45.4.

06
AOAC Official Method 976.26
Cholesterol
in Multicomponent Foods
Gas Chromatographic Method
First Action 1976
Final Action 1977

A. Principle

Lipid is extracted from sample by mixed solvent and saponified.

Unsaponifiable fraction containing cholesterol and other sterols is
extracted with benzene. Sterols are derivatized to form trimethylsilyl
(TMS) ethers which are determined quantitatively by GC, using
5-cholestane as internal preparing.
B. Apparatus

(a) Centrifuge tubes.Pyrex No. 13, 15 mL. Silanize tubes as

follows: Rinse clean tubes with anhydrous methanol and dry 30 min
at 110. Transfer tubes to desiccator. Fill tubes with 10% solution of
dimethyldichlorosilane (DMCS) in toluene, stopper tubes, and let
stand 1 h. Drain tubes and rinse thoroughly with anhydrous methanol. Dry in 110 oven before use. After use, clean tubes with
methanol, H2O, and methanol, in that order. Dry tubes in 100 oven
before use. Tubes can be re-used without silylation as long as strong
alkali wash is avoided.
(b) Gas chromatograph.With H2 flame ionization detector,
on-column injection system, and 2.4 m (8) 3 mm id U-shaped
glass column packed with 0.5% Apiezon L (No. 08304, Alltech-Applied Science) on 80100 mesh Gas-Chrom Q (No. 02002, AlltechApplied Science Laboratories, Inc.). Alternative column: 1.8 m (6)
4 mm id U-shaped glass column packed with 1% SE-30 on
100120 mesh Gas-Chrom Q (No. 12409, Alltech-Applied Science
Laboratories, Inc.). Operating conditions: temperatures ()flash
heater 275, detector 275, column 230; flow rates (mL/min)N2
(ultra high purity grade) ca 50, to elute cholesterol in 911 min, H2
ca 35, air 350; electrometer sensitivity 1 109 amp full-scale
deflection with 1 mV recorder.
(c) Homogenizer.Sorvall Omnimixer (DuPont Instrument Co.,
Sorvall Operations, Pecks Ln, Neweighton, CT 06470), or equivalent, for use with 12 oz (350 mL) wide-mouth screw-cap jars.
(d) Magnetic stirrer-hot plate.With variable speed and heat
controls.
(e) Rotary evaporator.With glass condenser flask between
concentration flask and metal shaft.
(f) Test tube mixer.Vortex-Genie mixer (No. 12-812, Fisher
Scientific Co.), or equivalent
C. Reagents

(Caution: Silanes are toxic. Avoid contact with skin and eyes. Use
effective fume removal device.)
(a) Cholesterol standard solutions.Standard cholesterol available as No. 21502, Alltech-Applied Science Laboratories, Inc. (1)
Stock solution.1.0 mg/mL DMF. (2) Working solutions.Dilute
stock solution with DMF to obtain concentration range from 0.05 to
0.5 mg/mL.
(b) 5-Cholestane internal standard solutions.Standard 5cholestane available as No. 19505, Alltech-Applied Science Laboratories, Inc. (1) Stock solution.1.0 mg/mL n-heptane. (2) Working
solution.0.2 mg/mL. Dilute stock solution with n-heptane to obtain concentration of 0.2 mg/mL.

(c) Dimethyldichlorosilane.No. 18008, Alltech-Applied Science, or equivalent.

