Analysis of protein-bound metabolites of furazolidone and furaltadone in pig liver by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

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-morpholino-methyl-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.