LC–MS/MS assay method development and validation for the simultaneous quantification of Solifenacin and Mirabegron in human plasma

Abstract

The present study intended to develop a simple and novel Liquid Chromatography–Mass Spectrometry/ Mass spectrometry (LC–MS/MS) method for the simultaneous quantification of solifenacin and mirabegron, a combined medication used for the treatment of overactive bladder symptoms in human plasma. Tolterodine drug is used as an internal standard in the study. Both the analytes and internal standard were isolated from 100 μL plasma samples by liquid-liquid extraction and then chromatographed on Zorbax C18 (4.6 mm×50 mm, 5 μm) column with a mobile phase consisting of methanol and 5mM ammonium formate in the ratio of 25:75 (v/v) pumped at 0.3 mL/min. The method had a chromatographic total run time of 5 min. The developed method gave symmetric peak at a retention time of 2.50 min, 2.99 min and 1.18 min respectively for solifenacin, mirabegron and nifedipine satisfying all the peak properties as per USP guidelines. The mass spectral characterization of separated analytes in the LC method was performed using mass detector operated at Multiple Reaction Monitoring mode with precursor-toproduct ion transitions at m/z of 363 to m/z of 110 as MH+ ion for solifenacin, m/z of 397 to m/z of 239 as MH+ ion for mirabegron. A very sensitive limit of detection of 7.5 ng/mL was observed and showed calibration curve linear over the concentration range of LLOQ to 1000 ng/mL. The other validation parameters were found to have acceptable accuracy, precision, linearity and selectivity. The mean extraction concentration was acceptable and very high for both the analytes in HQC, MQC and LOQ levels. Various stability studies of solifenacin and mirabegron such as freeze-thaw, short term, long term, auto-sampler and dry extract stability proved that the method was stable. Based on the results, it can be proved that the method was accurate, precise and specific for the simultaneous analysis of solifenacin and mirabegron in human plasma.