Simultaneous bioanalysis of a phosphate prodrug and its parent compound using a multiplexed LC-MS method

Background: Bioanalytical support of drug-discovery efforts increasingly requires more complex multiple component analysis, including the bioanalysis of drugs, prodrugs and metabolites. Just as the physiochemical properties of these components may differ widely from each other, optimal LC and MS conditions, including polarity, can also vary greatly among the analytes of interest, thus presenting significant challenges during quantitative LC-MS-based bioanalysis. A single compromised method for the determination of all analytes may sacrifice sensitivity or chromatographic conditions for one analyte in order to achieve adequate results for another. Manually switching between assay conditions to analyze samples under separately optimized conditions for individual compounds can be time consuming. Results: The method presented here addresses the problem of differential analyte optimization using a multiplexed approach for simultaneous quantitative bioanalysis of multiple analytes in the same sample, employing a mixed mode of both turbulent- and laminar-flow chromatography. Conclusion: The approach is illustrated with the quantitation of a lipophilic drug and its hydrophilic phosphate ester prodrug in a biological matrix under individually optimized LC-MS conditions.