Alan Breau

Plasma protein binding of celecoxib in mice, rat, rabbit, dog and human

The plasma protein binding of celecoxib was determined for animals and humans using in vitro and ex vivo methods. Eight, healthy, human volunteers (three male, five female, 20–39 years) received celecoxib (600 mg) BID for 7 days, blood samples were collected and concentrations of bound and unbound celecoxib determined. The fraction of bound drug in the volunteers was constant (97.4±0.1%) at total celecoxib plasma concentrations ranging from 0.01 to 4.02 μg/mL. The ex vivo plasma protein binding of celecoxib in the animals was concentration‐independent up to approximately 12, 8 and 10 μg/mL for mouse, rat and dog, respectively. The plasma protein binding of celecoxib after a single oral dose of 10 and 300 mg/kg to mice was 98.3±0.2%, of 1 and 400 mg/kg to rats was 98.3±0.2% and of 1 and 100 mg/kg to dogs was 98.5±0.1%. The percent binding of celecoxib to plasma proteins in vitro was slightly lower than those values determined ex vivo. The in vitro binding of celecoxib to plasma protein was constant over the concentrations of 0.1–10 μg/mL for all species, except rat. Copyright

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.

Determination of free levels of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry.

A liquid chromatography combined with tandem mass spectrometry assay for the determination of free levels of the highly protein bound drug phenytoin (5,5-diphenylhydantoin) in human plasma is described. The assay was demonstrated to be reliable, accurate and precise, and specific for phenytoin. The procedure involves isolation of the unbound drug from the drug/protein complex by ultrafiltration. Liquid-liquid extraction was employed to extract the resultant ultrafiltrate. PHT was separated on a 50 x 3 mm reversed-phase column using isocratic mobile phase conditions that yielded a run time of 1.5 min, enabling high throughput sample analysis. Linearity was obtained over the range 5.00 to 500 ng/ml. Both between-run and within-run coefficients of variation were less than 15% and accuracys across the assay range were all within 100 +/- 10%. The assay was successfully implemented to support a clinical interaction study with phenytoin.

Formation of a Global Contract Research Organization Council for Bioanalysis

Background Over the last year, the bioanalytical community strongly expressed their need for international harmonization of bioanalytical guidances through numerous international meetings and publications, and this need was acknowledged by several regulatory agencies [1–5]. Following the 4th Calibration and Validation Group (CVG) Workshop on Regulated Bioana lysis hosted in Montreal (April 2010), a unanimous consensus was reached for the global bioanalytical community to identify non-prescriptive, s ciencebased language that could form the basis of a proposed guidance document on bioana lysis [6]. Ideally, such guidance language would describe the rationale behind each bioanalytical requirement and would be presented for consideration by agencies and industry worldwide. A recent concrete action taken towards such global harmonization of bioanalytical guidances was the creation of the Global Bioana lysis Consortium (GBC). Drawing from representatives of scientific associations with involvement in regulated bioana lysis across the globe, the objective of the GBC is to merge existing or emerging bioanalytical guidances and create a unified document that can be presented to the regulatory authorities in various countries and regions. The development of the GBC is currently in process, with the intention to present plans and updates at upcoming bioanalytical meetings and to seek input from the scientists conducting bioana lysis [7]. At the 4th CVG Bioana lysis Workshop in Montreal, Canada, several Contract Research Organizations (CROs) highlighted the importance of having a strong and cohesive CRO contribution to the global harmonization process. Having CROs, academic laboratories and pharma ceutical industries appropriately represented was viewed as critical to identifying optimum language. Consequently, the proposal to build a Global CRO Council (GCC) arose, designed to be a distinct group consisting exclusively of bioanalytical CRO members conducting r egulated bioana lysis business. To initiate creation of the GCC, a special closed forum was held in Montreal on 14 September 2010, where 41 executive representatives from 32 bioanalytical CROs were present to discuss scientific harmonization issues and the institution of the GCC. This first CRO Formation of a Global Contract Research Organization Council for Bioana lysis

Recommendations on the interpretation of the new European Medicines Agency Guideline on Bioanalytical Method Validation by Global CRO Council for Bioanalysis (GCC)

) Guideline on Bioanalytical Method Validation (BMV), during the 4th GCC (23 October 2011, Washington DC, USA) and 5th GCC (14 November 2011, Barcelona, Spain) Closed Forums. These North American and European events provided a unique opportunity for CRO leaders to openly share opinions and perspectives and to agree on unified bioanalytical recommendations specifically in relation with the new EMA guideline.The Global CRO Council for Bioanalysis (GCC)

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry.

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.

Development and validation of a liquid chromatography–tandem mass spectrometric assay for Eplerenone and its hydrolyzed metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantify the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human plasma. The analytes (I, II) and their stable isotope-labeled analogues as internal standards were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was carried out on a narrow-bore reversed-phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile/water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). The analytes were ionized using negative-to-positive switch electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 was used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-2500 ng/ml of plasma for both I and II. The lower limit of quantification was 10 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A throughput of 80 human plasma standards and samples per run was achieved with run time of 5 min for each injection. The assay has been successfully used in analyses of human plasma samples to support clinical studies.

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine.

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.

Conference Report: 4th Global CRO Council for Bioanalysis: coadministered drugs stability, EMA/US FDA Guidelines, 483s and Carryover

The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters. Recent 483s and agency findings, as well as issues on method carryover, were also part of the topics discussed.