Assessment of the biotransformation of the cardiotonic agent piroximone by high-performance liquid chromatography and gas chromatography-mass spectrometry

• Published in: Journal of Chromatography A 1992-02, Vol.593 (1), p.1-7
• Main Authors: Berg-Candolfi, Martine, Borlakoglu, Jürgen T., Dulery, Bertrand, Jehl, François, Haegele, Klaus D.
• Format: Article
• Language: English
• Subjects: Analysis; Biological and medical sciences; General pharmacology; Medical sciences; Pharmacology. Drug treatments
• ISSN: 0021-9673
• DOI: 10.1016/0021-9673(92)80257-U

14C-labelled piroximone was administered to rats at a dose of 10 mg/kg body weight. Of the total radioactivity administered, 74.9 ± 7.9% ( n=4) and 87.8 ± 1.7 ( n=3) were recovered in the 8-h urine collection after oral and intravenous administration, respectively. Two major metabolites, M 1 and M 2, were detected in methanol extracts and accounted for 7.1 ± 1.2% ( n=4) (M 1 and 4.3 ± 0.4% ( n=4) (M 2) in response to oral administration and 5.7 ± 0.8% ( n=3) (M 1) and 6.7 ± 2.0% ( n=3) (M 2) in response to intravenous administration. In addition, three minor metabolites were detected; M 3 and M 4 in the 8-h urine collection and M 5 in the 12-h urine collection. Separation of piroximone and metabolites was achieved by high-performance liquid chromatography on a C 18 column by gradient elution with 0.05 M ammonium acetate (pH 7) using 0–60% methanol over 20 min at a flow-rate of 1 ml/min, followed by isocratic elution with 60% methanol for 10 min. M 1 and M 2 were isolated by fraction collection following the addition of 1 m M tetrabutylammonium acetate in the mobile phase. Between each injection a column re-equilibration time of 45 min was necessary to achieve optimum collection of M 1 and M 2 fractions. Gas chromatography-mass spectrometry of M 1 provided evidence for a molecular structure consistent with isonicotinic acid methyl ester. Corroborative evidence for this identification was obtained by comparison with a synthetic standard. Isonicotinic acid is assumed to be the actual metabolite while esterification with methanol had occurred as a result of the work-up procedure. In vitro studies carried out with rat liver microsomes resulted in a mean total metabolite formation rate of 94.3 pmol/mg microsomal protein/min.