Richard Houghton

Generic approach to validation of small-molecule LC–MS/MS biomarker assays

BACKGROUND While the regulatory guidelines that describe the validation requirements for small molecules are very comprehensive, they are written primarily for xenobiotic drug molecules. However, the presence of endogenous analyte in control matrix presents an added analytical challenge that must be overcome if small-molecule biomarker assays are to be developed and characterized, especially where downregulation of analyte concentrations is expected. EXPERIMENTAL A generic surrogate matrix calibration protocol has been successfully applied to the measurement of a number of small-molecule exploratory biomarkers using LC-MS/MS. The use of analyte-free matrix enables conventional calibration curves to be constructed across the anticipated range of sample concentrations. The evaluation of matrix effects is carried out using an experiment similar to the parallelism experiment used in ligand-binding assays. CONCLUSION There is currently no published consensus approach to validation of small-molecule biomarker methods. This paper presents a generic approach to endogenous method validation for consideration as bioanalytical best practice for this type of assay.

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)

Conference Report: The 3rd Global CRO Council for Bioanalysis at the International Reid Bioanalytical Forum

The 3rd Global CRO Council Closed Forum was held on the 3rd and 4th July 2011 in Guildford, United Kingdom, in conjunction with the 19th International Reid Bioanalytical Forum. In attendance were 21 senior-level representatives from 19 CROs on behalf of nine European countries and, for many of the attendees, this occasion was the first time that they had participated in a GCC meeting. Therefore, this closed forum was an opportunity to increase awareness of the aim of the GCC and how it works, share information about bioanalytical regulations and audit findings from different agencies, their policies and procedures and also to discuss some topics of interest and aim to develop ideas and provide recommendations for bioanalytical practices at future GCC meetings in Europe.

Efficacy of plasma phospholipid removal during sample preparation and subsequent retention under typical UHPLC conditions

BACKGROUND Several new products have been marketed with the specific capability of removing phospholipids. These products were evaluated alongside more traditional extraction techniques, using UHPLC and precursor-ion scanning (pre m/z 184), which detects glycerophosphocholines (GPCho), lyso-GPCho and sphingomyelins. Using this technique the plasma GPCho profile of human, dog and rat plasma is briefly compared. RESULTS Precursor-ion scanning detected more of the phospholipid profile in extracts than a SRM experiment (m/z 184-184). Products designed for the purpose were the most efficient at removing phospholipids and reversed-phase SPE was better than mixed-mode cation exchange. A comparison of different UHPLC columns demonstrated that C(8) or phenyl phases would help manage the elution of GPChos. CONCLUSION Phospholipid removal plates are useful where no sample concentration is required; however for more challenging LOD it will be necessary to enrich the original sample. In these cases build-up of phospholipids can be avoided with a thoughtful choice of UHPLC column.

Recommendations on incurred sample stability (ISS) by GCC

The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.

Determination of avibactam and ceftazidime in human plasma samples by LC-MS.

AIM Avibactam, a novel non-β-lactam β-lactamase inhibitor co-administrated with the β-lactam antibiotic ceftazidime, is in clinical development. The need to evaluate its PK and PD requires accurate and reliable bioanalytical methods. METHODS We describe LC-MS/MS methods for the determination of avibactam and ceftazidime in human plasma. Avibactam was extracted using weak anionic exchange solid-phase extraction and analyzed on an amide column. Ceftazidime was extracted using protein precipitation and analyzed on a C18 column. Calibration curves were established over 10-10,000 ng/ml (avibactam) and 43.8-87,000 ng/ml (ceftazidime). RESULTS & CONCLUSION Method validation, cross-validation between three laboratories and incurred sample re-analysis demonstrated method robustness. The methods were successfully applied to multiple clinical studies.

7th GCC Insights: incurred samples use; fit-for-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing

The Global CRO Council for Bioanalysis (GCC), a global independent consortium bringing together many senior level CRO representatives, was created in 2010 in order to openly discuss and share opinions on scientific and regulatory issues specific to the bioanalytical field [1]. Since its formation, the GCC members have met on a regular basis to discuss various topics and challenges faced by bioanalytical CRO companies. Several conference reports of past GCC meetings were published to share the discussions held during these events [2–4]. In addition, the GCC also published several influential White Papers on topics of widespread interest in bioanalysis [5–9]. These White Papers provide unified GCC recommendations that are helpful to the global bioanalytical community. The 7th GCC Closed Forum for Bioanalysis took place in Long Beach, CA, USA on 8 April 2013, one day before the start of the 7th Workshop on Recent Issues in Bioanalysis (WRIB). In attendance were 46 senior-level participants from six countries, representing 37 bioanalytical CRO companies/sites. This event represented a good opportunity for bioanalytical experts from CROs to share and discuss these issues of concern for the outsourcing industry. The 7th GCC Closed Forum was chaired by Mario Rocci, who started the meeting by communicating the official admonition statement, as has been done in all previous editions [1]. Prior to initiating discussions, participants introduced themselves. As in previous GCC meetings, multiple topics of 7th GCC Insights: incurred samples use; fitfor-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing

Conference Report: Analytical challenges in the qualification and validation of pharmacodynamic biomarkers

This 1-day workshop, held in association with the Royal Society of Chemistry Analytical Biosciences Group, discussed current concepts in the qualification and validation of biomarker assays for the measurement of pharmacodynamic responses to drugs and vaccines. The venue was Burlington House, the prestigious home of the Royal Society of Chemistry, with delegates drawn from academia, pharmaceutical companies and CROs.

The 20th International Reid Bioanalytical Forum

The 20th International Reid Bioanalytical Forum University of Surrey, Guildford, UK, 9-12 September 2013The 20th International Reid Bioanalytical Forum was attended by a little over 100 participants from pharma, contract research, instrument vendors and academia. The Forum was divided into six broad themes, which included collaborative partnerships between CROs and pharmaceutical companies, technology developments, problem solving, the bioanalytical toolbox, biomarker analysis and regulations in bioanalysis.