Journal of Chromatography B: Biomedical Sciences and Applications
Short communicationAssessment of microsomal tolbutamide hydroxylation by a simple thin-layer chromatography radioactivity assay
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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