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Determination of aliphatic thiols by fluorometric high-performance liquid chromatography after precolumn
1Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Abstract:
A sensitive, fluorometric high-performance liquid chromatographic method for the detection of aliphatic thiols, following pre-column derivatization with 2-(4-N-maleimidophenyl)-6-methylbenzothiazole, has been developed. The N-maleimide, the acid and the methyl ester derivatives of the commercially available 2-(4-aminophenyl)-6-methylbenzothiazole were synthesized and found to be equally effective for the precolumn derivatization procedure. The resulting fluorescentic derivatives of aliphatic thiols were separated on a reversed-phase column (Ultrasphere-ODS) using 0.1% hexane-sulfonic acid in 10 mM potassium hydrogen phosphate: acetonitrile (65:35) as a mobile phase and were detected fluorometrically (excitation 320 nm; emission 405 nm). The method is highly sensitive (femtomole range) and is easily applied for determination of SH-containing drugs and endogenous thiols in biological samples.