Kim Hakmin, Kim Kyeong-Seog, Kim Sihyun, Kang Jihyun, Kim Hyun Chul, Hwang Sejung, Chung Jae-Yong, Yoon Hyuk, Cho Joo-Youn
Department of Clinical Pharmacology and Therapeutics, Seoul National University Hospital, Seoul National University College of Medicine, Seoul 03080, Korea.
Department of Biomedical Sciences, Seoul National University Hospital, Seoul National University College of Medicine, Seoul 03080, Korea.
Transl Clin Pharmacol. 2025 Jun;33(2):66-79. doi: 10.12793/tcp.2025.33.e7. Epub 2025 Jun 23.
Methotrexate (MTX), a folate antagonist, is commonly administered at low doses for the treatment of Crohn's disease (CD). Anti-inflammatory effects of MTX are facilitated by its intracellular conversion to MTX polyglutamates (MTX-PGs). Because plasma-based monitoring of therapeutic response does not accurately reflect the therapeutic efficacy of MTX, quantifying intracellular MTX-PGs, potential biomarkers of the MTX response, is crucial. However, it is challenging to routinely monitor intracellular MTX metabolites in patients with CD due to the low concentrations of MTX-PGs. Therefore, quantitating MTX-PGs in clinical samples with a high-sensitivity method is necessary. We established a high-sensitivity method to quantify three MTX-PGs using perchloric acid deproteinization followed by high-performance liquid chromatography-tandem mass spectrometry. Calibration curves were generated using human red blood cells as biological matrix. This method was applied to analyze MTX-PGs in red blood cells (RBCs) from patients with CD undergoing MTX therapy. The method achieved a lower limit of quantification of 1 ng/mL for individual MTX-PGs. A nine-point calibration curve covering 1-400 ng/mL showed excellent linearity. Precision (relative standard deviation < 15%) and accuracy (93.41-109.37%) were satisfactory in both intra- and inter-day assays. Plasma MTX levels were not significantly correlated with any individual RBC MTX-PG level ( = 0.998, 0.640, and 0.587, respectively). The lack of correlation supports our conclusion that plasma MTX levels may not reliably represent intracellular accumulation. The developed quantitative method provides a useful tool to improve our understanding of MTX metabolism and may facilitate therapeutic drug monitoring in MTX therapy.
甲氨蝶呤（MTX）是一种叶酸拮抗剂，通常以低剂量用于治疗克罗恩病（CD）。MTX的抗炎作用是通过其在细胞内转化为甲氨蝶呤多聚谷氨酸盐（MTX-PGs）来实现的。由于基于血浆监测治疗反应不能准确反映MTX的治疗效果，因此对MTX反应的潜在生物标志物——细胞内MTX-PGs进行定量至关重要。然而，由于MTX-PGs浓度较低，对CD患者的细胞内MTX代谢物进行常规监测具有挑战性。因此，采用高灵敏度方法对临床样本中的MTX-PGs进行定量是必要的。我们建立了一种高灵敏度方法，通过高氯酸脱蛋白，然后采用高效液相色谱-串联质谱法对三种MTX-PGs进行定量。使用人红细胞作为生物基质生成校准曲线。该方法应用于分析接受MTX治疗的CD患者红细胞（RBC）中的MTX-PGs。该方法对单个MTX-PGs的定量下限为1 ng/mL。覆盖1-400 ng/mL的九点校准曲线显示出良好的线性。日内和日间测定的精密度（相对标准偏差<15%）和准确度（93.41-109.37%）均令人满意。血浆MTX水平与任何单个红细胞MTX-PG水平均无显著相关性（分别为 = 0.998、0.640和0.587）。缺乏相关性支持了我们的结论，即血浆MTX水平可能无法可靠地代表细胞内蓄积情况。所建立的定量方法为增进我们对MTX代谢的理解提供了一个有用的工具，并可能有助于MTX治疗中的治疗药物监测。
