Assessment of microsomal tolbutamide hydroxylation by a simple thin-layer chromatography radioactivity assay.
A radio thin-layer chromatographic method is described for in vitro measurement of tolbutamide methylhydroxylation as an alternative to the commonly used HPLC assay. After the incubation experiments of [14C]tolbutamide with human liver microsomes, the supernatants were directly spotted onto standard silica gel TLC plates and developed in a horizontal chamber using a solvent system consisting of toluene-acetone-formic acid (60:39:1, v/v). Dried TLC plates were exposed to a phosphor imager plate and quantificated by use of a phosphor imager. Reaction rates were calculated from the ratio of labelled metabolite to the total radioactivity. The correlation coefficient between HPLC and the TLC method was 0.978 (n=14). The described method provides a valuable tool for the determination of tolbutamide hydroxylation activity in human liver microsomes.
Ludwig
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Department of Pharmacokinetics and Drug Metabolism, Dr. Karl Thomae GmbH, Biberach an der Riss, Germany.
Wolfinger
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Ebner
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D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006801:Humans; D006900:Hydroxylation; D007004:Hypoglycemic Agents / Q000032:analysis* / Q000378:metabolism*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008862:Microsomes, Liver / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D012997:Solvents; D014044:Tolbutamide / Q000032:analysis* / Q000378:metabolism*
D007004:Hypoglycemic Agents; D007202:Indicators and Reagents; D012997:Solvents; D014044:Tolbutamide