H 200. 1C%O.nBtleenntd olf h5t±
o en
e v a p o r a t i n g d i s h , a n d r e m o v e s o l v e n t o n s t e a l n b a t h . R e ssure
i d u e complete
may H20 diSdbution and store in sealed container.Deter‐
c o n t a i n E ax cばt D & C O m g e N o , 4 a n d D & C G r e e n N o . 6 . R e n o v e mine H20 COntent of attusted silica gel as above,and rea
fomer by dissolving in 3-5 mL PoHionS 60%alcohol and Fllttng necesttly.
each portion trough small paper.Identify color spectrophotometri‐
a PreparatrOn Or Test sampre
cally as in chapter on color additives.
(TO aVOid rearrangement of Partial glycendes,use extreme cau―
Dissolve residue on paperin 2-5 mL portiolls petroleuneher,coト
t i o n i n a p p l y i n g h e a t t o t e s t s a mC P. l) e s . D o n o t h
lecdngiltrateinorittnal evapoFatingdish.Remove solvenlon stealn
°
batl.Blue residueよぅ i駅 D&C Creen No.6,Dissolve residue in(a)nttr sa“ メ2s″″″れど う2わッ 5θ C一 現宮加 ゼ
ates ヶ war″ れg αr
15 mL alccholand 10 mL H20,and add O.5 nL CH3C00H・ Iよ加ti母 く50°Cル rsれθtt Pヶ θ閉あ 加a厳協″切″).
わ`体rヨ
color spectrophotometFiCaly as in c h a p t t r (b)挽o n cSro sa々 r さaれ2"宅
l o pル d d i dα
vあeθッ
sタ50°
. Cttdnd ca 10 g ill mortar
Hnk colorin the various acid extractt u s u a l l y and
i n dPestle.If
i c a t e s necesstty,chili
synhenc samples in solld C02・
dye.However,com oilsomedmes produces faint pinkcolorin these Weigh O,9-1.l g Prepared test PortiOn to l mg in 100 nL beaker.
extracts.This is readily differentiatedfronsynthett colors spectro‐ Add 15 mLCHC13andWarmifnecesstt forCOmplete soludon.Use
p h o t o m e t r i c a l l y . C h l o r o p h y l l m a y a P p e a r a s g r e e n s c u n a t honly
t e r fminimum
ace heat and do not heat y40° C.

◎ 2000 AOAC tNTERNATtONAL

0 にS A N D F A T AOAC OFFtCIAL METHODS OF ANALYSlS(2000)

Chapter 41,p.56

, P r e P a r a f f o n o r C O r t r m a Monoglyceride,%=g monogiycende x 100/g test portion

Assemble chromatographic mbe but witlout nservdr.Do no4
ReFerences:ユ A秘.0″ 9Lθれ Scc.35,325(1958).
grettejdnts.Weigh30 gptpared stta gelinto 150 mLbeaker and ンにXC 48,々 “(1965),
add 50rm mLpetroleumether.Str slowly wihglass rodundi all air
bubbles are expelled.Place powderfunnelintllbe andmnsfer り. sl囲
O p e n s t o P c o C k a n d l e t l i q u i d l e v d d r o p t o c a 2 c m a b o v e s i l t t a g e41.1.62
l.To
mnsfer any silica gd slury remlmg in beakerDinvert beaker over AOAC Offtctal Method 966.18
powder fumel at 45°C and wash into tube witl wash bode,uttng 1‐ Monogけcendes
minimurn amount petroleum edler.闘 nse funnel and sits of mbe: :n Monogtycer:de Concentrates
when solvent Lvel dFOpst0 2 cmabove silica gel,close bottom stop‐
Oxidation Method
cock.Remove Powder mnnel and carettly add smple.Open stoP‐ Firat Actlon 1966
cockandadiuStaowt。 2 mWmin.Rinsebeakerwid15 mLCHC13and Final ActBon 1982
add ttnse to colum wttn level drops to 2 cmお ove silica gel.
(Keep temperature ofwork areabelow bp ofmostvolatile solvent AOCSrAOAC MernoJ
C).Higherに mperature will cause separation
used,ethyl etler(34,6° A . P r f n c r P r e
ofcoluHln Packing and pemit solventto channel.
l‐
Monoglycendes aredeteminedfromH104COnSunedindlecx‐
Neverletcolumnbecomedry ontoP,andmttntaln 2 mL/min flow
idationofadiacenthydroxyi groups.2,Monoglycerides are not oxi‐
Fate mOughOutelution.Ifnecesstt tOintemptelution,dosowhen
dzedby H104・ MChOdis applicable tomonog,ceFide COncentrate
leglyceFide iS passing httughbottomstopccck.Avoidsuch
very li“
contAning≧ 15%1‐ monoglyo胡 dei not appl的品le to samples con‐
interuptions,since solvent above bottom stoPCOCk may cause pres‐
taining CHC13‐S01uble substances withと 2 adiacent hydttxyl
surebulldup and resultinleakage through stopcock or cracks in sil‐
ica gel pacttng.) groups.

i swararron or crycerfreg a Feagentg

(Ca″rわれiSC2Appendix B,safetynctesondistill江拘n,fl― ble (ゆ 乃だ材をacir sθ 山所:θ払― Reagent grade(available tom GFS

solvents,toxic solvents,benzene,and diedlyl ether.) Chemicalslnc.).Testasfollowtt ToO.5rO.6g glycerolin50mLH20,
r e p m Ыt t m d d 鴫
a d d t t n L H 1 0 4 S O l u t i o n t ot mP 距 50mL
Attach reservoir to column,add 200 mL benzene,and collect
oh〉When ali ben‐ H20・Let s的岨 301Bin andtttrate asinD。 ■ terofsolution containing
eluate in tared 250 mL nask(triglycende fracは
zene has been added from sepamor and level in coluIIln drops to glyc卸ol伸ter Ofblank=0,StO.76 rreagent is satisfactoFy.
2 cm above sllica gel,add 200 HL 10陽(v/V)ether in benzene and
collect eluate in second tared250 mL nask(diglyceridefree
Ⅲ fatty
Dissolve 5.4 g H104in l∞ mL H20,add!1900 mL CH3C00H,
stoppered bottle in dark.
and mix dlorougHy.Store in giass‐ (
acid[FFA]fFaCtiOnj.When ali benzene‐ edler solvent has been (b)Srarchれ ガ'c″θ ,wr″ われ _Make h6mogeneous paste of
addedmm separatOr andlevelin cohmndroPs的 2 cmabove siltta l.O g soldble starchwidl H20・ Addto 100 mLboiling H20,Strrap‐
障d tared 250 mL flask,and col"
gel,add 200 mL edler,lhange to d村 乱 Ⅲm a y b e a d d e d a s p r e ‐
1a2c5■ g r l ∞
i d l y , a n d c o o l . S a i c y l i c0 。
lect monoglycedde fraction. servative.Soluttm keeps longer if stored in rettgerator.Cclor of
(Attlition ofehyl etherto coluElnoften creates intemal pressure, 2 HL starch soluは on diluted wid1 100 mL H20 COntttning O,05 mL
resul位 ng in increased flow rate and cracks in sllica gel packing be‐ O・ lM L must be discharged町 0。 05 nL O。lM Na2S203・ Discard if
fore fraction is completely eluted.Avoidby slightly separating FeS‐ end point from blue to cdorless is no longer shu・
二
f l o w i n t o c o l u m (C)働 わ沌力 月比 ReagentorUSP.BlanksonH104S01uは
e r v o i r f r o m c o l u m n f o r c a 3 0 nsg asnodl vleenttは onwitl
狂 same rate tt duateおcdに cted.) and without CHC13 muStCheck wtthn O.5 mL.
To ensure quandtative separation oftacdons,inse dP ofcolumn
a PrepantfOn orSamPres
into receiverwithsame solventusedineluttonjustbefore changing
(Do not subiect Samples to excessive temperatures or
nasks for next eluate. monoglyceride content may be reduced.)
Evaporate collecにd ti―,d‐ Plus FFA,and lnolloglyceride frac‐ rpLttMよ wihout nelti理.
(a)肋 脇 れル 印
tions on stealn bath under sttcam ofN2 0r dry air.Let aasks c001 at
●)肋 riA″ ,れ,α確 力 rPL―Melt at玉 10°C above mp,mix thor‐
roon tempeFatuFe≧ 15mよ n and weigh.Reheat smples on steam
ougtty,and take smple.Do nol俺 stsmples condning so much
bath 5 minunder N2 0r dry ar,Ltc001 15航 n,andreweigh.Repe斑
free glycerol hatit separates on sohdiflcation.
5 Hlin evaporation,cooling, and reweighing unt1 2 consecutive
weights agree within 2 ng. (OS初 筋 ′ ″ 泳 . ―P r O C e e d t t h ●
泳 α″ ′ ).
A n y f r e e f a t t y a c i d p r e s e n t i s e l u t e d w i t l d i g l y c t t d e faIたr a c trermrnatrOn
ion.Tc
d e t e m i n e f r e e f a t t y a c i d c o n t e n t o f w e l g h e d d i g l y c e r i d e , aAccurately
d d 2 5 m L weigh O.3-2gに 並
pOrtiOn caculated mmequ筑 10n:g
warm neutral alcohol and l drop phenolphthalein indicator,and ti‐
=31J/%monoglyceride h smple,dssolvein CHC13,and ttansfer to
trate with O,05M NaOH. 100 mL glass―stoppered volumetric nask.Dllute to volune witl
見 carcura的 何。 stoppered
CHC13 and航 X.Transferentire solutton to 500 mL giass‐
Erlenmeyer(dOnot五 nse andadd 100 mLH20・ StOpper,and shake
FFA(as oleiC),%=mL NaOH x Molarity x28.21g test pOrtion
′

v i g o r o u s lny. 晩
lt航
standuntiltters sepanteandCHCLlaye
′ ｒ ヽ ヽヽ

dearoronly slighdycloudy(1-3め .Ifemulsions fom,causingpoor

、
、

Triglycedde,79=g ttglycende x loo/g test portion

separttonDrepett deに rmin■ lon,using 100 mL CH3COOH(5+95)
Diglycende,%=(g diglyCeide x 100/g testportioo-7o FFA insにad of H20・

