Analysis of Aflatoxins in Ground Peanuts and Peanut Butter by HPLC with Photochemical Derivatization

September 1, 2010

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2010, Volume 0, Issue 0

Aflatoxins occur naturally in peanuts, cottonseed, corn, and dried chili pepper, as well as many mixed or processed foods and feeds.

Maria Ofitserova, Pickering Laboratories, Inc.

Aflatoxins occur naturally in peanuts, cottonseed, corn, and dried chili pepper, as well as many mixed or processed foods and feeds. A simple, sensitive, and robust HPLC method with post-column photochemical derivatization and fluorescence detection was used to analyze Aflatoxins B1, B2, G1, and 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.

Table I: Peanut butter (NIST SRM2387) – control sample

The extracts were cleaned by using AflaCLEAN™ (LCTech, Germany) Immunoaffinity columns that offer high recoveries for Aflatoxins even with the most challenging matrices. No matrix interferences were present after the sample clean up. AcceCLEAN™ (LCTech, Germany), an automated system that processes three samples at a time, was used for handling the Immunoaffinity columns. Instrumentation allows for quick and efficient detection of Aflatoxins at low ppb level.

Table II: Naturally contaminated ground peanuts

Sample Preparation

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 using HPLC with photochemical derivatization.

Figure 1: Chromatograms of a) naturally contaminated peanuts; b) NIST SRM2387 peanut butter sample. All samples are part of NIST Exercise E (April 2010).

Analytical Method

Analytical column: Mycotox™ (Pickering Laboratories, Inc), C18, 4.6 × 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 and Discussion

The 6-point calibration curves were build 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.

Pickering Laboratories, Inc.

1280 Space Park Way, Mountain View, CA 94043

tel. (800) 654-3330; fax (408) 694-6700

Website: www.pickeringlabs.com