A novel validated UPLC method for quantitation of lopinavir and ritonavir in bulk drug and pharmaceutical formulation with its impurities

A simple gradient Ultra Performance liquid chromatographic method (UPLC) was developed for determination of lopinavir and ritonavir from its related impurities and assay for the first time. This method involves the use of a C18 (Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 µm) column thermostated at 30 oC using triethylamine (pH 2.2): 0.1% H3PO4 in acetonitrile and methanol (85:15) as mobile phase in gradient elution mode. A Photo Diode Array (PDA) detector set at 215 nm was used for detection with flow rate 0.4 mL/min. This method was validated over the range of limit of quantitation (LOQ) to 50 to 150% of impurity specification limit and of working concentration for assay. The developed method was validated for linearity, range, precision, accuracy and specificity. This method was successfully applied for content determination of lopinavir and ritonavir in pharmaceutical formulations. This method can be conveniently used in quality control laboratory for routine analysis for assay and related substances as well as for evaluation of stability samples of bulk drugs and pharmaceutical formulations.

本研究首次建立了一种简单的梯度超高效液相色谱法（Ultra Performance liquid chromatographic method, UPLC），可用于洛匹那韦（lopinavir）与利托那韦（ritonavir）及其有关物质的分离测定与含量测定。该方法采用C18色谱柱（Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 µm），柱温设定为30 ℃；以三乙胺（以0.1%磷酸调节至pH 2.2）与乙腈-甲醇（体积比85:15）的混合溶液作为流动相，采用梯度洗脱模式。采用流速为0.4 mL/min的光电二极管阵列检测器（Photo Diode Array, PDA），于215 nm波长处进行检测。该方法的验证范围覆盖定量限（limit of quantitation, LOQ）至杂质限度的50%~150%，以及含量测定的工作浓度区间，并完成了线性、范围、精密度、准确度及专属性的方法学验证。该方法已成功应用于药物制剂中洛匹那韦和利托那韦的含量测定。本方法可便捷应用于质量控制实验室的常规含量测定与有关物质分析，同时可用于原料药及药物制剂的稳定性样品评价。