Circulating microRNAs as promising testicular translatable safety biomarkers: current state and future perspectives.

Drug-induced testicular injury (DITI) is one of the often-observed and challenging safety issues seen during drug development. Semen analysis and circulating hormones currently utilized have significant gaps in their ability to detect testicular damage accurately. In addition, no biomarkers enable a mechanistic understanding of the damage to the different regions of the testis, such as seminiferous tubules, Sertoli, and Leydig cells.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs ( - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.

Identification of Glycochenodeoxycholate 3-O-Glucuronide and Glycodeoxycholate 3-O-Glucuronide as Highly Sensitive and Specific OATP1B1 Biomarkers.

The aim of this study was to investigate the sensitivity and specificity of endogenous glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) as substrates for organic anion transporting polypeptide 1B1 (OATP1B1) in humans. We measured fasting levels of plasma GCDCA-3G and GDCA-3G using liquid chromatography-tandem mass spectrometry in 356 healthy volunteers. The mean plasma levels of both compounds were ~ 50% lower in women than in men (P = 2.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K.

A Multiplexed HILIC-MS/HRMS Assay for the Assessment of Transporter Inhibition Biomarkers in Phase I Clinical Trials: Isobutyryl-Carnitine as an Organic Cation Transporter (OCT1) Biomarker.

There is a growing interest in using endogenous compounds as drug transporter biomarkers to facilitate drug-drug interaction (DDI) risk assessment in early phase I clinical trials. Compared to other drug transporters, however, no valid biomarker for hepatic organic cation transporter (OCT) 1 has been described to date. The present work represents the first report of an endogenous compound, isobutyryl-l-carnitine (IBC), as a potential clinical OCT1 biomarker for DDI assessment.

Evaluation of OptiFlow™-MS/MS for bioanalysis of pharmaceutical drugs and metabolites.

Microflow tandem mass spectrometry-based methods have been proposed as options to improve sensitivity and selectivity while improving sample utility and solvent consumption. Here, we evaluate a newly introduced microflow source, OptiFlow™, for quantitative performance. We performed a comparison of the OptiFlow and IonDrive™ sources, respectively, on the same triple quadrupole mass spectrometer.

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. 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 bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches.

Dose-Dependent Inhibition of OATP1B by Rifampicin in Healthy Volunteers: Comprehensive Evaluation of Candidate Biomarkers and OATP1B Probe Drugs.

To address the most appropriate endogenous biomarker for drug-drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate-3-glucuronide, glycochenodeoxycholate-3-sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose-dependent change in area under the plasma concentration-time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR vs.

2018 White Paper on Recent Issues in Bioanalysis: 'A global bioanalytical community perspective on last decade of incurred samples reanalysis (ISR)' (Part 1 - small molecule regulated bioanalysis, small molecule biomarkers, peptides & oligonucleotide bioanalysis).

The 2018 12 Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in 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 bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches.

LC-MS/MS assay for N-methylnicotinamide in humans, an endogenous probe for renal transporters.

Background: N-methylnicotinamide (1-NMN) has been proposed as a potential clinical biomarker to assess drug-drug interactions involving organic cation transporters (OCT2) and multidrug and toxin extrusion protein transporters.

Results: A hydrophilic interaction liquid chromatography-MS/MS assay, to quantify 1-NMN, in human plasma and urine is reported.

Materials & Methods: A hydrophilic interaction chromatography (HILIC)-tandem mass spectrometry (MS/MS) assay to quantify 1-NMN in human plasma and urine is reported.

A multiplex HRMS assay for quantifying selected human plasma bile acids as candidate OATP biomarkers.

Aim: Selected bile acids (BAs) in plasma have been proposed as endogenous probes for assessing drug-drug interactions involving hepatic drug transporters such as the organic anion-transporting polypeptides (OATP1B1 and OATP1B3).

Materials & Methods: Plasma extracts were analyzed for selected BAs using a triple TOF API6600 high-resolution mass spectrometer.

Results: Glycodeoxycholic acid 3-sulfate, glycochenodeoxycholic acid 3-sulfate, glycodeoxycholic acid 3-O-β-glucuronide and glycochenodeoxycholic acid 3-O-β-glucuronide are presented as potential OATP1B1/3 biomarkers.

HPLC-high-resolution mass spectrometry with polarity switching for increasing throughput of human in vitro cocktail drug-drug interaction assay.

Aim: Evaluation of HPLC-high-resolution mass spectrometry (HPLC-HRMS) full scan with polarity switching for increasing throughput of human in vitro cocktail drug-drug interaction assay.

Materials & Methods: Microsomal incubates were analyzed using a high resolution and high mass accuracy Q-Exactive mass spectrometer to collect integrated qualitative and quantitative (qual/quant) data.

Results: Within assay, positive-to-negative polarity switching HPLC-HRMS method allowed quantification of eight and two probe compounds in the positive and negative ionization modes, respectively, while monitoring for LOR and its metabolites.

A fully automated and validated human plasma LC-MS/MS assay for endogenous OATP biomarkers coproporphyrin-I and coproporphyrin-III.

