Data integrity in regulated bioanalysis: a summary from the European Bioanalysis Forum Workshop in collaboration with the MHRA.

In this conference report, we summarize the main findings and messages from a workshop on 'Data Integrity'. The workshop was held at the 11th European Bioanalysis Forum Open (EBF) Symposium in Barcelona (21-23 November 2018), in collaboration with the Medicines and Health products Regulatory Agency to provide insight and understanding of regulatory data integrity expectations. The workshop highlighted the importance of engaging with software developers to address the gap between industry's data integrity needs and current system software capabilities. Delegates were also made aware of the importance of implementing additional procedural controls to mitigate the risk associated with using systems that do not fully meet data integrity requirements.

Collaborative Method Performance Study of the Measurement of Nicotine, Its Metabolites, and Total Nicotine Equivalents in Human Urine.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements.Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry.Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was < + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results.Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine.Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083-90. ©2018 AACR.

'Real-world' compensatory behaviour with low nicotine concentration e-liquid: subjective effects and nicotine, acrolein and formaldehyde exposure.

AIMS: To compare the effects of (i) high versus low nicotine concentration e-liquid, (ii) fixed versus adjustable power and (iii) the interaction between the two on: (a) vaping behaviour, (b) subjective effects, (c) nicotine intake and (d) exposure to acrolein and formaldehyde in e-cigarette users vaping in their everyday setting. DESIGN: Counterbalanced, repeated measures with four conditions: (i) low nicotine (6 mg/ml)/fixed power; (ii) low nicotine/adjustable power; (iii) high nicotine (18 mg/ml)/fixed power; and (iv) high nicotine/adjustable power. SETTING: London and the South East, England. PARTICIPANTS: Twenty experienced e-cigarette users (recruited between September 2016 and February 2017) vaped ad libitum using an eVic Supreme™ with a 'Nautilus Aspire' tank over 4 weeks (1 week per condition). MEASUREMENTS: Puffing patterns [daily puff number (PN), puff duration (PD), interpuff interval (IPI)], ml of e-liquid consumed, changes to power (where permitted) and subjective effects (urge to vape, nicotine withdrawal symptoms) were measured in each condition. Nicotine intake was measured via salivary cotinine. 3-Hydroxypropylmercapturic acid (3-HPMA), a metabolite of the toxicant acrolein, and formate, a metabolite of the carcinogen formaldehyde, were measured in urine. FINDINGS: There was a significant nicotine concentration × power interaction for PD (P < 0.01). PD was longer with low nicotine/fixed power compared with (i) high nicotine/fixed power (P < 0.001) and (ii) low nicotine/adjustable power (P < 0.01). PN and liquid consumed were higher in the low versus high nicotine condition (main effect of nicotine, P < 0.05). Urge to vape and withdrawal symptoms were lower, and nicotine intake was higher, in the high nicotine condition (main effects of nicotine: P < 0.01). While acrolein levels did not differ, there was a significant nicotine × power interaction for formaldehyde (P < 0.05). CONCLUSIONS: Use of a lower nicotine concentration e-liquid may be associated with compensatory behaviour (e.g. higher number and duration of puffs) and increases in negative affect, urge to vape and formaldehyde exposure.

E-cigarette puffing patterns associated with high and low nicotine e-liquid strength: effects on toxicant and carcinogen exposure.

BACKGROUND: Contrary to intuition, use of lower strength nicotine e-liquids might not offer reduced health risk if compensatory puffing behaviour occurs. Compensatory puffing (e.g. more frequent, longer puffs) or user behaviour (increasing the wattage) can lead to higher temperatures at which glycerine and propylene glycol (solvents used in e-liquids) undergo decomposition to carbonyl compounds, including the carcinogens formaldehyde and acetaldehyde. This study aims to document puffing patterns and user behaviour associated with using high and low strength nicotine e-liquid and associated toxicant/carcinogen exposure in experienced e-cigarette users (known as vapers herein). METHODS/DESIGN: A counterbalanced repeated measures design. PARTICIPANTS: Non-tobacco smoking vapers; have used an e-cigarette for ≥3 months; currently using nicotine strength e-liquid ≥12mg/mL and a second or third generation device. INTERVENTION: This study will measure puffing patterns in vapers whilst they use high and low strength nicotine e-liquid under fixed and user-defined settings, each for a week. The 4 counterbalanced conditions are: i) low strength (6mg/mL), fixed settings; ii) low strength user-defined settings; iii) high strength (18mg/mL) fixed settings; iv) high strength user-defined settings. Biomarkers of exposure to toxicants and carcinogens will be measured in urine. In the second phase of this study, toxicant yields will be measured in aerosol generated using a smoking machine operated to replicate the puffing behaviours of each participant. PRIMARY OUTCOMES: i) Puffing patterns (mean puff number, puff duration, inter-puff interval and mL of liquid consumed) and user behaviour (changes to device settings: voltage and air-flow) associated with using high and low strength nicotine e-liquid. ii) Toxicant/carcinogen exposure associated with the puffing patterns/device settings used by our participants. SECONDARY OUTCOMES: i) Subjective effects. ii) comparisons with toxicant exposure from tobacco smoke (using documented evidence) and with recommended safety limits. SAMPLE SIZE: Twenty participants. DISCUSSION: The findings will have important implications for public health messaging regarding the relative risks and subjective effects associated with using high and low strength nicotine e-liquid, and for policy makers regarding regulations on nicotine concentrations in e-liquids.

