Simultaneously measure the concentrations of busulfan and phenytoin in human plasma using an HPLC-MS/MS method: Application to the TDM for children underwent hematological stem cell transplantation

Abstract

In this work, a simple and rapid high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to carry out the simultaneous measurement of busulfan (BU) and phenytoin (PHT) in the plasma of children. In this method, plasma sample could be prepared by one-step protein precipitation using 1 mL of methanol/water (1:1, v/v). After centrifugation (14,500 rpm, 5 min, 4 °C), 10 μL of the supernatant was injected into a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm, Thermo Fisher Scientific) for separation by gradient elution. Quantification was carried out using multiple reactions monitoring (MRM) under positive scan mode. In the method verification, the calibration curves of BU and PHT showed satisfactory linearity (r > 0.99) at the concentration ranging from 0.02 to 20 μg mL−1. The accuracy and precision were tested at four concentration levels (including the LLOQ level) with the relative error (RE) ranging from −0.80% to 11.45% and coefficient of variation (CV) between 0.93% and 7.74%. There was no pronounced matrix effect to interfere with the quantitative analysis. Compared to determine BU and PHT using two individual methods, less pre-treatment process, labor and blood sample volume are required in this proposed method. Finally, this method was successfully applied to the therapeutic drug monitoring of BU and PHT for children underwent hematological stem cell transplantation.

PEG 400 Ion Suppression in Busulfan Detection by High-Performance Liquid Chromatography—Tandem Mass Spectrometry

“…These parameters are related to clinical outcomes, and pharmacodynamic studies have indicated that the optimal Bu target range depends on the conditioning regimen, age of the HSCT recipient, and the underlying disease. 7 Several analytical methods have been used to measure Bu plasma levels, including gas chromatography coupled with mass spectrometry, [7][8][9] high-performance liquid chromatography (HPLC) coupled with ultraviolet spectroscopy, and fluorescence detection. More recently, methodologies based on liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) have been developed that result in high analytical specificity and sensitivity, significantly shorter turnaround times, and a simplified workflow.…”