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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
A. Apparatus
(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).
Table 980.20
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
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:
Corrected g/kg =
apparent g kg
1 [(p P) + (q Q)]
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)