1β-Hydroxydeoxycholic acid as an endogenous biomarker in human plasma for assessment of drug-drug interaction with moderate CYP3A inhibitor.

1β-Hydroxydeoxycholic acid (1β-OH DCA) in plasma has been shown to be a promising biomarker to assess drug-drug interaction (DDI) with a strong CYP3A inducer or a strong CYP3A inhibitor. The changes in total 1β-OH DCA (sum of 1β-OH DCA, 1β-OH glycine deoxycholic acid, and 1β-OH taurine deoxycholic acid equivalents) were more significant than those observed from 4β-hydroxycholesterol, which has been limited to the identification of CYP3A inducers, not CYP3A inhibitors. The significant reduction in total 1β-OH DCA in response to strong CYP3A inhibitors led us to further explore its utility as a biomarker for DDI with moderate CYP3A inhibitors.

Emerging applications of quantitative supercritical fluid chromatography-tandem mass spectrometry for chiral bioanalysis.

Individual stereoisomers of a chiral drug can possess different pharmacokinetic (PK)/pharmacodynamic (PD) properties, leading to different therapeutic/toxicological effects. Therefore, chiral bioanalytical methods are required for individual stereoisomers to assess their PK properties and potential chiral inversion in vivo. Supercritical fluid chromatography (SFC) has not been a mainstay in bioanalytical labs due to limitations of robustness/reliability of old generations of SFC instrumentation.

1β-Hydroxydeoxycholic Acid as an Endogenous Biomarker in Human Plasma for Assessment of CYP3A Clinical Drug-Drug Interaction Potential.

4-Hydroxycholesterol (4-HC) in plasma has been used as a biomarker to assess CYP3A drug-drug interaction (DDI) potential during drug development. However, due to the long half-life and narrow dynamic range of 4-HC, its use has been limited to the identification of CYP3A inducers, but not CYP3A inhibitors. The formation of 1-hydroxydeoxycholic acid (1-OH DCA) from deoxycholic acid (DCA) is mediated by CYP3A, thus 1-OH DCA can potentially serve as an alternative to 4-HC for assessment of CYP3A DDI potential.

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.

Highly selective and sensitive measurement of active forms of FGF21 using novel immunocapture enrichment with LC-MS/MS.

Aim: Recombinant FGF21 analogs are under wide ranging investigations as a potential therapeutic agent for Type 2 diabetes, as well as other metabolic disorders. The endogenous FGF21 is often used as a surrogate pharmacodynamic(PD) biomarker to assess drug efficacy and safety. Results & methodology: Immunocapture was performed using a monoclonal antibody which had been generated to bind to specific domain of native FGF21 as the capture reagent.

A multiplexed immunocapture liquid chromatography tandem mass spectrometry assay for the simultaneous measurement of myostatin and GDF-11 in rat serum using an automated sample preparation platform.

Myostatin, also known as growth differentiation factor 8 (GDF-8), is a protein acting as a negative regulator in skeletal muscle growth. Inhibition of myostatin by therapeutic agents provides opportunities for current unmet medical needs. In order to better understand drug engagement to aid the drug development, we have developed a hybrid LC-MS/MS method which can differentially measure myostatin and another protein from the same GDF family, GDF-11.

Strategy for the Quantitation of a Protein Conjugate via Hybrid Immunocapture-Liquid Chromatography with Sequential HRMS and SRM-Based LC-MS/MS Analyses.

With the development of modern instrumentation and technologies, mass spectrometry based assays have played an important role in protein bioanalysis. We have developed a novel strategy by combining the "bottom-up" and "top-down" approaches using both high-resolution (HRMS) and selected reaction monitoring (SRM) based mass spectrometric detection to quantify a positron emission tomography (PET) detection tracer for an oncology marker. Monkey plasma samples were processed by immunocapture purification, followed by liquid chromatography (LC) with HRMS full scan analysis.

A validated LC-MS/MS method for the quantitative measurement of creatinine as an endogenous biomarker in human plasma.

Background: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations.

Dried blood spot analysis without dilution: Application to the LC-MS/MS determination of BMS-986001 in rat dried blood spot.

Sample dilution is one major challenge in dried blood spot (DBS) bioanalysis. To resolve this issue, we applied a no-dilution strategy for DBS analysis by using a calibration curve with very wide linear range. We developed an LC-MS/MS DBS assay with a linear range of 5 orders of magnitude (50-5000,000ng/mL) for BMS-986001, an HIV drug under development, by simultaneously monitoring two selective reaction monitoring transitions of different intensity.

A UHPLC-MS/MS bioanalytical assay for the determination of BMS-911543, a JAK2 inhibitor, in human plasma.

Herein we report a rapid, accurate and robust UHPLC-MS/MS assay for the quantitation of BMS-911453, a Janus kinase 2 inhibitor under clinical development for the treatment of myeloproliferative disorders, in human plasma. A systematic method development approach was used to optimize the mass spectrometry, chromatography, and sample extraction conditions, and to minimize potential bioanalytical risks. The validated method utilizes stable-isotope labeled (13)C4-BMS-911543 as the internal standard.

