Benzaldehyde Formation fromAspartame in the Presence of

Ascorbic Acid and Transition Metal Catalyst

Gl en D. Lawrence* and Dongmei Yuan
Chemi stry Department, Long I sl and Uni versi ty, Brookl yn, New York 11201
Benzal dehyde was produced from aspartame i n aqueous aci di c sol uti ons contai ni ng ascorbi c aci d
and Cu(I I ) or Fe(I I I ) i on. Benzal dehyde was i denti fi ed i n the system by GC-MS. The yi el d of
benzal dehyde decreases dramati cal l y as the pH of the medi um i ncreases above 2.0. EDTA and
DTPA compl etel y i nhi bi ted benzal dehyde producti on, whi l e desferri oxami ne i nhi bi ted onl y the
Fe(I I I )-catal yzed reacti on. Benzal dehyde i s not produced under anaerobi c condi ti ons unl ess H
2
O
2
i s added to reacti on mi xtures. H
2
O
2
i s produced by reducti on of atmospheri c oxygen under aerobi c
condi ti ons. Benzal dehyde producti on was dependent on ascorbi c aci d concentrati on, but the yi el d
of benzal dehyde decreased as the concentrati on of ascorbi c aci d exceeded that of aspartame. Addi ti on
of ethanol to the reacti on mi xture had l i ttl e or no effect on benzal dehyde producti on, suggesti ng a
mechani sm that may not i nvol ve free hydroxyl radi cal . A mechani sm i s proposed for the reacti on.
Keywords: Aspartame; benzaldehyde; ascorbic acid; freeradical degradation products; copper(I I )
I NTRODUCTI ON
The arti fi ci al sweetener aspartame (N-L-R-aspartyl -
L-phenyl al ani ne methyl ester) has been thoroughl y
studi ed for safety and stabi l i ty (Stegi nk and Fi l er, 1984),
and several of i ts hydrol yti c decomposi ti on products
have been i denti fi ed (Homl er, 1984). Because i t i s a
di pepti de of natural l y occurri ng ami no aci ds (asparti c
aci d and phenyl al ani ne), i ts hydrol ysi s products or
metabol i tes, other than methanol , are nontoxi c i n the
amounts that woul d be consumed by normal use of thi s
sweetener. However, l i ttl e i s known regardi ng i ts
i nteracti on wi th other food components, such as natural
nutri ents or food addi ti ves.
Mazur (1976) has reported the pH stabi l i ty profi l e
for aspartame i n aqueous sol uti on, i denti fyi ng among
the hydrol ysi s products 3-benzyl -2,5-pi perazi nedi one-
6-aceti c aci d, the di ketopi perazi ne deri vati ve of as-
partame. These products were subsequentl y i denti -
fi ed i n several soft dri nks that were stored for 6 or 36
months (Tsang et al ., 1985). Stamp and Labuza (1989)
showed that -aspartyl phenyl al ani ne, -aspartame, and
phenyl al ani ne methyl ester are products of aspartame
degradati on i n di l ute aci d sol uti on. Hussei n et al .
(1984) found that several food-fl avori ng al dehydes, such
as benzal dehyde, ci nnamal dehyde, and vani l l i n, wi l l
react wi th the free ami no group of aspartame i n ab-
sol ute al cohol to produce the correspondi ng Schi ff base.
Ascorbi c aci d (vi tami n C) i s a natural component of
many foods and i s often added to foods and beverages
as a vi tami n suppl ement and anti oxi dant. Several
studi es have addressed the stabi l i ty of ascorbi c aci d i n
aqueous sol uti on i n the presence of other food compo-
nents, i ncl udi ng aspartame (Ho et al ., 1994; Hsi eh and
Harri s, 1987, 1991). Earl i er work i n thi s l aboratory
(Gardner and Lawrence, 1993) showed aerobi c sol uti ons
of ascorbi c aci d coul d produce benzene from the wi del y
used food preservati ve sodi um benzoate under condi -
ti ons that are commonl y found i n beverages. These
studi es have been extended i n an attempt to i denti fy
possi bl e products of ascorbi c aci d dependent free radi cal
degradati on of aspartame.
EXPERI MENTAL PROCEDURES
Reagents. Aqueous stock sol uti ons of the fol l owi ng re-
agents were prepared from anal yti cal reagent grade chemi cal s
usi ng di sti l l ed dei oni zed water and stored i n the refri gerator:
1-50 mM aspartame (L-aspartyl -L-phenyl al ani ne methyl ester;
Si gma) i n 0.05 N HCl ; 52 mM phenyl al ani ne (Si gma) i n 0.05
N HCl ; 9.3 mM aspartyl phenyl al ani ne (Asp-Phe; Si gma) i n
0.10 M phosphate buffer, pH 2.0; 1.6 mM copper(I I ) sul fate
pentahydrate (G.F. Smi th); 150 mM hydrogen peroxi de (Fl u-
ka); 8 mM benzyl al cohol (Si gma); 8 mM benzal dehyde
(Si gma); 4 mM sodi um benzoate (Si gma); 100 mM ascorbi c aci d
(Fl uka) i n 0.10 N HCl ; 0.10 M monochl oroaceti c aci d buffer,
