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

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Abstract

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.

Introduction

In vitro investigations for the identification of cytochrome P450 enzymes that are involved in the metabolism of drug candidates are an important issue for rational drug development. Therefore, robust routine methods are needed to measure the in vitro metabolism of standard test drugs that are regarded to be specific for a single cytochrome P450 enzyme. Cytochrome P450 2C9 (CYP 2C9) is involved in the metabolism of many xenobiotics 1, 2, 3, 4. Methylhydroxylation of the hypoglycaemic agent tolbutamide (Fig. 1) has been proposed as a specific probe for CYP 2C9 [5]. In the present study we have developed a thin-layer chromatographic method for in vitro measurement of CYP 2C9 activity as an alternative to the commonly used high-performance liquid chromatography (HPLC) assay. This thin-layer chromatography (TLC) assay employs [14C]tolbutamide, a commercially available radiolabelled compound, is easy to establish, and allows a rapid and sensitive measurement of hydroxytolbutamide formation.

Section snippets

Reagents and chemicals

Tolbutamide was purchased from Sigma (Deisenhofen, Germany) and hydroxytolbutamide from Ultrafine Chemicals (Manchester, UK). [Ring-U-14C]tolbutamide (60 mCi/mmol) was obtained from Amersham (Buckinghamshire, UK), and NADPH from Boehringer Mannheim (Mannheim, Germany). Solvents of analytical grade (Merck, Darmstadt, Germany) were used for HPLC and development of TLC plates (silica gel 60 F-254, 10×10 cm, Merck). All other reagents were of the highest quality available.

Human liver microsomes

Results and discussion

Fully automated solid-phase sample extraction showed a mean recovery of hydroxytolbutamide from incubation mixtures performed with heat inactivated microsomes (30 min, 80°C) of 101±2.45% (n=4) over the concentration range of 1 to 12 nmol. Total recovery of 14C radioactivity in incubation mixtures (100 μM tolbutamide, 1 mg/ml microsomal protein) was 98.9% (n=2). The rate of formation of hydroxytolbutamide was linear with incubation time up to 2 h and with microsomal protein concentration from

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