Simultaneous analysis of 2-methoxyphenylmetyrapone and its seven potential metabolites by high-performance liquid chromatography

• Published in: Journal of chromatography. B, Biomedical sciences and applications Biomedical sciences and applications, 1997-12, Vol.704 (1), p.315-323
• Main Authors: Damani, Lyaquatali A, Tsai, Mui-Chiung, Lin, Ge, Mitterhauser, Markus, Zolle, Illse
• Format: Article
• Language: English
• Subjects: 2-Methoxyphenylmetyrapone; Analysis; Animals; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; General pharmacology; Male; Medical sciences; Metyrapone - analogs & derivatives; Metyrapone - blood; Metyrapone - metabolism; Metyrapone - urine; Microchemistry; Pharmacology. Drug treatments; Quality Control; Radioactive Tracers; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Solvents
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(97)00491-X

A sensitive and specific high-performance liquid chromatographic (HPLC) assay has been developed for the quantification of 2-methoxyphenylmetyrapone (2-MPMP) and its seven potential metabolites in rat urine and whole blood. 2-MPMP, 2-hydroxyphenylmetyrapone and their N-oxides, together with 2-methoxyphenylmetyrapol, 2-hydroxyphenylmetyrapol and their N-oxides were separated on an Isco Spherisorb ODS-2 reversed-phase column (250×4.6 mm, I.D., 5 μm), with an Isco Spherisorb ODS-2 guard cartridge (10×4.6 mm I.D.). A gradient elution was employed using solvent system A (acetonitrile–water–triethylamine–acetic acid, 27.3:69.1:0.9:2.7%, v/v) and solvent system B (methanol), the gradient program being as follows: initial 0–4 min A:B=74:26; 4–10 min linear change to A:B=50:50; 10–16 min maintain A:B=50:50; 16 min return to initial conditions (A:B=74:26). Flow-rate was maintained at 1.25 ml/min, and the eluent monitored using a diode array multiple wavelength UV detector set at 260 nm. Most of the analytes were baseline resolved, and analysis of samples recovered from blood or urine (pH 12, 3×5 ml of dichloromethane, recovery ∼20–95%) revealed no interference from any co-extracted endogenous compounds in the biological matrices, except for 2-hydroxyphenylmetyrapol N-oxide (2-OHPMPOL-NO) at low concentrations. The calibrations ( n=6) were linear ( r≥0.996) for all analytes (∼0.5–100 μg/ml), with acceptable inter- and intra-day variability. Subsequent validation of the assay revealed acceptable precision, as measured by coefficient of variation (C.V.) at the low (0.5 mg/ml), medium (50 μg/ml) and high (100 μg/ml) concentrations. The limits of detection for 2-MPMP and their available potential metabolites, except 2-OHPMPOL-NO, in rat urine and blood were both 0.5 μg/ml, respectively.