Department of Clinical Biochemistry and Pathology, Wythenshawe Hospital, Manchester University NHS Foundation Trust, Manchester, UK.
Ann Clin Biochem. 2021 Nov;58(6):599-604. doi: 10.1177/00045632211031284. Epub 2021 Jul 16.
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.
To develop a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for monitoring serum methotrexate in patients on high-dose chemotherapy.
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 min. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2, respectively.
Mean recovery was 111% for three different concentrations of methotrexate spiked into seven different patient samples, with ion suppression <1%. Between-batch and within-batch coefficient of variations 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, while mean bias compared to weighed in targets in external quality assessment samples was 1.6%.
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.
甲氨蝶呤被高剂量用于治疗多种癌症，特别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中甲氨蝶呤药代动力学的变化，监测血清中甲氨蝶呤的浓度非常重要。
建立一种快速液相色谱-串联质谱（LC-MS/MS）法，用于监测高剂量化疗患者的血清中甲氨蝶呤浓度。
在甲醇沉淀蛋白前，向样品中加入同位素标记的内标。稀释上清液注入 Waters Acquity UPLC 系统，与 TQS-Micro 质谱仪相连。色谱分离采用 Waters Phenyl Vanguard，保留时间约为 0.5 min。甲氨蝶呤的定量和定性转换分别为 455.2>134.1 和 455.2>175.2。
在 7 个不同患者样本中，三种不同浓度的甲氨蝶呤分别加入 3 个不同浓度的甲氨蝶呤，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批内和批间变异系数均<5%。定量下限为 0.02 µmol/L，该测定法在约 25 µmol/L 处呈线性。与免疫测定相比，LC-MS/MS 测定法的平均偏差为-8.6%，而与外部质量评估样本中加权目标的平均偏差为 1.6%。
已建立并验证了一种快速的甲氨蝶呤 LC-MS/MS 测定法。与更广泛应用的免疫测定相比，LC-MS/MS 方法可能具有更高的准确性和特异性。
