Recommendations and feedback from the European Bioanalysis Forum Workshop: 1 year into ICH M10 - keeping our finger on the pulse.

The ICH M10 guideline on bioanalytical method validation and sample analysis is being adopted since 2023. However, and inevitably, some paragraphs or requirements remain ambiguous and are open for different interpretations. In support of a harmonized interpretation by the industry and health authorities, the European Bioanalysis Forum organized a workshop on 14 November 2023 in Barcelona, Spain, to discuss unclear and/or ambiguous paragraphs which were identified by the European Bioanalysis Forum community and delegates of the workshop prior to the workshop. This manuscript reports back from the workshop with recommendations and aims at continuing an open scientific discussion within the industry and with regulators in support of a science-driven guideline for the bioanalytical community and in line with the ICH mission - that is, achieve greater harmonization worldwide to ensure that safe, effective and high-quality medicines are developed and registered in the most resource-efficient manner.

What are effective policies for promoting physical activity? A systematic review of reviews.

The importance of policy for promoting physical activity (PA) is increasingly recognized by academics, and there is a push by national governments and international institutions for PA policy development and monitoring. However, our knowledge about which policies are actually effective to promote PA remains limited. This article summarizes the currently available evidence by reviewing existing reviews on the subject. Building on results from a previous scoping review on different types of PA-related evidence, we ran searches for combinations of the terms "physical activity", "evidence", "effect", "review", and "policy" in six different databases (PubMed, Scopus, SportDiscus, PsycInfo, ERIC, and IBSS). We used EPPI Reviewer 4 to further process the results and conduct an in-depth analysis. We identified 57 reviews providing evidence on 53 types of policies and seven broader groups of policies. Reviews fell into four main categories: 1) setting- and target group-specific; 2) urban design, environment and transport; 3) economic instruments; and 4) broad-range perspective. Results indicate that there is solid evidence for policy effectiveness in some areas (esp. school-based and infrastructural policies) but that the evidence in other areas is insufficient (esp. for economic policies). The available evidence provides some guidance for policy-makers regarding which policies can currently be recommended as effective. However, results also highlight some broader epistemological issues deriving from the current research. This includes the conflation of PA policies and PA interventions, the lack of appropriate tools for benchmarking individual policies, and the need to critically revisit research methodologies for collating evidence on policies.

How Can Physical Activity Be Promoted Among Children and Adolescents? A Systematic Review of Reviews Across Settings.

Introduction: A vast majority of children and adolescents are physically inactive. As a result, high obesity rates and related diseases have made physical activity promotion a politically relevant topic. In order to form the basis for political decision making, evidence is required regarding the efficacy and effectiveness of interventions for physical activity promotion. In contrast to previous research, this systematic review of reviews targets three key settings (family and home, childcare, school), and is among the largest to have been conducted. Methods: A systematic review of reviews was conducted as part of a large-scale project to develop national recommendations for physical activity promotion in Germany. Six electronic databases were searched and inclusion criteria were defined. Two independent reviewers screened the titles and abstracts of potentially relevant literature. 213 reviews were identified and categorised by target group. A total of 74 reviews were identified dealing with children and adolescents. Each review underwent a quality assessment. Results: 39 reviews with the highest quality and relevance were analysed. Three reviews focused on the family and home setting, 4 on the childcare setting, 28 on the school setting and 4 on other settings. Evidence revealed the key role played by parents in promoting physical activity in children within each setting. Furthermore, evidence pointed toward the efficacy of multi-component interventions in the childcare and school setting. Several evidence-based intervention strategies were identified for childcare facilities and schools. Discussion: The review of reviews identified a number of promising strategies for PA promotion among children and adolescents. Among reviews, multi-component interventions in childcare facilities and schools stand out prominently. At the same time, the review of reviews indicated that there is still a lack of studies on the efficacy of interventions that go beyond the individual level. We recommend that future research should also target community and policy level interventions and interventions other than the school setting. In order to make more specific recommendations regarding the scale-up of promising intervention strategies, further knowledge about the effectiveness, health equity and cost effectiveness of interventions is needed.

Reinforcing the Importance and Feasibility of Implementing a Low-dose Protocol for CT-guided Biopsies.

