A Novel Method for Precolumn Derivatization of A atoxin B1, B2, G1, and G2 Prior to HPLC Analysis Using the XcelVap System as a Thermostatically Controlled Heated Water Chamber

December 1, 2014
Toni Hofhine

Dr. Cheri A. Barta

Dr. Pamela Doolittle

Robert Buco

Michael Ye


Zoe Grosser

Elizabeth Krantz

Jennifer Claus

Kenneth Espenschied

Special Issues

Special Issues, Special Issues-12-01-2014, Volume 32, Issue 12

Aflatoxins, a mold largely produced by Aspergillus flavus and Aspergillus parasiticus, are commonly tested mycotoxins found naturally in a wide range of agriculture crops and food products. Due to their harmful effects on human health, animal health, and global trade, aflatoxins are regulated in most countries and have established global limits in a wide variety of matrices. Regulations for the maximum limits vary for the reported aflatoxin B1 and total aflatoxins (sum of B1, B2, G1, and G2); however, most countries importing food and agriculture products perform testing to approve the safety of products.

There are several methods for detecting and quantifying aflatoxins; however, detecting all aflatoxins using the same method can be challenging. The limited response for B1 and G1 to naturally absorb UV light or fluoresce at the levels many countries need to quantify has created the need to add a derivatization step. To assist with detection at lower levels, derivatization of the aflatoxin standards using an acid solution aids in the fluorescence of both aflatoxin B1 and G1. Fluorescence is the more preferred reverse phase HPLC detection method for its ability to offer increased sensitivity at lower levels of aflatoxin.

A novel method was developed using an enclosed, dark, and moist heated environment to allow consistent linearity results to be obtained for all four Aflatoxin standards (B1, B2, G1, G2). This application focuses on the successful use of general equipment to accurately detect and report a linear seven-point calibration curve of aflatoxin B1, B2, G1, and G2 using the XcelVap as a thermostatically-controlled heated water chamber for derivatization. Figure 1 shows the resulting well-shaped peaks after successful derivatization.


The XcelVap Evaporator/Concentrator System was successfully used to optimize precolumn derivatization of all four aflatoxin standards (B1, B2, G1, G2). The enclosed, dark, and moist heated environment allowed for consistent linearity results to be obtained prior to fluorescence HPLC detection. Chromatography was used as an indicator of full derivatization, and peak fronting was been shown to be a reasonable factor in considering whether the derivatization process was complete.


"A Novel Method for Pre-Column Derivatization of Aflatoxin B1, B2, G1, and G2 Prior to HPLC Analysis Using the XcelVap® System as a Thermostatically-Controlled Heated Water Chamber," Horizon technology Application Note 0991410, available at http://www.horizontechinc.com/Application_Notes/4_SPE_Cartridge_Food_and_Agriculture_Landing_Page.htm.

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