Simultaneous measurement of the cell-differentiating agent hexamethylene bisacetamide and its metabolites by gas chromatography

Abstract

Hexamethylene bisacetamide (HMBA) is a potent in vitro differentiating agent that has clinical potential as an anticancer drug both as a single agent and as a component of combination therapy. A sensitive and efficient GC method for the isolation, derivatization, and measurement of both HMBA and its two major metabolites in plasma and urine in a single analysis is described. In situ carbamylation of the biological sample with diethylpyrocarbonate forms the urethane derivative of the basic N-acetyl diaminohexane metabolite and allows analyte isolation and concentration by solid-phase extraction. Subsequent formation of the n-butyl ester of 6-acetamidohexanoic acid, the major metabolite, provides a derivatized biological extract that can be rapidly analyzed by temperature-programmed GC. The quantitative extraction and the efficient derivatization steps provide a limit of quantitation of 0.05 m M (10 μg/ml) for all analytes with a precision better than 8% for the range of in vitro activity (0.1–2.0 m M). This method is amenable to automation and is well-suited for the analysis of clinical samples.