Simultaneous Determination of Bosentan, Glimepiride, HYBOS and M1 in Rat Plasma by UPLC-MS-MS and its Application to Pharmacokinetic Study.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the simultaneous determination of bosentan (BOS), glimepiride (GLP), hydroxyl bosentan (HYBOS) and hydroxyl glimepiride (M1) in rat plasma using one-step protein precipitation was developed and validated. After addition of ambrisentan as an internal standard (IS), protein precipitation by acetonitrile was used in sample preparation. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 mu m particle size, Waters Corp., Milford, MA, USA) and inline 0.2 mu m stainless steel frit filter (Waters Corp.) with acetonitrile-0.1% formic acid as the mobile phase at a flow rate of 0.4 mL/min with gradient elution. The column temperature was maintained at 40 degrees C. Only 4 min was needed for an analytical run. The retention times were similar to 3.29 min for BOS, 3.56 min for GLP, 1.42 min for HYBOS, 1.53 min for M1 and 3.22 min for IS. Electrospray ionization source was employed and operated in positive-ion mode; multiple reaction monitoring mode was applied to target fragment ions m/z 552 --> 202, m/z 568 --> 202, m/z 491 --> 352, m/z 507 --> 352 and m/z 379 --> 347 for BOS, HYBOS, GLP, M1 and IS, respectively. The assaywas validated over concentration ranges of 25-5,000 ng/mL (r(2) = 0.9984) for BOS, 1-200 ng/mL (r(2) = 0.9999) for GLP, 0.5-100 ng/mL (r(2) = 0.9999) for HYBOS and 0.1-20 ng/mL (r(2) = 0.9984) for M1. Intra- and interday precision values for replicate quality control samples were within 14.2% for all analytes during the assay validation. Mean quality control accuracy values were within -3.3 to 14.4% of nominal values for all analytes. The mean recoveries of BOS, GLP, HYBOS, M1 and ambrisentan from the plasma exceeded 90.4%. The analytes were stable in rat plasma for at least 2 h at room temperature, 30 days at -40 degrees C and following at least three freeze-thaw cycles (-40 degrees C to room temperature). This method was successfully applied to a pharmacokinetic study of coadministeration of BOS and GLP in rats.