Department of Pharmaceutical Analysis, School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, China.
Department of Pharmaceutics, Faculty of Pharmacy, Bahauddin Zakariya University, Multan, 60800, Pakistan.
J Chromatogr Sci. 2023 Aug 19;61(7):678-687. doi: 10.1093/chromsci/bmac057.
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.
本研究旨在建立一种经验证的反相高效液相色谱法（RP-HPLC），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）。色谱分离采用 C18 柱进行研究。对色谱条件，如组成、pH 值、流动相流速、柱温、吸收波长和样品进样体积进行了研究。该方法经过验证，符合国际协调会议（ICH）的指导原则，以确认其特异性、线性和准确性。分离的最佳条件包括：0.05 M 单磷酸缓冲液：乙腈（65:35 v/v），缓冲液 pH 值调至 6.2，流速为 1 mL/min。柱温最佳为 35°C，吸收波长为 265nm，进样体积为 50μL。实现了所有四种药物的基线分离，具有良好的灵敏度、分辨率和少于 15 分钟的运行时间。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 1.2ng/mL，定量限（LOQ）值分别为：42.6、26.8、5.6 和 6.2ng/mL。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敏感、精确、准确，可用于血浆、房水和药物中这些化合物的定量。