RATIONALE AND OBJECTIVES: This study sought to more definitely illustrate the impact and feasibility of implementing a low-dose protocol for computed tomography (CT)-guided biopsies using size-specific dose estimates and multivariate analyses. MATERIALS AND METHODS: Fifty consecutive CT-guided lung and extrapulmonary biopsies were reviewed before and after implementation of a low-dose protocol (200 patients total, mean age 61 ± 15 years, 128 women). Analyses of variance with Bonferroni correction were used to compare standard and low-dose protocols in terms of patient demographics, physician experience, target lesion size, total dose-length product, total acquisitions, size-specific dose estimate, signal-to-noise ratio, contrast-to-noise ratio, and lesion conspicuity ratings. All procedures were performed on the same 16-slice CT scanner. RESULTS: Voluntary protocol adherence was 100% (lung) and 89% (extrapulmonary). The low-dose protocol achieved significantly lower total average dose-length product [(lung) 735.6 ± 599.4 mGy × cm to 252.1 ± 101.9 mGy × cm, P < .001; (extrapulmonary) 724.7 ± 545.0 mGy × cm to 392.9 ± 239.5 mGy × cm, P < .001] and size-specific dose estimate [(lung) 5.2 ± 0.8 mGy × cm to 4.3 ± 1.5 mGy, P < .001; (extrapulmonary) 10.1 ± 6.7 mGy to 6.5 ± 2.7 mGy, P < .001]. Only the change in protocol was independently associated with lower size-specific dose estimates when controlling for the other variables (P < .0001). This was achieved with no significant differences in signal-to-noise ratio, contrast-to-noise ratio, or lesion conspicuity. CONCLUSIONS: Implementation of a low-dose protocol for CT-guided biopsies resulted in 21% and 36% of size-specific dose estimate reduction for lung and extrapulmonary biopsies, respectively, with excellent adherence. Interventional and body radiologists should implement low dose CT-guidance protocols aiming to improve patient safety.

LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.

AIM: We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies. METHODOLOGY: We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested. CONCLUSION: Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.

Alternative strategies for mass spectrometer-based sample dilution of bioanalytical samples, with particular reference to DBS and plasma analysis.

BACKGROUND: The analysis of bioanalytical samples has required a physical dilution of high-concentration samples to bring concentrations into the validated calibration range of an assay. RESULTS: A reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry method for the quantitative analysis of pioglitazone in dried blood spots has been used to partially validate two novel techniques to analyze sample concentrations that lie above a particular calibration range. The first of the two techniques is mass spectrometer signal dilution, which consists of lowering the signal that reaches the detector. The second technique designated isotope signal ratio monitoring looks at [M+2]+1 ions (caused by naturally occurring isotopes) for samples above the upper limit of quantification. CONCLUSIONS: The newly developed methods have the potential to simplify the analysis of bioanalytical samples for which previously a physical dilution of the sample was required to bring analytes within the calibration range of an assay.

Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites.

BACKGROUND: LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. RESULTS/METHODOLOGY: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5´n-1, 5´n-2, 5´n-3, 3´n-1, 3´n-2 and 3´n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. CONCLUSION: We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3´ and 5´ metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.

Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.

Development of a bioanalytical method for quantification of a 15-mer oligonucleotide at sub-ng/ml concentrations using LC-MS/MS.

LC-MS/MS provides a powerful technique for the selective quantification of therapeutic oligonucleotides; however, the LOQ (typically >1 ng/ml) may be higher than desirable for clinical bioanalysis. A method has been developed to allow quantification of a 15-mer unmodified DNA oligonucleotide in human plasma using SPE and UHPLC with MS/MS detection. The LOQ of this assay was 0.05 nM (∼250 pg/ml). This method was then further developed by the inclusion of online SPE to increase loading and apply additional sample cleanup. This allowed for improved assay precision at lower concentrations and increased signal, thus allowing the method to be validated over the range of 10-4000 pM (approximately 50-20,000 pg/ml). The method is accurate, precise and selective and it provides proof-of-concept for sub-ng/ml, high-throughput quantification of oligonucleotides using online SPE coupled to ion-pair, reversed-phase LC-MS/MS.

Bioanalytical approaches to analyzing peptides and proteins by LC--MS/MS.

Peptides and proteins have been utilized as therapeutic agents for over 40 years. Traditional approaches to quantify these molecules in biological matrices have utilized immunoassay approaches that can be time inefficient, lack assay specificity and have limited analytical ranges. The advances in sample preparation technologies, chromatographic systems and their chemistries, mass spectrometers and their software over the last decade have meant that LC-MS/MS approaches to peptide and protein quantification are feasible and can overcome the problems associated with quantification by immunoassay. In this article we present an overview of the challenges and approaches to overcome them when performing quantitative bioanalysis of peptides and proteins by LC-MS/MS.

Validation of a bioanalytical method for the quantification of a therapeutic peptide, ramoplanin, in human dried blood spots using LC-MS/MS.

A method has been developed and validated for the quantification of ramoplanin, a 2554 Da peptide antibiotic, in human dried blood spots using high-performance liquid chromatography with tandem mass spectrometric detection. The validation data meet FDA acceptance criteria for bioanalytical assays and cover the quantification of ramoplanin over the range 10-5000 ng/mL. The assay determines ramoplanin at the same lower limit of quantification as conventional liquid sample methods. Dried blood spot analysis provides an approach for quantification of peptide therapeutics and delivers significant benefits for sample collection and handling and also sample cleanup over conventional plasma and serum assays.