pH 2.1-3.3; and 0.10 M sodi um di hydrogen phosphate buffer,
pH 1.6-7.7, adjusted to desi red pH wi th NaOH. Aspartame
stock sol uti ons were prepared fresh every few days. Hydrogen
peroxi de concentrati on i n stock sol uti ons and reacti on mi xtures
was determi ned col ori metri cal l y wi th saturated Ti OSO4 sol u-
ti on i n 2 M H2SO4 ( ) 717 M
-1
cm
-1
at 410 nm; El l i s and
Sykes, 1973).
ReactionConditions. A typi cal reacti on mi xture (1.0 mL)
contai ned 50 mM buffer (buffer components and pH vari ed),
5.0 mM aspartame, 0.16 mM CuSO4, 8 mM H2O2, and 5.0 mM
ascorbi c aci d. The pH was checked at the end of a reacti on
because the presence of HCl i n the aspartame and ascorbi c
aci d stock sol uti ons caused some shi ft i n pH of the buffer when
these were added to the reacti on mi xtures. The reacti on
mi xtures were pl aced i n a 40 C water bath (except where
i ndi cated) and anal yzed by di rect i njecti on of an al i quot i nto
the l i qui d chromatograph 15 mi n after the reacti on was
i ni ti ated (except where i ndi cated). The reacti on condi ti ons
were vari ed to study the effects of pH and concentrati on of
reagents. I n some cases Fe2(SO4)3 was substi tuted for CuSO4,
and oxygen was purged from some reacti on mi xtures by
bubbl i ng wi th water saturated, prepuri fi ed N2.
The metal -chel ati ng agents di sodi um ethyl enedi ami netet-
raaceti c aci d (Na2EDTA), di ethyl enetri ami nepentaaceti c aci d
(DTPA), gl yci ne, and desferri oxami ne (desferal mesyl ate; a gi ft
of Ci ba Pharmaceuti cal Co.) were added to some reacti on
mi xtures to determi ne thei r effect on the metal catal ysts.
Ethanol was added as a competi ti ve hydroxyl radi cal scavenger
to determi ne whether free hydroxyl radi cal i s i nvol ved i n
these reacti ons.
The concentrati on of ascorbi c aci d was fol l owed duri ng the
progress of the reacti on by di l uti ng a 20 L al i quot of the
* Author to whom correspondence shoul d be ad-
dressed [e-mai l , gl awrenc@hornet.l i unet.edu; fax, (718)
488-1465].
3461 J. Agric. Food Chem. 1996, 44, 34613466
S0021-8561(96)00079-9 CCC: $12.00 1996 American Chemical Society
reacti on mi xture i n 1.98 mL of 0.10 M phosphori c aci d at a
gi ven ti me and absorbance measured at 245 nm ( ) 7943 M
-1
cm
-1
; Grasel l i , 1973). Other reactants gave l i ttl e or no
contri buti on to thi s absorbance (aspartame 245 ) 102 M
-1
cm
-1
; thi s study). Benzal dehyde produced i n the reacti on has
a strong absorbance at 245 nm (247 ) 11 482 M
-1
cm
-1
;
Grasel l i , 1973), and i ts contri buti on at any ti me i n the reacti on
coul d be cal cul ated from the chromatographi c quanti tati on.
Other products of the reacti on, such as aromati c hydroxyl ati on
products, woul d absorb at 245 nm, but thei r contri buti on i s
probabl y not si gni fi cant at l ow pH (judgi ng from chromato-
graphi c resul ts). The concentrati on of hydrogen peroxi de was
moni tored col ori metri cal l y duri ng the progress of the reacti on
as descri bed above.
Liquid Chromatography. The LC system consi sted of a
Knauer model 64 pump, Rheodyne 7125 i njecti on val ve wi th
20 L i njecti on l oop, and Knauer vari abl e wavel ength UV
detector at 255 nm wi th response recorded on ei ther a Ki pp
and Zonen BD41 stri p chart recorder or a Hewl ett-Packard
3396B i ntegrator. A Hewl ett-Packard model 1050 l i qui d
chromatograph wi th di ode array detector became avai l abl e at
the end of thi s study (gi ft of the Hewl ett-Packard Educati onal
Grants Program) and was used for some anal yses. The
anal yti cal col umn for anal ysi s of aspartame and benzal dehyde
was a 4.6 100 mm Spheri -5 (5 m) cyano col umn, wi th 4.6
30 mm guard cartri dge (Brownl ee). The mobi l e phase was
0.05 M sodi um di hydrogen phosphate, 50 M DTPA contai ni ng
8% (v/v) acetoni tri l e. The mobi l e phase was fi l tered through
0.2 m membrane fi l ters pri or to use and pumped at a fl ow
rate of 1.3 mL/mi n. Retenti on ti mes were aspartame, 1.9 mi n;
benzal dehyde, 2.8 mi n. These anal ytes were quanti fi ed by
compari son of peak hei ght wi th known standards. A product
of aspartame degradati on was not wel l resol ved from the
aspartame peak and i nterfered wi th quanti tati on by peak area;
consequentl y peak hei ght was used for quanti tati on of both
aspartame and benzal dehyde.
