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J Chromatogr Sci. 2019 Mar 1;57(3):195-203. doi: 10.1093/chromsci/bmy100.

Development and Validation of LC-MS-MS Assay for the Determination of the Emerging Alkylating Agent Laromustine and Its Active Metabolite in Human Plasma.

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Department of Internal Medicine, School of Medicine, Yale University, New Haven, CT, USA.
Department of Chemistry, 55 N. Eagleville Rd., University of Connecticut, Storrs, CT, USA.
West Campus Analytical Core, Yale University, West Haven, CT, USA.
Department of Molecular Biophysics and Biochemistry, Yale-Keck MS and Proteomics Resource, Yale University, New Haven, CT, USA.
Metastagen, Inc., Wilmington, DE, USA.


The objective of this study was to validate a method for the determination of laromustine (VNP40101M) and short-lived its active metabolite (VNP4090CE) that has a half-life in human blood of <90 s in human plasma by liquid chromatography (LC) with tandem mass spectrometric (MS/MS) detection. We overcome the stability dilemma by acidified the human plasma with citric acid. Laromustine "breaks" down on the source of mass spectrometry to give m/z 249 which is the same m/z for VNP4090CE. Because VNP4090CE and laromustine elute at approximate retention time of 1.93 and 2.94 min, respectively, we were able to quantify both of them in one method. VNP40101M, VNP4090CE and the internal standards were extracted from human plasma by liquid-liquid extraction into ethyl ether. The ethyl ether layer was evaporated, reconstituted and analyzed using LC with MS/MS detection. Validation parameters such as selectivity, limit of quantitation, linearity, precision, accuracy, recovery, autosampler viability, freeze-thaw cycles and compounds stability are evaluated for this method. Results were calculated using peak area ratios, and calibration curves were generated using a weighted (1/x2) linear least-squares regression. Calibration curves for VNP40101M and VNP4090CE in human plasma ranged from 1.00 to 1,000 ng/mL. In this study, both intra- and inter-assay results demonstrated a relative standard deviation for calibration standards (inter-assay) and quality control samples (intra- and inter-assay) to be ≤15.0%. In this method, there is ~1.79% isotopic interference of VNP40101M to VNP40101M-IS, and ~3.76% isotopic interference of VNP4090CE to VNP4090CE-IS. It was concluded that there was no significant carryover.

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