Search

While low-volume sampling technologies offer numerous advantages over venipuncture, implementation in clinical trials poses technical and logistical challenges. Bioanalytical methods were validated for measuring the concentration of crenezumab and etrolizumab in dried blood samples collected using Mitra and Tasso-M20. The data generated demonstrate that the concentrations of crenezumab and etrolizumab in dried blood collected by either device could be determined using calibrators prepared in serum. Drug concentrations from dried blood were converted to serum concentrations using patient hematocrit levels. Contract Research Organization experience in sample handling and analysis allowed us to compare differences between various low-volume sampling technologies. This study evaluated challenges and presented potential solutions for use of different low-volume sampling technologies for pharmacokinetic analysis.

Background: Alternatives to phlebotomy in clinical trials increase options for patients and clinicians by simplifying and increasing accessibility to clinical trials. The authors investigated the technical and logistical considerations of one technology compared with phlebotomy. Methodology: Paired samples were collected from 16 donors via a second-generation serum gel microsampling device and conventional phlebotomy. Microsamples were subject to alternative sample handling conditions and were evaluated for quality, clinical testing and proteome profiling. Results: Timely centrifugation of blood serum microsamples largely preserved analyte stability. Conclusion: Centrifugation timing of serum microsamples impacts the quality of specific clinical chemistry and protein biomarkers. Microsampling devices with remote centrifugation and refrigerated shipping can decrease patient burden, expand clinical trial populations and aid clinical decisions.

Aim: A sensitive and selective method for the determination of PF-07059013 in dried blood collected by Mitra™ tips was developed and qualified from 50 to 50,000 ng/ml. Materials & methods: PF-07059013 is isolated from 10 μl of human dried blood by extraction with methanol and analyzed by HPLC-MS/MS. Results & conclusions: In addition to routine validation elements, impact of hematocrit and Mitra tip's lot-to-lot variation on assay accuracy were evaluated. The qualified method was used in one clinical study with excellent performance. Correlation coefficient between blood concentrations obtained from liquid-incurred blood samples and dried-incurred blood samples is 0.95. Clinical Trial Registration: NCT04323124 (ClinicalTrials.gov).

Aim: Develop and validate a volumetric absorptive microsampling (VAMS)-based LC-MS/MS method to support the bioanalysis of amino acid and carboxylic acid biomarkers in mouse whole blood. Method: Mouse whole blood was collected using a 10 μl VAMS device. The analytes in VAMS were extracted and analyzed using an LC-MS/MS method. Results: The VAMS-based LC-MS/MS assay exhibited a linearity range of 10.0-10,000 ng/ml with acceptable precision and accuracy and consistent recovery. The analyte stability in mouse whole blood VAMS was shown for 7 days at ambient conditions and at -80°C, as well as with three freeze/thaw cycles. Conclusion: A simple and robust VAMS-based LC-MS/MS method was developed and further validated for simultaneous bioanalysis of nine biomarkers in mouse whole blood.

Background: Capillary microsampling (CMS) has been used for quantitative bioanalysis of small molecules; however, there is no report of applying this technique in the bioanalysis of antisense oligonucleotides (ASOs). Results: A CMS liquid chromatography-tandem mass spectrometry method was successfully developed and validated for the quantification of ASO1 in mouse serum. The validated method was applied in a safety study in juvenile mice. Equivalent performance between CMS samples and conventional samples was demonstrated in the mouse study. Conclusion: This work is the first to report using CMS for liquid chromatography-tandem mass spectrometry quantitative bioanalysis of ASOs. The validated CMS method was successfully applied to support good laboratory practice safety studies in mice and the CMS strategy has subsequently been applied to other ASOs.

Aim: To develop a nondestructive method for the estimation of hematocrit (HCT) in dried blood spots (DBSs). Materials & methods: Standards and controls were created (HCT range: 0.20-0.50 l/l) and DBS scanned using a flatbed scanner. Gray values and pixel areas were analyzed with open-source software to estimate HCT and volume, respectively. HCT obtained in whole blood using hematological analyzer was compared with DBS scanner method (n = 50). Results: Between-run precision was 4.7-10.2% and between-run accuracy was 89.6-102.1%. In the hematological instrument comparison, 96% of the patient sample results were within ±15%. Conclusion: The nondestructive method can be used to exclude patient DBS samples with extreme HCT levels from further analysis and avoid bias on measured concentration.

