Search

RATIONALE A two-layered polymeric membrane is employed for the formation of separated dried plasma spots from whole blood as an alternative to the direct analysis of whole dried blood spots (DBS). This dried plasma spot (DPS) analysis procedure precludes potential issues of hematocrit differences in whole blood samples while providing pharmokinetic data from plasma rather than whole blood. The described procedure is also semi-automated thus providing a simpler work flow for LC/MS/MS bioanalysis procedures. METHODS Molecular filtration of red blood cells (RBC) from applied microsamples of whole blood fortified with guanfacine and its stable isotope internal standard was accomplished with a two-layer polymeric membrane substrate. The lower membrane surface containing the separated plasma spot was physically separated from the upper membrane followed by semi-automated direct elution of the sample to an online solid-phase extraction (SPE) cartridge followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). RESULTS A two-layer membrane sample preparation substrate produced plasma from whole blood without centrifugation which could be directly eluted for semi-automated LC/MS/MS bioanalysis. Standard curves were constructed by plotting peak area ratios between the analyte and the stable isotope labeled internal standard (SIL-IS) versus the nominal concentration in whole blood. A weighted 1/x2 linear regression was applied to the data from DPS samples. Standard curves were linear over the range 0.25–250 ng/mL human whole blood. The representative regression equation was y = 0.0142x + 0.00248 (R2 = 0.995) for the described DPS assay. CONCLUSIONS The described work demonstrates proof-of-principle using membrane sample preparation techniques to form DPS samples from whole blood for subsequent bioanalysis by LC/MS/MS. This approach has the potential to eliminate the hematocrit issues from the current controversy surrounding validation of DBS assays. Copyright © 2012 John Wiley & Sons, Ltd.

The 4th Open Symposium of the European Bioanalytical Forum entitled ‘Less is More’ was held on 16–18 November 2011 at the Hesperia Tower Hotel, Barcelona, Spain. More than 50 interesting presentations were delivered covering areas with interest for the small- and large-molecule community – biomarker validation; regulations, including an update on new and emerging guidelines and on Global harmonization; technology updates; incurred sample stability; microdosing; dried blood spots and microsampling; challenges of ‘free’ and ‘total’ macromolecule quantification; stability issues in ligand binding assays or anomalous results. In excess of 450 delegates from more than 170 institutes and companies (industry, regulators and academia) from all global regions participated in the open and stimulating discussions. This manuscript provides an overview of the highlights discussed at the meeting.

Background: Dried blood spot (DBS) technology was evaluated for the quantitative determination of guanfacine in human blood in clinical studies. A very sensitive DBS assay has been developed using HPLC coupled with an AB Sciex 5500 QTRAP® (Applied Biosystems/MDS Sciex, ON, Canada) MS system (LC–MS/MS) with a linear calibration range of 0.05 to 25 ng/ml. High-resolution MS using an Exactive Orbitrap® (ThermoFisher, LLC., CA, USA) was compared with the QTRAP using extracted exact mass ion current profiles for guanfacine and its stable-isotope-incorporated internal standard. The sample preparation employed liquid–liquid extraction with methyl t-butyl ether of 5 mm punched DBS card disks, followed by reversed-phase HPLC separation coupled with either MS/MS or high-resolution MS. Routine experiments were performed to establish the robustness of the DBS assay, including precision, accuracy, linearity, selectivity, sensitivity and long-term stability of up to 76 days. In addition, several factors that potentially affect quantitation were investigated, including blood volume for DBS spotting, punch size and punch location. Results: A sensitive research assay with a LLOQ of 0.05 ng/ml was developed and subjected to several components of a method validation common to a regulated bioanalysis procedure employing DBS. This method development and partial validation study determined that spot volume, punch size or punch location do not affect assay accuracy and precision. The DBS approach was successfully applied to a clinical study (a Phase I, randomized, double-blind, placebo-controlled, crossover study to assess the effect of varying multiple oral doses of guanfacine on the pharmacokinetic, pharmacodynamic, safety, and tolerability profiles in healthy adult subjects). The pharmacokinetic profiles for 12 volunteers generated from the DBS assay and from a previously validated plasma assay were compared and were found to be comparable. DBS incurred samples collected from finger prick blood and directly applied to the DBS cards were also analyzed for comparison. Conclusion: From a bioanalytical perspective, DBS sample collection and analysis is a potentially viable alternative for guanfacine determination in clinical studies, utilizing approximately 100 µl of blood per subject profile compared with a few millilitres of blood drawn for conventional plasma bioanalysis.

