HPLC-MS/MS法同时测定微量血浆中甲氨蝶呤、左乙拉西坦、拉莫三嗪浓度及其临床应用

Simultaneous determination of Methotrexate,Levetiracetam,and Lamotrigine in small volume of plasma by HPLC-MS/MS and its clinical application

目的 建立HPLC-MS/MS法测定微量血浆中甲氨蝶呤、左乙拉西坦和拉莫三嗪的浓度,同时应用于儿童治疗药物监测。方法 取含药血浆10μl,用蛋白沉淀法对样本进行前处理。色谱柱为Agilent C18(50 mm×2.1 mm,1.8μm),流动相为0.1%甲酸水和甲醇,梯度洗脱;流速为0.4 ml/min。采用电喷雾离子源正离子模式,监测甲氨蝶呤m/z 455.1→308.2,甲氨蝶呤-D3 m/z 458.1→311.1(内标);左乙拉西坦m/z 171.1.4→126.2,左乙拉西坦-D6 m/z177.2→132.2(内标);拉莫三嗪m/z 256.1→211,拉莫三嗪-13C3-D3 m/z 262.1→217.1(内标)。结果 甲氨蝶呤在25~1 500 ng/ml线性关系良好,标准曲线方程:Y=0.251 6X+0.003 4(r=0.998 9),定量下限为25 ng/ml;左乙拉西坦在1~50μg/ml线性关系良好,标准曲线方程:Y=16.687 6X+0.000 2(r=0.991 1),定量下限为1μg/ml;拉莫三嗪在0.5~25.0μg/ml线性关系良好,标准曲线方程:Y=46.369 5X-0.059 9(r=0.999 1),定量下限为0.5μg/ml。样本批内、批间准确度和精密度符合要求、基质效应符合规定。本方法验证后成功用于1 348例临床患儿样本的检测。结论 本方法操作简便、专属性强、灵敏度高,可用于人血浆中甲氨蝶呤、左乙拉西坦及拉莫三嗪的分析,适用于儿童治疗药物监测。

Objective To established a HPLC-MS/MS method for determination the concentration of Methotrexate,Levetiracetam,and Lamotrigine in small volume of plasma and to monitor therapeutic drugs in children.Methods Drugcontaining plasma 10 μl was obtained,and the samples were pretreated by protein precipitation.The chromatography was performed on Agilent C18column(50 mm×2.1 mm,1.8 μm),the mobile phase was 0.1% formic acid and methanol,and gradient elution;the flow rate was 0.4 ml/min.Electrospray ion source positive ion mode was used,ions monitored were m/z455.1 →308.2 for Methotrexate,m/z 458.1 →311.1 for Methotrexate-D3(internal standard),m/z 171.1.4 →126.2 for Levetiracetam,m/z 177.2→132.2 for Levetiracetam-D6(internal standard),and m/z 256.1 →211 for Lamotrigine,m/z262.1 →217.1 for Lamotrigine-13C3-D3(internal standard),respectively.Results Methotrexate had a good linear relationship between 25-1 500 ng/ml,the standard curve equation was Y=0.251 6X+0.003 4(r=0.998 9),the lower limit of quantitation was 25 ng/ml;Levetiacetam had a good linear relationship between 1-50 μg/ml,the standard curve equation was Y=16.687 6Y+0.000 2(r=0.991 1),and the lower limit of quantitation was 1 μg/ml;Lamotrigine has a good linear relationship between 0.5-25.0 μg/ml,the standard curve equation was Y=46.369 5X-0.059 9(r=0.999 1),the lower limit of quantitation was 0.5 μg/ml.The accuracy within and between batches and precision of samples met the requirements,and the matrix effect met the requirements.This method was fully verified and successfully applied to therapeutic drug monitoring of 1 348 pediatric patients.Conclusion The method is simple,convenient,and sensitive,and can be applied to the analysis of Methotrexate,Levetiracetam,and Lamotrigine in human plasma,and is suitable for the monitoring of therapeutic drugs in children.