The chromatographi c system for anal ysi s of benzyl al cohol ,
benzoi c aci d, and benzal dehyde i n the presence of aspartame
consi sted of a Spheri -5 C8 (octyl si l ane) col umn (4.6 100
mm) wi th C8 guard cartri dge (Brownl ee) and mobi l e phase
contai ni ng 0.05 M sodi um di hydrogen phosphate, 0.01%
(w/v) sodi um octyl sul fate, and 8% (v/v) methanol adjusted to
pH 3.9 wi th phosphori c aci d sol uti on and pumped at a fl ow
rate of 3.0 mL/mi n. Retenti on ti mes were benzyl al cohol , 2.0
mi n; benzoi c aci d, 3.1 mi n; benzal dehyde, 4.3 mi n; aspartame,
6.3 mi n.
GasChromatography-MassSpectrometry. A Hewl ett-
Packard 5890/5971 GC-MS (gi ft of the Hewl ett-Packard
Educati onal Grants Program) was equi pped wi th a 30 m HP-1
(nonpol ar) capi l l ary col umn. GC parameters as fol l ows: i ni ti al
temperature, 100 C; i ni ti al ti me, 2.0 mi n; rate, 10 C/mi n;
fi nal temperature, 160 C; fi nal ti me, 2.0 mi n; i njector tem-
perature, 200 C. MS parameters as fol l ows: l ow mass, 50;
hi gh mass, 200; threshol d, 150; sampl i ng rate, 2/s. A hi gh
yi el d of a major product observed i n the l i qui d chromatographi c
anal ysi s was obtai ned by pl aci ng the standard reacti on
mi xture i n a di sti l l ati on apparatus and col l ecti ng the di sti l l ate
at 85 C. Thi s product coul d be extracted i nto organi c sol vents,
such as cycl ohexane, but attempts to concentrate i t by evapo-
rati on of the sol vent resul ted i n l oss of product as wel l . A smal l
(0.5 L) al i quot of the di sti l l ate was i njected (spl i t rati o 60:1)
i nto the GC-MS for anal ysi s. The major gas chromatographi c
peak (m/z > 50) was i denti fi ed as benzal dehyde.
Solid PhaseExtraction (SPE). Once benzal dehyde was
i denti fi ed as a product of aspartame oxi dati on i n the hydroxyl
radi cal -generati ng system, attempts were made to i sol ate and
i denti fy rel ated benzyl deri vati ves (benzyl al cohol and benzoi c
aci d). A systemati c i nvesti gati on of SPE adsorbents was
undertaken, usi ng octadecyl (C18), octyl (C8), cyano (CN), and
phenyl packi ngs i n 3 mL cartri dges (Supel co or Baker).
Reversed-phase packi ngs (C18, C8, phenyl , and cyano) were
ri nsed sequenti al l y wi th water, 50% aqueous methanol , 100%
methanol , 50% methanol , and then water pri or to addi ti on of
2 mL of reacti on mi xture. After the reacti on mi xture was
l oaded, the cartri dges were ri nsed sequenti al l y wi th 2 mL of
water, wi th 2 mL of 5% methanol , and several ti mes wi th 2
mL of 50% methanol . Each 2 mL el uent was anal yzed by
HPLC for benzyl al cohol , benzal dehyde, benzoi c aci d, and
aspartame, usi ng the C8 anal yti cal col umn. The bul k of the
benzyl deri vati ves was el uted i n the fi rst 50% methanol
fracti on.
RESULTS
Identifying Benzaldehyde as a Product of As-
partame Degradation. Prel i mi nary studi es of the
ascorbi c aci d-hydrogen peroxi de-Cu(I I ) free radi cal -
generati ng system i n the presence of aspartame re-
veal ed many uni denti fi ed peaks i n the l i qui d chromato-
grams of the reacti on mi xtures. There was a noti ceabl y
l arge peak under aci di c reacti on condi ti ons wi th a
retenti on ti me cl ose to that for aspartame that di sap-
peared al most compl etel y upon extracti on of the reacti on
mi xture wi th nonpol ar organi c sol vents (Fi gure 1). An
attempt to evaporate the organi c sol vent and di ssol ve
the extracted materi al i n mobi l e phase resul ted i n l oss
of the uni denti fi ed product, suggesti ng i t was a vol ati l e
compound. Even parti al evaporati on of organi c sol vent
resul ted i n a nearl y l i near l oss of the product. Conse-
quentl y, sol i d phase extracti on wi th octadecyl or octyl
SPE cartri dges showed that a very nonpol ar product
Figure1. Li qui d chromatograms of (top) aspartame (5.1 mM)
and benzal dehyde (45 M) standard, (mi ddl e) reacti on mi xture
contai ni ng 0.05 M phosphate buffer, pH 2.0, 5.1 mM aspar-
tame, 0.16 mM CuSO4, 8.4 mM H2O2 and 5.0 mM ascorbi c aci d
i ncubated for 30 mi n at room temperature, and (bottom)
reacti on mi xture after extracti on twi ce wi th 0.5 mL of cycl o-
hexane/mL of reacti on mi xture. Chromatographi c condi ti ons
on the Hewl ett-Packard 1050 LC as descri bed under Experi -
mental Procedures, detector response at 255 nm.
3462 J. Agric. Food Chem., Vol. 44, No. 11, 1996 Lawrence and Yuan
