Ann Lévesque

Analyte and internal standard cross signal contributions and their impact on quantitation in LC–MS based bioanalysis

Cross signal contributions between an analyte and its internal standard (IS) are very common due to impurities in reference standards and/or isotopic interferences. Despite the general awareness of this issue, how exactly they affect quantitation in LC-MS based bioanalysis has not been systematically evaluated. In this research, such evaluations were performed first by simulations and then by experiments using a typical bioanalytical method for tiagabine over the concentration range of 1-1000 ng/mL in human EDTA K(3) plasma. The results demonstrate that when an analyte contributes to IS signal, linearity and accuracy can be affected with low IS concentration. Thus, minimum IS concentrations have been obtained for different combinations of concentration range, percentage of cross contribution, and weighting factor. Moreover, while impurity in analyte reference standard is a factor in cross signal contribution, significant systematic errors could exist in the results of unknown samples even though the results of calibration standards and quality controls are acceptable. How these systematic errors would affect stability evaluation, method transfer, and cross validation has also been discussed and measures to reduce their impact are proposed. On the other hand, the signal contribution from an IS to the analyte causes shifting of a calibration curve, i.e. increase of intercept, and theoretically, the accuracy is not affected. The simulation results are well supported by experimental results. For example, good inter-run (between-run) accuracy (bias: -2.70 to 5.35%) and precision (CV: 2.07-10.50%) were obtained when runs were extracted with an IS solution containing 1-fold of the lower limit of quantitation.

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: 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.

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.

Impact of hemolysis during sample collection: how different is drug concentration in hemolyzed plasma from that of normal plasma?

Hemolysis is a common phenomenon in clinical studies. Despite the growing interest in hemolysis matrix effect, how hemolysis impacts the representability of hemolyzed plasma samples was rarely evaluated. The purpose of this research is to perform such an evaluation by theoretical consideration and experiment. A formula for estimating the impact is proposed, which includes the degree of hemolysis and the drugs red blood cell (RBC): plasma concentration ratio. The impact of hemolysis on the representability of hemolyzed plasma samples is compound-dependant. Given the same degree of hemolysis, the stronger a drug binds to RBCs, the more significant the impact of hemolysis. For a drug with high affinity to RBCs, the results of hemolyzed plasma samples may not be useful even though they are accurate. There is an overall agreement between theoretical predication and experimental results. Among the ten different drug compounds tested, only methazolamide, which binds strongly to RBCs, showed significant change in plasma concentration due to hemolysis.

Beyond successful ISR: case-by-case investigations for unmatched reassay results when ISR passed

Incurred sample reanalysis (ISR) is now commonly practiced in regulated bioanalytical laboratories. With an average ISR success rate of 95% or higher and an increasing number of ISR tests being conducted, more and more situations deserve scientific evaluation or investigation for the unmatched reassay results revealed in ISR tests even though they meet the acceptance criteria. First, should an investigation be initiated when an ISR test is acceptable? How large a discrepancy or what situation would warrant an investigation? What would be the impact on a study? How would investigations regarding unmatched reassay results be conducted? What are the main root causes identified? Can normal random errors cause a large discrepancy in unfavorable combinations? How could the timeline and cost be affected? All these questions are addressed in this paper with five real case examples.

8th GCC: Consolidated feedback to US FDA on the 2013 Draft FDA Guidance on Bioanalytical Method Validation

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this years closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industrys best practices and the conclusions from the discussion of these topics is included in this meeting report.

Internal Standards for Quantitative LC-MS Bioanalysis

Internal standards play critical roles in ensuring the accuracy of reported concentrations in LC-MS bioanalysis. How do you find an appropriate internal standard so that analyte losses and experimental variations during sample preparation, chromatographic separation, and mass spectrometric detection could be corrected? How is the concentration of an internal standard determined? Should internal standard responses be monitored during the analysis of incurred samples? What are the main causes for internal standard response variations? How do they impact the quantitation? Why are stable isotope labeled internal standards preferred? And yet one should still have an open-mind in their usage for the analysis of incurred samples. All these questions are addressed in this chapter supported by theoretical considerations and practical examples.

The importance of choosing the appropriate matrix to validate a bioanalytical method according to the study needs

Sylvain Lachance is a Bioanalytical Scientific Expert in the Bioanalytical Division of inVentiv Health Clinical Quebec Citys (Canada) site, a CRO offering clinical, commercial and consulting services to the healthcare industry. He is responsible for following up on the conduct of bioanalytical method development activities by enhancing the scientific and technical knowledge of the researchers, bioanalytical project coordinators and of the laboratory technicians. He assists bioanalytical project coordinators in investigations during bioanalyses and method validations. He has been working in the Bioanalytical Division of inVentiv Health Clinical for over 16 years, working as a Research Scientist, Chromatographic Specialist and Scientific Expert. He has worked on multiple method developments in HPLC and LC-MS/MS, specifically on troubleshooting. He has been involved in more than 70 posters and publications in the bioanalytical field for different scientific meetings. Ann Lévesque obtained her PhD in Biochemistry at the Université Laval in Québec City in 1994 studying the biological actions of peptide analogs of the gastrin releasing peptide in the growth inhibition of cancer cells. Prior to joining inVentiv Health Clinical, she held management positions at other Contract Research Organizations. Her publications include over 100 posters, 17 scientific articles and book chapters in the clinical biochemistry and bioanalytical fields. Within inVentiv Health, Dr. Lévesque is responsible for managing the R&D and sample analysis teams performing bioanalytical analysis of small molecules and peptides. She is also acting as the Biomedical Laboratory Director accountable for the oversight of all activities related to the safety testing of samples from subjects enrolled in early stage clinical trials. Since joining the Bioanalytical Division, Dr. Lévesque has been instrumental in the great success of the laboratory by developing a culture of quality, innovation and value. Validation guidelines from different agencies mainly recommend that matrix effect should be studied with hemolyzed and hyperlipidemic samples, while the European agency requires also to investigate matrix effect on special population. When studies are done in countries with different dietary habits, or when a medication is administered to decrease the concentration of the endogenous compounds, should the matrix effect in these conditions be evaluated? Herein, three case studies are described to show the importance of choosing the appropriate matrix for the bioanalytical method validations and for their use to analyze the study samples according to the conditions required by the clinical trials. The case studies presented are related to the use of the testosterone, Omega-3 and cortisol methods.