The AAPS Workshop 2008 on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples was the defining moment in establishing incurred sample reanalysis (ISR) as a mandatory exercise in demonstrating assay reproducibility using incurred (study) samples. The importance of ISR can be envisaged from its role in clinical as well as non-clinical studies. Incurred samples can differ significantly in their composition when compared with the calibration standards and quality control samples that are used to validate the developed method. The present article attempts to summarize five troubleshooting cases encountered in the analyses of incurred samples for bioanalytical methods developed in our laboratory for mesalamine, hydrochlorothiazide, clopidogrel, sildenafil and rabeprazole. The issues identified were related to: sample inhomogeneity, sample processing error, impact of buffer pH during sample preparation, instability of metabolite and change in laboratory environment. The steps taken to trace and correct these incidents are discussed with adequate data. These examples will further broaden the scope and emphasize the significance of ISR. We believe this investigation will help to develop more reliable and efficient bioanalytical methods.

Incurred sample reanalysis (ISR): a decisive tool in bioanalytical research

Developmental toxicity in vitro assays have hitherto been established as stand-alone systems, based on a limited number of toxicants. Within the embryonic stem cell-based novel alternative tests project, we developed a test battery framework that allows inclusion of any developmental toxicity assay and that explores the responses of such test systems to a wide range of drug-like compounds. We selected 28 compounds, including several biologics (e.g., erythropoietin), classical pharmaceuticals (e.g., roflumilast) and also six environmental toxicants. The chemical, toxicological and clinical data of this screen library were compiled. In order to determine a non-cytotoxic concentration range, cytotoxicity data were obtained for all compounds from HEK293 cells and from murine embryonic stem cells. Moreover, an estimate of relevant exposures was provided by literature data mining. To evaluate feasibility of the suggested test framework, we selected a well-characterized assay that evaluates 'migration inhibition of neural crest cells.' Screening at the highest non-cytotoxic concentration resulted in 11 hits (e.g., geldanamycin, abiraterone, gefitinib, chlorpromazine, cyproconazole, arsenite). These were confirmed in concentration-response studies. Subsequent pharmacokinetic modeling indicated that triadimefon exerted its effects at concentrations relevant to the in vivo situation, and also interferon-β and polybrominated diphenyl ether showed effects within the same order of magnitude of concentrations that may be reached in humans. In conclusion, the test battery framework can identify compounds that disturb processes relevant for human development and therefore may represent developmental toxicants. The open structure of the strategy allows rich information to be generated on both the underlying library, and on any contributing assay. © 2014 The Author(s). Chemicals/CAS: abiraterone, 154229-19-3; arsenic trioxide, 1303-24-8, 1327-53-3, 13464-58-9, 15502-74-6; chlorpromazine, 50-53-3, 69-09-0; gefitinib, 184475-35-2, 184475-55-6, 184475-56-7; geldanamycin, 30562-34-6; triadimefon, 43121-43-3

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Profiling of drugs and environmental chemicals for functional impairment of neural crest migration in a novel stem cell-based test battery

Intra-articular drug delivery from an optimized topical patch containing teriflunomide and lornoxicam for rheumatoid arthritis treatment: does the topical patch really enhance a local treatment?

Quantitation of total and free teriflunomide (A77 1726) in human plasma by LC-MS/MS.

This study describes a comparison between LC-UV and LC–MS method for the simultaneous analyses of a few disease-modifying agents of multiple sclerosis. Quantitative determination of fampridine (FAM), teriflunomide (TFM) and dimethyl fumarate (DMF) was performed in human plasma with the recovery values in the range of 85–115%. A reversed-phase high-performance liquid chromatography (HPLC) with UV as well as MS detection is used. The method utilizes an XBridge C18 silica column and a gradient elution with mobile phase consisting of ammonium formate and acetonitrile at a flow rate of 0.5 mL min−1. The method adequately resolves FAM, TFM and DMF within a run time of 15 min. Owing to low molecular weights, the estimation of DMF and FAM is more versatile in UV than MS detection. With LC-UV, the detection limits of FAM, TFM and DMF were 0.1, 0.05, 0.05 μg and the quantification limit for all the analytes was 1 μg. With LC–MS, the detection and quantification limits for all of the analytes were 1 and 5 ng, respectively. The two techniques were completely validated and shown to be reproducible and sensitive. They were applied to a pharmacokinetic study in rats by a single oral dose. Copyright © 2016 John Wiley & Sons, Ltd.

Comparison of LC-UV and LC-MS methods for simultaneous determination of teriflunomide, dimethyl fumarate and fampridine in human plasma: application to rat pharmacokinetic study.

Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion (HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 μL human plasma was carried out by solid-phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax Eclipse XDB C18 (150 × 4.6 mm, 5 µm) analytical column using isocratic mobile phase consisting of 20 mm ammonium acetate–methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of 0.1–350 ng/mL for BUP and 0.1–600 ng/mL for HBUP. The matrix effect was assessed by post-column infusion and the mean process efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to a bioequivalence study of 150 mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian male subjects under fed conditions. Copyright © 2011 John Wiley & Sons, Ltd.

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC-MS/MS: application to a bioequivalence study in healthy Indian subjects.

Rajendrasinh N. Vaghela

Papers

Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry.

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC-MS/MS: application to a bioequivalence study in healthy Indian subjects.