Volumetric Absorptive Microsampling Research Articles

Measurement of 5- fluorouracil, capecitabine and its metabolite concentrations in blood using volumetric absorptive microsampling technology and LC-MS/MS

5-fluorouracil (5-FU) and its oral formulation, capecitabine, are widely used in treating a range of malignancies, either alone or in combination with other antineoplastic drugs. Body surface area-based dosing is used for these agents, despite this approach leading to substantial variability in drug exposure and often resulting in either toxicity or treatment failure. Tailoring therapeutic regimens for individual patients using therapeutic drug monitoring (TDM) has been shown to significantly reduce toxicity and improve cancer outcomes. However, for optimum TDM, sample timing is crucial, along with the need for a venepuncture blood sample to obtain the plasma currently used for 5-FU measurement. In addition to complex blood sample handling requirements, large sample volume and frequent sampling required for pharmacokinetic analysis is another barrier to successfully implementing TDM in a healthcare setting. Microsampling is an alternative collection method to venepuncture, which, combined with the now readily available liquid chromatography mass spectrometry (LC-MS/MS) technology, overcomes the plasma-associated issues. It also has the significant advantage of enabling at home and remote sampling, thus facilitating 5-FU TDM in clinical practice.A LC-MS/MS method for simultaneous measurement of capecitabine, 5′-deoxy-5-fluorocytidine, 5′-deoxy-5-fluorouridine and 5-FU using Mitra® microsampling devices for sample collection was developed. A Shimadzu 8060 LC-MS/MS equipped with electrospray ionisation source interface, operated in positive and negative ion modes, with reversed-phase chromatographic separation was employed for sample analysis. Samples were extracted from Mitra® devices using acetonitrile containing stable isotope-labelled internal standards, sonicated, evaporated under vacuum and resuspended in 0.1 % formic acid before injection into the LC-MS/MS. Chromatographic separation was on a Luna Omega Polar C18 (100 × 2.1 mm, 1.6 µm) column with gradient elution of 0.1 % formic acid in water and acetonitrile. Total run time was 5 min, with the injection volume of 1 µL. The intra and inter-day imprecision ranged from 3.0 to 8.1 and 6.3–13.3 % respectively. Accuracy ranged from 95 −114 % for all analytes. Lower limit of quantification with imprecision of < 19 % and accuracy between 89 and 114 % was 0.05 mg/L for 5-FU and 10 µg/L for other analytes. Assays were linear from 0.05 to 50 mg/L for 5-FU and 10–10,000 µg/L for all other analytes. Analytes were stable on Mitra® devices for up to 9 months at room temperature, 2 years at −30 ℃ and 3 days at 50 ℃. The method was successfully applied for the analysis of samples from patients undergoing cancer treatment with 5-FU and capecitabine. Microsampling using volumetric absorptive microsampling proved to be as reliable as conventional blood collection for 5-FU and capecitabine. This sampling technique may lead to less invasive and better-timed sample collection for TDM, supporting dose optimization strategy.

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.

ANALYTICAL METHOD VALIDATION OF DOXORUBICIN AND DOXORUBICINOL IN VOLUMETRIC ABSORPTIVE MICROSAMPLING BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Doxorubicin is a broad-spectrum anthracycline antibiotic which has antineoplastic activity. Doxorubicin can cause cardiotoxic effects due to the formation of doxorubicinol as its main metabolite. One of the latest bio sampling methods, Volumetric Absorptive Microsampling (VAMS), has various advantages which include blood sampling by finger-prick, hematocrit does not influence it, and it can be stored at room temperature. This study aims to determine the optimum analysis conditions and sample preparation method in VAMS with daunorubicin as an internal standard, and to develop sensitive, specific measurements as well as validate the analytical method using Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). Analytical quantification analysis using mass spectrometry with a mass analyzer type is a triple quadrupole with a positive type of electrospray ionization (ESI). The separation was carried out with the Acquity® UPLC BEH C18 column (2.1 x 100 mm; 1.7 μm), with a flow rate of 0.2 mL/min, and gradient elution using 0.1% formic acid in water and 0.1% formic acid in acetonitrile for 7 minutes. Multiple reaction monitoring (MRM) values were set at m/z 544.22 &gt; 396.9 for doxorubicin; m/z 546.22 &gt; 398.9 for doxorubicinol; and m/z 528.5 &gt; 362.95 for daunorubicin. Sample preparation used protein precipitation with methanol as the extracting solution, the drying time of the VAMS was 2 hours, and the sonication time was 30 minutes. LLOQ values obtained were 8 ng/mL for doxorubicin and 3 ng/mL for doxorubicinol with the linearity of 0.9904 for doxorubicin and 0.9902 for doxorubicinol.