coul d be parti al l y puri fi ed but al ways contai ned some
detectabl e i mpuri ti es i n the el uent contai ni ng thi s
product.
Si nce thi s product was found to be vol ati l e, attempts
to col l ect i t i n concentrated form by di sti l l ati on of the
aqueous reacti on mi xture were successful . A hi ghl y
concentrated sol uti on of the unknown product of i nterest
was obtai ned i n the 85 C di sti l l ate, devoi d of noti ceabl e
i mpuri ti es. Thi s di sti l l ate was anal yzed by GC-MS,
and the si ngl e peak (m/z > 50) gave a mass spectrum
that cl osel y matched that of benzal dehyde. A standard
sol uti on of benzal dehyde was found to gi ve a peak i n
the l i qui d chromatogram wi th the same retenti on ti me
as that of the unknown peak of i nterest i n the free
radi cal reacti on mi xture contai ni ng aspartame. A stan-
dard addi ti on of benzal dehyde to the reacti on mi xture
further i ndi cated the unknown peak of i nterest was
benzal dehyde. The UV absorbance spectrum of the
peak, usi ng a di ode array detector, i ndi cated benzal de-
hyde was the product.
OptimumConditionsfor BenzaldehydeProduc-
tion. A ti me dependence study for the reacti on (Fi gure
2) i ndi cates there i s a rapi d phase for benzal dehyde
producti on at pH 2.6 that i s compl ete i n about 30
mi n at room temperature or wi thi n 15 mi n at 40 C.
There i s a sl ower conti nuous producti on of benzal dehyde
and consequent di sappearance of aspartame over sev-
eral hours, al though thi s l atter phase accounts for a
rel ati vel y smal l amount of the overal l reacti on. Sub-
sequentl y, reacti on mi xtures stood for 15 mi n at 40 C
or for 30 mi n at room temperature for anal ysi s of
products as reacti on condi ti ons vari ed. The i ni ti al rapi d
phase of the reacti on i s compl ete before the concentra-
ti ons of ascorbi c aci d and hydrogen peroxi de are com-
pl etel y di mi ni shed. The H
2
O
2
and ascorbi c aci d remai n-
i ng i n the reacti on mi xture conti nue to decrease at a
rel ati vel y sl ow rate (Fi gure 2). Thi s may be due to
chel ati on of the Cu(I I ) i on by one of the products (see
Di scussi on).
The producti on of benzal dehyde from aspartame i n
the free radi cal -generati ng system showed a strong
dependence on pH and buffer composi ti on. There was
a sharp decrease i n yi el d of benzal dehyde as pH of the
reacti on mi xture i ncreased from 2 to 3 (see Fi gure 3).
Onl y trace amounts of benzal dehyde were produced i n
the neutral pH range, wi th a broad, l ate el uti ng peak
(uni denti fi ed) appeari ng i n the l i qui d chromatograms
i n the hi gher pH range.
Under aci di c condi ti ons, there was a much greater
yi el d of benzal dehyde when phosphori c aci d/phosphate
buffer was used compared to chl oroaceti c aci d or gl yci ne
buffers (Tabl e 1). The l atter buffers may act as radi cal
scavengers or bi nd the Cu(I I ) i on, i nacti vati ng or chang-
i ng the rel ati ve reacti vi ty of thi s catal yst for free radi cal
producti on. Phosphate and gl yci ne buffers were pre-
pared wi th constant i oni c strength (I ) 0.10 M wi th
added NaCl O
4
) i n the aci di c pH range and found to gi ve
the same yi el d of benzal dehyde as those wi thout added
NaCl O
4
at any gi ven pH (data not shown), i ndi cati ng
the decrease i n yi el d of thi s product wi th i ncreasi ng pH
was not due to a nonspeci fi c i on effect.
Figure2. Rate of di sappearance of reactants and formati on
of benzal dehyde i n reacti on mi xtures contai ni ng 0.05 M
phosphate buffer, pH 2.6, 5.0 mM aspartame, 0.16 mM CuSO4,
8.4 mM H2O2, and 5.0 mM ascorbi c aci d, i ncubated at 40 C.
Ascorbi c aci d (9) and H2O2 (2) were measured as descri bed i n
the Experi mental Secti on; aspartame (0) and benzal dehyde
() were determi ned chromatographi cal l y.
Figure 3. Dependence of benzal dehyde producti on on pH of
the medi um. Each reacti on mi xture contai ned 0.05 M phos-
phate buffer at pH i ndi cated; other condi ti ons were as i n
Fi gure 1, mi ddl e, except reacti on mi xtures were i ncubated for
15 mi n at 40 C.
Table 1. Benzaldehyde Production fromAspartame
under Varying Reaction Conditions
addi ti ons to reacti on mi xture
[benzal dehyde]
produced (M)
compl ete reacti on mi xture 110
anaerobi c condi ti ons 111
+ 32 mM ethanol 103
+ 500 M DTPA nd
+ 200 M Na2EDTA nd
+ 500 M gl yci ne 120