◎ 2000 AOAC,NTERNAT10NAL
AOAC OFttC,AL METHODS OF ANALYSIS(2000) OILSAND FAT
Chapter 41,p.57

Pipet 50 mL H104 SOmtiOn htt sttes of tt mL beakers.Add glycerol and HsI06‐ methyl alcohol solution oxidizes
50 mL CHClato 2 and 50 mL H20 tO third as blanks tBc)].Hpet monogiyceride plus glycerol. 1」 nused H5106 iS deterHlined
50 mL CHC13 Sample soludon into fo曲 ,avdding any aqueous iodometrically by reachng with KI and titrating with O.0125M shn_
・
phase,and shake gendン 05°F[32°C],
(S01utiOns mustbe cooledto“ dard arsenite sclution.
ifnecessaFy.)cOVer wih watch glass and leCsmd 30 min. Calculation ofmonoglycende is based on amount ofH5106 COn‐
Add20mL 157o KIsolution,IIllx by gentle shaking,andlet stand sumed in oxidation of monoglyceFide plus glycerol cOrected for
≧l min but≦5 min,away from strong sudight.Add 100 mL H20, amount consumed in ox的 鯨m of free glycerol alone.
Na2S203,Stirring continucusly
andはtrate with standardized O.1〃 Metlod is applicable to fats,olls,shoHenings,monoglycttd
with lnechanica stirer.After L CO10r disappears from aqueous
and blends containing<15%1‐ monoglycendes.
iayer,add 2 mL starch soludon and condnue dmiOn tO disappear‐
ユ Reagen的 卸 どAPPara拘 3
如CeOfbluefromaqueouslayer.Vigorous stirring isnecessary to re‐
mOVe L mm cHc131ayer. (0乃 れ材たactt sracた sθれ炊ル.―Dissolve 12.O g H5106(GFS
Iftiter of sample 0.8
isくdterofblanks,repeat deterEInation,us‐ Chemictts lnc"reagent grade)in 100 mL H20。 StOre atroom ttm‐
ing smaller aliquotofCHC13 on or snallerに
SOluは slsample weを h e, perature in brown」ass‐StOppeFed bOtde.
to assure adequate excess ofH104・ 0乃 湖 たaC″ 筋 労 %肥 rha″ ユ■ Mix 25 mL stock sdution
with 475 nL methyl alcchol.Store at room temperature in brown
―め Xtt X17.927/W
拒as monostearin,%=●
Monoglyce魚 glass,stoppered bottle and Prepare fFeSh eVery 3-5 days.
(C)R伊 わ閉C aciFれりα脅ぇ― Mix 50mL stock solution,(a),Widl
where β=はterofCHC13bla■k,S=dteroftestsmple,M=molanけ 950 mL H20・
o f N a 2 助0 3 ' W = g に s t p O r t i o n i n C H C 1 3 a l i q u c t , a n d 1 7 . 9 2 7 = M W 触 St切 肋 脇 術 θ!″:θ れ.二Dissolve 75 g KI and 50 g
0乃
monosteann/20. NaHC03in H20・
If content is to be calculated to molloglyceride other than
(￨)Sてガ:閉″arsを 円:!2.―
●,0125M.S22939.12 Gee Appendix A).
e r斌既独
m o n o s t e a n , s u b s t i m t e f o r 1 7 . 9 2 7 M W o f o t lぼ m的 Al‐
O SttrCれ tttta。 ぇ― Makehomogeneouspaste ofO.50gpotato
temttvely,detemine MW as followtt Sepme tt acidS as h ttt
starchwith5 nL H20・ Addto200mLboilingH20andboili5min,
2p_hs of勲 肱18C位 241.1.10.A― ly weigh Ca 2 呼 g仙
封厳ingconstantly.Cool andstorein glass‐stopperedbottle.Prepare
acidsinto 250 mL Erle―yer and add20r30 mLhot alcchol neuml‐
飾esh dよly.
佐 d b 価 証 p i n k o f P h n o l P h M c i n . G D A f o n a 3 0 o r 3 A i s 調厳
f a c t o r y . ) A d d 0 5 m L p h e n o l p h M e i n a n d etdiimntleyi ― whiに (D CttθR】ら円".一12 abSCrption should beく
0。l mLO.lM
shakingwlt105MNaOHtopinktttPAIStS30s.Calculaclidnum‐
Na2S20y30mL solvent.
bertmg KOH requiled to neudize fawお 赴 in l g― plej=nL い)ヽ 「‐ D筋 ″rlyrra″幽 材,ル (DM恥 ,-25%solunon in cHc13
NaOH x mol価 虚 y x561/g smple.Avewe MW offatwお ds= ( 9 . ( 6 例 , ″: T h i s m a t e d a l i s t o x i c . A v o i d c o n t a c t w i h s k
ο
5610守 add nunber=舟 + 9 2 ■
生M W I n o n o g l y c e r i d e = o ″ , ) - 1 8 . 0 2 . latiぃ of vaPors.)

D u p l i c a t e d e t e m i n a t i o n s m a d e o n s a n e d a y bOy rrorPrarc l malyst should

n o t d i r e r e 5 % c o n a d e n c e l i m i t s ) b y m O r e t h a n c a O . 5 a, a nPdr el .P 2a%r a t r m r s a n P n g
1‐
Hlonoョ veヽsingle deteminations and
ycttde■40 and 90%に ビ妬 。
Sβ 18C G″
41.1.62).
average ofduplicatedeteminations madein2 dfferentlaboFatOries
ShOuldROtdifferbymorethanca l.2 and3.2%,and l.l and 3.1%,re‐ a DeremrnatrOn
spectvely,at same levels. “務″位加fざββAppendix B,safeけ nOtes on plpets,toxic solvents,
dinedlyl‐
fommide,and chlorofom.)
References:ユ A秘.θJ'C確 加.S征 .34,301(1957).
AOCS Method Cd ll-57 corected 1991). Weigh saH耳〕
leinto 100 mL beaker,calculadng g requil●
d as:
JAOAC 49,816(1966),55,352(1972).
S=紹 ぱ +8o=50/は キ8o‐ 471=2W2
CAS-31566‐31‐1(mOnOstearin)
where n″ =%mOnOglycende expected;Ci%free glycerol ex‐
pected;【 = constant which determines % excess reagent
41.1.63 =
[100 TIK,1-Tl)h Wi=aliquot weight for monoglyceide;晩
AOAC Officia,Method 069.34 aliquct weight for free glycer01; lβ= blank titration for
loMono01yceride3 in Fats, monoglyceride plus glyceroL 32=blank tttation for fre
Shortenings,and Monog:ycerides T l = にs t s a n P l e t i t r a t i o n f o r m o n o g l y c e r i d e p l u s g l y c e
Revised Miner Mtthod test smple htration for free glycerol.
First Action 1969 For 20%excess,【 =70;50%ettcess,【 =50(preferreo,and
Final Actlon 1972 =42.Maxinum S shouldbe 40 g/100 mL or,iFpre,
100%excess,【
A . 印 呂 ferred,10 g can be weighed direcdy into reacton vessel.
Periodic acid oxidizes vicinal hydroxyi groups.Compounds witl (a)MO力 θg,c2,'どタメ″sg,Cを ア οr._Dissolve weighed test
=0,-OH,― NIIR,and― NH2 attaChed to瑚 acentC atOns willbeox‐ portion in 20 mL25%DMF.Use CHC13tO transferquantitat
血 z e d b y H 5 1 0 t t p h o s p h o l i p i d s c a n i n t e r t t r e . S u c h s u b1s0t0a nmcLe sv o al rm e t r i c f l a s k a n d d i l u t e t o v o l u m e . A l l q u c t s c a n b
absent from usutt fat smples. foranalysis ofbothmonoglycerideplusglycerol andfree glycer
Samples are dissolved in CIIC13 60ntaining 57o dilnethyト Pipet 25 mL aliquotinto 500 HlL Erlenlneyer and record smple
fomanide(tO assure soluはon ofglycerol)and aliquots are cxidzed weightin aliquot as yl.Prepare 2 blanks h sinllarnasksby addi
wih excess Hs106・HS10「 H20 SOludon extracts and oxidizes free 5 mL25%DMF and 20 nL CHC13tO eaCh.

