2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 2 - hybrid LBA/LCMS and input from regulatory agencies).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event - a full immersion bioanalytical week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed at providing the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 2 covers the recommendations for hybrid LBA/LCMS and regulatory agencies' inputs. Part 1 (small molecule bioanalysis using LCMS) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in volume 7 of Bioanalysis, issues 22 and 24, respectively.

Robust, fit-for-purpose method transfer: why we should apply equivalence testing.

Efficient method transfer is the key in advancing drug candidates through development and requires careful planning and communication between the two laboratories. Successful transfers require robust bioanalytical methods that allow for some small deviations from the method in sender laboratory; however, by keeping the fundamentals of the sender method intact. The equivalence of data produced at both sides should be tested by mutually prepared and analyzed QCs and by analyzing incurred samples at both sides and assessed by a statistical equivalence testing. Current regulatory bodies' guidances provide very limited direction for experimental setup and evaluation of transfer. This perspective paper gives an overview of the available approaches and proposes a way forward for the bioanalytical community.

Technical aspects of inductively coupled plasma bioanalysis techniques.

This White Paper is focused on the technical aspects regarding quantifying pharmaceutically derived inorganic elements in biomatrices in support of GLP nonclinical and clinical studies using inductively coupled plasma (ICP) techniques. For decades ICP has been used in support of Environmental Protection Agency analyses and has more recently been applied for use in the pharmaceutical industry. Current bioanalytical method validation and sample analysis regulatory guidance applies to chromatographic platforms used for analysis of large- and small-molecule PK and TK assessments; however, it is not directly applicable to all aspects of various ICP techniques. Increasingly, quadrupole and high-resolution ICP-MS methods of analysis are being used to quantify inorganic elements contained in pharmaceutical compounds and biomatrices. Many elements occur endogenously in biomatrices, affecting quantification of blanks, standard curve samples, QC samples, and the selection of appropriate levels for the LLOQ.

IS addition in bioanalysis of DBS: results from the EBF DBS-microsampling consortium.

BACKGROUND: In the framework of wider exploration of the application of dried blood spots (DBS) in bioanalysis, by the DBS consortium of the European Bioanalytical Forum, one team of five laboratories investigated the merits of the various ways of IS addition prior to LC-MS/MS analysis. A set of 22 pharmaceutical compounds with log P in the range of 0-10 was selected for this purpose. Assessments were made of precision, recovery, and of the effects of prolonged storage. RESULTS: Assay precision was not significantly different for 3 month-aged samples as compared with 'fresh' samples stored for 7-22 days. Extraction recovery from 3 month-aged spots decreased for some of the analytes; the most widely employed addition of IS in the extraction solvent does not compensate for recovery in such cases. CONCLUSION: From the overall results, it is clear that there is no 'one size fits all' approach to IS addition in DBS bioanalysis.

Recommendations on biomarker bioanalytical method validation by GCC.

The 5th GCC in Barcelona (Spain) and 6th GCC in San Antonio (TX, USA) events provided a unique opportunity for CRO leaders to openly share opinions and perspectives, and to agree upon recommendations on biomarker bioanalytical method validation.

Inductively coupled plasma-MS in drug development: bioanalytical aspects and applications.

The vast majority of today's modern bioanalytical methods for pharmacokinetic, pharmacodynamic and immunogenicity purposes are based on LC-MS/MS and immunoanalytical approaches. Indeed, these methodologies are suitable for a wide range of molecules from small to large. For a smaller but not insignificant group of compounds, LC-MS/MS is not suitable - or in some cases much less suitable - as a reliable bioanalytical methodology, and inductively coupled plasma (ICP)-MS is a more appropriate methodology. ICP-MS is one of these less widely used techniques in drug development. This methodology is predominantly used for elemental bioanalysis for pharmacokinetics, for imaging purposes, for mass-balance, food-effect and biomarker studies. In addition, in the last couple of years an increasing number of applications has been published, where ICP-MS and its various hyphenations (LC-ICP-MS, CE-ICP-MS) have been used for speciation/metabolism and proteomics studies. Here, the analytical potential, the quantitative bioanalytical aspects, the various modes of operation and the challenges of the application of ICP-MS in life sciences applications are given. This includes an overview of recent applications in this area in scientific literature, the various hyphenation possibilities and their application areas and the analysis of the various sample matrices applicable to these fields. It also provides a brief outlook of where the potential of this technique lies in the future of regulated bioanalysis and drug development.

Development and validation of automated SPE-HPLC-MS/MS methods for the quantification of asenapine, a new antipsychotic agent, and its two major metabolites in human urine.

To support the evaluation of the pharmacokinetic parameters of asenapine (ASE) in urine, we developed and validated online solid-phase extraction high-performance liquid chromatography methods with tandem mass spectrometry detection (SPE-LC-MS/MS) for the quantification of ASE and two of its major metabolites, N-desmethylasenapine (DMA) and asenapine-N⁺-glucuronide (ASG). The linearity in human urine was found acceptable for quantification in a concentration range of 0.500-100 ng/mL for ASE and DMA and 10.0-3000 ng/mL for ASG, respectively.

Quantification of asenapine and three metabolites in human plasma using liquid chromatography-tandem mass spectrometry with automated solid-phase extraction: application to a phase I clinical trial with asenapine in healthy male subjects.