Aim: With microsamples of blood, full pharmacokinetic profiles from individual animals can be obtained as an alternative to the sparse-sampling approach, where conventional volume samples from several animals are required. However, microsamples require assays that are more sensitive. Methods: The sensitivity of the LC-MS assay was increased 47-fold using microflow LC-MS. Results & conclusion: By analyzing both microsamples and conventional samples from the same animals, it is demonstrated that sparse-sampling profiles can be nonrepresentative of the full profiles. This bias can affect the tested treatment by increasing or reducing its apparent effect. Microsampling enables unbiased results compared with sparse-sampling. An increase in assay sensitivity to balance the low sample volumes was achievable by microflow LC-MS.

Aims: An automated method for the measurement of blood tacrolimus on volumetric absorptive microsampling (VAMS) devices was developed. Materials & methods: VAMS devices prepared by the automated method were compared with those prepared by the existing manual method (n = 284; mean concentration: 8.0 μg/l; range: 0.6-18.1). Results: The performance of both methods was comparable. Passing-Bablok regression demonstrated an acceptable correlation (y = -0.449 + 1.06x). Bland-Altman analysis demonstrated acceptable agreement (mean bias: -0.007 μg/l; standard deviation: 1.536). Automation reduced operator touch time by 40 min (48-sample batch). Conclusion: Automated preparation of VAMS devices reduced touch time and improved process consistency, facilitating high-throughput testing and transformation of existing laboratory workflows. Automation did not improve precision for VAMS devices but did so for liquid blood samples.

Volumetric absorption microsampling devices offer minimally invasive and user-friendly collection of capillary blood in volumes as low as 10 μl. Herein we describe the assay validation for determination of the selective estrogen receptor degrader giredestrant (GDC-9545) in dried human whole blood collected using the Mitra ® and Tasso-M20 devices. Both LC-MS/MS assays met validation acceptance criteria for the linear range 1-1000 ng/ml giredestrant. Mitra and Tasso-M20 samples were stable for 84 and 28 days at ambient conditions, respectively, and for 7-9 days at 40 and -70°C. Blood hematocrit, hyperlipidemia and anticoagulant did not impact quantitation of giredestrant. These validated assays are suitable for the determination of giredestrant in dried blood samples collected using Mitra and Tasso-M20 microsampling devices.

Background: Dried blood spot sampling has been reported for on-site collection of specimens, but measurements are affected by blood hematocrit, and special handling is required, especially for forensic applications. The hemaPEN ® blood collection device was developed to produce spots with constant volume. Results: Linearity between 1 and 500 ng/ml was shown for cocaine and the metabolites benzoylecgonine and cocaethylene. The assay demonstrated acceptable precision and accuracy, and analytes were stable for 7 days when kept inside hemaPEN devices. Accuracy of the assay was affected by hematocrit but was within acceptable limits. Conclusion: Use of the hemaPEN, which retains dried blood within the device, could be advantageous for the quantification of illicit drugs in capillary blood compared with conventional dried blood spot collection.

COVID-19 led to changes in the way blood samples are collected. As societies were isolated to control viral spread, access to facilities became limited. Remote sample collection with a volumetric microsampling approach, using Mitra ® devices based on VAMS ® technology, proved to be highly effective. It allowed people to collect high-quality samples at home and post them to a laboratory. This enabled scientists to conduct large serosurveillance studies, with results showing that seroprevalence of COVID-19 was higher than initially expected. Furthermore, remote microsampling studies by several institutions were conducted to measure the relationship between antigen levels and antibody response and duration. VAMS technology was also used in COVID-19 clinical trials. In summary, the independent research reviewed in this paper proved that VAMS is an effective sample collection alternative.

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: Low volume sampling technologies have come a long way; however, their uptake has been slow due to logistical and perceived implementation challenges. Additional studies are needed to overcome these barriers. Materials & methods/results: Here we present two studies where different sampling technologies were evaluated to determine the feasibility of their implementation. First, we evaluated pharmacokinetic profiles for anti-gD in rats using three tail bleed sampling methods, glass capillary tubes, Shimadzu MSW2 ®  and the Neoteryx Mitra ® microsampler. Second, we evaluated two low volume-sampling methods to measure drug levels from patients treated with anti-A therapeutic. This evaluation used whole blood finger pricks for Neoteryx Mitra and plasma from capillary blood using TASSO OnDemand technology to compare results to established venipuncture collection method. Conclusion: These studies evaluate the feasibility and considerations for implementation of different low volume sampling technologies.

