JOURNAL OF

FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 21 (2008) 306314
Original Article
A simplied method for cholesterol determination in meat and
meat products
T.T.N. Dinh
a
, J.R. Blanton Jr.
b
, J.C. Brooks
a
, M.F. Miller
a
, L.D. Thompson
a,
a
Department of Animal and Food Sciences, Texas Tech University, Box 42141, Lubbock, TX 79409-2141, USA
b
Supachill Technologies, 9714 4th Drive, Lubbock, TX 79416, USA
Received 28 August 2007; received in revised form 2 February 2008; accepted 2 February 2008
Abstract
The objectives of this study were to develop an accurate and precise method for cholesterol quantication in meat samples based on
modications made to an existing procedure (AOAC Ofcial Method 994.10), and to apply this modied method to evaluate cholesterol
levels in longissimus muscles (LM) from Angus (AN, n 5), Brahman (BR, n 4), and Romosinuano (RM, n 9) breeds. Validation of
this method was performed using a meat homogenate (Standard Reference Material 1546) from National Institute of Standards and
Technology (NIST), and LM samples from the three breeds with fat contents ranging from 2.4% to 9.3%. The results indicated that the
modied method was accurate with cholesterol recovery exceeding 95%. The method was also found to be precise with an average
coefcient of variation of 3.12%. The modication reduced 90% of chemicals used and eliminated time-consuming steps that hindered
high throughput application of the traditional method. The application of this method to quantify cholesterol contents of LM samples
revealed differences among the three breeds evaluated. The Angus LM with a higher fat content (50% higher) was associated with a
signicantly higher cholesterol concentration (70.25 mg/100 g) as compared to LM from Brahman and Romosinuano purebreds
(64.77 and 65.76 mg/100 g; P 0.005 and 0.006, respectively). Cholesterol concentration was found to be correlated with the i.m. fat
content of LM muscle from the three breeds (r 0.90, Po0.001). Cholesterol concentrations of LM determined in this study were
comparable to those reported in the USDA National Nutrient Database for Standard Reference for separable lean from Choice rib-eye
steaks. This modied method was reliable and should be evaluated for adoption as an appropriate method for cholesterol quantication
in meat samples.
r 2008 Elsevier Inc. All rights reserved.
Keywords: Cholesterol determination; Gas chromatography; Method validation; Longissimus muscle; Beef cattle; Cattle breeds; Angus; Brahman;
Romosinuano
1. Introduction
Cholesterol is an integral lipid component that has been
villanized for its perceived negative effects on health. Public
concern is more specically related to meat products,
especially red meat (Li et al., 2005). The concern over the
effects of dietary cholesterol on heart disease, and the
obligatory nutritional labeling in the United States (FDA,
1993) led to the need for an accurate and efcient
cholesterol determination technique. As the source of the
most validated and trusted analytical methods, the AOAC
adopted the rst cholesterol analysis procedure for foods in
1976 (AOAC Ofcial Method 976.26). A modication
using direct saponication was made in 1994 and later
adapted to the AOAC Ofcial Method 994.10. This
progress was a signicant change because it eliminated
the lipid extraction step, and therefore, reduced analysis
time and solvent use (Klatt et al., 1995). However, this
method is still complicated, using large amount of
chemicals for post-extraction of unsaponied materials,
and includes many other preparation steps such as drying,
reconstitution, and derivatization of cholesterol prior to
gas chromatographic (GC) evaluation (AOAC, 1996a, b).
Hence, many laboratories developed their specic methods
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Corresponding author. Tel.: +1 806 742 2805x224;

fax: +1 806 742 0898.
E-mail address: leslie.thompson@ttu.edu (L.D. Thompson).
for cholesterol determination (Thompson and Merola,
1993). However, most of these methods were developed to
quantify cholesterol content in blood serum, milk, lipid, or
other lipid-based samples (Fletouris et al., 1998; Russo
et al., 2005; Kaneda et al., 1980; Punwar, 1975; Hwang
et al., 2003; Galanos et al., 1964; Stein et al., 1991;
Cardenas et al., 1995; Abidi, 2004; Abidi, 2001; Lohninger
et al., 1990).
Due to the development of GC capillary columns with
high heat tolerance (280300 1C) and increased resolution,
the separation of free cholesterol became possible. Most
attention is now directed toward development of methods
to increase recovery efciency and stability (Thompson and
Merola, 1993). Because the direct saponication technique
of the AOAC Ofcial Method 994.10 is relatively effective
(Klatt et al., 1995; Adams et al., 1986), this study was
performed to modify this AOAC procedure and to
eliminate unnecessary steps to create a simple, precise,
accurate, and productive method to quantify cholesterol in
meat products. The modied procedure was then tested
with the longissimus muscle (LM) from three divergent
breeds of cattle.
