甲氨蝶呤注射液含量及其有关物质的高效液相色谱电化学检测方法的研究

HPLC-electrochemical detection of methotrexate and related substances in methotrexate injection

目的:建立高效液相色谱电化学方法检测甲氨蝶呤注射液含量及其色谱柱有关物质。方法:以乙腈-0.2mol·L^(-1)磷酸盐缓冲溶液(pH 6.0)(10:90)为流动相,A TSK-GEL~@ODS-100SP 柱(150mm×4.6mm,5μm),流速为1.0mL·min^(-1),柱温35℃时,在+0.8V 电位处,以玻碳圆盘电极为工作电极,银-氯化银电极为参比电极,研究了电化学安培检测中电位与检测量的关系,比较了电化学安培检测和紫外检测2种不同的检测方法。结果:实现了甲氨蝶呤及其有关物质的分离检测。甲氨蝶呤的线性范围为9.1～91μg·mL^(-1),r=0.9999;平均回收率为100.4%,RSD 为1.0%(n=9)。氨蝶呤、甲蝶呤和甲氨蝶呤的最低检出量分别为3.3,11,8.6ng。结论:电化学安培检测与紫外检测相比,具有较高的灵敏度,能有效地应用于甲氨蝶呤注射液中极微量有关物质的检测。

Objective：To establish an HPLC -electrochemical detection method to separate and determine methotrexate and its related substances on an ODS column. Method：A TSK - GEL ODS - 100SP column（ 150 mm ×4. 6 mm,5 μm） was used as analytical column with a mobile phase consisted of acetonitrile -0. 2 mol· L^-1 phosphate buffer（ pH 6.0） （10： 90）. The flow rate was 1.0 mL·min^-1 ,the column temperature was 35 ℃ ,and the working potential was 0. 8 V when using the glassy carbon disc electrode as the working electrode. The condition of electrochemical detection was studied and the difference between electrochemical detection and UV detection was comared. Results： Methotrexate, aminopterin, and methopterin were separated. The linear range of methotrexate was 9. 1 -91 μg·mL^-1 （r =0. 9999） ,the average recovery of the method was 100. 4% （ RSD = 1.0% ,n =9） . The detection limits of aminopterin, methopterin and methotrexate were 3.3,11 and 8.6 ng, respectively. Conclusion： Compared with the UV detection method,the sensitivity of electrochemical dection is higher. It can be further employed in the detection of methotrexate in biosamples.