Immunoaffinity Extraction of 4-Hydroxy-2-(4-methylphenyl) benzothiazole and Its Metabolites for Determination by Gas Chromatography-Mass Spectrometry

Immunoaffinity extraction of 4-hydroxy-2-(4-methylphenyl) benzothiazole and its metabolites, together with the corresponding meta-isomers has been achieved by the use of an antibody raised against an immunogen, an O-carboxymethyloxime-bovine serum albumin conjugate of 4-hydroxy-2-(4-formylphenyl) benzothiazole. The antibody produced exhibited a broad spectrum of affinity, not only for metabolites oxidized at the 4-methyl group of the benzene moiety but also for the corresponding meta-isomers. Up to 4 μg in total of these benzothiazoles could be extracted on the immunoaffinity adsorbent and recovered almost quantitatively by elution with 90% methanol. The resulting chromatogram was free from any interference. The eluted compounds were derivatized by conversion to their methyl esters and/or trimethylsilyl ethers, and subsequently separated into individual benzothiazoles by means of gas chromatography-mass spectrometry. The derivatized compounds were monitored using a characteristic ion, [M-CH3]+, and the limit of detection was 10 fmole. The peak height ratio of each metabolite to its corresponding meta-isomer internal standard was plotted against the concentration of the former and good linearity was observed over the range 0.2-5 ng/ml.