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.

RA Koster currently works as Associate Director of Bioanalytical Science at the LC-MS/MS department at PRA Health Sciences in the Laboratory in Assen, The Netherlands. He is responsible for the LC-MS/MS analytical method development and leads a team of method development analysts and scientists. As global microsampling specialist within PRA he is interested in all developments regarding microsampling and aims to continuously improve microsampling techniques. He has been working in the field of bioanalysis for 19 years, in which he performed and supervised numerous analytical method developments using LC-MS/MS. He started his career in 2001 at Pharma Bio-Research (before it was acquired by PRA) as an LC-MS/MS analyst. In 2005, he moved to the University Medical Center Groningen where he focused on the development and validation of analytical methods for drugs and drugs of abuse in matrices like blood, plasma, hair, saliva, dried blood spots and volumetric absorptive microsampling with LC-MS/MS. In 2015 he obtained his PhD on the subject 'The influence of the sample matrix on LC-MS/MS method development and analytical performance'. In 2017, he started as Senior Scientist at PRA Health Sciences and in 2019, he accepted his current role of Associate Director of Bioanalytical Science. He is a (co-)author of more than 35 publications.

Aim: Evaluation of a novel microsampling device for its use in clinical sample collection and biomarker analysis. Methodology: Matching samples were collected from 16 healthy donors (ten females, six males; age 42 ± 20) via K2EDTA touch activated phlebotomy (TAP) device and phlebotomy. The protein profile differences between sampling groups was evaluated using aptamer-based proteomic assay SomaScan and selected ELISA. Conclusion: Somascan signal concordance between phlebotomy- and TAP-generated samples was studied and comparability of protein abundances between these blood sample collection methods was demonstrated. Statistically significant correlation in selected ELISA assays also confirmed the TAP device applicability to the quantitative analysis of protein biomarkers in clinical trials.

Background. Systemic glucocorticoids are prohibited in-competition by the World Anti-Doping Agency. Here, we describe an original microsampling workflow for the quantitation of three endogenous (cortisol, corticosterone and cortisone) and three exogenous (dexamethasone, methylprednisolone and fludrocortisone) corticosteroids in 30 μl of human urine. Materials & methods. Microsampling was carried out by dried urine spot (DUS) sampling and volumetric absorptive microsampling (VAMS), followed by solvent extraction and LC-MS/MS analysis. Results & conclusion: Good linearity (r 2  > 0.9989) was obtained for all analytes; extraction yields (>81%), precision (RSD < 8.6%) and matrix effect (<12%) were satisfactory. Microsample stability at room temperature was good (analyte loss <15% after 3 months). Data obtained from real urine microsample analysis were compared with those of fluid urine, providing very good agreement (r 2  > 0.9991).

Aim: A novel automated serial dried blood spot (DBS) sampler, 'Fluispotter', was tested for its sampling performance. Materials & methods: An LC-MS/MS method was developed for the analysis of cortisol in DBS samples serially spotted by Fluispotter. The cortisol concentrations in 148 paired DBS and plasma samples were compared across a hematocrit (HCT) range of 22-55%. Results: The interassay accuracy and precision were <10%. Overall assay bias was negligible across the HCTs tested when analyzing the whole-spot DBS samples. The accuracy and precision of the blood volume in 10 μl DBS samples spotted by Fluispotters and micropipettes were within 3%. Deming regression and Bland-Altman analysis showed a good agreement of DBS-predicted and measured plasma cortisol. Conclusion: The Fluispotter performed serial sampling with high accuracy and precision of the sample blood volume.

Aim: Microflow tandem mass spectrometry-based methods have been proposed as options to improve sensitivity and selectivity while improving sample utility and solvent consumption. Here, we evaluate a newly introduced microflow source, OptiFlow™, for quantitative performance. Results/methodology: We performed a comparison of the OptiFlow and IonDrive™ sources, respectively, on the same triple quadrupole mass spectrometer. The comparison used a neat cocktail of commercially available drugs and extracted plasma samples monitoring midazolam and alprazolam metabolites. Microflow produced a 2-4× signal increase for the neat drug cocktail and a 5-10× increase for extracted plasma samples. Conclusion: The OptiFlow method consistently gave increased signal response relative to the IonDrive method and enabled a better lower limit of quantitation for defining phamacokinetics.

