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Aim: Determination of plasma protein binding ( PPB) is considered vital for better understanding of pharmacokinetic and pharmacodynamic activities of drugs due to the role of free concentration in pharmacological response. Methodology & results: Solid-phase microextraction (SPME) was investigated for measurement of PPB from biological matrices and compared with a gold standard approach (rapid equilibrium dialysis [RED]). Discussion & conclusion: SPME-derived values of PPB correlated well with literature values, and those determined by RED. Respectively, average protein binding across three concentrations by RED and SPME was 33.1 and 31.7% for metoprolol, 89.0 and 86.6% for propranolol and 99.2 and 99.0% for diclofenac. This study generates some evidence for SPME as an alternative platform for the determination of PPB.

Background: The hemaPEN is a liquid microsampling device for the reproducible collection and storage of blood samples as dried blood spots, for subsequent quantitative analysis. Materials & methods: We examined the device’s ability to collect accurate and precise blood volumes, at different hematocrit levels, via in vitro studies using acetaminophen in human blood. We also investigated the impact of different user training approaches on device performance. Results: The hemaPEN demonstrated acceptable volumetric accuracy and precision, regardless of the training medium used. Issues with apparent hematocrit-dependent bias were found to be associated with the extraction process, rather than the volumetric performance of the device. Conclusion: The hemaPEN is capable of readily producing high quality blood microsamples for reproducible and accurate quantitative bioanalysis.

There is growing interest in the implementation of microsampling approaches for the quantitation of circulating concentrations of analytes in biological samples derived from nonclinical and clinical studies involved in drug development. This interest is partly due to the ethical advantages of taking smaller blood volumes, particularly for studies in rodents, children and the critically ill. In addition, these technologies facilitate sampling to be performed in previously intractable locations and occasions. Further, they enable the collection of samples for additional purposes (extra time points, biomarkers, sampling during a clinical event, etc). This article gives a comprehensive insight to the utilization of these approaches in drug discovery and development, and provides recommendations for best practice for nonclinical, clinical and bioanalytical aspects.

A previous Senior Editor, and the present Senior Editor of Bioanalysis reflect on their journeys in the field of bioanalysis, and with the journal. They discuss the evolution and progress of journal since its launch 10 years ago, and where they would like to see it heading in the future.

There is continuing interest in the development and application of various microsampling technologies for drug development. The AAPS bioanalytical community microsampling subgroup and the European Bioanalysis Forum conducted a survey of their members (39 individual organizations). This gives a snapshot of current practices and demonstrates that implementation of microsampling approaches is becoming increasingly commonplace, but not universal. Greater adoption was observed for nonclinical studies, particularly nonregulatory. A number of respondents reported that they have included microsampling data in regulatory submissions. Another important observation was that where microsampling is employed for clinical studies, dried blood approaches predominate, reflecting the interest in their use where they enable sample collection which is not feasible with standard approaches or to derive richer data sets.

Aim: The quality of quantitative analytical measurements is dependent on the quality of the sample collected, and dried blood spots (DBS) are no exception. As the use of DBS has matured into late-stage clinical drug-development studies, it has become apparent that a simple and straightforward approach in a controlled single-site, first-time-into-human clinic, does not always translate into multicenter clinical studies. Using synthetic blood, a method of training and assessing clinical laboratory staff has been developed to ensure the quality of sampling. Methods: A test kit comprising of synthetic blood, a pipetting aid, blank blood spot card, drying rack and training manual was sent to each clinical site for each technician to assess and approve prior to spotting PK samples. Results: The development of a DBS training kit along with a step-by-step guide has been successfully implemented. Conclusion: The training kit has been 100% successful across three large multisite clinical studies.

