Validation of analytical method for determination of ochratoxin A in cereals by C₁₈ solid phase extraction and high-performance liquid chromatography with fluorescence detection

Author: Emad R. Attallah ^*, Ahmed M. Gomaa, Mohamed E. Amer and Sohair A. Gad

Received 12 December 2007, accepted 21 March 2008.

Abstract

A HPLC method was validated for determination of ochratoxin A (OTA) in cereals. Samples were extracted with chloroform in presence of phosphoric acid solution, OTA was isolated by liquid–liquid partitioning into aqueous bicarbonate solution. The sample solution is cleaned-up on a C₁₈ column and OTA is eluted with ethyl acetate–methanol – acetic acid mixture. OTA is identified by reversed phase liquid chromatography and quantified by fluorescence detector. The limit of quantification (1 µg/kg) was found to be significantly lower than current regulatory EU limits. The average recovery of OTA from several different levels varied between 70 and 120%. The reproducibility expressed as relative standard deviation was 10.3%. The method showed to be linear from the limit of quantification up to 40 µg/kg level. The method accuracy was confirmed by participating in 7 proficiency testing (PT) programs organized by FAPAS, CSL, UK during the last 3 years (2004-2007). Two approaches were used for estimating measurement uncertainty; using validation data and PT results. The measurement uncertainty in terms of expanded uncertainty expressed as relative standard deviation (at 95% confidence level and coverage factor K= 2) was less than ±29%. The proposed method was applied to 78 samples of cereals and grains collected from the local Egyptian markets; OTA was detected in 3 samples.

Key words: Food analysis, method validation, uncertainty, mycotoxins, ochratoxin A, SPE, HPLC, cereals.