Development and Validation of Bioanalytical Method for Simultaneous Estimation of Nebivolol Enantiomers in Human Plasma Using Liquid Chromatography-tandem Mass Spectrometry

The present study describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of S-RRR and R-SSS nebivolol (nebivolol enantiomers) in human plasma using solid phase extraction technique. Method of both S-RRR and R-SSS nebivolol (nebivolol enantiomers) has been developed and validated using racemic nebivolol D۴ as an internal standard. Analytes from human plasma were extracted by ion exchange cartridges and subsequently separated on chiral column using acetonitrile: ammonium carbonate in water, ۱۵۸ mg/L ۸۰:۲۰% v/v as a mobile phase, at a flow rate of ۰.۹ mL/min. Quantification of S-RRR and R-SSS nebivolol and R-nebivolol D۴ was performed using multi-reaction monitoring mode (MRM) in positive mode. The calibration curve was linear (r۲ > ۰.۹۹) over the concentration range of ۲۰.۰ to ۶۰۰۰ pg/mL for S-RRR and R-SSS nebivolol. The intra-day and inter-day assay precision revealed within ±۱۵% (at LLOQ level ±۲۰%) with accuracy within ۸۵%-۱۱۵% (at LLOQ level ۸۰% -۱۲۰%). The LC-MS/MS method was fully validated for all the validation parameters as per current regulatory requirement (US FDA, EU) such as selectivity, matrix effect, recovery and stability (in solution and in matrix stability).

Keywords:

Bioanalytical method , Nebivolol enantiomers , Human plasma , LC-MS/MS