2. Materials and methods
2.1. Validation of cholesterol quantication technique
2.1.1. Standard reference material (SRM)
Standard Reference Material 1546 (SRM-1546) was
purchased from National Institute of Standards and
Technology (NIST). The certied cholesterol content of
this material was 7577.2 mg/100 g, which was quantita-
tively analyzed using an isotope dilution/gas chromato-
graphy/mass spectrometry (ID/GC/MS) method developed
at NIST (2004). SRM was carefully mixed, frozen in
liquid nitrogen, and homogenized to a powder to use for
procedure development.
2.1.2. Beef longissimus muscle samples
Three samples of beef LM with fat content determined to
be from 2.4% to 9.3% were used. Muscle samples were
collected between the 10th and 13th ribs, frozen immedi-
ately using liquid nitrogen and stored in a 80 1C freezer
for subsequent sample preparation. Frozen samples were
later trimmed of all external fat, leaving only white ecks of
marbling within muscle bundles. Trimmed samples were
chopped, homogenized to nely divided muscle powder,
and stored at 80 1C for subsequent analysis.
2.1.3. Standard materials
Cholesterol standards were used at concentration of
0.0125, 0.025, 0.05, and 0.1 mg/mL to construct the
standard curve for cholesterol quantication. An internal
standard, 5a-cholestane (Sigma-Aldrich, MO, USA), was
used as a correction factor to standardize injection
errors. All standards were diluted in high-grade toluene
(Sigma-Aldrich).
2.1.4. Experimental design
Two SRM-1546 samples (designated SRM-1546-1 and
SRM-1546-2) and three LM samples (designated LM-1,
LM-2, and LM-3) were evaluated as nine sets of replicates.
Samples used for the recovery test received 1 mg of free
cholesterol (Sigma-Aldrich) added to each of the control
samples and were characterized with letter R.
Six replicates were withdrawn per every set of samples
used for validation, with a sample size of 1 g being utilized.
All samples were analyzed over four consecutive days. If
more than one set of samples was prepared on the same
day, they were processed separately with at least a 6-h
interval in between. The extractable fat content of samples
varied from 2.4% (LM-3) to 21.0% (SRM-1546).
2.1.5. Method accuracy
The accuracy of the method was determined using both
non-addition and addition of free cholesterol (Sigma-
Aldrich) to sample matrices. For the non-addition of free
cholesterol, two sets of SRM-1546 samples (SRM-1546-1
and SRM-1546-2) were analyzed for comparison with the
certied concentration (NIST, 2004). The other four sets of
samples, designated SRM-1546R, LM-1R, LM-2R, and
LM-3R, were used to determine the recovery performance
of the method. These four sets were all treated as unknown
samples and the actual identity was unknown to the analyst
until the data was collected.
2.1.6. Method repeatability precision
Five sets of SRM-1546 (SRM-1546-1 and SRM-1546-2,
of which data were previously collected) and LM
(LM-1, LM-2, and LM-3) samples were used to calcu-
late the repeatability of the modied procedure using
coefcient of variation (CV, %) of each data set to
compare with the expected precision, which is a function
of concentration (mass fraction, %), according to the
AOAC Guidelines for Single Laboratory Validation
of Chemical Methods for Dietary Supplements and
Botanicals (AOAC, 2002).
2.1.7. Method detection limit
The detection limit was determined using dilution of
standard solution. The cholesterol standard was diluted
and subjected to GC analysis until the responding signal
was twice as high as the noise signal and detectable at the
retention time corresponding to that of free cholesterol
standard. The cholesterol detection limit was calculated as
if the cholesterol amount was from 1 g of fresh tissue.