Comparison of conventional dried blood spots and volumetric absorptive microsampling for tacrolimus and mycophenolic acid determination

Therapeutic drug monitoring (TDM) of immunosuppressants is essential to avoid either rejection or toxicity after solid organ transplantations. Capillary microsampling approaches are an outstanding alternative to conventional venous sampling for TDM (easy and non-invasive collection, enabling self-sampling, and cost-saving shipment, processing and storage). Volumetric absorptive microsampling (VAMS) has gained importance in the last years, as it was meant to overcome the hematocrit (Hct) related issues commonly associated to DBS analysis. Despite all the benefits, microsampling techniques performance (including a thorough clinical validation) should be set up before their implementation in clinical practice. The aim of this study was to perform a clinical validation for both tacrolimus (TAC) and mycophenolic acid (MPA) in both DBS and Mitra™ VAMS. For the clinical validations, two different requirements were set up: analytical (following EMA and FDA guidelines) and clinical (following the Royal College of Pathologists of Australasia -RCPA- recommendations) acceptance criteria. For DBS, both analytical and clinical acceptance criteria were fulfilled for TAC, with 98.7% and 95% of the paired samples within the preset limits, respectively. For MPA, the analytical criterion was met (70.6% of paired specimens), although only half of the pairs were within the clinical limits. For VAMS, the clinical validation for both TAC and MPA showed good correlations but significant lower concentrations in VAMS compared to the routine matrices. After VAMS concentrations correction, the analytical requirement was fulfilled for both analytes (71.1% for TAC, 75% for MPA), although the more restrictive criteria recommended by the RCPA were not met for any analyte (half of the samples fell within the acceptance area). In addition, no significant Hct impact on the quantification was found in any case. Also, a preliminary home-sampling trial was set up, showing promising results. Moreover, a comparison between VAMS vs. DBS analytical and clinical performances was carried out, including a home-sampling trial, sample quality results and costs. Although the analytical performance for both VAMS and DBS was similar, DBS were superior regarding clinical criteria, sampling quality and cost.

Volumetric absorptive microsampling as a suitable tool to monitor tyrosine kinase inhibitors

Therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) shows significant potential in guiding personalized anticancer treatment. Dried blood microsampling could be a valuable alternative for traditional plasma sampling to provide TDM results faster and to reach a wider audience. Sample collection is easy and patient friendly as only a small volume of blood is collected via a fingerprick. This enables the possibility of home sampling by the patients themselves. Therefore, an LC-MS/MS method was developed and validated for the quantification of bosutinib, dasatinib, gilteritinib, ibrutinib, imatinib, midostaurin, nilotinib and ponatinib in dried blood samples collected via volumetric absorptive microsampling (VAMS). A VAMS device collects a fixed volume of blood (± 10 µL), irrespective of the sample’s hematocrit (Hct). During method validation, special attention was paid to the possible impact of Hct (range 0.18–0.55) on matrix effect (ME), robustness of the extraction, and accuracy of the method.The method was successfully validated based on international guidelines in terms of calibration curves, precision (within-run CV 2.20–14.8%; between-run CV 2.40–12.3%), accuracy (within-run bias 0.34–12.5%; between-run bias −0.15 to 16.2%), carry-over and selectivity. IS-compensated ME and recovery were Hct independent and no significant impact of Hct on the accuracy of the TKI quantifications was observed. All TKIs were stable in VAMS samples stored at −20 °C, 4 °C and room temperature for at least 4 weeks and for 2 days at 60 °C (except ibrutinib). Lastly, we demonstrated a good agreement between liquid blood obtained from patients on TKI treatment and VAMS samples prepared from that venous blood. As this implies that there is no methodological impact of liquid versus dried blood analysis, the presented method can be applied in clinical follow-up studies for determining TKIs in (capillary) VAMS samples with varying Hct levels.

