ORIGINAL PAPER Simultaneous LC–MS/MS determination of aflatoxin M 1 , ochratoxin A, deoxynivalenol, de-epoxydeoxynivalenol, α and β-zearalenols and fumonisin B 1 in urine as a multi-biomarker method to assess exposure to mycotoxins Michele Solfrizzo & Lucia Gambacorta & Veronica M. T. Lattanzio & Stephen Powers & Angelo Visconti Received: 16 June 2011 /Revised: 17 August 2011 /Accepted: 22 August 2011 /Published online: 4 September 2011 # Springer-Verlag 2011 Abstract Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M 1 (AFM 1 ), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha- zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumoni- sin B 1 (FB 1 ) can be used as urinary biomarkers. A liquid chromatographic–tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed- phase chromatography using a multi-step linear methanol– water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC– ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03–12 ng mL -1 , using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3–20%. Enzymatic digestion with β- glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time. Keywords Mycotoxins . Urine . Biomarkers . LC–MS/MS . Immunoaffinity cleanup Introduction Aflatoxin B 1 (AFB 1 ), deoxynivalenol (DON), ochratoxin A (OTA), fumonisin B 1 (FB 1 ), and zearalenone (ZEA) have been recognised as major mycotoxins that can contaminate foods and beverages all over the world [1]. Although these toxins belong to different classes of mycotoxins and are produced by different fungal species, mixtures of AFB 1 , DON, OTA, FB 1 , and ZEA occur naturally in cereals [2]. M. Solfrizzo (*) : L. Gambacorta : V. M. T. Lattanzio : A. Visconti Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Via Amendola, 122/O – 70126 Bari, Italy e-mail: michele.solfrizzo@ispa.cnr.it S. Powers Vicam, A Waters Business, 34 Maple Street, Milford, MA 01757, USA Anal Bioanal Chem (2011) 401:2831–2841 DOI 10.1007/s00216-011-5354-z