© 2013 AOAC INTERNATIONAL

AOAC Official Method 2013.05 Aflatoxins B1, B2, G1, and G2

in Olive Oil, Peanut Oil, and Sesame OilImmunoaffinity Column Cleanup,

Postcolumn Derivatization, and LC with Fluorescence Detection

First Action 2013[Applicable to the determination of total aflatoxins (AFs; sum of

AFB1, AFB2, AFG1, and AFG2) in olive oil, peanut oil, and sesame oil at 2–20 µg/kg and AFB1 in the matrixes at 1–10 µg/kg.]Caution:�The International Agency for Research on Cancer (IARC)

has classified AFs as a human carcinogen (group 1A; 1). Protective clothing, gloves, and safety glasses should be worn at all times, and all standard and sample preparation stages should be carried out in a fume hood. Clean accidental spills of AFs with diluted bleach (1:10 dilution) and let stand 10 min. After wiping the bleach solution off the area, wipe with 5% aqueous acetone. Rinse all glassware with diluted bleach before washing. Methanol and acetonitrile are hazardous and must be poured in a fume cupboard. All analyses should be carried out inside the fume hood. Dispose waste solvents according to applicable environmental rules and regulations.

See Tables 2013.05A–C for the results of the interlaboratory study supporting acceptance of the method.A.  Principle

A test portion is extracted with methanol–water (55 + 45, v/v). After shaking and centrifuging, the extract is filtered, diluted with water, and applied to an immunoaffinity column (IAC) containing antibodies specific for AFs. After washing with methanol–water (10

+ 90, v/v), the AFs are eluted from the column with methanol and quantified by LC with fluorescence detection. For AF postcolumn derivatization, a photochemical derivatization device or Kobra cell is used.B.  Materials

Olive oil, peanut oil, and sesame oil were purchased from local grocery stores. AF naturally contaminated peanut oils were provided by Shan Dong Exit-Entry Inspection and Quarantine Bureau of China (Qing Dao, China).C.  Apparatus

(a) Orbital� shaker.—VWR DS-500E (VWR International, Bridgeport, NJ), or equivalent shaker capable of 400 rpm.

(b) Centrifuge� tubes.—50 mL, polypropylene, plug seal cap (Corning 430290; Corning, NY).

(c) Centrifuge.—Allegra X-22R (Beckman Coulter, Inc., Brea, CA).

(d) Filter�paper.—Qualitative folded filter papers, Grade 597½, 185 mm (Whatman, Dassel, Germany), or equivalent.

(e) Glass� microfiber� filter� paper.—Whatman 934-AH, GF/B circles, 90 mm (Whatman, Maidstone, UK), or equivalent.

(f) IAC.—AflaTest WB columns (G1024; VICAM, Watertown, MA). The aflatoxin IACs contain monoclonal antibodies that are cross reactive with AFB1, B2, G1, and G2. The columns should have capacity of not less than 100 ng total AF and should give a recovery of not less than 80% for AFB1, B2, G1, and G2 when 5 ng of each AF is applied in 10 mL methanol–PBS (10 + 90, v/v). The columns should have a shelf life of 18 months at 4°C or 12 months at room temperature.

(g) Column� manifold.—VICAM G1104; 12-position pump stand, or equivalent.

Table 2013.05A. Interlaboratory study results for AFsa in olive oil samples

AFLevel, µg/kg

Average, µg/kg

Mean recovery, % Sr, µg/kg SR, µg/kg RSDr, % RSDR, % HorRat

Predicted SD, 0.22c HorRatb No. labs Outliers

B1 1 0.86 86 0.07 0.10 8.09 11.18 0.24 0.19 0.51 14 1

B2 0.25 0.23 93 0.01 0.02 5.40 7.52 0.13 0.05 0.34 13 2

G1 0.5 0.45 90 0.05 0.06 11.75 13.18 0.26 0.10 0.60 14 1

G2 0.25 0.21 82 0.01 0.02 5.08 7.57 0.13 0.05 0.34 13 2

AFs 2 1.73 87 0.12 0.16 6.89 9.47 0.23 0.38 0.43 14 1

B1 2 1.77 89 0.07 0.27 3.98 15.35 0.37 0.39 0.70 15 0

B2 0.5 0.46 92 0.02 0.06 4.18 13.62 0.27 0.10 0.62 14 1

G1 1 0.88 88 0.07 0.16 8.12 18.12 0.39 0.19 0.82 15 0

G2 0.5 0.40 80 0.03 0.07 7.43 17.56 0.34 0.09 0.80 14 1

AF 4 3.56 89 0.12 0.52 3.42 14.52 0.39 0.78 0.66 13 2

B1 10 8.61 86 0.57 1.00 6.67 11.62 0.35 1.89 0.53 14 1

B2 2.5 2.23 89 0.14 0.25 6.20 11.15 0.28 0.49 0.51 14 1

G1 5 4.25 85 0.28 0.63 6.60 14.72 0.40 0.93 0.67 14 1

G2 2.5 1.90 76 0.13 0.22 6.68 11.45 0.28 0.42 0.52 13 2

AFs 20 16.89 84 0.96 1.79 5.70 10.59 0.36 3.72 0.48 14 1 a AFs = Sum of AFB1, B2, G1, and G2.

b Calculated using predicted SD of 0.22c.

