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In contrast to quantification of biotherapeutics, endogenous protein biomarker and target quantification using LC–MS based targeted proteomics can require a much more stringent and time-consuming tryptic signature peptide selection for each specific application. While some general criteria exist, there are no tools currently available in the public domain to predict the ionization efficiency for a given signature peptide candidate. Lack of knowledge of the ionization efficiencies forces investigators to choose peptides blindly, thus hindering method development for low abundant protein quantification. Here, the authors propose a tryptic signature peptide selection workflow to achieve a more efficient method development and to improve success rates in signature peptide selection for low abundant endogenous target and protein biomarker quantification.

While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost–effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.

Background: To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. Results: Strategies were developed to overcome two major bioanalytical challenges: poor drug tolerance of the anti-drug antibodies assay and very low cut point observed in the screening and confirmatory assays. By using acid dissociation (400 mM glycine solution at pH 2.0), drug tolerance of 200 μg/ml drug was achieved for both the screening and confirmatory assays. Effects of biological matrix (disease state vs normal serum) and assay conditions (capture/detector reagent concentration, minimum required dilution, acid pretreatment) on assay cut points were systematically evaluated. Conclusion: A bridging immunogenicity assay for detecting anti-cabiralizumab antibodies in human serum has been successfully developed, validated and applied to clinical studies.

Glucuronides, especially acyl glucuronides, were often found to be unstable in vitro and in vivo. Acyl glucuronide metabolites can convert back to the parent drugs at physiological pH through hydrolysis. Glucuronides can also undergo in-source fragmentation during MS ionization to form the same ions as those of the parent compounds, which could cause interference to the analysis of the parent compounds. All of these may cause significant challenges in developing LC–MS/MS bioanalytical assays of labile glucuronides or parent compounds in the presence of glucuronide metabolites. In this manuscript, we will discuss these challenges and summarize recommended strategies and practices for fast and efficient method development. Critical considerations in assay development will also be discussed.

Background: Currently, no regulatory guidelines are available for parallelism assessment for LC-MS biomarker quantification. Spike recovery, standard addition and dilutional linearity are recommended with no mention of the implications of applying these approaches. Results: Here, using human urine creatinine, the authors compared spike recovery and standard addition in LC-MS biomarker quantification, and evaluated a new hybrid approach: parallelism QCs. The authors drew different conclusions based on which approach was used (

Aim: The result of investigation on the procedure of sample handling and bioanalysis of small volume of plasma sample for nonclinical studies stored in 0.5-ml micronic tubes was reported. Results/methodology: Sample integrity of the small volume (25 μl) during long-term storage and the feasibility and data reliability of performing multiple re-assays on the small volume sample using 5 μl aliquot per analysis was evaluated. Conclusion: Integrity was maintained in samples (25 μl) stored for up to 1 month in 0.5-ml micronic tubes at -20°C. A 25 μl sample is sufficient for four-times of re-assays. This evaluation demonstrated the feasibility of this workflow of handling and bioanalysis on small volume plasma sample for GLP studies under the US FDA guidance.

Bioanalytical methods evolve throughout clinical development timelines, resulting in the need for establishing equivalency or correlation between different methods to enable comparison of data across different studies. This is accomplished by the conduct of cross validations and correlative studies to compare and describe the relationship. The incurred sample reanalysis acceptance criterion seems to be adopted universally for cross validations and correlative studies; however, this does not identify any trends or biases between the two methods (datasets) being compared. Presented here are graphing approaches suitable for comparing two methods and describing equivalence or correlation. This article aims to generate awareness on graphing techniques that can be adopted during cross validations and correlative studies.

The 18th Annual Land O'Lakes Bioanalytical Conference, titled ‘Cutting-Edge Bioanalytical Technologies and Concepts - Issues, Solutions and Practical Considerations for Applications in Novel and Emerging Modalities’, was held 10–13 July 2017 in Madison, WI, USA. This Land O'Lakes Conference is presented each year by the Division of Pharmacy Professional Development within the School of Pharmacy at the University of Wisconsin-Madison (USA). The purpose of this conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. This report summarized the presentations at the 18th Annual Conference.

Aim: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7β-hydroxy-DHEA, cortisone and 6β-hydroxycortisone as potential biomarkers to predict CYP3A activity. Results: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7β-hydroxy-DHEA. Conclusion: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug–drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

Aims: A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. Materials & methods: An average blood volume of 10.6 μl was absorbed by the samplers across the different HCTs investigated (20–65%). Results: No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. Conclusion: The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

The University of Wisconsin bioanalytical conference is presented each year by the Extension Services in Pharmacy, the professional development department within the School of Pharmacy. The purpose of this 3-day conference was to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference was designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference the program was composed of a mixture of lectures, interactive discussions, poster sessions and roundtables. This paper summarizes the presentations at the Fourteenth Annual Conference, offered in a new venue.

Background: Increasing assay sensitivity is critical for promoting the application of LC–MS/MS quantitative bioanalysis of therapeutic proteins. A sample processing method that can selectively remove the abundant background peptides in the serum tryptic digests and retain the target peptides can greatly improve the assay sensitivity. Results: Mixed-mode strong-cation exchange SPE was systematically investigated as an orthogonal sample separation technique to reversed-phase UHPLC for the analysis of a test monoclonal antibody, BMS-986012, in monkey serum after pellet digestion. Strong cation exchange SPE efficiently removed most of the background peptides and reduced the matrix effect and background level in the monitored mass transition channels. As a result, improved sensitivity was observed for the surrogate peptides VVSV and SLIY. Conclusion: This orthogonal approach provides a simple and easy-to-develop sample preparation method that can selectively remove most background peptides and extract the target peptides, therefore, improving the LC–MS/MS assay sensitivity.

