液相色谱串联质谱法检测去乙酰毛花苷、地高辛血浆浓度及其临床应用

Liquid chromatography-tandem mass spectrometry for the determination of plasma Deslanoside and Digoxin levels and its clinical application

目的:建立一种检测血浆中去乙酰毛花苷(cedilanid D,CDL)和地高辛(digoxin,DIG)浓度的液相色谱串联质谱法(LC-MS/MS)并探索CDL的合理监测指标及安全治疗范围。方法:质谱采用ESI正离子模式,以Kinetex®2.6μm C_(18)100·作为分析柱,以地高辛-D3为内标,流动相为10 mmol·L^(-1)乙酸铵(0.1%甲酸)-甲醇(0.1%甲酸),梯度洗脱;样品前处理为乙腈蛋白沉淀法结合氮气吹干后复溶;对该LC-MS/MS法进行较为系统的方法学验证。EMIT法使用VIVA-E 2000进行检测,使用Siemens®地高辛检测试剂盒。根据制定的纳入标准和排除标准收集了122例使用CDL患者的血样,均使用LC-MS/MS法和EMIT法检测。结果:该LC-MS/MS法经方法学验证,CDL在0.16~10.3 ng·mL^(-1)、DIG在0.16~9.89 ng·mL^(-1)范围内线性良好,且精密度、准确度、提取回收率及基质效应符合检测要求。对两种方法检测结果的分析显示EMIT法所测DIG浓度(E-DIG浓度)高于LC-MS/MS法所测DIG与CDL浓度之和[L-(DIG+CDL)浓度](P<0.01),且E-DIG浓度与L-(DIG+CDL)浓度对于评估CDL相关不良反应的效果相当(ROC分析的AUC分别为0.90、0.93,P>0.05),截断值分别为2.46 ng·mL^(-1)、1.78 ng·mL^(-1)。结论:对于使用CDL的患者,以CDL与DIG浓度之和反映CDL的安全性可能更为合适,而EMIT法检测结果高于LC-MS/MS法,其中毒浓度须根据不同检测方法作相应调整。

OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of Deslanoside(CDL)and Digoxin(DIG)concentrations in plasma and to explore reliable monitoring indicators and a safe therapeutic range for CDL.METHODS Mass spectrometry was conducted in ESI positive ion mode,utilizing Kine⁃tex®2.6μm C_(18)100Åas the analytical column.Digoxin-D3 served as the internal standard.The mobile phase consisted of 10 mmol·L^(-1)ammonium acetate(0.1%formic acid)-methanol(0.1%formic acid),applied in a gradient elution.Samples were prepared by redissolving after nitrogen drying followed by protein precipitation with acetonitrile.Systematic methodological valida⁃tion of the LC-MS/MS method was performed.For the EMIT method,Siemens®Digoxin Assay Kit was used based on VIVAE 2000.Blood samples of 122 patients who had received CDL were collected according to specific inclusion and exclusion criteria.The samples were analyzed using both LC-MS/MS and EMIT methods.RESULTS The LC-MS/MS method showed good lin⁃earity for CDL in the concentration range of 0.16-10.3 ng∙mL^(-1)and for DIG in the range of 0.16-9.89 ng∙mL^(-1).The preci⁃sion,accuracy,extraction recovery rate and the matrix effect of CDL and DIG met the assay requirements for biological sample analysis.The results of comparative analysis showed that the DIG concentrations measured by EMIT(E-DIG level)were higher than the sum of DIG and CDL concentrations measured by LC-MS/MS(L-(DIG+CDL)level)(P<0.01).Furthermore,the effectiveness of E-DIG concentrations and L-(DIG+CDL)concentrations in assessing CDL-related adverse reactions was equiva⁃lent(AUCs of ROC analysis were 0.90 and 0.93,respectively,P>0.05),and the cutoff values were 2.46 ng∙mL^(-1)and 1.78 ng∙mL^(-1),respectively.CONCLUSION For patients using CDL,it may be more appropriate to assess the safety of CDL by the combined concentrations of CDL and DIG.Since the EMIT method yields higher results than the LC-MS/MS method,the toxic⁃ity concentration needs to be adjusted according to different detection methods.