The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products J.V. Samsonova a, * , A.J. Douglas b , K.M. Cooper a , D.G. Kennedy b , C.T. Elliott a a Queen’s University Belfast, University Road Belfast, BT7 1NN, Northern Ireland, UK b Agri-Food and Biosciences Institute, Stoney Road, Belfast, UK Received 15 April 2006; accepted 10 December 2007 Abstract Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitro- furazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative bio- markers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as pre- parative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC–MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Nitrofurazone; Semicarbazide; Alternative biomarker; Rat liver; Albumin 1. Introduction Four drugs – furazolidone, furaltadone, nitrofurazone and nitrofurantoin – belong to a group of nitrofuran anti- bacterial agents which were widely used for treatment of gas- trointestinal infections of cattle, pigs and poultry. Due to the potential carcinogenicity and mutagenicity of nitrofurans and theirs metabolites since mid 1990s using of any nitrofu- ran in any food-producing animal within the EU, or in any animal destined for export into the EU has been banned (Commission Regulation, 1995). EU Member States are required to ensure compliance with this ban through their annual residue monitoring plans (Council Directive, 1996). The nitrofuran antibiotics are characterised by quick metabolism, half-life time in vivo is a few hours (McCracken et al., 1995). Therefore determination of the parent com- pounds is unsuitable for monitoring purposes. Recently it was found that high concentrations of nitrofuran metabo- lites accumulate in the retina of pigs (Cooper and Kennedy, 2005) and that parent nitrofurazone accumulates in avian eyes (Cooper et al., 2005a). It was suggested that discovery of these residue depots may allow monitoring of parent drugs instead of theirs metabolites for the detection of drug abuse. However this will only be the case where samples of eyes are available for analytical work. 0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.12.019 Abbreviations: DMFA, dimethylformamide; GSH, glutathione; GST, glutathione S-transferase; IEF, isoelectric focusing; SDS, sodium dodecyl sulfate; SEM, semicarbazide. * Corresponding author. Present address: Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia. Tel.: +7 495 939 3407; fax: +7 495 939 2742. E-mail address: jvs@enz.chem.msu.ru (J.V. Samsonova). www.elsevier.com/locate/foodchemtox Available online at www.sciencedirect.com Food and Chemical Toxicology 46 (2008) 1548–1554