A low flow ionization technique to integrate quantitative and qualitative small molecule bioanalysis

摘要

Mass spectrometry-based assays are used in drug discovery and development to detect, characterize and quantify drugs, metabolites, impurities and degradants. Recently, high resolution-based mass spectrometers have begun to emerge as a platform with potential for performing integrated qualitative and quantitative assays in order to streamline the drug discovery and development process. However, the widely different LC-MS response observed for a drug and its metabolites limit the direct use of LC-MS responses for relative quantitative determination of metabolites. This in turn limits the use of conventional LC-ESI-MS methods, in the absence of reference standards, as an integrated technique for detection, characterization and quantification of drugs and metabolites. The goal of this study was to explore the use of LC-captive spray ionization (CSI)-mass spectrometry for detection, characterization and quantification of drugs and metabolites. CSI allows the use of conventional HPLC or uHPLC columns and flow rates of 0.35-0.6 mL/min (before post-column flow splitting) and can be considered as a technique which can function as a nanospray or microspray. Also, in comparison to conventional nanospray ionization (NSI) techniques, setup and maintenance of CSI do not require: (1) X, Y, and Z positioning or cameras to guide the spray positioning, (2) difficult to control splitters to deliver nano-flow ratios and difficult to maintain nanospray nozzles. Evaluations using equimolar mixture of buspirone and four monoxy metabolites present in human plasma show that LC-CSI-MS is a highly sensitive technique that gives a near equimolar response for the compounds used in this example. Comparisons of LC-ESI-MS data with that obtained using LC-CSI-MS show that reasonable quantification of metabolites may be achievable without using reference standards or administration of radiolabeled drugs.