Volumetric Absorptive Microsampling Samples Research Articles

Is the stability of folates in dried blood microsamples sufficient to perform home-sampling studies?

Dried blood microsampling is increasingly used for home-sampling and epidemiological studies because of its multiple advantages, including an often greatly improved analyte stability. However, a critical assessment of the stability under realistic conditions should always be performed as part of the validation, especially for unstable molecules like folates (vitamin B9). Here, the objective was to determine whether folate stability in dried blood microsamples is sufficient to allow the set-up of home-sampling studies for the monitoring of folate status in e.g., women of reproductive age. An extensive set of stability experiments was performed to evaluate the stability of the main folate vitamer 5-methyltetrahydrofolate (5MTHF), its oxidation product MeFOX and the minor non-methyl folate vitamers 10-formylfolic acid (10FoFA), 5,10-methenyltetrahydrofolate (5,10CH+THF) and tetrahydrofolate (THF) in dried blood microsamples using volumetric absorptive microsampling (VAMS) or regular dried blood spots (DBS). The evaluations included (EDTA-anticoagulated blood was collected from a single donor measured in four replicates per condition and time point): (i) the effect of temperature (-20 °C, 4 °C, ambient temperature and 37 °C), (ii) the effect of light (during drying and storage) and humidity, and (iii) the effect of storage under vacuum and pretreatment of the microsamples with stabilizing agents on folate stability. At -20 °C and 4 °C, all folate levels were within 85 to 115% of the baseline value up till two weeks of storage in both VAMS samples and DBS. However, at room temperature the stability of the analyzed folates was only consistently observed up till three days in VAMS samples, and for none of the folates at 37 °C. Humidity had a major impact on 5,10CH+THF stability, but this could be easily improved by using desiccant. Both vacuum treatment and pretreatment of microsamples with 0.1% DL-dithiothreitol and 5% butylated hydroxytoluene improved the stability at room temperature in VAMS samples, but these effects were limited at 37 °C and in DBS. Overall, the stability of the individual folate vitamers proved to be challenging and strongly temperature- and time-dependent. Nonetheless, if controlled transport (temperature and duration) can be assured, the set-up of home-sampling studies to evaluate the folate status using dried blood microsamples can still be beneficial.

Therapeutic drug monitoring of mycophenolic acid (MPA) using volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients: ultra-high-performance liquid chromatography-tandem mass spectrometry analytical method development, cross-validation, and clinical application

BackgroundMycophenolic acid (MPA) is widely used in posttransplant pharmacotherapy for pediatric patients after renal transplantation. Volumetric absorptive microsampling (VAMS) is a recent approach for sample collection, particularly during therapeutic drug monitoring (TDM). The recommended matrix for MPA determination is plasma (PL), and conversion between capillary-blood VAMS samples and PL concentrations is required for the appropriate interpretation of the results.MethodsThis study aimed to validate and develop a UHPLC-MS/MS method for MPA quantification in whole blood (WB), PL, and VAMS samples, with cross and clinical validation based on regression calculations. Methods were validated in the 0.10–15 µg/mL range for trough MPA concentration measurement according to the European Medicines Agency (EMA) guidelines. Fifty pediatric patients treated with MPA after renal transplantation were included in this study. PL and WB samples were obtained via venipuncture, whereas VAMS samples were collected after the fingerstick. The conversion from VAMSMPA to PLMPA concentration was performed using formulas based on hematocrit values and a regression model.ResultsLC–MS/MS methods were successfully developed and validated according to EMA guidelines. The cross-correlation between the methods was evaluated using Passing-Bablok regression, Bland–Altman bias plots, and predictive performance calculations. Clinical validation of the developed method was successfully performed, and the formula based on regression was successfully validated for VAMSMPA to PLMPA concentration and confirmed on an independent group of samples.ConclusionsThis study is the first development of a triple matrix-based LC–MS/MS method for MPA determination in the pediatric population after renal transplantation. For the first time, the developed methods were cross-validated with routinely used HPLC–DAD protocol.Graphical

VAMS-Based Blood Capillary Sampling for Mass Spectrometry-Based Human Metabolomics Studies

