Determination of Busulfan and Melphalan in Plasma by Turbulent Flow Chromatography-Tandem Mass Spectrometry

Melphalan and busulfan are DNA-alkylating agents that are often used concurrently in hematopoietic stem cell transplant (HCT) conditioning regimens. Studies have demonstrated that this combination of alkylating agents is very effective and well-tolerated prior to HCT. This combination is widely used for acute leukemia, advanced lymphoid malignancies, and multiple myeloma. Our goal was to develop an assay for the rapid measurement of both compounds simultaneously in the hopes that rapidly measuring their concentrations could possibly shorten the length of hospital stay. It would also simplify specimen handling in the clinic and the laboratory, reduce the amount of blood drawn, and allow for rapid reporting of the drug levels, thereby facilitating rapid dose adjustments.This chapter describes a validated method that measures both compounds simultaneously. Melphalan and busulfan were extracted from plasma with methanol containing deuterated internal standards. Turbulent flow chromatography coupled with reversed-phase HPLC was used for separation, while the mass spectrometer was set in the positive ion mode. This method has proven accurate and rapid and allowed for timely dose adjustments. The assay was linear over the clinically relevant ranges; the analytical measurement range for busulfan and melphalan was 10-5000 ng/mL and 10-15,000 ng/mL, respectively. Specimens containing elevated drug levels were diluted yielding a final clinically reportable range of 10-25,000 ng/mL for busulfan and 10-75,0000 ng/mL for melphalan. This method is very suitable for simultaneous measurements of these drugs and is currently being used to support pharmacokinetic studies.