Combined phenyl silane and immunoaffinity column cleanup with liquid chromatography for determination of ochratoxin A in roasted coffee: Collaborative study

A collaborative study was conducted to evaluate a liquid chromatography (LC ) method for ochratoxin A using sequential phenyl silane and immunoaffinity column cleanup, The method was tested at 3 different levels of ochratoxin A in roasted coffee, which spanned the range of possible future European re gulatory limits, The test portion was extracted with methanol and sodium bi carbonate by shaking for 30 min, The extract was filtered, centrifuged, and then cleaned Lap on a phenyl silane column before being eluted from the wa shed column with methanol-water. The eluate was diluted with phosphate-buff ered saline (PBS) and applied to an ochratoxin A immunoaffinity column, whi ch was washed with water. The ochratoxin A was eluted with methanol, the so lvent was evaporated, and the residue was redissolved in injection solvent, After injection of this solution onto a reversed-phase LC apparatus, ochra toxin A was measured by fluorescence detection, Eight laboratory samples of low-level naturally contaminated roasted coffee and 2 laboratory samples o f blank coffee (< 0.2 ng/g ochratoxin A at the signal-to-noise ratio of 3:1 ), along with ampules of ochratoxin A calibrant and:spiking solutions, were sent to 15 laboratories in 13 different European countries, Test portions of the laboratory samples were spiked at levels of 4 ng/g ochratoxin A, and recoveries ranged from 65 to 97%. Based on results for Spiked blank materi al (blind duplicates) and naturally contaminated material (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) ran ged from 2 to 22% and the relative standard deviation for reproducibility ( RSDR) ranged from 14 to 26%. The method showed acceptable within- and betwe en-laboratory precision, as evidenced by HORRAT values, at the low lever of determination for ochratoxin A in roasted coffee.