HPLC测定氯氮平有关物质的方法

Determination of Related Substances of Clozapine by HPLC

目的:建立 HPLC 测定氯氮平有关物质的方法。方法:采用 Waters XSelect CSH Phenyl-Hexyl 色谱柱(4.6 mm×150 mm,3.5 μm),以含 0.08 %三氟乙酸和 0.12 %三乙胺的水溶液-0.08 %三氟乙酸的乙腈溶液为流动相进行梯度洗脱,柱温 20 ℃,流速 1 m L·min^(-1),检测波长 257 nm,进样体积为 5 μL。结果:氯氮平与各已知杂质及强制破坏产生的降解杂质均分离良好,氯氮平、杂质 A、杂质 B、杂质C 和杂质 D 浓度分别在 0.25130~5.02593 μg·m L^(-1)(r=0.9999)、0.25272~5.05435 μg·m L^(-1)(r=1.0000)、0.24670~4.93393 μg·m L^(-1)(r=0.9999)、0.24153~7.18828 μg·m L^(-1)(r=0.9999)、0.24950~4.98998 μg·m L^(-1)(r=1.0000)范围内与峰面积呈良好的线性关系。已知杂质(杂质 A、杂质 B、杂质 C、杂质 D)的平均回收率分别为 100.59 %、99.46 %、99.42 %和 101.59 %,RSD 依次为 1.1 %、0.6 %、1.1 %和 3.0 %(n=9)。杂质A、杂质 B、杂质 C、杂质 D、其他最大单杂、总杂质精密度试验的绝对差值分别为 0.011 %、0.022 %、0.024 %、0.025 %、0.009 %、0.082 %(n=12)。结论:本方法专属性强、准确、灵敏,可用于氯氮平的有关物质检测和质量控制。

Objective:To establish an HPLC method for the determination of related substances of clozapine.Methods:The determination was performed on a Waters XSelect CSH Phenyl-Hexyl(4.6 mm×150 mm,3.5 μm),with a mobile phase consisting of a mixture of 0.08 % TFA+0.12 % TEA in water and 0.08 % TFA in ACN by gradient elution.The column temperature was set at 20 ℃ with a flow rate of 1 ml·min^(-1),the detective wavelength was 257 nm,and the injection size was 5 μL.Results:The known impurities and degraded products by forced destruction were completely separated from clozapine.The calibration curves for clozapine,impurity A,impurity B,impurity C and impurity D revealed good linearities over the ranges of 0.25130~5.02593 μg·m L^(-1)(r=0.9999),0.25272~5.05435 μg·m L^(-1)(r=1.0000),0.24670~4.93393 μg·m L^(-1)(r=0.9999),0.24153~7.18828 μg·m L^(-1)(r=0.9999),0.24950~4.98998 μg·m L^(-1)(r=1.0000),respectively.The recoveries of the impurities(impurity A,impurity B,impurity C,impurity D)were 100.59 % with RSD of 1.1 %,99.46 % with RSD of 0.6%,99.42 % with RSD of 1.1 % and 101.59 %with RSD of 3.0 %,respectively.The absolute difference of precision of impurity A,impurity B,impurity C,impurity D,other maximum unknown individual impurity,total impurity were 0.011 %,0.022 %,0.024 %,0.025 %,0.009 %,0.082 %,successively.Conclusion:The method is specific,accurate and sensitive,which can be used for the test of the related substances of clozapine and quality control.