建立血清叶酸及代谢物和甲氨蝶呤血药浓度同时检测的方法

Establishment of a method for simultaneous detection of serum folate and metabolites,as well as methotrexate blood concentration

目的基于超高效液相色谱串联质谱(UPLC-MS/MS)技术,建立同时检测血清叶酸及代谢物5-甲基四氢叶酸、甲酰四氢叶酸和甲氨蝶呤浓度的分析方法。方法通过固相萃取将样品中目标分析物浓缩富集,经Acquity UPLC HSS T3色谱柱(2.1 mm×100 mm,1.8μm)分离,以0.1%甲酸水和乙腈为流动相进行梯度洗脱,使用超高效液相色谱-串联四极杆质谱联用仪(UPLC I-Class Xevo TQ-S micro IVD)建立检测方法,并依据CLSI C62-A等文件对该方法的定量限、线性范围、精密度、准确度、稀释效应、携带污染以及特异度和干扰试验等进行性能评价。结果该方法中叶酸检测定量限为1 ng/mL,5-甲基四氢叶酸、甲酰四氢叶酸和甲氨蝶呤定量限为5 ng/mL。叶酸在1~200 ng/mL质量浓度范围内线性关系良好(R^(2)>0.99),5-甲基四氢叶酸、甲酰四氢叶酸和甲氨蝶呤线性范围为5~2000 ng/mL(R^(2)>0.99)。4种目标分析物日内精密度为1.7%~13.9%,日间精密度为2.7%~14.6%,样品加标回收率为93.6%~108.9%,精密度和准确度良好。稀释效应、携带污染以及特异度等其他考察结果均符合评价要求。结论建立了一种准确高效的UPLC-MS/MS方法,一针进样可以同时完成血清中叶酸及代谢物和甲氨蝶呤血药浓度的检测。该方法不仅帮助临床实现了对甲氨蝶呤血药浓度和解救药物甲酰四氢叶酸钙浓度的同步监测,还可动态观察治疗过程中患者体内总叶酸和活性叶酸的水平变化,为制订合理的甲酰四氢叶酸钙解救方案,进一步提升甲氨蝶呤治疗的安全性和有效性奠定了基础。

Objective To establish an analytical method bas ed on ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)technology for simultaneous detection of serum folate and its metabolites 5-methyltetrahydrofolate,formyl tetrahydrofolate,and methotrexate concentration.Methods Target analytes in samples were concentrated and enriched using solid-phase extraction,separated by an Acquity UPLC HSS T3 chromatographic column(2.1 mm×100 mm,1.8μm),and eluted with a gradient using 0.1%formic acid water and acetonitrile as the mobile phase.The method was established using an ultra-high performance liquid chromatography-tandem quadrupole mass spectrometer(UPLC I-Class Xevo TQ-S micro IVD)and according to CLSI C62-A and other documents,the performance of this method in terms of quantification limit,linear range,precision,accuracy,dilution effect,carrying contamination,specificity,interference testing and so on was evaluated.Results The quantitation limit for folate was 1 ng/mL,and the quantitation limits for 5-methyltetrahydrofolate,formyltetrahydrofolate,and methotrexate were 5 ng/mL.Folate had a good linear relationship in the concentration range of 1-200 ng/mL(R^(2)>0.99),and 5-methyltetrahydrofolate,formyltetrahydrofolate,and methotrexate had linear ranges of 5-2000 ng/mL(R^(2)>0.99).The intra-day precision ranged from 1.7%to 13.9%,and the inter-day precision ranged from 2.7%to 14.6%.The spiked sample recovery rates ranged from 93.6%to 108.9%,showing good precision and accuracy.Other examination results,such as dilution effect,carryover,and specificity,met evaluation requirements.Conclusion This study established an accurate and efficient UPLC-MS/MS method that allows for the simultaneous detection of serum folate and its metabolites and methotrexate blood concentrations.This method not only aids clinicians in simultaneously monitoring methotrexate blood concentration and calcium formyl tetrahydrofolate concentration,but also enables dynamic observation of changes in total folate and active folate levels in patients during the treatment process.It lays the foundation for developing a reasonable rescue plan for calcium folinate and further improving the safety and effectiveness of methotrexate treatment.