Volumetric Absorptive Microsampling Device Research Articles

Analytical Validation of a Volumetric Absorptive Microsampling Method for Therapeutic Drug Monitoring of the Oral Targeted Anticancer Agents, Abiraterone, Alectinib, Cabozantinib, Imatinib, Olaparib, and Sunitinib, and Metabolites.

Volumetric Absorptive Microsampling (VAMS) is a useful tool for therapeutic drug monitoring (TDM) of oral targeted anticancer agents. VAMS aims to improve safety and efficacy by enabling at-home blood sample collection by patients. This study aimed to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and the metabolites, Δ(4)-abiraterone (D4A), alectinib-M4, imatinib-M1, and N-desethyl sunitinib, in dried whole blood samples using VAMS to support TDM. After the collection of 10 μL of whole blood sample using the VAMS device, the analytes were extracted from the tip using methanol with shaking, evaporated, and reconstituted in acetonitrile:0.1 mol/L ammonium hydroxide in water (1:1, vol/vol). The extracts were then analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. Validation experiments based on the ICH M10 guideline were carried out, and stability was evaluated under shipping and storage conditions. VAMS specimens were collected in the outpatient clinic to demonstrate the applicability of the assay. The validated range of the method was considered accurate and precise for all analytes. Accordingly, the validation experiments met the relevant requirements, except for cross-analyte interference. Based on the stability data, shipment can be performed at room temperature within 14 days after sample collection and the VAMS specimen can be stored up to 9 months at -20 and -70°C. Samples from 59 patients were collected at the hospital. The developed method could be used to successfully quantify the concentrations of abiraterone, D4A, alectinib, alectinib-M4, cabozantinib, imatinib, imatinib-M1, olaparib, sunitinib, and N-desethyl sunitinib within the validated range using VAMS. Therefore, the method can be used to estimate the dried whole blood-to-plasma ratios for TDM in the clinic.

A Novel, Rapid, and Accurate Quantitative Hydroxyurea Assay

Introduction: Hydroxyurea is a highly effective treatment for children and adults with sickle cell anemia (SCA) and is now considered standard-of-care therapy in high-resource countries. There is wide interpatient variability in the optimal dose, which is defined as the daily dose that provides maximal induction of protective fetal hemoglobin to decrease erythrocyte sickling while causing only mild marrow suppression. The time and cost of hydroxyurea dose titration are barriers to its widespread use in low-resource settings. The titration process required to identify an optimal dose can be abbreviated by measuring individualized hydroxyurea pharmacokinetics (PK) to guide personalized precision dosing, which could be especially useful in low-resources settings with limited laboratory monitoring. However, measurement of hydroxyurea in serum and other physiological fluids is not commonly performed during routine clinical treatment. Current methodology to quantify hydroxyurea concentrations requires complex and time-consuming chemical derivatization followed by detection by colorimetric spectrophotometry, high performance liquid chromatography (HPLC), or liquid chromatography mass spectrometry (LC-MS). To consider performing PK-guided dosing in low-resource settings, a simple, accurate, and efficient hydroxyurea assay is needed. Methods: A commercially available open-system HPLC unit for hemoglobin separation (SmartLifeLC, PolyLC Inc, Columbia MD) was equipped with a 3.5-micron, 4.6 mm × 50 mm Zorbax Eclipse XDB-C18 column with matching guard column (Agilent Technologies, Santa Clara, CA); mobile phases of 46% methanol and 100% acetonitrile; and a 200-600nm multi-wavelength detector. The chemical derivatization process was modified from previously published techniques that use xanthydrol (Sigma-Aldrich, St. Louis MO) for detection of primary amides. N-methylurea (Sigma-Aldrich) was included as an internal standard. Hydroxyurea (Sigma-Aldrich) was diluted in whole blood collected from healthy controls to create a calibration curve encompassing the typical pharmacological range of hydroxyurea at 80, 40, 20, 10, and 5 mg/mL. Quality Control (QC) samples were prepared at 50, 25, 12.5, and 6.25 mg/mL to validate the calibration curve before testing patient samples. Blood was applied to 20mL volumetric absorptive microsampling devices (Mitra, Trajan Scientific and Medical, Torrance CA) and dried for future elution and analysis. HPLC results were compared to LC-MS results to assess accuracy. Results: Blood was eluted from the Mitra microsampling device and derivatized in a xanthydrol solution before HPLC analysis. Using a 10-minute run with 210nm wavelength detection, the xanthydrol-modified hydroxyurea reproducibly yielded a linear calibration curve with r 2 >0.95 (Figure), and the actual concentrations were within 20% of the theoretical values. Repeated testing of control and patient samples revealed a typical intra-day coefficient of variation <15% and inter-day coefficient of variation <20%. The agreement between nominal values and assay results determined for a wide range of QC samples from both methods exceeded the FDA-recommended threshold of 67% for chromatographic assay validation with 86% agreement on LC-MS and 85% with the new method. The entire elution and derivatization procedure could be completed using dried Mitra blood samples in less than 60 minutes. Conclusions: Quantitative hydroxyurea measurement of blood or serum can now be accomplished using a novel, rapid, HPLC technique that features xanthydrol-based derivatization of hydroxyurea, an internal standard, and UV wavelength detection. This technique is accurate, reproducible, and relatively simple, and thus deemed suitable for hydroxyurea concentration measurements needed for PK-guided dosing calculations for children with SCA living in low-resource settings. This novel technique will be added to the multi-country Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) trial that will feature PK-guided precision dosing for young children with SCA starting hydroxyurea treatment.