Volumetric absorptive microsampling (VAMS) is a recently developed sample collection method that enables single-drop blood collection in a minimally invasive manner. Blood biomolecules can then be extracted and processed for analysis using several analytical platforms. The integration of VAMS with conventional mass spectrometry (MS)-based metabolomics approaches is an attractive solution for human studies representing a less-invasive procedure compared to phlebotomy with the additional potential for remote sample collection. However, as we recently demonstrated, VAMS samples require long-term storage at -80 °C. This study investigated the stability of VAMS samples during short-term storage and compared the metabolome obtained from capillary blood collected from the fingertip to those of plasma and venous blood from 22 healthy volunteers. Our results suggest that the blood metabolome collected by VAMS samples is stable at room temperature only for up to 6 h requiring subsequent storage at -80 °C to avoid significant changes in the metabolome. We also demonstrated that capillary blood provides better coverage of the metabolome compared to plasma enabling the analysis of several intracellular metabolites presented in red blood cells. Finally, this work demonstrates that with the appropriate pre-analytical protocol capillary blood can be successfully used for untargeted metabolomics studies.

Therapeutic Drug Monitoring of Tacrolimus Based on Volumetric Absorptive Microsampling Technique (VAMS) in Renal Transplant Pediatric Recipients-LC-MS/MS Method Development, Hematocrit Effect Evaluation, and Clinical Application.

Tacrolimus (TAC) is post-transplant pharmacotherapy's most widely used immunosuppressant. In routine clinical practice, frequent uncomfortable venipuncture is necessary for whole-blood (WB) collection to check trough TAC levels. Volumetric absorptive microsampling (VAMS) is an alternative strategy to WB collection. In this study, we aimed to validate and develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for TAC quantification in WB and VAMS samples. After extraction with water and protein precipitation, the samples were directly analyzed using LC-MS/MS. Whole-blood and VAMS capillary-blood samples were collected from 50 patients treated with TAC during the follow-up visits. The cross-correlation between the developed methods was evaluated using Passing-Bablok regression and a Bland-Altman bias plot. The matrix effect (ME) and carry-over were insignificant for both scenarios. There was a high correlation between the processes and no significant clinical deviation. LC-MS/MS methods were successfully developed and validated in the 0.5-60 ng/mL calibration range. This study demonstrated and confirmed the utility of VAMS-based TAC monitoring in the pediatric population. This is the first study to directly develop and validate the VAMS LC-MS/MS method for evaluating the hematocrit effect in the pediatric population. The statistical correlation between immunochemical and VAMS-based methods was satisfactory.

Analytical and clinical validation of dried blood spot and volumetric absorptive microsampling for measurement of tacrolimus and creatinine after renal transplantation

BackgroundSerial monitoring of tacrolimus and serum creatinine after renal transplantation is of vital importance. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the estimation of tacrolimus and creatinine, obtained from dried blood spots (DBS) or by volumetric absorptive microsampling (VAMS) was validated and the two sampling strategies were compared with traditional venous sampling. MethodsThe LC-MS/MS assay was validated using a shared extract for the estimation of tacrolimus and creatinine from DBS and VAMS independently. The relationship between the concentrations in DBS/VAMS specimens and in venous samples was assessed using Passing-Bablok (PB) analysis and the bias between the two methods was determined by the Bland Altman (BA) analysis. ResultsThe imprecision and bias of tacrolimus and creatinine estimated from DBS and VAMS samples was <12% and was independent of the hematocrit (Hct). Samples were stable for five days at ambient temperature. From the PB regression analysis, correction equations were generated for the prediction of tacrolimus and creatinine values from DBS and VAMS samples. In a separate cohort of patients for validation, the corrected DBS and VAMS concentrations had a mean (95% CI) bias for tacrolimus of −0.64 (−2.98 to 1.70)% and −0.92 (−3.69 to 1.85)% respectively and for creatinine of 1.00 (−2.73 to 4.72)% and −0.71 (−3.74 to 2.32)% respectively. Using DBS and VAMS respectively, for tacrolimus, 91.8 and 89.8% of patient values and for creatinine, 69.4 and 81.6% of patient values were within the limits of clinical acceptance (within 15% agreement against the venous samples). ConclusionWe conclude that VAMS is the preferred single sampling option for estimating tacrolimus and creatinine in renal transplant patients.