(Note: Blank samples were found to contain <0.1 µg/kg AFs.)

© 2013 AOAC INTERNATIONAL

(h) LC�system.—Model 2690 Alliance system (Waters, Milford, MA) with Waters Model 2475 FLD, or an equivalent LC system could also be used. LC operating conditions: flow rate, 0.8 mL/min; detector set at excitation (Ex) wavelength of 362 nm and emission (Em) wavelength of 440 nm; column, YMC AQ12S03-1546WT, YMC ODS-AQ S-3, 3 mm, 150 × 4.6 mm id, or equivalent.

(i) Postcolumn�derivatization�systems�for�AFs.—(1) Postcolumn derivatization device: Cat. No. 1100-3347; UVE (Pickering Laboratories Inc., Mountain View, CA), photochemical postcolumn derivatization of AF in a special reactor loop with UV light, standard reactor volume is 1.0 mL. (2) PHRED cell (postcolumn photochemical derivatization cell (AURA Industries, New York, NY). (Caution: Avoid visual exposure to the UV light). (3) Kobra cell; electrochemical cell, postcolumn bromination derivatization cell (R-Biopharm Inc., Marshall, MI). (Caution: Set at 100 mA. Do not turn on current until LC pump is operating to avoid overheating the cell membrane.)D.  Reagents

(a) Solvents� and� sodium� chloride.—LC-grade methanol and acetonitrile; ACS-grade sodium chloride.

(b) Extraction� solvent.—Methanol–H2O (55 + 45, v/v), mix, equilibrate to room temperature.

(c) Washing� solution.—Methanol–H2O (10 + 90, v/v), mix, equilibrate to room temperature.

(d) AFs.—Nos. A6636, A9887, A0138, and A0263 (Sigma Aldrich). Prepare stock standard solutions of each of the four AFs at 10 µg/mL in acetonitrile according to 971.22.

(1) Preparation� of� 10� µg/mL� individual� AF� stock� standard�solution.—Weigh 10 mg each AF into a separate 100 mL volumetric flask. Add 50 mL acetonitrile, mix, dilute to mark with additional acetonitrile, and mix again. Pipet 10 mL of this solution into another 100 mL volumetric flask and dilute to mark with additional acetonitrile, and mix. Record UV spectrum of each AF solution. Determine concentration of AF solution by measuring absorbance (A) at wavelength of maximum absorption close to 360 nm and using equation in 971.22:

µg AF/mL = (A × MW × 1000)/ε

where MW is molecular weight and e is molar absorptivity. The concentration should be approximately 10 µg/mL.

(2) Preparation�of�400�ng/mL�AF�second�stock�standard�solution�(mixture�of�AFB1,�B2,�G1,�and�G2�at�200,�50,�100,�and�50�ng/mL).—Add appropriate amount of each AF stock standard to the same volumetric flask and dilute to volume with acetonitrile. Use the 400 ng/mL AF second stock standard as the spiking solution for the recovery study. Store stock standard solution at –18°C. Equilibrate to room temperature before use.

(3) Preparation� of� working� AF� calibrant� solution.—Prepare daily six calibrates in separate 5 mL volumetric flasks according

