ANALYSIS OF AFLATOXINS

Introduction

Aflatoxin (AF) is the strongest known naturally occurring chemical structures of B1, B2, G1, G2, M1, and M2
carcinogen. Animal feed and food products are strictly aflatoxins.
inspected for AF contamination. Figure 1 shows the

Figure 1 – Aflatoxin chemical structures

Analysis of AF in Animal Feed

Figures 2 to 4 show chromatograms of AF found in
naturally contaminated peanut meal, corn, and poultry
feed. The samples were prepared for HPLC injection
according to the CB Method of the Association of Official
Analytical Chemists (AOAC).
Figure 5 shows the analysis of contaminated cheese
containing aflatoxins M1 (0.1 – 0.9 ppb) and M2 (0.01
ppb). As shown in Figure 6, the sample was pretreated by
an acetone extraction of AF.
Table 1 shows the percent recovery of aflatoxins in milk
and dairy products using this method. The percent
recovery was low when solvents other than methanol and
the mixed methanol/acetone solvents were used.

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Analytical Conditions

Column : Zorbax SIL

Mobile phase : Toluene-ethylacetate-formic acid-methanol
(89+7.5+2.0+1.5)
Flow rate : 1.0ml/min
Column temp. : 40°C
Detection : Shimadzu spectrofluorophotometer
(Em=365nm, Ex=425nm)

Figure 2 – Peanut meal aflatoxin

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Analytical Conditions

Column : Zorbax SIL

Mobile phase : Toluene-ethylacetate-formic acid-methanol
(89+7.5+2.0+1.5)
Flow rate : 1.0ml/min
Column temp. : 40°C
Detection : Shimadzu spectrofluorophotometer
(Em=365nm, Ex=425nm)

Figure 3 – Chromatogram of aflatoxins extracted from contaminated corn

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Analytical Conditions

Column : Silica gel (5.5µm particle size), 4mm i.d. x 100mm

Mobile phase : Toluene-ethylacetate-formic acid-methanol
(89+7.5+2.0+1.5)
Flow rate : 1.1ml/min
Detection : Shimadzu spectrofluorophotometer
(Em=365nm, Ex=425nm)

Figure 4 – Chromatogram of mixed feed for chicken (aflatoxins B1, B2, G1, and G2, 33 ppb each added)

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Analytical Conditions
Column : Silica gel (5.5µm particle size), 4mm i.d. x 100mm
Mobile phase : Toluene-ethylacetate-formic acid-methanol
(90 : 5 : 2.5 : 2.5)
Flow rate : 1.1ml/min
Column temp. : 40°C
Detection : Shimadzu spectrofluorophotometer
(Em=365nm, Ex=425nm)
(a) Standard of aflatoxins : AFB1, B2, G1 and G2,
5ng each; AFM1 and M2 10 ng each.
(b) Aflatoxins from contaminated cheese :
injection amount, 20 µl
Figures 5 – Chromatograms of aflatoxins extracted from contaminated cheese

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Sample (75g)
water
acetone (215ml)
Blended for 30 min
saturated sodium sulfate (10ml)
diet earth (10g)
Stirred
Filtered
Filtrate (200ml)
5% sodium chloride (100ml)
hexane (100ml)
Shaken for 5 min

hexane layer aqueous acetone layer

5% sodium chloride (50ml)
chloroform (100ml, 50ml)
Shaken for 3 min

chloroform layer aqueous acetone layer

5% sodium chloride (100ml)
Shaken for 3 min

aqueous layer chloroform layer

Dehydrated through an anhydrous sodium sulfate column
Concentrated to about 10ml
Acidic alumina-silica gel-sodium sulfate column
(5g, 15g, 15g layer)
Washed with benzene-acetic acid (9 : 1) (100ml)
ether-hexane (3 : 1) (150ml)
Eluted with chloroform-methanol (97 : 3) (200ml)
Evaporated to dryness in an N2 stream
Residue
Dissolved in chloroform (100ul)
Test solution for HPLC

Figure 6 –Extraction procedure for milk products

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 Recovery of aflatoxins (%)
AFB1 AFB2 AFG1 AFG2 AFM1 AFM2
Milk 98 90 86 93 93 95
Butter 88 92 99 96 92 94
Cheese 60 80 67 85 93 97

Table 1 - Recovery of AF from milk and dairy products

This Application Note was edited with the Food Research Institute, Ministry of Agriculture,
cooperation of Dr. Tetsuya Goto of the National Forestry, and Fisheries, Japan.

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