Therapeutic drug monitoring of busulfan and cyclophosphamide in hematologic malignancies: An LC-MS/MS development, validation and application.

Background: Busulfan and cyclophosphamide (Bu-Cy) is a widely used conditioning regimen for hematopoietic stem cell transplantation (HSCT) in the treatment of hematologic malignancies. To explore the exposure-efficiency relationships of Bu-Cy regimen and evaluate the necessity of relative therapeutic drug monitoring (TDM), we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify the busulfan and cyclophosphamide in human plasma.

Methods: A simple LC-MS/MS bioassay for simultaneous determination of busulfan and cyclophosphamide using isotope dilution internal standardization has been established. Plasma samples were subjected to protein precipitation prior to their injection into a column for subsequent separation (Thermo Accucore aQ C₁₈, 50 × 2.1 mm, 2.6 μm). Positive electrospray ionization (ESI) mode with selected reaction monitoring (SRM) was used for analytes determination.

Results: Busulfan and cyclophosphamide could be measured within 3 min, with the calibration ranges of 80-4000 ng/mL for busulfan and 25-2000 ng/mL for cyclophosphamide. The validated method showed good sensitivity and linearity. Accuracies and precisions were less than 15 %. The results of stability showed that busulfan and cyclophosphamide were stable in the plasma under room temperature for 16 h, 4 °C for 24 h, -80 °C for 2 weeks, and after three freeze-thaw processes.

Conclusion: The bioassay was simple, sensitive and successfully implemented in the realm of TDM for Bu-Cy, ultimately leading to improved dosing precision.