HPLC-MS/MS法同时测定难治性癫痫患者血浆中托吡酯、左乙拉西坦和卡马西平的浓度

Simultaneous determination of topiramate,levetiracetam and carbamazepine in plasma of patients with refractory epilepsy by HPLC-MS/MS

目的建立快速准确的HPLC-MS/MS方法,同时测定难治性癫痫患者内托吡酯、左乙拉西坦和卡马西平的血药浓度。方法色谱柱为Phenomenex Kinetex XB-C18,(2.6μm,50×3 mm);流动相为甲醇(A)-水(含2%的甲酸)(B),按以下梯度进行洗脱(0~2 min,30∶70→80∶20;2~4 min,80∶20;4~5 min,80∶20→30∶70)。流速为0.4 ml/min,柱温为40℃,进样量为5μl,ESI源,离子源电压和温度分别为5500 V和600℃,气帘气压力为20 psi,辅助气压力为55 psi,雾化气压力为55 psi,碰撞气压力为High;毛细管入口和出口的电压都为10 V。对该方法的标准曲线、专属性、精密度、定量下限、回收率、稳定性和基质效应进行了检验。选择了32名接受口服托吡酯、左乙拉西坦和卡马西平治疗的难治性癫痫患者。托吡酯从0.5~1.0 mg·kg^(-1)·d^(-1)开始,并滴定到4~8 mg·kg^(-1)·d^(-1),左乙拉西坦从10~20 mg·kg^(-1)·d^(-1)开始,并滴定到30~40 mg·kg^(-1)·d^(-1),卡马西平从5 mg·kg^(-1)·d^(-1)开始,并滴定到15~20 mg·kg^(-1)·d^(-1)。在获得所有3种药物的稳定浓度后,于早晨服药前30 min从肘部静脉抽取5 ml血液,用HPLC-MS/MS测定托吡酯、左乙拉西坦和卡马西平的血浆浓度。结果托吡酯在0.1~20μg/ml范围内线性良好,定量下限(LOQ)为0.01μg/ml;左乙拉西坦在1~70μg/ml范围内有良好的线性关系,最低定量下限为0.1μg/ml;卡马西平在0.1~20μg/ml范围内有良好的线性关系,最低定量下限为0.01μg/ml;3种药物的准确度为±15%,日内精密度及日间精密度的RSD均<15%,回收率在91.57%~107.24%,并显示出有良好的稳定性。结论本检测方法可以准确、快速地测定癫痫患者中托吡酯、左乙拉西坦和卡马西平的血药浓度。

Objective To establish a rapid and accurate HPLC-MS/MS method for the determination of topiramate,levetiracetam and carbamazepine in the plasma of patients with epilepsy,and to detect the serum drug concentration in patients with epilepsy.Methods The chromatographic column was phenomenex Kinetex XB-C18,(2.6μm,50×3mm).The mobile phase consisted of methanol(A)and water(containing 2%formic acid)(B),and elution was carried out in the following gradient(0~2min,30∶70→80∶20;2~4 minutes,80∶20;4~5 minutes,80∶20→30∶70).The flow rate was 0.4ml/min,the column temperature was 40℃,the injection volume was 5μl.ESI source,positive ion detection mode,multi reaction monitoring mode scanning;The ion source injection voltage and ion source temperature were 5500V and 600℃,respectively.The pressures of air curtain gas,atomization gas,auxiliary gas and collision gas were 20psi,55psi,55psi,high,respectively.Both capillary inlet voltage and Capillary outlet voltage were 10V.The specificity,standard curve,quantitative lower limit,precision,recovery,stability and matrix effect of the method were investigated.Thirty-two patients with epilepsy were treated by topiramate,levetiracetam and carbamazepine orally.The initial dose of topiramate was 0.5~1.0mg·kg^(-1)·d^(-1)and gradually increased to 4~8mg·kg^(-1)·d^(-1).The initial dose of levetiracetam was 10~20mg·kg^(-1)·d^(-1)and gradually increased to 30~40mg·kg^(-1)·d^(-1).The initial dose of carbamazepine was 5mg·kg^(-1)·d^(-1)and gradually increased to 15~20mg·kg^(-1)·d^(-1).After the above three drugs reached the maintenance dose,5ml of venous blood of the elbow was taken half an hour before the morning medication.The plasma concentrations of topiramate,levetiracetam and carbamazepine were detected by HPLC-MS/MS method.Results The linearity of topiramate in the range of 0.1~20μg/ml was good,and the lower limit of quantification(LOQ)was 0.01μg/ml.Levetiracetam showed good linearity in the range of 1 to 70μg/ml,and the lower limit of quantification was 0.1μg/ml.Carbamazepine showed good linearity in the range of 0.1~20μg/ml,and the lower limit of quantification was 0.01μg/ml.The accuracy of the three drugs was±15%,the RSDS of intraday and intraday precision were below 15%,and the recoveries ranged from 91.57%to 107.24%,showing good stability.Conclusion This method can accurately and rapidly detect the blood drug levels of topiramate,levetiracetam and carbamazepine in patients with epilepsy.