2.1.8. Modied cholesterol analysis method
The AOAC Ofcial Method 994.10, Cholesterol in
Foods, Direct Saponication-Gas Chromatographic
Method (First Action 1994) was used with the following
modications of the saponication and extraction of
free cholesterol, and was specically tested using raw beef
LM samples. Sample weight was reduced to 1 g and all
other chemicals, except anhydrous sodium sulfate, were
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T.T.N. Dinh et al. / Journal of Food Composition and Analysis 21 (2008) 306314 307
decreased to 10% as compared to the amounts stipulated in
the original procedure. Meat samples were accurately
weighed to 1.0000 g and placed into a 125-mL boiling
ask, followed by the addition of 2 mL of 50% potassium
hydroxide in water and 10 mL of 95% ethanol. The
mixture was stirred, boiled, and reuxed for 15 min, as
compared to 80 min in the original procedure. The boiling
mixture was then cooled to room temperature (25 1C) prior
to the addition of 10 mL of high-grade toluene (Sigma-
Aldrich). A 30-s mixing was required before the solution
was transferred to a 250-mL (or 125-mL, if applicable)
separatory funnel. At least ve washes of toluene extract to
eliminate aqueous components were carried out. Except the
rst vigorous wash and vortex with 10 mL of 1.0 N
potassium hydroxide, the second mixture with 5 mL of
0.5 N potassium hydroxide and other three with 5 mL
of distilled water were gently swirled. The amounts of
washing solutions (1.0 N, 0.5 N KOH, and distilled water)
were dramatically reduced due to the reduction of toluene
solvent used. For all washes, it was important that the
toluene layer be allowed to separate completely before the
aqueous layer was discarded. The nal toluene layer, which
could be cloudy, was poured into a 50-mL (25-mL if
applicable) test tube containing about 57 g of anhydrous
sodium sulfate. The mixture of toluene and anhydrous
sodium sulfate was shaken so that all moisture associated
with toluene could be removed. The toluene in test tube
was clear after shaking.
The 0.5 mL of crystal-clear toluene solution containing
extracted cholesterol was mixed with 0.5 mL of internal
standard solution in a 2.0-mL vial before being subjected to
the GC system. There was no evaporation, reconstitution,
and derivatization needed, as compared to the original
procedure.
2.2. Cholesterol determination of longissimus muscle from
purebred cattle
The cattle used in this study had been assembled,
weighed, blocked by breeds, assigned to feedlot pens, and
fed with the identical commercial nishing diet that were
isonitrogenous and isocaloric prior to harvest at a
commercial processing plant in Amarillo, TX. After a 48-
h chill, the carcasses were assigned a USDA Quality grade
and transferred to the Texas Tech University G.W Davis
Meat Science Laboratory for further fabrication. Samples
of LM were obtained from 18 purebred steers which
consisted of Angus (AN, n 5), Brahman (BR, n 4), and
Romosinuano (RM, n 9) breeds. The LM samples were
collected between the 10th and 13th ribs, frozen immedi-
ately in liquid nitrogen, and stored in a 80 1C freezer for
sample preparation.
Frozen LM were later trimmed of all external fat, which
left only white ecks of marbling within muscle bundles.
Trimmed samples were chopped, homogenized to nely
divided powder, and stored at 80 1C for subsequent
extraction and analysis.
The method for cholesterol quantication was modi-
ed from the AOAC Ofcial Method 994.10 and validated
using matrices of meat samples. The intramuscular
fat content of LM was determined using the Soxhlet
technique.
2.2.1. Crude fat determination
Crude fat was determined using Soxhlet extraction
technique (AOAC Ofcial Method 991.36). About 45 g
of frozen muscle powder was placed in an aluminum pan
and accurately weighed. The sample was dried for 18 h at
100110 1C. After moisture data were collected, the dried
sample was covered by layer of non-absorbent cotton, and
the pan was folded, weighed, and placed in Soxhlet
apparatus for 18 h. The sample pan was then dried for
1 h at 100110 1C before the dried weight was recorded.
2.3. Gas chromatographic analysis
The liberated cholesterol was quantied using the
Agilent 6890N gas chromatographic system and the DB-
17 capillary column (30 m0.250 mm0.15 mm, Agilent
Technologies Inc., CA, USA). The DB-17 has mid-polarity
and is suitable for analysis of free steroids. One microliter
(1.0 mL) of analyte mixture was injected into GC system
with split/splitless injector and ame ionization detector.
The inlet temperature was 250 1C and split ratio was 10:1.
The carrier gas was helium at 2.5 mL/min constant ow.
The oven was programmed initially at 250 1C, held for
5 min, followed by increases of 5 1C/min up to 260 1C, and
held for 8 min. Total time for gas chromatographic
determination was 15 min. The detector was set at 300 1C
with 200 mL/min airow, 80 mL/min hydrogen ow, and
40 mL/min helium makeup ow.
2.4. Data calculation and statistical analysis
Data obtained from chromatograms were calculated
using the standard curve of the cholesterol standard
solutions. The ratios of standard peak areas to the
corresponding internal standard peak areas were plotted
against standard concentrations, and the slope of standard
curve was calculated using method of least squares due to
the linear relationship. The internal standard concentration
was the same in all standard and sample solutions.
The resolution of column (R
s
) provides a quantitative
measure of a columns ability to separate two analytes. The
resolution coefcient was calculated by the following
equation:
R
s
2
t
R;cholesterol
t
R;5a-cholestane

W
cholesterol
W
5a-cholestane

,
where t
R
is the peak retention time, min and W is the peak
width, min.