Background: Control blood plasma is regularly used in bioanalysis, biomarkers and proteomics, and is often obtained from commercial sources. It has always been assumed that this plasma will be comparable to plasma drawn during a drug development study. Results: When compared using total protein concentrations, plasma from only one species (dog) demonstrated statistical comparability, plasma from all other species tested (human, rabbit, mouse and rat) shows a statistically significant difference. Conclusion: If endogenous components of blood plasma are being measured, or if an assay technique does not significantly limit matrix effects, any assay controls should be prepared using control plasma from the drug development site, or using commercial plasma that has been screened against drug development site plasma.

Aims: A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. Materials & methods: An average blood volume of 10.6 μl was absorbed by the samplers across the different HCTs investigated (20–65%). Results: No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. Conclusion: The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years’ editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies’ input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.

The portfolios of pharmaceutical companies have diversified substantially over recent years in recognition that monotherapies and/or small molecules are less suitable for modulating many complex disease etiologies. Furthermore, there has been increased pressure on drug-development budgets over this same period. This has placed new challenges in the path of bioanalytical scientists, both within the industry and with contract research organizations (CROs). Large pharmaceutical, biotechnology and small-medium healthcare enterprises have had to make important decisions on what internal capabilities they wish to retain and where CROs offers a significant strategic benefit to their business model. Our journey has involved asking where we believe an internal bioanalytical facility offers the greatest benefit to progressing drug candidates through the drug-development cycle and where externalization can help free up internal resources, adding flexibility to our organization in order to deal with the inevitable peaks and troughs in workload.

Background: Dried blood spot (DBS) sampling is emerging as a valuable technique in a variety of fields, including clinical and preclinical testing of pharmaceuticals. Despite this popularity, current DBS sampling and analysis processes remain laborious and time consuming. Digital microfluidics, a microscale liquid-handling technique, characterized by the manipulation of discrete droplets on open electrode arrays, offers a potential solution to these problems. Results: We report a new digital microfluidic method for multiplexed extraction and analysis of pharmaceuticals in DBS samples. In the new method, four DBS samples are extracted in microliter-sized droplets containing internal standard, and the extract is delivered to dedicated nanoelectrospray ionization emitters for direct analysis by tandem mass spectometry and selected reaction monitoring. Conclusion: The new method allows for an order of magnitude reduction in processing time and approximately three-times reduction in extraction solvent relative to conventional techniques, while maintaining acceptable analytical performance for most drugs tested.

Background: The aim of the work was to evaluate the sensitivity and reproducibility of real-time reverse transcriptase PCR for quantitative analysis of an oligonucleotide in a biological matrix. A novel approach for the identification of outliers when assessing the suitability of calibration standards and QC samples is investigated. Results: A suitable assay was established for the determination of the oligonucleotide in human plasma over a range of 0.5–100 ng/ml. Conclusion: In these preliminary investigations, the precision and accuracy of the method was established for the quantification of the oligonucleotide in human plasma. It was established that the method was precise and accurate for quantification of the oligonucleotide in human plasma. The acceptability of the data was assessed using a novel three-step process to identify any outliers, involving the use of the Grubbs’ test. The analytical method only requires a small sample volume (

This article discusses the benefits of dried blood spot sampling and the recent issues that have emerged when this technique is used in the regulated quantitative bioanalytical environment. The author explores what the way forward might be for this important technique and what some of the unexpected benefits of this change in sampling methodologies have been.

