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 a nd its active metabolite 3-morpholinosydnonimine (Sin-l) in the ng/ml range in plasma. The method developed is based on on-line LC-MS-MS usi ng pneumatically assisted electrospray ionisation as an interface, pre ceded by off-line solid-phase extraction (SPE) on disposable extractio n cartridges (DECs). The SPE operations were performed automatically b y means of a sample processor equipped with a robotic arm (automated s ample preparation with extraction cartridges; ASPEC system). The DEC, filled with phenyl-modified silica, was first conditioned with methano l and water. The washing step was performed with water. Finally, the a nalytes 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 mu m). The mobi le 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 atmosphe ric pressure ionisation interface in the positive ion mode. The chroma tographed analytes were detected in the multiple reaction monitoring m ode. The MS-MS ion transitions monitored were (m/z) 243-->86 for molsi domine 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 metho d was found to be linear in the 0.5-50 ng/ml concentration range for t he two analytes (r(2)=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 devel oped was successfully used to investigate the bioequivalence of oral d oses of molsidomine between a generic tablet and a reference product. (C) 1998 Elsevier Science B.V. All rights reserved.