47.3.05
Repeat extrac-
AOAC Official Method 983.16
Benzoic Acid and Sorbic Acid in Food
Gas Chromatographic Method
First Action 1983
NMKL–AOAC Method
(Collaboratively tested on apple juice, almond paste, and fish ho-
mogenate [at 0.5–2 g/kg levels], representing carbohydrate-rich,
pasty and rich in fat and carbohydrates, and protein-rich foods.)
A. Principle
Benzoic acid and sorbic acid are isolated from food by extraction
with ether and successive partitionings into aqueous NaOH and
CH2Cl2. Acids are converted to trimethylsilyl (TMS) esters and de-
termined by GC. Phenylacetic acid and caproic acid are used as in-
ternal standards for benzoic acid and sorbic acid, respectively.
B. Apparatus
(a) Gas chromatograph.—With linear oven temperature pro-
grammer, flame ionization detector, and recorder. Following condi-
tions have been found satisfactory: column, 1.8 m × 2 mm (id) coiled
glass with 3% OV-1 on 100–120 mesh Varaport 30. Operating tem-
peratures: oven 80–210°C, 8°C/min; injection port 200°C, detector
280°C. N2 carrier gas 20 mL/min. Approximate retention times 2.5,
4, 5, and 6 min for caproic acid, sorbic acid, benzoic acid, and
phenylacetic acid, respectively.
(b) Centrifuge.—With head for 30 and 200 mL centrifuge flasks.
(c) Mechanical shaker.—Bühler SM-B, or equivalent.
C. Reagents
Use analytical reagents throughout.
(a) Internal standard solution.—Dissolve 250 mg phenylacetic
acid and 250 mg caproic acid in 100 mL 3% aqueous KOH solution.
(b) Silylating agent.—N-Methyl-N-trimethylsilyl-trifluoracetamide
(MSTFA), available from Pierce Chemical Co, Rockford, IL, USA,
or Machery-Nagel Co, Neumann-Neander-Str, PO Box 307,
D-5160 Düren, Germany.
(c) Standard solutions.—Prepare mixed standard solutions in
CHCl3 of benzoic acid, sorbic acid, phenylacetic acid, and caproic
acid, respectively, of following concentrations: (1) 200, 200, 750,
and 750 µg/mL; (2) 400, 400, 750, and 750 µg/mL; (3) 600, 600,
750, and 750 µg/mL; (4) 800, 800, 750, and 750 µg/mL; (5) 1000,
1000, 750, and 750 µg/mL
D. Preparation of Test Sample
Homogenize test sample in mechanical mixer. If consistency of
laboratory sample makes mixing difficult, use any technique to en-
sure that the material will be homogeneous.
E. Extraction
(a) General method.—Accurately weigh 5.0 g homogenized test
portion into 30 mL centrifuge tube with Teflon-lined screw cap. Add
3.00 mL internal standard solution, 1.5 mL H2SO4 (1 + 5), 5 g sand,
and 15 mL ether. Screw cap on tightly to avoid leakage. Me-
chanically shake 5 min and centrifuge 10 min at 1500 × g. Transfer
ether layer with disposable pipet to 250 mL separator.
tion twice with 15 mL ether each time.
Extract combined ether phases twice with 15 mL 0.5M NaOH and
10 mL saturated NaCl solution each time. Collect aqueous layers in
250 mL separator, add 2 drops of methyl orange, and acidify to pH 1
with HCl (1 + 1). Extract with CH2Cl2, using successive portions of
75, 50, and 50 mL. If emulsion forms, add 10 mL saturated NaCl so-
lution. Drain CH2Cl2 extracts through filter containing 15 g anhy-
drous Na2SO4 into 250 mL round-bottom flask. Evaporate CH2Cl2
solution in rotary evaporator at 40°C just to dryness.
(b) Cheese and food products with paste-like consistency.—Ac-
curately weigh 5.0 g homogenized test portion into 200 mL centri-
fuge flask. Add 15 mL H2O and stir with glass rod until test portion is
suspended into aqueous phase. Add 3.00 mL internal standard solu-
tion, 1.5 mL H2SO4 (1 + 5), and 25 mL ether. Stopper flask carefully
and check for leakage. Mechanically shake 5 min and centrifuge
10 min at 2000 × g. Transfer ether layer with disposable pipet to
250 mL separator. Repeat extraction twice with 25 mL ether each
time. Continue as in (a), beginning “Extract combined ether
phases…”.
F. Derivatization and Gas Chromatography
Add 10.0 mL CHCl3 to residue in 250 mL round-bottom flask.
Stopper and shake manually 2 min. Transfer 1.00 mL CHCl3 solu-
tion to 8 mL test tube with Teflon-lined screw cap and add 0.20 mL
silylating agent. Cap and let stand 15 min in oven or H2O bath at
60°C. Inject duplicate 1 µL portions of residue solution into gas
chromatograph. Start temperature program when solvent peak
emerges. Measure peak heights and calculate peak height ratios of
benzoic acid/phenylacetic acid and sorbic acid/caproic acid. Use av-
erage of duplicate ratios. Peak height ratios for duplicate injections
should differ ≤5%.
G. Preparation of Standard Curves
Transfer 1.00 mL standard solutions to five 8 mL test tubes with
Teflon-lined screw caps. Add 0.20 mL silylating agent to each tube,
cap, and let stand 15 min in oven or H2O bath at 60°C. Inject dupli-
cate 1 µL portions of standard solutions into gas chromatograph. Use
same conditions as for test portion solution. Measure peak heights
and calculate peak height ratios of benzoic acid/phenylacetic acid
and sorbic acid/caproic acid, respectively. Peak height ratios for du-
plicate injections should differ ≤5%. Plot weight ratios (x) vs. aver-
age peak height ratios (y) for each preservative. Calculate slope and
intercept of standard curve by method of least squares.
H. Calculation
y− a W ′
Preservative, mg/kg = × ×1000
b W
where b = slope of standard curve; a = intercept; y = average peak
height ratio of preservative/internal standard; W = weight of test por-
tion in g; and W¢ = weight of internal standard in mg.
Reference: JAOAC 66, 775(1983).
CAS-65-85-0 (benzoic acid)
CAS-110-44-1 (sorbic acid)
© 2000 AOAC INTERNATIONAL