⑥ 2000 AOAC tNTERNATtONAL

01LS AND FAT AOAC OttlclAL METHODS OFANttYSS(2000)
Chapter 41,p.58

Hpetexactly 25 mLH5106二 methyl alccholintothe 25 mL aliquct R e f e r e n cユA e革

開. 0 】C 陸恥 最た。31,46R195o.
s 似 Cり ,81“1966j;S■409,602(1969),
a n d i n t o e a c h b l a n k . ( F o F t e S t S m p l e s l o w i n m o n o g l y c e F i d e , S u c h a泌 r
直」y C e n t t f a t O r 0 1 1 , v o l u m elHd5C1O0h6o lmmea的
ybereduced SS,352(1972).
from 25コ lL to 10,5,or even 3 mLi use same anount ofreagentfor
CAS‐31566t31‐
l KInOnosteariゅ
blanks.)Add 2-3 glass beads to each smple and heatjustto bp on
mes duing heating.KDo not heat
hot plate,swining severalは
bla■ks.)Lβ t stand 30 min to cool,protected m sunlight
aЮ or odler 41.1.64
s t r o n g l l l u m i n a t i o n . A d d 2 0 0 m L Hs 2C 0 S to Ol u e‐a c h b l a n AOAC
k a Offic:at
ndに Method 966.19
はon,andswH seveFalは mes.Lettestsolutionstand5mine鉛 5 1ninis G:ycerides in Shortening
障 miSttЫ O・酎anks may be altla町 発di― ediately.Addtt nL組 cmmatographic Memod
solu6on fromgraduate and swirltomix.Letstand l min;thendtrate Fi『
tt Acdon 1966
withsodumarsenite.After12CO10rfadestopaleye1low,add2-3mL Final Action 1380
starch indicaor and condnueは t出 12C010r dsap‐
荘lon until starch…
tNot generally applicabに when emulttners other dlan mono‐ and
pears.Record average dtrationof2blanks as,l andofsample as Tl.
‐
When>l smpleisrun on salne day widlsame reagents,averageは diglrerides are present.)
trationof2 blanks whichcheck withinO.l mLcanbe usedh allcal‐
A ApParatws
culations,If2blanksdonctcheckwidMnO.lmL,thirdblankmustbe
挽2 9 6 5 3 5 A ●α4 1 . 1 . 6 1 ) .
run.欧cessHsI06muStbe20-100%.If100Tttpl― Tl)isく 狗%,降‐
peat witt smller saHIPlet if excess is much>100枠 ,repeat wi血 島 PrcParatrOn Orsfrta cゴ
largersmpleげ u s e L s s H 5m1e0的「 l d C C h o l . T o c o m p l e t e H 5 1 0品
6 ″96SaSB Gec 41.1.61).CheCk each ttw lot of adscrbent for
oxidation, Hlinimum of 20% excess is required. If excess satisfactory separation of glycendes.Collect last 15 mL of each
H5106 methyl alcchol is much>1007う , high apparent eluate h wdghed nask.ェf residue after evaporation is>2 mg,in‐
sult.
monoglycerlde mtty I● crease amount ofeluate until last 15 mL collected contains(2 ng.
OnCe aE10unts of eluates needed for compleに separation of
ω F″ gt燿 減 ― Free g,CerOl nust tt extracted comple悔 呼
8om DMF layer into aqueous layer,If glycerol is not in aqueous
g,Ceridettactionsareestablished,only occasionalcheckingofeach
lot of silica gel is Fequired.
iayer when Hs10「 H20 iS added,or during smding dme,results
my below. a PrepararOn Ormmpre
Melt entire sample in hot H20 atC 50°
to avoid overheating,and
Pipet50mLaliquotofwelghedtestportionfrome)into 500 mL
Erlenmeytt Recodに st POrtiOn wttght in dttuol岱W2・Prepare
2 blanksin similar nasks by adding 10 mL 25%DMF and 40 mL
mix wdl.Weigh 4.9-5,l g prepared shortening to l mg in 100 mL
beaker.Add 10 mLbenzene andwagnifnecessary forcomplete so‐ (
lution.Use caly minimum heat and db■ot heat>50°C.
C H C 1 3 ・
a Pppara怖 孤 加 励
Addca100mLH20tOtestsolutionandblanks.Swirlca l min,re‐
Prepare coluHln as in 96535D(s資 41.1.61)and adiust aow tO
versing direcion several dmes.Pipet exacdy 25 mL H5106 H20
2 mL7min.Beghcollectingeluateinweighed4001nLbeaker.Rinse
intt each nask.Letstand atroom temperature 30min,swirling vlg‐
beaker with 10 mLbenzene and add to column when Lvel dropsto
orous,と 30s■ begilttng and every 5 min duttg this脚的 d.Add
2 cm above ttlica gel.Repeatinttng wih 10 mL pOrtions benzene,
40 mL KIsolutton from graduate,swin,and ld stand l航 n.Titate
using total of40 mL.
w i t h O . 0 1 2 5 M s o d i u m 1 2a r es ne dn i pt Oe i nt to as st a ir nc h c―) , i n ‐
Observe precautions of iast 2 Paragraphs of 96S.35D Gを タ
l宜
c l u d i n g bには調adons.Record test solution dtration as T2 and aVeF‐
4 1 , i 6 1 ) .
a g e o f b l a n k t tA rn ay t ie ox nc e as s > 32 20 ・
% i s t t m i s s i b l e f o r
H510「 H20・ E L P a r a r r o n翻 徳 a r 卸
Attach reservoiF SepaFatOr to colum,add 300 nL benzene,and
E Carcwrarran
collect eluate in weighed 400 mL beakerくtriglyceFide缶抵tioゅ.
Monoglyceride,7)= When ali benzene has beenaddedand levelincolulmdropsto 2 cm
above silica gel,add 250 mL 10%(v/V)etherinbenzene and collect
【乳-1)―tW1/L)幌 ―孔河X4Mx〃 W x100 eluate in second weighed 400 mL beaker(diglyc筑能 fracttonj.
Wlx2 x1000 When all benzeneredler solventhas beenadded andlevelincolumn
dropstoca2cmabovesilicagel,add200mLetheFandC01lecteluate
w h e r e 〃 i s m o l a F i t y O f sほ
側 n d 〃W i s m o l e c u ‐
u n a r s e n i t e s o l u d,oaは de ttactioけ
in third weighed 400 mLbeaker(monOg,ceぶ ObserVe
lar weight ofmonoglyceride. 夕
on ofsecond paragraph,%53SE●
precauは 241.1.61).
Monoglycende content ctt be calculated as monostearin.Using
T o e n s u r e q u a n vt ei t sぷe p a r a t i o n o血職
,よ ,ofcOlum f f r 笹n s e 的
into receiver before changing beakers for next eluate.げeighed
exactly O.0125M sodium arsenite,358 as MW ofmonosteaFin,and
sample weight of free glycerol lqual to twice alnount used for 300 nL Soxhletflasks can be used fbrcollecting fracdons,but2 will
monoglyceFide plus glycerol,fo1lowing fomula can be appliedi be required for triglyceFide duate.)
酌 aporate d‐ ,andmonoglycttde tactions on sに alnbadlun‐
,di‐
Monoglyce由 拒 (as mOnostearin),%= derstreamofcleanN2 0Fdry Ar.晩 tbeakeA coolttroomtemperture
15 min and weigh.Replace beakers on stealn bath 5 min,remove,
[(38-ra)一Q5(亀 ―T2)]XQ895 cool≧15 min,and reweigh.Repeat 5 min evaporation,cooling,and
比 reweighing unti1 2 consecutive weights agree within 2 mg.

◎ 2000 AOAC,NTERNATiONAL
AOAC OFFICiAL METHODS OF ANALYStS(2000) 0 1 L S A N D F A T
Chapter 41,p.59

i carcwrarrOns ユ A p p a r a r w s

Tiglycedde,%=g triglycttde x 100/g smple ( a ) C C S y s , 2.閉

―E q u i p p e d w i t h s p l i tOinn iOerC は
ocno‐
lulnn in‐
Jecはon,oven temperature pttgra― ng,and flame‐ lonizattOn de‐
Diglyceride,7o=g diglycel宝L x 100/g smple tector.Operating conditiOntt split itteCti飢
(split Fati0 1,1041:5o;
drectiniecdOn(splitless,hold for l min),itteCtiOn Port,320°
C(Or
%Monoglyce他 =g monoglycedde x 100/g smple for on‐
column inieCtOn,60°C)i C01um,initial,80°C(or fOr
on‐coluHln,3)°C);program rate,10° 《 y血 ni td temperature,
H i g h t t e f a t t y a c i d c o n t e n t t w i l l g i v ,e s ih ni cg eh r ae ps pu rl o低x l ‐ 360°C,hold 15 mini detec的 ,350° C;cz口trgas flow,5 mL He/min
m a t e l y 2 0 % o f f r e e f a t t y t t i d i s e l u t e d w i t h e a ca hn d o f t h(at e t80°r iC‐
);iniecdOn vOlune,1-5 μ L.o西θ確:For on‐cOlumn inieC‐
monoglyceride fracは ons and 60%witl diglycende fraction.With はon,or dttct inieCtiOn,dilute 50卜 L reaction mixture widl i mL
mos:shortenings,effect is insigniicant,since level offree fatty ac‐
hexane and iniect l μ L.When aPplying on‐ c01umn inieCtOns,a
ids is“ 3.2%.
precolumn my be used to lenghen colum life.On‐ colum iniec‐
N o n h y d r o x yd‐e r i v e d g l y c e r i d e s , w h i c h H l a y b e f o r m e d b y don t h egives
r ‐ nore consistant resPonse Facttrs.)
mal exposure,may interfere.側はs material is found Primarily in
(b)監 C。だJ″ g pθrを″rjθ"g″ ,α,″りrgたor″ぇた れrcgraゎL
diglyceride f岡 じはono Signiflcant alnounts are not present in fresh
(C)CC∽ r四“″._o.25-0。35 mm id x15-25 m glass or ttsed sil‐
shortenings.Wm used fats,digly∝ide ccntent ofthat fraction can
ica,surace fully deacttvated by silylation agent,dinethyisll通 one
be obtained mm hydrOxyi value.
sP‐2100j or phenyinethyldinethylsilicone,10%phenyl(OV‐ 3)
Reference:泌 OAC 49,812(1966). coating(orOtherphase withsimilarpolariけ ),0。 ltO.2卜 mnlmdlick_
n e s s ,t障 c : U S e c O l u m n i e n g d l a s r e q Ou rl r e d
d i g l y c e F i d e S . I n d i v i d u d u n s a t u r a taendd mdoingol‐
yceFideS my
41.1.65 not separate from saturated or less‐ unsaturated mono‐Or
AOAC Off:olal Method 993.18 diglycttdes,■ 直n layer chromatophy on sllica gelimpregnated
MonoH and Di口 :ycerides in Fats and 01:s widl boric acit i― editly prioF tO derivitization,can be used to
Gas ChttmatographBc Method resolve glycerol‐ 2‐ monoesters ton giycerol‐ 1‐ monoesters.)
First Actlon 1333
口りAガ θ,施ric s卯 夕■― Optiod.orθ ttf For automatic sam‐
Fina,Act3on 1998
pltts with 2 mL crimp‐ top vials,double sample and reageni
brnc」 anounts.)
rt/PACヽA00SrAOAC‖
(O SC″ W‐Capッ ねh-2.5mLtor2.OmLcrimpHtop vials forauto
(Applicお にto detenninぶ on of mono‐ a nd diglyceFideS in concen‐ s m p l e ,めw i h T e n o n _ f a c e d s e p t a e
trates and fats and oils.Other emulsiflers and components of fats ―Capable 6f maintaining
(f)″を α!:れg ″●ッ:cc rbr ッ :a′s.―
and oils lgiyCer01,fatty acids,steF01S,etc.】 may be converted to
70± 0.5° C.
t i m e 的1 韻
lyにt h e r d e F i V a t i v e s a n d a n d y z e) d b y t h i s p F O C e d u r e 。
a Feagentg
挽2 Tables 993.18A― C for the results ofthe interlaboratoFy study
e)siゅ :ar:町ag御 な 一〈r)Bisttrimedlyl豆
lyl)trinuorOacet倒
耐 de
supporting acceptatlce ofthe etlod.
l■ (BSTFA).o Trimettlchlorsnane tTMcS),
A P r f n c r p r e (b)ル 勉 :陥 ―‐
Store over KOH.
C)閉 ‐
M o n o ‐a n d d i g l y u i m a r e c o n v e F t e d W i h b i s m 距 船 1 メ) 苗n u O r 0 宮初 a
acetamideい 1■ A ) a n d t i m h y l c H o r s i h e n C s ) h 財 占d t t t O v o l ‐ C ) = 確, 肥r s 胸 滋 た" _ T e t r a d e c a n e , 9 9 % m i n purit
池 輸闘W S i l y l e m r . 断 v 筑
t t v a t i v 岱鴎 a r e s e p a W e d " g a s
(0'確 所距JS腕 沼 SOrrtth_Accurately weigh ca 100 mg
c h r o M o p p h y c o a n d t t e c t e d b y n血 a m.en_T‐ie od ne ic 奴
am ″‐tetradecane,to nearestO。
2 mg,into 10mLvoluHletric naskanddト
i s u s e d a s a n i n t e m a l s m lute to volume with pyridine.