A resolution coefcient greater than 1.0 indicates the
acceptable separation between two analytes (Skoog et al.,
1998).
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T.T.N. Dinh et al. / Journal of Food Composition and Analysis 21 (2008) 306314 308
The recovery efciency (R) was calculated using ratio of
cholesterol content (mg/100 g) recovered to the cholesterol
content (mg/100 g) added. The calculation was carried out
using the following equation:
R% C
r
100
1
C
a

,
where C
r
is the cholesterol content recovered (mg/100 g),
which was derived from subtraction of non-addition
concentration from the cholesterol-added concentration
and C
a
is the cholesterol content added, which was
expressed in mg/100 g of sample.
The CV (%) of the data set was calculated and used as
relative standard deviation (RSD, %) as in the AOAC
Guidelines for Single Laboratory Validation of Chemical
Methods for Dietary Supplements and Botanicals (AOAC,
2002). To determine the repeatability precision, the
Horwitz ratio (HORRAT), dened as ratio of determined
CV (%) to calculated CV (%) and presented in the Study
Directors Manual of AOAC International (AOAC, 2002),
was calculated.
The differences in cholesterol concentrations of LM
samples from 18 purebred cattle were analyzed by one-way
analysis variance (P 0.05), using a completely rando-
mized design with cattle breed serving as treatment. The
relationship between intramuscular fat content and cho-
lesterol concentration was also determined using Pearson
correlation coefcient. The statistical analysis was accom-
plished using the GLM, UNIVARIATE, and CORR
procedures of the Statistical Analysis System (SAS)
Institute version 9.1. Means were separated using protected
t-test with LSMEANS/PDIFF option of PROC GLM
(P 0.05).
3. Results
Free cholesterol without derivatization displayed a single
peak at 10.06 min from injection (Fig. 1). The smallest
sample peak that could be determined was twice as high as
the noise signals and was equivalent to concentration of
0.00054 mg/mL solvent (0.54 ppm) or 1.07 mg/100 g of
fresh muscle. Increasing the injection temperature, oven-
programmed temperature, and ow rate of carrier gas
accelerated the peak appearance (the retention times at
other conditions not shown, Po0.001). Despite the
acceleration, the cholesterol peak was well separated from
the internal standard peak (5a-cholestane at 5.15 min)
(Fig. 1, R
s
25.9). The chromatograms were very clean
without any peak interfering with cholesterol or the 5a-
cholestane peaks (Fig. 2). Hence, the results from this study
indicated that saponication by KOH/water resulted in
removal of all interferences in this matrix caused by fatty
acid esters. Moreover, the peak ratio was highly linear to
cholesterol standard concentration (R
2
0.99).
The method accuracy was proven by both the non-addi-
tion (Table 1) and the addition of free analyte (Table 2).
The analyses of SRM-1546 samples yielded concentrations
within the expected range of the certied concentration
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Fig. 1. Typical chromatogram of free cholesterol determination by GC.
Fig. 2. Typical chromatogram of cholesterol separation with peak area
report embedded, using a longissimus muscle sample.
Table 1
Method accuracy of a modied cholesterol determination procedure for
meat products determined by comparing analyzed concentrations of
standard reference material to the certied concentrations without adding
free cholesterol
SRM Certied
concentration
(mg/100 g)
Recovered
concentration
(mg/100 g)
Recovery
(%)
CV (%)
SRM-1546-1
a
77.1273.03 102.83 3.75
SRM-1546-2
b
7577.2 75.5774.03 100.76 5.08
Average 76.3572.12 101.80 3.34
Data represent recovery efciency.
a
SRM-1546-1: Standard Reference Material from the rst sample at
day 1 (n 6).
b
SRM-1546-2: Standard Reference Material from the second sample at
day 2 (n 6).
T.T.N. Dinh et al. / Journal of Food Composition and Analysis 21 (2008) 306314 309
(76.3572.12 mg/100 g as compared to 7577.2 mg/100 g;
P 0.08). These results were produced with high precision
(CV 3.34%) and there was no statistical difference
(P 0.45) in cholesterol content among SRM-1546 sam-
ples prepared on different days. The data of cholesterol
recovery tests (Table 3) revealed high recovery perfor-
mance (97.97100.17%) with high precision (CV 3.19
4.58%) of the modied procedure. Acceptable recovery
efciency is 90108% for concentrations around 0.1% or
100 mg/100 g (AOAC, 2002). There was, again, no sig-
nicant difference in recovery efciency among the four
sets of sample (P 0.79), regardless of sample type
(meat homogenate or LM), sources (different breeds of
cattle). It was more interesting that the recovery efciency
did not vary although fat content of samples was
signicantly different (from 2.4% to 21.0%; Po0.001).
