Determination of Theophylline Metabolites in Human Liver Microsomes by High-Performance Liquid Chromatography

✍ Scribed by B.B. Rasmussen; K.K. Nielsen; K. Brosen

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 347 KB
• Volume: 222
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1446

✦ Synopsis

A method for the quantitation of three metabolites of theophylline, 1-methylxanthine (1MX), 3-methylxanthine (3MX), and 1,3 -dimethyluric acid (13DMU) in human liver microsomes has been developed. The method is based on a simple one-step extraction followed by isocratic, reversed-phase high-performance liquid chromatography with uv detection (detection wavelength: (273 \mathrm{~nm}) ). The detection limit was 0.03 nmol (\cdot \mathrm{mg}^{-1} \cdot \mathbf{h}^{-1}), which corresponds to 10 pmol per sample for all three metabolites. Linear standard curves were obtained for all three compounds within a concentration range of (0.6-6.0 \mathrm{nmol} \cdot \mathrm{mg}^{-1} \cdot \mathrm{h}^{-1}) for (3 M X) and (1 \mathrm{MX}) and 2.4-24.0 (\mathrm{nmol} \cdot \mathrm{mg}^{-1} \cdot \mathrm{h}^{-1}) for 13DMU. The absolute recoveries ranged from 61 to (80 %, 68) to (74 %), and 74 to (85 %) for (1 M X, 3 M X), and (13 D M U), respectively, within the concentration range of the standard curve. The reproducibility and repeatability showed a coefficient of variation (<12 %) at five concentrations within the standard curve range. The accuracy for all three metabolites was within (\pm 5 %) at three concentrations, except for (1 \mathrm{MX}) in the lowest concentration ((12 %)). The simple but sensitive method developed is highly suitable as a probe for cytochrome P4501 A2 (CYP1A2) and probably also CYP2E1 function in human liver microsomes. (c) 1994 Academic Press, Inc.