T a b l e 9 9 3 . 1 8 A i n t e r g a b o r a t o r y s t u d y r e s u 3 t s f o r d e t e r m i n a,tyicoenr lodfe 3m o3nnc じ

c2 aamnnodd『 dd ii 口
gtycer3de concentrates
(eXpre38ed ag percent of ma38 0fteSt eampBej
Mean・ 9る SP SR RSDr.% RSD。 .%
碑３
０ １
５ ７

16MyriState 0,04 0.04 9.2

．４
︲

0,05 0.1 5.7

．３
７
１

0。7 ■9 10.9
３
．
９ ８
０ ０

４
３

l‐
Palmitate 27.2 2.4 8.9
．３

23.6 3.3 14.1

５
．
２
１ ３

６
４

４
８

16Stearate 60.1 10,7

．４
０

１
０

6.2 15.8
５
．

釣２ ︲２ 勢
０２ ８２ ８
０ ７
２ ４

０ ６
０ ０

０
０
６

３
４

l,3‐
Dipalmitate‐3‐stearate
．６

４ ７３
４
１

。
．
０ ３
８ ０

０ ０
０ ４
５

０ ３
１ ０

３
︲

Palmitate‐
l‐ 3‐stearate
．６
３
．
１
１

０
３

l,3‐
Distearate

0 2000 AOAC iNTERNATiONAL

01LS AND FAT AOAC OFttCIAL METHODS OF ANALYSIS(20001
Chapter 41,p.60

T a b l e 9 9 3 . 1 8 B l n t e r t a b o r a t o r y s t u d y r e s u i t s t o r d e t e r m i n a t i o n o f m o n o n a ne dX p dr it gt tS ye cd e ra igにe
dぃen3t iOnf 2 f o R l f l e d
ma88 0f eamplo r
Splke・% Mean rec.・
t%) sn RSD"% RSDR,%
1‐
Palmitate 1.00 0.96(96.0) 0,03 0.12 3.3 12.0
1.77 1.72(97.幼 0,03 0,23 4.8 13.4
l‐
Stearate 1.00 0.98(98.0) 0.03 0.14 3.4 13.8
2.85 2.78(97.5) 0,14 0.40 4.9 14.5
l,2‐
Dipaimltate 1,00 0.97(97.り 0,04 0.24 4.0 24.4
2.06 1.98(96.1) 0.06 0.53 2.8 26.9
t,3‐
Dipalmilate 1.00 0.93(98.0) 0.02 0.19 2.5 20,2
2‐Distearate
t・ 1.00 0,97(97.0) 0,06 0。
19 6.2 19.8
0.68 076(11分 0.06 0.20 8.0 26。 2

二 腔脇rFcatrOn Or a n印
wOn,助 g例融随
(9魚 衆″″2 Srattα ras._Glycerol,Palmidc acid,1‐
PaltttOyl
31ycerol, 1‐ stearoyl glycerol, 1,2‐dipalmitoyl glycerol, Analyze reference solution undeF Sane Operating ccnditions as
1,3‐ distearoyi glycerol.畑
dipahitoyl glycerol,1,2‐ 基)9%purity fortestsolution ldentify peaksby compansonofretentiondme widl
仰 u Chek PreP,Inc.,Elysian,MN,USA,is sdttble sou距). known substances(or apply coupled CCrMS)。 s″ Rgure"ユ 18.

lgj Rて力″″確 WrZ`醜 ・ _For each reference stantt accu‐

A CattrrarfOn
rately weigh,to ttarest O.2 mg,ca 100 mg refeFenCe Smdard,o,
and ca 100 mg″‐ tetradecane,(d),int0 10 mL voluEletric flask and ぇ‐
(a)ReSP9附eracrθ二usingrefeFenCeSolutionchromtogmm,
dlute to voluEle withpyndine;。 r welghca 100ng ofmixture con‐ calculate response Facto阜
阜 ,of reference standard vs intemal
胡 n i n g s e v e r a l ( e . g " 5 ) r e f e r e n c e s t a n d a r d s , e a c standard.
h component being
presentin abouts卸障 quantiけ,and 100ng tttetmmane mt0 2 nL
volunetric nask and dluにto volume with P」 戸dne。 随 を:One Cr 氏 = いヽプ鴫 ) X は/ A θ
more reference solutions can alsO be prepared without
閉‐ e,Sllylationofreference duは
tetradeca■ o ns is then caTriedoutas where岳 =resPOnse factorofreference standardヌ;鴫 8=ngintema (
forsample soludon,D(a),ateradditonofinteFnalStandardsoludon standard,sx=mg reference stmdard x:A】 =peak aFeaCfreference
and silylating reagents.] standard】i andAi3=peak aFea OFintemal standard.
a DerermrnatrOn cmk resPonse ttmls periodically.RespOnse facFS ShOuld be
対 .5.LowerrespoN factorsindicate somelossordecowosition,Use
(a)Sa印 ル Sθ ル=ioな― Accurately weigh,to nearest O.2 mg,ca
O.S-10 mymLcomponentsinbotllef― ce and smple solutions.
10 mg homogenized emulsifler concenmm,or ca 50 mg oils and
fats cont宜 減ng emulsiflers,intt vial,B● ).Add O,2 mL BSTFA, JCWrarヵ″ザ sttβた Cattθ″打 ∽確 班 ヨ 3dCulate con‐
0働
Ca)(r),and O.l mLTMCS,C(a)(り ,and dlen o.l nL intemlstan‐ にnt ofSample componentぁ mxl(in ng%)aS f01low蛍
dard solutton,C(0,tO Sample.Cap vial securely and shake vigor‐
o u s l y . H e a t r e a c t i o n m i x t u r e 3 0 m iCnhiena7t0i°
ngdevice,Wihout m'x=(1爪ゆXぶ 、 /mり x(A生′
A佑)
delay,inieCt l-5 11L reaction nixture into CC tpreVbusly equili‐
brated to stablebaseline).C卸 げOutreacdon2、 duplicateiniectiOns where m'x=mg%componentメ inに斑smplα =円 spOnse factor 馬
per FeaCtion. ofcomponentxinsmplα m'“=mg mtemalstandardin smplα m'3
●)呼 RttkをSοれ'ど 加.工BtttO。 10mLFeference solution,C(9,
て =mgtteiAF諄 孤醜OfCompoLntxm― plei andA'“=peak
into vial and add O.2 nL BSttA andO.l mL TMCS.Heat reaction area of internal standard in test samples
m i x t t r e a n d i t t e c t i n t O C UC s ea s ci on n ce e) n・t r a t i o n r a n g eReference:P″
ofreft
″ A,ュ C陸 "・63,1153(1991).
e r e n c e s t a n d a r d s s i m i l a r t o r a n g e o f c o m p o n e n t s t to i fb le e dq ―i n
ユA O A C れ, 7 7 , 6 7 7 ( 1 9 9 4 j .
sample solution.Check line菰 け by p10ttingresPonse factoF VS COn‐
centration ofreference solutions. Rをッ
な宏 March r997

Table 993.18C interaaboratOry study resuits for determination of lyceride3 di。bt3nd dup!tcates fted
monoD and in 8un■
of fo問 OWer
o3,(eXpressed as percent of ma38 0f Samplo

Splke.% Mean rec,。 (%) S, SR RSDp比 _ __

1‐
Patm陥 崎 0.30 0,74(91.9) 0・ 07 0.14 10.0 18.5
1‐
Stearate 1.54 1.44(93.2) 0.24 0。
33 17.0 23,4
l,2‐Dipat輌 4 10:4
nitate
l,3‐Dbaimilate
1,09
1.39
1.02(93.6)
1.36(97.5) 0.07
0.09 0.11
0.12
8。
5。 3 9.2
《
1,2‐
Distearate 2.18 2.38て 110 0,30 0.94 11,3 36,3

◎ 2000 AOAC iNTERNAT10NAL

AOAC OFFICIAと METHODS OF ANALYSlS(2000) OtLS AND FAT
Chapter 41,p.61

里言費けｇ 占岳０●０

Reterは"Sme tmi0

Figure 093.18-Typical chromatograms oftttmethyisilytether derivatives of monぃ and dig:ycerides:A,reference

standardo;B,8nOnO・ and digtycende emutsitter.The sttytation procedure,cotumn epecl■ cation3,Operating oondト
tions,and peak identl■ cation are ae f●:icws:(a)SfryraH研 ._sampte sizo,10 mg,reagents,0.18■ L pyridine containing
l.O ntetradecane,0.2 mL BSTFA,0.l mL TMCSireaction time,30 min at 70。C.(b)COrtrmn.-25 m xO。 31 mm 3d fused
silica:0,17 μm f8:m thickness(5%pheny,methy,oこ !icon,U:tra No.2,HewiettaPacttrd)。 (c)Operarrng● 。nd随 "s「―in‐
iector 320°C;carrter gas,He,5 mWmin,80° C.(d)Peat rrerPrrrra何 。何「→S,matetradecane(interna3 standard),1,olyぃ
orol,2,digtycero:;3,hexadecanolc acld,4,octadecanolc acid;5,口 lycero1 ltetradecancate;6,01ycero:
2‐hexadecanoate,7,gtycerot l‐ hexadecanoatei 8,gtycero1 2‐ ctadecanoate,9,gtycero:1‐ octndecanoate5 10,01yCerol
l口:oosanoate,11,。 lycerol l‐ docosanoates 12,giycero:lctetradecanoate<卜 hexadecanoate5 13,03ycerot
t,2‐dihexadecanoate,14,Olycero!1算 卜dtheXadecanoate,15,gtycerot l‐ hexadecanoater2toctadecanoate,16,giycero:
1‐hexadecanoater3roctadecancate,17,gryoer。 :1,2く対的 decanoate,18,glycerol,1,3rdioctadecanoate;19,
triglycertde C48;20,trigtyceride C50,21,trigty● ●ride C52i and 22,trtg,y● ●『[de C54.

