Bioanalysis is complex with many personnel, regulations, instruments and processes involved. Bioanalytical (BA) laboratories use a range of technologies and procedures to ensure that samples are processed efficiently and accurately and quality results are delivered in a timely manner. The BA laboratory is facing constant pressure to reduce operation costs while optimizing efficiency to be competitive. This chapter focuses on the vital role of four major components in a GLP bioanalytical laboratory: facility, infrastructure, compliance, and documentation. In addition, quality assurance (QA) and other supporting functions such as quality control (QC), sample management, archivist, technical support, planning, and reporting also play pivotal roles. Monitoring BA activities in CROs has also become more and more important in recent years.
Abstract A gas chromatographic/mass spectrometric procedure has been developed for the quantiation in human plasma of the enatiomers of rimanatadine and its three hydroxylated metabolites. The assay utilized derivatization of all analytes with the optically active reagent S‐α‐methyl‐α‐methoxy(pentafluorpheyl)acetic acid, selective ion monitoring, methane negative ion chemical ionization mass spectrometry and stable isotope dilution techniques. This method has been used to meausure plasma concentrations of the enantiomers of rimantadine, m ‐hydroxyrimantadine and p ‐hydroxyrimantadine (equatorial and axial epimers) in the ranges 2.5‐250, 2.5‐50, 1.25–62.5 and 1.25–62.5 ng/mL, respectively, in six subjects given a single 200 mg dose of racemic rimantadine. Although there are no significant differences in the concentration‐time profiles of R ‐ and S ‐rimantadine, large stereospecific differences in the disposition of their metabolites are observed.
High throughput-solid phase extraction tandem mass spectrometry (HT-SPE/MS) is a fully automated system that integrates sample preparation using ultrafast online solid phase extraction (SPE) with mass spectrometry detection. HT-SPE/MS is capable of conducting analysis at a speed of 5–10 s per sample, which is several fold faster than chromatographically based liquid chromatography–mass spectrometry (LC–MS). Its existing applications mostly involve in vitro studies such as high-throughput therapeutic target screening, CYP450 inhibition, and transporter evaluations. In the current work, the feasibility of utilizing HT-SPE/MS for analysis of in vivo preclinical and clinical samples was evaluated for the first time. Critical bioanalytical parameters, such as ionization suppression and carry-over, were systematically investigated for structurally diverse compounds using generic SPE operating conditions. Quantitation data obtained from HT-SPE/MS was compared with those from LC–MS analysis to evaluate its performance. Ionization suppression was prevalent for the test compounds, but it could be effectively managed by using a stable isotope labeled internal standard (IS). A structural analogue IS also generated data comparable to the LC–MS system for a test compound, indicating matrix effects were also compensated for to some extent. Carry-over was found to be minimal for some compounds and variable for others and could generally be overcome by inserting matrix blanks without sacrificing assay efficiency due to the ultrafast analysis speed. Quantitation data for test compounds obtained from HT-SPE/MS were found to correlate well with those from conventional LC–MS. Comparable accuracy, precision, linearity, and sensitivity were achieved with analysis speeds 20–30-fold higher. The presence of a stable metabolite in the samples showed no impact on parent quantitation for a test compound. In comparison, labile metabolites could potentially cause overestimation of the parent concentration if the ion source conditions are not optimized to minimize in-source breakdown. However, with the use of conditions that minimized in-source conversion, accurate measurement of the parent was achieved. Overall, HT-SPE/MS exhibited significant potential for high-throughput in vivo bioanalysis.
Glycosylation is one of the most important post-translational modifications to mammalian proteins. Distribution of different glycoisoforms of certain proteins may reflect disease conditions and, therefore, can potentially be utilized as biomarkers. Apolipoprotein C3 (ApoC3) is one of the many plasma glycoproteins extensively studied for association with disease states. ApoC3 exists in three main glycoisoforms, including ApoC3-1 and ApoC3-2, which contain an O-linked carbohydrate moiety consisting of three and four monosaccharide residues, respectively, and ApoC3-0 that lacks the entire glycosylation chain. Changes in the ratio of different glycoisoforms of ApoC3 have been observed in pathological conditions such as kidney disease, liver disease, and diabetes. They may provide important information for diagnosis, prognosis, and evaluation of therapeutic response for metabolic conditions. In this current work, a liquid chromatography (LC)-high-resolution (HR) time-of-flight (TOF) mass spectrometry (MS) method was developed for relative quantitation of different glycoisoforms of intact ApoC3 in human plasma. The samples were processed using a solid-phase extraction (SPE) method and then subjected to LC-full scan HRMS analysis. Isotope peaks for each targeted glycoisoform at two charge states were extracted using a window of 50 mDa and integrated into a chromatographic peak. The peak area ratios of ApoC3-1/ApoC3-0 and ApoC3-2/ApoC3-0 were calculated and evaluated for assay performance. The results indicated that the ratio can be determined with excellent reproducibility in multiple subjects. It has also been observed that the ratios remained constant in plasma exposed to room temperature, freeze-thaw cycles, and long-term frozen storage. The method was applied in preliminary biomarker research of diabetes by analyzing plasma samples collected from normal, prediabetic, and diabetic subjects. Significant differences were revealed in the ApoC3-1/ApoC3-0 ratio and in the ApoC3-2/ApoC3-0 ratio among the three groups. The workflow of intact protein analysis using full scan HRMS established in this current work can be potentially extended to relative quantitation of other glycosylated proteins. To our best knowledge, this is the first time that a systematic approach of relative quantitation of targeted intact protein glycoisoforms using LC-MS has been established and utilized in biomarker research.
Significant differences in the pharmacodynamic activity and pharmacokinetic properties could exist for a pair of enantiomeric drugs. In order to evaluate the activity, toxicity, absorption, distribution, metabolism, and excretion properties of the individual enantiomers, and any potential for chiral inversion caused by the biotransformation process, chiral bioanalytical assays are necessary for individual enantiomers and/or their metabolites for in vivo samples. However, development and validation of chiral quantitative assays are highly challenging in comparison to typical nonchiral assays. Therefore, a tiered approach should be used to address specific needs arising in different scenarios of chiral drug development, including development of racemate or fixed-ratio (nonracemic) enantiomers, development of a single enantiomer, racemic switches, and quantitation of enantiomeric metabolites. The choice of a nonchiral quantitative assay, a chiral qualitative assay, or a chiral quantitative assay should be based on the development strategy and on the molecular properties of the drug candidate.
Bioanalysis supports a broad range of activities in drug discovery and development. While certain bioanalytical activities are governed by regulatory guidelines, others reside outside the scope of regulations. It has become a consensus in the bioanalytical community that ' fit-for-purpose' method development, assay performance verification and study implementation should be considered for different stages of drug discovery and development to ensure cost and time efficiency. The key notion of the fit-for-purpose approach is that bioanalytical activities should be tailored to meet the intended purpose of the study, with a level of stringency consistent with the intended use of the data. Fit-for-purpose bioanalytical practice can be reflected in four aspects: flexible exclusion/inclusion of validation parameters to improve efficiency while ensuring the scientific quality of the assay, suitable experiment design for verification of assay performance for specific purposes, broader/narrower acceptance criteria in recognition of the challenges for some types of work while meeting the intended requirements, and an appropriate level of recording keeping and compliance for a certain degree of reconstructability of the work. The application of fit-for-purpose approaches in different types of bioanalytical activities, such as biomarker measurement, metabolite quantitation, chiral compound analysis, assay transfer/cross-validation and so on, are also discussed in this chapter.