Aim: Collection and quantitative analysis in dry blood using volumetric absorptive microsampling (VAMS™) potentially offers significant advantages over conventional wet whole blood analysis. This manuscript explores their use for pediatric sampling and explores additional considerations for the validation of the bioanalytical method. Results: HPLC-MS/MS methods for the determination of midazolam and its major metabolite 1-OH midazolam in both whole wet blood, and dry blood collected on VAMS were developed, validated, and used to support an observational clinical study to compare pharmacokinetic parameters in pediatric patients. Conclusion: Validation data met internationally accepted guideline criteria. A strong correlation was observed in calculated concentrations between wet and dry test samples, indicating that VAMS is a suitable technique for use in pediatric clinical studies.

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.

Following the completion of a detailed experimental protocol into the potential inhomogeneity of capillary liquid microsamples, which was performed at seven European Bioanalysis Forum member companies, the summary and conclusion on the data are reported here. It has been demonstrated that it is possible to generate homogeneous samples using these microsampling techniques; that the resultant microsamples can be accurate and precise and that capillary liquid microsampling data can be consistent with conventional larger volume plasma samples. However, the data contain some variability which is contributed to by the different range of experiences that each investigating site had with these techniques. Therefore, knowledge of the compounds, well-designed experiments and experience with these techniques are essential for the delivery of high quality data.

Aim: Microsampling in preclinical pharmacokinetics (PK) studies is currently widely adopted across the pharmaceutical industry. Materials & methods: The European Bioanalysis Forum liquid microsampling consortium member companies assessed the accuracy and precision of handheld pipettes and microcapillaries at volumes of less than 10 μl. The following key factors on pipetting performance were also evaluated: Pipette type (positive displacement, air displacement and microcapillary), experience of user and the liquid type. Water was selected as a best-case scenario for accuracy and precision determination and blood plasma as a 'real world' bioanalysis sample type. Conclusion: Accuracy and precision on the pipetted volume decreased at lower volumes and experienced laboratory technicians performed better compared with the infrequent users. With respect to the pipetting devices used, microcapillaries showed better or equivalent accuracy and precision compared with handheld pipettes across the volume range 1-8 μl independent of the matrix used.

Aim: The aim of this study was to develop and validate a LC-MS/MS assay for tacrolimus, sirolimus, everolimus, cyclosporin A and mycophenolic acid using volumetric absorptive microsampling tips as a sampling device and to investigate the effect on the recoveries of the analyte concentration in combination with the hematocrit (HT), which included temsirolimus (a structural analog). Results: The maximum observed overall bias was 9.6% for the sirolimus LLOQ, while the maximum overall coefficient of variation was 8.3% for the everolimus LLOQ. All five immunosuppressants demonstrated to be stable in the volumetic absorbtive microsampling tips for at least 14 days at 25°C. Biases caused by HT effects were within 15% for all immunosuppressants between HT levels of 0.20 and 0.60 l/l, except for cyclosporin A, which was valid between 0.27 and 0.60 l/l. Reduced recoveries were observed at high analyte concentrations in combination with low HT values for sirolimus, everolimus and temsirolimus. Conclusion: A robust extraction and analysis method in volumetric absorptive microsampling tips was developed and fully validated. HT- and concentration-related recovery effects were observed but were within requirements of the purpose of the analytical method.

Aim: Alkaptonuria is a disorder of tyrosine metabolism where elevated circulating homogentisic acid causes progressive dysfunction of collagenous tissues. Logistical, financial and patient experience concerns with collection and transport of specimens to central laboratories makes evaluation of microsampling attractive., Methodology: Volumetric absorptive microsampling devices were evaluated for accuracy, precision and drying time. Elution methods were evaluated for several urinary metabolites of interest and stability assessed under multiple conditions. Comparison was performed between dried and liquid specimens via LC-MS/MS., Conclusion: Volumetric absorptive microsampling was found to be highly accurate and precise. Elution methods showed good recovery and reproducibility. Dried and liquid samples compared favorably. Analyte stability was variable, presenting barriers to implementation into routine use in this context.