⑥ 2000 AOAC iNTERNAT10NAL

0,LS AND FAT AOAC OFFtCttL METHODS OF ANALYS,S(2000)
Chapter 41,p.62

41.1.66 detected by differendalactometry.Quandtati。

re■ ■is by area nor‐
AOAC Offtciat Method 993.24 HYdization.
Trigtycerides(by Partiticn Number3) `イ
酎uはcn order is礎 節 航 礎 d by cdculating equivdent carbon
in Vegetable Ot,s
numbers,ECN,often deaned as cN-2ら where CN is carbonnum‐
Ltqu3d Chromatognphic Method
ber and■ is number of double bonds.To calculate ECN more pre‐
First Actlon 1993
cisely,origin oftle double bond is taken intO account.
Final ActBon 1998

′
JPA● ツ10CStAOAC‖ 修mod ECN=CN― a辞 。一Jrar-4ぼ ,ぁ

( A p p l i c a b l e t o d e tneamt遺
i o n o f d g l y c e d d e s o f l ocnhga―
i n t t t y a c ‐ whereち ,,!,and″h are number of double bonds of Oleic,linoleic,
kh in vegetable oils.) and linolenic acids,respective町
,and cOefrlcien低 毛,4,andtt are
calculated mm reference tFiglycerides.Under condidons in dlis
Stt Table 993泌 for tt results ofthe interlaboratory study sup‐ medlot賢 N approxlmate雛
ethod.
porting acceptance ofthe l■ ECN=CN― (2.30x″。
)―(2.35x,′
)―(2.17x″h)
A P r r n c r P r e a A p P a r a t w s

Triglycerides in vegetablepils are scParated according to equlva‐ ( a ) ' ?材統" “ ο t t r ? g rた L C p w s―

a Pr れ t eE m4 ・
uiwed widl dle r‐
lent carbon numbtt by ttvげ seいphase Lquid chromatography md m o s t a t i c c o n t r o l o f c o l u n n t e m p e rLaitnuireec,d1O0nμ
unitidiffeゃ

Tabie 993.24 3ntenabOratory s tf uo dr y d re et se ur lm ほ

inatlon ot t r i g l y w i d oe 3s 1 8i ,n b av ge eg de t oa nb l t●t s u t t e trom 2 3tudlee,1
and 18 1aboratorle8
Tast samnto ECNe40 ECN‐ 42 ECNe44 ECN346 ECN‐ 48
Sowboan o‖

０
７
８
２

Mean,0/。 25.5 31.0 21.6 i5

７
３
３
４

RSDr 1.8 1.3 3.5 ９

５
14.5
６
７

RSDR 3.5 3.9 4.3 71.7

２
２
０
８

r 1.3 1 . 1 2.1 0.6

２
９

R l_6 2.6 3.4 2.7 3.1

Almond oll
的
４ ６ ７ ３ ９

４
４
Mean,°/。 0,1 2.2 13,0 30.7
１ ２ ２ ３

４
５
RSDr ―- 18.8 4.4 1.7
９
RSDR - 27.8 6.9 2.3 １
０
７

r - 1.2 1.6 1,6

１
１

R ― - 1ハ 8 2.6 2.0
Suttower oit
Mean,% 0.3 29.4 33,4 22.5 3.2 1.3
RSDr 21.4 ■8 1.2 1.6 10,7 37.0
RSDR 70.4 3.2 3.4 2.4 11.7 92.6
r O.5 1.5 1.3 1.0 2.5 1.4
R n6 27 a_7 1_6 2_7 34
01ive o出
９
０
６

７
２

Maan,% 21,2 64,7

︲
６
２

１
５

３
６

RSDr 1.3 0.8

０
２
︲
１

８
５

RSDR ■6 1.6
３
。
３

０
７

r 0 . 8 1.5
。
３

０
５

１
７

R ■0 2.9
Rn6ぶ Oad O‖
３ ３
０・２・８・１・４・
９
３

９
３

２
１

Mean,°/6 28.7 31.2

︲ 範
１
８

０
７
５
３

RSDr 1.7 ■7
８
５

２
３

２
３

RSDR 2.4 2.5

１ ３
０

０
５
５

r 1.4 1.5
０

０
６

１
８
７

R 2ぃ
0 2.2
Palm oit
餌
７ ２ ３ ３ ２

９
４

Mean,°/。 3.3 22.1

１ ２ ２ ４

６
９

RSDr 8.1 2.3

９
６

RSDR 16.2 2.8

２
５

r 0.8 1.4
２
５

R lS 18

◎ 2000 AOAC!NTERNAT10NAL
AOAC OFFICIAL MFMODS OF ANALYSiS(2000) OILS AND FAT
Chapter 41,p.63

Figure 993.24A― Typlcat LC chromatogram for soybean o13 aS reference sample fortdentittcatton of trigty●
●rides:
0,oletc acid,P,pal,■■,c acid;L:inoLic aCidi St eteartc actd;Ln,tino:enic ndd.Thu3 000 represents trSote:n.

e n t a l r e f r a c t o m e t e Ls cwailteh sfeunlsi,i‐
at tv i lけe a s tu dlt(O声
f
refracは ve index,and recorder andrOr integratoL

(D LCC。 れ閉限-250x4.5 mln id stainless steel,packed witl

5‐μm―dalneter silica, FeaCted with ocladecylsilane to obtain
22-23%carbonloading。 (LichrosorbRP18A■ 50333,LichrosPhere
100CH18 A■ 50377,E.Merck,DaHnstadt,Cermany,are suitable.)

a Feagents

a)sary御 な.一Chlorofolll,acetone,andacetonitrilαLC grade.

(b)'所 餌 Jθ′ ,鶴,― Acetonitrile― acettnei begin with 50+

50 mixture andadiuStprOwilonSt00btAndesiredsepmiOn.Elu‐
tionsolvents maybedegassedandrecycledseveraltimes wihoutef‐
fect on sepmtions.

(C)肋 ′ ″う'′
協 rjθ ″ Jθルタ″,一 Acetone or acettnerchioroform
航 xture(1+1,V/V).

(d)Rチ ″″C2'宅 般 r:』 夕S・一 Use commercial ttigiyceddes

(tripalmiは ■,triolein,etc.),plottingretentiondmes vsequivalentcar‐
bon number:or,altematively,prepare reference chromatogram
fromsoybeanoilG″ ngure993.%A).肘 θ確:Withseveral reference
d g t t C e F i d e S O , r e s O l uc ta in o nb (e のc a c u l a t e d w i t l r e s p e c t to
triolein:

=端
的 Figure
Nunber ot Oouble,3ng・

993.24Bttraph of,og =KO

α
tn'

Where α tS FeSCe
lution of reference trig[ycertde with r●
spect to trlotein
usingreducedretendonhneR4・ =RTr― 即 的ivent・DeterIIllne reten‐ (α /P■
l=d、 市。leh)and,ls number of doubte bondein
はo n v a l u e s f o r a l l t t g l y c e r i d e s o f f a t t y t t i d s c o n t a i n tFigtycerid●
e d i n r e f e r.Graph
e n c e determines retention va3ues for a‖
岨glycettdes(sを夕rFlgure 993洲 )by graphing logはvs″ Knumber trigtycerides of fatty acids contained ln reference trt‐
of double bonds). 91ycerides.

⑥ 2000 AOAC tNTERNATtONAL

01LSAND FAT AOAC OF同 OAL METHODS OF ANALYSs(2000)

Chapter 41,p.34

a sampre PrwaratrOn Table 993.25 interiaboratory study resultre for deten■

ination
A c c u r a t e l y w e i0g.h0O1. 5g± t e s t p o H i o n t t n e a r e s t O . 0 0 1 gof ipotymeri2ed tiglycerides in vegetabee otis
10 mL graduated aask and dilute tt volume with solubilization sola
nto
and fat3,oel per― tion‖qurd r
chromatographic method
vent,C(c).
Sample
島 DetemrnatrOn No.a Mean,% Sr SR RSD"% RSDR,%
Pump ehtton solvent,C(め le baseline is
,at l.5 mWmin undlstお 3.6 0.1 0.5 3.3 12.5
obtained. 2 5.2 0.1 0.3 2.3 6.7
町 e C t 1 0 1Oれ
f s m p l e s o lo岨
n,D.
3 9.7 0。 3 0.4 3.2 4.6
見 carcwrarran 4 10.O o.2 1.2 2.2 12.2
on C to identify triglyceidest Calculate 5tri‐
Use graph from secは 22.0 0.2 1.5 0.8 6.8
glyCeFideSby areano―lization,1.e"assume dlatsumofPeak are容 8 1are伯
●限mtte問
封倒輸肝柵瑞掲 h
coHesponding to vanous triglyceFideS equals 100%.Calculate rela― 騨樹海予
辮 肥
l o w p 0 1 y m e r c o n t e n twin O3 ョ
diW
叩 ng。1!With an average polymer content and presenung R,301u付 On d“■‐
はve percentage of each tnglycende t0 1 decimal place,using for‐culties between rnonomers and p。 1即 付zed tHgiン
oondesi and 5=ex‐
mula: hausted frying oli greaty damged.

