LC–MS/MS method development for quantification of busulfan in human plasma and its application in pharmacokinetic study
Graphical abstract
Introduction
Busulfan (1,4-busulfantanediol dimethanesulfonate) (Fig. 1) has been commonly used for the treatment of chronic myelogenous leukemia and for bone marrow transplantation. Busulfan has a narrow therapeutic index, and acute toxicity may be related to absorption and disposition of the drug and metabolites. High systemic exposure of busulfan has been shown to contribute to transplantation-related toxicities, such as veno-occlusive disease, interstitial pneumonia, or neurotoxicity [1], [2]. The toxic effects were strongly related to high drug exposure by the steady-state plasma concentrations and/or area under the curve of busulfan. Therefore, therapeutic drug monitoring (TDM) has been considered of benefit for individual optimization of busulfan therapy.
Several methods have been developed for the determination of busulfan in plasma, including gas chromatography–mass spectrometry (GC–MS), gas chromatography with electron capture detection (GCECD), and high performance liquid chromatography with ultraviolet detection (HPLC-UV) [3], [4], [5], [6]. Recent studies have described simpler and rapid HPLC-MS/MS method for busulfan analysis in plasma [7], [8]. In the present study a new HPLC- tandem mass spectrometry method for the analysis of busulfan levels in plasma. LC–ESI MS/MS (liquid chromatography and tandem mass spectrometry with electron spray ionisation) technique was used in positive ionization mode with deuterated internal standard (busulfan d8) and was quantified using multiple reaction monitoring (MRM) method (Fig. 2).
Section snippets
Chemicals and reagents
Busulfan and busulfan d8 were procured from Symed Labs Ltd. Acetonitrile (HPLC grade) was purchased from Merck Millipore India Ltd. Milli-Q water from Millipore water system (Billerica, MA, USA) was used throughout the experiments. Analytical grade Ammonium Formate was obtained from Sigma–Aldrich (Bangalore, India). Blank plasma was purchased from Kaveri blood bank (Hyderabad, India).
Equipment
The equipments used in the present study are vortex mixer CM-101 plus (Remi Laboratory Instruments, Mumbai,
Results and discussion
The main aim of this work was to develop a rapid, precise, selective and sensitive LC–MS/MS method for quantification of busulfan in human plasma. busulfan d8 was used as internal standard. Chromatographic separation was achieved on Phenomenex Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) with acteonitrile: 10 mM ammonium formate buffer (80:20 v/v) as an isocratic mobile phase with a flow rate of 0.5 mL min−1. Quantitation was performed by transition of 264.1 → 151.1 (m/z) for busulfan and 272.1 → 159.1 (m/z)
Conclusion
The developed method is simple, cost effective rugged and a high throughput method for estimation of busulfan in human plasma. The method consists of a simple sample pretreatment by protein precipitation to give consistent and reproducible recoveries of busulfan. In the present work a new LC–MS/MS method for the quantification of busulfan in human plasma with less processing volume, sensitive and less run time developed for clinical study sample analysis. The proposed method is suitable for
Conflict of interest
The authors have no conflict of interest.
Acknowledgements
The authors thankful to the management of Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Guntur for providing the facilities to carry out the research work.
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