The European Bioanalysis Forum is a bioanalytical nonprofit organization comprised of European pharmaceutical companies (27 members to date) and currently expanding to include CROs as well. The European Bioanalysis Forum provides a broad European bioanalytical network for the discussion of scientific, technological and regulatory topics of bioanalytical interest. The 3rd Annual Open Symposium was again much anticipated after the two previous successful meetings. The symposium included sessions on thinking outside the ‘commodity’ box, bioanalytical challenges with blood, global harmonization, assay platforms, dried blood spots, immunogenicity, matrix effects, anomalous results, biomarkers and two plenary technology sessions hosted by the Platinum sponsors. Experts and key opinion leaders were invited as guest speakers. A total of 424 delegates registered from 113 companies representing a large percentage of the European bioanalytical community. In addition to 48 oral presentations, 88 posters were presented and there was a vendor exposition of 40 companies.

The European Bioanalysis Forum is a non-profit organization comprised of European pharmaceutical companies (25 members to date). Their activities focus on bringing together managers and scientists in the broad field of bioanalysis to discuss topics related to science, process and regulations. There has been much interest over the past few years in the potential application of dried blood spots as an alternative to traditional plasma collection in pharmacokinetic studies. The success of the technique has been highlighted by several companies. We know that seven of the European Bioanalysis Forum member companies are using dried blood spots intensively and that 22 out of 25 companies are using it or plan to use it very soon, initially in nonregulated studies. However, most companies have less than 1 year of experience with dried blood spots and, beyond the scientific merit, it is less clear just how the technique is perceived by key client groups, such as toxicology, clinical and regulatory authorities. The objective of this symposium was to bring together representatives from all of these client groups as well as a broad bioanalytical audience to discuss the various perspectives on dried blood spots and to try to provide answers to potential issues that still remain. The symposium included sessions on dried blood spots in the nonregulated environment, toxicology and regulatory/QA perspectives, clinical use and bioanalytical applications and tools. There was plenty of time for discussion within the program in tutorials, poster and break-out sessions and the degree of delegate participation reflected the high level of engagement with the topic. A total of 190 delegates attended from more than 80 organizations. In addition to 21 oral presentations, more than 20 posters were presented and there was a vendor exposition of ten sponsor companies.

The European Bioanalysis Forum is a bioanalytical discussion group comprised of European pharmaceutical companies (27 members to date). The membership share a common vision to advance the shared understanding of topical concerns through discussion of scientific, technological and regulatory issues of bioanalytical interest. The 2nd Annual Open Symposium was much anticipated after the success of the first one. The symposium included sessions on the European Medicines Agency draft bioanalytical guidance, challenges in the analysis and validation of biomarkers, metabolite quantification issues, dried blood spot technology, the analysis of proteins and peptides by MS, latest hot topics in ligand-binding assays, unresolved issues in method validation and a plenary session from the Platinum sponsors. Experts and key opinion leaders were invited as guest speakers. A total of 420 delegates (up from 251 delegates in 2008) attended from 160 organizations, representing a large percentage of the European and international bioanalytical community. In addition to 40 oral presentations, 72 posters were presented and there was a vendor exposition of 35 companies.