徒% = 面
T F i J y c e F i沖 Xl∞
詳 舘 漁 杭 悪
0助 recめ e n 車―
, 二 R t t a c t i v e i n d e x d e t e c t o r wmmu mm is市
References,酪 .F,6θ ?s.C,岱 卸 ,279(1983). 山vity atfuusscale,≧
1.10x lド re施 血 n index units;capableofbe‐
P″″ とAPP'C力 ι秘.631173(1991). ing maintained■ a few degrees above ambientに mperame(lf
detector is not equipped with heater, use recirculating, con‐
ユAOACれ ,77,954(199o,
stanいtemperature waterbathj.
Rタッis2ど
f MarcLゴ997
(O Reccrたぇぺ uitable to disPlay and tify
q― peak areas,re‐
SPOnSe dme,1.5s oO%reSPOnse with 100%signa
41.1.67 0.1-10嗣 mini sensitivity,1-100 mV;accepts signal output from
AOAC Ottlcial Method 993.25 detector(d).
Potymertzed Tr`gtyceHdes in Oi:s and Fat8 (O carc12脇″ あ 筋θ ″商eg″ルぇ
Gel‐Permeation Llqu:d Chromatographic Method 0降 腕 ‐防rra加 抑 虚 -250 mL,widl heating mnde capable
Ftrat Actlon 1003 ofmaintaining nask cOntents at 180±
2°C.
Final Action 1996
(h)用 !負 打付.一‐l μm pore size,medium porosity,p01ytetra‐
n u O r O e t t l eぼ
距e a O n T F E j o r c e m u l o s e re(sctoeHrl mnelrに
c i d
ryPA朗 OCStACAC‖ b輸口ど
LC dsposable wnnge mter units aFe Suitablo.
い ppliCabletodeは航 nationof濁 %EW/W]p01ymerizedtrigttC胡 隆 S a Feageara
in vegetable fats and oiほ
:heatea notheated,and used for frying.)
(a)〃 θうfrβ″れαs2.―Tetrahydrofuran(perOXide‐ free,
Ca″ Jο
″:Tettdrofumn and toluene are higmy na_able and nOnsmilizedj.

toxic by ingesdon and inhalation.Use h a fuHle hood ( め S t o r a g c w r r.r二

筋TOluene.
at all dmes.
(Oθ 】Sranra,4_モIeat ca 185 mL reined soybean ollin aask,
BQめ,t0180±2° Cuntilpolymerizedtriglycendelevelis 10-15%,as
挽βTable 993:おfor dle results ofthe interlaboratory sttdy sup‐
analyzed in F.
POrting acceptance oftle method.
A P r r n c r p r e
(0鋭 脇 S″ t角絃― Attdrous.
, P p p a r a t r o n O r c P とc s y S R 封"
01l andfats aredissolvedin tetrahydrofurani polymenzedtriglyc‐
erides in smples are separated on basis of molecular size byTo condidon CPLC colum,change solvent stepwise from tolu―
gel―
Pemeatlon liquid chromatography(GPLC)and quantifled by e n e , C ● ) ( s o l V e n t B ) , t o t e t a h y d r o f u r a n ,く
Cs(Oつ
IVent A).PuHlp
retactive index detecは
cn, 1 0 m L p o r t i o n s o f s o l v e n t s a t l S m i n a s fs oにl,l1o,w2ま 5%A in
B i s t e p 2 ,脇5 〔A i n B ; s t e p 3 , 7 5 % A i n B : s t e p 4 , 1 0 0 % A . C o n t i n u c
ユ APParartrs
t o p u m p t e t r a h y d r o f u r a n 1f2o rh≧t o s t a b i l i z e c o l u H l n .
(う CPLC Sy並閉.―〈r)s。 ル2江ァ 2s研的:L―Equipped with mo‐
Connect colum to iniecttOn valve and wash widl ca 30 mL
bile‐ r
phase Hne nlに tpOre size l向
.2)LC PZ″ ― hiSdess,
にt r a h y d r o f u r a n . C o n n e c t c o l u H m t o d e t e c t o r s m p l e c d l . F n i
with aow o.7-1.5正
直ノmin.ぐめItted海″″ rr.二 With iow dead vol‐
ence cell with tetrahydrofun.Adiust f10W to O.8-l mminan
ume,equiplttd with 10L μ
sample looP.
wait for baseline stablization(typiCally ca 15 min),
(b)妙 ri4ga-50-100 μL comp■ ible widl iniectOr.
oわ 縁 Reverse sにpwise wash ofcolum for sttrage h toluene。
)
(C)CPLC御 ル閉み―‐ 7.8 mmidx300 mm,stainless steel,packed
with spheical pOlystyrene―divinylbenzene dcroporous paFtiCles, is/gren s“ 佑宮朗町
ca 5 μm paticle dameter,100A POre siZe(PL Gel,Phenomenex, Analyze oil standard as じ in F,打detector sensitvity to
usdng
To「 ance,CA,or Polymer LAttxatts,Ltd,Stretton,Shropshire, achieve chromatograln with mononeic ttglycende peak tpea
UK,is suitable)。さs″ :StOre coluttl in toluene.) m o r e t h a n h a l f c h a r t w iFditgtu rGe″9 9 3 . 2 5 ) .

◎ 2000 AOAC tNTERNAT10NAL

 AOAC OFF,CIAL METHODS OF ANALYS,S(2000)
01LS AND FAT
Chapter 41,p.65

s u s p e n d e d 脚血 c l e s , a v o i d b 1 0 c k a g e o f p o r o u s f r i t t e d t t i t e r a e c o l u I I l n
top by flltering fat trough l ltm pore size fliten

h器 差 配 托 継 !管 撚 縛 堀 苫 培 比 沼 泌 総 諮 .粘 惜 寸 紺
a °W r a t e ,5は
■ 耐 前 nぃ 1耐 航 、 如aytts
艦 鮒 檻 ギ

C,Resurts

Chromatogrm(s22 ngure 993.25)shows main peak(mOnomenc

航 glyceides,MW ca900)and 1 0rmOre smaler peaks with shOier

鞘艦盤謎
萎 蘭 古 毬
を鯛
比 car●urarrOn

Use areanormalittdon to calculate percentage ofpolymeizedtrit

g l y c e n d e s , a s s u t t n g t h a t a l i s a m P l e c O m p O n e n t sd .a r e e l u に

Retention distance(dR) 騨 襴
a s f o l l o w s :
Figure 093.2rTypicai gel permea付 on ilquid
c h r o m a t o g r a m o f o i : g t t n d a r d t h e a にd ●。y b e a n o i りf O r =tAtt xloo
Iy「
calcutaJon3 0f reSolu付 on and theoretSca:pla敵 ,numbeL
l and 2,po:ymerlzed宜 高giycerSdesi 3,trigry● 。百desi 4, WheFeAPr=ulmof-OfPOlwind宜
出 摩 魚 魅 郎 ; コ ヽ= S l l m o f
f R 靖 伯町 a c i d G . 狐 述 of測 瞬 速 にR 町 碇 F 「 的 1 色 輸 d 出 現 .

F o r c a l c u l唯述A P r 9 2 c a s e s a r e p o(srs)iGboloα
d resolution be‐

Use chromatogrm to check efFlciency and resoludon Power of 樹縄協脳盟駄投騨協毬猟::l翌

columm.Perfomance should be atleast as good as shOwn in Fig―
ure"船 .
C』 culate theoretical Piale number,″,and resolution,Pで,as
認塩!撒離艦期縄撒冊紹撤 F絡
gratiOn, attust integrator carefully (baCkWard hOriz
follows:
integrate di surfaces incl
″=ゴ 5 1trr″]2 艦 r)的

縛翻艦麟鑑縦鞘翻 艦
R=[」 W]

where』R=retentlon dstance from stat Of chroHlatograln to peak

mttmum fortriglycerides,in mlni w=width oftiglyceddes peak polymehzed triglycerides as fO110ws:
atbaseliコ峰,measuredbetween tangents andbaseline,in mmiand△ =
distance between peak inaxim oftriglyco点 des peak and neighbOr
P T = “ 仰欧 ] X 1 0 0
peak of polymerized triglycendes(Peaks 3 and 2,respectivdy,in
whereA缶 =areaabOvebaseline measured fromstartuntilretention
Figure 993.25),in mm.
C h o o s e a n a l y s i s c o nodnisはs u c h t h aits″≧6 0 0 0 a n d R i 1s .≧

え Deね 何協旧rfOn
鞘 盟t晋曲齢船罷 輔摘艦:温就!
lffatis not completely melted at ambient temperature,heat until
no more dlan lo° c above its melting temperature and mix thOr‐
ougtty.
Accllately weigh O.2± 0.01 g prepared test portiOn into cOnical References:P″
″α ″
ホぷ薦鑑
どれqβ
憚
i Cれ を加.63,1163(1991).
flask.Add 15 mLpunttedtetrahydrofuran,which has been collecに d IUPAC Standard Methods for Analysis Of oils,Fats
after last peaks have eluted from column dttng a chromatOgraphic and Dettvatives,7th Ed.,Blackwell Scientirlc
run.Swid aask,and leave wttl fatis cOmpletely diss01ved.Add ca Publicadons,1987,Method 2.508,
50 mg soduln sulfate,C(d),tO dssolved fat,shake,and wait ca ユ Aθ ACn,r.77,957(1994).
2 min.Filにrttmugh l μ m pOre size f11に
r,B(h)。When fat contains Rタッな?″,March F997

◎ 2000 AOAC tNTERNAT30NAと

01LS AND FAT AOAC OFF,C:AL METHODS OF ANALYSiS(2002)

