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.
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