C.

Preparation of Sample

49.2.19
AOAC Official Method 980.20
Aflatoxins
in Cottonseed Products
Thin Layer and Liquid Chromatographic Methods
First Action 1980
Final Action 1988

Grind whole seed or kernels in Wiley mill, or equivalent, to pass

No. 10 sieve. For seed containing lint, screen ground sample on 4/64
in. screen to remove coarse lint. Grind meals to pass No. 18 sieve.
Quarter or riffle ground sample to obtain 50100 g analytical sample.
D. Extraction and Cleanup

A. Apparatus

(a) High speed blender.Waring, with qt (1 L) jar, or equivalent,

or wrist action shaker, Burrell, or equivalent.
(b) Liquid chromatograph.With septumless injector; UV A
detector, 360365 nm, 0.005 or 0.01 A units full scale (AUFS)
sensitivity; and fluorescence detector, 360365 nm excitation, 400
410 nm cutoff glass filter, or 430 nm cutoff interference filter,
containing silica gel-packed flowcell in excitation path.
(c) LC column.Normal phase, silica gel, microparticulate column (10 m particle size). Waters Associates -Porasil, 300 4 (id)
mm, or Whatman Partisil-10, 250 4.6 (id) mm long, are satisfactory. Other types of columns producing essentially complete resolution of aflatoxins in reference standard are satisfactory.
(d) Cleanup columns.Glass columns, 300 10 (id) mm, with
35 m porous polyethylene frit, Nylon stopcock, and polypropylene
funnel reservoir.
(e) Butt tube.Corning Glass Works, or equivalent.
B. Reagents

(a) Solvents.Stored in glass: methanol, hexane, toluene, anhydrous ethyl ether (0.01% ethyl alcohol), CH2Cl2 (dichloromethane), acetone. Distilled-in-glass: CHCl3, cyclohexane,
CH3CN, absolute alcohol, isopropanol.
(b) Cleanup column eluting solvents.Toluene-CH3COOH (9 +
1), ether-hexane (3 + 1), CH2Cl2-acetone (9 + 1).
(c) LC developing solvent.Prepare H2O-saturated CHCl3 by
shaking 1 L CHCl3 with four ca 100 mL portions H2O, ca 1 min each
time. After each extraction, let phases separate, and discard upper
aqueous layer. Drain clear lower layer after last extraction into
amber bottle. Mix H2O-saturated CHCl3-cyclohexane-CH3CN (25
+ 7.5 + 1.0), and add either 1.5% absolute alcohol or 2.0% isopropanol.
(d) Extraction solvent.Acetone-H2O (85 + 15).
(e) Lead acetate solution.Dissolve 200 g Pb(CH3COO)23H2O in
1 L H2O with warming, add 3 mL CH3COOH, and dilute to 1 L.
Zinc acetatealuminum chloride solution.Optional. Dissolve 200
g Zn(CH3COO)2 and 5 g AlCl3 in H2O to 1 L.
(f) Diatomaceous earth filter aid.Acid-washed Hyflo SuperCel or Celite analytical (Celite Corporation).
(g) Silica gel for column chromatography.Silica Gel 60 (E.
Merck, No. 7734) 0.0630.200 mm (80230 mesh), or equivalent.
Dry ca 2 h at 105, cool in vapor-tight container, add 1.0% H2O, mix
well, and equilibrate overnight. Store in vapor-tight container.
(h) Silica gel for thin layer chromatography.See 970.43B(d)
(see 49.1.01).
(i) Sodium sulfate.Anhydrous, granular.
(j) Aflatoxin reference standards.See 970.44CD (see 49.2.02)
and 971.22 (see 49.2.03). (1) LC working standard solution.1.0
g each B1 and G1 and 0.3 g each B2 and G2/mL. Dilute solution
from 971.22A (see 49.2.03) to volume with LC developing solvent,
(c). (2) TLC working standard solution.See 971.22D (see 49.2.03).

Transfer 50 g sample to blender jar, add 250 mL acetone-H2O (85

+ 15), and blend 1 min at slow speed and 3 min at high speed.
Alternatively, transfer sample to 500 mL glass-stoppered Erlenmeyer flask and shake 30 min with acetone-H2O (85 + 15) on
wrist-action shaker. (For ammoniated meal or seed, add 50 mL 0.1N
HCl, let soak 5 min, add 200 mL acetone, and extract as above.)
Filter through folded or fluted 24 cm Whatman No. 4 or 2 V paper,
or equivalent; collect 100 mL filtrate.
Measure 100 mL filtrate into 250 mL beaker, add 20 mL
Pb(CH3COO)2 solution and 80 mL H2O, stir, and let stand ca 5 min
to flocculate. Add ca 5 g filter aid, stir, and refilter as above.
Optionally, replace Pb(CH3COO)2 solution with Zn(CH3COO)2
AlCl3 solution.
Measure 100 mL filtrate into 250 mL separator, add 25 mL
CH2Cl2, and shake vigorously ca 1 min, and let phases separate
cleanly. Plug constriction at bottom of Butt tube with glass wool.
Add ca 4 cm layer Whatman CF-11 cellulose powder or anhydrous
granular Na2SO4. Drain CH2Cl2 phase through Butt tube, collecting
filtrate in 150 mL beaker. Repeat partition with another 25 mL
CH2Cl2. Wash Butt tube with additional 25 mL CH2Cl2. Evaporate
solvent on steam bath. Remove beaker from steam bath as soon as
solvent disappears to prevent overheating of extract. Hold for column chromatography.
E. Column Chromatography