A sensitive liquid chromatographic-mass spectrometry method for the quantification of vincristine in whole blood collected with volumetric absorptive microsampling

Vincristine is a well-established cytotoxic drug. In paediatric populations blood collection via venipuncture is not always feasible. Volumetric absorptive microsampling (VAMS) is a less invasive method for blood collection. Furthermore, VAMS lacks the haematocrit effect on the recovery known with dried blood spots. Therefore, a liquid chromatography tandem-mass spectrometry method was developed and validated for the quantification of vincristine in whole blood collected with VAMS devices. Sample preparation consisted of solid-liquid extraction with 0.2% formic acid in water and acetonitrile. The final extract was injected on a C18 column (2.0 ×50 mm, 5 µm). Gradient elution was used and quantification was accomplished with a triple quadruple mass spectrometer operating in the positive mode. The validated concentration range was from 1 to 50 ng/mL with an intra- and inter-accuracy and precision of ± 10.3% and ≤ 7.3%, respectively. This method was able to successfully quantify vincristine concentrations in whole blood collected with VAMS from paediatric oncology patients. Vincristine concentrations in whole blood were non-linearly associated with plasma concentrations, which could be described with a saturable binding equilibrium model.

HPLC-DAD quantification of favipiravir in whole blood after extraction from volumetric absorptive microsampling devices

Favipiravir is a prodrug of T-1105 made by modifying the pyrazine group as a COVID-19 therapy. During the pandemic, a safe and comfortable biosampling technique is needed for the subject or patient. Volumetric Absorptive Microsampling (VAMS) is a biosampling technique with a small blood volume and minimum hematocrit effect. The aims of this study were to develop and validate an analytical method for quantifying favipiravir extracted from VAMS using High Performance Liquid Chromatography – Photodiode Array with remdesivir as an internal standard. Analysis of favipiravir was performed using a C18 column (Waters, Sunfire™ 5 µm; 250 × 4.6 mm), with injection volume of 50 µL, flow rate of 0.8 mL/min, column temperature 30 ℃, and wavelength 300 nm. The separation was conducted under gradient elution with mobile phase consists of acetonitrile-0.2 % formic acid-20 mM sodium dihydrogen phosphate pH 3.5 and run time 12 min. Sample preparation was carried out using a protein precipitation method with 500 µL of methanol as precipitating agent. Samples were mixed on vortex for 30 s, sonicated for 15 min, and centrifuged at 10,000 rpm for 10 min. Lower Limit of Quantification (LLOQ) obtained was 0.5 µg/mL and the calibration curve ranged from 0.5 to 160 µg/mL. Sensitivity, linearity, selectivity, carry-over, accuracy, precision, recovery, and stability were validated by the guideline from Food and Drug Administration 2018. The method developed has successfully met the full validation requirements by FDA 2018 with the LLOQ obtained was 0.5 µg /mL.

