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49.2.15 (c) Vacuum apparatus.

—Equipped with vacuum gauge/flow

AOAC Official Method 993.17 controller and manifold fitted with 10 female Luer connectors
Aflatoxins in Corn and Peanuts (Baker extraction system, J.T. Baker Chemical Co., is suitable).
Thin-Layer Chromatographic Method (d) Vials.—2 dram (8 mL), with foil or Teflon-lined screw caps.
First Action 1993 (e) TLC plate.—20 ×20 cm glass plate coated with 0.25 mm thick
Final Action 1996 silica gel without fluorescent indicator (precoated, silica gel
60 plates, E. Merck, Gibbstown, NJ, USA, are suitable).
(Applicable to determination of 5–50 ng B1/g corn, 3–15 ng B2/g
(f) Viewing cabinet.—270 × 270 mm base minimum, equipped
corn,10–50 ng G1/g corn, 3–15 ng G2/g corn, 5–25 ng B1/g raw
with 15 W longwave ultraviolet (UV) lamp (Chromato-Vue, Ul-
peanuts and 1.5–7.5 ng B 2/g raw peanuts by densitometry;
tra-Violet Products, Inc., San Gabriel, CA, USA, is suitable).
10–50 ng B1/g corn, 50 ng G1/g corn, 10–25 ng B1/g raw peanuts,
7.5 ng B2/g raw peanuts, and 10–25 ng G1/g raw peanuts by visual (g) Fluorodensitometer (TLC scanner).—Capable of scanning in
comparison.) reflectance mode by fluorescence, equipped with high-pressure Hg
lamp, monochrometer for adjustment to excitation 366 nm, and
See Tables 993.17A and B for results of the interlaboratory emission cutoff filter 420 nm (CAMAG TLC Scanner, Applied Ana-
study supporting the acceptance of the method. lytical Industries, Wilmington, NC, USA, is suitable).

Caution: Grinding of dry samples may result in airborne dust. C. Reagents

Even if no toxin is present, there is potential harm (a) Solvents.—Methanol, hexane, chloroform, anhydrous ethyl
from inhalations of mold spores or from allergic re- ether (100%), dichloromethane, acetone, and isopropanol.
sponse to inhaled dust. Use protective mask and/or ( b ) A f l a t o x i n s t a n d a r d s o l u t i o n .— P r e p a r e i n b e n-
dust collector. Prepare samples in area separate from zene–acetonitrile (98 + 2) as in 971.22 (see 49.2.03) to contain
analytical laboratory. 0.5 µg/mL each B1 and G1 and 0.15 µg/mL each B2 and G2.

A. Principle D. Extraction and Partition

Aflatoxins are extracted from samples with methanol–water. Fil- Weigh 50 g (ground to pass No. 20 sieve) corn or peanuts into
trate is diluted with NaCl solution and defatted with hexane. 500 mL glass-stoppered Erlenmeyer flask. Add 200 mL metha-
Aflatoxins are partitioned into chloroform which is then removed by nol–H2O (85 + 15) and secure stopper with masking tape. Shake vig-
evaporation. Aflatoxins are purified by chromatography on 0.5 g sil- orously by hand until samples show no clumps. Shake 30 min on
ica gel column, and quantitated by thin layer chromatography (TLC) wrist-action shaker, B(a), and filter mixture through medium fluted
on silica gel 60 plate with densitometry or visual estimation. paper. Collect 40 mL filtrate in 50 mL graduated cylinder. Transfer
filtrate to 125 mL separatory funnel. Add 40 mL 10% NaCl solution,
B. Apparatus mix, and add 25 mL hexane. Shake 1 min. Let phases separate, drain
(a) Wrist-action shaker.—Capable of holding four to eight lower (aqueous) phase into second 125 mL separatory funnel, and
250 mL flasks (Burrell Corp., Pittsburgh, PA, USA, is suit- discard upper phase.
able source). Extract aflatoxins from aqueous phase with two 25 mL portions
(b) Silica gel column.—6 mL disposable column, packed with CHCl3; shake 1 min each time. Combine CHCl3 fractions in 125 mL
40 µm (60Å) silica gel. Erlenmeyer flask and evaporate to dryness on steam bath.