Self-titration by experienced e-cigarette users: blood nicotine delivery and subjective effects.

RATIONALE: Self-titration is well documented in the tobacco literature. The extent to which e-cigarette users (vapers) self-titrate is unknown. OBJECTIVE: This study explored the effects of high and low nicotine strength liquid on puffing topography, nicotine delivery and subjective effects in experienced vapers. METHODS: Eleven experienced male vapers completed 60 min of ad libitum vaping under low (6 mg/mL) and high (24 mg/mL) nicotine liquid conditions in two separate sessions. Measurements included puffing topography (puff number, puff duration, volume of liquid consumed) and changes in plasma nicotine levels, craving, withdrawal symptoms, self-reported hit, satisfaction and adverse effects. RESULTS: Liquid consumption and puff number were higher and puff duration longer, in the low nicotine strength condition (all ps < 0.01). The mean difference in nicotine boost from baseline in the low condition was 8.59 (7.52) ng/mL, 16.99 (11.72) ng/mL and 22.03 (16.19) ng/mL at 10, 30 and 60 min, respectively. Corresponding values for the high condition were 33.77 (34.88) ng/mL, 35.48 (28.31) ng/mL and 43.57 (34.78) ng/mL (ps < 0.05). There were no statistically significant differences between conditions in self-reported craving, withdrawal symptoms, satisfaction, hit or adverse effects. CONCLUSIONS: Vapers engaged in compensatory puffing with lower nicotine strength liquid, doubling their consumption. Whilst compensatory puffing was sufficient to reduce craving and withdrawal discomfort, self-titration was incomplete with significantly higher plasma nicotine levels in the high condition.

Bioanalysis for plasma protein binding studies in drug discovery and drug development: views and recommendations of the European Bioanalysis Forum.

Plasma protein binding (PPB) is an important parameter for a drug's efficacy and safety that needs to be investigated during each drug-development program. Even though regulatory guidance exists to study the extent of PPB before initiating clinical studies, there are no detailed instructions on how to perform and validate such studies. To explore how PPB studies involving bioanalysis are currently executed in the industry, the European Bioanalysis Forum (EBF) has conducted three surveys among their member companies: PPB studies in drug discovery (Part I); in vitro PPB studies in drug development (Part II); and in vivo PPB studies in drug development. This paper reflects the outcome of the three surveys, which, together with the team discussions, formed the basis of the EBF recommendation. The EBF recommends a tiered approach to the design of PPB studies and the bioanalysis of PPB samples: 'PPB screening' experiments in (early) drug discovery versus qualified/validated procedures in drug development.

In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium.

BACKGROUND: At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity. RESULTS: The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels. CONCLUSION: The results demonstrate that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled.

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.

Interlaboratory comparability of serum cotinine measurements at smoker and nonsmoker concentration levels: a round-robin study.