Following intensive discussions, review, alignment of procedures and multiple surveys among their member companies, the European Bioanalysis Forum (EBF) is providing a recommendation on how to integrate incurred sample reproducibility (ISR) in the bioanalytical process. The recommendation aims to provide guidance throughout the lifecycle of a validated method, including the application of the method in study support. In its recommendation, the EBF considers both the internal discussions with EBF member companies, as well as the input provided in international meetings where ISR was discussed. The ultimate goal of the EBF recommendation is to ensure that bioanalytical methods can provide accurate and reproducible concentration data for pharmacokinetic and/or toxicokinetic evaluation, without any compromise, while safeguarding the optimal use of laboratory resources.

In bioanalysis of small molecules, the analyte concentration in the measured samples should reflect the concentration during sample collection. Precautions may be needed to prevent over- or under-estimation of the obtained result. This might require the addition of stabilizers to prevent degradation or nonspecific binding. For unstable drugs, it is essential to know how analytes can be stabilized before the start of the clinical study. Although the stabilization methods are well documented, the impact of the stabilization on the clinical workflow is not properly addressed. Already during method development, the clinical implications in terms of personnel safety, ease of use, training possibilities and staff capacity should be taken into account, and validation of the bioanalytical method should reflect collection procedures.

Accelerator mass spectrometry (AMS) is being used more widely to provide PK data for early decision making or to generate absolute bioavailability data in later phases of development. Presently, there is no clear consensus on the level of the scientific validation required for these assays. The European Bioanalysis Forum (EBF) has conducted two surveys with its members and presented the results at its 4th Open Symposium. With AMS being used for discrete scientific assessment, method establishment of AMS assays should focus on science rather than trying to fit the assay parameters into validation criteria used for Regulated Bioanalysis guidance, and an amount of freedom of execution and interpretation is needed. Hence, the EBF focuses their recommendation on introducing terminology around scientific qualification or validation to be used in relation to AMS. Guidance is given on which parameters should be investigated when a qualified method is required. The recommendations of the EBF for scientific validation are described herein. The scientific validation of AMS assays will be different to that applied for LC–MS/MS assays, and an example is that accuracy and precision limits, as used for ligand-binding assays, would be more appropriate.

Background: Direct analysis of dried blood spot (DBS) samples was investigated using a prototype semi-automated robotic device that allows the direct elution of sample spots from a DBS paper card to an online SPE cartridge. The eluted SPE samples were analyzed with high-performance LC–MS/MS. Results: A LLOQ of 0.01 ng/ml was achieved with a linear calibration range from 0.01 to 25 ng/ml. Optimal performance data were obtained from spotting the internal standard solution on the card before blood spotting. Internal standard addition from the system injector loop produced intra-assay inaccuracy of -9.0–7.3% and precision of 1.3–8.2%, and inter-assay inaccuracy of -3.5–3.9% and precision of 4.4–8.7%. Conclusion: Results demonstrated the feasibility of a semi-automated online rapid direct elution method that avoids manual extraction for DBS sample analysis using the online DBS-SPE system coupled to LC–MS/MS.

The European Bioanalysis Forum (EBF) is a bioanalytical discussion group comprised of European pharmaceutical companies (26 members to date). The membership shares a common vision to advance the shared understanding of topical concerns through discussion of scientific, technological and regulatory issues of bioanalytical interest. The objective of the open symposium was to reach out to the broader European and global bioanalytical community, to report back on discussions EBF member companies have during their biannual closed meetings and, going forward, to provide guidance and recommendations to the European and global bioanalytical community. Pharmaceutical and biotechnology companies, contract research organisations (CROs), academia and instrument vendors were invited to attend to exchange scientific ideas and information on topical issues of mutual concern. The symposium included sessions on assay validation, metabolite quantification, instrumentation developments and protein quantification and immunogenicity. Experts and key opinion leaders were invited as guest speakers. A total of 251 delegates attended, representing a large percentage of the European bioanalytical community. The majority of attendees represented pharmaceutical companies (36%), CROs (42%), instrument vendors (15%) and academia (4%). In addition to 18 oral presentations, 51 posters were presented and there was a well-supported vendor exposition.