The paradigm shift of globalized research and conducting clinical studies at different geographic locations worldwide to access broader patient populations has resulted in increased need of correlating bioanalytical results generated in multiple laboratories, often across national borders. Cross-validations of bioanalytical methods are often implemented to assure the equivalency of the bioanalytical results is demonstrated. Regulatory agencies, such as the US FDA and European Medicines Agency, have included the requirement of cross-validations in their respective bioanalytical validation guidance and guidelines. While those documents provide high-level expectations, the detailed implementation is at the discretion of each individual organization. At Bristol-Myers Squibb, we practice a fit-for-purpose approach for conducting cross-validations for small-molecule bioanalytical methods using LC–MS/MS. A step-by-step proposal on the overall strategy, procedures and technical details for conducting a successful cross-validation is presented herein. A case study utilizing the proposed cross-validation approach to rule out method variability as the potential cause for high variance observed in PK studies is also presented.

Aim: Serum 7α-hydroxy-cholesten-3-one (C4) has been reported as a biomarker to assess CYP7A1 enzyme activity and bile acid synthesis. To support a clinical program, a sensitive and reliable assay without derivatization was required for the analysis of C4 in human serum. Methodology & results: A systematic approach was used to optimize mass spectrometry, LC and sample extraction conditions, therefore, significantly improved assay sensitivity, and achieved the required quantification limit without derivatization. A surrogate matrix approach was used to overcome the interference from endogenous C4. A stable isotope-labeled C4 was used as internal standard. The samples were extracted using a simple protein precipitation method with 2% formic acid in acetonitrile. Conclusion: A simple, fast, sensitive and robust UHPLC-MS/MS method for the quantification of 0.50 ng/ml C4 in 100 µl human serum was developed and fit for purpose validated. The method was successfully applied to the bioanalysis of C4 in a clinical study.

Background: Rapidly developing LC–MS/MS assays with high-throughput and quality are challenging yet desired. Methodology & results: A simple method development approach was reported and demonstrated with the quantitative bioanalysis of BMS-984478, a hepatitis C virus nonstructural protein 5A inhibitor. An accurate, precise and robust LC–MS/MS method for the quantitation of BMS-984478 in rat and monkey plasma was developed and validated. Incurred sample reanalysis evaluation passed with 100% of samples meeting the acceptance criteria. The validated assay was successfully applied in toxicology studies without any failed runs. Conclusion: The approach was successfully applied to the bioanalysis of BMS-984478 in toxicology and clinical studies. This approach was shown to be effective and reliable in speeding the development of high-throughput and reliable LC–MS/MS assays.

Distribution of drugs into tissues is an important determinant of the overall PK and PD profile. Thus, bioanalysis of drugs and their metabolites in tissues can play an important role in understanding the pharmacological and toxicological properties of new drug candidates. Unlike liquid matrices, bioanalysis in tissues offers unique challenges such as proper tissue sampling, appropriate tissue sample preparation, efficient extraction of the analytes from the tissue homogenates, and demonstration of stability and recovery of analytes in intact tissues. This article provides a systematic review of tissue sample analysis for small molecules using LC–MS/MS. The authors provide rationale for tissue sample analysis, and discuss strategies for method development, method qualification or validation, and sample analysis. Unique aspects of method development and qualification/validation are highlighted based on authors’ direct experiences and literature summary. Analysis using intact tissue samples such as MALDI imaging is also briefly discussed.

This University of Wisconsin School of Pharmacy bioanalytical conference is presented each year by the Extension Services in Pharmacy, the professional development department within the School. The purpose of this 4-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program was a mixture of lectures, poster sessions, round table discussions and workshops. This article summarizes the presentations at the 13th Annual Conference.

This University of Wisconsin School of Pharmacy bioanalytical conference is presented each year by the Extension Services in Pharmacy, the professional development department within the school. The purpose of this 4-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program was a mixture of lectures, poster sessions, round table discussions and workshops. This article summarizes the presentations at the 12th Annual Conference.

This University of Wisconsin School of Pharmacy Bioanalytical Conference is arranged by the Extension Services in Pharmacy, the professional development department within the School. The purpose of this 4 day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program was a mixture of lectures, poster sessions, round-table discussions and workshops. This paper summarizes the presentations at the Eleventh Annual Conference.

Representing the first industry-led workshop on this topic, the Drug Information Association (DIA) and Pharmaceutical Research and Manufacturers of America (PhRMA) cosponsored a workshop on dried blood spots (DBS) sampling in the pharmaceutical industry. Sampling from DBS on specialty papers is a novel methodology in the area of pharmacokinetics and toxicokinetics that serves to replace conventional plasma analysis for pharmaceutical development. As the awareness around this technology has increased exponentially in recent years, this 1-day symposium aimed to share current best practices for DBS use and implementation, sample collection, handling and associated bioanalysis. The event drew over 150 industry and agency participants; an impressive attendance for a relatively new technology within the industry. Overall, the day was divided into six separate technical sessions plus poster sessions, and included concurrent breakout sessions dedicated to three key areas surrounding DBS implementation: bioanalytical, toxicology/safety assessment and clinical/pharmacokinetics.

This conference was arranged by the Extension Services in Pharmacy at the University of Wisconsin School of Pharmacy. The purpose of this annual 4-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics and metabolites in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program will be a mixture of lectures, poster sessions, round table discussions and workshops. This paper summarizes the presentations at the Tenth Annual Conference.