Aim: The purpose of this work was to determine the feasibility of supporting a clinical microdose study for PF-06882961 (danuglipron), an oral small molecule agonist of the GLP-1 receptor, by LC-MS/MS. Methodology: Statistical instrument parameter optimization using response surface methodology was employed to develop a LC-MS/MS method for the analyte, PF-06882961. Results: An LC-MS/MS method was developed and validated to support a proof of concept microdose pharmacokinetics preclinical study in monkeys, administered PF-06882961 (0.005 mg total, average dose = 0.0007 mg/kg) via intravenous bolus injection. Conclusion: The present study demonstrated the feasibility of analyzing human microdose plasma samples for PF-06882961 by LC-MS/MS, instead of accelerator mass spectrometry, thereby reducing cost and eliminating synthesis and exposure to 14C labeled material.
[Box: see text].

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on \"EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations\" and on \"US FDA/OSIS Remote Regulatory Assessments (RRAs)\" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.

Managing ocular microbial infections typically requires pharmacotherapy using antibiotic eye drops, such as moxifloxacin hydrochloride (MFX), combined with an antifungal agent like amphotericin B (AB). We carried out and validated an LC-MS/MS assay to quantify these compounds in rabbit tear fluid in order to look into the pharmacokinetics of these two drugs. We employed a protein precipitation technique for the extraction of drugs under examination. A Waters Symmetry C18 column was used to separate the analytes and internal standard. The composition of the mobile phase was like (A) 0.1% v/v formic acid in water and (B) methanol. The detection of MFX and AB was accomplished through the utilization of positive ion electrospray ionization under multiple reaction monitoring mode. The linearity curves for both analytes exhibited an acceptable trendline across a concentration range of 2.34-300 ng/mL for MFX and 7.81-1000 ng/mL for AB in surrogate rabbit tear fluid. The lower limit of quantitation for MFX was 2.34 ng/mL, while for AB, it was 7.81 ng/mL. The approach was strictly validated, encompassing tests of selectivity, linearity (with r2 > 0.99), precision, accuracy, matrix effects, and stability. Consequently, we employed this method to evaluate the pharmacokinetics profiles of MFX and AB in rabbit tear fluid following single topical doses.

UNASSIGNED: A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for estimation of bedaquiline (BDQ) in human plasma using the deuterated analogue of the analyte, bedaquiline-d6 (BDQ-d6) as the internal standard.
UNASSIGNED: The plasma sample of 50 µL was extracted by liquid-liquid extraction using methyl tertiary butyl ether (MTBE). The separation was achieved on Zodiac C18 (50 x 4.6 mm, 5 µm) column with a mobile phase consisting of methanol and 5 mM ammonium formate in 0.1 % formic acid (w/v) (90:10, v/v) at a flow rate of 1.0 mL/min. Protonated analyte and internal standard were detected on a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) mode.
UNASSIGNED: The linearity of the method was established in the concentration range of 5---1800 ng/mL with correlation coefficient, r2 ≥ 0.99. All the validated parameters were found well within the limits.
UNASSIGNED: The method was applied for the first time to evaluate the pharmacokinetic parameters after single oral dose of BDQ 100 mg under fed conditions in healthy human volunteers, and the results were further authenticated by incurred sample reanalysis.

Approximately 25% of breast cancer patients with HER2 overexpression tend to have a high risk of disease progression and death. Various HER2-targeting therapies have been approved for treatment. Recently, a novel antibody-drug conjugate, SHR-A1201, is being researched and developed. For the pharmacokinetic study of SHR-A1201, suitable bioanalytical methods are needed for quantifying unconjugated cytotoxin, cytotoxin-conjugated antibodies and total antibodies. In this research, bioanalytical methods involving a highly sensitive LC-MS/MS assay for unconjugated cytotoxic payload DM1 in human plasma, ELISA strategies for DM1-conjugated trastuzumab and total trastuzumab in human serum were developed, validated and successfully applied to a phase I dose-escalation pharmacokinetic study of SHR-A1201. The pharmacokinetic properties and exposure-to-dose proportionality was evaluated for SHR-A1201. According to the bioanalytical method validation guidance, the bioanalytical methods were fully validated and the validation results met the acceptance criteria. The nonspecific binding of DM1 and dimer was avoided for the LC-MS/MS assay. In the dose-escalation pharmacokinetic study of SHR-A1201, a potential dose-proportional pharmacokinetics was observed over the dose from 1.2 mg/kg to 4.8 mg/kg. The validated bioanalytical strategies are robust and reproducible and these bioanalytical methods will contribute to better understanding of the pharmacokinetic properties of SHR-A1201.

