Over 400 professionals representing pharmaceutical companies, CROs, and multiple regulatory agencies participated in the 6th Workshop on Recent Issues in Bioanalysis (WRIB). Like the previous sessions, this event was in the format of a practical, focused, highly interactive and informative workshop aiming for high-quality, improved regulatory compliance and scientific excellence. Numerous 'hot' topics in bioanalysis of both small and large molecules were shared and discussed, leading to consensus and recommendations among panelists and attendees representing the bioanalytical community. The major outcome of this year's workshop was the noticeable alignment of multiple bioanalytical guidance/guidelines from different regulatory agencies. This represents a concrete step forward in the global harmonization of bioanalytical activities. The present 2012 White Paper acts as a practical and useful reference document that provides key information and solutions on several topics and issues in the constantly evolving world of bioanalysis.

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myo-Inositol is being investigated as a biomarker to monitor disease states involving the central nervous system. We have developed and validated a quantitative method to study endogenous myo-inositol metabolism in rat brain tissue. Tissue samples were homogenized, and their myo-inositol content was determined using spiked calibration curves and mass spectrometry. The assay was validated on an LC/MS/MS platform, and specificity was evaluated using accurate mass measurements. A novel chiral LC/MS/MS method was also developed to resolve myo-inositol from other endogenous inositol epimers and confirm the selectivity of the quantitative procedure. The validated method is selective, convenient, precise (<15% RSD), accurate (<15% RE), and sensitive over a linear range of 0.100-100 microg/mL. This method could potentially be used as an instrument for monitoring pathological conditions related to psychotherapeutics, as well as a tool for screening curative pharmaceuticals for efficacy.

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Biomarkers play an increasingly important role for drug efficacy and safety evaluation in all stages of drug development. It is especially important to develop and validate sensitive and selective biomarkers for diseases where the onset of the disease is very slow and/or the disease progression is hard to follow, i.e., osteoarthritis (OA). The degradation of Type II collagen has been associated with the disease state of OA. Matrix metalloproteinases (MMPs) are enzymes that catalyze the degradation of collagen and therefore pursued as potential targets for the treatment of OA. Peptide biomarkers of MMP activity related to type II collagen degradation were identified and the presence of these peptides in MMP digests of human articular cartilage (HAC) explants and human urine were confirmed. An immunoaffinity LC/MS/MS assay for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues was developed and clinically validated. The assay has subsequently been applied to analyze human urine samples from clinical studies. We have shown that the assay is able to differentiate between symptomatic OA and normal subjects, indicating that uTIINE can be used as potential biomarker for OA. This chapter discusses the assay procedure and provides information on the validation experiments used to evaluate the accuracy, precision, and selectivity data with attention to the specific challenges related to the quantification of endogenous protein/peptide biomarker analytes. The generalized approach can be used as a follow-up to studies whereby proteomics-based urinary biomarkers are identified and an assay needs to be developed. Considerations for the validation of such an assay are described.

A simple plasma extraction method coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous mevalonic acid (MVA), a biomarker indicative of the rate of cholesterol biosynthesis, in human plasma samples. The analyte was extracted from the plasma matrix using a straightforward liquid-liquid sample preparation procedure. The extract supernatants were evaporated, reconstituted in aqueous solvent and injected into the LC/MS/MS system without further processing. The chromatographic separation was achieved on a reverse-phase high-performance liquid chromatography column. The accuracy and precision of the method was determined over the concentration range 0.25-25 ng/mL MVA from human plasma extracts in three validation batch runs. Inter-assay precision (%CV) and accuracy (%RE) of the quality control samples were ≤7.00% (at lower limit quality control) and ≤6.10%, respectively. The sensitivity and throughput of this assay was significantly improved relative to previously published methods, resulting in smaller sample requirements and shorter analysis time. Assay results from a clinical study following the oral administration of an exploratory statin demonstrate that this procedure could potentially be used in the investigation of therapies associated with hypercholesterolemia.

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