The present study aimed to develop a validated RP-HPLC method for the simultaneous determination of timolol maleate (TM), moxifloxacin hydrochloride (MOXI), diclofenac sodium (DS) and dexamethasone (DEXA) in human plasma, bovine aqueous humor and pharmaceutical preparations. The chromatographic separation was studied using the C18 column. The chromatographic conditions, such as composition, pH, the flow rate of mobile phase, the temperature of column, wavelength of absorption and injection volume of the sample, were studied. The method was validated to confirm specificity, linearity and accuracy in accordance with an International Conference on Harmonization guidelines. The optimum conditions for separation included mobile phase 0.05 M monobasic phosphate buffer: acetonitrile (65:35 v/v), pH of buffer adjusted to 6.2 and the flow rate of 1 mL/minute. The optimum temperature of the column was found to be 35°C, absorption wavelength 265 nm and injection volume 50 μL. The baseline separation of all four drugs with good sensitivity, resolution, and a less than 15 min run time was achieved. The retention time of TM, MOXI, DS and DEXA were 4.3,5.7,9.9 and 13.5 minutes respectively. The limit of detection (LOD) values were 6.2, 4.8, 0.8 and 1.2 ng/mL for TM, MOXI, DS and DEXA, respectively, whereas their respective limit of quantification (LOQ) values was: were 42.6, 26.8, 5.6 and 6.2 ng/mL. The coefficient of variation for intra-day and inter-day were in the range of 0.32–1.57 and 1.29–3.07%, respectively. The method was found to be sensitive, precise and accurate in human plasma and bovine aqueous humor and can be applied for the quantification of these compounds in plasma, aqueous humor and pharmaceuticals.

中文翻译：

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用于同时测定血浆、房水和药品中马来酸噻吗洛尔、盐酸莫西沙星、双氯芬酸钠和地塞米松的简易 RP-HPLC 方法的开发和验证

本研究旨在开发一种经过验证的 RP-HPLC 方法，用于同时测定人血浆、牛房水和药物制剂中马来酸噻吗洛尔 (TM)、盐酸莫西沙星 (MOXI)、双氯芬酸钠 (DS) 和地塞米松 (DEXA)。采用C18柱进行色谱分离研究。研究了样品的组成、pH、流动相流速、柱温、吸收波长和进样量等色谱条件。根据国际协调会议指南对该方法进行了验证，以确认特异性、线性度和准确性。最佳分离条件为流动相0.05 M磷酸二氢缓冲液:乙腈(65:35 v/v)，缓冲液pH调节至6.2，流速1 mL/min。最佳柱温为35℃，吸收波长265 nm，进样量50 μL。所有四种药物均实现了基线分离，具有良好的灵敏度、分辨率，运行时间不到 15 分钟。TM、MOXI、DS和DEXA的保留时间分别为4.3、5.7、9.9和13.5分钟。TM、MOXI、DS 和 DEXA 的检测限 (LOD) 值分别为 6.2、4.8、0.8 和 1.2 ng/mL，而它们各自的定量限 (LOQ) 值为： 分别为 42.6、26.8、5.6 和 6.2纳克/毫升。日内和日间变异系数分别在0.32-1.57和1.29-3.07%范围内。该方法在人血浆和牛房水中灵敏、精确且准确，可用于血浆、房水和药物中这些化合物的定量。