Automation of tacrolimus measurement on volumetric absorptive microsampling devices by tandem mass spectrometry.

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: 8.0μg/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: -0.007μg/l; standarddeviation: 1.536). Automation reduced operator touch time by 40min(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 dried blood microsampling for monitoring imatinib mesylate therapy: Method development and clinical application in patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematologic neoplasia, characterized as a proliferative disease of the hematopoietic system. Imatinib mesylate (IM), a selective tyrosine kinase inhibitor, is considered a first-line therapy for CML, indicated for both adult and pediatric patients presenting the Philadelphia chromosome (Ph+). However, patients in treatment with IM may show different responses due to interindividual pharmacokinetic variability. Therapeutic drug monitoring should be routinely performed to identify treatment response profile, adherence to treatment, or possible drug interactions, thus supporting better treatment management. Volumetric absorptive microsampling (VAMS) are innovative devices for blood collection whose advantages include the possibility of home collection by the patient or at the physician's office. The assay was fully validated according to bioanalytical validation guidelines. Estimated plasma concentrations of IM were not statistically different between groups according to adherence (p = 0.15), with median of 789 ng ml-1 in the group with some level of non-adherence versus 1141.9 ng ml-1 in the group with adherence, classified with the Morisky-Green questionnaire. This study included 33 patients with CML in treatment with IM. These patients answered socioeconomic, sociodemographic, and adherence profile (Morisky-Green) questionnaires. Patients also received instructions for home blood collection with VAMS devices. Afterwards, the samples were analyzed by LC-MS/MS. The mean age of the patients was 52 years, 84.8% were ingesting doses of 400 mg/day and the majority were male (69.7%). IM and its metabolite NIM were extracted from VAMS with an aqueous solution with 0.1% formic acid, followed by protein precipitation with acetonitrile. The methodology developed in this study was satisfactory for the determination of IM and NIM in VAMS and can be used in hospital and office routines for the therapeutic monitoring of patients with CML.

Blood simultaneous determination of PEth 16:0/18:1, PEth 16:0/20:4 and ethylglucuronide using DBS: Application to a clinical study of patients hospitalized for withdrawal

