Assessment of microsomal tolbutamide hydroxylation by a simple thin-layer chromatography radioactivity assay

J Chromatogr B Biomed Sci Appl. 1998 Apr 10;707(1-2):347-50. doi: 10.1016/s0378-4347(97)00603-8.


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.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Humans
  • Hydroxylation
  • Hypoglycemic Agents / analysis*
  • Hypoglycemic Agents / metabolism*
  • In Vitro Techniques
  • Indicators and Reagents
  • Microsomes, Liver / chemistry*
  • Microsomes, Liver / metabolism*
  • Reproducibility of Results
  • Solvents
  • Tolbutamide / analysis*
  • Tolbutamide / metabolism*


  • Hypoglycemic Agents
  • Indicators and Reagents
  • Solvents
  • Tolbutamide