Slurry 2 g silica gel for column chromatography with ca 10 mL

ether-hexane (3 + 1) in 15 mL beaker, and pour into column. Wash
beaker with ca 5 mL ether-hexane (3 + 1), and pour into column.
When gel settles, top with ca 1.5 g (10 mL beaker filled to top)
Na2SO4, and drain solvent to top of Na2SO4.
Dissolve extract in ca 2 mL CH2Cl2, and pour on column. Wash
beaker twice with ca 1 mL portions CH2Cl2; add washes to column.
Drain solvent to top of Na2SO4 and wash walls of column with ca 1
mL CH2Cl2.
Wash column with 25 mL toluene-CH3COOH (9 + 1); then with
50 mL ether-hexane (3 + 1). Discard washes. Elute aflatoxins with
60 mL CH2Cl2-acetone (9 + 1), and collect in 100 mL beaker. Add
each successive solvent when previous solvent has reached top of
Na2SO4 cap. Collect final solvent from time of addition. Evaporate
to near dryness on steam bath. (Time for column cleanup, from
beginning of first wash to end of elution, should be ca 30 min.)
Dissolve extract in ca 1 mL CH2Cl2, and pour through 2 mL
Bchner funnel with coarse porosity fritted disc overlaid with tightly
pressed circle of Whatman GS/A glass fiber paper slightly larger than
fritted disc. (No. 9 cork borer is optimum for cutting circles.) Collect
filtrate in 5 mL screw cap vial. Wash beaker with three ca 1 mL
portions CH2Cl2, adding washes to funnel. (This filtration removes
particulate matter which could plug LC lines or column filter. Any
other system for removing particulate matter without loss of sample
is satisfactory.) Evaporate to dryness under stream of N2. Hold for
LC or TLC. If LC determinative chromatography is not done same
day, add 0.51.0 mL CH2Cl2, cap with foil or Teflon lined screw cap,
and store in freezer until needed. Re-evaporate when ready to use.

Copyright 1998 AOAC INTERNATIONAL

F. Preliminary Thin Layer Chromatography

Dissolve dry sample extract (equivalent to 10 g original sample)

in 200 L benzene-CH3CN (98 + 2), or, if approximate aflatoxin
level is known, to give B1 concentration of ca 0.5 g/mL, and proceed
as in 968.22F(a) and (b) (see 49.2.08).
Spray plate with fine mist of H2SO4 (1 + 3) and view under
longwave UV. Aflatoxins B1 and B2 should now have yellow fluorescence, and G1 and G2, yellow-blue fluorescence. While G aflatoxins are rarely found in cottonseed, blue fluorescent spots are
sometimes found in cottonseed extracts at or near Rf of G1 or G2 that
will not exhibit characteristic color change. Test is not confirmatory,
but will rule out presumptive aflatoxins which do not show color
change.