This study aims to present a LC-MS/MS method for simultaneous determination of phosphatidyléthanol (PEth)16:0/18:1, PEth16:0/20:4 and ethylglucuronide (EtG) using dried blood spots (DBS) and to report preliminary results of PETHOXICO, a monocentric non-interventional non-randomized cross-sectional clinical study (ClinicalTrials.gov NCT04557631 ) in a population of patients hospitalized for withdrawal. Blood samples can alternatively consist in 10 μL from usual blood sampling tubes (e.g. fluoride preserved) transferred on DBS card, or direct DBS sampling using Volumetric Absorptive MicroSampling devices (VAMS; Hemaxis™, Neoteryx™). For PETHOXICO, PEth16:0/18:1, PEth16:0/20:4 and EtG were analyzed simultaneously together with urinary EtG and blood alcohol concentration (BAC), determined using immunoassay and GC-FID, respectively. The fully validated LC-MS/MS method exhibits a LLOQ of 2 μg/L for the 3 analytes. A total of 37 chronic and excessive ethanol consumers (ethanol per week consumptions ranging from 560 to 1750 g) were included in PETHOXICO [sex ratio 11/26 (F/M); 30 to 67 YO with a median at 46 YO]. At D0, the mean concentration of PEth16:0/18:1 was 1180 μg/L (range: 53–3889 μg/L) with 95% of participants above the 210 μg/L threshold. PEth16:0/20:4-to-PEth16:0/18:1 mean ratio was 0.46 with PEth16:0/20:4 concentrations ranging from 9 to 2353 μg/L. The mean BAC was 1060 mg/L (range: undetected–3540 mg/L) and for 78% of participants, urinary EtG was above the 500 μg/L threshold. At D7, no BAC nor EtG (urine and blood) were found as expected. The mean concentration of PEth16:0/18:1 dropped to 527 μg/L (range: 16–1655 μg/L) with still 78% of participants above the 210 μg/L threshold. PEth16:0/20:4 concentrations dropped more significantly (range: undetected–923 μg/L) with a mean PEth16:0/20:4-to-PEth16:0/18:1 ratio of 0.26. This decreased ratio (0.46 at D0 and 0.26 at D7) is coherent with the expectation of a shortened detection window for PEth16:0/20:4. Blood elimination half-life of PEths were calculated with the assumption of a single elimination phase: mean half-life were 6.9 days (range: 2.2–18.6 days) and 4.6 days (range: 1.3–9.3 days) for PEth16:0/18:1 and PEth16:0/20:4, respectively. PEth16:0/18:1 observed half-life is consistent with literature data (around 7 days with a significant inter-individual variability). Observed PEth16:0/20:4 half-life is higher than the very scarce data from literature (2 days). In this study, the use of an analytical method combining 3 direct metabolites of ethanol allows, in one run, to discriminate a very recent consumption (< 48 h) from a recent consumption (< 2 weeks) and an older consumption (2–4 weeks). PEth16:0/18:1 sensitivity to detect chronic and excessive alcohol consumers was 95% with a 210 μg/L threshold. However, these preliminary results need yet to be confronted to detailed clinical data.

Added value evidences of a simultaneous determination of two phosphatidylethanol isoforms and ethylglucuronide in dried blood spots in forensic contexts