Opnurasib (JDQ-443) is a highly potent and promising KRASG12C inhibitor that is currently under clinical investigation. Results of the ongoing clinical research demonstrated the acceptable safety profile and clinical activity of this drug candidate as a single agent for patients with NSCLC harboring KRASG12C mutations. In this early stage of development, a deeper insight into pharmacokinetic properties in both preclinical and clinical investigations of this drug is very important. Thus, a reliable quantification method is required. To date, no quantitative bioanalytical assay of opnurasib was publicly available. In this study we present a validated assay to quantify opnurasib in mouse plasma and eight mouse tissue-related matrices utilizing liquid chromatography-tandem mass spectrometry. Erlotinib was used as internal standard and acetonitrile was utilized to treat 10 µl of the sample with protein precipitation in a 96-well plate format. Separation and detection were achieved using a BEH C18 column under basic chromatographic conditions and a triple quadrupole mass spectrometer, respectively. We have fully validated this assay for mouse plasma and partially for eight mouse tissue-related matrices over the range of 2-2000 ng/ml. The accuracy and precision of the assay fulfilled international guidelines (EMA & U.S. FDA) over the validated range. The method was proven selective and sensitive to quantify opnurasib down to 2 ng/ml in all investigated matrices. The recoveries of both analyte and internal standard in mouse plasma were ∼100 % with no significant matrix effect in any of the matrices. Opnurasib in mouse plasma was stable up to 12 h at room temperature, and up to 8 h at room temperature in tissue homogenates (except for kidney up to 4 h). This presented method has been successfully applied to quantify opnurasib in preclinical samples from a mouse study and demonstrated its usability to support preclinical pharmacokinetic studies.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies covalently bound to cytotoxic drugs by a linker. They are designed to selectively bind target antigens and present a promising cancer treatment without the debilitating side effects of conventional chemotherapies. Ado-trastuzumab emtansine (T-DM1) is an ADC that received US FDA approval for the treatment of HER2-positive breast cancer. The purpose of this study was to optimize methods for the quantification of T-DM1 in rats. We optimized four analytical methods: (1) an enzyme-linked immunosorbent assay (ELISA) to quantify the total trastuzumab levels in all drug-to-antibody ratios (DARs), including DAR 0; (2) an ELISA to quantify the conjugated trastuzumab levels in all DARs except DAR 0; (3) an LC-MS/MS analysis to quantify the levels of released DM1; and (4) a bridging ELISA to quantify the level of anti-drug antibodies (ADAs) of T-DM1. We analyzed serum and plasma samples from rats injected intravenously with T-DM1 (20 mg/kg, single dose) using these optimized methods. Based on these applied analytical methods, we evaluated the quantification, pharmacokinetics, and immunogenicity of T-DM1. This study establishes the systematic bioanalysis of ADCs with validated assays, including drug stability in matrix and ADA assay, for future investigation on the efficacy and safety of ADC development.

The term \"bioanalytical\" encompasses a much greater breadth of analytical deliverables than ever before. Circulating drug concentration data are complemented by experimental evidence of drug in biophase, immunogenicity, target engagement and subsequent pathway modulation. Many bioanalytical assays bridge the traditional divide across discovery and development. Our approach is the Bioanalytical Hub model bringing together a wide breadth of bioanalytical support (GxP and non-GxP), multiple end points (pharmacokinetics, anti-drug antibodies and biomarkers) and analytical platforms (LC/MS, immunoassay, flow cytometry, genomics, immunohistochemistry) onto a common lab footprint. This maximizes instrument utilization, facilitates workforce agility and enhances data interpretation capability while reducing the number of hand-offs as assays evolve from their origins as exploratory end points to fully characterized to support primary and secondary end points.

Cefepime (CEF) is a cephalosporin and can be administered in secondary peritonitis together with metronidazole to treat sepsis. This study aimed to develop and validate an LC-ESI-QTOF-MS method for the quantification of cefepime in the plasma and peritoneal microdialysate of healthy Wistar rats. Chromatographic separation was performed using a CLC-ODS C18 column (250 × 4.6 mm), a C18 pre-column (4 mm, 5 μm) and isocratic elution. Gallic acid was used as the internal standard. The mobile phase consisted of (A) ultrapure water (pH adjusted to 3.5) and (B) acetonitrile (80:20, v/v) at 0.8 ml/min. Quantification was performed using a mass spectrometer with electrospray ionization in positive mode to monitor ions with m/z 481.1322 (CEF) and m/z 171.0288 (IS). The method was validated for selectivity, precision, accuracy, linearity, stability, lower limit of quantification, carryover, recovery and matrix effect. Calibration was done in the ranges 1-40 and 1-100 μg/ml for the peritoneal microdialysate and plasma, respectively. Plasma extraction recovery ranged from 93.9 to 99.9%. The technique was validated and successfully applied in a pilot pharmacokinetic study for estimating the free concentration of CEF in the peritoneal microdialysate of rats for the first time.

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of procyclidine hydrochloride in human plasma using Procyclidine D11 hydrochloride as internal standard. Liquid-liquid extraction technique with methyl tertiary butyl ether was used for the extraction of plasma samples. Chromatographic separation of the analyte and the internal standard from the endogenous components was done on Zodiac C18 column (50 × 4.6 mm, 5 µm) using a mixture of methanol and 0.1% formic acid in water (70:30, v/v) as mobile phase at a flow rate of 1 mL/min with the run time of 2 min. The detection of the eluents was done using multiple reaction monitoring (MRM) in positive ion mode. Linearity of the method was established in the concentration range of 0.5 to 120 ng/mL. Full validation of the method was done as per USFDA guidelines and the results were well within the acceptance limits. The successful application of the method was done on healthy human subjects under fasting conditions, proving it to be used for bioequivalence and bioavailability (BA/BE) studies of procyclidine.