Evaluation and analytical applicability of a novel volumetric absorptive microsampling strategy for adherence monitoring of antihypertensive drugs by means of LC-HRMS/MS

Volumetric absorptive microsampling (VAMS), an emerging microsampling technique, is expected to overcome some disadvantages of dried blood spots. This study aimed to develop and evaluate a VAMS-based strategy for quantifying ten frequently prescribed antihypertensive drugs (AHD) (amlodipine, bisoprolol, candesartan, carvedilol, lercanidipine, losartan carboxylic acid, metoprolol, nebivolol, telmisartan, valsartan) in finger prick blood (FPB) within the scope of adherence monitoring. The straightforward workflow consisted of VAMS tip hydration and subsequent precipitation. Samples were analyzed by using reversed phase ultra-high performance liquid chromatography coupled to orbitrap mass spectrometry operating in parallel reaction monitoring mode. The analytical procedure was successfully validated based on international recommendations for most of the analytes. Selectivity and within/between-run accuracy and precision were in accordance with the recommendations. Internal standard normalized matrix factor met recommended criteria for all analytes at HT 20%, 40%, and 60% except for amlodipine were the CV exceeded 15% at HT 20% (CV 18%). Dilution integrity was given for all substances, covering the quantification in the upper part of the therapeutic range of selected AHD. Long-term stability in VAMS tips was tested and revealed degradation of lercanidipine after one week of storage at 24 °C. A proof of concept of the analytical applicability was done by quantification of selected AHD in VAMS tips and matched plasma samples. Results revealed that determined concentration in FPB by VAMS and plasma cannot be used interchangeably, and thus that specific reference ranges have to be established. However, a novel VAMS application was implemented in the context of adherence monitoring for at least the investigated AHD.

Review of the Preanalytical Errors That Impact Therapeutic Drug Monitoring.

Preanalytical errors comprise the majority of testing errors experienced by clinical laboratories and significantly impact the accuracy of therapeutic drug monitoring (TDM). Specific preanalytical factors in sample timing, collection, transport, processing, and storage that lead to errors in TDM were reviewed. We performed a literature search using several scientific databases including PubMed, ScienceDirect, Scopus, Web of Science, and ResearchGate for human studies published in the English language from January 1980 to February 2021, reporting on TDM and the preanalytical phase. Blood collection errors (ie, wrong anticoagulant/clot activator used, via an intravenous line, incorrect time after dosing) delay testing, cause inaccurate results, and adversely impact patient care. Blood collected in lithium heparin tubes instead of heparin sodium tubes produce supertoxic lithium concentrations, which can compromise care. Specimens collected in serum separator gel tubes cause falsely decreased concentrations due to passive absorption into the gel when samples are not processed and analyzed quickly. Dried blood spots are popular for TDM as they are minimally invasive, allowing for self-sampling and direct shipping to a clinical laboratory using regular mail. However, blood collection techniques, such as trauma to the collection site, filter paper fragility, and hematocrit (Hct) bias, can adversely affect the accuracy of the results. Volumetric absorptive microsampling is a potential alternative to dried blood spot that offers fast, volume-fixed sampling, low pain tolerance, and is not susceptible to Hct concentrations. The identification of preanalytical factors that may negatively impact TDM is critical. Developing workflows that can standardize TDM practices, align appropriate timing and blood collection techniques, and specimen processing will eliminate errors.

Development and Validation of the Quantification Method for Hydroxychloroquine in Volumetric Absorptive Microsampling (VAMS) Using High-Performance Liquid Chromatography-Photodiode Array.