i n 0.05 M chl oroacetate buffer, pH 2.3
a
66
i n 0.05 M gl yci ne buffer, pH 2.1
a
30
wi th 4.6 mM Asp-Phe i nstead
of aspartame
b
166
wi th 5.2 mM Phe i nstead of aspartame
b
61
-H2O2, aerobi c
c
18
-H2O2, anaerobi c
c
nd
no metal s added
d
15
+ 880 M desferri oxami ne nd
+ 500 M DTPA nd
+ 500 M Na
2EDTA nd
+ 500 M Gl yci ne 6
wi th varyi ng [Cu(I I )] added
e
+ 16 M Cu(I I ) 52
+ 48 M Cu(I I ) 82
+ 96 M Cu(I I ) 98
+ 316 M Cu(I I ) 120
wi th added Fe(I I I ) i n pl ace of Cu(I I )
d
67.5 M Fe2(SO4)3; [Fe(I I I )] ) 135 M 18
+ 200 M Na2EDTA nd
+ 32 mM ethanol 16
i n 0.05 M chl oroacetate buffer, pH 2.3
a
9
a
Each reacti on mi xture contai ned 0.05 M phosphate buffer, pH
2.3 (except those wi th footnote a), 5.0 mM aspartame (except those
wi th footnote b), 8 mM H2O2 (except those wi th footnote c), 168
M CuSO4 (except those wi th footnotes d and e), and 5 mM
ascorbi c aci d. Reacti on mi xtures were i ncubated i n a seal ed vi al
for 15 mi n at 40 C pri or to removal of an al i quot for di rect
i njecti on i n the l i qui d chromatograph. Al l reacti ons performed i n
dupl i cate or tri pl i cate. nd ) not detectabl e.
Benzaldehyde from Aspartame Free Radical Oxidation J. Agric. Food Chem., Vol. 44, No. 11, 1996 3463
There i s a nonl i near dependence on i ni ti al hydrogen
peroxi de concentrati on i n the reacti on mi xture that may
be parti al l y due to the l i mi ti ng amount of ascorbi c aci d
present or to bi ndi ng of the Cu(I I ) catal yst to products.
A si gni fi cant amount of benzal dehyde was produced
when hydrogen peroxi de was omi tted (see Tabl e 1). Thi s
producti on of benzal dehyde i n the absence of hydro-
gen peroxi de coul d be el i mi nated by deaerati ng the
reacti on mi xture wi th ni trogen. Thi s i ndi cates hydro-
gen peroxi de coul d be formed from ascorbi c aci d de-
pendent reducti on of ambi ent oxygen i n the sol uti on.
Purgi ng the compl ete reacti on mi xture wi th ni trogen
(i .e., when hydrogen peroxi de was present) had l i ttl e or
no effect on benzal dehyde producti on (see Tabl e 1).
There i s a compl ex dependence of the reacti on on
ascorbi c aci d concentrati on (Fi gure 4). The yi el d of
benzal dehyde i ncreases wi th i ncreasi ng concentrati on
of ascorbi c aci d i n the reacti on mi xture up to 5 mM but
then decreases wi th i ncreasi ng concentrati on of ascorbi c
aci d i n excess of 5 mM. Thi s can be expl ai ned by the
fact that the concentrati on of aspartame i n the reacti on
mi xture was 5 mM, and ascorbi c aci d can compete wi th
aspartame as a free radi cal scavenger i n thi s system.
I t shoul d be noted that there was not detectabl e ben-
zal dehyde producti on when ascorbi c aci d was absent
from the reacti on mi xture, and hydrogen peroxi de was
present, even after 24 h at room temperature. Al though
Cu(I I ) catal yzes the di sproporti onati on of H
2
O
2
, thi s rate
i s extremel y sl ow i n aci di c medi a. There was no
detectabl e l oss of H
2
O
2
i n 3 h i n 0.05 M phosphate buf-
fer at pH 2.6, i n ei ther the presence or absence of
aspartame.
When Cu(I I ) i on was omi tted from the reacti on
mi xture, there was a dramati c decrease i n benzal dehyde
producti on, but i t was sti l l measurabl e (Tabl e 1). Thi s
background producti on of benzal dehyde i n the absence
of any added metal i on catal yst was l i kel y due to the
presence of trace amounts of i ron i n dei oni zed, di sti l l ed
water and reagents. When desferri oxami ne, a strong
Fe(I I I ) i on chel ator, was added to the Cu(I I )-defi ci ent
reacti on mi xture, benzal dehyde was not detectabl e.
Addi ti on of EDTA or DTPA, two other metal i on
chel ators, al so compl etel y i nhi bi ted benzal dehyde pro-
ducti on, whether Cu(I I ) was added or omi tted (Tabl e
1). Thi s i ndi cates these l atter chel ati ng agents are very
effecti ve at i nhi bi ti ng both Fe(I I I ) and Cu(I I ) i ons from
catal yzi ng the ti tl e reacti on. Addi ti on of 500 M gl yci ne
to reacti on mi xtures contai ni ng 168 M CuSO
4
had no
si gni fi cant effect on benzal dehyde producti on. However,
when 50 mM gl yci ne was used to buffer the reacti on
mi xture, there was a si gni fi cant decrease (-76%) i n
benzal dehyde producti on (Tabl e 1).
The addi ti on of 32 mM ethanol to the reacti on mi xture
had no effect on benzal dehyde producti on. Ethanol i s
a competi ti ve OH

