Determination of molsidomine and its active metabolite in human plasma using liquid chromatography with tandem mass spectrometric detection.

Pharmacokinetic studies of molsidomine require a sensitive analytical method to allow the determination of concentrations of this compound and its active metabolite 3-morpholinosydnonimine (Sin-1) in the ng/ml range in plasma. The method developed is based on on-line LC-MS-MS using pneumatically assisted electrospray ionisation as an interface, preceded by off-line solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (automated sample preparation with extraction cartridges; ASPEC system). The DEC, filled with phenyl-modified silica, was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol containing formic acid (0.2%) and water. The liquid chromatographic separation of molsidomine and Sin-1 was achieved on an RP-8 stationary phase (5 microns). The mobile phase was a mixture of methanol-water-formic acid (65:35:0.1, v/v/v). The HPLC system was then coupled to a MS-MS system with an atmospheric pressure ionisation interface in the positive ion mode. The chromatographed analytes were detected in the multiple reaction monitoring mode. The MS-MS ion transitions monitored were (m/z) 243-->86 for molsidomine and 171-->86 for Sin-1. The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 74 +/- 3 and 55 +/- 5% for molsidomine and Sin-1, respectively. The method was found to be linear in the 0.5-50 ng/ml concentration range for the two analytes (r2 = 0.999 for both molsidomine and Sin-1). The mean RSD values for repeatability and intermediate precision were 3.4 and 4.8% for moldsidomine and 3.1-7.7% for the metabolite. The method developed was successfully used to investigate the bioequivalence of oral doses of molsidomine between a generic tablet and a reference product.

RCT Entities:   Population Interventions Outcomes