Methotrexate (MTX) is a folate antagonist drug used for several diseases, such as cancers, various malignancies, rheumatoid arthritis (RA) and inflammatory bowel disease. Due to its structural features, including the presence of two carboxylic acid groups and its low native fluorescence, there are some challenges to develop analytical methods for its determination. MTX is metabolized to 7-hydroxymethotrexate (7-OH-MTX), 2,4-diamino-N10-methylpteroic acid (DAMPA), and the active MTX polyglutamates (MTXPGs) in the liver, intestine, and red blood cells (RBCs), respectively. Additionally, the drug has a narrow therapeutic range; hence, its therapeutic drug monitoring (TDM) is necessary to regulate the pharmacokinetics of the drug and to decrease the risk of toxicity. Due to environmental toxicity of MTX; its sensitive, fast and low cost determination in workplace environments is of great interest. A large number of methodologies including high performance liquid chromatography equipped with UV–visible, fluorescence, or electrochemical detection, liquid chromatography-mass spectroscopy, capillary electrophoresis, UV–visible spectrophotometry, and electrochemical methods have been developed for the quantitation of MTX and its metabolites in pharmaceutical, biological, and environmental samples. This paper will attempt to review several published methodologies and the instrumental conditions, which have been applied to measure MTX and its metabolites within the last decade.

甲氨蝶呤 (MTX) 是一种叶酸拮抗剂药物，用于治疗多种疾病，如癌症、各种恶性肿瘤、类风湿性关节炎 (RA) 和炎症性肠病。由于其结构特征，包括存在两个羧酸基团及其低天然荧光，开发其测定的分析方法存在一些挑战。 MTX 在肝脏、肠道和红细胞 (RBC) 中代谢为 7-羟基甲氨蝶呤 (7-OH-MTX)、2,4-二氨基-N10-甲基蝶呤酸 (DAMPA) 和活性 MTX 聚谷氨酸盐 (MTXPG) ， 分别。此外，该药物的治疗范围较窄；因此，治疗药物监测（TDM）对于调节药物的药代动力学和降低毒性风险是必要的。由于MTX的环境毒性；其在工作环境中的灵敏、快速和低成本测定引起了人们的极大兴趣。已开发出大量方法用于 MTX 及其代谢物的定量，包括配备紫外可见、荧光或电化学检测的高效液相色谱、液相色谱-质谱、毛细管电泳、紫外可见分光光度法和电化学方法用于制药、生物和环境样品。本文将尝试回顾过去十年中用于测量 MTX 及其代谢物的几种已发表的方法和仪器条件。