scavenger that woul d be expected to

di mi ni sh products of OH

attack at thi s concentrati on

i n the reacti on mi xture. The l ack of i nhi bi ti on of
benzal dehyde producti on by ethanol suggests a mech-
ani sm that may i nvol ve an aspartame-Cu i on compl ex
that undergoes an i ntramol ecul ar, si te-speci fi c attack
on aspartame.
DI SCUSSI ON
The present study shows that benzal dehyde i s among
the products of aspartame degradati on i n the presence
of ascorbi c aci d autoxi dati on, catal yzed by Cu(I I ) or
Fe(I I I ) under aci di c condi ti ons. Addi ti on of hydrogen
peroxi de to the system augments the producti on of
benzal dehyde, wher eas metal i on-chel ati ng agents
strongl y i nhi bi t the reacti on. We have not found any
report of benzal dehyde as a decomposi ti on product of
aspartame i n the l i terature, and i ts i denti fi cati on as a
major product under these condi ti ons was qui te unex-
pected.
Buettner (1986) found compl ete oxi dati on of ascorbi c
aci d (0.1 mM) i n 15 mi n i n ai r-saturated sol uti ons at
pH 7. Al l chel ati ng agents used i n that study resul ted
i n i nhi bi ti on of the copper i on-catal yzed autoxi dati on
of ascorbi c aci d, al though EDTA augmented the i ron i on-
catal yzed reacti on. Hsi eh and Harri s (1991) found that
aspartame woul d augment the copper i on-catal yzed
autoxi dati on of ascorbi c aci d, al though they di d not
report measuri ng any aspartame decomposi ti on prod-
ucts. The present study has i denti fi ed a new decom-
posi ti on product of aspartame i n sol uti ons contai ni ng
ascorbi c aci d under condi ti ons that mi ght prevai l i n
foods.
When ascorbi c aci d was omi tted from reacti on mi x-
tures, there was no detectabl e formati on of benzal de-
hyde from aspartame, even when hydrogen peroxi de and
Cu(I I ) were present. Cu(I I ) i s an effecti ve catal yst for
the di sproporti onati on of H
2
O
2
by the fol l owi ng scheme
(Gutteri dge and Wi l ki ns, 1983):
However, i n thi s study i t was found that there was no
detectabl e decomposi ti on of H
2
O
2
by Cu(I I ) i on i n aci di c
medi a over several hours. There was no detectabl e
producti on of benzal dehyde even after 24 h i n the
absence of ascorbi c aci d.
The presence of ascorbi c aci d i n the reacti on mi xture
resul ts i n reducti on of Cu(I I ) to Cu(I ) i n the system (eq
5), whi ch woul d faci l i tate reducti on of H
2
O
2
to OH

(or
Cu(OH)
2+
or CuO
+
, vide infra) by a copper-catal yzed
Fenton reacti on (eq 4).
I t i s l i kel y that the Cu(I I ) present i n the reacti on
mi xtures i s coordi nated to aspartame, whi ch may al ter
Figure4. Dependence of benzal dehyde producti on on i ni ti al
ascorbi c aci d concentrati on. Condi ti ons were the same as i n
Fi gure 1, mi ddl e, except concentrati on of ascorbi c aci d was
vari ed. Reacti on mi xtures were i ncubated for 15 mi n at 40 C.
Cu(I I ) + H
2
O
2
f Cu(I ) + HO
2