Aims Dried blood spots (DBS) alongside micro-analytical techniques are a potential solution to the challenges of performing pharmacokinetic (PK) studies in children. However, DBS methods have received little formal evaluation in clinical settings relevant to children. The aim of the present study was to determine a PK model for caffeine using a ‘DBS/microvolume platform’ in preterm infants. Methods DBS samples were collected prospectively from premature babies receiving caffeine for treatment of apnoea of prematurity. A non-linear mixed effects approach was used to develop a population PK model from measured DBS caffeine concentrations. Caffeine PK parameter estimates based on DBS data were then compared with plasma estimates for agreement. Results Three hundred and thirty-eight DBS cards for caffeine measurement were collected from 67 preterm infants (birth weight 0.6–2.11 kg). 88% of cards obtained were of acceptable quality and no child had more than 10 DBS samples or more than 0.5 ml of blood taken over the study period. There was good agreement between PK parameters estimated using caffeine concentrations from DBS samples (CL = 7.3 ml h−1 kg−1; V = 593 ml kg−1; t1/2 = 57 h) and historical caffeine PK parameter estimates based on plasma samples (CL = 4.9–7.9 ml h−1 kg−1; V = 640–970 ml kg−1; t1/2 = 101–144 h). We also found that changes in blood haematocrit may significantly confound estimates of caffeine PK parameters based on DBS data. Conclusions This study demonstrates that DBS methods can be applied to PK studies in a vulnerable population group and are a practical alternative to wet matrix sampling techniques.

Background: The surge in interest in switching from traditionally used wet plasma to dried matrix spot (DMS) sampling and analysis to support pharmaceutical drug development is due to the significant ethical, financial and data quality advantages on offer. Unfortunately these advantages do not extend to sample bioanalysis, as DMS extraction is more complex than the protein precipitation method typically used for wet plasma analysis. Direct elution techniques coupled to HPLC–MS/MS have been identified as a potential means to counter this additional complexity. Results: The robustness and reproducibility of DMS HPLC–MS/MS data generated using a CAMAG DBS–MS 16 prototype automated direct elution instrument has been demonstrated to meet or exceed results obtained using a conventional manual extraction methodology. Conclusion: The data generated suggest that a simple and fast direct elution method of DMS samples that does not require additional sample or extract clean-up, offers sufficiently robust performance to be compatible with high-sample-throughput quantitative analysis. Further evaluation of the technique and the development of more advanced fully automated direct elution instrumentation is fully warranted.

Background: Dried blood spot (DBS) sampling, the collection of whole blood samples on paper, is being evaluated for its use in quantitative analysis. The aim of this investigation was to evaluate the impact of sample hematocrit (HCT) and punch location on assay bias in the measurement of compounds with varying physicochemical properties, when a portion of a DBS sample is analyzed. In addition, the statistical significance of factors such as compound, card type, HCT level, spot position on a card and the location of the punch on the spot were evaluated. Results: The results of this work indicate that for quantitative analysis using a portion of the DBS, notable bias (>15%) exists due to the effect of HCT and nonhomogeneous distribution of compound across the spot. The statistical analysis of results from a split-split-plot designed study showed that the factors of compound, card type and HCT were statistically significant (p < 0.05) both individually and in complex interactions, which affect the accuracy of quantitative concentrations obtained from DBS samples for each compound on each card type. Conclusion: To achieve the acceptance criteria (±15%) for accuracy when removing a portion of the DBS spot for quantitative analysis, HCT and punch location for each selected card type will need to be assessed during validation and sample analysis.

Background: Before shipping and storage, dried blood spot (DBS) samples must be dried in order to protect the integrity of the spots. In this article, we examine the time required to dry blood spot samples and the effects of different environmental conditions on their integrity. Results: Under ambient laboratory conditions, DBS samples on Whatman 903®, FTA® and FTA® Elute substrates are dry within 90 min of spotting. An additional 5% of moisture is lost during subsequent storage with desiccant. When exposed to elevated conditions of temperature and relative humidity, the DBS samples absorb moisture. DBS samples on FTA lose this moisture on being returned to ambient conditions. DBS samples on 903 show no visible signs of deterioration when stored at elevated conditions. However, these conditions cause the DBS to diffuse through the FTA Elute substrate. Conclusion: Blood spots are dry within 90 min of spotting. However, the substrates examined behave differently when exposed to conditions of high relative humidity and temperature, in some cases resulting in the integrity of the substrate and DBS sample being compromised. It is recommended that these factors be investigated as part of method development and validation.

