Simultaneous determination of busulfan, fludarabine, phenytoin, and posaconazole in plasma from patients undergoing hematopoietic stem cell transplantation

A simple, fast, sample-saving, and sensitive liquid chromatography-tandem mass spectrometry method was established with a linear range adjusted by in-source collision-induced dissociation. Notably, this could simultaneously determine busulfan, fludarabine, phenytoin, and posaconazole in plasma from children, each having unique physical and chemical properties. The procedure necessitated only 20 μL of plasma and involved a simple protein precipitation process. Chromatographic separation was accomplished on a reversed-phase column (C18, 50 × 2.1 mm, 2.6 μm) through gradient elution utilizing water (containing 0.1 % formic acid and 2 mM ammonium acetate) and acetonitrile (containing 0.1 % formic acid) as the mobile phase. An injection volume of 2 μL was utilized, with a total run time of 3.6 min. Mass spectrum acquisition was performed on a Triple Quad™ 4500MD tandem mass spectrometer with an electrospray ionization source in positive mode. Moreover, in-source collision-induced dissociation was used to adjust the linear range of phenytoin due to its excessive response. The calibration curves ranged from 20 to 2560 ng/mL for busulfan, 10-1280 ng/mL for fludarabine, 0.4-51.2 μg/mL for phenytoin, and 0.1-12.8 μg/mL for posaconazole, with mean r² greater than 0.997. In addition, the method underwent rigorous validation following the European Medicines Agency guidelines, demonstrating exceptional accuracy (90.5 %-106.7 %) and precision (2.0 %-13.0 %). Furthermore, its applicability to atypical matrices, including hemolytic and hyperlipidemic plasma, was thoroughly assessed. As such, this approach was effectively utilized for the therapeutic drug monitoring of busulfan, fludarabine, phenytoin, and posaconazole for children undergoing hematopoietic stem cell transplantation.