高效液相色谱法测定人血浆中甲氨蝶呤的浓度

Determination of methotrexate in human plasma by high performance liquid chromatography detector

目的建立灵敏、特异、快速、稳定可靠的甲氨蝶呤高效液相色谱检测方法,对患者血浆中甲氨蝶呤的血药浓度进行监测。方法色谱柱:SB-C18柱(4.6 mm×150 mm,5μm);流动相:甲醇-0.05mol·L^(-1)磷酸二氢钾缓冲液=26∶74(p H 2.4);流速:1.0 m L·min^(-1);检测波长:306nm;柱温:室温;进样量:40μL;甲硝唑为内标。结果血浆中甲氨蝶呤在0.04~4.0μg·m L^(-1)线性相关程度良好(R^2均大于0.99);低、中、高质量浓度质控样品(0.1,0.5,3.0μg·m L^(-1))的日内相对标准偏差(RSD)为2.12%~3.93%,日间RSD为3.48%~6.16%,绝对回收率在60.63%~75.72%。结论本研究建立的方法有良好的特异性、灵敏度、精密度和准确度,适用于临床患者甲氨蝶呤的血药浓度测定。

Objective To establish a method for determination of methotrexate（ MTX） concentration in human plasma by high performance liquid chromatography detector method（ HPLC）. Methods MTX and the internal standard（ IS） metronidazole were extracted from human plasma after protein precipitation by 20% perchloric acid and acetonitrile,then detected with detection wavelength set at 293 nm after separation by an Eclipse XDB- C18（ 4. 6 mm × 150 mm,5 μm）. The mobile phase consisted of methanol- 0. 05 mol·L-1potassium phosphate buffer p H 2. 4 = 26 ∶ 74,with a flow rate of 1. 0 m L · min-1. The method was validated for specificity,precision and accuracy,recovery as well as stability. Results Linear calibration curves were obtained in the range of0. 04- 4. 00 μg·m L-1（ R2〉 0. 99）. Intra- day RSD were in the ranges of 2. 12%- 3. 93% and inter- day RSD were in the ranges of3. 48%- 6. 16%. Conclusion The method provides a sensitive,accurate,precise and reliable analytical procedure for clinical monitoring of methotrexate in plasma.