HPLC法测定拉莫三嗪中的有关物质

Determination of related substances of lamotrigine by HPLC

目的:建立拉莫三嗪原料药有关物质的HPLC测定方法。方法:采用Feini Gen Red ClassicalⅡC18(250 mm×4.6 mm,5μm)色谱柱,对拉莫三嗪的有关物质进行定量分析,以磷酸二氢钾缓冲液(取磷酸二氢钾2.7 g,加水800 m L使溶解,加三乙胺6.7 m L,用磷酸调节p H至2.0,加水至1 000 m L)为流动相A,乙腈为流动相B,梯度洗脱,流速为1.5 m L·min-1,柱温为35℃,检测波长为210 nm(用于检测2,3-二氯苯甲酸)和270 nm(用于检测除2,3-二氯苯甲酸外的其他杂质)。用HPLC-MS鉴定氧化降解溶液中未知杂质;采用Thermo AcclaimTM120 C18(250 mm×4.6 mm,5μm)色谱柱,以甲醇-水(50∶50)为流动相,ESI离子源,正离子扫描方式,检测离子m/z为272。结果:主峰与各有关物质峰分离度良好,拉莫三嗪及各有关物质在质量浓度为0.3~4.0μg·m L-1范围内呈良好的线性关系(r2=0.998 7),拉莫三嗪的定量下限为0.2μg·m L-1(270 nm)、杂质Ⅰ的定量下限为0.2μg·m L-1(210 nm)。3批样品有关物质测定结果显示,已知杂质和未知杂质均未检出。结论:本法可为拉莫三嗪质量控制提供参考。

Objective:To establish an HPLC method for determination of related substances in lamotrigine.Methods:The test for related substances of lamotrigine was performed on the FeiniGen RedClassical Ⅱ C18(4.6 mm×250 mm,5 μm)column.The mobile phase A was potassium dihydrogen phosphate buffer solution(dissolve 2.7 g potassium dihydrogen phosphate in 800 mL water,add 6.7 mL triethylamine,adjust to pH 2.0 with phosphoric acid and dilute to 1 000 mL with water)and the mobile phase B was acetonitrile with gradient elution at the flow rate of 1.5 mL·min-1;the column temperature was 35 ℃ and the detection wavelength was 210 nm for detecting 2,3-dichlorobenzoic acid and 270 nm for detecting impurities except 2,3-dichlorobenzoic acid.The unknown impurity in oxidative degradation solution was identified by HPLC-MS;the separation was performed on the Thermo AcclaimTM120 C18 column(250 mm×4.6 mm,5 μm)with methanol-water(50∶50)as the mobile phase.The mass spectrometer was operated in positive electrospray ionization modes with the transition m/z of 272.Results:The main peak and the peaks of the related substances were well separated.A good linearity was observed over the range of 0.3-4.0 μg·mL-1(r2=0.998 7),the LOQ of lamotrigine was 0.2 μg·mL-1(270 nm)and the LOQ of impurity Ⅰ was 0.2 μg·mL-1(210 nm).The test results of three batches of samples showed that each of the impurities was not detected.Conclusion:The established method can be used for quality control of lamotrigine.