MA 215 / CLEAN-UP AND DETERMINATION OF AFLATOXINS IN PEANUTS AND PEANUT BUTTER

Aflatoxins occur naturally in peanuts, cottonseed, corn, and dried chili pepper as well as many mixed or processed foods and feeds. Of significant assistance is the cleanup of extracts by an Immunoaffinity column containing antibodies specific to the Mycotoxin of interest. We used a simple, sensitive and robust HPLC method with post-column photochemical derivatization and fluorescence detection to analyze Aflatoxins B1, B2, G1, G2 in peanut butter and ground peanuts. The UVE™ (LCTech, Germany) photochemical reactor requires no additional reagents and is easy to install between the HPLC column and FLD detector. This method and instrumentation allows for quick and interference-free detection of Aflatoxins at the low ppb level.

PROJECT OVERVIEW

As participants in an NIST study, we analyzed samples of peanuts and peanut butter (table 1, 2). Four other laboratories that use other HPLC methods for analysis of Aflatoxins participated in this study. Community results for peanuts are presented in table 2. The extracts were cleaned up using the AflaCLEAN™ (LCTech, Germany) Immunoaffinity columns for Aflatoxin B1, B2, G1, G2.

For the handling of the columns, we used the AcceCLEAN™ automated system that processes three samples simultaneously. The prepared samples were analyzed by HPLC with post-column photochemical derivatization using the UVE™ photochemical reactor.

METHOD

Isolation of Aflatoxins B1, B2, G1, G2

Blend 20 g of sample at high speed with extraction solution (100 mL of Methanol/water 80/20, 50 mL of Hexane, 2 g NaCl) and filter through fluted paper. Dilute 14 mL of aqueous layer with 86 mL of PBS buffer (pH 7.2), filter and apply 11 mL of solution on AflaCLEAN™ Immunoafinity column. The toxins are eluted with 2 mL of Methanol and analyzed as described.

Analytical Conditions

Analytical Column: Mycotox™ (Pickering Laboratories, Inc), C18, 4.6 x 250 mm
HPLC Eluent: Sodium Phosphate buffer (Cat #1700-1108)/Methanol/ Acetonitrile (57/28/15)
Flow Rate: 1 mL/min
Injection Volume: 30 μL
FLD: Excitation 365 nm, Emission 430 nm

 

RESULTS & DISCUSSION

The 6-point calibration curves were built in a range of 11.49 – 0.24 ppb for B1, 3.29 – 0.07 ppb for B2 and G2, 9.85 – 0.21 ppb for G1 with R2 exceeding 0.999.X

There were no matrix interferences present after the sample clean-up using the Immunoaffinity columns. The results for all Aflatoxins are in good agreement with certified NIST values and with the results obtained by other methods.