+ H
+
(1)
HO
2

f H
+
+ O
2
-
(2)
Cu(I I ) + O
2
-
f Cu(I ) + O
2
(3)
Cu(I ) + H
2
O
2
f Cu(I I ) + OH

+ OH
-
(4)
Cu(I I ) + H
2
Asc f Cu(I ) + HAsc

(5)
3464 J. Agric. Food Chem., Vol. 44, No. 11, 1996 Lawrence and Yuan
the rates and speci fi ci ti es of these reacti ons rel ati ve to
the aquated Cu(I I ) i on. A Cu(I I )-aspartame compl ex
has been used for resol uti on of ami no aci d enanti omers
by capi l l ary el ectrophoresi s (Gozel et al ., 1986) and
HPLC (Gi l on et al ., 1979). Cu(I I ) readi l y forms 1:1 and
1:2 compl exes wi th aspartame, wi th the 1:1 compl ex
predomi nati ng bel ow pH 4 (Ai hara et al ., 1992). I t
appears that the coordi nati on of Cu(I I ) resul ts i n a
l i gand-di rected, si te-speci fi c free radi cal attack on the
aspartame, si nce hi gh concentrati ons of ethanol , a
noncoordi nati ng OH

scavenger, had no effect on ben-

zal dehyde producti on. Furthermore, the effect of vary-
i ng buffer i ons (phosphate, chl oroacetate, and gl yci ne,
Tabl e 1) shows that stronger coordi nati ng l i gands
i nhi bi t benzal dehyde producti on.
Gutteri dge and Wi l ki ns (1983) showed that protei ns,
i n general , i nhi bi t Cu(I I )-medi ated (but not Fe(I I I )-
medi ated) OH

generati on from H
2
O
2
di sproporti onati on
and subsequent degradati on of deoxyri bose. However,
the Cu(I I ) i ons appeared to bi nd to the protei ns and
generate OH

i n a si te-speci fi c attack on the protei n

(spari ng the deoxyri bose). Czapski et al . (1983) have
shown a si mi l ar ascorbi c aci d (or superoxi de) dependent,
Cu(I I )-catal yzed si te-speci fi c damage to protei ns i n the
presence of H
2
O
2
, whi ch i s not i nhi bi ted by hydroxyl
radi cal scavengers. The nature of the oxi di zi ng radi cal
speci es i n these systems has been proposed to be a Cu-
(I I )OH

speci es rather than free OH

for reacti ons

i nvol vi ng Cu(I ) i on wi th H
2
O
2
. The pK
a
for the probabl e
Cu(OH)
2+
has been esti mated to be l ess than 3.5, from
the product yi el d observed for the Cu(I I )-H
2
O
2
reacti on
wi th methanol (Johnson et al ., 1985). There i s a si mi l ar
decrease i n benzal dehyde yi el d i n the pH range of 2-3.5,
i ndi cati ng a pK
a
near 2.5 for one of the reacti ve speci es
(possi bl y a Cu-aspartame-acti ve oxygen i ntermedi ate).
Snook and Hami l ton (1974) have shown that benzal -
dehyde i s the major product of OH

attack on 1-phen-
yl al kanol s (PhCHOHR) when R was i sopropyl or tert-
butyl , i ndi cati ng the radi cal -stabi l i zi ng effect of the
substi tuent al kyl groups favored cl eavage, whereas a
methyl group favored oxi dati on to acetophenone. OH

attack on di pepti des resul ts i n H

abstracti on from the

C-H bond adjacent to the ami de ni trogen rather than
near the protonated ami no termi nal (Tani guchi et al .,
1970).
We propose a si mi l ar mechani sm, whereby a Cu(I I )-
OH compl ex attack on aspartame resul ts i n H

