Simultaneous quantification of methotrexate, 7-hydroxymethotrexate and creatinine in serum by LC-MS/MS for predicting...

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-C₁₈ 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 (R² ≥ 0.997) for MTX and 7-OHMTX and 1-70 μg/mL (R² ≥ 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_48 h, 7-OHMTX_48 h, the 7-OHMTX/MTX_48 h ratio, creatinine_48 h 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_48 h exhibited superior predictive accuracy with sensitivity of 87.5 %, specificity of 93.1 % and AUC of 0.914 compared to 7-OHMTX_48 h with a sensitivity of 50.0 %, specificity of 89.7 % and AUC of 0.683. This finding suggesting that MTX_48 h is a better predictor of delayed elimination than 7-OHMTX_48 h. The 7-OHMTX/MTX_48 h ratio provide a more reliable prediction of delayed elimination compared to 7-OHMTX_48 h 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.