Determination of Ochratoxin A in Black and White Pepper, Nutmeg, Spice Mix, Cocoa, and Drinking Chocolate by High-Performance Liquid Chromatography Coupled with Fluorescence Detection: Collaborative Study.

A method validation study for the determination of ochratoxin A in black and white pepper (Piper spp.), nutmeg (Myristica fragrans), spice mix (blend of ginger, turmeric, pepper, nutmeg, and chili), cocoa powder, and drinking chocolate was conducted according to the International Harmonized Protocol of the International Union of Pure and Applied Chemistry. The method is based on the extraction of samples with aqueous methanol, followed by a cleanup of the extract with an immunoaffinity column. The determination is carried out by reversed-phase LC coupled with a fluorescence detector. The study involved 25 participants representing a cross-section of research, private, and official control laboratories from 12 European Union (EU) Member States, together with Turkey and Macedonia. Mean recoveries ranged from 71 to 85% for spices and from 85 to 88% for cocoa and drinking chocolate. The RSDr values ranged from 5.6 to 16.7% for spices and from 4.5 to 18.7% for cocoa and drinking chocolate. The RSDR values ranged from 9.5 to 22.6% for spices and from 13.7 to 30.7% for cocoa and drinking chocolate. The resulting Horwitz ratios ranged from 0.4 to 1 for spices and from 0.6 to 1.4 for cocoa and drinking chocolate according to the Horwitz function modified by Thompson. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, and it conforms to requirements set by current EU legislation.