Development and validation of a bio-analytical method for simultaneous quantification of nebivolol and labetalol in aqueous humor and plasma using LC-MS/MS and its application to ocular pharmacokinetic studies.

Beta blockers is the class of choice of drugs in treatment of open angle Glaucoma. However, many of these drugs suffer from systemic side effects due to their absorption into systemic circulation via nasolachrymal duct. To evaluate the safety and efficacy of nebivolol and labetalol for the treatment of open angle glaucoma, it is important to have a bioanalytical method for measuring the drug concentrations both in aqueous humor and plasma. A simple, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation technique was developed for simultaneous quantification of nebivolol and labetalol using nebivolol-d4 and metoprolol, respectively, as internal standards in aqueous humor and plasma. Nebivolol and labetalol were monitored in electrospray positive ionization (ESI) mode at transition 406.2/151.1 and 329.2/162.0, respectively. Mobile phase comprised of mixture of aqueous buffer (solvent A) and organic phase (solvent B) (mixture of A:B in the ratio of 30:70, v/v). The aqueous buffer was 5 mM ammonium acetate buffer adjusted to pH 3.5 ±â€¯0.05 with formic acid while the organic phase was a mixture of methanol and acetonitrile in the ratio of 25:75, v/v. Chromatographic separation was achieved using reverse phase Zorbax SB-C18 column (4.6 × 100 mm, 3.5 µm). Method was linear in both the matrices in the concentration range of 0.43-750 ng/mL for nebivolol and 0.39-668 ng/mL for labetalol with r2 > 0.99. Accuracy values, expressed in terms of bias (%), for nebivolol in aqueous humor and plasma were ≤9.6% and ≤11.4% and for labetalol were ≤8.6% and ≤5.9%, respectively. Inter-day and intra-day precision values, expressed in terms of RSD (%), for both the drugs were within 11.4%. No interference was obtained due to matrix components. Mean recovery (%) values in aqueous humor and plasma were 72.4% and 73.0% for nebivolol and 56.7% and 54.4% for labetalol, respectively. No significant degradation was observed in both the drugs in both the matrices when stored at -20 °C for 1 month. Aqueous humor and plasma samples of nebivolol and labetalol on bench top were stable for 18 h and 8 h, respectively. The developed method was applied for determining pharmacokinetic parameters of both drugs in aqueous humor following single dose ocular administration in rabbits.