Hydroxychloroquine is an antimalarial drug used for systemic lupus erythematosus, rheumatoid arthritis, and malaria treatment. However, hydroxychloroquine has several side effects such as ocular toxicity, neurotoxicity, gastrointestinal disorder, and also severe toxicity such as cardiotoxicity. Therefore, therapeutic drug monitoring of high dose or long-term use of hydroxychloroquine is needed. This study aims to obtain an optimum and validated analysis and preparation method for hydroxychloroquine in volumetric absorptive microsampling (VAMS) using the high-performance liquid chromatography–photodiode array detector based on the Food and Drug Administration guidelines (2018). Hydroxychloroquine quantification was performed using HPLC-PDA with Waters Sunfire™ C18 (5 µm; 250 × 4,6 mm) column. Mobile phase consists of acetonitrile-diethylamine 1% (65 : 35, v/v) (isocratic elution) and delivered at a flow rate of 0.8 mL/min throughout the 12 minutes run. Sample in VAMS is extracted by liquid-liquid extraction with ammonia 1% and n-hexane-ethyl acetate (50 : 50 v/v) as a extraction solvent. This method has successfully qualified the Food and Drug Administration (2018) parameters, with 2 ng/mL of LLOQ, range of calibration curve 2–6500 ng/mL, and coefficient of correlation 0.9993–0.9997.

Direct analysis of Volumetric Absorptive Micro Sampling (VAMS) devices by ATR-FT-MIR and chemometric analysis: A new challenge

Volumetric Absorptive Micro Sampling (VAMS) strategy, in its simplicity, has made a major contribution to the development of at-home sampling strategies. Mainly used for blood analysis, it absorbs a fixed volume of sample. Folded into its cover, the VAMS device dries, and it can be sent to a lab via mail.In this article, for the first time in our knowledge, we explored the possibility to use this sampling strategy to expand the scope of VAMS to other samples than clinical ones. In this way we used VAMS to sample and analyze milk, which is one of the most important and analyzed samples all over the world. VAMS devices were employed to sample commercial milk samples from Italy, Switzerland and Spain, and for the first time the device was directly analyzed by ATR-FT-IR to predict protein, carbohydrate and fat content in the milk samples. Samples were collected in different sessions from different persons and analyzed by different lab operators to include in the models these sources of variability. Multivariate regression was used to correlate ATR-FT-IR spectra with the investigated properties: models were validated with external validation.

Determination of paracetamol and its metabolites via LC-MS/MS in dried blood volumetric absorptive microsamples: A tool for pharmacokinetic studies

Paracetamol (acetaminophen, APAP) is the most frequently used analgesic and antipyretic worldwide. Nonetheless, APAP induced hepatotoxicity is the most common cause of acute liver failure in the western world. This hepatotoxicity is related to the metabolism of APAP, via the formation of the electrophilic oxidation product N-acetyl-para-benzoquinone imine. To investigate differences in APAP metabolism in specific patient populations and to optimize dosing regimens, quantification of metabolites from the different metabolic pathways is needed to perform pharmacokinetic (PK) studies. For this purpose, sensitive and short liquid chromatography-tandem mass spectrometry methods were developed for the quantitation of APAP and four of its metabolites (APAP-glucuronide, APAP-sulfate, APAP-mercapturate, and APAP-cysteine) in plasma, whole blood and dried blood microsamples collected via 10 µL volumetric absorptive microsampling (VAMS) devices. The methods were successfully validated based on internationally accepted guidelines (EMA, FDA), encompassing selectivity, evaluation of the calibration model, matrix effect and recovery, accuracy and precision, stability, and dilution integrity. In addition, for the VAMS samples, the effect of the hematocrit on the recovery was evaluated. Successful application on whole blood and plasma, as well as on VAMS samples prepared from venous or capillary blood of patients, demonstrated that the methods were fit-for-purpose and can be used for future PK studies.

