Simultaneous determination of amlodipine and bisoprolol in rat plasma by a liquid chromatography/tandem mass spectrometry method and its application in pharmacokinetic study

A sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the quantitative determination of amlodipine and bisoprolol, using clenbuterol as the internal standard (IS). The analytes and IS were isolated from 100 μL plasma samples by a simple liquid–liquid extraction (LLE). Reverse-phase high performance liquid chromatography (RP-HPLC) separation was accomplished on a Diamonsil C18 column (50 mm × 4.6 mm, 5 μm) with a mobile phase composed of methanol–water–formic acid (75:25:0.01, v/v/v) at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 3 min. Multiple reacting monitoring (MRM) transitions of m/z [M+H]+ 409.1 → 237.9 (amlodipine), m/z [M+H]+ 326.2 → 116.0 (bisoprolol) and m/z [M+H]+ 277.0 → 203.0 (clenbuterol, IS) were used to quantify amlodipine, bisoprolol and IS, respectively. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.2 ng/mL for both amlodipine and bisoprolol, and the linear range was 0.2–50 ng/mL for both amlodipine and bisoprolol (r2 > 0.9961). All the validation data, such as accuracy, precision and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic studies of amlodipine and bisoprolol in Sprague–Dawley (SD) rats.