Table 980.20

Aliquots for Visual and Densitometric Analysis

Visual Analysis
Densitometric Analysis

Approximate
B1 Content
from Prelim.
TLC, g/kg

Dilution
of Extract,
mL

Aliquots
on Plate,
L

Dilution
of Ext,
mL

Aliquots
on Plate,
L

010
1025
2550
5075
75125
125150
150175
175200

0.125
0.125
0.50
0.75
1.00
1.25
1.50
2.00

357
357
357
357
357
357
357
357

0.125
0.125
0.25
0.50
0.50
0.75
1.00
1.25

1010
55
66
66
55
55
66
66

G. Quantitative Thin Layer Chromatography

Evaporate remaining extract from 980.20F and dissolve in appropriate volume benzene-CH3CN (98 + 2) as in (a), for either visual
or densitometric analysis.
(a) Sample dilution and aliquots for visual and densitometric
analysis.See Table 980.20.
(b) Visual analysis.Spot 3, 5, and 7 L sample extract on plate,
along with 2, 3, 4, and 5 L aflatoxin standard, 971.22D (see
49.2.03). Spot and develop plates as in 968.22F(b) (see 49.2.08).
Interpret chromatogram as in 968.22F(d) (see 49.2.08).
(c) Densitometric analysis.Spot suggested sample aliquots, (a), on
plate, along with duplicate aliquots of mixed aflatoxin standard to
provide 5 ng aflatoxin B1 and G1 and 1 ng each B2 and G2/spot. Also
adjust separate sample aliquots to these values for B2, G1, and G2 by
diluting, and spot separately. Place spots along imaginary line ca 4 cm
from bottom of plate. Develop as in 968.22F(b) (see 49.2.08). Do not
damage gel layer during spotting, as significant errors may be
introduced. If plate is visually inspected before densitometry, use
low wattage UV source and minimum exposure time.
Scan plate with densitometer according to instructions of manufacturer. Irradiate with 365 nm source: observe for emission at
420460 nm.
Calculate concentration of aflatoxin B1 in g/kg as follows:
g/kg = (B Y S V)/(Z X W)
where B = average area aflatoxin B1 peaks in sample aliquots; Y =
concentration aflatoxin B1 standard, g/mL; S = L aflatoxin B1
standard spotted; V = final dilution of sample extract, L; Z = average
area aflatoxin B1 peaks in standard aliquots; X = L sample extract
spotted; and W = g sample represented by final extract (See
980.20F).
Repeat calculation for each of other aflatoxins observed.
Correct aflatoxin concentration values from densitometric measurement for amount of sample extract removed for either Preliminary TLC, 980.20F, o r Visual analysis, (b), if used before
densitometric measurements, as follows:

H. TLC Confirmation of Identity of Aflatoxin G1 and/or G2

Aflatoxins G1 and G2 are rarely observed in cottonseed products,

but some extracts may contain bluish fluorescent non-aflatoxin spot
at or near Rf of G1 or G2, depending on TLC conditions. If G1 or G2
is judged to be present, confirm by respotting two 5 L aliquots of
sample extract on new plate. Spot 5 L aflatoxin standard on top of
one sample aliquot and develop plate in 150 mL CHCl3-methanol
(95 + 5). Although aflatoxins are not well resolved in this solvent,
bluish fluorescent nonaflatoxin component will be resolved from
aflatoxins G1 and G2 as indicated by comparison of sample aliquots
with and without internal standard.
I. Liquid Chromatography

Stabilize flow rate at 1.0 mL/min with LC development solvent;

set UV A detector wavelength, and detector sensitivity at 0.005 or
0.01 AUFS.
Quantitatively inject 10 L LC working standard for 0.005 AUFS
sensitivity, or 20 L (20 ng) for 0.01 AUFS sensitivity. Record
detector response at chart speed of 0.2/min (0.5 cm/min) until all 4
aflatoxins elute (1011 min).
Dissolve dry sample extract (equivalent to 10 g original sample)
in 200 L LC developing solvent, or, if approximate aflatoxin level
is known, to give ca 0.51.0 g B1/mL. Inject 10 L sample extract
for 0.005 AUFS sensitivity, or 20 L for 0.01 AUFS sensitivity, and
record detector response. Although only aflatoxins B1 and B2 are
normally present in cottonseed products, record response beyond
point where G1 and G2 elute to confirm their presence or absence.
For fluorescence detection, follow same operating technique as
above, except set excitation and observation wavelengths, and either
inject less volume standard and sample, or suitably attenuate detector
to give B1 peak response 5060% of full scale for standard.
In all cases, use same AUFS sensitivity setting for both standards
and samples. If peaks from sample injection go off scale, suitably
redilute and repeat injection.
From either integrator area count, or peak height measured from
baseline to apex of peak, calculate B1 as follows:
B1 (g/kg) = (P C V D)/(P V W)

Corrected g/kg =

apparent g kg
1 [(p P) + (q Q)]

wher ep = total L sample extract spotted in 980.20F; P = L sample

extract prepared in 980.20F; q = total L sample extract spotted in
(b) (if done prior to densitometric analysis); Q = L sample extract
prepared for (b).

where P = area, or peak height B1 sample peak; C = concentration

B1 in standard, g/mL; V = L standard injected; D = dilution
sample extract, in L; P = area, or peak height B1 standard peak; V
= L sample extract injected; W = sample weight (10 g in original
solution; recalculate for amount removed, if redilution required).
Repeat calculation for other aflatoxins, substituting appropriate
areas, or peak heights, and standard concentrations. Correct afla-

Copyright 1998 AOAC INTERNATIONAL

toxin concentration values as in 980.20G, if extract has been removed for TLC.
References: JAOAC 63, 899(1980); 66, 418(1983); 69, 240,
294(1986).
CAS-1162-65-8 (aflatoxin B1)
CAS-7220-81-7 (aflatoxin B2)
CAS-1165-39-5 (aflatoxin G1)
CAS-7241-98-7 (aflatoxin G2)

Copyright 1998 AOAC INTERNATIONAL