The recovery efciency was analyzed to be independent of
the fat content of the samples (P 0.3).
The method stability was proven by its high repeatability
precision (CV 2.155.08%). The fact that Horwitz
ratios for all sample sets fell in the range of 0.52.0
(HORRAT 0.711.73) highlighted the precision of the
modied procedure (AOAC, 2002). Additionally, accord-
ing to the CV (%) obtained (Table 3), it was realized that
the method performance on high-fat samples (SRM-1546,
21.0% fat) had numerically higher coefcients of variation
than did the lower fat samples (LM samples, 2.49.3%).
However, this phenomenon was not statistically analyzed.
The modied method for cholesterol quantication was
applied to the analysis of cholesterol content of 18 LM
samples from Angus (AN, n 5), Brahman (BR, n 4),
and Romosinuano (RM, n 9) steers (Table 4). Choles-
terol content (mg/100 g) in these LM samples was found to
be signicantly different among the three breeds
(P 0.007). The cholesterol concentrations of LM samples
from Angus purebreds (70.25 mg/100 g) was higher than
was those of LM samples from Brahman and Romosinua-
no purebreds (64.77 and 65.76 mg/100 g; P 0.005 and
P 0.006, respectively). The Brahman and Romosinuano
LM samples did not differ in cholesterol content
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Table 2
Method accuracy of a modied cholesterol determination procedure for
meat products determined by adding free cholesterol into unknown
samples and standard reference material
Sample Added
amount per
sample
(mg)
Recovery (%) Acceptable
recovery
a
(%)
Range Mean CV, %
SRM1546-R
b
1.00 96.68107.22 100.17 3.81
90108
LM-1R
c
1.00 96.76104.99 99.22 3.19
LM-2R
d
1.00 92.86105.50 98.59 4.58
LM-3R
e
1.00 94.48103.35 97.97 4.11
Data represent recovery efciency.
a
Acceptable recovery for concentration of 0.1% (AOAC, 2002).
b
SRM-1546-R: Recovery sample using SRM1546 (standard reference
material) sample from the rst sample at day 1 (n 6). Samples were
treated as if their concentrations were unknown.
c
LM-1R: Recovery sample using the rst longissimus muscle (LM)
sample (n 6).
d
LM-2R: Recovery sample using the second LM sample (n 6).
e
LM-3R: Recovery sample using the third LM sample (n 6).
Table 3
Precision of a modied cholesterol determination procedure in meat products determined by comparing experimentally determined coefcient of variation
(CV, %) to calculated CV
Sample Fat (%) Cholesterol
concentration (%)
Calculated CV (%)
a
Determined CV (%) HORRAT
b
SRM-1546-1
c
21.00 0.077 2.93 3.75 1.28
SRM-1546-2
d
21.00 0.075 2.94 5.08 1.73
LM-1
e
9.34 0.074 2.95 2.22 0.75
LM-2
f
6.16 0.069 2.98 2.41 0.81
LM-3
g
2.39 0.063 3.02 2.15 0.71
a
Calculated CV: function of concentration: CV (%) [Concentration/100]
0.15
.
b
HORRAT: Horwitz ratio presented in the Study Directors Manual of AOAC International; dened as ratio of determined CV (%) to calculated CV
(%). The acceptable ratio falls into range of 0.52.0.
c
SRM-1546-1: Standard Reference Material from the rst sample at day 1 (n 6).
d
SRM-1546-2: Standard Reference Material from the second sample at day 2 (n 6).
e
LM-1: The rst longissimus muscle (LM) sample (n 6).
f
LM-2: The second LM sample (n 6).
g
LM-3: The third LM sample (n 6).
Table 4
Cholesterol concentration as determined by a modied cholesterol
procedure in longissimus muscle samples from Angus (n 5), Brahman
(n 4), and Romosinuano (n 9) in relation to intramuscular (i.m.) fat
content
Breed Cholesterol content
(mg/100 g)
Fat (%) Fatness
correlation
Angus (n 5) 70.25a 7.00 r 0.90,
Po0.001 Brahman (n 4) 64.77b 3.56
Romosinuano (n 9) 65.76b 3.53
Within column of cholesterol content, means without common letter (a, b)
differ (Po0.05). Pearson coefcient of correlation is signicant if Po0.05.