(d) Dimethylformamide.Distilled in glass (Burdick & Jackson
Laboratories, Inc.; Anspec Co., Inc., PO Box Ann Arbor, MI 48107;
or equivalent).
(e) Glass wool.Silane-treated (No. 14502, Alltech-Applied
Science, or equivalent).
(f) n-Heptane.Distilled in glass (Burdick & Jackson Laboratories, Inc., Eastman Kodak Co., No. 2215, or equivalent).
(g) Hexamethyldisilazane (HMDS).No. 18006, Alltech-Applied Science, Pierce Chemical Co., or equivalent.
(h) Concentrated potassium hydroxide solution.Dissolve 60 g
KOH in 40 mL H2O.
(i) Reagent alcohols.ethyl alcohol-methanol-isopropanol (90
+ 5 + 5). Following reagent alcohols are satisfactory: EM Diagnostics, A Div. of EM Industries Inc., 480 Democrat Rd, Gibbstown, NJ
08027 (no longer available); Wilkens-Anderson Co., 4525 W Division St, Chicago IL 60651; No. 7019 or No. 7006, Mallinckrodt
Chemical Works.
(j) Toluene.Nanograde, distilled in glass (Mallinckrodt Speciality Chemical Co., or equivalent).
(k) Trimethylchlorosilane (TMCS).No. 18010, Alltech-Applied Science, or equivalent.
(l) Trimethylsilyl (TMS) reagent.HMDS-TMCS-pyridine (9 +
6 + 10).
(m) Adsorbent.Celite 545, acid-washed (Johns-Manville Products Corp.), or equivalent, is usually suitable for column chromatography. When interfering materials are present, purify as follows:
place pad of glass wool in base of chromatographic tube 100 mm
diameter and add siliceous earth to height ca 5 times diameter. Add
volume HCl equal to ca 13 volume of earth, and let percolate. Wash
with methanol, using small volumes at first to rinse walls of tube,
and then until washings are neutral to moistened indicator paper.
Extrude into shallow dishes, heat on steam bath to remove methanol,
and dry at 105 until material is powdery and methanol free. Store
in tightly closed containers.
D. Preparation and Packing of Gas Chromatographic Column

(Caution: See Appendix B, safety notes on hydrofluoric acid and

isooctane.)
Attach empty column to aspirator and draw through 5% HF
solution. Stop vacuum with pinch clamp, quickly cap both ends of
column with rubber stoppers, and let column stand filled with 5%
HF solution 10 min.
Attach column to aspirator again, draw off 5% HF solution, and
rinse with ca 150 mL H2O followed by 150 mL anhydrous methanol.
Finally, rinse column with 150 mL isooctane. Draw air through
column until dry. Fill column with TMS reagent, (l), by pulling it
through slowly with aspirator. Plug both ends of column and let stand
30 min. Draw TMS reagent through and rinse immediately with 100
mL anhydrous methanol, followed by 200 mL isooctane. Let column
dry under vacuum.
Use commercially prepared column packing of 0.5% Apiezon L
on 80100 mesh Gas-Chrom Q (Alltech-Applied Science Laboratories, Inc.), or prepare as follows: Weigh 0.5 g Apiezon L into 100
mL beaker, add 80 mL toluene, stir magnetically until it dissolves
completely, and transfer to 500 mL Erlenmeyer, rinsing beaker with
four 5 mL portions toluene. Weigh 10 g 80100 mesh Gas-Chrom
Q and add to Apiezon L solution. Stopper flask and shake to make

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slurry. Immediately pour slurry through buchner-type fritted glass

Pyrex filter (medium porosity) under vacuum, stirring continuously
until all liquid is drawn off. Measure filtrate in graduate and determine amount Apiezon L adsorbed. Let stand under vacuum, stirring
occasionally until almost dry. Transfer packing to porcelain evaporation dish and dry completely in 110120 oven. Store in glass
bottle until ready to use.
Heat packing 15 min in 100 oven. Plug detector end of silanized
column with 6 mm silanized glass wool and attach to aspirator. Add
warm packing through funnel attached to column and gently tap
column. Finally, plug injection port end with silanized glass wool.
Condition column 24 h at 235 with N2 flow.

pentane and shake 1 min. Let layers separate. Drain aqueous (lower)
layer into second separator. Repeat extraction with 100 mL ether and
100 mL pentane, shaking 1 min after each addition. If layers do not
separate, add 40 mL reagent alcohol, gently rotate end over end 10
times, and let stand 5 min. Discard aqueous layer. Filter combined
ether extracts through column of anhydrous Na2SO4 into 600 mL
beaker. Evaporate to ca 10 mL under gentle N2 stream on 70 H2O
bath. Transfer extract to 300 mL glass-stoppered Erlenmeyer, rinsing
beaker with pentane. Evaporate to dryness under gentle N2 stream
on steam bath, and proceed as in 976.26G, paragraph 2.

