Busulfan is a chemotherapy drug widely used as part of conditioning regimens for patients prior to bone marrow transplantation (BMT). Busulfan dosing is challenging due to a narrow therapeutic window and wide inter- and intra-patient variability. Therapeutic drug monitoring (TDM) of plasma busulfan concentrations using repeated measurements and subsequent adjustment of dosage has been shown to reduce busulfan-related toxicity and improve treatment outcomes. Despite this, busulfan TDM is not widely available due to lack of available methods.
To evaluate a newly developed, simple, and rapid high-performance liquid chromatography-coupled electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for measuring busulfan concentrations over a large analytical range with small sample volumes. Method: Patient heparinized plasma from leftover clinical samples on which busulfan was ordered were collected and either analyzed immediately or frozen at -80°C until analysis. Busulfan was isolated from 100 µL of plasma after internal standard (busulfan-D8)-spiked methanol extraction, centrifugation, and dilution of the supernatant with mobile phase. The diluted supernatant is injected into the HPLC-ESI-MS/MS (Waters Alliance HT 2795 Separation Module using an XTerra MS C-8, 35µm, 21x50mm analytical column and Micromass Quatro Micro API equipped with Masslynx software), and quantified using a six-point standard curve.
The HPLC-ESI-MS/MS method was linear from 0.025 µg/mL (~ 0.1 µmol/L) to at least 6.2 µg/mL (~ 25 µmol/L) with a correlation coefficient of >0.99. Recoveries ranged from 90 – 105%, with intra- and inter-assay coefficients of variation (CVs) of <5% over the entire linear range. No significant ion suppression or interference from several common anticoagulants, hemolysis, lipemia, or icterus was identified (>5%). No carryover was observed following repeated injections of samples containing high concentrations of busulfan (> 6 µg/mL). Correlation with an established GC-ECD method using patient samples (n = 40) revealed a slope of 0.95, an intercept of 0.1, and r2 of 0.99 using Deming regression analysis.
The HPLC-ESI-MS/MS assay is a simple and quick (2-minute total analysis time per sample) method for accurate and precise TDM of plasma busulfan concentrations.