High-Throughput Quantitative LC–MS- MS Assays by On-Line Extraction Using a Monolithic Support

Please direct correspondence to Raymond Xu at [email protected] oday, due to its inherent specificity and sensitivity, the most prevalent bioanalytical technique in drug development is liquid chromatography tandem mass spectrometry (LC–MS-MS). This technique is regarded widely as the preferred tool for quantitative determination of drug and metabolites from biological matrices including plasma, blood, urine, and tissues (1). Samples from biological matrices usually are not compatible with direct LC–MSMS analyses. Conventional sample preparation techniques for LC–MS-MS traditionally employ protein precipitation, liquid–liquid extraction, or solid-phase extraction (SPE). Manual operations associated with these processes are labor intensive and time-consuming. An alternative sample extraction method that has generated much interest in recent years is the direct injection of plasma using on-line extraction (2–9). A major advantage of on-line SPE over off-line extraction techniques is that the sample preparation steps are embedded into the chromatographic separation and, thus, it eliminates much of the time required for sample preparation. Different extraction supports have been developed, allowing direct injection of biological fluids or extracts for various applications. These extraction supports include restricted-access media (RAM) (2,3), large-size particle (4,5), monolithic material (6–8), and disposable cartridges utilizing traditional packings such as those used in Spark-Holland (Plainsboro, New Jersey) systems (9). In some techniques, a Liquid chromatography tandem mass spectrometry (LC–MS-MS) has become a widely used technique for the fast and sensitive quantitation of small molecules, especially pharmaceutical compounds, in complex matrices such as biological fluids. Among various sample preparation techniques used for bioanalytical applications, on-line solid-phase extraction (SPE) utilizing column-switching techniques is rapidly gaining acceptance to reduce both time and labor required to produce LC–MS-MS results. An automated procedure using monolithic support has been described previously for pharmaceutical component analysis in human plasma by LC–MS-MS. In this article, this approach has been extended to high-throughput quantitative LC–MS-MS analysis under GLP applications for a drug candidate in development from preclinical animal studies through clinical development. Assays in rat, monkey, and human plasma have been developed and validated based upon a generic approach to sample processing procedures. On-line concentration provided for highly sensitive determinations of the analyte in human plasma with a lower limit of quantitation of 0.077 ng/mL. All three assays exhibited high recovery and good tolerance to matrix effect. The results show that quantitative LC–MS-MS analysis by on-line extraction using the monolithic support is reproducible, sensitive, and accurate enough to satisfy the requirements of both preclinical and clinical drug development.