Abstract 2759: Validated liquid chromatography-mass spectrometry assay for determination of busulfan: Application to clinical pharmacokinetics studies
Authors: Lin Hongxia , Gounder Murugesan K. , Goodin Susan , Bertino Joseph R. , DiPaola Robert S. , Strair Roger K. 
Published: February 2011

Abstract
Busulfan (BU), an alkylating agent, also known to have anti-leukemic efficacy, is commonly used in preparative regimens for allogenic and autologous stem cell marrow transplantation. The efficacy of BU is related to systemic levels and the area under concentration-time curve (AUC), and hence, a timely pharmacokinetic (PK) report is critical for subsequent dose adjustment. We developed two highly sensitive methods: a rapid, accurate, and sensitive liquid chromatography-mass spectrometry (LC-MS) method using direct inject tandem mass spectrometry with SIM mode, and an alternate equally sensitive high performance liquid chromatographic fluorometric (HPLC-FL) method for the quantitation of BU in human plasma. Method: BU concentrations were quantified in 50 μL of plasma spiked with [2H8]-busulfan, followed by liquid-liquid extraction with ethyl acetate. The solvent extract was dried under nitrogen and the residue was dissolved in mobile phase and analyzed using LC-MS. The mobile was 10mmol/L ammonium acetate and 10mL/L acetic acid in water and acetonitrile, (70:30) and flow rate was 0.5ml/min. Separation of BU was performed on a Gemini C18 column (4.6 × 150 mm, 3μm) at 30°C. BU and IS were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2 at the retention time of 5.8 min. When plasma samples were analyzed by HPLC-FL method, 200μL plasma spiked with 1,6 (methanesulfonyloxy) octane was extracted in ethyl acetate and dried under air at 45°C. The residue was dissolved in 200μL ethanol and derivatized with 2-naphthelenethiol according to Nara S., et al (Analytical Sci. 2000,16, 287). The samples were analyzed on a HPLC system with fluorescence detector set at excitation wavelength 255 nm and emission wavelength 370 nm. The mobile was a mixture of methanol: acetonitrile: sodium acetate buffer (0.1M, pH 7.0) using gradient elution, and flow rate was 1.0 mL/min. Results: Both assay methods are comparable in precision, accuracy, linearity, and sensitivity. The calibration curve for LC-MS assay was linear at 31.25-1000ng/ml. The LOD of assay was 7.8ng/ml and the LOQ was 31.25ng/ml. The correlation coefficient for calibration curve was 0.997±0.003 (n =4). The intra and inter-assay precision was less than 3.0% and the accuracy ranged from 95.4% to 101.7%. Busulfan AUClast calculation comparison with HPLC-FL derivatization method showed an average difference between the assays of 7.7%. Conclusion: The method LC-MS is highly accurate, reproducible, and requires less specimen, sample preparation, and analysis time over the HPLC-FL derivitization method. The new LC-MS assay is rapid and sensitive and provides an appropriate method for quantification of BU in human plasma, making therapeutic drug monitoring of BU faster and easier in clinical practice.
Acknowledgement: The study is supported by funds from Century for the Cure and U01 grant from NCI.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2759.