Volumetric absorptive microsampling (VAMS) as a reliable tool to assess thiamine status in dried blood microsamples: a comparative study

Although populations from low- and middle-income countries are at higher risk for thiamine (vitamin B-1) deficiency, accurate data on the global prevalence of thiamine deficiency are still lacking due to the difficult blood collection in remote regions. Volumetric absorptive microsampling (VAMS) from finger prick blood, generating dried blood microsamples, could simplify blood collection and allow the setup of epidemiological studies to improve the diagnosis, treatment, and prevention of thiamine deficiency. To explore the potential of VAMS to serve as an alternative, patient-centric sampling strategy to evaluate the thiamine status. Venous liquid, venous VAMS, and capillary VAMS samples were collected from 50 healthy volunteers to compare thiamine diphosphate results, as a marker of thiamine (vitamin B-1) status, in the different sample types. In addition, capillary VAMS samples were sent through regular mail to evaluate the influence of noncontrolled transport on the final results. All samples were analyzed using previously described fully validated LC-MS/MS methods. A good agreement (94-100% of the results lying within 20% of their mean) was obtained for all comparisons: venous VAMS compared with venous liquid blood samples, capillary VAMS compared with venous VAMS samples, and capillary VAMS compared with venous liquid blood samples, with no significant bias (maximum mean bias of -1.0%; 95% CI: -4.1%, 2.0%) observed between the different methods. Finally, we demonstrated that VAMS samples can be safely transported through regular mail without affecting the final results. VAMS sampling can be used as a reliable alternative tool to evaluate the thiamine status, starting from only one drop of finger prick blood, in both developed and developing countries.

Volumetric absorptive microsampling: its use in COVID-19 research and testing.

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.

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.

Enhanced urinary stability of peptide hormones and growth factors by dried urine microsampling

Volumetric absorptive microsampling (VAMS) and dried urine spot (DUS) strategies were applied for the collection of dried microsamples for anti-doping testing of low-stability peptide hormones and growth factors prohibited by the World Anti-Doping Agency (WADA). Drying, storage and transport conditions, as well as pretreatment steps, were optimised before liquid chromatography - tandem mass spectrometry (LC–MS/MS) analysis. The analytical method has been fully validated in terms of sensitivity (limits of quantitation 0.3−10 ng/mL), precision (RSD% < 6.6 %) and extraction yields (78–91 %). Dried microsample stability studies (90 days) have been performed and compared to fluid urine stability. Significantly higher losses have been observed in fluid urine stored at −20 °C (up to 55 %) and −80 °C (up to 29 %) than in dried urine microsamples stored at room temperature (< 19 %). The final microsampling and analysis protocols allow the collection of urine microvolumes, unlikely to be tampered, stably storable and shippable with no particular precautions for possible anti-doping testing of prohibited peptides and hormones.

Therapeutic Drug Monitoring of Antiseizure Medications Using Volumetric Absorptive Microsampling: Where Are We?

Therapeutic drug monitoring (TDM) of antiseizure medications (ASMs) represents a valuable tool to establish an appropriate patient therapy, to collect important information about drugs’ interactions and to evaluate patient’s metabolic capabilities. In recent years, a new volumetric absorptive microsampling technique using VAMS® technology and Mitra® devices, consisting of a sampling technique for the collection of fixed-volume capillary blood, was developed. These new devices provide a new home-sampling technique for whole blood that has been spread out to simplify sample collection from finger-pricks. This review is aimed to compare published articles concerning the application of VAMS® in epilepsy and to identify the strengths and improvement points for the TDM of antiseizure medications. VAMS® allowed a minimally invasive blood sampling even in the absence of trained personnel. Good stability data have indicated that storage and delivery can be facilitated only for specific ASMs. Trueness and precision parameters have been evaluated, and the hematocrit (HCT) effect was minimized.

Pharmacokinetics of Hydroxyurea Therapy in African Children with Sickle Cell Anemia:Â Noharm Ancillary PK Study

