Stereoselective analysis of nebivolol isomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry: Application in pharmacokinetics

• The article describes the stereoselective analysis of nebivolol in human plasma.
• The validated method showed quantification limit of 25 pg of each isomer/mL plasma.
• The method was applied in a pharmacokinetic study of a single dose of nebivolol.
• Plasma nebivolol isomers were quantified until 48 h after drug administration.

Nebivolol is available for clinical use as a racemic mixture. Isomer d-nebivolol (SRRR) is a β1 adrenergic receptor blocker and its antipode, l-nebivolol (RSSS) is responsible for endothelium-dependent NO liberation. This report describes the development and validation of a method of analysis of nebivolol isomers in human plasma by high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). Nebivolol isomers were extracted from 2 mL aliquots of plasma spiked with tramadol as internal standard, alkalinized and added of sodium chloride and diisopropyl ether:dichloromethane (70:30, v/v). Nebivolol isomers were resolved on a Chirobiotic® V column using methanol:acetic acid:diethylamine (100:0.15:0.05, v/v/v) as mobile phase. Protonated ion and respective ion product were monitored in transitions 406 > 151 for nebivolol and 264 > 58 for internal standard tramadol. There was no racemization of nebivolol isomers during the procedures of sample preparation and chromatographic analysis and matrix effect was absent. Analysis of nebivolol isomers showed linearity for plasma concentrations of 25–2500 pg/mL of each isomer. The quantification limit was 25 pg of each isomer/mL of plasma. Variation coefficients and inaccuracy calculated in precision and accuracy determinations were lower than 15%. Nebivolol was stable in human plasma after three successive cycles of freezing and thawing, during 4 h at room temperature and after processing during 12 h in the auto sampler at 5 °C showing deviation values lower than 15%. The method was applied in a study of the kinetic disposition of nebivolol in plasma samples collected until 48 h after administration of an oral single dose of 10 mg of racemic nebivolol hydrochloride to a patient with systemic arterial hypertension. The clinical study demonstrated that the nebivolol pharmacokinetics is stereoselective. Isomer l-nebivolol showed higher AUC0–∞ (9.4 ng/h/mL vs. 4.7 ng/h/mL) and smaller apparent clearance (Cl/f) (531.8 L/h vs. 1304.4 L/h) when compared to antipode d-nebivolol.