An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC-MS/MS: Application to clinical pharmacokinetics

- Indirect analysis of nebivolol glucuronide isomers in plasma is sensitive.
- Nebivolol pharmacokinetics is stereoselective with plasma accumulation of l-isomer.
- Nebivolol glucuronidation is stereoselective with plasma accumulation of d-isomer.
- Plasma concentrations of glucuronides are higher than unchanged nebivolol.

Nebivolol is a racemate of the d-isomer responsible for β1 adrenergic receptor antagonism and the l-isomer responsible for the release of nitric oxide from endothelial cells. Nebivolol is mainly metabolized to nebivolol glucuronide, which also contribute to the nebivolol β1 adrenoreceptor antagonism. This study reports the development and validation of an indirect stereoselective method of analysis of nebivolol glucuronides in plasma by LC-MS/MS. The method was applied to the investigation of stereoselectivity in the glucuronidation of nebivolol in elderly hypertensive patients (n = 11) CYP2D6 phenotyped as EM and treated with a single oral dose of the racemate. One-milliliter plasma aliquots spiked with internal standard (S)-(−)-metoprolol were incubated with 25 μL of β-glucuronidase (final concentration 2500 unit/mL) at pH 5.0 for 16 h at 37 °C. Linearity for total nebivolol was 0.2-125 ng of each isomer per mL plasma and permitted analysis of nebivolol glucuronide isomers up to 48 h after administration of a single oral dose of 10 mg racemate. Regarding to the nebivolol glucuronide isomers, higher plasma concentrations of the d-isomer were observed compared to the l-isomer (d/l AUC = 5.4), explaining at least in part the plasma accumulation of unchanged l-nebivolol (l/d AUC = 1.8). This study also showed metabolic glucuronide nebivolol/unchanged nebivolol ratios of approximately 6.5 for the l-isomer (AUC 65.3 vs 10.1 ng h/mL) and approximately 62.1 (335.2 vs 5.4 ng h/mL) for the d-isomer. Considering that d-nebivolol glucuronide also contributes for β1 adrenergic receptor antagonism, future studies regarding PK-PD of nebivolol should evaluate not only plasma concentrations of unchanged nebivolol isomers but also glucuronide nebivolol isomers.

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