A rapid isocratic LC-MS/MS method for the quantification of busulfan in human plasma and its clinical application in pediatric bone marrow transplantation.

A rapid isocratic LC-MS/MS method for quantifying busulfan in human plasma was developed and validated. Plasma samples were processed by protein precipitation with acetonitrile using busulfan-d8 as the internal standard. Separation was performed on a Kinetex C18 column (2.1 × 50 mm, 5 µm; Phenomenex) with isocratic elution (water : acetonitrile = 70 : 30, v/v, containing 1 mM formic acid and 1 mM ammonium acetate) at 0.5 mL min-1, with a total run time of 1 min. Detection was conducted using positive electrospray ionization in multiple reaction monitoring mode (m/z 264.1 → 151.1 for busulfan; m/z 272.1 → 159.1 for busulfan-d8). The assay was linear over the concentration range of 0.100-10.0 µg mL-1 (r = 0.9998). Intra- and inter-batch RSD values were <5.5% and bias ranged from -3.6% to 5.0%. Recovery and matrix effect values were 100.1-101.3% and 101.4-101.8%, respectively. The method was successfully applied to 85 pediatric patients undergoing bone marrow transplantation. AUC0-∞ could be reliably estimated in 70 of the 85 patients (82.4%). Of these, 61.4% achieved the target AUC0-∞ (900-1500 µmol min L-1) after the first dose, while 18.6% fell below and 20.0% exceeded the target range, highlighting the substantial pharmacokinetic variability and the inadequacy of weight-based dosing in pediatric patients. This simple, rapid (1 min per sample) method is suitable for busulfan therapeutic drug monitoring in pediatric patients.