Table 2013.05B. Interlaboratory study results for AFsa in peanut oil samples

AFaLevel, µg/kg

Average, μg/kg

Mean recovery, %

Sr,µg/kg

SR,µg/kg

RSDr,%

RSDR,% HorRat

Predicted SD, 0.22c HorRatb No. labs Outliers

B1 1 0.86 86 0.07 0.08 8.39 8.72 0.19 0.19 0.40 13 2

B2 0.25 0.24 94 0.02 0.02 7.94 10.51 0.19 0.05 0.48 15 0

G1 0.5 0.47 94 0.05 0.05 11.4 11.65 0.23 0.10 0.53 15 0

G2 0.25 0.21 85 0.01 0.03 6.08 12.22 0.21 0.05 0.56 14 1

AFs 2 1.80 90 0.17 0.19 9.27 10.36 0.25 0.40 0.47 15 0

B1 2 1.77 89 0.19 0.22 10.48 12.34 0.30 0.39 0.56 15 0

B2 0.5 0.47 94 0.04 0.05 8.30 10.50 0.21 0.10 0.48 15 0

G1 1 0.93 93 0.09 0.12 9.72 13.22 0.29 0.20 0.60 15 0

G2 0.5 0.42 84 0.03 0.05 7.64 10.97 0.21 0.09 0.50 14 1

AFs 4 3.56 89 0.32 0.35 8.91 9.94 0.27 0.78 0.45 15 0

B1 10 8.70 87 0.30 0.65 3.50 7.45 0.23 1.91 0.34 15 0

B2 2.5 2.28 91 0.07 0.17 3.24 7.36 0.18 0.50 0.33 15 0

G1 5 4.40 88 0.29 0.48 6.51 10.80 0.30 0.97 0.49 15 1

G2 2.5 2.00 80 0.10 0.19 4.82 9.61 0.27 0.44 0.44 14 1

AFs 20 17.29 87 0.65 1.09 3.76 6.30 0.21 3.80 0.29 15 0

B1 NC 10.70 NA 1.17 1.57 10.93 14.66 0.46 2.35 0.67 15 0

B2 NC 1.73 NA 0.16 0.25 9.51 14.59 0.35 0.38 0.66 15 0

G1 NC 0.62 NA 0.07 0.09 10.73 14.35 0.30 0.14 0.65 14 1

G2 NC 0.11 NA 0.01 0.02 14.17 23.68 0.38 0.02 1.08 14 1

AFs NC 13.17 NA 1.34 1.89 10.15 14.34 0.47 2.90 0.65 15 0a AFs = Sum of AFB1, B2, G1, and G2.

b Calculated using predicted SD of 0.22c.

(Note: Blank samples were found to contain <0.1 µg/kg AFs.) NA = Not applied; NC = naturally contaminated.

© 2013 AOAC INTERNATIONAL

to Table 2013.05D. Dilute to volume with methanol–water (1 + 1, v/v). Store in refrigerator and equilibrate to room temperature before use. Prepare working calibration solutions daily.

(e) Mobile�phase.—Isocratic, 0.8 mL/min.(1) For�AF�postcolumn�derivatization�with�PHRED�cell�or�UVE

device.—Methanol–acetonitrile–water (25 + 17 + 60, v/v/v).(2) For� AF� postcolumn� derivatization� with� Kobra� cell.—1 L

methanol–acetonitrile–water (25 + 17 + 60, v/v/v) + 350 µL 4 M nitric acid (M of concentrated nitric acid is 15.9) + 120 mg potassium bromide, and mix.E.  Extraction

Weigh 5.0 g test portion in a 50 mL centrifuge tube. Add 1.0 g NaCl and 25 mL extraction solvent. Vortex until sample particles and extract solvent are well mixed. Shake at 400 rpm for 10 min. Centrifuge at 7000 rpm (g value = 5323 mm/s2) for 10 min. Aspirate and discard the upper oil layer. Pass the lower aqueous methanol layer through folded filter paper. Measure 15 mL filtrate with a 25 mL graduated cylinder and place in a 50 mL centrifuge tube. Add 30 mL water, mix, and filter through glass microfiber paper. Collect 30 mL filtrate (equivalent to 2 g test portion) into a 50 mL graduate cylinder and proceed immediately with IAC chromatography.

(Note: Silanized vials were used for storage of AF stock standard solutions. All other glassware used was nonsilanized.)F.  Immunoaffinity Column Cleanup

After removing from storage at 4°C, IACs are equilibrated at room temperature for at least 15 min before use. Remove top cap from column and connect to reservoir of column manifold. Remove bottom cap from column and let liquid in column pass through until liquid reaches 2 mm above the column packing. Add 30 mL filtrate into column reservoir. Let filtrate flow through IAC by gravity force until the liquid level reaches 2 mm above the column packing. Add 10 mL washing solution to column reservoir. Let column run dry and then force 10 mL air through column with a syringe. Place a 2 mL volumetric flask under column. Elute with 0.6 mL LC grade methanol, and collect AFs in a 2 mL volumetric flask; let drip freely. Let column run dry. Elute with additional 0.6 mL methanol and collect into the same volumetric flask. Let column run dry and force 10 mL air through column. Dilute eluate to volume with water and perform LC analysis.G.  LC Analysis

Inject 50 µL reagent blank (calibrate 1), AF working standards, or test sample into LC column. Identify AF peaks in test sample by comparing retention time with those of standards. AFs elute in the