Mercury biomonitoring in German adults using volumetric absorptive microsampling

Mercury (Hg) is a global pollutant and a danger to human health. Human biomonitoring of Hg using a dried blood matrix instead of venous blood sampling for exposure assessment is of growing interest. This study aims to develop, validate, and evaluate the application of volumetric absorptive microsampling (VAMS) for Hg biomonitoring in humans. Sampling, drying, and storage conditions were evaluated during method development. Storage in pre-cleaned glass vials after drying for 2 h in a desiccator ensured analyte stability for at least 4 weeks. Sixty-eight paired capillary VAMS and venous blood samples from volunteers in Munich, Germany, were used for method validation. Hg levels in VAMS and venous blood samples were determined by direct mercury analysis. The limits of detection and quantitation for VAMS were 0.18 and 0.61 µg/l, respectively. However, sensitivity could be improved by using two microsamples for analysis instead of one. Hg levels in VAMS samples correlated very well with Hg levels in venous blood samples (R2 = 0.958). Furthermore, VAMS showed a high accuracy (median recovery: 117%) and precision (median relative standard deviation: 8.7%), especially for Hg concentrations above 1.0 µg/l. In fact, accuracy and precision of VAMS improved with increasing Hg concentrations. In conclusion, VAMS in combination with direct mercury analysis is an accurate and viable alternative for human biomonitoring of Hg.Graphical abstract

Stability of acetylsalicylic acid in human blood collected using volumetric absorptive microsampling (VAMS) under various drying conditions.

Acetylsalicylic acid (ASA) is one of the most commonly used medications in global market, with a risk of intoxication in certain patients. However, monitoring blood drug concentration often requires frequent hospital visits; hence there is an unmet need to increase patient-centricity by conducting blood sampling at home. Volumetric absorptive microsampling (VAMS) is a device that allows collection of homogenous and accurate volume of blood without venipuncture, and can be utilized by patients who are not in hospital settings; but because ASA is prone to hydrolysis and stabilizing reagents cannot be added to VAMS samples, a way to improve sample stability must be developed. The objective of this study was to identify the cause of instability with ASA samples collected by VAMS, and to evaluate ways to improve sample stability. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used for analysis of ASA concentration in whole blood. Samples collected with VAMS were kept under different drying conditions (desiccator, pressurized, nitrogen gas and household vacuum sealer) and were compared to the control samples collected by conventional venous sampling. The recovery of ASA was about 31% of the control when VAMS sample was dried at room temperature, whereas VAMS samples under humidity controlled conditions showed more than 85% of recovery. Our results suggest that adequate level of humidity control was critical to ensure sample stability of ASA, and this humidity control could also be achieved at home using household vacuum sealer, thus enabling patient-centric clinical trials to be conducted.

1104. Comparison of Antibiotic Sampling Techniques: Predicting Plasma Vancomycin Concentrations Using Volumetric Absorptive Microsampling (VAMS) from Capillary and Venous/Arterial Whole Blood

BackgroundTherapeutic drug monitoring (TDM) is paramount to optimize the safety and efficacy of vancomycin (VAN). In children, TDM is challenged by difficulty in obtaining venous samples, impeding timely sampling. We assessed the ability of volumetric absorptive microsampling (VAMS) as a novel, whole blood sampling technique to predict plasma VAN concentrations in plasma.MethodsWe conducted a prospective pilot study among critically ill children prescribed VAN for clinical care. Coincident with VAN TDM in plasma (P), we collected 20 µL of capillary whole blood (C) and venous/arterial whole blood (V) using VAMS. Paired VAMS-P samples drawn >5 mins apart and VAMS samples with over- or under-loaded filter tip on visual inspection were excluded. Plasma concentrations were measured via chemiluminescent immunoassay in the Chemistry Laboratory. VAMS C and V concentrations were measured using LC/MS in the Bioanalytic Core Laboratory. Plasma concentrations were predicted from whole blood VAMS with Passing-Bablok regression using 3 methods: 1) uncorrected VAMS measures, 2) hematocrit-corrected VAMS, and 3) lab-corrected VAMS (Figure 1). We then assessed bias, imprecision, and accuracy of plasma predictions from VAMS (C and V) as compared to coincident P concentrations for each technique (Figure 1).Figure 1. Methods for relating whole blood vancomycin concentrations collected via VAMS to plasma concentrations and measure to evaluate predictive performance. ResultsPaired samples were collected from 31 enrolled subjects (Figure 2), with a median age of 3.3 years (range 0.1-17.9). Measured P concentrations ranged from 4.6 - 54.9 mg/L. 11 C samples (29%) and 3 V samples (10%) were excluded due to collection issues. Prediction results are shown in Figure 3. The 3 prediction techniques had similar performance characteristics, with each method displaying minimal bias (-0.4-2.0%) and reasonable imprecision (13.7-20.2%). The accuracy of prediction of P concentrations using VAMS was better for V than C samples.Figure 2. Flow diagram from sample collection to evaluation. Abbreviations: C-P, capillary VAMS-plasma; V-P, venous/arterial VAMS-plasma; VAMS, volumetric absorptive microsampling.Figure 3. Performance of 3 techniques to predict plasma vancomycin concentrations using whole blood collected via VAMS. ConclusionOur pilot highlights the challenges of using VAMS for TDM. Sample collection issues were common. When VAMS is used, education on collection techniques is imperative. The predictive performance of VAMS was modest and V sampling had higher accuracy than C, although our sample size was small. Larger studies will be needed to further evaluate the predictive performance of the regression equations derived by our study.Disclosures Kevin J. Downes, MD, Merck, Inc. (Grant/Research Support)

