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 ± 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.

中文翻译：

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使用LC-MS / MS同时测定房水和血浆中奈必洛尔和拉贝洛尔的生物分析方法的开发和验证，并将其应用于眼药代动力学研究。

β受体阻滞剂是治疗开角型青光眼的药物选择类别。然而，由于这些药物通过鼻泪管吸收到全身循环中，因此许多这类药物具有全身性副作用。为了评估奈必洛尔和拉贝洛尔治疗开角型青光眼的安全性和有效性，重要的是要有一种生物分析方法来测量房水和血浆中的药物浓度。建立了一种简单，灵敏，高通量的液相色谱-串联质谱（LC-MS / MS）方法和蛋白质沉淀技术，分别使用nebivolol-d4和美托洛尔作为房水中的内标，同时定量了奈比洛尔和拉贝洛尔等离子体。奈比洛尔和拉贝洛尔分别在电喷雾正离子化（ESI）模式406.2 / 151.1和329.2 / 162.0时进行监测。流动相由水性缓冲液（溶剂A）和有机相（溶剂B）（A：B比例为30：70，v / v的混合物）组成。水性缓冲液是用甲酸调节至pH 3.5±0.05的5 mM乙酸铵缓冲液，而有机相是甲醇和乙腈的比例为25：75，v / v的混合物。使用反相Zorbax SB-C18色谱柱（4.6×100 mm，3.5 µm）进行色谱分离。奈比洛尔和拉贝洛尔的浓度范围均为0.43-750 ng / mL，拉贝洛尔为0.39-668 ng / mL时，两种方法的线性均r2> 0.99。房水和血浆中奈必洛尔的准确度值以偏差（％）表示，分别为≤9.6％和≤11。拉贝洛尔的4％≤8.6％，拉贝洛尔≤5.9％。两种药物的日间和日内精度值（以RSD（％）表示）均在11.4％以内。由于基质成分，未获得干扰。奈比洛尔的房水和血浆中的平均回收率（％）分别为72.4％和73.0％，拉贝洛尔为56.7％和54.4％。当在-20°C下保存1个月时，两种基质中的两种药物均未观察到明显降解。台式奈必洛尔和拉贝洛尔的房水和血浆样品分别稳定18 h和8 h。所开发的方法用于测定兔眼内单剂量给药后房水中两种药物的药代动力学参数。两种药物的比例均在11.4％以内。由于基质成分，未获得干扰。奈比洛尔的房水和血浆中的平均回收率（％）分别为72.4％和73.0％，拉贝洛尔为56.7％和54.4％。当在-20°C下保存1个月时，两种基质中的两种药物均未观察到明显降解。台式奈必洛尔和拉贝洛尔的房水和血浆样品分别稳定18 h和8 h。所开发的方法用于测定兔眼内单剂量给药后房水中两种药物的药代动力学参数。两种药物的比例均在11.4％以内。由于基质成分，未获得干扰。奈比洛尔的房水和血浆中的平均回收率（％）分别为72.4％和73.0％，拉贝洛尔为56.7％和54.4％。当在-20°C下保存1个月时，两种基质中的两种药物均未观察到明显降解。台式奈必洛尔和拉贝洛尔的房水和血浆样品分别稳定18 h和8 h。所开发的方法用于测定兔眼内单剂量给药后房水中两种药物的药代动力学参数。当在-20°C下保存1个月时，两种基质中的两种药物均未观察到明显的降解。台式奈必洛尔和拉贝洛尔的房水和血浆样品分别稳定18 h和8 h。所开发的方法用于测定兔眼内单剂量给药后房水中两种药物的药代动力学参数。当在-20°C下保存1个月时，两种基质中的两种药物均未观察到明显降解。台式奈必洛尔和拉贝洛尔的房水和血浆样品分别稳定18 h和8 h。所开发的方法用于测定兔眼内单剂量给药后房水中两种药物的药代动力学参数。