Department of Pharmacy, University of Peshawar, Peshawar, Pakistan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 15;879(30):3434-43. doi: 10.1016/j.jchromb.2011.09.021. Epub 2011 Sep 16.
A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.
建立了一种同时测定药物、人血浆和房水中噻吗洛尔（TM）、瑞舒伐他汀（RST）和双氯芬酸钠（DS）的新型 HPLC-UV 方法，以萘普生钠为内标（IS）。采用 Hypersil BDS C（18）柱（250mm×4.6mm，5μm），以 0.2%三乙胺（TEA）和乙腈（ACN）（40:60，v/v）为流动相，在 pH 2.75 条件下，用 85%磷酸调至 25℃，流速为 1ml/min，等度洗脱。柱温箱温度保持在 45℃，进样量为 50μl，HPLC 系统在 284nm 处检测峰响应。采用甲醇沉淀蛋白后，以流动相作为提取溶剂，直接进行液-液萃取，处理人血浆和牛房水样品。根据标准指南优化了不同的 HPLC 实验参数，并对方法进行了验证。建议的方法在人血浆中的回收率分别为 TM、RST 和 DS 的 98.72%、96.04%和 95.14%，而在房水中分别为 TM 和 DS 的 94.99%和 98.23%。TM、RST 和 DS 的检测限分别为 0.800、0.500 和 0.250ng/ml，定量限分别为 2.00、1.50 和 1.00ng/ml。日内和日间研究的变异系数（CV）分别在 0.1492-1.1729%和 1.0516-4.0104%之间。该方法在人血浆和牛房水中准确，将应用于动物模型和药物中血浆和房水样品中这些化合物的定量。