In clinical or postmortem forensic medicine, acute or chronical alcohol abuse are of main concern and direct/indirect biomarkers are widely use in order to assess recent or retrospective alcohol abuse. We aim to illustrate that a simultaneous determination of 3 directs ethanol biomarkers [two phosphatidylethanol isoforms, 16:0/18:1 (PEth18:1) and 16:0/20:4 (PEth20:4), and ethylglucuronide (EtGB)] using dried blood spot (DBS) can be a useful additional tool in many situations. (Case#1) Sexual assault of a 17-year-old girl with negative urine and blood ethanol concentration (BAC) (but urine EtG concentration was 10 mg/L) sampled 19 hours after the facts. (Case#2) Only blood sampling (negative BAC) was performed 14 hours after a 36-year-old man (with heavy psychiatric disorders) tried to kill 3 persons. He was reported drunk at the time of the attack by witnesses. (Case#3) A 27-year-old man was implicated in a car-related fatality that occurred 11 days before sampling. Blood and hair sampling were performed in order to investigate his alcoholic status: hair EtG concentration was < 5 pg/mg. (Case#4) The alcoholic status of a 43-year-old woman killed in a car accident had to be established. BAC was 140 mg/L in the postmortem peripheral blood (only available biological sample; sampled less than one hour after death and kept refrigered until time of analysis, 2 days later). (Case#5) A low BAC (70 mg/L) was found in postmortem cardiac blood (only sample available) sampled on the corps of a 42-year-old man found submerged in the sea. (Case#6) Peripheral blood sampling (for BAC determination) and direct DBS sampling (MitraTM) were performed at the time of discovery of the 36-year-old man corpse, allegedly heavy drinker according to the testimonies (BAC was 2810 mg/L). Blood samples can alternatively consist in 10 μL from usual blood sampling tubes (e.g. fluoride preserved) transferred on DBS card, or direct DBS sampling using Volumetric Absorptive MicroSampling devices (VAMS; HemaxisTM, MitraTM). These DBS are analyzed using a LC-MS/MS (routinely used in clinical toxicology) method (LLOQ: 2 μg/L for the 3 analytes). Results (μg/L; PEth18:1, PEth20:4 and EtGB, respectively) were: – (Case#1) 22, 2 and 300, in coherence with both a recent alcohol intake hypothesis [EtGB above the 45 μg/L threshold supported an alcohol consumption [> 2 standard units (SU)] in the last 24 to 48 hours] and that this girl was not an excessive drinker [owing to the low PEths concentrations]; – (Case#2) 98, 103 and 71, consistent with an alcohol intake hypothesis arround the time of the attack by this man who had a significant consumption of alcohol in the last 10 days as PEth 20:4 concentration was similar to the PEth18:1 one; – (Case#3) 9, < 2 and < 2, and did not contradict the negative EtG hair result as these low values were, at most, compatible with an “occasional” or “social drinker” status (< 14 SU per week); – (Case#4) 817, 44 and 130. EtGB positive result supported a recent alcohol consumption and the very high PEth18:1 value strongly suggested chronic excessive alcohol consumption; – (Case#5) negative (< 2 μg/L) for the 3 analytes in postmortem cardiac blood, in agreement with both the hypothesis of a light postmortem ethanol production, and a probable “abstinent” or “occasional consumer” status of the victim; – (Case#6) 3470, 1030 and 13,400, supporting the massive alcohol intoxication hypothesis close to the death of a heavy drinker. This analytical tool is of high interest when only blood samples are available, although postmortem use (due to PEths neoformation issues) should be limited to cases with negative BAC, or when blood sampling is performed close to death using a VAMS or a refrigerated preserved (< 4 days) tube.

Quantification of vincristine and tariquidar by liquid chromatography-tandem mass spectrometry in mouse whole blood using volumetric absorptive microsampling for pharmacokinetic applications.

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of vincristine and tariquidar in 10 μL of mouse whole blood using volumetric absorptive microsampling devices. Samples were extracted from the devices and quantified against calibrators prepared in a human blood plasma matrix. Separation of vincristine and tariquidar was achieved using a Shimpack XR ODS III C18 stationary phase and H2 O and methanol mobile phase solvents containing 0.1% formic acid, running a gradient elution at a flow rate of 0.2 mL/min over 6.0 min. The method was linear up to 1200 ng/mL (R2 > 0.99 for both analytes), with calibrator accuracy within ± 15% of the nominal concentrations and analyte coefficient of variance <15% for both vincristine and tariquidar. Pharmacokinetic assessment of both analytes was successfully applied in mice as both single-agent therapy and combination therapy over a 24-h period, and a 2.3-fold increase in vincristine drug exposure was observed in combination with tariquidar. This study validates the use of this approach for longitudinal analysis of drug exposure in animal studies.

Clinical Validation of a Volumetric Absorptive Micro-Sampling Device for Pharmacokinetic Studies With Tranexamic Acid.