Chapter 41,p.66

41.1.68
AOAC Omcial Method 927.08★ に e.
st sam】 r
,dentifBcatlon of Fiax30ed O11 a . A P P a n t t r s
on
03i tt Refrac付 rc,:rttr′″泌 αr″crθ
″.―Producing a pressure of
(a)S呼 夕
Ftrst Ac宙on 1927 .
51.7 kPa and a tempedure of 100° C;equipped widl a heated
Final Ac宙on
Surptus 1965 restrictor(adiuStable or ttxed aow)kecPing the tempertture of he
evolving C02 abOVe40° C.Forextractions withC02+15%ethanol,
Sを226.111-2&115,10th配 . use an on‐line modifler pump.Low oll values ctt result from leaks
in the SFE insmnent Or inefflcient collection of dle extracted oil.
B e f o r e i n i t i a l u s e o f h e e x m c t O r a n d p e r i o d
41.1.69 tract I●
plicate 400 mg test portions of soybean oll on granular
AOAC Omcial Method 999.02 diatomceous etth using he desired set of conditions and drying
01:in O,lseeds procedure to ttfy eqttpment condition,collectbn ettciency,and
Superc付宙cal F3uld Extractlon tSFE)MethOd PrOCedure.Recovery ofthP od shOuld be>99ワ みand no oll aerosol
First Act3on 1999 should be observed rising above tle glass wool dwing ext
な「も-10 mL intemal volume.
(め mracrゎれα′
AOCS―AOAC tte命oJ
を 一S u i t a b l e f o r e x t a c m n s y S ―
O c r a s s a r r a c r 切 ! ″c r f t t w s sな.
s o y b e a n s , c o t t o n s e e dtem.Vessels
a t i o n o f o i l nctoonfに , mustbe capable ofholding≧ 1.O g 10osely packed glass
( A p p l i c a b l e t or E dI en に
canola seed,saFaower sect and Sunacwer胡 .) wool.
江 院 比閉竹・ ― A c c u r a tにo 主0,0001g.
( d ) A ″け, た
ca″r:θ ″: SFE uses compressed gases as an extraction fluid.S22 鶴 a白″″幽4P.― Maintaning d.rying conditions as
(e)｀物C″四"θ け
Appendx B,Laboratory Safety,for safe handling and outlined in 9拓12ぃ o241.1.02)with pettssble oven temperature
storage of conPressed gas cylinders.SFE equipment
20-25°Cお ove bP of H20 at WOrking pressure≦ 100 Hlm Hg
must be consmcted and mlntalned in proPer working
(13.3 kPa:72-77° ± 1° C at 100 mm Hg working pressurei
order to minimize dle chance ofany exPlosiVe decoma
5 8 - 6 3と1
°°C at 50 Hlm Hg working pressure).
pression ofextraction auid.Although C021S a nOn‐
toxic gas,adequate ventilation in he lttratory must o乃 鳳 み αル θ″″.―Operating at i30± 1° C.Use if vacuun
of diSPlaCing too
be maintained to avoid dle possibiliけ oven is not availablet
s o be man‐
much oxygen.Adequate ventilation nust』 a脱
胡 配d when using modiners tO avOid dle toxic effects (
脇 を競 ._welding研 曲 sdd grade宙 血 dP髄 。
ofbrea皿 ng organic vapors.
Pwr grades of C02 may be used,but are not requlred.
'`あSθ
●)Er施脇伊 r″確._IIPE grade or equivalenti moisture
挽2 Tables現".tD2A―C Forthe resultt ofthe intedaboratory smdy
血 contentく1コ唱A嘔.Denatllring solvent type is unimportant.
supporting dle acceptance of dle碇
一
OD脇 `"Cθ 四 “″L gra″ ″ねえ HydromatrixTM(Smple
A.財 ,仰 re
Oil is extracted from prepared oilseed witl a supercntical fluid

collectお
ned in a
(1lquid C02)and deposited direcdy onto giass wool contお
n apparams.ARerevaporationofttsture andany residua SI.Jose,L M149085,USA),or
馴 胡二 1撒網 淵 equivdent,are suit
on
modifler fromglass wool and Collecは aPPmtus,the weightofex‐ (d)orass wθ ′
.―Borosllicate giass.

Table 999.02A intertaboratory study resultts(17 1aboratories)ofAOCS SFE Method Am 3a96 u33ng Co2a:One for deteHminaば on of oi3
content of o:iseeds

Sttdy samHe dにα対° Labs accepted No.of ou付 ねrs x 革 「 SR R RSD角 % RSDR,%
A/M(Soy 4) 14 3 19.2 0.30 0.33 0,45 1.27 16 2.4

E/1(Soy 7) 14 3 19.2 0、 90 2.51 0.91 2.55 4.7 4.8

G/R(Sun l) 15 2 38.9 1,02 2.86 1.68 4.70 26 4.3

C7P(Sun 3) 15 2 41.6 1.66 4.62 2.30 6.45 4.0 5。 5

K/Q(Can 4) 15 2 39.8 0,45 1.26 0,90 2.51 1.1 2.3

D/J(Can 3) 15 2 37.7 0.67 1.88 1.03 2.89 18 2.7

Brs(saf 2) 14 3 36.6 1.39 2.25 1.66 4.65 3.8 46

H州 (Saf l) 14 3 357 0.86 240 1,08 303 2.4 3.0

F汗 (Cot l) 14 3 19。 1 0.46 1.28 0.51 144 2.4 2.7

L70(Cot 2) 14 3 18.4 0.34 0,95 0,38 1.06 1.9 2.1

8 0‖ seeds from 1995-1996 AOCS Smalley LabOratory Prondency Program Soy,sOybeani Sun,sunacweri Cant can。 lai Sari samOwe,cOt.cottonseed

◎ 2002 AOAC tNTERNAT,ONAL

 AOAC OFFiCIAL METHODS OFANALYSiS(2002)
C)!LS AND FAT
Chapter 41,p.67

Table 999.o2B interlaboratory study results t15 1aboratories)ofAOCS SFE Method Am 3‐ 96 using Co2 Wlth EtOH modmerfOr
deteHninatlon of oit content of ol130ed3

Study samp崎 o‖se筑 3 Labs accepted No`of outl● 時 、 返 %RSD酌 %

sr r SR R RSD“
A/M(Soy 4) 12 3 20.5 0.43 1.19 o.77 2.16 2.1 3.8
E′1(Soy 7) 13 2 20.4 0.58 1.63 o.87 2.43 2.9 43
G′R(Sun l) 12 3 39。 7 044 1.22 1.65 4.63 1.1 42
C/P(Sun 3) 12 3 430 0。
63 1.78 1.64 4.59 1.5 3.8
K/Q(Can 4) 12 3 43.5
o,37 1.04 1.32 3.68 0.9 3.0
D/J(Can 3) 14 1 40.2 0.86 2.42 1.43 4.00 2.2 3.6
Brs(saf 2) 12 3 38.0 0,77 2.15 1.14 3.13 2.0 3.0
H7N(Saf l) 12 3 36.7 0.67 1.86 1.48 4.15 1.8 4.0
F灯 (Cot l) 12 3 197 0.18 0.50 0.52 1.46 0.9 2.7
L70 1 19 0,35 2`7 146 1
・
Oilseeds from 1995-1996 AOCS Sma‖
ey LabOratory Prondency Program_Soy,soybeani Sun.sunnowe,can,canola:sat samowe,cOt cOttonseed.

a Deremrna節 。"
Take≧l g glass w001,C(d),and Pack into each collection vessel
(a)Ce‐ θ"り ar確 ごrj例 !θs,"ガ αtt P!α″rp″ cをss clrrac_ B(C),unditop ofresuldng plug is atleast 。 2m.O5fcvme sf―r o m b o 性
肋 ■,―Prepareに st皿 叩 le as desttbed in section A ofthe AOCS sel. Accurately weigh collection vessel with glass wool tO
の C朋 材統 拡 sa財 絶 cθ閉由 dP″ 胡 αS,5血 醐 .,Ameican 却 .0001g.o,θ姥:CaPs or septaon collection vessel are notrequired
Oil Chemists'Socieけ ,CttHwttgn,L,USA,using he IIlethod sPe― unless dle SFE hsmment requitts tler use.)
ciflc for oilseed being malyコ
働エ
InstalleachcollectiOnvessel onthecollecは
onreel ofthe extractor
Accurately weigh 2± 0.0001gに st pOrtion into extraction cell, (autOmaはc systero or inStall each collection vessel with he
B(b),Wid10utletendcaPinPlaCe.TaPcellSeveraldmes on work sur― restrictor or transFer mbe leading tOm the restFiCtOr inserted as
face to pack material htc an evenly Packed plug in Oudetenddeeply ofthe as PoSSお Lin的 悔 giass wool plug(manualloading syst鉱
ゅ.
c e l l . U p p e r s u r f a c e o f P l u g s h o u l d b e e v e n . n l l r e mSolneinsmments
t t n i n g v o i d i n may requlFe ttuSment Ofhe restricto
c e l l w i t h g r a n u l a r d a t o m a c e o u s, Ce(抽
c ) . W i P e a n y s P l l l e d n i l e raddiはonal glass w001 tO ensure deep posidOrung of the restrictor or
間は画 al from outside of extraction cell. transfer tube wihin the glass w001.

Table 999802C Cornpar3son of methOds:sFE versu3 80臣 ent exhction

Oiiseed°
Ψ 汚 u g 1and
WS〔 method
8u 81に Wluu x sr : sR RSDP,D/c RSDR,%

4
SFEoC02 a10ne 19.2 0.30 0083 0.45 1.6 2.4
SFEtC02 Ⅲ 15%ethand 20.5 0.43 1.19 0,77 2.1 3.8
AOCS Ac3‐ 44め 19.0 0,03 0.36 0。
2 1

7
SFE‐C02a10ne 19。
2 0.90 2.51 0.91 4,7 4.8
SFE‐C02・ 15%ethanol 20.4 0.58 1.63 0.87 2.9 4.3
1 0.05 0.3 2.0
Sunlower seed l
SFE‐C02a10ne 38.9 1.02 2.86 1.68 2.6 4.3
SFE‐
C 0 2 Ⅲ1 5 % e t h a n o l 397 0.44 1.22 1.65 ■2 4.2
AOCS Ai3‐ 75う 38.5 0.10 0.28 1.24 0.3 3.2
AOCS Am 2‐ 93・ 41.8

seed 3
SFE‐C02a10ne 416 1.65 4.62 230 4.0 55
S F E C‐0 2 Ⅲ1 5 % e t h a n o l 43.0 0.63 1.78 1.64 15 3.8
AOAC A,3t75め 42.7 0.09 0.25 0.50 0.2 12
AOCS Am
a Olseeds ttm 199st1996 AOCS Sma‖
ey Laboratory Prottdency Program.
' Soivent extractbn,But tube extradbn method,AOCS Ottdtt Methods approwate tO。
lSeed.Srrla‖
ey Laboratory Prondency Program resute rOr 1 995r1 996.
C Soivent otaclion,AOCS O師
間al Method Am 2‐
931 FOSFA intemattonal Hlethoc.

