Separation methods for methotrexate, its structural analogues and metabolites

Federico Maria Rubino
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引用次数: 81

Abstract

Methotrexate (MTX) is the prototype folate antagonist cytotoxic drug, employed in the therapy of solid tumors and leukaemias, and recently also as an immunosuppressive agent in organ transplantation, in the treatment of some autoimmune diseases and in the therapy of severe asthma. MTX is one of the very few antineoplastic drugs the therapeutic concentration monitoring of which is currently employed in clinical practice and can be routinely measured in biological samples by a number of different analytical techniques, among which are immunoenzymatic and chromatographic methods. Each technique has of course its own advantages in terms of sensitivity, specificity, speed, cost and level of expertise required. Along with therapeutic drug concentration monitoring and clinical pharmacology, fundamental research into the mechanism of action of antifolate drugs is still a field which requires the measurement of MTX, of its new analogues and of their metabolites in biological samples. This review summarizes the instrumental conditions and the performance of several published chromatographic methods employed to measure MTX, its metabolites and some analogues in clinical and biological research. More than 70 papers describing chromatographic assays for MTX and its metabolites have been published in the literature between 1975 and 2000. A wide array of experimental conditions for sample preparation, analyte separation and detection have been employed. According to their chemical properties, MTX, its metabolites and analogue drugs present in several biological samples (plasma, serum, saliva, urine, cerebrospinal fluid, tissue specimens) can be extracted, separated and detected under a variety of chromatographic conditions, i.e. on different stationary phases, under a wide choice of mobile phase conditions (acidic or neutral, employing ion-pair or micellar chromatography), followed by several detection techniques (UV–Vis spectrophotometry, pre- or post-column oxidation and fluorimetry, electrochemistry, mass spectrometry). Optimized methods allow simultaneous measurement within a few minutes of the plasma levels of MTX and its main metabolites at concentrations in the low-nM range. One special field which needs sensitive, fast and inexpensive methods for the detection and measurement of MTX is the monitoring of contamination in workplace environments, such as pharmaceutical industries and oncological hospital pharmacies, and in sewage waters. The measurement of the intracellular γ-oligo-glutamate metabolites of biological folates, of MTX and of some analogue drugs is of great importance in basic pharmacological research. The existence of empirical quantitative relationships between the retention of individual oligomers under different chromatographic conditions and the number of added glutamic acid units allows identification of the metabolites even when authentic standards are not available.

甲氨蝶呤及其结构类似物和代谢物的分离方法。
甲氨蝶呤(MTX)是叶酸拮抗剂细胞毒性药物的原型,用于治疗实体瘤和白血病,最近也作为器官移植的免疫抑制剂,用于治疗一些自身免疫性疾病和治疗严重哮喘。MTX是目前临床上为数不多的治疗浓度监测的抗肿瘤药物之一,可以通过多种不同的分析技术在生物样品中常规测量,其中包括免疫酶法和色谱法。当然,每种技术在灵敏度、特异性、速度、成本和所需专业知识水平方面都有自己的优势。随着治疗药物浓度监测和临床药理学的发展,抗叶酸药物作用机制的基础研究仍然是一个需要在生物样品中测量MTX、其新的类似物及其代谢物的领域。本文综述了几种已发表的用于测定MTX及其代谢物和一些类似物的色谱方法在临床和生物学研究中的仪器条件和性能。从1975年到2000年,有70多篇论文描述了MTX及其代谢物的色谱分析方法。广泛的实验条件阵列样品制备,分析物分离和检测已被采用。根据其化学性质,存在于几种生物样品(血浆、血清、唾液、尿液、脑脊液、组织标本)中的MTX及其代谢物和类似物可以在多种色谱条件下进行提取、分离和检测,即在不同的固定相上,在多种流动相条件下(酸性或中性,采用离子对或胶束色谱),然后采用几种检测技术(紫外-可见分光光度法,柱前或柱后氧化和荧光法,电化学,质谱法)。优化的方法可以在几分钟内同时测量MTX及其主要代谢物在低nm范围内的血浆水平。需要灵敏、快速和廉价的方法来检测和测量MTX的一个特殊领域是监测工作场所环境中的污染,例如制药工业和肿瘤医院药房,以及污水。生物叶酸、甲氨蝶呤和一些类似药物的细胞内γ-寡谷氨酸代谢物的测定在基础药理学研究中具有重要意义。在不同色谱条件下单个低聚物的保留与添加的谷氨酸单位的数量之间存在经验定量关系,即使在没有可靠标准的情况下,也可以对代谢物进行鉴定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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