High-Throughput Validated Method for the Quantitation of Busulfan in Plasma Using Ultrafast SPE-MS/MS.

Abstract

Busulfan is an alkylating agent used to ablate bone marrow cells before hematopoietic stem cell transplantation. Because of its highly variable pharmacokinetics, studies have shown that therapeutic drug monitoring is clinically useful for patients undergoing bone marrow transplant so that toxic effects associated with high drug exposure could be reduced and improve clinical outcomes. Current methods for assaying busulfan include the use of gas chromatography mass spectrometry (GC/MS), high-performance liquid chromatography, and liquid chromatography mass spectrometry. The clinical need for faster turnaround times and increased testing volumes has required laboratories to develop faster methods of analysis for higher throughput of samples. Therefore, we present a method for the quantification of busulfan in plasma using an ultrafast solid-phase extraction/tandem mass spectrometry, which has much faster sample cycle times and similar analytical results to GC/MS. Calibration standards, quality controls, and patient samples after addition of busulfan-d4 internal standard were extracted into n-butyl chloride from plasma. The organic fraction was dried and reconstituted in 600 μL of water containing ammonium acetate, trifluoroacetic acid, and formic acid. Sample analysis was performed at a rate of less than 20 seconds per sample using a Rapidfire 300 system coupled to an Agilent 6490 MS/MS using electrospray ionization in positive ion mode. Concentrations were calculated based on a 5-point calibration curve using a 1/x linear curve fit. The analytical method shows excellent precision, sensitivity, and specificity. Minimal ion suppression or enhancement due to the matrix effect was observed. No significant carryover was seen following a sample containing 15,000 ng/mL of busulfan. Seventy-two patient samples were cross-validated with a current GC/MS method. All patient results throughout the analytical range correlated within the acceptance criteria of ±20%. The linear regression demonstrated the following: slope = 1.0067, r = 0.9964, and intercept = -6.2. A simple, fast, and robust method was developed for the quantitation of busulfan in plasma with solid-phase extraction/tandem mass spectrometry cycle times of <20 seconds per sample.