Antibody Mediated Immunity to SARS-CoV-2 and Human Coronaviruses: Multiplex Beads Assay and Volumetric Absorptive Microsampling to Generate Immune Repertoire Cartography.

The COVID-19 pandemic is caused by SARS-CoV-2, a novel zoonotic coronavirus. Emerging evidence indicates that preexisting humoral immunity against other seasonal human coronaviruses (HCoVs) plays a critical role in the specific antibody response to SARS-CoV-2. However, current work to assess the effects of preexisting and cross-reactive anti-HCoVs antibodies has been limited. To address this issue, we have adapted our previously reported multiplex assay to simultaneously and quantitatively measure anti-HCoV antibodies. The full mPlex-CoV panel covers the spike (S) and nucleocapsid (N) proteins of three highly pathogenic HCoVs (SARS-CoV-1, SARS-CoV-2, MERS) and four human seasonal strains (OC43, HKU1, NL63, 229E). Combining this assay with volumetric absorptive microsampling (VAMS), we measured the anti-HCoV IgG, IgA, and IgM antibodies in fingerstick blood samples. The results demonstrate that the mPlex-CoV assay has high specificity and sensitivity. It can detect strain-specific anti-HCoV antibodies down to 0.1 ng/ml with 4 log assay range and with low intra- and inter-assay coefficients of variation (%CV). We also estimate multiple strain HCoVs IgG, IgA and IgM concentration in VAMS samples in three categories of subjects: pre-COVID-19 (n=21), post-COVID-19 convalescents (n=19), and COVID-19 vaccine recipients (n=14). Using metric multidimensional scaling (MDS) analysis, HCoVs IgG concentrations in fingerstick blood samples were well separated between the pre-COVID-19, post-COVID-19 convalescents, and COVID-19 vaccine recipients. In addition, we demonstrate how multi-dimensional scaling analysis can be used to visualize IgG mediated antibody immunity against multiple human coronaviruses. We conclude that the combination of VAMS and the mPlex-Cov assay is well suited to performing remote study sample collection under pandemic conditions to monitor HCoVs antibody responses in population studies.

Patient-Centric Assessment of Thiamine Status in Dried Blood Volumetric Absorptive Microsamples Using LC-MS/MS Analysis.

Many populations in low- and middle income countries are at a higher risk of thiamine deficiency, mainly due to the lack of dietary diversification and their reliance on staple crops low in thiamine content, such as polished rice. Unfortunately, symptoms of thiamine deficiency are variable and clinical determination of thiamine status is essential for early diagnosis. Currently, the diagnosis of thiamine deficiency in remote regions is hampered due to several drawbacks related to venous blood collection, for example, cold chain transport. Therefore, we here describe the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of thiamine diphosphate (TDP) in dried blood, using volumetric absorptive microsampling (VAMS). Moreover, by setting up an additional method in liquid blood, the results in VAMS samples could be compared to liquid blood samples. Both methods, employing a simple one-step extraction and fast (2 min) chromatography, were fully validated based on international guidelines. Accuracy (% bias) was below 6.5% for all QC levels. The total imprecision (% CV) was below 13% for both QCs and native blood samples. The recovery of the VAMS samples was not impacted by the hematocrit, within the hematocrit range of 0.20-0.60. Additionally, we showed improved TDP stability in dried blood compared to liquid blood. VAMS samples were stable for 1 week at 60 °C or at high humidity (80%) and for at least 1 month at room temperature. Finally, we demonstrated the commutability of commercial calibrators with authentic blood samples. The validity and applicability of both methods were demonstrated via their successful application on blood samples from healthy volunteers.