Conventional sampling techniques for clinical pharmacokinetic studies often require the removal of large blood volumes from patients. This can result in a physiological or emotional burden, particularly for neonates or pediatric patients. Antibiotic pharmacokinetic studies are typically performed on healthy adults or general ward patients. These may not account for alterations to a patient's pathophysiology and can lead to suboptimal treatment. Microsampling offers an important opportunity for clinical pharmacokinetic studies in vulnerable patient populations, where smaller sample volumes can be collected. This systematic review provides a description of currently available microsampling techniques and an overview of studies reporting the quantitation and validation of antibiotics using microsampling. A comparison of microsampling to conventional sampling in clinical studies is included.

Aim: GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated., Methodology: After accurate sampling of 10 μl blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated., Conclusion: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

Aim: Sample collection and preparation are important steps in the metabolomics workflow. Any improvement should be aimed toward making them simpler, faster and more reproducible. This paper describes the evaluation of different types of whole blood microsampling techniques applied in a metabolic fingerprinting study of breast cancer patients., Results: A total of 139, 124 and 128 metabolites were identified in protein precipitation, dried matrix on paper discs and Mitra ® volumetric absorptive microsampling, respectively in 80% of the sample sets, where the quality control samples had a relative standard deviation of <30%. Ten metabolites in breast cancer samples were detected as being altered significantly (p < 0.05)., Conclusion: Our results suggest that whole blood microsampling techniques do not obtain statistically different results in comparison with the metabolomics applied standard reference method of protein precipitation, in terms of the number of detected compounds, the reproducibility and modeling of differences between the groups.

Microsampling has the 3R benefits of refining blood collection techniques while reducing the number of animals required for rodent safety assessment studies. There are significant scientific benefits of correlating study findings with systemic exposure and consequently, there is an industry drive to utilize microsampling in regulatory toxicology studies. This article will focus on capillary microsampling and will discuss the initial considerations before using capillary microsampling, study design and some practicalities of sample collection. From a bioanalytical perspective, the impact of different sample collection and storage workflows on the design of method validation experiments, the suitability of using microsampling for unstable analytes and aspects supporting analysis of peptides, large molecules and for biomarker applications will be discussed.

Aim: Volumetric absorptive microsampler (VAMS) was designed to sample a fixed volume of blood regardless of the hematocrit (HCT) levels. Model compounds with a wide range of hydrophobicity were evaluated for their extraction recoveries from VAMS dried blood samples., Results: For the highly hydrophobic compounds, recoveries with methanol or methanol/acetonitrile extraction were higher compared with using the aqueous mixture of methanol or acetonitrile. Extraction with methanol/acetonitrile (1:1) yielded more consistent recovery across the HCT range of 20-70% than using methanol alone, with good linearity, accuracy and precision achieved from 1 to 2000 ng/ml., Conclusion: An organic solvent sample preparation approach was developed to optimize extraction recovery and minimize the HCT effect on the analysis of VAMS dried blood samples.

The reliability of extraction recovery of an analyte in bioanalysis is fundamentally important for downstream analytical testing. For dried format microsamples, if the recovery changes with time the concentration in clinical samples, derived from calibration standards and alongside quality control samples prepared following different drying protocols, may not reflect the true result. The purpose of this paper was therefore to evaluate changes to extraction recovery across time for one analyte, the glycopeptide antibiotic vancomycin, in plasma using two dried microsampling formats, dried plasma spots and volumetric absorptive microsampling.

Aim: Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS., Methods & Results: We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold., Conclusion: Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.

Background: Quantitative DBS LC-MS/MS assay for midazolam was used to compare two sample collection techniques (venipuncture and finger prick) and the midazolam concentrations measured in plasma samples, DBS and dried plasma spots., Methodology: Midazolam was extracted from DBS cards and compared with whole blood collected from usual venipuncture. Dried plasma spots were also compared with plasma. The blood volume used as well as the temperature impact during the blood and plasma deposits was evaluated. Midazolam was administrated to six healthy subjects during a clinical trial to obtained blood and plasma samples for the statistical comparison., Conclusion: The method for midazolam using DBS was validated and showed an excellent performance. Excellent correlations were observed when the same collection procedures were used.

This Land O'Lakes Conference is presented each year by the Division of Pharmacy Professional Development within the School of Pharmacy at the University of Wisconsin-Madison (USA). The purpose of this 3-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics, metabolites, biologics and biomarkers in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program is a mixture of lectures, interactive discussions and a poster session. This report summarized the presentations at the 16th Annual Conference. 6th Annual Land O'Lakes Bioanalytical Conference, Fluno Center Madison, WI, USA, 13-16 July 2015.