Background: A study was performed to evaluate the suitability of stored EDTA-treated control whole blood for use in the preparation of calibration standards and quality control samples for quantitative bioanalytical methods employing dried blood spot (DBS) samples to support pharmaceutical exposure studies. Results: It has been demonstrated that a storage time of 14 days for control human and animal blood is suitable for producing quantitative analytical results within internationally recognised acceptance criteria for two analytes. Furthermore, blood hemolysis and chill–thaw cycles have been evaluated and shown not to affect bioanalytical results notably. Aggressive mixing techniques can result in rat blood coagulation; however, this does not occur with other species tested and can be affected by the method of blood collection. Conclusion: Control whole blood handled and stored using the recommendations generated from this study will not notably affect quantitative bioanalytical results when used for the preparation of calibration standards and quality control samples for DBS assays. It was demonstrated that control human and animal blood can be stored for periods long enough to effectively eliminate wastage.

Background: A clinical investigation was performed into the practicalities of the collection of blood samples for the determination of drug exposures on filter paper, known as dried blood spot (DBS) sampling using a two-period, single-dose, open-label trial conducted in 11 healthy volunteers who received a single oral dose of paracetamol. Questionnaires relating to the blood sampling and spotting process and tolerability were completed by staff and volunteers. Paracetamol concentrations in DBS samples obtained by venous cannula (DBS-Can) were compared against those from fingerprick (DBS-FP) and fresh whole blood obtained from a cannula (WB-Can). Results: The questionnaires demonstrated that FP and blood spotting was easy to perform and well tolerated and compared favorably with cannula sampling. Paracetamol concentrations in DBS-Can were greater than those in WB-Can (positive bias) except below 8000 ng/ml when both were interchangeable. When comparing DBS-FP to DBS-Can, both the bias and variability differed significantly across the five sampling time points. Conclusion: The study has shown that the DBS technique is practical in the context of a clinical trial. Interchangeability of drug concentrations between blood sampling site and mode of blood collection has to be checked and taken into account when designing pharmacokinetic studies for other compounds.

The CAMAG thin-layer chromatography mass spectrometer (TLC-MS) interface has been assessed as a tool for the direct quantitative bioanalysis of drugs from dried blood spot (DBS) samples, using an MS detector, with or without high-performance liquid chromatography (HPLC) separation. The approach gave acceptable sensitivity, linearity, accuracy, and precision data for bioanalytical validations with and without the inclusion of HPLC separation. In addition, the direct elution technique was shown to increase assay sensitivity for a range of analytes representing a wide "chemical space" for pharmaceutical-type molecules over that obtained by conventional manual extraction of samples (punching of DBS and elution with solvent prior to HPLC-MS analysis). Investigations were performed to optimize extraction time, minimize sample-to-sample carry-over, and compare chromatographic performance. On the basis of this preliminary assessment, it has been demonstrated that the TLC-MS interface has the potential to be an effective tool for the direct analysis of drugs in DBS samples at physiologically relevant concentrations, an approach that could provide significant time and cost savings and greatly simplify bioanalytical procedures compared to current manual practices. Further, the increased sensitivity compared to that of manual extraction may enable the analysis of analytes not currently amenable to DBS sampling due to limitations in assay sensitivity.

