Simultaneous Determination of Methotrexate Concentrations in Human Plasma and Cerebrospinal Fluid Using Two-Dimensional Liquid Chromatography: Applications in Primary Central Nervous System Lymphoma.

Abstract

In this study, we aimed to develop a method for the simultaneous quantification of methotrexate (MTX) samples extracted from human plasma and cerebrospinal fluid (CSF), using two-dimensional liquid chromatography (2D-LC). Furthermore, we intended to verify whether intravenous mannitol could increase MTX concentration in the CSF of patients. The mobile phase of PUMP1 consisted of 10.0 mmol/L ammonium acetate and acetonitrile. PUMP2 solution consisted of an aqueous solution of 10.0 mmol/L ammonium acetate. The mobile phase of PUMP3 comprised 50.0 mmol/L ammonium acetate and acetonitrile, with a flow rate of 1.0 mL/min. The developed method was successfully employed to simultaneously determine drug levels in plasma and CSF from the patients treated with MTX. CSF samples were obtained by lumbar puncture 0.5 - 2 h after starting the high-dose methotrexate (HD-MTX) infusion (over 4 h) and immediately before the intrathecal (IT) administration of MTX. Venous blood samples were drawn 4 h after the start of infusion. The calibration curve was linear, with a range of 0.07 - 2.38 µmol/L for CSF samples and a range of 0.11 - 5.51 µmol/L for plasma samples. Precision (> 95%) and accuracy (> 97%) were within the acceptance criteria for each quality control (QC) level. Inter- and intra-day accuracy and precision values met the acceptance criteria for each QC level. The correlation between MTX concentrations in the plasma and CSF was moderate (r = 0.502). No significant difference was observed in MTX concentration in CSF between patients using intravenous mannitol and those not using intravenous mannitol (P = 0.682). The developed method was useful for therapeutic drug monitoring of MTX and suitable for assessing the risks and benefits of chemotherapy in patients with primary central nervous system lymphoma. Intravenous mannitol did not increase MTX concentration in the CSF of patients.