Endogenous plasma riboflavin is not a viable BCRP biomarker in human.

Recent reports suggest that plasma riboflavin may serve as a biomarker for BCRP inhibition in humans. However, the clinical data supporting this claim have been limited, with only two studies showing modest increases in riboflavin levels after administration of a BCRP inhibitor. We have recently demonstrated that co-administration of 375 mg once daily (q.

Rapid LC-MS/MS Evaluation of Collagen and Elastin Crosslinks in Human and Mouse Lung Tissue with a Novel Bioanalytical Surrogate Matrix Approach.

Alterations to post-translational crosslinking modifications in the extracellular matrix (ECM) are known to drive the pathogenesis of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Thus, the methodology for measuring crosslinking dynamics is valuable for understanding disease progression. The existing crosslinking analysis sample preparation and liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are typically labor-intensive and time-consuming which limits throughput.

High throughput bioanalysis of serum 7a-hydroxy-4-cholesten-3-one (C4) using LC-MS/MS: Devising an end-to-end single vial solution for a sample limited application.

Crohn's disease (CD) is characterized by chronic ileal/ileocolonic inflammation, and in some cases, can result in bile acid malabsorption (BAM) and subsequent bile acid diarrhea (BAD). Although BAD is common in CD, diagnosis is difficult. In patients with CD who had ileal resection (IR), elevated serum 7a-hydroxy-4-cholesten-3-one (C4), a cholesterol-derived stable intermediate in bile acid synthesis, is associated with diarrhea attributable to BAM and therefore, may have diagnostic utility.

High-Throughput SFC-MS/MS Method to Measure EPSA and Predict Human Permeability.

Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability.

Signature peptide selection workflow for biomarker quantification using LC-MS-based targeted proteomics.

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.

Integrity and efficiency: AbbVie's journey of building an integrated nonregulated bioanalytical laboratory.

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.

Use of in-sample calibration curve approach for quantification of peptides with high-resolution mass spectrometry.

Rationale: The in-sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked-in stable isotope labeled internal standard (SIL-IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high-resolution mass spectrometers, quantitation on high-resolution mass spectrometer with product ions has not been tested.

Comparison of three parallelism assessment methods of biomarker quantification by LC-MS/MS: a case study of the bioanalysis of creatinine in human urine samples.

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. 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.

Fit-for-purpose LC-MS/MS quantification of leukotoxin and leukotoxin diol in mouse plasma without sample pre-concentration.

LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes.

A bridging immunogenicity assay for anti-cabiralizumab antibodies: overcoming the low assay cut point and drug tolerance challenges.

To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. 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.

Land O'Lakes Workshop on Microsampling: Enabling Broader Adoption.

The microsampling workshop generated recommendations pertaining to blood sampling site (venous blood versus capillary blood), when to conduct a bridging study, statistical approaches to establish correlation/concordance and deciding on sample size, opportunities and challenges with patient-centric sampling, and how microsampling technology can enrich clinical drug development. Overall, the goal was to provide clarity and recommendations and enable the broader adoption of microsampling supporting patients' needs, convenience, and the transformation from clinic-centric to patient-centric drug development. The need and adoption of away-from-clinic sampling techniques has become critical to maintain patient safety during the current COVID-19 pandemic.

Challenges and recommendations in developing LC-MS/MS bioanalytical assays of labile glucuronides and parent compounds in the presence of glucuronide metabolites.

Glucuronides, especially acyl glucuronides, were often found to be unstable and 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.

Integration of Acoustic Liquid Handling into Quantitative Analysis of Biological Matrix Samples.

Acoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution.

Evaluation of correlation between bioanalytical methods.

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.

Investigation on the Effect of Capillary Microsampling on Hematologic and Toxicokinetic Evaluation in Regulatory Safety Studies in Mice.

Microsampling techniques enable the minimization of blood collection volume from animals and subsequent handling of the blood samples or their derived plasma or serum samples. This offers advantages over conventional large-volume sampling, such as eliminating the need for satellite animals and improving animal welfare aspects, and providing the opportunity for additional assessments in small animals where blood volume constraints limit endpoints. This study evaluated the feasibility of implementation of capillary microsampling (CMS) in a single-dose study in mice with the ultimate goal of enabling its use in toxicology studies.

Application of in-sample calibration curve methodology for regulated bioanalysis: Critical considerations in method development, validation and sample analysis.

Traditionally, for a liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC-MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification.

A Fit-for-Purpose Method for the Detection of Human Antibodies to Surface-Exposed Components of BMS-986263, a Lipid Nanoparticle-Based Drug Product Containing a siRNA Drug Substance.

ND-L02-s0201/BMS-986263 is a lipid nanoparticle (LNP) drug product containing a heat shock protein 47 (HSP47)-specific small interfering ribonucleic acid (siRNA) and being developed for the treatment of liver and idiopathic pulmonary fibrosis. To address immunogenicity-related issues, we developed a robust, fit-for-purpose (FFP) three-tier electrochemiluminescent (ECL) anti-drug antibody (ADA) assay for the detection of antibodies (Abs) generated to surface-exposed components of BMS-986263. The drug was coated directly on plates, and several Abs specific for polyethylene glycol (PEG) and other surface components were tested for use as positive quality controls (QCs).

A practical workflow for capillary microsampling in nonclinical studies.

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. 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.

A convenient strategy to overcome interference in LC-MS/MS analysis: Application in a microdose absolute bioavailability study.

Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes.

Development of a Chemiluminescent ELISA Method for the Detection of Total Anti-Adeno Associated Virus Serotype 9 (AAV9) Antibodies.

Recombinant adeno associated viruses (rAAV) have become an important tool for the delivery of gene therapeutics due to long-standing safety and success in clinical trials. Since humans often become exposed to AAVs and develop anti-AAV antibodies (Abs), a potential impediment to the success of gene therapeutics is neutralization of the viral particle before it has had a chance to bind and enter target cells to release the transgene. Identification of subjects with preexisting Abs having neutralizing potential, and exclusion of such subjects from clinical studies is expected to enhance drug efficacy.

Overcoming the stability, solubility and extraction challenges in reversed-phase UHPLC-MS/MS bioanalysis of a phosphate drug and its prodrug in blood lysate.

BMS-986104 is a S1PR modulator drug candidate under development and has been evaluated in Phase I clinical trials. BMS-986104 functions as a prodrug and undergoes enzymatic transformations in vivo to form the pharmacologically active phosphate drug, BMS-986104-P. Here, we report approaches to overcome the stability, solubility and extraction challenges in developing a sensitive, accurate and rugged LC-MS/MS method for the simultaneous quantification of the phosphate drug and its prodrug in blood lysate.

Report on the 18th Annual Land O'Lakes Bioanalytical Conference.

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.

Discovery, identification and mitigation of isobaric sulfate metabolite interference to a phosphate prodrug in LC-MS/MS bioanalysis: Critical role of method development in ensuring assay quality.

Metabolite interferences represent a major risk of inaccurate quantification when using LC-MS/MS bioanalytical assays. During LC-MS/MS bioanalysis of BMS-919194, a phosphate ester prodrug, in plasma samples from rat and monkey GLP toxicology studies, an unknown peak was detected in the MRM channel of the prodrug. This peak was not observed in previous discovery toxicology studies, in which a fast gradient LC-MS/MS method was used.