反相高效液相色谱法同时测定人血浆中亚叶酸、5-甲基四氢叶酸及甲氨蝶呤的浓度及临床应用

Determination of Folinic Acid,5-Methyltetrahydrofolate and Methotrexate in Human Plasma by High-Performance Liquid Chromatographic and Its Clinical Application

目的建立一种同时测定人血浆中亚叶酸、5-甲基四氢叶酸及甲氨蝶呤(MTX)浓度的反相高效液相色谱法。方法乙腈沉淀后,上清液用氮气吹干,残留物流动相复溶后进样。色谱条件:分析柱为SpherisobC18反相柱,流动相为0.05mol·L-1醋酸钠+0.07mmol.L-1EDTA缓冲液(pH4.4):甲醇梯度洗脱,流速:1.0mL·min-1,紫外及荧光同时检测。结果亚叶酸、5-甲基四氢叶酸及MTX的保留时间分别为11.6,16.0,27.0min;检测限分别为0.5,0.05,0.05mg·mL-1;线性关系良好,r分别为0.9980、0.9992、0.9989(n=6);低、中、高浓度的回收率、日内及日间精密度均符合方法学要求。结论本方法结果稳定,重现性好,可以用于临床大剂量MTX化疗体内药物浓度监测。

OBJECTIVE To develop a HPLC method using a reversed-phase C18 column for the determination of folinic acid, 5- methylenetetrahydrofolate and methotrexate for plasma drug monitoring. METHODS The samples were precipitated with CH3 CN, dried with N2, then dissolved with mobile phase and injected into HPLC. The separation was performed on a Spherisob C18 column with gradient elution. The mobile phase constitute of 0. 05 mol·L^-1 msodium acetate, 0. 07 mmol· L^-1 EDTA buffer （pH 4. 4） and methanol with the flow rate of 1.0 mL·min^-1. The samples were determined by ultraviolet and fluorescence detection at the same time. RESULTS The retention time of folinic acid, 5-methyltetrahydrofolate and methotrexate were 11.6, 16. 0 and 27.0 min. The detection limits were 0. 5,0. 05 and 0.05 mg· L ^-1. The linearity was obtained in the quantitative range, r were 0. 998 0, 0. 999 2 and 0. 998 9 （ n ： 6） , respectively. CONCLUSION It is a repeatable and stable method for simultaneous determination of folinic acid, 5-methylenetetrahydrofolate and methotrexate in plasma.