FOOD CHEMICAL CONTAMINANTS Determination of Fumonisins B 1 and B 2 in Corn by Liquid Chromatography/Mass Spectrometry with Immunoaffinity Column Cleanup: Single-Laboratory Method Validation HAMIDE Z. SENYUVA and SUREYYA OZCAN TUBITAK, Ankara Test and Analysis Laboratory (ATAL), Konya Yolu No. 67, Besevler, 06530, Ankara, Turkey DILEK CIMEN Gazi University, Department of Chemistry, Besevler, 06500, Ankara, Turkey JOHN GILBERT Central Science Laboratory, Food and Rural Affairs, Department for Environment, Sand Hutton, York, YO41 1LZ, United Kingdom A single-laboratory method validation was conducted to establish the effectiveness of an immunoaffinity column cleanup procedure followed by liquid chromatography/mass spectrometry (LC/MS) for the determination of fumonisins B 1 and B 2 (FB 1 + FB 2 ) in corn. The test portion is extracted with acetonitrile–methanol– water (25 + 25 + 50). The extract is filtered, diluted with phosphate-buffered saline solution, and applied to an immunoaffinity column. FB 1 + FB 2 are removed with methanol and directly determined by reversed-phase LC with MS detection using selected-ion monitoring of 2 characteristic ions in each case. Test portions of blank corn samples were spiked with a mixture of FB 1 + FB 2 to give total levels of 200 and 500 ng/g, respectively. Recoveries of both FB 1 and FB 2 from spiked samples averaged 90.4–101%. Based on results for spiked raw corn (triplicates at 2 levels), the relative standard deviation for repeatability ranged from 2.8 to 7.1%. The accuracy of the method was demonstrated by analysis of Food Analysis Performance Assessment Scheme (FAPAS ® ) test material. The method was also applied to a small survey of processed corn products such as corn chips, cornflakes, and popcorn. F umonisins are a class of mycotoxins produced by several Fusarium fungi, occurring predominantly in corn and frequently at mg/kg levels (1, 2). The fumonisins are sufficiently stable to survive wet and dry milling and also subsequent processing such as extrusion cooking (3); thus, they can be found in finished products such as corn flour, snack foods, and breakfast cereals, although at significantly lower levels than are found in raw corn (4–6). There is some evidence that fumonisins become matrix-bound after such processing, making them more difficult to extract from naturally contaminated processed corn products (7, 8). Therefore, particular care is required in optimizing extraction conditions in methods to be used for analyzing heat-processed corn products. The European Union (EU) has recently regulated levels of fumonisins in corn and corn-based foods (9), basing regulatory limits on the sum of fumonisin B 1 (FB 1 )+ fumonisin B 2 (FB 2 ). For unprocessed corn, with the exception of unprocessed corn intended to be processed by wet milling, the sum of FB 1 + FB 2 should not exceed 4000 mg/kg. For maize intended for direct human consumption and maize-based foods for direct human consumption, with the exception of breakfast cereals, snacks, and baby foods, levels of FB 1 + FB 2 should not exceed 1000 mg/kg. For corn-based breakfast cereals and corn-based snacks, a limit of 800 mg/kg for FB 1 + FB 2 is applied, and for processed corn-based foods and baby foods for infants and young children, the regulatory limit is lower, i.e., 200 mg/kg. Milling fractions of corn with particle size of >500 mm that fall within the Combined Nomenclature (CN) code, in accordance with the EU common customs tariff CN code 1103 13 or 1103 20 40, and other corn milling products with particle size of >500 mm that are not used for direct human consumption and fall within CN code 1904 10 10 have a limit of 1400 mg/kg. Milling fractions of corn with particle size of £500 mm that fall within CN code 1102 20 and other corn milling products with particle size of £500 mm that are not used for direct human consumption and fall within CN code 1904 10 10 have a limit of 2000 mg/kg (9). In contrast, in the United States there are recommended maximum levels (rather than regulatory limits) for fumonisins in human foods and in animal feeds (10) that the U.S. Food and Drug Administration (FDA) considers achievable with the use of good agricultural and good manufacturing practices. The recommended FDA limits are similar to the regulatory limits of the EU, in that both are expressed as total fumonisins; however, the FDA total = FB 1 + FB 2 + fumonisin 598 SENYUVA ET AL.:JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 3, 2008 Received January 9, 2008. Accepted by AP February 26, 2008. Corresponding author's e-mail: hamide.senyuva@tubitak.gov.tr Downloaded from https://academic.oup.com/jaoac/article/91/3/598/5656058 by guest on 01 March 2023