Validation of LC-MS/MS method for determination of rosuvastatin concentration in human blood collected by volumetric absorptive microsampling (VAMS).

In light of the shift toward patient-centric clinical trials, a measure of simplifying blood collection process and minimizing the volume of blood samples is on the rise. Volumetric absorptive microsampling (VAMS) is a microsampling device developed for blood sampling in non-hospital settings, which enables accurate hematocrit-independent collection of 10 or 20 µL of whole blood with a simple finger prick. In this study, liquid chromatography (LC)-tandem mass spectrometry workflow for quantification of rosuvastatin after VAMS sampling was developed and validated. The VAMS sample was stabilized by matrix drying and the optimum LC conditions and extraction methods were used to reach adequate sensitivity with lower limit of quantification verified at 1 ng/mL in 10 µL of blood. The bioanalytical method to quantify rosuvastatin from 1 to 100 ng/mL in VAMS sample was qualified by specificity, carryover, linearity, within-run and between-run reproducibility and stability. Inaccuracy was less than ± 6% and imprecision was less than 10% after analyzing the samples on 5 different days at all concentration levels. In addition, the feasibility of delivery to the analytical laboratory after home sampling during the guaranteed stability period of 10 days at room temperature was confirmed by evaluating concentration changes after VAMS sampling without adding pH buffer. Our results suggest that VAMS sampling did not have an effect on the stability of rosuvastatin, and it is a viable option for simple and accurate blood collection at home.

Dried Blood Microsampling-Based Therapeutic Drug Monitoring of Antiepileptic Drugs in Children With Nodding Syndrome and Epilepsy in Uganda and the Democratic Republic of the Congo.

Nodding syndrome is a highly debilitating, generalized seizure disorder, affecting children in subregions of sub-Saharan Africa. Despite numerous efforts to uncover the etiology, the exact cause of this syndrome still remains obscure. Therefore, to date, patients only receive symptomatic care, including the administration of first-generation antiepileptic drugs for seizure control. As data on the efficacy of drugs within this population are completely lacking, the aim of this study was to explore how therapeutic drug monitoring could help to understand the differential response to therapy. Considering the challenging environment in which sampling had to be performed (remote areas, devoid of electricity, running water, etc), dried blood matrices [ie, dried blood spots (DBSs)] and volumetric absorptive microsampling (VAMS) were considered fit-for-purpose. In addition, owing to the similarities between the syndrome and other forms of epilepsy, samples originating from patients suffering from (onchocerciasis-associated) epilepsy were included. In total, 68 patients with Nodding syndrome from Uganda, 58 Ugandan patients with epilepsy, and 137 patients with onchocerciasis-associated epilepsy from the Democratic Republic of the Congo were included. VAMS samples and DBS were analyzed using validated methods, involving manual extraction or fully automated extraction, respectively, before quantification using liquid chromatography coupled with tandem mass spectrometry. Analysis revealed that serum concentrations (calculated from DBS) within the respective reference ranges were attained in only 52.9% of the 68 Nodding syndrome patients treated with valproic acid, in 21.4% of the 56 Ugandan epilepsy patients treated with carbamazepine, and in 65.7% of the 137 onchocerciasis-associated epilepsy patients from the Democratic Republic of the Congo treated with phenobarbital. In all other instances, concentrations were subtherapeutic. Furthermore, on comparing DBS with VAMS concentrations, an inexplicable overestimation was observed in the latter. Finally, no obvious link could be observed between the obtained drug concentrations and the number of seizures experienced during the last month before sampling, elaborating the fact that the level of improvement in some patients cannot simply be linked to reaching therapeutic concentrations.

Application of volumetric absorptive microsampling (VAMS) to measure multidimensional anti-influenza IgG antibodies by the mPlex-Flu assay.