Table 2013.05C. Interlaboratory study results for AFsa in sesame oil samples

AFaLevel, µg/kg

Average, µg/kg

Mean recovery, %

Sr,µg/kg

SR,µg/kg

RSDr,%

RSDR,% HorRat

Predicted SD, 0.22c HorRatb No. labs Outliers

B1 1 0.91 91 0.04 0.07 4 8.05 0.18 0.20 0.37 13 2

B2 0.25 0.24 94 0.01 0.02 3.91 7.13 0.13 0.05 0.32 13 2

G1 0.5 0.49 97 0.07 0.08 14.16 15.92 0.32 0.11 0.72 15 0

G2 0.25 0.2 78 0.01 0.04 7.63 18.12 0.31 0.04 0.82 14 1

AFs 2 1.82 91 0.06 0.11 3.23 6.08 0.15 0.40 0.28 13 2

B1 2 1.85 93 0.13 0.2 7.03 10.82 0.26 0.41 0.49 14 1

B2 0.5 0.48 95 0.03 0.04 6.16 8.32 0.20 0.10 0.38 14 1

G1 1 0.96 97 0.14 0.15 14.89 15.09 0.33 0.21 0.69 15 0

G2 0.5 0.39 78 0.03 0.07 6.77 17.03 0.33 0.09 0.77 15 0

AFs 4 3.69 92 0.25 0.3 6.64 8.19 0.22 0.81 0.37 14 1a AFs = Sum of AFB1, B2, G1, and G2.

b Calculated using predicted SD of 0.22c.

(Note: Blank samples were found to contain <0.1 µg/kg AFs.)

Table 2013.05D. Preparation of working AF calibration solutions

Working standard solution

400 ng/mL AFs second stock standard solution, µL

Final aflatoxin concentration of working standard solution, ng/mL

AFB1 AFB2 AFG1 AFG2 AFs

1 0 0 0 0 0 0

2 10 0.4 0.1 0.2 0.1 0.8

3 25 1.0 0.25 0.5 0.25 2.0

4 50 2.0 0.50 1.0 0.5 4.0

5 100 4.0 1.0 2.0 1.0 8.0

6 250 10.0 2.5 5.0 2.5 20.0

© 2013 AOAC INTERNATIONAL

order of G2, G1, B2, and B1. After passing through the UVE device, PHRED cell, or Kobra cell, the AFG1 and AFB1 are derivatized to form G2a (derivative of G1) and B2a (derivative of B1). The retention times of AFG2, G2a, B2, and B2a are between about 11 and 21 min using the PHRED cell (Figure 2013.05); retention times are shorter using the Kobra cell. The peaks should be baseline resolved. Construct standard curves of each AF. Determine concentration of each AF in test solution from calibration curve.

Calibration curves should be prepared for each AF using the working calibration solutions containing the four AFs described. These solutions cover the range of 0.4–10.0 ng/mL for AFB1, 0.1–2.5 ng/mL for AFB2, 0.2–5.0 ng/mL for AFG1,

and 0.1–2.5 ng/mL for AFG2. Make the calibration curves prior to analysis according to Table 2013.05D and check the curve for linearity. If test portion area response is outside (higher) the calibration range, then the purified test extract should be diluted with methanol–water (50 + 50, v/v) and reinjected into the LC column.

(a) Quantitation�of�aflatoxins.—Quantitation of AFs should be performed by measuring peak areas at each AF retention time and comparing them with the relevant calibration curve.

(b) Calculations.—Plot peak area (response, Y-axis) of each AF standard against concentration (ng/mL, X-axis) and determine slope (S) and Y-intercept (a). Calculate level of toxin in test sample with the following equation, where R is the test solution peak area, V is the final volume (mL) of the injected test solution, F is the dilution factor (F is 1 when V is 2 mL), and W is 2 g test sample passed through the IAC. The total AFs is the sum of the AFB1, AFB2, AFG1, and AFG2.

Toxin, µg/kg = {[(R – a)/S] ×V/W} × F

References: (1) International Agency for Research on Cancer (1993) Monographs�on�the�Evaluation�of�Carcinogenic�Risks�to�Humans:�Heterocyclic�Amines�and�Mycotoxins, Vol. 56, IARC, Lyon, France

J.�AOAC�Int. 95, 1689(2012)http://dx.doi.org/10.5740/jaoacint.12-199

J.�AOAC�Int. 96, 1017(2013)DOI: 10.5740/jaoacint.13-129

Figure 2013.05. Liquid chromatograms of AF standard solution (AFs 4.0 ng/mL; retention time 11–21 min, postcolumn derivatization with a PHRED).

AF

G2 -

11.8

72

AF

G2a

- 14.3

66

AF

B2 -

16.4

01

AF

B2a

- 20.2

27

EU

-2.00

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Figure 1. Liquid chromatograms of AF standard solution (AFs 4.0 ng/mL) (retention time 11-21 min, postcolumn derivatization with a PHRED.