Table 993.17A Interlaboratory study results for determination of aflatoxins B1, B2, G1, and G2 in corn and peanuts by thin-layer
chromatographic method with densitometer estimation

Product Aflatoxin Spike, ng/g Mean rec., % sr sR RSDr, % RSDR, %

Corn B1 5 88 1.98 1.98 56.6 56.6
10 83 2.58 2.58 41.7 41.7
50 76 14.28 14.25 47.5 47.3
B2 3 94 0.64 1.18 26.8 49.3
15 118 5.37 6.32 45.5 53.6
G1 10 102 3.31 4.61 43.6 60.7
50 91 13.07 15.42 36.1 42.6
G2 3 90 1.12 1.44 48.8 62.8
15 104 4.68 4.68 45.0 45.0
Raw peanuts B1 5 78 0.66 0.82 21.3 26.4
10 83 2.31 2.31 37.3 37.3
25 84 4.36 4.83 26.1 28.9
1.5 103 0.38 0.50 38.1 50.3
B2 3 74 0.67 0.67 37.3 37.3
7.5 123 2.31 2.31 37.2 37.2


Table 993.17B Interlaboratory study results for determination of aflatoxins B1, B2, G1, and G2 in corn and peanuts by thin-layer
chromatographic method with visual estimation

Product Aflatoxin Spike, ng/g Mean rec., % sr sR RSDr, % RSDR, %

Corn B1 10 100 0.86 4.25 11.4 56.7
50 92 12.66 12.66 34.6 34.6
G1 50 96 11.67 11.67 30.3 30.3
Raw peanuts B1 10 101 0.96 2.71 12.6 35.7
25 87 5.53 5.53 31.8 31.8
B2 7.5 120 1.67 2.69 27.8 44.9
G1 10 69 0.63 1.77 12.1 34.0
25 76 6.52 6.54 42.9 43.0

E. Silica Gel Column Chromatography and standard spots are dispersed evenly. To avoid errors, prepare spot-
Attach silica gel column, B(b), to extraction system, B(c), (or ting plan, either on plate or in notebook, prior to spotting.
clamp to stand if using gravity flow only). Condition column by Develop plate 1 h with CHCl3–acetone (9 + 1). Evaporate solvent
washing with 3 mL hexane, then 3 mL dichloromethane using vac- 5 min in fume hood followed by 2 min in 50°C forced draft oven.
uum (flow rate 6 mL/min), or let drip freely unassisted by suction. Examine plate under longwave UV light to determine presence or
Check column suitability by adding aflatoxin B1 standard (3 mL absence of aflatoxins.
dichloromethane containing 100 ng aflatoxin B1) to 0.5 g silica gel Quantitate by fluorodensitometric measurement. Scan test and
column. Recovery must be ≥90% by this method. aflatoxin reference spots (transmission or reflectance mode, exci-
Dissolve residue, from D, in 3 mL dichloromethane and add to tation 365 nm and emission cutoff 430 nm). At end of plate scan,
column. Let drip freely (flow rate ca 3 mL/min, apply vacuum if rescan 1st or 2nd lane. Scans of test spots should be within ±5%; if
needed). Rinse residue container with two 1 mL portions of di- not, rescan entire plate.
chloromethane and add rinses to column. Wash column with 3 mL G. Calculation
hexane, 3 mL anhydrous ethyl ether, and then 3 mL dichloro-
Calculate concentration of aflatoxin B1 in test portion, using fol-
methane. (Use vacuum, flow rate 6 mL/min, or use syringe and
lowing formula:
adapter to apply pressure to increase solvent flow if necessary. Do
not pull up syringe plunger while it is still attached to column.) Turn
250 × Ru
off vacuum, remove extraction system cover, and place vial, B(d), B1, ng/g =
under each column (test tube rack can be used to hold vials). 5 × Rs × 10
Elute aflatoxins (without vacuum) with two to four 3 mL por-
tions (according to results of column suitability test) of CHCl3–ac- where 250 = µL test solution volume; Ru = average densitometer re-
etone (9 + 1). Evaporate eluate to dryness on steam bath under sponse for B1 spots of test solution duplicates; 5 = µL test solution
stream of nitrogen. spotted; Rs = calculated average densitometer response/ng for 4 B1
F. Thin-Layer Chromatography–Fluorodensitometry
standard spots; 10 = g corn or peanut represented by extract.
Determination Calculate concentrations of aflatoxins B2, G1, and G2 similarly.
Dissolve residue from E in 250 µL CHCl3. Spot plate, B(e), with H. Thin-Layer Chromatography–Visual Estimation
5 µL CHCl3 test solution in duplicate and 2, 5, 10, and 20 µL aflatoxin Dissolve sample extract from E in 250 µL CHCl3 and proceed as
standard solution, C(b). Randomize standard and test solution spots in 968.22F(a)–(d) (see 49.2.08).
across plate so duplicate test solution spots are not next to each other
Reference: J. AOAC Int. 77, 637(1994).
Revised: March 1997