45.4.06 (c) Dimethyldichlorosilane.No.

18008, Alltech-Applied Sci-

AOAC Official Method 976.26 ence, or equivalent.
Cholesterol (d) Dimethylformamide.Distilled in glass (Burdick & Jackson
in Multicomponent Foods Laboratories, Inc.; Anspec Co., Inc., PO Box Ann Arbor, MI 48107;
Gas Chromatographic Method or equivalent).
First Action 1976 (e) Glass wool.Silane-treated (No. 14502, Alltech-Applied
Final Action 1977 Science, or equivalent).
(f) n-Heptane.Distilled in glass (Burdick & Jackson Laborato-
A. Principle
ries, Inc., Eastman Kodak Co., No. 2215, or equivalent).
Lipid is extracted from sample by mixed solvent and saponified. (g) Hexamethyldisilazane (HMDS).No. 18006, Alltech-Ap-
Unsaponifiable fraction containing cholesterol and other sterols is plied Science, Pierce Chemical Co., or equivalent.
extracted with benzene. Sterols are derivatized to form trimethylsilyl (h) Concentrated potassium hydroxide solution.Dissolve 60 g
(TMS) ethers which are determined quantitatively by GC, using KOH in 40 mL H2O.
5-cholestane as internal preparing. (i) Reagent alcohols.ethyl alcohol-methanol-isopropanol (90
B. Apparatus + 5 + 5). Following reagent alcohols are satisfactory: EM Diagnos-
tics, A Div. of EM Industries Inc., 480 Democrat Rd, Gibbstown, NJ
(a) Centrifuge tubes.Pyrex No. 13, 15 mL. Silanize tubes as
08027 (no longer available); Wilkens-Anderson Co., 4525 W Divi-
follows: Rinse clean tubes with anhydrous methanol and dry 30 min
sion St, Chicago IL 60651; No. 7019 or No. 7006, Mallinckrodt
at 110. Transfer tubes to desiccator. Fill tubes with 10% solution of
Chemical Works.
dimethyldichlorosilane (DMCS) in toluene, stopper tubes, and let
(j) Toluene.Nanograde, distilled in glass (Mallinckrodt Speci-
stand 1 h. Drain tubes and rinse thoroughly with anhydrous metha-
ality Chemical Co., or equivalent).
nol. Dry in 110 oven before use. After use, clean tubes with
(k) Trimethylchlorosilane (TMCS).No. 18010, Alltech-Ap-
methanol, H2O, and methanol, in that order. Dry tubes in 100 oven
plied Science, or equivalent.
before use. Tubes can be re-used without silylation as long as strong
(l) Trimethylsilyl (TMS) reagent.HMDS-TMCS-pyridine (9 +
alkali wash is avoided.
6 + 10).
(b) Gas chromatograph.With H2 flame ionization detector,
(m) Adsorbent.Celite 545, acid-washed (Johns-Manville Prod-
on-column injection system, and 2.4 m (8) 3 mm id U-shaped
ucts Corp.), or equivalent, is usually suitable for column chromatog-
glass column packed with 0.5% Apiezon L (No. 08304, Alltech-Ap-
raphy. When interfering materials are present, purify as follows:
plied Science) on 80100 mesh Gas-Chrom Q (No. 02002, Alltech-
place pad of glass wool in base of chromatographic tube 100 mm
Applied Science Laboratories, Inc.). Alternative column: 1.8 m (6)
diameter and add siliceous earth to height ca 5 times diameter. Add
4 mm id U-shaped glass column packed with 1% SE-30 on
100120 mesh Gas-Chrom Q (No. 12409, Alltech-Applied Science volume HCl equal to ca 13 volume of earth, and let percolate. Wash
Laboratories, Inc.). Operating conditions: temperatures ()flash with methanol, using small volumes at first to rinse walls of tube,
heater 275, detector 275, column 230; flow rates (mL/min)N2 and then until washings are neutral to moistened indicator paper.
(ultra high purity grade) ca 50, to elute cholesterol in 911 min, H2 Extrude into shallow dishes, heat on steam bath to remove methanol,
ca 35, air 350; electrometer sensitivity 1 109 amp full-scale and dry at 105 until material is powdery and methanol free. Store
deflection with 1 mV recorder. in tightly closed containers.
(c) Homogenizer.Sorvall Omnimixer (DuPont Instrument Co., D. Preparation and Packing of Gas Chromatographic Column
Sorvall Operations, Pecks Ln, Neweighton, CT 06470), or equiva-
lent, for use with 12 oz (350 mL) wide-mouth screw-cap jars. (Caution: See Appendix B, safety notes on hydrofluoric acid and
(d) Magnetic stirrer-hot plate.With variable speed and heat isooctane.)
controls.
(e) Rotary evaporator.With glass condenser flask between Attach empty column to aspirator and draw through 5% HF
concentration flask and metal shaft. solution. Stop vacuum with pinch clamp, quickly cap both ends of
(f) Test tube mixer.Vortex-Genie mixer (No. 12-812, Fisher column with rubber stoppers, and let column stand filled with 5%
Scientific Co.), or equivalent HF solution 10 min.
C. Reagents Attach column to aspirator again, draw off 5% HF solution, and
rinse with ca 150 mL H2O followed by 150 mL anhydrous methanol.
(Caution: Silanes are toxic. Avoid contact with skin and eyes. Use Finally, rinse column with 150 mL isooctane. Draw air through
effective fume removal device.) column until dry. Fill column with TMS reagent, (l), by pulling it
through slowly with aspirator. Plug both ends of column and let stand
(a) Cholesterol standard solutions.Standard cholesterol avail- 30 min. Draw TMS reagent through and rinse immediately with 100
able as No. 21502, Alltech-Applied Science Laboratories, Inc. (1) mL anhydrous methanol, followed by 200 mL isooctane. Let column
Stock solution.1.0 mg/mL DMF. (2) Working solutions.Dilute dry under vacuum.
stock solution with DMF to obtain concentration range from 0.05 to Use commercially prepared column packing of 0.5% Apiezon L
0.5 mg/mL. on 80100 mesh Gas-Chrom Q (Alltech-Applied Science Laborato-
(b) 5-Cholestane internal standard solutions.Standard 5- ries, Inc.), or prepare as follows: Weigh 0.5 g Apiezon L into 100
cholestane available as No. 19505, Alltech-Applied Science Labo- mL beaker, add 80 mL toluene, stir magnetically until it dissolves
ratories, Inc. (1) Stock solution.1.0 mg/mL n-heptane. (2) Working completely, and transfer to 500 mL Erlenmeyer, rinsing beaker with
solution.0.2 mg/mL. Dilute stock solution with n-heptane to ob- four 5 mL portions toluene. Weigh 10 g 80100 mesh Gas-Chrom
tain concentration of 0.2 mg/mL. Q and add to Apiezon L solution. Stopper flask and shake to make

