HPLC测定人血清中甲氨蝶呤浓度

Determination of Methotrexate Concentration in Human Plasma by HPLC

目的 建立高效液相色谱法测定人血清中甲氨蝶呤（methotrexate，MTX）浓度的方法。方法 以茶碱为内标，色谱柱为Agilent Eclipse XDB-C18（4.6 mm×250 mm，5 μm），流动相为磷酸盐缓冲液（pH 6.6）-甲醇（82∶18），检测波长为MTX 306 nm和茶碱254 nm，流速为1.0 mL·min^-1，柱温为30 ℃。结果 MTX浓度分别在0.03～1.65 μmol·L^-1（r=0.999 9）和1.65～66.08 μmol·L^-1（r=0.995 8）内线性关系良好，平均方法回收率97.6%。平均萃取回收率68.5%，日内、日间RSD均〈5%，血清最低检测浓度为0.03 μmol·L^-1。结论 该方法灵敏、准确，线性范围宽，适用于临床甲氨蝶呤的血药浓度监测。

OBJECTIVE To develop an HPLC method to determine the concentration of methotrexate（MTX） in human plasma. METHODS Theophyline was used as the internal standard. The analytical column was Agilent Eclipse XDB-C18（4.6 mm×250 mm, 5 μm）. The mobile phase was phosphate buffer solution with pH 6.6-methanol（82∶18）. The UV determine wavelength of MTX was 306 nm and theophyline was 254 nm. The flow rate was 1.0 mL·min^-1, and the column temperature was 30 ℃. RESULTS The calibration curves of MTX were linear at a concentration range from 0.03 to 1.65 μmol·L^-1（r=0.999 9） and 1.65 to 66.08 μmol·L^-1（r=0.995 8）. The intra-day and inte-day precision were both less than 5%. The average method recovery was 97.6% and the average extraction recovery was 68.5%, with a lowest limit of 0.03 μmol·L^-1. CONCLUSION The method is sensitive, accurate, wide linear range, suitable for plasma concentration monitoring of MTX.