A rapid and sensitive LC-MS/MS method for simultaneous determination of melphalan and its monohydroxy and dihydroxy metabolites in human plasma

Abstract

As a hydrolysis mediated drug in vivo, the pharmacokinetics of melphalan are highly variable in patients. Few methodologies could simultaneously measure the concentrations of melphalan and its hydrolyzed metabolites in plasma. The aim of this study was to develop a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of melphalan and its hydrolyzed metabolites, monohydroxy melphalan (MOH melphalan) and dihydroxy melphalan (DOH melphalan). A simple protein precipitation was employed for sample preparation and melphalan-d8 was used as internal standard. Baseline separation of target analytes was achieved using an XSelect HSS T3 column (2.1 × 50 mm, 5 µm) with a gradient elution at a flow rate of 0.5 mL/min in 5 min. The monitored transitions were m/z 305.1 → 287.7 for melphalan, m/z 287.1 → 228.0 for MOH melphalan, m/z 269.3 → 251.8 for DOH melphalan, and m/z 313.1 → 295.7 for melphalan-d8. The method was fully validated in accordance with the FDA guideline. The calibration curves were established over the range of 5.22–5220 ng/mL for melphalan, 7.94–1588 ng/mL for MOH-melphalan, and 15.0–3000 ng/mL for DOH-melphalan with the regression coefficients greater than 0.99. The intra- and inter-day coefficients of variation for the analytes were ≤11.0% and all the biases were less than 8.3%. The method has been successfully applied to the quantification of melphalan and its metabolites in clinical plasma samples obtained from hematopoietic stem cell transplantation patients who received a dose of melphalan for pre-transplant conditioning.