An ultrahigh‐performance liquid chromatography–tandem mass spectrometry method for quantification of methotrexate and 7‐hydroxy‐methotrexate and application for therapeutic drug monitoring in patients with central nervous system lymphoma

Abstract

A method using ultrahigh‐performance liquid chromatography–tandem mass spectrometry was developed, validated, and applied to simultaneously determine plasma methotrexate (MTX) and 7‐hydroxy‐methotrexate (7‐OH‐MTX) in 117 patients with central nervous system (CNS) lymphoma. The ion transitions utilized were m/z 455.2 > 308.2 for MTX and m/z 471.2 > 324.1 for 7‐OH‐MTX. Samples were prepared through protein precipitation using methanol. Chromatographic separation was achieved within 3.0 min on a CMS9030 column (Ruixi, 2.1 × 50 mm, 3 μm) through a gradient elution of methanol and a 10% ammonium acetate solution at a flow rate of 0.4 mL/min. The method demonstrated linearity in the concentration range of 0.05–10 μM for MTX and 0.25–50 μM for 7‐OH‐MTX. The intra‐ and inter‐day inaccuracy ranged from –7.38% to 7.83%, and the imprecision was less than 6.00% for both analytes. The recovery and matrix effect normalized by the internal standard (MTX‐D3) remained consistent. Both analytes remained stable under nine different storage conditions. In patients with CNS lymphoma, MTX levels at 12 h and 7‐OH‐MTX levels at 12, 36, and 60 h after dosing in individuals with impaired renal function were significantly higher compared with those with normal renal function. 7‐OH‐MTX could potentially serve as a superior indicator for nephrotoxicity compared with MTX.