T.T.N. Dinh et al. / Journal of Food Composition and Analysis 21 (2008) 306314 310
(P 0.52). Finally, the cholesterol content of LM samples
increased with an increase in intramuscular fat due to
its signicant positive correlation to the muscle fatness
(r 0.9, Po0.001).
4. Discussion
The method for cholesterol quantication in multi-
component foods was adopted in 1976 (AOAC, 1996a, b;
Punwar, 1976). This method involved lipid extraction,
saponication, extraction of unsaponied matter with
benzene, derivatization, and GC determination with
5a-cholestane as the internal standard. Cholesterol recov-
ery from this method fell into the range of 85.891.4% with
CV (%) of 12.514.4% for several types of food samples
(Punwar, 1976). It has been conrmed that saponication
or hydrolyzation is a very critical step when determining
total cholesterol (Hwang et al., 2003; Rodriguez-Palmero
et al., 1994; Kaneda et al., 1980; Naeemi et al., 1995; Klatt
et al., 1995). Saponication not only liberates cholesterol,
but also puries it from other components such as fatty
acids. Despite the fact that lipids in samples were formerly
extracted before cholesterol liberation was performed, Van
Elswyk et al. (1991) concluded that direct saponication
was the most accurate method to release free cholesterol.
This conclusion was previously stated by Adams et al.
(1986) when these authors evaluated direct saponication
using ethanolic KOH to prepare meat sample, and they
suggested that the lipid extraction step could be eliminated.
Their data showed excellent repeatability (CV 1.74%)
and recovery (99.8%), which was rarely seen in the current
studies. Direct saponication in their study yielded higher
recovery efciency than did other methods, even the
AOAC Ofcial Method 43.235. The saponication techni-
que can be performed with KOH in either water (AOAC,
1996a, b; Klatt et al., 1995), or alcohol (Fenton, 1992;
Hwang et al., 2003). In this study, the saponication using
KOH in water with addition of ethanol was found to be a
very effective mean to remove all fatty acids in the form of
soaps in the aqueous phase, making them separable during
extraction and purication. Slightly different from this
study, De La Huerga and Sherrick (1972) concluded that
the saponication procedure of Abell et al. (1952) was most
suitable by using 0.33 or 0.5 M KOH solution in ethanol.
However, their conclusion was based on the test of serum
cholesterol, not food samples or materials with high
protein content (LM samples: 2021% protein, data not
shown) or complex cell structure like muscle tissue. In
contrast, to saponify food samples, other studies used
aqueous KOH with signicant addition of ethanol (Indyk,
1990; Beyer and Jensen, 1989; Patton et al., 1990; Kovacs
et al., 1979; Van Elswyk et al., 1991; Kaneda et al., 1980).
Especially, Indyk (1990) and Van Elswyk et al. (1991) used
the same KOH-in-water (50%)/ethanol ratio (1:5) as did
this study, which resulted in good separation and recovery;
while the ratio reported by Kovacs et al. (1979) was slightly
different (1:4). Although Fenton (1992) reported some
peaks of free fatty acid, it was interesting that no additional
peaks overlapped the cholesterol and 5a-cholestane peaks
in this study. Hwang et al. (2003) suggested the use of
KOH/methanol saponication and BF3 methylation to
eliminate fatty acid interferences together with ether to
extract cholesterol; however, the chromatogram in their
study showed some signicant peaks other than those of
cholesterol and the internal standard. The existence of
those unnecessary peaks limits the ability to quicken peak
appearance (by increasing carrier gas velocity or tempera-
ture over the optimum value for theoretical plate number)
due to possible peak overlap caused by a decrease in
number of theoretical plates and resolution (Skoog et al.,
1998).
The cleanness of resultant chromatogram was caused
not only by saponication but also by extraction and
purication. Cholesterol with very low polarity in saponi-
ed mixture had to be extracted using a solvent that could
mix well in a waterethanol environment and create a
homogeneous-phase extraction. Other studies used either
ether (Hwang et al., 2003) or hexane (Fenton, 1992; Fenton
and Sim, 1991) to extract cholesterol, claiming that no
extra clean-up steps required. Their data showed that the
recovery efciencies were comparable to those from this
study. The use of ether as a solvent, however, resulted in
the formation of peroxides that could cause degradation of
sterols (Fenton, 1992). Indyk (1990) and Fenton and Sim
(1991) extracted cholesterol only once with hexane and
reported that it was sufcient for an accurate recovery.
However, Patton et al. (1990), Kovacs et al., (1979), and
Al-Hasani et al. (1990, 1993) all had to use multiple hexane
extractions to obtain adequate recoveries. Lognay et al.