E. Moisture Determination

(Caution: See Appendix B, safety notes on distillation, pipets, benzene, and petroleum ether.)

Accurately weigh ca 5.0 g sample into tared Al dish, place in

circulating-type 100 air oven, and dry overnight or 3 h at 110.
Cover, and let cool in desiccator. Weigh accurately and determine
moisture content to adjust for H2O to be added in 976.26F.
F. Extraction of Lipid

(Caution: See Appendix B, safety notes on distillation, diethyl

ether, chloroform, methanol, and pentane.)
(a) For foods other than dried whole egg solids, mayonnaise, and
nonfat dry milk.Accurately weigh known amount sample containing ca 0.51 g fat and transfer quantitatively to homogenizer cup
with 100.0 mL anhydrous methanol. On basis of moisture determination, add enough H2O to bring total H2O content in extraction to
40 mL. Add 50 mL CHCl3 and blend 3 min at high speed. (Ratio of
CHCl3-methanol-H2O must be 5010040 in this single-phase extraction.) Add additional 50 mL CHCl3 and blend 0.5 min at medium
speed. Then add 50 mL H2O and again blend 0.5 min at medium
speed. Filter homogenate under vacuum into 1 L suction flask
through Bchner fitted with Whatman No. 1 paper containing 2 g
diatomaceous earth. Pour filtrate into 500 mL graduate. Re-extract
filter cake and paper with ca 90 mL CHCl3 and filter extract without
diatomaceous earth. Rinse cup and filter cake with two 15 mL
portions CHCl3. Add these rinses to original filtrate and let layers
separate. (If emulsion develops, centrifuge filtrate 5 min at 2500
rpm.) Record volume of CHCl3 (lower) layer and aspirate aqueous
alcohol layer. (Total volume of CHCl3 layer should be ca 200 mL.)
Proceed as in 976.26G.
(b) For dried whole egg solids.Use acid hydrolysis,
925.32A(b) (see 34.1.07), and proceed as in 976.26G, paragraph 2.
(c) For mayonnaise.Accurately weigh ca 1.21.5 g sample and
transfer quantitatively to homogenizer cup with 100.0 mL anhydrous methanol. Add 40 mL H2O and 50 mL CHCl3 and blend 3
min at medium speed. Add additional 50 mL CHCl3 and blend 0.5
min at medium speed. Then add 50 mL H2O and again blend 0.5 min
at medium speed. Transfer homogenate to 500 mL separator. Rinse
cup with three 20 mL portions CHCl3 and add these rinses to
separator. Mix by gently rotating separator end to end. Let layers
separate. Drain CHCl3 (lower) layer into graduate. Rinse aqueous
methanol layer with 40 mL CHCl3, add rinse to graduate, and mix.
Record volume of CHCl3 layer. Proceed as in 976.26G, using 150
mL aliquot CHCl3-lipid extract and 250 mL beaker.
(d) For nonfat dry milk.Accurately weigh ca 25 g sample and
transfer quantitatively to 300 mL Erlenmeyer containing 100 mL
H2O. Stir to mix thoroughly, and refrigerate overnight. Pour reconstituted milk into 1 L separator, add 100 mL reagent alcohol, (i), and
shake 1 min. Add 100 mL ether and shake 1 min. Add 100 mL

