ASSESSMENT OF MICROSOMAL TOLBUTAMIDE HYDROXYLATION BY A SIMPLE THIN-LAYER CHROMATOGRAPHY RADIOACTIVITY ASSAY

A radio thin-layer chromatographic method is described for in vitro me asurement of tolbutamide methylhydroxylation as an alternative to the commonly used HPLC assay. After the incubation experiments of [C-14]to lbutamide with human liver microsomes, the supernatants were directly spotted onto standard silica gel TLC plates and developed in a horizon tal chamber using a solvent system consisting of toluene-acetone-formi c acid (60:39:1, v/v). Dried TLC plates were exposed to a phosphor ima ger plate and quantificated by use of a phosphor imager. Reaction rate s were calculated from the ratio of labelled metabolite to the total r adioactivity. The correlation coefficient between HPLC and the TLC met hod was 0.978 (n = 14). The described method provides a valuable tool for the determination of tolbutamide hydroxylation activity in human l iver microsomes. (C) 1998 Elsevier Science B.V.