Preanalytical approaches to improve recovery of amyloid-β peptides from CSF as measured by immunological or mass spectrometry-based assays.

Background: Amyloid-β 1-42 (Aβ) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aβ are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aβ recovery and quantification accuracy.

2017 White Paper on recent issues in bioanalysis: aren't BMV guidance/guidelines 'Scientific'? (Part 1 - LCMS: small molecules, peptides and small molecule biomarkers).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches.

Quantitation of levodopa and carbidopa in rat plasma by LC-MS/MS: The key role of ion-pairing reversed-phase chromatography.

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC-MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV) (Part 1 - small molecules, peptides and small molecule biomarkers by LCMS).

The 2016 10 Workshop on Recent Issues in Bioanalysis (10 WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity.

Validation of a biotherapeutic immunoaffinity-LC-MS/MS assay in monkey serum: 'plug-and-play' across seven molecules.

Background: Biotherapeutics development requires validated assays in biological matrices for pharmacokinetic assessment. Historically, ligand-binding assays have been the predominant platform available. Recently, alternative hybrid methods, combining ligand-binding analyte enrichment with LC-MS detection have emerged.

CSF Aβ - an excellent but complicated Alzheimer's biomarker - a route to standardisation.

The 42 amino acid form of amyloid β (Aβ-) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimer's disease. Several immunoassays for CSF Aβ are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

Aim: There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development.

Results: Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects.

Development and validation of HILIC-ESI/MS/MS methods for simultaneous quantitation of several antipsychotics in human plasma and blood.

Background: Knowledge of antipsychotic drug levels at point of care (POC) may significantly aid therapeutic decision-making. To support the development of future POC devices and to validate the use of fingerstick capillary blood sampling, two robust hydrophilic interaction LC-ESI/MS/MS methods were developed and validated. Two PK studies were completed evaluating the correlation between fingerstick blood and plasma concentrations with corresponding venous blood and plasma concentrations for several commonly prescribed atypical antipsychotics and selected metabolites.

Ultrasensitive sub-pg/ml determination of tiotropium bromide in human plasma by 2D-UHPLC-MS/MS: challenges and solutions.

Background: To adequately support pharmacokinetic evaluations of tiotropium bromide in planned clinical studies. It was desirable to measure it with a LLOQ of sub pg/ml.

Results: A sensitive bioanalytical method for the determination of tiotropium in human plasma sample was successfully developed and validated in the range of 0.

Comparison of a stable isotope labeled (SIL) peptide and an extended SIL peptide as internal standards to track digestion variability of an unstable signature peptide during quantification of a cancer biomarker, human osteopontin, from plasma using capillary microflow LC-MS/MS.

Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide 'GDSVVYGLR' was used as a signature peptide for this method.

Scientific or regulated validation: a tiered approach? Meeting report from a joint EBF/DVDMDG workshop.

Tiered approach is rapidly gaining interest in the regulated bioanalytical community. Alternative approaches to the workflows as proposed in the regulatory Guidance (US FDA, EMA) are being used in discovery and early drug development, but with a growing array of assay types and studies requiring bioanalytical support in early drug development, the bioanalytical community is discussing how to bring best value to support these studies. Recently, international industry groups like European Bioanalysis Forum and Global Bioanalysis Consortium have discussed and published on the opportunity and need to include tiered approach more systematically in the early drug development support.

Round robin test on quantification of amyloid-β 1-42 in cerebrospinal fluid by mass spectrometry.

Introduction: Cerebrospinal fluid (CSF) amyloid-β 1-42 (Aβ42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative.

Bioanalytical method validation considerations for LC-MS/MS assays of therapeutic proteins.

This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC-MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC-MS/MS-based assays where proteins are quantified using the LC-MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC-MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria.

Workshop Report: AAPS Workshop on Method Development, Validation, and Troubleshooting of Ligand-Binding Assays in the Regulated Environment.

A novel format was introduced at the recent AAPS NBC Workshop on Method Development, Validation and Troubleshooting in San Diego on 18th May 2014. The workshop format was initiated by Binodh De Silva; Marie Rock and Sherri Dudal joined the initiative to develop and chair the workshop. Questions were solicited by a variety of avenues, including a Linked-In Discussion Group.

An extended stable isotope-labeled signature peptide internal standard for tracking immunocapture of human plasma osteopontin for LC-MS/MS quantification.

A stable isotope-labeled signature peptide, whose sequence corresponds to the human osteopontin (hOPN) specific antibody epitope, was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity-coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well from 150 to 4500 ng and analysis was carried out with internal standards added before and after the immunocapture step. The immunocapture variability ranged from -80.

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites.

Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics.

This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion.

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science.

Development and optimization of on-line 2-dimensional chromatographic approaches for eliminating matrix effects and improving bioanalysis of peptides in human plasma using UHPLC-MS/MS.

Online 2-dimensional chromatographic approaches for eliminating matrix effects and optimizing bioanalysis of peptides using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were studied. Three therapeutic peptides (octreotide, desmopressin, and vasopressin) were selected as model analytes. Human plasma was precipitated with acetonitrile; peptides were analyzed on C(8), C(18), Phenyl and HILIC ACQUITY UPLC columns.

Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies.

Background: There is a need for general and reliable LC-MS assays capable of supporting the bioanalysis of a variety of human monoclonal antibody-based therapeutic drug candidates in animal PK/TK studies.

Results: We present herein improvements in our previously reported universal peptide approach to the bioanalysis of human monoclonal antibody protein drug candidates in animal studies. These improvements include incorporation of a second, light chain-based universal peptide into the assay, thus introducing the concept of a dual universal peptide assay; and incorporation of a universal stable isotope-labeled monoclonal antibody into the assay.

Investigation of endogenous blood lipids components that contribute to matrix effects in dried blood spot samples by liquid chromatography-tandem mass spectrometry.

Dried blood spot (DBS) sampling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a rapidly developing approach in the field of biopharmaceutical analysis. DBS sampling enables analysis of small sample volumes with high sensitivity and selectivity while providing a convenient easy to store and ship format. Lipid components that may be extracted during biological sample processing may result in matrix ionization effects and can significantly affect the precision and accuracy of the results.

Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.