ABSTRACT A rapid and robust method for measuring methotrexate (MTX) and its two primary metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA), was developed for use in pharmacokinetic studies of plasma and cerebrospinal fluid samples collected from infants with malignant brain tumors. Sample aliquots (100 µL) were prepared for bioanalysis of MTX and metabolites using a Waters Oasis HLB microelution solid-phase extraction (SPE) plate. Chromatography was performed using a Phenomenex Synergi Polar-RP 4 µ 75 × 2.0 mm ID column heated to 40°C. A rapid gradient elution on a Shimadzu HPLC system was used, with mobile phase A consisting of water/formic acid (100/0.1 v/v) and mobile phase B consisting of acetonitrile/formic acid (100/0.1 v/v). Column eluent was analyzed using AB Sciex QTRAP 5500 instrumentation in electrospray ionization mode. The ion transitions (m/z) monitored were 455.2 → 308.1, 471.1 → 324.1, and 326.2 → 175.1 for MTX, 7-OHMTX, and DAMPA, respectively. The method was linear over 0.0022–5.5 µM for MTX, 0.0085–21 µM for 7-OHMTX, and 0.0031–7.7 µM for DAMPA. The method was applied to the analysis of serial plasma samples obtained from infants diagnosed with malignant brain tumors receiving high-dose methotrexate and results were compared to MTX concentrations from an immunoassay based on fluorescence polarization. GRAPHICAL ABSTRACT

中文翻译：

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LC-MS/MS 测定血浆和脑脊液中的甲氨蝶呤、7-羟基甲氨蝶呤和 2,4-二氨基-N10-甲基蝶酸及其在大剂量甲氨蝶呤药代动力学分析中的应用

摘要 开发了一种用于测量甲氨蝶呤 (MTX) 及其两种主要代谢物 7-羟基甲氨蝶呤 (7-OHMTX) 和 2,4-二氨基-N10-甲基蝶呤酸 (DAMPA) 的快速而可靠的方法，用于血浆药代动力学研究以及从患有恶性脑肿瘤的婴儿身上采集的脑脊液样本。使用 Waters Oasis HLB 微量洗脱固相萃取 (SPE) 板制备样品等分试样 (100 µL)，用于 MTX 和代谢物的生物分析。使用加热至 40°C 的 Phenomenex Synergi Polar-RP 4 µ 75 × 2.0 mm 内径色谱柱进行色谱分析。使用 Shimadzu HPLC 系统上的快速梯度洗脱，流动相 A 由水/甲酸 (100/0.1 v/v) 组成，流动相 B 由乙腈/甲酸 (100/0.1 v/v) 组成。使用 AB Sciex QTRAP 5500 仪器在电喷雾电离模式下分析柱洗脱液。对于 MTX、7-OHMTX 和 DAMPA，监测到的离子跃迁 (m/z) 分别为 455.2 → 308.1、471.1 → 324.1 和 326.2 → 175.1。该方法在 MTX 的 0.0022–5.5 µM、7-OHMTX 的 0.0085–21 µM 和 DAMPA 的 0.0031–7.7 µM 上呈线性。该方法用于分析从接受高剂量甲氨蝶呤治疗的被诊断患有恶性脑肿瘤的婴儿获得的连续血浆样本，并将结果与​​基于荧光偏振的免疫测定的 MTX 浓度进行比较。图形概要 0031–7.7 µM 用于 DAMPA。该方法用于分析从接受高剂量甲氨蝶呤治疗的被诊断患有恶性脑肿瘤的婴儿获得的连续血浆样本，并将结果与​​基于荧光偏振的免疫测定的 MTX 浓度进行比较。图形概要 0031–7.7 µM 用于 DAMPA。该方法用于分析从接受高剂量甲氨蝶呤治疗的被诊断患有恶性脑肿瘤的婴儿获得的连续血浆样本，并将结果与​​基于荧光偏振的免疫测定的 MTX 浓度进行比较。图形概要