Abstract

Background

Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen.

Objective

To develop a rapid liquid chromatography-tandem mass spectrometry method for monitoring serum methotrexate in patients on high-dose chemotherapy.

Method

Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 minutes. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2 respectively.

Results

Mean recovery was 111% for 3 different concentrations of methotrexate spiked into 7 different patient samples, with ion suppression <1%. Between-batch and within-batch CVs were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, whilst mean bias compared to weighed in targets in EQA samples was 1.6 %.

Discussion

A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.

摘要

背景

甲氨蝶呤以高剂量用于治疗多种癌症，尤其是某些血液系统恶性肿瘤。由于甲氨蝶呤的潜在毒性和不同患者在相同治疗方案下甲氨蝶呤药代动力学的差异，监测血清甲氨蝶呤浓度很重要。

客观的

开发一种用于监测大剂量化疗患者血清甲氨蝶呤的快速液相色谱-串联质谱方法。

方法

在用甲醇沉淀蛋白质之前，将同位素标记的内标添加到样品中。将稀释的上清液注入与 TQS-Micro 质谱仪相连的 Waters Acquity UPLC 系统中。使用 Waters Phenyl Vanguard 进行色谱分离，保留时间约为 0.5 分钟。甲氨蝶呤的量词和限定词转换分别为 455.2>134.1 和 455.2>175.2。

结果

将 3 种不同浓度的甲氨蝶呤加标到 7 个不同的患者样本中的平均回收率为 111%，离子抑制 <1%。在新鲜冷冻血浆中三种不同浓度的甲氨蝶呤时，批次间和批次内的 CV 均 <5%。定量的下限为 0.02 µmol/L，检测结果呈线性至大约 25 µmol/L。与免疫测定相比，LC-MS/MS 测定的平均偏差为 -8.6%，而与 EQA 样品中称重的目标相比，平均偏差为 1.6%。

讨论

已开发并验证了甲氨蝶呤的快速 LC-MS/MS 测定法。LC-MS/MS 方法可能为更广泛使用的免疫测定提供卓越的准确性和特异性。