abstrac-
ti on from the R-CH of the phenyl al anyl moi ety fol l owed
by sci ssi on to yi el d the benzyl radi cal and an oxi di zed
pepti de backbone (aspartyl dehydrogl yci ne methyl ester,
ADGME), whi ch i s hi ghl y stabi l i zed by a conjugated
-system (Fi gure 5). There i s a sl ow, conti nual forma-
ti on of benzal dehyde over several hours, wi th conse-
quent decrease i n the three major reactants after the
i ni ti al rapi d burst of product. I t was i ni ti al l y suspected
that ascorbi c aci d was compl etel y di mi ni shed after the
i ni ti al burst of product, and excess hydrogen peroxi de
was accounti ng for the sl ow phase of the reacti on.
However, l ack of any benzal dehyde producti on i n the
absence of ascorbi c aci d suggested thi s was not so.
Measurement of ascorbi c aci d i n the reacti on mi xture
i ndi cated there was sti l l a si gni fi cant amount of ascorbi c
aci d l eft when the i ni ti al rapi d phase of the reacti on was
over. Consequentl y, chel ati on of Cu(I I ) i on by one of
the products of the reacti on (ADGME?) i s proposed to
be the cause of thi s sl owi ng of the reacti on.
When phenyl al ani ne repl aced aspartame as the scav-
enger, the yi el d of benzal dehyde was onl y about 55% of
the yi el d wi th aspartame. Thi s may be due to l ess
-stabi l i zati on of the nonaromati c product i n the phen-
yl al ani ne reacti on. Asp-Phe augmented the producti on
of benzal dehyde (+51%); the nonaromati c product of thi s
scavenger woul d have greater -stabi l i zati on than as-
partame due to i ncreased resonance i n the carboxyl ate
group. Addi ti on of H
2
O to the benzyl radi cal yi el ds
benzyl al cohol , whi ch woul d be rapi dl y oxi di zed by
Cu(I I ) to benzal dehyde under the reacti on condi ti ons.
A concerted mechani sm for sci ssi on and oxi dati on of the
benzyl radi cal may be i mpl i ed, si nce coordi nati on of the
Cu(I I ) catal yst by the nonaromati c product has been
i nvoked. Benzal dehyde i s not readi l y oxi di zed to ben-
zoi c aci d by Cu(I I ) (March, 1968).
Wal l i ng (1975) has proposed a phenyl (ri ng-centered)
radi cal cati on i n equi l i bri um wi th the hydroxycycl o-
hexadi enyl radi cal i n aci di c medi a, i n the case of OH

attack on phenyl aceti c aci d. I t was suggested the

phenyl radi cal cati on i ntermedi ate can undergo decar-
boxyl ati on to yi el d benzyl radi cal , whi ch i s oxi di zed to
benzyl al cohol and ul ti matel y benzal dehyde i n the
presence of Cu(I I ) i on. However, the hydroxycycl o-
hexadi enyl radi cal i ntermedi ate can undergo oxi dati on
to yi el d the correspondi ng phenol i n the presence of
Cu(I I ) and O
2
. Phenol i c products from OH

attack on
aspartame have been i denti fi ed under neutral condi -
ti ons (unpubl i shed resul ts). The pH of the medi um
strongl y i nfl uences the di stri buti on of products i n thi s
reacti on, i .e., benzal dehyde vs phenol i c products. There
appears to be a decrease i n yi el d of phenol i c products
bel ow pH 3 (i n conjuncti on wi th the i ncreased benzal -
dehyde yi el d).
Attempts to measure benzyl al cohol and benzoi c aci d
i n the reacti on mi xtures showed there was l i ttl e i f any
of these products formed. There was no benzyl al cohol
detected i n the GC-MS anal ysi s of the di sti l l ed reacti on
mi xture. The octyl (C8) col umn descri bed i n the Ex-
peri mental Secti on gave good resol uti on of these benzyl
deri vati ves and aspartame, but chromatograms of the
Figure 5. Proposed reacti on scheme.
Benzaldehyde from Aspartame Free Radical Oxidation J. Agric. Food Chem., Vol. 44, No. 11, 1996 3465
reacti on mi xtures moni tored at 220 nm contai ned many
peaks, and trace amounts of benzyl al cohol and benzoi c
aci d coul d have been obscured by other peaks. I t was
esti mated by standard addi ti on that as l i ttl e as 30 M
benzyl al cohol or benzoi c aci d coul d be detected i n these
reacti on mi xtures, but thi s l evel was not observed. I t
shoul d be noted that UV detecti on for benzal dehyde i s
possi bl e at 254 nm wi th a l i mi t of detecti on near 1 M,
whereas benzyl al cohol has no si gni fi cant absorpti on at
wavel engths > 225 nm, and benzoi c aci d absorbs onl y
weakl y above 230 nm.
Thi s study shows that aspartame, i n the presence of
ascorbi c aci d and a transi ti on metal catal yst, such as
Cu(I I ) or Fe(I I I ), under aerobi c condi ti ons can produce
benzal dehyde vi a a free radi cal attack on the aspartame.
Al though benzal dehyde i s a commonl y used fl avori ng
agent (al mond fl avori ng), and the ti tl e reacti on woul d
have l i ttl e or no si gni fi cant i mpact on publ i c heal th,
these resul ts show that ascorbi c aci d, i n the presence
of trace amounts of metal catal ysts, can i ni ti ate some
very i nteresti ng chemi cal reacti ons i n commonl y used
food addi ti ves.
ACKNOWLEDGMENT
G.D.L. thanks Long I sl and Uni versi ty for rel ease ti me
from teachi ng duti es to perform thi s research.
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Recei ved for revi ew February 2, 1996. Revi sed manuscri pt
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X
JF960079K
X
Abstract publ i shed i n AdvanceACS Abstracts, Oc-
tober 1, 1996.
3466 J. Agric. Food Chem., Vol. 44, No. 11, 1996 Lawrence and Yuan