We assessed the accuracy of tranexamic acid (TXA) concentrations measured in capillary whole blood using volumetric absorptive micro-sampling (VAMS) devices. Paired venous and VAMS capillary blood samples were collected from 15 healthy volunteers participating in a pharmacokinetic study of alternative routes (oral, IM and IV) of administering TXA. To assess accuracy across a range of concentrations, blood was drawn at different times after TXA administration. We measured TXA concentrations in plasma, whole blood from samples collected by venepuncture and whole blood from venous and capillary samples collected using VAMS devices. TXA was measured using a validated high sensitivity liquid chromatography - mass spectrometry method. We used Bland-Altman plots to describe the agreement between the TXA concentrations obtained with the different methods. In the 42 matched samples, the mean plasma TXA concentration was 14.0 mg/L (range 2.6–36.5 mg/L) whereas the corresponding whole blood TXA concentration was 7.7 mg/L (range 1.6–17.5 mg/L). When comparing TXA concentrations in VAMS samples of venous and capillary whole blood, the average bias was 0.07 mg/L (lower and upper 95% limits of agreement: −2.1 and 2.2 mg/L respectively). When comparing TXA concentrations in venous whole blood and VAMS capillary whole blood, the average bias was 0.7 mg/L (limits of agreement: −2.7 and 4.0 mg/L). Volumetric absorptive micro-sampling devices are sufficiently accurate for use in pharmacokinetic studies of tranexamic acid treatment in the range of plasma concentrations relevant for the assessment of fibrinolysis inhibition.

Remote Fingerstick Blood Collection for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibody Testing

The rapid worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has propelled the rapid development of serologic tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, and helping establish a recent diagnosis of coronavirus disease 2019 (COVID-19), and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serologic assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large-scale SARS-CoV-2 serologic studies by combining self or office collection of fingerprick blood with a volumetric absorptive microsampling device (Mitra, Neoteryx LLC) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys, Roche Diagnostics Inc) that is emergency use authorization approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Mitra devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies compared with neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Mitra dried blood collection device with the Roche Elecsys SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.

Novel LC-MS/MS method for the determination of selumetinib (AZD6244) in whole blood collected with volumetric absorptive microsampling.

Aim: A method has been developed and validated for quantitation of selumetinibin human whole blood collected using a Mitra™ volumetric absorptive microsampling device. This device is patient-friendly, affording less-invasive sampling with broad applicability to clinical and diagnostic applications - specifically in pediatric populations. Materials & methods: In this method, drug is extracted from the Mitradevice via sonication in methanol: Ammonium hydroxide, then analyzed by LC-MS/MS. The linear rangefor selumetinib analysis is 2.00-2000ng/ml. Results: All validation parameters met acceptance criteria established in agreement with current regulatory guidance for bioanalytical method validation. The stability of selumetinib in Mitratips was established at both ambient and frozen conditions. Conclusion: A simple method has been developed and validated for determination of selumetinib from human whole blood, collected using volumetric absorptive microsamplingand analyzed by LC-MS/MS.

Tranexamic acid quantification in human whole blood using liquid samples or volumetric absorptive microsampling devices.

Background: Recent clinical trials demonstrate the benefits of the antifibrinolytic drug tranexamic acid but its pharmacokinetics remain to be investigated more in depth. Although pharmacokinetics studies are usually performed with plasma, volumetric absorptive microsampling devices allow us to analyze dried whole blood samples with several advantages. Materials & methods: High-sensitivity LC-MS/MS methods for the quantification of tranexamic acid in human whole blood using liquid samples or dry samples on volumetric absorptive microsampling devices were developed and validated based on International Association from Therapeutic Drug Monitoring and Clinical Toxicology, European Medicines Agency and US FDA guidance. Conclusion: The method performances were excellent across the range of clinically relevant concentrations. The stability of tranexamic acid in blood samples stored up to 1 month at +50°C was demonstrated. The methods' suitability was confirmed with clinical samples.

Blood and Plasma Volumetric Absorptive Microsampling (VAMS) Coupled to LC-MS/MS for the Forensic Assessment of Cocaine Consumption.

