Tandem Mass Spectrometry Method for the Quantification of Serum Busulfan

Abstract

Busulfan, an alkylating agent, is most commonly used as a component of bone marrow transplantation preoperative regimens. Significant interpatient and intrapatient variations in pharmacokinetics require individualizing the dosage based on area under the time-versus-concentration curve. Timely result reporting is critical to dose adjustment to reduce morbidity and mortality associated with the regimen. The authors developed a rapid, accurate, and sensitive method for the quantification of serum busulfan using direct inject tandem mass spectrometry. Plasma samples (50 microL) are extracted in 1 mL of methanol containing 1,6-bis-(methanesulfonyloxy)hexane as an internal standard. The supernatant is dried under nitrogen (40 degrees C, 30 minutes) and then dissolved in 200 microL methanol and transferred into a clean glass vial suitable for LC/MS/MS analysis. The sample is delivered using an HPLC pump that delivers 0.2 mL of methanol per minute, and 20 microL of sample is injected into a turbo ion spray-equipped tandem mass spectrometer. Total analysis time is 5 minutes. The Q1/Q3 transition for busulfan (BU) is monitored at 269/55 and 297.1/55.1 for the internal standard. The assay is linear to 10 micromol/L and sensitive to at least 0.5 micromol/L. The interassay reproducibility at 1, 2.2, and 4.4 micromol/L were 4.2%, 5.6%, and 6.3%, respectively. Within-run precision using 3 different control samples was 3.9%, 3.9%, and 6.9%. Mean recovery of 4 different BU concentrations spiked into 10 different BU free plasma samples was 98%. Correlation with an established HPLC-UV method revealed a slope of 0.98, an intercept of 0.1, and r = 0.95 (n = 48). No significant interfering substances or ion suppression was identified. This method is a significant improvement over the existing HPLC-UV method for BU determination. The method is highly accurate, reproducible, and requires less specimen, sample preparation, and analysis time.