0 2002 AOAC tNTERNATtONAL

0にS AND FAT AOAC OFFICtAL METHODS OFANALYSiS(2002)
Chapに,41,p68

had each extraction cell on the instrument reel(automated sys‐ (1982),Vol,3(ediにd by Applewttte)(1985):6θ rrα
町を2″α冴
tem),Or install each extraction cell onto the extracttr(manua1load‐6θrr9酒β″ Prttcrs,Interscience Publishers,Inc.,New York,
i n g s y s t e m ) ,e0nは t i o n i s i n d l e o u d e t eNY(1948).
t t h e c e l l s s os tt hpaotrに nd。
欧 r a c t f o r 3 0 価n w i t h C 0 2 u n d e r t h e f o l l o w i n g c o n d i t i oBOEKENOOGEN,A″
nH rysおα材 伽 把c姥挽″ われ ゲ θjh施 な,α材
Cino航nalliquid aow rate,3.4ぎ
51,7 kPa;100° ミn(2・
6r3.4g/min Far P陶 ど″cぉ,Vol.1(1964),Vol.2(1968),Joh Wileyも しSons,
is pemissible):and resthctor temperature such chat C02 eVOIVes at Inc.,New York,NY.
B R t t S H S T A N I D A R D S I N S T R t夕
t施I OおN ,ザ材A ぇa r y s ザ
な O f なα材
between 80-100° C(40-130° C is acceptable).The temperature of
Fαな,British Standard 684,London,UK(1950).
the exPandng C02 in the collecdon vessel will direcuy affect tle
CHAPMAN,m2説 rttcr″ ″ ヴ ,レ fガ
S,JOhn Wiley&Sons,Inc.,New
alnount of moisture and7or modifler entralned in he extracted oil
York,NY(1965).
and the subsequenttime required to remove it before the weight can
CHRISTE,A益 邸 c奮 れ 白レガ 材2れ。所θ!θ =メー θ″2,■ に Oily Press,
be detemined.Overheating can cause loss ofthe more voladle olls.
Ayrp SCodand(1992).
Consuit instrtlment manufacturer fbr resttctor/collection tempera‐
CttsTB Cas勧 畑 昭 rο araPり α′ゼ とtiPjな,The Oily Press,Ayr,
ture scは ngs that will価 n imize rloisture(and mOdineL ifuseo re‐
Scodand(1989).
movallimc.A lowerexpanding C02temperamre may be used widl
CHRISTIB,HigれP"筋 α"確 とj?″芝 勧 死加 αrograpり α材 ,,どな ,
insmments thatincludc heating ofthe collection vessels.Somein―
Pergalnon Press,New York,NY(1987).
smments may notrequire subsequentremoval ofmoisl町 9or mod‐ CHRIST配 ,L"″ A″αい な,2nd Ed.,Pergalnon Press,New York,NY
ner from the exract,Vedfy by drying the extracts according to
(1982).
926.12G2241.1,02)to enSure that no signiflcant weightloss is ob‐ C O C K S & V A N R E D E , 協 防 ″わヮ 打α芝 防 o た拘 r θ】α泌 月α! A ″ り‐
s e r v e d b e f o r e e l i m i n t t i n g hP e. d r y h g s に sお ,Academic Press,New York,NY(196o.
Remove each collection vessel froln the instmment and,ifneces‐ DEUEL ttc町 泳 ,VOl,1,Inters,lence Publishers,Inc.,New York,
S 叩 , r e m o v e r e s i d u a l m o i s t u r e b y t t i n g t o c o n sNY(1951).
ttt weight using
vacuum oven,B(e),(preferred),or fOrced‐ なr oven,Bo,with rec_ DEUTSCttE CESELLSCHAF「 問 R nttTWIssENSCHAFF E.V.,後 閉竹昭
ommendedに mperatures and pressuresち Record flnal colに
ction ves‐ S 勧 滋 ″ M a 施 お ヵ ″! ル A ″ r y s 体 ザ n な 滅 o r 2 2 , 1 妙施 ,
sel weightto± o.o001g. (English transiation),Munsに r,RG(1988).
ω Cθ 2+ゴ 5%を r脇″ :餌 racri伽め sf閉 “rara a脇匹だ″ 研 rac‐ EcKEY,“ Vegetable Fats and Oils,"Reinhold Pubushing c。 .,New
rJθ
L ― Prepare test smple,weigh test poilon into 研
exmcは
cell, York,NY(1954).
and i】●pare and install extraction cell and coll∝ はon vessel in the C h a r a c r施
i C aα
ど泌 勧卸 泣 r
2 ホ ザ 0 : な, 施 句
F I R E S T O N E , PS り
αtt W後 確s,AOAC Press,Champaign,IL(1999).
S F E i n s t e―n t a s d e s c d b e d a b o v e Of no lr yt h e x mC c0 d2 O‐n . A f ‐
terf11ling cell void withdiatomaceous 配h,depress
eセ uppeFSufaCe ca GuNSTONE&HAMILTON・ Aと ,返 CtOSSaヮ ,The Oil Press,Ays,
3 HIIn to tighten packing and installinlet endcap.Exmtfbr60 min Scodand(1992). (
With C02+15%emaol,c(b),under the following condiは on車 GuNSTOtt A″ あ!Rガ“crわ″!θれを働 を閉前 ヮ aゼ 'focれcttsリ ザ
Faり Actts滅 挽 fr crycctts,2nd】 丑 ,ChapElan and H』 1
51.7 kPa;100。C;noEinal auid aOw raに ,2.lg/min(1.6r2.6g/min
Ltd.,London,UK(1967).
is Pemlssible>and restrictor temperature such dlat C02 eV01Ves at
的 TCN&HERSL6F,と tipttA″rysお=A Pracr,c″:App″ 延 れ,Ox―
between 80t100。C K40-130°C is acceptable).挽2 cOmments on
ford University Press,New York,NY(1992).
resmctor/collecよonに mperature in Procedure for C02‐ Only extrac‐
IIAMILTON&RossEL Attry品 ヴ θJrs a材免 な,Elsevier Applied
don.Remove each collecton vessei from insmment and,if neces‐
Science Publishers,New York,NY(198o.
sary,remove residual ndsture by drying to constant weight as
的 O N D ・働 ″醒 O g r a P り 乃 r れ c A ″ r y s おげ 砕 施 , c R C P r e s s ,
descdbed above for C02‐ Only extraction.Record rlnal c011ection
Boca Raton,FL(1993).
vessel weight to±0.0001g. HILDFrCH&WTlLAMS,ロ セく
助 β閉た】 め Ari!“rゎれヴ 肘″“rar Faな ,
E carcura育。n3 4th Ed.,John Wiley&Sons,Inc.,New York,NY(1964j.
Calculate percent oil as follows: HOLMAN,Prog″s s れ, 権勧 を開なリ ザ F a r s a t t θ
! 姥r ん" f お, V O l . 1
(1952)Pergamon Press,New York,NY.
= 刈 ∞ Hul,3at!り な r材 “srria′θ】 α角″月α,Pぇ ガ“crs,Vols.1-5,Jchn
呻
Wiley&Sons,Inc.,New York,NY(1996).
INTERNAT10NALUNIONOFPUREANDAPPLIEDCHEMISTRY,Sran沈 舛
′ど
ReferencαAOCS ttCj】 筋夕陥 ふ αtt R“θ"切例姥 どPracric2s, 〃夕r 肋な ヵ r r t t A ″
rysお ザ θJ h F a t t材
α肋aPs,7th revised and
5th Bと,1988,Ameican Oil CheEttStS'Society,Cham‐ enlaFged editon,Blackwell ScientiFlc Publications,Boston,MA
pおgn,L61826,USA,Ofrlcial Mchod Am 3‐ 96. (1987);lst Supplement(1992),
KAUFMANN,A″ αりsを どタアFcせ確 “れど F夕rrP″ど″たた,2 Volume,
R2フな夕″f Marc力 2ω 2
Springer‐Verlag,Berlin,GFR(1958).
LuNDBERG.A″ rθ 】jど α′jοtt ″
αガA″rjο IJtta″
rs,Vol.1(1961);Vol.
SPEClAL REFERENCES 2 ( 1 9 6 2 ) , I n t e r s c i e n c e P u b l i s h e r s ,,INnecw。 Y o r k , N Y .
MARKLEY,FarryAご 比なっ動 をど rCれ を″:H,ProP夕 rr,2島Pr9ど″cr:ο た,
AMBRICAN OIL CHEMISTS'SOCIETY,QttC筋 ど財夕 抗θて な αtt R夕●οれ‐ α4tr r/sを
s,2nd Ed.,Part l(1960):Part 2(1961);Part 3(1964);
れで,どをどPracr,ces,Ch倒 阻PAgn,IL(1999 incmding additions and Part4(1967),Part S(1968);Interscience Publishers,Inc.,New
revislons) !York,NY.
脇 r b E y ' s″
加なr r i aθ ′, r 御どF a r P r t t c r s , 4 h E d . , e d i t e d b y S wMcDoNALD&MOSSoBA.打
em, β″ 1膨cん,セ″容 `Mtrン,P'cα ガa,sれ
J o h n W i l e y & S O n s , N e w Y o r k , N Y ( 1 9 6 4 j i V o l . 1 ( 1 9 7 9 ) , V oLilp″
l . 2 A″ rysな,AOAC Press,Champaign,IL(1997).

◎ 2002 AOAC tNTERNATiONAL

AOAC OFFICtAL MttMODS OF ANALYSiS(2000)
0 1 L S A N O F A T
Chapter 41,p.69

MBHLENBACHER,何 竹 A,α 研 な ゲ Fars a″ど 0:な ,The Carard Press, RAPPE,CHAUDHARY,&KEITH(Eds)Cれ わrれa姥 ″ Dあ ガ四 切 ″
Champagn,IL.(1960), Dibettrazsれ 乃 ,ヮ 2cr挽 ,Le琥 s Publisheぉ ,Inc.,cheisea
M I N & S M O U s R F r a t t r t t β 閉なリ ザ F a r s 研 ″θ! r s , A m e d c a n 0 1 1 MI(1980・
Chemists'Society,Champaign,IL(1985).
PARDUN,A″ !距夕どを,N品 脇″gsrgr々,vol.16 ofseries R勧 はRdlagen
und Fortschritte der LebensHlitteluntersuchung und s t 協料酬 総摺
仏, 正
当獣翠盈摺路軽笛踏難F滅亜 S確
れA4aい
容
Lebensmitttitechn。10git,"Verlag Paul Parey in Berlin/Ham‐
burg,GFR(1976). ,
PATISON,乃 り Actt aゼ 恥 夕jrれ あ srrtt APガ ∽ !加 S,Marcd
WEISS,Fθ ο″ θ:な α材 恥 2:rr/scs,Avi Publishing CO.,Inct,
Dekker,Inc.,New York,NY(1968).
Westport,9「 (197o.
PERKNS,A″ rysな ゲ Fah θ ,4α ガ LilP?″ 確 ;西 ,Amencan Oil WILLIAMS,α :s,r七馬 αガFar9 F∽ 』s,4th Ed,Amedcan Elsevier
C h e m i s t s ' S o c i,eCけ
hmpaign,L(1991). Publおhng Co.,New York,NY(19(拓 ).

0 2000 AOAC tNTERNATiONAL

`