Determination of Aflaochra Prep® in Conjunction with a Single HPLC Fluorescent Method for the Determination of Total Aflatoxin and Ochratoxin

Aflatoxin and ochratoxin A can be found in a wide variety of commodities including cereals, nuts, dried fruit, herbs, spices and coffee when such foods are stored under adverse conditions of temperature and humidity. Over the years legislation for mycotoxins in food has increased to incorporate additional matrices and toxins, resulting in an increased demand for faster and less labour intensive tests to allow more analyses to be performed in a shorter time period. This has placed pressure on laboratories to perform multiple mycotoxin analysis on an ever-increasing range of commodities.

Since immunoaffinity columns are the official standard method for analysis for specific mycotoxins, there is a need for immunoaffinity columns, which can offer multi-mycotoxin analysis in conjunction with either HPLC or LC-MS/MS using a single extraction method. R-Biopharm Rhône Ltd has developed a number of new multi-mycotoxin immunoaffinity columns for use with HPLC or LC-MS/MS, these include EASI-EXTRACT® T-2 & HT-2 and the DZT immunoaffinity columns for simultaneous detection of deoxynivalenol, zearalenone, T-2 and HT-2 columns.

In many countries joint legislation already exists for aflatoxins and ochratoxin A in cereals, spices, baby food and feed, in response R-Biopharm Rhone Ltd has produced AFLAOCHRA PREP®, a new immunoaffinity column, which enables the isolation and concentration of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), aflatoxin B2 (AFB2), aflatoxin G2 (AFG2) and ochratoxin A (OTA) from a food sample. In addition RBR have selected suitable HPLC conditions, based on use of the KOBRA® CELL for derivatisation of AFB1 and AFG1, and capable of quantifying all five mycotoxins in a single run.

The AFLAOCHRA PREP® method was validated in-house for the determination of total aflatoxins and ochratoxin A in cereals, dried fruit and spices. FAPAS certified reference materials were also analysed to determine the accuracy of the method. Total aflatoxins and ochratoxin A were extracted using methanol: water (80:20 (v/v)). The extract was clarified and passed through AFLAOCHRA PREP® containing antibodies with high affinity and specificity to aflatoxins and ochratoxin A in order to isolate the toxins of interest. Subsequently the immunoaffinity column was washed with 10 ml water and the toxin released from the immunoaffinity column with 1ml of methanol and 1ml of water. The toxins were quantified by HPLC-fluorescence using an Inertsil ODS-3V analytical column with a gradient elution consisting of water and methanol.

Recovery data for spiked extract of cereals, spices and dried fruit ranged from 86-108% (RSD 2-4%), while recovery data for spiked matrix of cereals, spices and dried fruit ranged from 77-106% (RSD 4-6%). Four FAPAS samples were analysed and values were found to lie within the acceptable range for all test samples. The FAPAS samples were subsequently spiked with all 5 toxins and recoveries ranged from 83-102% (RSD 2-4%). AFLAOCHRA PREP® was shown to perform in line with current EU recommendations for method performance with regard to recovery (>70% and < 110%) and method repeatability (RSD <30%).

In conclusion, AFLAOCHRA PREP® was found to enable quantitative determination of aflatoxin and ochratoxin A using a single sample preparation method and a single set of HPLC conditions. This led to a greater sample throughput in addition to a reduction of consumables, reagents and associated costs. AFLAOCHRA PREP® was found to provide a rapid and robust method from a wide range of commodities (cereals, dried fruit and spices) enabling accurate, low level (<2ppb) quantification of all five mycotoxin groups. The performance of AFLAOCHRA PREP® with regard to percentage recovery and method repeatability were in line with EU legislation.