Copyright 1998 AOAC INTERNATIONAL

slurry. Immediately pour slurry through buchner-type fritted glass pentane and shake 1 min. Let layers separate. Drain aqueous (lower)
Pyrex filter (medium porosity) under vacuum, stirring continuously layer into second separator. Repeat extraction with 100 mL ether and
until all liquid is drawn off. Measure filtrate in graduate and deter- 100 mL pentane, shaking 1 min after each addition. If layers do not
mine amount Apiezon L adsorbed. Let stand under vacuum, stirring separate, add 40 mL reagent alcohol, gently rotate end over end 10
occasionally until almost dry. Transfer packing to porcelain evapo- times, and let stand 5 min. Discard aqueous layer. Filter combined
ration dish and dry completely in 110120 oven. Store in glass ether extracts through column of anhydrous Na2SO4 into 600 mL
bottle until ready to use. beaker. Evaporate to ca 10 mL under gentle N2 stream on 70 H2O
Heat packing 15 min in 100 oven. Plug detector end of silanized bath. Transfer extract to 300 mL glass-stoppered Erlenmeyer, rinsing
column with 6 mm silanized glass wool and attach to aspirator. Add beaker with pentane. Evaporate to dryness under gentle N2 stream
warm packing through funnel attached to column and gently tap on steam bath, and proceed as in 976.26G, paragraph 2.
column. Finally, plug injection port end with silanized glass wool.
G. Saponification and Extraction of Unsaponifiable Fraction
Condition column 24 h at 235 with N2 flow.
E. Moisture Determination (Caution: See Appendix B, safety notes on distillation, pipets, ben-
Accurately weigh ca 5.0 g sample into tared Al dish, place in zene, and petroleum ether.)
circulating-type 100 air oven, and dry overnight or 3 h at 110.
Cover, and let cool in desiccator. Weigh accurately and determine Filter 100 mL aliquot CHCl3-lipid extract through glass funnel
moisture content to adjust for H2O to be added in 976.26F. containing small pledget of glass wool and ca 25 g anhydrous
Na2SO4 into 150 mL beaker. Rinse Na2SO4 with 15 mL CHCl3 and
F. Extraction of Lipid evaporate extract to dryness under gentle N2 stream on 90o H2O bath
or steam bath. Dissolve residue in ca 70 mL petroleum ether and
(Caution: See Appendix B, safety notes on distillation, diethyl
filter through Whatman No. 1 paper containing ca 20 g anhydrous
ether, chloroform, methanol, and pentane.)
Na2SO4 into 300 mL glass-stoppered Erlenmeyer. Rinse beaker and
(a) For foods other than dried whole egg solids, mayonnaise, and Na2SO4 with several 10 mL portions petroleum ether. Evaporate to
nonfat dry milk.Accurately weigh known amount sample contain- dryness under gentle N2 stream on steam bath.
ing ca 0.51 g fat and transfer quantitatively to homogenizer cup Introduce magnetic stirring bar into Erlenmeyer and place on
with 100.0 mL anhydrous methanol. On basis of moisture determi- magnetic stirrer-hot plate. With gentle stirring, slowly add 8 mL
nation, add enough H2O to bring total H2O content in extraction to concentrated KOH solution, (h), and 40 mL reagent alcohol, (i).
40 mL. Add 50 mL CHCl3 and blend 3 min at high speed. (Ratio of Attach condenser, turn on magnetic stirrer-hot plate, and reflux
CHCl3-methanol-H2O must be 5010040 in this single-phase ex- solution 1 h. Turn off heat and add 60 mL reagent alcohol through
traction.) Add additional 50 mL CHCl3 and blend 0.5 min at medium condenser into saponified solution while stirring and cooling. When
speed. Then add 50 mL H2O and again blend 0.5 min at medium sample ceases to reflux, remove condenser, and pipet 100 mL
