Application of experimental design to the development of an HPLC method for the analysis of ochratoxin A in Triticum aestivum grain

Received May 8, 2003, accepted August 19, 2003Dr. M. Zecˇevic´, Ass. Prof., Institute of Pharmaceutical Chemistry and Drug Analysis, Faculty of Phar-macy, Vojvode Stepe 450, P.O. Box 146, 11000 Belgrade, Serbiamzecevic@pharmacy.bg.ac.yuPharmazie 59: 175–177 (2004)Ochratoxin A is a mycotoxin, a natural product of Aspergillus and Penicillium species. It can be pre-sent in grain from Triticum aestivum, (Graminae) and other starch-abundant cereals. This paper de-scribes the investigation of ochratoxin A in grain from Triticum aestivum using a statistically optimizedHPLC method. The assay was developed using two mathematical statistical models: factorial designand response surface mapping. The final step was to optimize the values of variables by responsesurface design. The analysis of variance ‘ANOVA’ method was applied to the analytical results inorder to construct an adequate model. The optimal experimental conditions obtained by the responsesurface diagram method were: pH ¼ 2.5, composition of the mobile phase acetonitrile: water 55:45 v/vand flow rate 1.0 ml/min. with a C18 column. Retention time and capacity factor for ochratotoxin Awere 7.46 min. and 1.19, respectively.1. IntroductionOchratoxin A is one of the mycotoxins which are toxicsecondary metabolites produced by fungi growing on agri-cultural commodities in the field or during storage. Itcauses nephrotoxicity, hepatotoxicity and carcinogenicityin animals, (Delacruz and Bach 1990; Kuiper and Scott1989). Several analytical methods for the quantification ofochratoxin A in biological samples (Pascale and Visconti2000), agricultural commodities (Scudamore and MacDo-nald 1998; Kwak and Shon 2000; Entwisle et al. 2000;Eskola et al. 2002) and food (Gareis and Scheuer 2000;Markaki et al. 2001) have been reported previously. All ofthese have been developed by traditional methodology.The aim of this work was the application of the experi-mental design technique to the development and optimisa-tion of an RP HPLC method for the determination ofochratoxin A in different wheat. A full factorial design,‘2