▪Background. Hydroxyurea is an important disease-modifying therapy for sickle cell anemia (SCA). Through induction of fetal hemoglobin and other mechanisms, hydroxyurea reduces the number and severity of vaso-occlusive painful crises and episodes of acute chest syndrome, while also lowering the frequency of transfusions and hospitalizations, and even reducing mortality. In the US, hydroxyurea is recommended widely for both children and adults with SCA, although variation in treatment response is well-recognized. In sub-Saharan Africa, which has the greatest global burden of SCA, hydroxyurea is not used widely, due in part to a lack of data regarding its safety and efficacy. Pharmacokinetics (PK) analysis of hydroxyurea has also not been performed to date in Africa, but would provide important data regarding the variable responses to hydroxyurea treatment in low-resource settings.Objectives. There were three main goals of the NOHARM Ancillary PK Study: (1) to evaluate the feasibility of hydroxyurea sparse sampling PK collection and analysis in a low-resource clinical setting; (2) to determine the PK parameters for a cohort of young African children with SCA receiving hydroxyurea at an average oral daily dose of 20 mg/kg; and (3) to compare the PK parameters of African children collected and analyzed in the NOHARM trial with those of US children with SCA from the earlier HUSTLE study that included African-American children with SCA.Methods. NOHARM (NCT01976416) is a prospective, randomized, placebo-controlled, double-blinded phase 3 trial that is investigating the effects of hydroxyurea on the incidence and severity of malaria in very young children living in Uganda with SCA. After a year of blinded treatment, all participants were offered open-label hydroxyurea at an average oral daily dose of 20 ± 2.5 mg/kg, using either a 5-day or 7-day per week treatment regimen. After written informed consent for the ancillary PK study, hydroxyurea analysis was performed using a published sparse-sampling technique based on Bayesian estimation with collections pre-dose and then at T=15 minutes, 60 minutes, and 180 minutes. These four timed finger-prick sample collections over 3 hours used a volumetric absorptive microsampling device that collects exact 10 uL aliquots. All samples were stored at -80°C until analysis of hydroxyurea by LC-MS/MS (ug/mL) using a stable isotope internal standard, performed at Cincinnati Children's Hospital. Population PK analysis was performed using nonlinear mixed effect modeling (NONMEM 7.2). Allometric scaling was used to correct for differences in size.Results. A total of 100 children were enrolled in the NOHARM ancillary PK study (average age 3.8 ± 0.9 years; 55 males), and 98 of these children had three evaluable post-dose samples. Individual PK profiles were best described by a one-compartment model with first order absorption. The dose-normalized hydroxyurea concentrations in NOHARM were comparable between the 5-day and 7-day dosing regimens and similar to published HUSTLE data for 96 children, except for higher T=180 hydroxyurea concentrations and overall higher hydroxyurea exposure in the NOHARM cohort. The higher blood hydroxyurea concentrations were the result of reduced drug clearance in NOHARM, possibly influenced by younger age. Population PK parameter estimates of oral clearance in the NOHARM and HUSTLE cohorts were 9.8 L/h/70kg versus 12.9 L/h/70kg, respectively (p<0.01).Conclusions. The NOHARM PK study demonstrates the feasibility of collecting timed samples for analysis of hydroxyurea exposure, even in the low-resource setting of Uganda. Using a sparse sampling technique, young children with SCA had individual PK parameters calculated and a population PK model established. The oral drug clearance was lower in NOHARM than HUSTLE, with resulting higher hydroxyurea exposure, perhaps reflecting differences in age, nutrition, glomerular filtration, and genetic modifiers that warrant further investigation. DisclosuresWare:Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Microsampling Assays for Pharmacokinetic Analysis and Therapeutic Drug Monitoring of Antimicrobial Drugs in Children: A Critical Review.

With the increasing prevalence of multidrug resistant organisms, therapeutic drug monitoring (TDM) has become a common tool for assuring the safety and efficacy of antimicrobial drugs at higher doses. Microsampling techniques, including dried blood spotting (DBS) and volumetric absorptive microsampling (VAMS), are attractive tools for TDM and pediatric clinical research. For microsampling techniques to be a useful tool for TDM, it is necessary to establish the blood-plasma correlation and the therapeutic window of antimicrobial drugs in the blood. DBS involves the collection of small volumes of blood (30-50 µL per spot) on a filter paper, whereas VAMS allows the accurate and precise collection of a fixed volume of blood (10-30 µL) with microsampling devices. One of the major advantages of VAMS is that it reduces or eliminates the volumetric blood hematocrit (HCT) bias associated with DBS. Liquid chromatography with tandem mass spectrometry is a powerful tool for the accurate quantification of antimicrobial drugs from small volumes of blood specimens. This review summarizes the recent liquid chromatography with tandem mass spectrometry assays that have used DBS and VAMS approaches for quantifying antimicrobial drugs. Sample collection, extraction, validation outcomes, including the interassay and intra-assay accuracy and precision, recovery, stability, and matrix effect, as well as the clinical application of these assays and their potential as tools of TDM are discussed herein. Microsampling techniques, such as VAMS, provide an alternative approach to traditional plasma sample collection for TDM.

