FOOD CHEMICAL CONTAMINANTS Combined Phenyl Silane and Immunoaffinity Column Cleanup with Liquid Chromatography for Determination of Ochratoxin A in Roasted Coffee: Collaborative Study A. CATHERINE ENTWISLE,ALISON C. WILLIAMS,PETER J. MANN,JOANNE RUSSELL, and PHILIP T. SLACK Leatherhead Food RA, Randalls Rd, Leatherhead, Surrey KT22 7RY, UK JOHN GILBERT Ministry of Agriculture, Fisheries and Food, Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK Collaborators: P. Burdaspal; E. Eklund; J. Gardikis; B. Hald; M-P. Herry; K. Jørgensen; H. Kandler; S. Patel; A. Pittet; M. Schuster; M. Solfrizzo; E. Strassmeier; R. Tiebach; T. Torgensen; A. Van der Stegen 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 possi- ble future European regulatory limits. The test por- tion was extracted with methanol and sodium bi- carbonate by shaking for 30 min. The extract was filtered, centrifuged, and then cleaned up on a phenyl silane column before being eluted from the washed column with methanol–water. The eluate was diluted with phosphate-buffered saline (PBS) and applied to an ochratoxin A immunoaffinity col- umn, which was washed with water. The ochratoxin A was eluted with methanol, the solvent was evaporated, and the residue was redissolved in injection solvent. After injection of this solution onto a reversed-phase LC apparatus, ochratoxin A was measured by fluorescence detection. Eight laboratory samples of low-level naturally contami- nated roasted coffee and 2 laboratory samples of blank coffee (< 0.2 ng/g ochratoxin A at the sig- nal-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 re- sults for spiked blank material (blind duplicates) and naturally contaminated material (blind dupli- cates at 3 levels), the relative standard deviation for repeatability (RSD r ) ranged from 2 to 22% and the relative standard deviation for reproducibility (RSD R ) ranged from 14 to 26%. The method showed acceptable within- and between-laboratory precision, as evidenced by HORRAT values, at the low level of determination for ochratoxin A in roasted coffee. S everal countries have regulatory or guideline limits for ochratoxin A in foods. Levels range from 2 to 50 ng/g ochratoxin A in a variety of commodities, although only in Greece is coffee specifically mentioned (1). In 1998, the European Commission established Regulation No. 1525/98 (2), setting maximum levels for aflatoxins in certain food- stuffs. This was accompanied by Commission Directive 98/53/EC (3), which laid down methods of sampling and per- formance criteria that have to be met for methods of analysis. It is envisioned that Europe-wide legislation for ochratoxin A will be produced in the near future as an amendment to these Regulations. To date, discussion has focused on a proposed regulatory limit for ochratoxin A, with suggestions that 8 ng/g ochratoxin A in green coffee and 4 ng/g ochratoxin A in roasted and instant coffees might be appropriate. Whatever level is ultimately prescribed, a fully validated analytical method will be required to support this legislation. The AOAC First Action Method for ochratoxin A dates back to 1975 (4) and applies only to green coffee rather than roasted coffee, which is acknowledged to be a more problem- atic matrix to analyze. The method uses chloroform for extrac- tion which is no longer acceptable in a number of countries and a thin-layer chromatography (TLC) quantitation which in- creases the detection limit to a level above current ng/g re- quirements for ochratoxin A in coffee. A number of methods have been used for analysis of ochratoxin A in roasted coffee and soluble coffee products. Methanol–bicarbonate has been the preferred extraction sol- vent for a number of procedures. When combined with C 18 Sep-Pak cleanup and fluorescence LC, this procedure had a 2 ng/g ochratoxin A detection limit for roasted coffee (5) al- 444 ENTWISLE ET AL.:JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, 2001 Submitted for publication July 2000. The recommendation was approved by the Methods Committee on Natural Toxins and Food Allergens and was adopted by the Official Methods Board of AOAC INTERNATIONAL. See “Official Methods Board Actions,” (1999) Inside Laboratory Management, November/December issue. Downloaded from https://academic.oup.com/jaoac/article-abstract/84/2/444/5656556 by guest on 22 July 2020