超高效液相色谱法测定人血清中甲氨蝶呤的浓度

Determination of methotrexate concentration in human serum by ultra-high performance liquid chromatography

目的:建立超高效液相色谱法测定人血清中甲氨蝶呤的浓度。方法:色谱柱为Shim-pack GIST-HP C18(2.1 mm×150 mm,3μm),保护柱为Shim-pack GIST-HP C18(2.1 mm×10 mm,3μm),流动相为乙腈-磷酸溶液(含0.4%三乙胺,pH 2.35,体积比为10.2∶89.8),流速0.4 mL/min,检测波长313 nm,柱温40℃,进样量10μL,内标为对氨基苯甲酸,分析时间9 min。结果:血清中内源性物质或外源性合并药物对样品的测定无干扰,甲氨蝶呤浓度在0.04~150μg/mL时的线性关系良好(r=0.998),批内和批间准确度为100%~110%,批内和批间精密度为4%~12%,提取回收率为59%~66%,样品在4℃放置5 d、处理后室温放置24 h、反复冻融3次与-70℃冷冻3个月的稳定性良好。结论:该方法快速简便,灵敏度较高,适用于临床患者体内甲氨蝶呤的血药浓度监测。

Objective:To establish an ultra-high performance liquid chromatography method for the determination of methotrexate concentration in human serum.Methods:The chromatographic column was Shim-pack GIST-HP C18(2.1 mm×150 mm,3μm),Shim-pack GIST-HP C18(2.1 mm×10 mm,3μm)as the guard column,the flowing phase was acetylene-phosphoric acid solution(including 0.4%tactimine,pH 2.35,volume ratio was 10.2∶89.8),the flow velocity rate 0.4 mL/min,detection wavelength of 313 nm,pillar temperature 40°C,10μL in the sample,internal standard substance was para-amino benzoic acid,and the analysis time was 9 minutes.Results:Endogenous substances in serum or exogenous combined drugs did not interfere with the determination of the samples.The linearity of methotrexate concentration was good at 0.04-150μg/mL(r=0.998),the intra-and inter-batch accuracy was 100%-110%,the intra-and inter-batch precision was 4%-12%,the extraction recoveries were 59%-66%,and the samples were stable at 4℃for 5 d,room temperature for 24 h after sample preparation,repeated freeze-thawing for 3 times and freezing at-70℃for 3 months.Conclusion:This method is fast and simple,and the sensitivity is high.It is suitable for monitoring of the blood concentration of methotrexate in clinical patients.