Further to the discussions on the bioanalysis of samples generated using microsampling techniques [1–6] and more specifically the ongoing work of the European Bioanalysis Forum (EBF) Liquid Microsampling Consortium [7], this article seeks to highlight some of the ‘philosophical’ aspects around liquid microsampling and to introduce some of the experimental elements that will form part of future efforts by the Consortium. Discussion will be focused on three major areas: sample manipulation, homogeneity of samples and validation of assays. The scope of this manuscript will be liquid microsampling and will not focus on adsorption techniques such as dried blood spots (DBS) and solid phase microextraction. Different microsampling techniques have created great interest from toxicokinetic (TK) and pharmacokinetic (PK) scientists, since they offer the potential to reduce sample volumes for exposure assessment in biofluids. For preclinical studies microsampling can facilitate the generation of serial profiles in rodent exposure evaluation studies, rather than working with composite designs. Microsampling has facilitated the removal of satellite animal groups leading to substantial reductions in the number of animals required and the reduction or elimination of rodent warming. Benefits in the clinical environment include the ability to take reduced sample volumes from pediatric, elderly and critically ill patient populations. In a recent publication the NC3R group proposes the following definition of a blood microsample: the sample should ideally not contain more than 50 μl whole blood [8]. As a consequence the subsequent plasma volumes are 20 μl or lower. Another, more philosophical consideration would be the impact of sampling volume on the animal or subject; for example, 50 μl of blood may not be considered as a ‘microsample’ in the context of juvenile animals or newborn babies. Two major liquid microsampling approaches can be envisaged

Volumetric absorptive microsampling (VAMS) is a novel approach to obtaining a dried blood sample for quantitative bioanalysis that overcomes the area bias and homogeneity issues associated with conventional dried blood spot (DBS) sample when a subpunch is taken. The VAMS sampler absorbs a fixed volume of blood (∼10 μL) in 2–4 s with less than 5% volume variation across the hematocrit range of 20–70% with low tip-to-tip variability. There is no evidence of selective absorption by the tip of the plasma component over whole blood. Recommendations for best practice when collecting samples were developed based upon the results of tests examining a number of potential abuse scenarios.

There are a number of key characteristics of Ukrainian agriculture which are central to the discussion of the reform process. These include the predominance of state ownership and control, lack of commercial management skills, uneven commitment to reform in a decentralized system of responsibility, monopoly, a low level of technology and limited access to resources that facilitate efficient operation of the market. Necessary restructuring will involve three categories of measures including the establishment of a competitive environment, augmenting enterprise response, and macroeconomic stabilization. At present reform has proceeded extremely slowly and speeding the process appears to require Western aid, both financial and technical, to provide a demonstration of the benefits of reform in order to stimulate its more rapid uptake.

Determining circulating drug concentrations in children is an ongoing obstacle to the development of age-appropriate dosing regimens. The requirement for small blood sample volumes in children compared with adults is a significant barrier to obtaining age-specific pharmacokinetic–pharmacodynamic data for this population and hence optimizing the efficacy and safety profile of medicines used by this group. This article discusses the potential for dried blood spot sampling to offer a solution to this issue.

The use of dried blood and dried plasma spots for storage and transportation of samples derived from clinical trials holds the promise to reduce cost, simplify storage and shipping as well as reducing animal usage. From the bioanalysts’ point of view, these dried-paper samples add an extra layer of complexity to the analysis introducing extra matrix effects from the paper itself and sometimes from antiviral treatments applied to the card. In this article we demonstrate the use of the sub-2-µm particle LC–MS/MS for the bioanalysis of samples derived from a dried blood spot. The higher resolution provided by these small-particle separations allowed for greater resolution of the analyte from the endogenous components in blood samples and from the card-treatment chemicals. The method -development process was enhanced by the use of MS, which could simultaneously acquire full scan and multiple reaction monitoring data, allowing resolution from metabolites and endogenous matrix components. The use of this approach produced sensitivity levels in the 50–100 pg/ml range and analysis times in the 1–2 min range, which was five-times more sensitive and three-times faster than HPLC. This throughput and sensitivity makes this approach ideal for the analysis of preclinical and clinical studies derived from dried blood spots.

Background: As hematocrit levels are known to vary between individuals and with disease state, its effect on the physical characteristics of dried blood spot (DBS) samples and on the accurate quantification of analytes within these samples is examined. Results: The area of DBS samples decreases with increasing hematocrit levels in a linear manner on the three cellulose paper substrates tested. Furthermore, a bias was observed in the concentrations of two analytes determined in DBS samples at different hematocrits, which in some cases exceeded acceptable values, particularly for hematocrits outside normal values. Conclusion: If it is expected that the hematocrit of study samples will vary from values considered normal, then its effect on the quantitative determination of an analyte in DBS samples should be investigated as part of the method development and validation. If an unacceptable effect is observed, then this will need to be addressed, by modification of the analytical method, or the inclusion of quality control samples at different hematocrit levels to show control of the assay.