Aim: A validated LC-MS/MS assay for the quantitation of coproporphyrin-I and -III (CP-I, CP-III) in human plasma has been developed to understand the utility of both as possible endogenous biomarkers for organic anion-transporting polypeptides (OATP)-mediated drug-drug interactions (DDIs).

Materials And Methods:  Human plasma extracts were analyzed for CP-I and CP-III using a Sciex API 6500+ mass spectrometer. Results: The assay was utilized for plasma samples from a clinical DDI study involving a new chemical entity that presented as an OATP inhibitor in vitro.

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.

A highly selective and sensitive LC-MS/HRMS assay for quantifying coproporphyrins as organic anion-transporting peptide biomarkers.

Aim: Coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) in plasma and urine have been proposed as biomarkers for assessing drug-drug interactions involving hepatic drug transporters such as organic anion-transporting peptides (OATP), 1B1 and 1B3. Materials & methods: Plasma and urine extracts were analyzed for CP-I/CP-III using a TripleTOF API6600 mass spectrometer. Results: Previously unreported, CP-I/CP-III doubly charged ions (m/z 328.

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.

Unconjugated payload quantification and DAR characterization of antibody-drug conjugates using high-resolution MS.

Aim: The application of high-resolution MS to antibody-drug conjugate (ADC) drug development may provide insight into their safety and efficacy. Quantification of unconjugated cytotoxic drug (payload) and characterization of drug-to-antibody ratio distribution were determined in plasma using orthogonal acceleration quadrupole-time-of-flight MS.

Results: Unconjugated payload quantification determined by quadrupole-time-of-flight-based MRM(highresolution) and triple quadrupole-based multiple reaction monitoring were comparable and achieved detection limits of 0.

Advances in high-resolution MS and hepatocyte models solve a long-standing metabolism challenge: the loratadine story.

Loratadine (LOR, Claritin(®)) is a long-acting antihistamine used to treat allergic rhinitis. The major active human metabolite, desloratadine (DL, Clarinex(®)), is extensively metabolized to 3-hydroxydesloratadine (3-OH-DL) (M40) and subsequently glucuronidated before elimination. This study revealed the ability of a novel, long-term hepatocyte micropatterned co-culture (MPCC) model to generate in vivo metabolites.

Integrated quantitative and qualitative workflow for in vivo bioanalytical support in drug discovery using hybrid Q-TOF-MS.

Background: UHPLC coupled with orthogonal acceleration hybrid quadrupole-TOF (Q-TOF)-MS is an emerging technique offering new strategies for the efficient screening of new chemical entities and related molecules at the early discovery stage within the pharmaceutical industry. In the first part of this article, we examine the main instrumental parameters that are critical for the integration of UHPLC-Q-TOF technology to existing bioanalytical workflows, in order to provide simultaneous quantitative and qualitative bioanalysis of samples generated following in vivo studies.

Material & Methods: Three modern Q-TOF mass spectrometers, including Bruker maXis™, Agilent 6540 and Sciex TripleTOF™ 5600, all interfaced with UHPLC systems, are evaluated in the second part of the article.

It is time for a paradigm shift in drug discovery bioanalysis: from SRM to HRMS.

It can be argued that the last true paradigm shift in the bioanalytical (BA) arena was the shift from high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection to HPLC with tandem mass spectrometry (MS/MS) detection after the commercialization of the triple quadrupole mass spectrometer in the 1990s. HPLC-MS/MS analysis based on selected reaction monitoring (SRM) has become the gold standard for BA assays and is used by all the major pharmaceutical companies for the quantitative analysis of new drug entities (NCEs) as part of the new drug discovery and development process. While LC-MS/MS continues to be the best tool for drug discovery bioanalysis, a new paradigm involving high-resolution mass spectrometry (HRMS) and ultrahigh-pressure liquid chromatography (uHPLC) is starting to make inroads into the pharmaceutical industry.

Novel MS solutions inspired by MIST.

To improve patient safety and to help avoid costly late-stage failures, the pharmaceutical industry, along with the US FDA and International Committee on Harmonization (ICH), recommends the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug-development process. LC-MS is the technique of choice for detection and characterization of metabolites, however, the widely different LC-MS response observed for a new chemical entity (NCE) and its structurally related metabolites limits the direct use of LC-MS responses for quantitative determination of NCEs and metabolites. While no method provides completely accurate universal response, UV, corona charged aerosol detection (CAD), radioactivity, NMR and low-flow (< 20 µl/min) nanospray approaches provide opportunities to quantify metabolites in the absence of reference standards or radiolabeled material with enough precision to meet the needs of early clinical development.

Conference report: Inaugural Applied Pharmaceutical Analysis: India 2010 conference.

The inaugural Applied Pharmaceutical Analysis-India (APA-India) conference was held between 21 and 24 February 2010 in Hyderabad, India. The theme of the 2010 APA-India meeting was "The best of bioanalytical science in India: the role of bioanalysis and absorption, distribution, metabolism and excretion in translational medicine". The conference brought together scientists from across India and the rest of the world to stimulate discussion in the areas of discovery bioanalysis, new technologies, regulated bioanalysis and biotransformation, as applied to pharmaceutical analysis in India, the USA and other parts of the world.