HPLC-ESI-MS/MS同时测定癫痫患儿血浆中丙戊酸、苯巴比妥和托吡酯的药物浓度

Simultaneous Determination of Valproic Acid, Phenobarbital and Topiramate in Plasma of Epileptic Children by HPLC-ESI-MS/MS

关键词:  丙戊酸  托吡酯  苯巴比妥  高效液相色谱串联质谱  血药浓度监测  儿童

Key words:  valproic acid  topiramate  phenobarbital  HPLC-ESI-MS/MS  therapeutic drug monitoring  children

摘要:

目的 建立基于高效液相色谱串联质谱技术(HPLC-ESI-MS/MS)同时测定癫痫患儿血浆中丙戊酸、苯巴比妥和托吡酯浓度的方法，用于癫痫患儿的血药浓度监测及结果分析。方法 样品用有机溶剂沉淀蛋白法进行前处理，分别以丙戊酸-D6、托吡酯-D12、苯巴比妥-D5为内标，采用Phenomenex Kinetex^® EVO C₁₈色谱柱(2.1 mm×50 mm，2.6 μm)，以0.07%甲酸-1 mmol乙酸铵-水(A)和甲醇(B)作为流动相进行梯度洗脱，流速为0.5 mL·min^-1。质谱检测系统采用电喷雾离子源负离子模式，多离子反应监测模式扫描。考察该方法的专属性、定量下限(LLOQ)、标准曲线、残留效应、稀释效应、准确度、精密度、基质效应和稳定性。结果 丙戊酸在0.6~120μg·mL^-1线性关系良好(r=0.998 9)，LLOQ为0.6 μg·mL^-1；苯巴比妥在0.25~50 μg·mL^-1线性关系良好(r=0.999 1)，LLOQ为0.25 μg·mL^-1；托吡酯在0.25~50 μg·mL^-1线性关系良好(r=0.999 0)，LLOQ为0.25 μg·mL^-1。批内和批间精密度RSD≤8.0%，稳定性良好，不受普通基质和溶血基质(溶血程度≤5%)的影响，方法学验证均符合中国药典2020年版规定。结论 本方法快速、简便、稳定、经济，具有较高的灵敏度和特异性，可应用于监测临床癫痫患儿丙戊酸、苯巴比妥和托吡酯的血药浓度。

Abstract:

OBJECTIVE To establish a method for simultaneous determination of valproic acid, phenobarbital and topiramate in plasma of epileptic children by high performance liquid chromatography-tandem mass spectrometry(HPLC-ESI-MS/MS). METHODS The samples were pre-treated by precipitation protein method with organic solvent. Valproic acid-D6, topiramate-D12, and phenobarbital-D5 were used as internal standard. Chromatographic column: Phenomenex Kinetex^® EVO C₁₈(2.1 mm×50 mm, 2.6 μm); flow phase: 0.07% formic acid-1 mmol ammonium acetate-water(A) and methanol(B), gradient elution; flow speed: 0.5 mL·min^-1. Ion source was electric spray ion source, multireaction monitoring mode for quantitative analysis, using negative ion monitoring mode. The specificity, lower limit of quantitation(LLOQ), standard curve, residual effect, dilution effect, accuracy, precision, matrix effects and stability were investigated. RESULTS The linear range of valproic acid was 0.6-120 μg·mL^-1 (r=0.998 9), and LLOQ was 0.6 μg·mL^-1. The linear range of phenobarbital was 0.25-50 μg·mL^-1 (r=0.999 1), and LLOQ was 0.25 μg·mL^-1. The linear range of topiramate was 0.25-50 μg·mL^-1 (r= 0.999 0), and LLOQ was 0.25 μg·mL^-1. The intra-batch and inter-batch precision RSD was ≤8.0%, with good stability, and this method was not affected by common matrix and hemolysis matrix(hemolysis degree ≤5%). The methodology validation was in line with the provisions of Chinese Pharmacopoeia(2020 edition). CONCLUSION This study determined the blood concentration of valproic acid, phenobarbital and topiramate in children with HPLC-ESI-MS/MS, which is rapid, simple, stable, economical, and high sensitivity and specificity. It can be applied to therapeutic drug monitoring of valproic acid, phenobarbital and topiramate in clinical children with epilepsy.