INTRODUCTION: Cotinine, the primary proximate metabolite of nicotine, is commonly measured as an index of exposure to tobacco in both active users of tobacco and nonsmokers with possible exposure to secondhand smoke (SHS). A number of laboratories have implemented analyses for measuring serum cotinine in recent years, but there have been few interlaboratory comparisons of the results. Among nonsmokers exposed to SHS, the concentration of cotinine in blood can be quite low, and extensive variability in these measurements has been reported in the past. METHODS: In this study, a group of seven laboratories, all experienced in serum cotinine analysis, measured eight coded serum pools with concentrations ranging from background levels of about 0.05 ng/ml to relatively high concentrations in the active smokers range. All laboratories used either gas-liquid chromatography with nitrogen-phosphorus detection or liquid chromatography with mass spectrometric detection. RESULTS: All seven laboratories reliably measured the cotinine concentrations in samples that were within the range of their methods. In each case, the results for the pools were correctly ranked in order, and no significant interlaboratory bias was observed at the 5% level of significance for results from any of the pools. DISCUSSION: We conclude that present methods of chromatographic analysis of serum cotinine, as used by these experienced laboratories, are capable of providing accurate and precise results in both the smoker and the nonsmoker concentration range.

Changes in environmental tobacco smoke (ETS) exposure over a 20-year period: cross-sectional and longitudinal analyses.

AIMS: To examine long-term changes in environmental tobacco smoke (ETS) exposure in British men between 1978 and 2000, using serum cotinine. DESIGN: Prospective cohort: British Regional Heart Study. SETTING: General practices in 24 towns in England, Wales and Scotland. PARTICIPANTS: Non-smoking men: 2125 studied at baseline [questionnaire (Q1): 1978-80, aged 40-59 years], 3046 studied 20 years later (Q20: 1998-2000, aged 60-79 years) and 1208 studied at both times. Non-smokers were men reporting no current smoking with cotinine < 15 ng/ml at Q1 and/or Q20. MEASUREMENTS: Serum cotinine to assess ETS exposure. FINDINGS: In cross-sectional analysis, geometric mean cotinine level declined from 1.36 ng/ml [95% confidence interval (CI): 1.31, 1.42] at Q1 to 0.19 ng/ml (95% CI: 0.18, 0.19) at Q20. The prevalence of cotinine levels < or = 0.7 ng/ml [associated with low coronary heart disease (CHD) risk] rose from 27.1% at Q1 to 83.3% at Q20. Manual social class and northern region of residence were associated with higher mean cotinine levels both at Q1 and Q20; older age was associated with lower cotinine level at Q20 only. Among 1208 persistent non-smokers, cotinine fell by 1.47 ng/ml (95% CI: 1.37, 1.57), 86% decline. Absolute falls in cotinine were greater in manual occupational groups, in the Midlands and Scotland compared to southern England, although percentage decline was very similar across groups. CONCLUSIONS: A marked decline in ETS exposure occurred in Britain between 1978 and 2000, which is likely to have reduced ETS-related disease risks appreciably before the introduction of legislation banning smoking in public places.

Relative bioavailability and pharmacokinetics of two oral formulations of docosahexaenoic acid/eicosapentaenoic acid after multiple-dose administration in healthy volunteers.

OBJECTIVES: To assess the comparative pharmacokinetic profile and bioavailability of docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) after multiple-dose administration of a new oral formulation (test formulation) and a commercially available reference formulation in healthy subjects. METHODS: Forty-eight healthy subjects received a 28-day oral treatment with DHA/EPA in the form of either the test or the reference product according to an open-label, randomized, parallel-group design. Both formulations were given t.i.d. at 8-h intervals at a dose of 3.0 g/day. Steady-state DHA and EPA concentrations in plasma and lysed whole blood were measured by gas-liquid chromatography at baseline and after 7, 14, 21 and 28 days of treatment. Kinetic parameters were compared both after subtraction of baseline concentrations and by using baseline concentrations as a covariate. RESULTS: For both DHA and EPA, plasma and RBC concentrations measured from day 7 to day 28 were significantly higher than at baseline and did not differ significantly between the two products. On day 28 the plasma DHA concentration on average doubled the baseline level after administration of test and reference product, while there was a 10-fold increase in EPA plasma concentration. When the assessment was performed using baseline values as covariate, test-to-reference ratios for area under the curve (AUCss(0-8)) and for peak concentration (Css(max)) after the last administration on day 28 met bioequivalence criteria (i.e., 90% confidence intervals within 0.80-1.25 for AUCss(0-8) ratios, and within 0.75-1.33 for Css(max) ratios). When the assessment was conducted after subtraction of baseline values, the 90% confidence intervals for Css(max) ratios were within the bioequivalence range, whereas the intervals for AUCss(0-8) ratio were borderline for bioequivalence. CONCLUSION: The two formulations tested were similarly effective in increasing DHA and EPA concentrations in plasma and lysed whole blood, and showed comparable bioavailability for both active components.