Douglas Fast

Workshop Report and Follow-Up—AAPS Workshop on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples—Implications of Crystal City Recommendations

The Conference Report of the 3rd AAPS/FDA Bioanalytical Workshop (Crystal City III) endorsed the concept that assay methods supporting bioanalytical data in submissions must demonstrate assay reproducibility by using incurred samples. The present Workshop was convened to provide a forum for discussion and consensus building about incurred sample assay reproducibility for both nonclinical and clinical studies. Information about current regulatory perspectives on incurred sample reanalysis (ISR) was presented, implications of ISR for both large and small molecules were discussed, and the steering committee put forth recommendations for performing ISR. These recommendations from the Workshop, along with the subsequent evolution of approaches leading to a robust ISR program, may be used by scientists performing bioanalytical assays for regulated studies to provide additional confirmation of assay reproducibility for incurred samples.

Perforated dried blood spots: a novel format for accurate microsampling

Dried blood spots (DBS) in their current format encounter challenges in bioanalysis using fixed areas, including but not limited to, waste of DBS samples (only a fraction is used for analysis), the need for sample punching leading to concerns of sample carryover, uncertainty for accurate recovery assessments and hematocrit (HCT) effects. Here we describe a novel concept, namely perforated dried blood spots (PDBS), for accurate microsampling that addresses previous challenges. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm. An accurate amount of blood sample (5-10 µl), was deposited, dried and stored on the PDBS discs. Upon sample analysis, PDBS samples are simply pushed by single-use pipette tips into 96-well plates. The proof-of-concept study was carried out on a PDBS LC-MS/MS assay development and validation under GLP criteria for the quantitation of lansoprazole in human whole-blood (K(3)EDTA). Particularly, the effect of HCT on the accuracy of quantitation was found to be related to recovery from PDBS samples. In all, PDBS was proved to be a viable alternative to conventional DBS, offering additional advantages of complete sample utilization, no requirement for punching, ease of recovery assessments, and elimination of sampling influence due to HCT levels.

Absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC-MS.

The advancement of biotechnology has led to an increase in biotherapeutic drugs, especially recombinant proteins and monoclonal antibodies. Ligand-binding assays or immunoassays are the standard methods of choice in pharmacokinetic studies in support of drug discovery and development for protein therapeutics. LC-MS-based methodologies are increasingly used as alternatives to immunoassays for absolute protein quantitation in biological samples. We review recent advancements in absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC-MS.

Development of a sensitive and selective method for the quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC-MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M+2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 microm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within +/-15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.

Comparison of fused-core and conventional particle size columns by LC–MS/MS and UV: Application to pharmacokinetic study

The chromatographic performance of fused-core (superficially porous) HPLC packing materials was compared with conventional fully porous particle materials for LC-MS/MS analysis of two pharmaceuticals in rat plasma. Two commercially available antidepressants, imipramine and desipramine, were assayed using a conventional analytical C(18) column (5 microm, 2.0 mm x 30 mm) and a fused-core C(18) column (2.7 microm, 2.1 mm x 30 mm). Retention time, column efficiency, pressure drop, resolution, and loading capacity were compared under the same operating conditions. The fused-core column demonstrated reduced assay time by 34% and 2-3-fold increased efficiency (N). Loading capacity up to 25 microl of extract injected on column showed no peak distortion. The registered back-pressure from a flow rate of 1.0 ml/min did not exceed 3400 psi making it compatible with standard HPLC equipment (typically rated to 6000 psi). Two mobile phases were examined, and morpholine as an organic base modifier yielded a 2-5-fold increase in S/N near the limit of detection over triethylamine. The 2.7 microm fused-core column was applied to the analysis of imipramine and desipramine in extracted, protein precipitated rat plasma by LC-MS/MS. The calibration curves were linear in the concentration range of 0.5-1000 ng/ml for both imipramine and desipramine. Intra-run precisions (%CV) and accuracies (%bias) were within +/-7.8% and +/-7.3% at three QC levels and within 14.7% and 14.4% at the LOQ level for both analytes. Following a single method qualification run, the method was applied to the quantitation of pharmacokinetic study samples after oral administration of imipramine to male rats.

Request for Global Harmonization of the Guidance for Bioanalytical Method Validation and Sample Analysis

The 2001 US FDA Bioanalytical Method Validation (BMV) guidance document has been widely accepted and adopted by the bioanalytical community worldwide. As such, it has become the cornerstone of regulated bioanalytical laboratory procedure. In recent years, clarifications to these FDA guidelines and subsequent enhancements were discussed at North American-and European-hosted meetings and conferences. The outcome of these meetings, published in White Papers, conference reports or recommendations, are currently being implemented in many bioanalytical laboratories around the world. Nevertheless, differences in expectations or interpretation of the guidelines from individual auditors/inspectors or regional health authorities are a growing concern for the bioanalytical community. Further globalization of the pharmaceutical industry is also impacting the bioanalytical community. Bioanalytical labs are booming in regions outside the EU and North America, and regional authorities are looking to accommodate this growth or being confronted with the lack of guidance within their own regulations. Consequently, this creates a stimulus for these countries/regions to draft or issue their own guidance documents. The European Medicines Agency (EU), Medicines and Healthcare Products Regulatory Agency (UK), Agência Nacional de Vigilância Sanitária (Brazil) and Therapeutic Goods Administration (Australia) are the most prominent and recent examples. Although the 2001 FDA BMV guidance is often the basis of the emerging guidelines, there is an inherent risk that new sets of quality standards or nuances to the existing guidance will become effective. Over the last few months, following discussions at international meetings that brought together health authorities and industry experts on bioanalysis, the industry has expressed their concerns that multiple regulations on a similar topic will not benefit data generated in bioanalytical laboratories worldwide. Bioanalysis has become a true global discipline and, as such, the bioanalytical community should be served with globally harmonized standards. Therefore, the undersigned would like to ask health authorities worldwide to consider a collaboration and work towards a global harmonization of the guidelines on bioanalytical method validation and sample analysis for preclinical and clinical studies. Standardization and harmonization will largely contribute to the quality, transparency and efficiency of the data generated. These aspects are clearly of immediate benefit for the health authorities (ease of review of data) and laboratories (one set of standards), but eventually also for the patient and the community. We are confident that all involved parties (health authorities and industry) will be equally supportive of this initiative. We are open to any suggestion on how to reach this goal …

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

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.

Conference Report: 6th GCC focus on LBA: critical reagents, positive controls and reference standards; specificity for endogenous compounds; biomarkers; biosimilars

The 6th Global CRO Council for Bioanalysis (GCC) Closed Forum was held on 27 March 2012 in San Antonio, TX, USA, the day before the start of the 6th Workshop on Recent Issues in Bioanalysis. The attendance consisted of 45 bioanalytical CRO senior-level representatives on behalf of 37 CRO companies/sites from six countries. In addition to following up on the issue of co-administered drugs stability and on recommendations regarding the European Medicines Agency guideline, this GCC Closed Forum discussed topics of current interest in the bioanalytical field with focus on ligand-binding assays, such as lot changes for critical reagents, positive controls and reference standards, specificity for endogenous compounds, qualification and validation of biomarker assays, approach for biosimilars and criteria for LC–MS assays of small versus large molecules.

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.