Reliable, feasible analytical methods are needed for forensic and anti-doping testing of cocaine and its most important metabolites, benzoylecgonine, ecgonine methyl ester, and cocaethylene (the active metabolite formed in the presence of ethanol). An innovative workflow is presented here, using minute amounts of dried blood or plasma obtained by volumetric absorptive microsampling (VAMS), followed by miniaturized pretreatment by dispersive pipette extraction (DPX) and LC-MS/MS analysis. After sampling 20 µL of blood or plasma with a VAMS device, the sample was dried, extracted, and loaded onto a DPX tip. The DPX pretreatment lasted less than one minute and after elution with methanol the sample was directly injected into the LC-MS/MS system. The chromatographic analysis was carried out on a C8 column, using a mobile phase containing aqueous formic acid and acetonitrile. Good extraction yield (> 85%), precision (relative standard deviation, RSD < 6.0%) and matrix effect (< 12%) values were obtained. Analyte stability was outstanding (recovery > 85% after 2 months at room temperature). The method was successfully applied to real blood and plasma VAMS, with results in very good agreement with those of fluid samples. The method seems suitable for the monitoring of concomitant cocaine and ethanol use by means of plasma or blood VAMS testing.

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.

SAT-011 Quantification of Testosterone, Androstenedione and 17-Hydroxyprogesterone Collected Using Mitra®Micro Sampling Devices

Introduction Measurement of total plasma testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17OHP) typically requires a venous sample. A highly sensitive and specific assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after extraction from a Mitra® volumetric absorptive microsampling (VAMs) device. VAMs devices are small absorptive polymer tips which absorb a fixed amount of blood (1); they can be dried after collection of finger prick blood and stored prior to analysis. Differences in binding in whole blood compared to serum require the quantification of haematocrit using a published protocol (2), thus allowing an estimated serum result to be generated. Method Pre-mixed calibrator/sample (10 µL) was combined with deionised water and mixed internal standard, after vortexing MTBE was added and mixed by inversion for 1 minute. The supernatant was transferred to a clean 2 mL deep-well plate, dried down and re-constituted with 100 µL 50:50 methanol/water. Mitra® tips were prepared from anonymised whole blood samples in the laboratory and compared to the corresponding plasma using the same method. Results Mean recovery was 86% for A4, 102% for T and 97% for 17OHP, the lower limit of quantification was 1 nmol/L for A4, 1 nmol/L for testosterone and 4 nmol/L for 17OHP. Ion suppression was within acceptable criteria for all the analytes measured. For the samples prepared in the laboratory, comparisons for T, A4 and 17OHP the R-squared values were 0.95, 0.93 and 0.95 respectively with a minimal bias. Summary We have developed a robust assay for LC-MS/MS measurement of VAMs for T, A4, and 17OHP in a routine clinical laboratory. It is simple, reproducible and easy to perform and may have implications for monitoring of T therapy in hypogonadal males or screening/monitoring of patients with congenital adrenal hyperplasia. In-house comparisons of T, A4 and 17OHP compared well when haematocrit was used to adjust for the space occupying effect of red cells.

Simultaneous determination of vancomycin and creatinine in plasma applied to volumetric absorptive microsampling devices using liquid chromatography-tandem mass spectrometry

Vancomycin (VCM) is a glycopeptide antibiotic, widely used against methicillin-resistant Staphylococcus aureus infections, and its clearance is highly correlated to creatinine (CRE) clearance. Usually, VCM and CRE levels are measured in serum or plasma specimens obtained from venous blood, after phlebotomy. As the majority of hospitals in Developing Countries do not have on-site access to VCM measurements, the availability of data on patient’s VCM levels would involve the transport of liquid samples to specialized laboratories. An alternative to overcome the logistic difficulties of liquid biological specimens is the use of dried microsamples, such as plasma applied to volumetric absorptive microsampling (VAMS) devices. The aim of this study was to develop and validate a LC–MS/MS assay for the simultaneous determination of VCM and CRE in plasma applied to VAMS devices. VCM and CRE levels were determined in clinical relevant ranges with acceptable precision and accuracy and both compounds were stable in VAMS for up to two weeks at 45 °C. Concentrations measured in VAMS differed from those measured in plasma and the use of estimation approaches was necessary to estimate plasma concentrations. VCM levels in plasma can be estimated by multiplying VAMS measurements by the correction factor of 0.934. The unbiased estimation of CRE plasma levels from VAMS required the use of the equation CREplasma = 0.9808 * CREVAMS + 1.6621. VAMS can be used as an alternative for short-term storage and transportation of plasma at ambient and high temperatures, allowing a more widespread access to VCM therapeutic drug monitoring in limited-resources settings.

