Quantitative determination of nebivolol from human plasma using liquid chromatography–tandem mass spectrometry

► Liquid–liquid extraction method was used to extract nebivolol from human plasma. ► Liquid chromatography with tandem mass spectrometry was used to analyze and quantify nebivolol. ► Method was validated in terms of accuracy, precision, selectivity, absolute recovery and stability. ► This method is free from ion suppression, ion enhancement and any type of abnormal ionization. ► Developed bioanalytical method is a highly sensitive as lower limit of quantification was proved 30pg/mL. In the present work, a rapid, sensitive, specific, precise and accurate liquid chromatography–tandem mass spectrometry method for determination of nebivolol in human plasma was developed and validated with a large calibration curve range (50–5000pg/mL) which can be used for routine drug analysis and bioequivalence studies. Liquid–liquid extraction method was used to extract the analyte from the human plasma. The separation was achieved using Waters symmetry, C18, 4.6×150mm, 5μm column with formic acid in water, 0.01%, v/v: Acetonitrile (40:60) as a mobile phase. A flow rate of 0.8mL/min, no splitting and run time 2.00min was used for the chromatographic analysis of nebivolol. Sensitivity of this method was found to be 30pg/mL. The analyte was analyzed by mass spectrometry in the multiple reaction monitoring mode. A Turbo-Ion spray source was interfaced between the HPLC and triple quadrupole mass spectrometer (MDS Sciex API 4000). The precursor-product ion m/z 406.00–151.00 for nebivolol and m/z 410.20–151.00 for nebivolol-D4 were used for quantification of an analyte and its IS. The method was validated in terms of accuracy, precision, selectivity, absolute recovery, freeze-thaw stability, bench-top stability, dry extract stability, short and long term stock solution stability, wet extract stability and re-injection reproducibility. The within- and between-batch accuracy was found to lie within the range of 87.00–100.40% and within- and between-batch precision was obtained within the range 0.33–8.67%. The mean recovery of all three concentration levels for drug was obtained 67.67% where as the mean recovery of IS was 68.74%. The %RSD value at higher concentration and lower concentration in all stability experiments was within 15%. This method is free from ion suppression, ion enhancement and any type of abnormal ionization.