45.4.

02
AOAC Official Method 983.23
Fat in Foods
Chloroform-Methanol Extraction Method
First Action 1983
Final Action 1984

(Method is applicable to composite foods and foods for which methods of analysis for fat or lipids are not specified. Method is for lipids,
not for fats [triglycerides and other ether-soluble materials.])
A. Apparatus and Reagents

(a) Blending assembly.Semi-micro, stainless steel blending

assembly for Waring blender (Scientific Products Co., Cat. No.
S8395-1, or equivalent).
(b) Enzyme solution.1% Clarase 40 000 in 0.5M sodium acetate solution. Suspend 10 g Clarase 40 000 (Miles Laboratories, Inc.
1127 Myrtle St, PO Box 70, Elkhart, IN 46514, USA) in 200 mL
0.5M sodium acetate solution in 1 L volumetric flask, dilute to volume with 0.5M sodium acetate solution, and mix thoroughly. (Stable
at least 7 days when stored at 4C.)
B. Determination

Accurately weigh ca 5 g well-minced test portion (3 g if >10% fat)

into 50 mL digestion tube, add enough enzyme solution so that total
H2O content (enzyme solution added plus original moisture in test
portion) is 32 mL. Shake gently until test portion is thoroughly
mixed with enzyme solution and place tube in 4550C H2O bath
1 h. Mix thoroughly and quantitatively transfer digest to blending assembly with 80.0 mL methanol and 40.0 mL CHCl3.
Cover and blend 2 min at high speed. Remove cover, add 40.0 mL
CHCl3, cover, and blend 30 s. Remove cover, add 40 mL H2O, cover,
and blend additional 30 s.
Transfer extract to 250 mL polypropylene centrifuge bottle and
centrifuge 10 min at ca 3000 rpm (when rotor diameter is 11.5 in.) to
clarify bottom CHCl3 layer. Pipet 20.0 mL CHCl3 extract into tared
100 mL beaker. (Care should be taken to prevent transferring aqueous phase.) Evaporate to dryness on steam bath in fume hood. Dry
fat residue in 101C oven to constant weight (ca 30 min), cool in desiccator to constant weight (ca 30 min), and weigh. Calculate
% fat = (g residue/g test portion) 4 100.
(Note: Ratio of CHCl3methanolH2O is critical for quantitative
extraction of fat. This may necessitate determination of moisture
content of product to optimize amount of H2O [as enzyme solution]
in initial extraction step. Reference source such as USDA Agriculture Handbook No. 8, Composition of Foods, or USDA Agriculture
Handbook No. 456, Nutritive Value of American Foods, is adequate
for this purpose. Also use as guide to pre-estimate fat content so that
proper test portion weight can be chosen for analysis.)
Reference: JAOAC 66, 927(1983).

2000 AOAC INTERNATIONAL