G. Saponification and Extraction of Unsaponifiable Fraction

Filter 100 mL aliquot CHCl3-lipid extract through glass funnel

containing small pledget of glass wool and ca 25 g anhydrous
Na2SO4 into 150 mL beaker. Rinse Na2SO4 with 15 mL CHCl3 and
evaporate extract to dryness under gentle N2 stream on 90o H2O bath
or steam bath. Dissolve residue in ca 70 mL petroleum ether and
filter through Whatman No. 1 paper containing ca 20 g anhydrous
Na2SO4 into 300 mL glass-stoppered Erlenmeyer. Rinse beaker and
Na2SO4 with several 10 mL portions petroleum ether. Evaporate to
dryness under gentle N2 stream on steam bath.
Introduce magnetic stirring bar into Erlenmeyer and place on
magnetic stirrer-hot plate. With gentle stirring, slowly add 8 mL
concentrated KOH solution, (h), and 40 mL reagent alcohol, (i).
Attach condenser, turn on magnetic stirrer-hot plate, and reflux
solution 1 h. Turn off heat and add 60 mL reagent alcohol through
condenser into saponified solution while stirring and cooling. When
sample ceases to reflux, remove condenser, and pipet 100 mL
benzene into sample while slowly stirring. Remove stirring bar,
stopper flask, and shake vigorously 30 s.
Pour into 500 mL separator without rinsing. Add 200 mL 1N KOH
and shake vigorously 10 s. Let layers separate and discard aqueous
(lower) layer (will be turbid). Wash benzene layer with 40 mL 0.5N
KOH, rotate gently end to end 10 s, and discard aqueous (lower)
layer. Pour benzene layer into 250 mL separator. Back-wash benzene
layer with 40 mL H2O by gently rotating separator end to end 10
times. Repeat H2O wash 3 more times. pH of last H2O wash should
be ca 7. Pour benzene extract from top of separator, filtering through
Whatman No. 4 paper containing ca 15 g anhydrous Na2SO4 into
125 mL glass-stoppered Erlenmeyer. Add ca 20 g anhydrous
Na2SO4; stopper and shake flask vigorously. Let stand 15 min.
Pipet 50 mL aliquot into 100 mL round-bottom glass-stoppered
flask and evaporate to dryness on rotary evaporator at 40. Add 3 mL
acetone and again evaporate to dryness. Dissolve residue in 3 mL
DMF.
H. Derivatization of Cholesterol Standards and Gas Chromatographic Calibration

Transfer 1.0 mL of each cholesterol working standard solution,

976.26C(a)(2), to 15 mL silanized centrifuge tube. (Keep DMCSsilanized centrifuge tubes clean and dry.) Add 0.2 mL HMDS and
0.1 mL TMCS. Stopper tube and shake vigorously on test tube mixer,
(f), or by hand for 30 s. Let solution stand undisturbed 15 min. Add
1 . 0 m L 5-cholestane internal standard working solution,
976.26C(b)(2), and 10 mL H2O to tube. Shake vigorously 1 min and
centrifuge 2 min.
Inject duplicate 3 L or other appropriate volumes (use same
volume throughout for all standards and samples) heptane layer into

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gas chromatograph. Adjust GC parameters to give retention times of

ca 5 min for 5-cholestane and 10 min for cholesterol. Determine
area of each peak by using height-width measurement or digital
integrator. Divide cholesterol peak area by internal standard peak
area to obtain standard response ratio. Average results for duplicate
determinations. Plot average response ratio (y-axis) against cholesterol concentration (mg/mL) (x-axis). Standard response ratio plot
should bracket sample response ratio.
I. Derivatization and Analysis of Samples

Transfer 1.0 mL sample solution, 976.26G, to 15 mL silanized

centrifuge tube and proceed as in 976.26H, beginning Add 0.2 mL
HMDS . . . If GC response is beyond scope of standard calibration,
dilute sample solution and derivatize again.
mg Cholesterol/100 g sample = (mg/mL cholesterol in sample from
standard curve 100)/ (g/mL sample used for derivatization)
References: JAOAC 58, 804(1975); 59, 46(1976).
CAS-57-88-5 (cholesterol)

Copyright 1998 AOAC INTERNATIONAL