(1989) showed a need for ve diethyl ether extractions to
obtain quantitative recovery of cholesterol. In this study,
these solvents, especially hexane, were also evaluated and it
was found to require at least double extractions, similar to
fatty acid extraction using hexane (Li and Watkins, 2003),
to obtain over 90% recovery (data not shown). This
phenomenon was probably due to their extremely low
polarity, which results in their exceptional exclusion from
water-based saponied mixture. The multiple-extraction
process requires evaporation of the solvent and reconstitu-
tion of dried residue, creating additional preparation steps.
Hence, single extraction using toluene was chosen due to its
desirable solvent properties and relatively low toxicity
(Li and Watkins, 2003). The 10-mL toluene extraction was
found to be appropriate due to high recoveries of the
method (Table 2), which were very dependent of solvent
nature. Oles et al. (1990) studied some factors affecting the
recovery of cholesterol from various food matrices: type of
alcohol, extraction solvent, use of antioxidants, time and
temperature of hydrolysis; and found that toluene extrac-
tion resulted in signicantly higher recoveries than a
mixture of hexanediethyl ether (85:15, v/v). This result
together with the ones in this study, again, conrmed the
suitability of toluene in extracting cholesterol. Occasionally
however, an emulsion may form which can be remedied by
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use of anhydrous sodium sulfate, which can absorb
moisture and dry the solvent. With adoption from the
AOAC 994.10 Ofcial Method, the saponication and
washing techniques were found to be very effective in
eliminating all unrecognizable peaks that might interfere
with the peaks of cholesterol and 5a-cholestane, allowing
an increase in analyzing speed without jeopardizing
separation quality.
The decrease of the sample size from 5 to 10 g (to obtain
at least 1 g of fat) to 1 g was proven to be time and cost-
effective without experiencing any uncertainty of method
performance. The modication signicantly reduced 90%
of the costs of chemicals and solvent use, as compared to
the AOAC Ofcial Method 994.10 (AOAC, 1996a, b).
Total time used in this method was 3045 min per sample,
as compared to at least 7080 min for saponication only,
and extra time for drying, reconstitution, and derivatiza-
tion in the original method (AOAC, 1996a, b). This
timesaving would lead to very high productivity and
decreased errors related to method complication. The
chromatographic separation of free cholesterol without
derivatization to trimethylsilyl ethers has been studied and
documented in recent years due to the development of
capillary GC columns with high resolution and heat
tolerance (Lee et al., 1998; Fenton, 1992; Wu et al., 1997;
Hwang et al., 2003; Thompson and Merola, 1993;
Al-Hasani et al., 1993). These studies have found that
cholesterol was not required to be transformed to more
volatile derivatives, such as silylated form to be quantita-
tively detected and quantied (Hwang et al., 2003). The
results from those studied were very comparable with the
traditional derivatization techniques. As with the current
study, although detector response for cholesterol was
slightly lower than its silylated ethers (Hwang et al.,
2003), the method sensitivity still allowed for quantitative
detection of cholesterol content in meat samples.
In a collaborative study, the AOAC 994.10 method had
an average repeatability of 4.81% and cholesterol recovery
of 100.03% when performed with NIST egg powder (Klatt
et al., 1995). Accordingly, the modication used in this
study compared favorably with its original method with an
average repeatability and recovery efciency of 3.12% and
98.99%, respectively. The decrease in saponication time
(from at least 70 to 15 min) did not produce any negative
effect on recovery efciency. The decision to decrease
saponication time was made because the large amount of
cholesterol in muscle is in free form, hydrogen-bonded or
non-covalently bonded, and packed into the cell membrane
rather than in esteried form with fatty acids (Karp, 2005).
The utilization of aqueous KOH with addition of alcohol
instead of alcoholic KOH as used in many studies
previously mentioned suggested that the presence of water
played some role in hydrolyzing cell membrane and
releasing cholesterol. Hence, using aqueous KOH with
later addition of ethanol was suitable for hydrolyzing
fresh meat samples. Hwang et al. (2003) suggested using
methylation as an additional treatment after saponication
to hydrolyze ester bonds between cholesterol and fatty
acids. However, this technique might be only useful for
lipid samples such as oil, extracted fat, of which ester
linkages are much more predominant than of muscle-based
samples, which contain more free cholesterol, a major
component of cell membrane.
Of most concern was that of lower repeatability
precision associated with high-fat samples. Most fatty
acids in the sample were converted to potassium salts or
soap, which could act as emulsifying agents and prevent the
complete separation of toluene from the rest of the
mixture. As observed, the solution cloudiness seemed to
appear in samples with higher fat content, especially in
SRM-1546 samples. Fenton and Sim (1991) also reported
that fat content could affect the extraction of cholesterol.