The development and validation of methods for determining concentrations of the antipsychotic drug asenapine (ASE) and three of its metabolites [N-desmethylasenapine (DMA), asenapine-N(+) -glucuronide (ASG) and 11-O-sulfate-asenapine (OSA)] in human plasma using LC-MS/MS with automated solid-phase extraction is described. The three assessment methods in human plasma were found to be acceptable for quantification in the ranges 0.0250-20.0 ng/mL (ASE), 0.0500-20.0 ng/mL (DMA and OSA) and 0.250-50.0 ng/mL (ASG).

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.

High temperature liquid chromatography hyphenated with ESI-MS and ICP-MS detection for the structural characterization and quantification of halogen containing drug metabolites.

In this paper we describe the hyphenation of high temperature liquid chromatography with ICP-MS and ESI-MS for the characterization of halogen containing drug metabolites. The use of temperature gradients up to 200°C enabled the separation of metabolites with low organic modifier content. This specific property allowed the use of detection methods that suffer from (significant) changes in analyte response factors as a function of the organic modifier content such as ICP-MS. Metabolites of two kinase inhibitors (SB-203580-Iodo and MAPK inhibitor VIII) produced by bacterial cytochrome P450 BM3 mutants and human liver microsomes were identified based on high resolution MS(n) data. Quantification was done using their normalized and elemental specific response in the ICP-MS. The importance of these kinds of quantification strategies is stressed by the observation that the difference of the position of one oxygen atom in a structure can greatly affect its response in ESI-MS and UV detection.

Incurred sample accuracy assessment: design of experiments based on standard addition.

It is commonly acknowledged that random and systematic analytical errors contribute to poor data quality, and moreover, to imprecise and inaccurate pharmacokinetic parameters. To investigate the random errors in GLP bioanalysis, common ground has been found in today's bioanalysis to assess the reproducibility of the method by reanalyzing part of the incurred samples. The undesired systematic errors in bioanalysis affecting the trueness of the method and leading to inaccurate data remain relatively unattended so far. In order to obtain both precise and accurate data it is suggested in this paper to apply standard addition experiments to calculate the relative systematic errors as an estimate for the incurred sample accuracy. This approach, which can be seen as an important extension to current guidelines in GLP bioanalysis, is illustrated by assessing the accuracy of the bioanalytical results for a bioequivalence study for alendronate.

Application of dried blood spot sampling combined with LC-MS/MS for genotyping and phenotyping of CYP450 enzymes in healthy volunteers.

An early clinical development study (phase I) was conducted to determine the usefulness of dried blood spot (DBS) sampling as an alternative to venous sampling for phenotyping and genotyping of CYP450 enzymes in healthy volunteers. Midazolam (MDZ) was used as a substrate for phenotyping CYP3A4 activity; the concentrations of MDZ and its main metabolite 1'-hydroxymidazolam (1-OH MDZ) were compared between the DBS method from finger punctures, plasma and whole blood (WB), drawn by venipuncture, whereby several methodological parameters were studied (i.e. punch width, amount of dots analyzed and storage time stability). Genotyping between DBS and venous WB samples was compared for CYP2D6 (*3, *4, *6), CYP2C19 (*2, *3), CYP3A4 (*1B) and CYP3A5 (*3C). In addition, the subject's and phlebotomist's satisfaction with venous blood sampling compared with the DBS method was evaluated using a standardized questionnaire. An LC-MS/MS method for the quantification of the MDZ and 1-OH MDZ concentrations in DBS samples was developed and validated in the range of 0.100-100 ng/mL. No compromises were made for the limits of quantification of the DBS-LC-MS/MS method vs the authentic plasma and WB methods.

Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers.

OBJECTIVE: In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose (600 mg twice daily), ritonavir also inhibits CYP2D6. The effect of low-dose ritonavir on CYP2D6 is unknown and was investigated in this study. METHODS: This was a 1-arm, 2-period, fixed-order study in 13 healthy male volunteers who were extensive metabolizers of CYP2D6. The first period examined baseline CYP2D6 activity by evaluating the pharmacokinetics of a single dose of desipramine and by metabolic phenotyping with dextromethorphan. During the second period, participants took ritonavir, 100 mg twice daily, for 2 weeks, followed by repeat assessment of desipramine pharmacokinetics and the dextromethorphan metabolic phenotype in the presence of ritonavir. RESULTS: Low-dose ritonavir (100 mg twice daily) significantly increased the exposure to single-dose desipramine, as reflected in a geometric mean ratio (with ritonavir/without ritonavir) of 1.26 (95% confidence interval, 1.13-1.40) for the desipramine area under the concentration versus time curve from time 0 to infinity (P < .001). Coadministration of low-dose ritonavir did not significantly affect the dextromethorphan/dextrorphan urinary metabolic ratio and did not convert any extensive metabolizer to a poor metabolizer. CONCLUSIONS: Low-dose ritonavir (100 mg twice daily) exerts a modest inhibitory effect on the activity of CYP2D6 in extensive metabolizers, as assessed with desipramine as the index substrate. This effect was not apparent with the dextromethorphan/dextrorphan metabolic ratio as an indicator for CYP2D6 activity. It is expected that the effect of low-dose ritonavir on CYP2D6 will not require standard dose reductions for CYP2D6 substrates.