Analysis of Protein-bound metabolites of Furazolidone and furaltadone in pig liver by high-performance liquid chromatography and liquid chromatography–mass spectrometry

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Abstract
Studies undertaken using radiolabelled furazolidone have demonstrated the covalent binding of residues of the drug to cellular protein in vivo. A portion of these bound residues and those formed by furaltadone, a related nitrofuran drug, possess intact side-chains, 3-amino-2-oxazolidinone (AOZ) and 5-morpholinomethyl-3-amino-2-oxazolidinone (AMOZ), respectively. These side-chains have molecular characteristics in common with the parent compounds and may be released from liver tissue under mild acidic conditions. Derivatization with 2-nitrobenzaldehyde (NBA) serves to isolate the released side-chains and the derivatives NPAOZ and NPAMOZ are chromophoric, thereby permitting UV detection. This paper reports the introduction of an extract clean-up step to the existing procedure which eliminates or decreases interference from NBA in the HPLC–UV determination of NPAOZ. The modified procedure was also applied to the determination of AMOZ. The development of an LC–MS method for the quantitative and confirmatory determination of AOZ and AMOZ extracted and derivatized according to the same procedure as that for HPLC–UV is described. The methods were validated for AOZ and AMOZ in fortified (intra-and inter-assay studies) and incurred (inter-assay studies) pig liver samples. The limit of determination for fortified control liver samples was 5 ng AOZ g–1 and 10 ng AMOZ g–1 by HPLC–UV and 10 ng AOZ or AMOZ g–1 by LC–MS. In addition, a study to determine the ratio of released AOZ to the total bound residues present in incurred liver samples from pigs treated with furazolidone is described.