高效液相色谱-串联质谱法同时测定人全血中硫胺素及其2种磷酸酯的浓度  

Simultaneous determination of thiamine and its 2 phosphate esters in human whole blood by high performance liquid chromatography-tandem mass spectrometry

作　　者：姜小梅 龚子珊 汪曣[1] JIANG Xiao-mei;GONG Zi-shan;WANG Yan(Department of Precision Instrument and Opto-Electronics Engineering,Tianjin University,Tianjin 300072,China)

机构地区：[1]天津大学精密仪器与光电子工程学院,天津300072

出　　处：《中国临床药理学杂志》2021年第19期2691-2694,共4页The Chinese Journal of Clinical Pharmacology

摘　　要：目的建立一种同时测定人全血中的硫胺素、一磷酸硫胺素(TMP)和焦磷酸硫胺素(TPP)浓度的高效液相色谱-串联质谱方法。方法以[2H3]-硫胺素为内标,样本用三氯乙酸沉淀蛋白后进高效液相色谱-串联质谱系统(HPLC-MS/MS)分析。色谱柱:Zorbax Extend-C18(50.0 mm×4.6 mm,3.5μm),流动相:含0.1%氨水的10 mmol·L^(-1)乙酸铵水溶液-80%的乙腈水溶液,流速:0.60 mL·min^(-1),梯度洗脱,柱温:35℃,进样量:10.0μL。用电喷雾离子源,多反应监测模式,正离子扫描检测。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、基质效应和稳定性。结果硫胺素和TMP在0.25~50.00 ng·mL^(-1),TPP在2.50~500.00 ng·mL^(-1)内,线性相关系数均大于0.99;硫胺素、TMP和TPP的定量下限分别为0.25,0.25和2.50 ng·mL^(-1);日内和日间精密度在3.27%~11.70%,准确度在86.56%~104.04%,提取回收率在85.54%~104.98%。内标对基质效应具有较好的校正作用,且稳定性良好。结论本方法操作简便快速,准确可靠,适用于3种维生素B1在人全血中浓度的检测。Objective To establish a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method to simultaneously determine the concentrations of thiamine,thiamine monophosphate(TMP) and thiamine pyrophosphate(TPP) in human blood.Methods [2 H3]-thiamine was used as internal standard and samples were treated by protein precipitation method using trichloroacetic acid,and then determined by HPLC-MS/MS.Chromatographic column was Zorbax Extend-C18(50.0 mm ×4.6 mm,3.5 μm),mobile phase was 0.1% ammonia in 10 mmo·L^(-1) ammonium acetate-80%acetonitrile,flow rate was 0.60 mL·min^(-1),gradient elution,column temperature was 35℃,injection volume was 10.0 μL.Electrospray ion source was used and the positive ions of analytes were detected in multiple reaction monitoring mode.The specificity,calibration curve and limit of quantitation,precision and accuracy,matrix effect and stability of the method were investigated.Results Calibration curves were linear over the range of 0.25-50.00 ng·mL^(-1) for thiamine and TMP,and2.50-500.00 ng·mL^(-1) for TPP(correlation coefficient>0.99).The limits of quantification were 0.25,0.25 and 2.50 ng·mL^(-1) for thiamine,TMP and TPP.The intra-day and interday precision were 3.27%-11.70%.The accuracy was 86.56%-104.04% and the extraction recovery was85.54%-104.98%.The matrix effect was corrected by the internal standard and the stability was good.Conclusion The described HPLC-MS/MS method allows simultaneous determination of thiamine,TMP and TPP in whole blood without intensive sample preparation.

关 键 词：硫胺素 一磷酸硫胺素 焦磷酸硫胺素 高效液相色谱-串联质谱法 全血 

分 类 号：R97[医药卫生—药品]