Application of the DBS methodology to a toxicokinetic study in rats and transferability of analysis between bioanalytical laboratories.

BACKGROUND: There are little published data on either the comparison of liquid blood and dried blood spots (DBS) analyses or the ability to generate comparable DBS data at different analytical laboratories. We assess the comparative results of samples stored as liquid blood and DBS. We also determine the transferability of DBS samples by comparing the analysis at two laboratories. RESULTS: Bioanalytical methods for the analysis of pioglitazone in DBS and liquid blood samples were validated to US FDA guidelines. Pharmacokinetic data generated from DBS and liquid blood samples demonstrated area under the time-concentration profile (0-24 h) values within 3% of each other and maximum plasma concentration values within 7% of each other. Comparing DBS sample results at different laboratories showed more than 99% of results agreeing within 20%. CONCLUSIONS: The results indicate that comparable concentration results are obtained from DBS and whole blood samples within the same laboratory, indicating that changing between the two matrices is viable. The comparable results of DBS samples analyzed at two laboratories using different analytical methodologies demonstrate that the technique is robust and transferable.

Evaluation of ultra-performance liquid chromatography in the bioanalysis of small molecule drug candidates in plasma.

Ultra-performance liquid chromatography (UPLC) combined with mass spectrometric detection (MS) is used successfully in the bioanalysis of small molecule drug candidates in plasma. UPLC-MS is shown to increase sample throughput by reducing run times over 3-fold, without compromising analytical sensitivity or analyte resolution. The technique is demonstrated to be practical and robust on a commercially available ultra-high pressure system when injecting extracts of plasma and has also shown to be a technique that can be used effectively on a conventional high-performance liquid chromatography system fitted with short columns (

Negative ion electrospray mass spectrometry of aminomethylphosphonic acid and glyphosate: elucidation of fragmentation mechanisms by multistage mass spectrometry incorporating in-source deuterium labelling.

Glyphosate and its main metabolite, aminomethylphosphonic acid, introduced by direct infusion in (2)H(2)O, appear in negative ion electrospray mass spectrometry (ES-MS) as triply deuteriated [M[bond]H](-) ions. Sites of deuterium residence and loss were established using the multistage (MS(n)) capabilities of an ion trap mass spectrometer to assist in the determination of fragmentation mechanisms. The study reveals specific mechanisms, common to each analyte, such as those involving a five-membered transition state between the amine and phosphonate group, as well as analyte specific transitions.

Analysis of glyphosate and glufosinate by capillary electrophoresis-mass spectrometry utilising a sheathless microelectrospray interface.

The potential of capillary electrophoresis combined with mass spectrometry for the simultaneous determination of two herbicides (glyphosate and glufosinate) and their metabolites (aminomethylphosphonic acid and methylphosphinicopropionic acid) as the native species is demonstrated utilising a simple microelectrospray interface. The interface uses the voltage applied to the CE capillary to drive separation and generate the electrospray, avoiding sample dilution associated with the use of a sheath liquid interface. The chemistry of the internal walls of the capillary has a marked influence on peak shape, and appropriate choice is essential to successful operation of the interface. A linear polyacrylamide coated capillary, which has no electroosmotic flow, gave best reproducibility, with precision of migration time and peak area in the range 1-2 and 7-12% RSD, respectively, for the four analytes. Limits of detection, low-pg on-column, are substantially better than for previous methods and calibration curves over the range 1-100 microM have R2 values greater than 0.97. The observed concentration limit of detection for glyphosate in water is 1 microM and for a water-acetone extract of wheat is 2.5 microM, allowing the underivatised herbicide to be detected at 10% of the maximum residue limit in wheat.

Tandem mass spectrometric analysis of glyphosate, glufosinate, aminomethylphosphonic acid and methylphosphinicopropionic acid.

A detailed MS(n) study of glyphosate, glufosinate and their main metabolites, aminomethylphosphonic acid and methylphosphinicopropionic acid, using an ion trap mass spectrometer, was performed. The analytes show good response in negative ion electrospray mass spectrometry (ES-MS) as [M-H](-) ions. Tandem-MS spectra reveal a wealth of structurally specific ions, allowing characterisation of the fragmentation pathways of the four analytes in their native form for the first time. The ions formed at each stage of fragmentation reveal ions common to each analyte, such as phosphinate, as well as analyte specific transitions. Simplex optimisation allows optimum trapping and fragmentation parameters to be determined leading to improved response for particular transitions and transition sequences, and revealing previously unseen ions.