Analysis of tamoxifen and its metabolites in dried blood spot and volumetric absorptive microsampling: comparison and clinical application

This research was conducted to develop the Dried Blood Spot (DBS) and Volumetric Absorptive Microsampling (VAMS) method in the analysis of Tamoxifen (TAM) and its metabolites endoxifen (END), 4-hydroxytamoxifen (4-HT), and N-desmethyltamoxifen (NDT) using Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). This method was then applied to monitor TAM and its metabolites in breast cancer patients. The UPLC-MS/MS method was developed and validated with propranolol as the internal standard. The recovery and matrix effects on DBS and VAMS were investigated. The validation requirements were fulfilled by the methodology of analysis and sample preparation described in this study. Both VAMS and DBS extraction recoveries were satisfactory, with low variability. Extraction recovery in the VAMS sample was found to be slightly higher than in the DBS sample. Sample stability in DBS and VAMS was demonstrated for up to 2 months. Both of these methods were successfully applied for the analysis of TAM and metabolites in clinical patients. The mean concentrations obtained from the two methods were not significantly different.

Volumetric Absorptive Microsampling as a New Biosampling Tool for Monitoring of Tamoxifen, Endoxifen, 4-OH Tamoxifen and N-Desmethyltamoxifen in Breast Cancer Patients.

IntroductionIn this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites.MethodsVAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C18 column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for N-desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol.Results and DiscussionThe lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.

Simultaneous LC-MS/MS quantification of oxycodone, tramadol and fentanyl and their metabolites (noroxycodone, oxymorphone, O- desmethyltramadol, N- desmethyltramadol, and norfentanyl) in human plasma and whole blood collected via venepuncture and volumetric absorptive micro sampling

IntroductionA range of opioids are commonly prescribed to manage chronic pain, but individual patient responses vary greatly, especially in older populations. One source of that variability are differences in absorption, metabolism and excretion, i.e. pharmacokinetics. Blood, plasma and serum concentrations of opioids allow that variability to be quantified and may be used to optimise opioid dosing. As an aid to that process, there is an unmet need to rapidly quantify several opioids and their metabolites in a single analytical method. AimsTo develop a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of tramadol, oxycodone, fentanyl and their major metabolites in various human matrices. MethodsSample preparation involved adding three deuterated internal standards followed by protein precipitation with 100 % acetonitrile, evaporation and reconstitution. Separation of analytes via LC was achieved on a reversed phase column via binary gradient elution using 0.005 % formic acid in water and 100 % acetonitrile as mobile phases. Analytes were detected via MS/MS with multiple reaction monitoring (MRM). ResultsThe method was accurate with the inter-day and intra-day accuracy of quality control samples (QCs) below 15 %. It was also precise with inter-day and intra-day coefficient of variation below 15 %. The lower limit of quantification (LLOQ) was 0.2 ng/mL for all analytes except tramadol and its metabolites, where the LLOQ was 10 ng/mL. Recovery was greater than 88 % for all analytes, except for O-desmethyltramadol (81 %). Analytes were stable over four freeze-thaw cycles, for 24 h on the bench top and for 24 h post-preparation. The inter- and intra-day variability of concentrations determined in blood and plasma were within 84–124%, whereas the inter- and intra-day variability for blood samples prepared using volumetric absorptive micro-sampling (VAMS) compared to those prepared from whole blood ranged between 83–122%. ConclusionA LC–MS/MS method is described that is able to accurately and precisely quantify a number of commonly prescribed opioids and their major metabolites in plasma and whole blood, including whole blood collected using VAMS.