By increasing the amount of soybean oil added to chole-
sterol standard (for 0170 mg), these authors observed the
efciency of rst extraction to be decreased from 98.2% to
94.1%. However, this study showed that higher fat content
only caused an increased variation in cholesterol determi-
nation while the average recovery efciency was not
inuenced due to no signicant difference in recoveries
among samples (P 0.79). Therefore, the samples with fat
contents of 920% should be processed with special care to
avoid the emulsication. Additionally, as cholesterol
content begins to approach the detection limit, some
analyte enrichment techniques such as increasing sample
size or solvent evaporation and reconstitution should be
performed to obtain sufcient cholesterol in nal solution
prior to GC analysis.
The validated method was used to quantify cholesterol in
LM samples from cattle. The signicantly fatter Angus LM
samples had signicantly higher cholesterol content
(70.25 mg/100 g) as compared with Brahman and Romosi-
nuano (P 0.005, 0.006, respectively). The cholesterol
content was also found to be positively correlated to
muscle fatness. Generally, cholesterol content in meat is
not necessarily increased with fatness because a large
amount of cholesterol is found in cell membranes as the
free form (Karp, 2005). However, cholesterol is also stored
(e.g. in cytosol) in esteried form with fatty acids,
especially with long-chained fatty acids (Kvilekval et al.,
1994; Xie et al., 2002; Lewis-Barned et al., 2000).
Consequently, it is possible that the stored cholesterol
becomes more signicant when fat content is increased
because it was hypothesized and proven that fatty acids,
especially some unsaturated fatty acids, regulate cholester-
ol esterication and secretion (Xie et al., 2002). Xie et al.
(2002) demonstrated that unsaturated fatty acids drove the
esterication reaction and enhanced lipoprotein cholesterol
secretion by the liver under conditions where cholesterol
balance across this organ was constant.
Regarding the cholesterol level in beef muscle, Padre
et al. (2006) reported the lower cholesterol content
(45.745.8 mg/100 g) in LM of bulls and steers nishing
in pasture systems as opposed to traditional nishing;
however, the intramuscular fat content was also much
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lower (1.713.38%) than that in LM from this study. They
also found no difference in cholesterol content among
animals despite the variation in fatness. Rule et al. (1997)
emphasizes that breed, nutrition, and gender do not affect
cholesterol concentration of bovine skeletal muscle. They
also suggested that changes in cholesterol content in muscle
might require marked changes in structure of muscle cells
associated with a marked redistribution of membrane fatty
acids. Additionally, Rule et al. (2002) reported slightly
higher cholesterol content of beef LM (5253 mg/100 g)
than those previously mentioned in Padre et al. (2006)
study, but their i.m. fat content was not reported.
However, by investigating lipid characteristics of long-
issimus thoracis between Angus and Wagyu steers fed to
the US and Japanese endpoints, Chung et al. (2006)
reported very similar levels of cholesterol in longissimus
thoracis from Angus (7278 mg/100 g) as compared to LM
samples from Angus in this study. They also indicated a
positive, but weak, relationship between cholesterol con-
tent in muscle and intramuscular fatness. Interestingly,
Rhee et al. (1982) found that cholesterol concentrations
were directly related to the USDA marbling scores.
Cholesterol was increased from 51.77 to 64.74 mg/100 g
with an increase in marbling scores from practically devoid
(2.73% i.m. fat) to moderately abundant (12.08% i.m. fat).
The results from studies previously mentioned indicated an
unpredictable relationship between fatness and cholesterol.
It might be hypothesized that genetic differences between
Angus and the other two breeds possibly cause a signicant
inuence on cell structure, especially of adipocytes, which
could result in higher cholesterol levels. However, the
relationships among cholesterol level and quantity, size,
and structure of adipose and muscle cells are not well
understood and need further study.
5. Conclusion
The modications, which were made to AOAC Ofcial
Method 994.10, revealed a new cholesterol quantication
procedure that was validated to be efcient and reliable.
The new procedure was not only faster, but also more cost-
effective. The procedure, which was previously outlined,
allowed a quick and quantitative detection of cholesterol
concentration in fresh meat samples. The results from this
study were comparable to most recent studies in precision
and accuracy, which were shown to be quantitatively
applicable. The cholesterol contents determined were
similar to those from other studies and to certied
reference materials. The fact that the SRM is a meat
homogenate of pork and chicken products blended
together in a commercial process (NIST, 2004) indicates
that the modied method was a precise and accurate
determination of the true cholesterol content of not only
fresh meat samples but also variety of meat products.
However, further validation is recommended and can be
useful for an application of this modied method in other
types of meat samples.
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