Rane Shaligram S, Ajameri Alkesh, Mody Rustom, Padmaja P
Department of Applied Chemistry, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India.
Intas Biopharmaceuticals Ltd., Ahmedabad, Gujarat, India.
J Pharm Anal. 2012 Apr;2(2):160-165. doi: 10.1016/j.jpha.2011.11.006. Epub 2011 Nov 29.
Rapid and sensitive reverse phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (UPLC) methods with UV detection for quantification of erythropoietin (EPO) in presence of human serum albumin (HSA) as a stabilizer in a pharmaceutical formulation of EPO have been developed and validated. Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and a flow rate of 1.5 mL/min for HPLC and 0.35 mL/min for UPLC. Quantification was accomplished with internal reference standard (qualified using EP reference standard). The methods were validated for linearity (correlation coefficient=0.99), accuracy, precision and robustness. Robustness was confirmed by considering three factors; percentages of TFA in mobile phase, age of test sample and mobile phase and column temperature. Intermediate precision was confirmed by different analysts, different equipments and on different days. The relative standard deviation (RSD) value (<2%, =30) indicated good precision of the developed method. The proposed RP-HPLC method had retention time less than 20 min while the developed UPLC method had retention time less than 4 min. Both the RP-HPLC and UPLC methods were simple, highly sensitive, precise and accurate, suggesting that the developed methods are useful for routine quality control.
已开发并验证了快速灵敏的反相高效液相色谱（RP-HPLC）和超高效液相色谱（UPLC）方法，用于在含有作为稳定剂的人血清白蛋白（HSA）的促红细胞生成素（EPO）药物制剂中定量EPO，并采用紫外检测。色谱分析使用流动相，其中在超纯水含有0.1%三氟乙酸（TFA），在乙腈中含有0.1% TFA，采用梯度程序，HPLC的流速为1.5 mL/min，UPLC的流速为0.35 mL/min。使用内标物（采用欧洲药典参考标准进行鉴定）进行定量。对这些方法进行了线性（相关系数 = 0.99）、准确度、精密度和稳健性验证。通过考虑三个因素来确认稳健性；流动相中TFA的百分比、测试样品的保存时间以及流动相和柱温。由不同分析人员、不同设备在不同日期确认中间精密度。相对标准偏差（RSD）值（<2%，n = 30）表明所开发方法具有良好的精密度。所提出的RP-HPLC方法的保留时间小于20分钟，而所开发的UPLC方法的保留时间小于4分钟。RP-HPLC和UPLC方法均简单、高度灵敏、精密且准确，表明所开发的方法可用于常规质量控制。
