目的 建立检测类风湿关节炎患者红细胞中多聚谷氨酸化甲氨蝶呤(methotrexate polyglutamates,MTXPGs)浓度的HPLC-MS/MS法。方法 采用间接测定法,以同位素甲氨蝶呤-d3为内标,分别测定游离甲氨蝶呤(methotrexate,MTX)和总MTX(MTX+MTXPGs),从而计算出红细胞中MTXPGs浓度。为测定游离MTX,全血样品经反复冻融、甲醇沉淀蛋白后,经LC-MS/MS进样分析,采用电喷雾离子源(ESI)正离子模式,多反应监测(MRM)。为测定总MTX,样品(反复冻融后)与200 mmol·L-1抗坏血酸37 ℃孵育2.5 h,酶促MTXPGs转化为MTX,蛋白沉淀后进样分析。结果 全血MTX质量浓度在1~300 ng·mL-1内线性关系良好(r=0.999 4),检测限(LOD)为0.5 ng·mL-1,准确度(RE)在±5%内,日内和日间精密度(RSD)分别低于7.96%和10.88%。结论 本方法快速、灵敏,具有高专属性和重现性,成功应用于临床上类风湿关节炎患者红细胞中MTXPGs浓度的监测。
Abstract
OBJECTIVE To establish an HPLC-MS/MS method for determining erythrocyte methotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA).METHODS The indirect method was applied to determine the concentrations of free methotrexate (MTX) and total MTX (MTX+MTXPGs) respectively by using isotope MTX-d3 as the internal standard, and then concentration of erythrocyte MTXPGs was calculated. To determine free MTX, whole blood (after freeze-thaw cycles to break blood cells) was precipitated with methanol, and then analyzed by LC-MS/MS. Multiple reactions monitoring (MRM) was performed with electrospray ionization (ESI) at a positive ion mode.To determine total MTX, whole blood (after freeze-thaw cycles) was incubated with 200 mmol·L-1 ascorbic acid at 37 ℃ for 2.5 h to enzymatically convert the MTXPGs to MTX,and the protein was precipitated for sample analysis.RESULTS The calibration curve of MTX was linear over the range of 1-300 ng·mL-1 (r=0.999 4) and the limit of detection (LOD) was 0.5 ng·mL-1. The accuracy (RE) was within ±5% and the intra-and inter-day precisions (RSD) were less than 7.96% and 10.88%, respectively.CONCLUSION This method is rapid, sensitive, highly specific and reproducible, which is successfully applied for the erythrocyte MTXPGs monitoring in patients with RA.
关键词
高效液相色谱-串联质谱法 /
多聚谷氨酸化甲氨蝶呤 /
甲氨蝶呤 /
红细胞 /
类风湿关节炎
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Key words
HPLC-MS/MS /
methotrexate polyglutamate /
methotrexate /
erythrocyte /
rheumatoid arthritis
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中图分类号：
R969.1
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参考文献
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脚注
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基金
福建省科技计划项目资助(2018J01394)
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