Introduction: Recently, volumetric absorptive microsampling (VAMS) has been used for accurate sampling of a fixed peripheral blood volume (10 µL) on a volumetric swab, and long-term sample storage. The mPlex-Flu assay is a novel, high-throughput assay that simultaneously measures the concentration of antibodies against the hemagglutinin (HA) proteins from multiple influenza virus strains with ≤5 µL of serum. Here we describe combining these two methods to measure multidimensional anti-influenza IgG activity in whole blood samples collected by a finger stick and VAMS, with correction for serum volume based on simultaneous hemoglobin measurement. Methods: We compared capillary blood samples obtained from a finger stick using a VAMS device with serum samples collected by traditional phlebotomy from 20 subjects, with the influenza antibody profiles measured by the mPlex-Flu assay. Results: We found that results with the two sampling methods were highly correlated within subjects and across all influenza strains (mean R 2 = 0.9470). Adjustment for serum volume, based on hemaglobin measurement, was used to estimate serum volume of samples and improved the accuracy. IgG measurements were stable over 3 weeks when VAMS samples were stored at room temperature or transported using a variety of shipping methods. Additionally, when volunteers performed finger-stick VAMS at-home by themselves, the comparison results of anti-HA antibody concentrations were highly consistent with sampling performed by study personnel on-site (R 2 = 0.9496). Conclusions: This novel approach can provide a simple, accurate, and low-cost means for monitoring the IgG anti-influenza HA antibody responses in large population studies and clinical trials.

Volumetric absorptive microsampling (VAMS) as an alternative to conventional dried blood spots in the quantification of miltefosine in dried blood samples

Miltefosine is an oral agent against the neglected tropical disease leishmaniasis, which is mostly endemic in resource-poor areas. Dried blood spot (DBS) sampling is an attractive alternative to plasma sampling for pharmacokinetic studies in these remote areas, but introduces additional variability in analyte quantification due to possible blood spot inhomogeneity and variability in blood spot volume and haematocrit values. Volumetric absorptive microsampling (VAMS) potentially overcomes a few of these issues as the VAMS device absorbs a fixed volume that is processed as a whole. We developed and validated an LC–MS/MS method for the quantification of miltefosine with this novel sampling technique with good performance in terms of linearity, selectivity, accuracy (bias within ±10.8%), precision (CV%≤11.9%), recovery, carry-over and matrix effect. VAMS samples were stable for at least one month at room temperature and 37°C. The impact of haematocrit on assay accuracy was reduced compared to conventional DBS sampling, but indicated a declining recovery with increased haematocrit due to haematocrit dependency in recovery from the sampling device. A clinical validation will be required to investigate whether VAMS is an appropriate and cost-effective alternative sampling method to conventional DBS sampling.

Does volumetric absorptive microsampling eliminate the hematocrit bias for caffeine and paraxanthine in dried blood samples? A comparative study

Volumetric absorptive microsampling (VAMS) is a novel sampling technique that allows the straightforward collection of an accurate volume of blood (approximately 10μL) from a drop or pool of blood by dipping an absorbent polymeric tip into it. The resulting blood microsample is dried and analyzed as a whole. The aim of this study was to evaluate the potential of VAMS to overcome the hematocrit bias, an important issue in the analysis of dried blood microsamples. An LC-MS/MS method for analysis of the model compounds caffeine and paraxanthine in VAMS samples was fully validated and fulfilled all pre-established criteria. In conjunction with previously validated procedures for dried blood spots (DBS) and blood, this allowed us to set up a meticulous comparative study in which both compounds were determined in over 80 corresponding VAMS, DBS and liquid whole blood samples. These originated from authentic human patient samples, covering a wide hematocrit range (0.21–0.50). By calculating the differences with reference whole blood concentrations, we found that analyte concentrations in VAMS samples were not affected by a bias that changed over the evaluated hematocrit range, in contrast to DBS results. However, VAMS concentrations tend to overestimate whole blood concentrations, as a consistent positive bias was observed. A different behavior of VAMS samples prepared from incurred and spiked blood, combined with a somewhat reduced recovery of caffeine and paraxanthine from VAMS tips at high hematocrit values, an effect that was not observed for DBS using a very similar extraction procedure, was found to be at the basis of the observed VAMS-whole blood deviations. Based on this study, being the first in which the validity and robustness of VAMS is evaluated by analyzing incurred human samples, it can be concluded that VAMS effectively assists in eliminating the effect of hematocrit.