Feng Yingying, Li Chunzi, Xia Wei, Pei Yumei, Sun Chang, Cao Haiwei, Ji Zhengchao, Huang Jing, Li Yanyan
Department of Laboratory Medicine, The First Hospital of Jilin University, 1 Xinmin Street, Changchun, Jilin Province 130061, China; College of Medical Technology, Beihua University, Jilin 132013, China.
Secretariat of Jilin Medical Association, 88 Yuandong Road, Changchun, Jilin Province 130041, China.
J Pharm Biomed Anal. 2025 Aug 15;261:116846. doi: 10.1016/j.jpba.2025.116846. Epub 2025 Mar 29.
High-dose methotrexate (HDMTX) is widely accepted as the first-line chemotherapeutic agent for pediatric acute lymphoblastic leukemia (ALL). However, it exhibits significant pharmacokinetic variability among individuals, which may lead to delayed elimination of MTX. In this study, we innovatively developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of serum MTX, 7-hydroxymethotrexate (7-OHMTX) and creatinine. The analytes were isolated using a protein precipitation method and separated on an Agilent Poroshell 120 SB-C column (4.6 × 50 mm, 2.7 µm) using a gradient elution with methanol (B) and 0.1 % formic acid in water (A), at a flow rate of 0.5 mL/min. The assay was linear over the range of 20-2000 ng/mL (R ≥ 0.997) for MTX and 7-OHMTX and 1-70 μg/mL (R ≥ 0.997) for creatinine. Intra- and inter-day accuracy for all analytes ranged from 88.1 % to 109.8 % with corresponding precision of 1.0-14.5 %. No significant matrix effects were observed, and analyses were extracted from human serum with recoveries exceeding 93.4 %. The predictive performance of the MTX, 7-OHMTX, the 7-OHMTX/MTX ratio, creatinine and the combined test value of the four indicators was evaluated using area under the curve (AUC). Receiver operating characteristic (ROC) analysis revealed that MTX exhibited superior predictive accuracy with sensitivity of 87.5 %, specificity of 93.1 % and AUC of 0.914 compared to 7-OHMTX with a sensitivity of 50.0 %, specificity of 89.7 % and AUC of 0.683. This finding suggesting that MTX is a better predictor of delayed elimination than 7-OHMTX. The 7-OHMTX/MTX ratio provide a more reliable prediction of delayed elimination compared to 7-OHMTX alone, emphasizing the importance of metabolic rates in addition to concentration levels. The combined test values of these four indicators at 48 h demonstrated higher predictive accuracy with sensitivity of 93.8 %, specificity of 96.6 % and AUC of 0.963 than any individual index with sensitivity (ranging from 50.0 % to 87.5 %), specificity (ranging from 79.3 % to 93.1 %) and AUC (ranging from 0.683 to 0.914). Notably, this combined test identified delayed elimination at 48 h rather than 72 h, thus enabling timely adjustments to calcium folinate rescue regimens.
大剂量甲氨蝶呤（HDMTX）被广泛公认为小儿急性淋巴细胞白血病（ALL）的一线化疗药物。然而，它在个体间表现出显著的药代动力学变异性，这可能导致甲氨蝶呤（MTX）消除延迟。在本研究中，我们创新性地开发了一种液相色谱 - 串联质谱（LC-MS/MS）方法，用于同时定量血清MTX、7-羟基甲氨蝶呤（7-OHMTX）和肌酐。采用蛋白沉淀法分离分析物，并使用甲醇（B）和0.1%甲酸水溶液（A）进行梯度洗脱，在安捷伦Poroshell 120 SB-C柱（4.6×50 mm，2.7 µm）上分离，流速为0.5 mL/min。该测定法在MTX和7-OHMTX的20 - 2000 ng/mL范围内（R≥0.997）以及肌酐的1 - 70 μg/mL范围内（R≥0.997）呈线性。所有分析物的日内和日间准确度范围为88.1%至109.8%，相应精密度为1.0 - 14.5%。未观察到显著的基质效应，从人血清中提取分析物的回收率超过93.4%。使用曲线下面积（AUC）评估MTX、7-OHMTX、7-OHMTX/MTX比值、肌酐以及这四个指标的联合测试值的预测性能。受试者工作特征（ROC）分析显示，与7-OHMTX相比，MTX表现出更高的预测准确性，其灵敏度为87.5%，特异性为93.1%，AUC为0.914，而7-OHMTX的灵敏度为50.0%，特异性为89.7%，AUC为0.683。这一发现表明，MTX比7-OHMTX更能准确预测消除延迟。与单独的7-OHMTX相比，7-OHMTX/MTX比值能更可靠地预测消除延迟，强调了除浓度水平外代谢率的重要性。这四个指标在48小时时的联合测试值显示出更高的预测准确性，灵敏度为93.8%，特异性为96.6%，AUC为0.963，高于任何单个指标的灵敏度（范围为50.0%至87.5%）、特异性（范围为79.3%至93.1%）和AUC（范围为0.683至0.914）。值得注意的是，这种联合测试在48小时而非72小时时识别出消除延迟，从而能够及时调整亚叶酸钙解救方案。