ISSN 1757-6180 10.4155/BIO.10.153 © 2010 Future Science Ltd Bioanalysis (2010) 2(11), 1781 Well here we are again! Following hot on the heels of the special focus issue of Bioana lysis dedicated to dried blood spots in August 2010, we have another special issue concentrating on the same subject. The large volume of highquality contributions in these two editions and the numerous dedicated conference sessions and workshops on this subject in the last 2 years is rapidly contributing to a broader understanding and acceptance of this technique by the drug bioana lysis community. However, the work of this community is increasingly highly regulated, which naturally leads to a conservative approach and in-depth questioning of any new technologies. This is resulting in a number of members of the community holding back on implementing any real change, whilst they wait for others to trip up, or for the regulators to make a proclamation etched in stone about the suitability of the technique. Whilst this may at times be ‘irritating’ to the leaders who just want to get out there and do it – “isn’t it obvious?” – this is an understandable reaction that must be heard. We need to take the time to thoroughly understand the technique and to perform the best science possible. However, it is my belief that we should not let all the understandable questions and reservations bury the pure fact that there are real benefits to be had with this technique. Standing still is not an option. The pressure from society to deliver ethical and environmentally friendly solutions is ever present. Added to this is the need of the pharmaceutical industry and healthcare providers to get better value for their money. Dried blood spot sampling delivers on all these aspects in spades and so will become an accepted part of the drug-development landscape of the future. The revolution is here! Anyway, enough of my rant! I welcome you to this special focus issue of Bioana lysis dedicated to dried blood spot sampling. I would like to thank the authors for sharing with us their high-quality science and discussion. I would also like to thank the dedicated and hard work of the editorial staff for putting together this special issue.

Exposure of female Nucella lapillus to tributyltin (TBT) in seawater at a concentration of 40 ng Sn/litre led to accumulation of TBT in the tissues, and an increase in penis length compared to control animals. Investigation of the steroid concentrations by radioimmunoassay revealed a significant increase in testosterone after 28 days exposure to TBT. No significant effect was noted upon the amounts of progesterone or estradiol-17β. When testosterone was injected into female N. lapillus in the absence of TBT, penis length was found to increase significantly. These observation suggest that there may be an association between the change in testosterone titre in response to exposure to TBT and the development of imposex in the dogwhelk.

This paper explores the linkages between structural adjustment and the liberalization of agricultural marketing in sub‐Saharan Africa (SSA). It examines the nature of structural problems, which have been manifested in stagnating output and balance of payments difficulties. Policy responses are described, in terms of short term macroeconomic stabilization and longer term growth‐oriented adjustment policies. The process of structural adjustment in Africa is surveyed, and policies and stages in the liberalization of markets are considered. Debates on agricultural marketing in SSA are reviewed. This leads into an analysis of the specific issues which arise while marketing policy reform proceeds as an integral part of structural adjustment strategies.

Toxicokinetic analysis is an essential part of nonclinical drug development. Advances in bioanalytical techniques have opened up the potential to use smaller sample volumes (microsamples) to assess drug exposure in blood, plasma and/or serum. Microsampling can increase the amount of nonclinical safety information available, improve its validity by linking toxic effects to drug exposure in individual animals and represents the most significant opportunity to reduce animal use in toxicology studies in the short term. In May 2013, a workshop was held with 80 delegates from 33 companies with the aim of sharing information and knowledge on microsampling technologies. This article covers the discussions at the workshop, current practice in the industry, regulatory experiences and the future direction of microsampling across drug development.