speed. Filter homogenate under vacuum into 1 L suction flask benzene into sample while slowly stirring. Remove stirring bar,
through Bchner fitted with Whatman No. 1 paper containing 2 g stopper flask, and shake vigorously 30 s.
diatomaceous earth. Pour filtrate into 500 mL graduate. Re-extract Pour into 500 mL separator without rinsing. Add 200 mL 1N KOH
filter cake and paper with ca 90 mL CHCl3 and filter extract without and shake vigorously 10 s. Let layers separate and discard aqueous
diatomaceous earth. Rinse cup and filter cake with two 15 mL (lower) layer (will be turbid). Wash benzene layer with 40 mL 0.5N
portions CHCl3. Add these rinses to original filtrate and let layers KOH, rotate gently end to end 10 s, and discard aqueous (lower)
separate. (If emulsion develops, centrifuge filtrate 5 min at 2500 layer. Pour benzene layer into 250 mL separator. Back-wash benzene
rpm.) Record volume of CHCl3 (lower) layer and aspirate aqueous layer with 40 mL H2O by gently rotating separator end to end 10
alcohol layer. (Total volume of CHCl3 layer should be ca 200 mL.) times. Repeat H2O wash 3 more times. pH of last H2O wash should
Proceed as in 976.26G. be ca 7. Pour benzene extract from top of separator, filtering through
(b) For dried whole egg solids.Use acid hydrolysis, Whatman No. 4 paper containing ca 15 g anhydrous Na2SO4 into
925.32A(b) (see 34.1.07), and proceed as in 976.26G, paragraph 2. 125 mL glass-stoppered Erlenmeyer. Add ca 20 g anhydrous
(c) For mayonnaise.Accurately weigh ca 1.21.5 g sample and Na2SO4; stopper and shake flask vigorously. Let stand 15 min.
transfer quantitatively to homogenizer cup with 100.0 mL anhy- Pipet 50 mL aliquot into 100 mL round-bottom glass-stoppered
drous methanol. Add 40 mL H2O and 50 mL CHCl3 and blend 3 flask and evaporate to dryness on rotary evaporator at 40. Add 3 mL
min at medium speed. Add additional 50 mL CHCl3 and blend 0.5 acetone and again evaporate to dryness. Dissolve residue in 3 mL
min at medium speed. Then add 50 mL H2O and again blend 0.5 min DMF.
at medium speed. Transfer homogenate to 500 mL separator. Rinse H. Derivatization of Cholesterol Standards and Gas Chroma-
cup with three 20 mL portions CHCl3 and add these rinses to tographic Calibration
separator. Mix by gently rotating separator end to end. Let layers Transfer 1.0 mL of each cholesterol working standard solution,
separate. Drain CHCl3 (lower) layer into graduate. Rinse aqueous 976.26C(a)(2), to 15 mL silanized centrifuge tube. (Keep DMCS-
methanol layer with 40 mL CHCl3, add rinse to graduate, and mix. silanized centrifuge tubes clean and dry.) Add 0.2 mL HMDS and
Record volume of CHCl3 layer. Proceed as in 976.26G, using 150 0.1 mL TMCS. Stopper tube and shake vigorously on test tube mixer,
mL aliquot CHCl3-lipid extract and 250 mL beaker. (f), or by hand for 30 s. Let solution stand undisturbed 15 min. Add
(d) For nonfat dry milk.Accurately weigh ca 25 g sample and 1 . 0 m L 5-cholestane internal standard working solution,
transfer quantitatively to 300 mL Erlenmeyer containing 100 mL 976.26C(b)(2), and 10 mL H2O to tube. Shake vigorously 1 min and
H2O. Stir to mix thoroughly, and refrigerate overnight. Pour recon- centrifuge 2 min.
stituted milk into 1 L separator, add 100 mL reagent alcohol, (i), and Inject duplicate 3 L or other appropriate volumes (use same
shake 1 min. Add 100 mL ether and shake 1 min. Add 100 mL volume throughout for all standards and samples) heptane layer into

Copyright 1998 AOAC INTERNATIONAL

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 choles-
terol 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