Strategies for improving sensitivity and selectivity for the quantitation of biotherapeutics in biological matrix using LC-MS/MS.

In recent years, the applicability of using LC-MS/MS as a complementary technique to traditional ligand binding assays in the absolute quantitation of therapeutic proteins in biologic matrix has been demonstrated. Protein quantitation workflow via LC-MS/MS is primarily based on a enzymatic digestion model and recent works seek to improve selectivity and sensitivity. This review focuses on recent innovations in this field and discusses the following in detail: the applicability of two-dimensional liquid chromatography and its use to improve sensitivity and alleviate matrix ion suppression; the use of derivatization agents after digestion to improve extraction and MS ionization efficiency; techniques to reduce excess protein background and their positive effects on sensitivity, selectivity, and extraction consistency; the application of immunoaffinity extraction of proteins to enrich the analyte(s) of interest while improving selectivity and sensitivity.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation.

Selective reaction monitoring of negative electrospray ionization acetate adduct ions for the bioanalysis of dapagliflozin in clinical studies.

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.

LC-MS/MS determination of apixaban (BMS-562247) and its major metabolite in human plasma: an application of polarity switching and monolithic HPLC column.

Background: apixaban (BMS-562247) (Eliquis(®)) is a novel, orally active, selective, direct, reversible inhibitor of the coagulation factor Xa (FXa). A sensitive and reliable method was developed and validated for the measurement of apixaban (BMS-562247) and its major circulating metabolite (BMS-730823) in human citrated plasma for use in clinical testing.

Methodology/results: A 0.

A novel and cost effective method of removing excess albumin from plasma/serum samples and its impacts on LC-MS/MS bioanalysis of therapeutic proteins.

We have developed an innovative method to remove albumin from plasma/serum samples for the LC-MS/MS quantitation of therapeutic proteins. Different combinations of organic solvents and acids were screened for their ability to remove albumin from plasma and serum samples. Removal efficiency was monitored by two signature peptides (QTALVELVK and LVNEVTEFAK) from albumin.

Sensitivity-based analytical approaches to support human absolute bioavailability studies.

The characterization of absolute bioavailability (BA) is useful for non-intravenous (iv.) formulations during drug development and is required by some health authorities. A study design of co-administrating an iv.

Bioanalysis of propylparaben and p-hydroxybenzoic acid, and their sulfate conjugates in rat plasma by liquid chromatography-tandem mass spectrometry.

Two rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of propylparaben, its major metabolite, p-hydroxybenzoic acid (pHBA), and their sulfate conjugates have been developed and validated in citric acid-treated rat plasma. To prevent propylparaben being hydrolyzed to pHBA ex vivo, rat plasma was first treated with citric acid; then collected and processed at a reduced temperature (ice bath). Stable isotope labeled internal standards, d4-propylparaben, (13)C6-pHBA, and the d4-labeled internal standards of their sulfate conjugates were used in the methods.

Improved ruggedness of an ion-pairing liquid chromatography/tandem mass spectrometry assay for the quantitative analysis of the triphosphate metabolite of a nucleoside reverse transcriptase inhibitor in peripheral blood mononuclear cells.

Rationale: Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents.

Fit-for-purpose bioanalytical cross-validation for LC-MS/MS assays in clinical studies.

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.

Overcoming bioanalytical challenges in an Onglyza(®) intravenous [(14)C]microdose absolute bioavailability study with accelerator MS.

Background: An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range.

Implementation of high-temperature superficially porous technologies for rapid LC-MS/MS diastereomer bioanalysis.

Background: The fast-paced nature of the pharmaceutical industry requires robust bioanalytical methods to endure the high-throughput sample demands of the production environment.

Results: A rapid, accurate and precise LC-MS/MS method was developed for the quantitation of a diastereomer quartet in human plasma. Virtually all of the phosphatidylcholine and most of the lysophosphatidylcholine from human plasma were removed using a phospholipid-removing protein precipitation 96-well plate.

Determination of a novel thrombin receptor antagonist (SCH 530348) in human plasma: evaluation of Ultra Performance Liquid Chromatography™-tandem mass spectrometry for routine bioanalytical analysis.

SCH 530348 is a safe and effective oral anti-platelet agent for patients with acute coronary syndrome. Clinical study results suggest that SCH 530348 dosage at 20 mg or 40 mg is feasible to achieve rapid maximum platelet inhibition following an acute coronary event or intervention procedure. To permit accurate determinations of circulating SCH 530348 in plasma following dosing, a method for measuring SCH 530348 concentrations in human plasma was validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Using temperature to optimize resolution and reduce analysis times for bioanalytical diastereomer LC-MS/MS separations.

A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method was developed for the quantitation of drug stereoisomers in human plasma. Column temperature was shown to be an important variable toward optimizing diastereomer selectivity, resolution and analysis cycle time. Non-linear Van't Hoff plots and changes in peak shape with temperature suggested that selectivity was governed by multiple retention mechanisms.