Evaluation of the Mitra microsampling device for use with key urinary metabolites in patients with Alkaptonuria.

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. 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. 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.

Volumetric absorptive microsampling as an alternative sampling strategy for the determination of paracetamol in blood and cerebrospinal fluid.

In the field of bioanalysis, dried matrix spot sampling is increasingly receiving interest, as this alternative sampling strategy offers many potential benefits over traditional sampling, including matrix volume-sparing properties. By using a microsampling strategy, e.g., volumetric absorptive microsampling (VAMS), the number of samples that can be collected from a patient can be increased, as a result of the limited sample volume that is required per sample. To date, no VAMS-based methods have been developed for the quantification of analytes in cerebrospinal fluid (CSF). The objective of this study was to develop and validate two LC-MS/MS methods for the quantification of paracetamol in dried blood and dried CSF, with both matrices sampled using VAMS. Both methods were fully validated based on internationally accepted guidelines. Paracetamol was chromatographically separated from its glucuronide and sulfate metabolites and no carry-over or unacceptable interferences were detected. The total precision (%RSD) was below 15% for all QC levels and accuracy (%bias) was below 7% (17% for the LLOQ of aqueous VAMS). The influence of the hematocrit on the recovery of blood VAMS samples appeared to be limited within the hematocrit range of 0.21 to 0.62. The blood VAMS samples were stable for 1week if stored at 50°C, and for at least 8months when stored between - 80°C and room temperature. The aqueous VAMS samples were stable for at least 9months when stored between - 80 and 4°C, and for 1month when stored at room temperature. Application of the methods on external quality control material and analysis of patient samples demonstrated the validity and utility of the methods and provided a proof of concept for the analysis of CSF microsamples obtained via VAMS devices. Graphical abstract ᅟ.

Microsampling Collection Methods for Measurement of C-peptide in Whole Blood.

Microsampling techniques are alternative methods to venous sampling for obtaining blood for measurement of circulating biomarkers, offering the convenience of reduced sample volume and elimination of the need for phlebotomists. Dried blood spot (DBS) microsampling methods have been used for many years while more recently a volumetric absorptive microsampling device (VAMS™) has been introduced. In diabetes mellitus, circulating C-peptide is commonly used as an indicator of endogenous insulin secretion and clinical measurement can aid in diagnosis as well as informing on therapy. This pilot study investigated the effectiveness of microsampling collection of capillary blood for measurement of C-peptide. Capillary blood was collected into capillary tubes and centrifuged for plasma samples. Simultaneous samples were also collected using both microsampling methods (DBS and VAMS). Blood from both microsamplers was extracted prior to assaying for C-peptide alongside the corresponding plasma samples, using specific immunoassays and results obtained from microsampling compared to the reference plasma concentrations. Stability was determined by collecting duplicate DBS and VAMS and assaying both in a single assay after storing one at -20°C immediately and one at room temperature for 48 hours post-collection. Good agreement was observed between C-peptide concentrations in plasma and equivalent DBS and VAMS samples ( R2 = .929 and .9231, DBS and VAMS, respectively), with mean differences of 75.7 and 8.4 pmol/L observed for DBS and VAMS. Small decreases in C-peptide of 11.6% and 0.1% were observed after 48 hours storage for DBS and VAMS, respectively. C-peptide collected using DBS and VAMS showed good agreement with reference plasma concentrations, suggesting both would be an effective microsampling method for collection and measurement of C-peptide.