Search Results (31)

Everolimus (EVE), an mTOR inhibitor, is widely used in solid organ transplantation (SOT) because of its immunosuppressive properties. Due to its narrow therapeutic window and significant pharmacokinetic variability, therapeutic drug monitoring (TDM) is essential for achieving optimal outcomes. We developed and thoroughly validated a robust LC-MS/MS method to measure EVE levels in venous whole blood (WB) and capillary blood collected using two microsampling devices: Mitra™ (volumetric absorptive microsampling, VAMS) and Capitainer^® (quantitative dried blood spot, qDBS). The validation followed EMA and IATDMCT guidelines, assessing linearity (1.27–64.80 ng/mL for WB and 0.50–60 ng/mL for VAMS/qDBS), as well as selectivity, accuracy, precision, matrix effects, recovery, stability, and incurred sample reanalysis. Clinical validation involved 66 matched samples from 33 adult SOT recipients. The method demonstrated high accuracy and precision across all matrices, with no significant carryover or matrix interference. Statistical analysis using Passing–Bablok regression and Bland–Altman plots showed excellent agreement between the microsampling methods and the venous reference. Hematocrit effects were tested both in laboratory conditions and on clinical samples and were found to be negligible. This study provides the first comprehensive analytical and clinical validation of the Mitra and Capitainer devices for EVE monitoring. The validated LC-MS/MS microsampling method supports decentralized, patient-centred TDM, offering a reliable alternative to conventional blood sampling in transplant care. Full article

Objectives: A simple method for quantifying fluconazole in small blood volumes has been developed using volumetric absorptive microsampling (VAMS^®) technology and isocratic high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Methods: For sample collection, Mitra^® devices are used to keep the sample volume at 10 µL. For the quantitative determination of fluconazole, the Mitra^® samples are extracted using acetonitrile as the extraction agent, containing 2-(4-chlorophenyl)-1,3-bis(1,2,4-triazol-1-yl)propan-2-ol as the internal standard. A Synergi 4 μm Polar-RP 80 Å (150 × 2 mm) column forms the stationary phase, and a mixture of acetonitrile and phosphate buffer is the mobile phase. The UV detection is set at a wavelength of 210 nm. The therapeutic concentration range of 5 to 160 mg/L is covered, and the linear equation with 1/x² weighting is used to determine unknown samples. This method has been validated according to the current EMA and FDA guidelines for bioanalytical methods. Results: The validation data obtained after analysing whole blood samples (EDTA) showed within- and between-run accuracy between 94.4% and 115% and precision between 0.4% and 9.4%, respectively. A lower limit of quantification (LLOQ) of 5 mg/L was sufficient for therapeutic drug monitoring in paediatric patients receiving fluconazole as antifungal prophylaxis after haematopoietic cell transplantation. Conclusions: So far, 211 samples from 49 patients were successfully analysed, and concentrations between 5.84 mg/L and 107 mg/L were determined for whole blood Mitra^® samples. To our knowledge, this is the first application of VAMS^® technology using simple and cheap HPLC-UV quantification. Full article

Background: The combination of ivacaftor, tezacaftor and elexacaftor (ETI) is approved for patients with cystic fibrosis (CF) aged two years and older and at least one F508del mutation in the CFTR gene. Variability in ETI treatment response has been repeatedly reported, and its reasons are unclear and understudied. Objectives: We present a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid and simultaneous quantification of ETI in plasma, dried plasma spots (DPS), and whole blood volumetric absorptive microsampling (VAMS). Methods: The method utilizes a rapid extraction protocol with 200 μL methanol after the addition of deuterated internal standards. Chromatographic separation was achieved using a reversed-phase Hypersil Gold aQ column (Thermo Fisher Scientific). The method was validated according to ICH (International Council on Harmonisation) guidelines M10 for bioanalytical method validation, demonstrating linearity in the concentration range 0.020–12.000 µg/mL. It was also proved accurate and reproducible with no matrix effect. This method was applied to anonymized samples from patients undergoing ETI treatment: eight plasma and DPS and five VAMS samples were analyzed. Results: ETI concentrations measured in plasma and DPS were interchangeable, whereas ETI concentrations in VAMS were lower than in plasma, as expected for molecules with high plasma protein binding (99%). A correction factor based on the hematocrit value was used to calculate the equivalent plasma concentration from VAMS concentrations. Conclusions: This method is suitable for pharmacokinetic (PK) studies and could facilitate the centralization of samples to specialized laboratories, supporting multicenter studies. Full article

Blood microsampling (BμS) has recently emerged as an interesting approach in the analysis of endogenous metabolites but also in metabolomics applications. Their non-invasive way of use and the simplified logistics that they offer renders these technologies highly attractive in large-scale studies, especially the novel quantitative microsampling approaches such as VAMs or qDBS. Objectives: Herein, we investigate the potential of BµS devices compared to the conventional plasma samples used in global untargeted mass spectrometry-based metabolomics of blood. Methods: Two novel quantitative devices, namely, Mitra, Capitainer, and the widely used Whatman cards, were selected for comparison with plasma. Venous blood was collected from 10 healthy, overnight-fasted individuals and loaded on the devices; plasma was also collected from the same venous blood. An extraction solvent optimization study was first performed on the three devices before the main study, which compared the global metabolic profiles of the four extracts (three BµS devices and plasma). Analysis was conducted using reverse phase LC-TOF MS in positive mode. Results: BµS devices, especially Mitra and Capitainer, provided equal or even superior information on the metabolic profiling of human blood based on the number and intensity of features and the precision and stability of some annotated metabolites compared to plasma. Despite their rich metabolic profiles, BµS did not capture metabolites associated with biological differentiation of sexes. Conclusions: Overall, our results suggest that a more in-depth investigation of the acquired information is needed for each specific application, as a metabolite-dependent trend was obvious. Full article

Volumetric absorptive microsampling (VAMS) is an emerging technique in clinical and forensic toxicology. It is recognized as a promising alternative to traditional sampling methods, offering an accurate and minimally invasive means of collecting small volumes of biological samples, such as blood, urine, and saliva. Unlike conventional methods, VAMS provides advantages in terms of sample stability, storage, and transportation, as it enables samples to be collected outside laboratory environments without requiring refrigeration. This review explores several VAMS methodologies, with a particular focus on its application for the quantification of drugs and other substances in clinical and forensic toxicology. It compares VAMS to other microsampling techniques, such as dried blood spots (DBSs), highlighting VAMS’s superiority in addressing issues related to sample volume consistency and environmental impact. Despite its advantages, VAMS also presents certain limitations, including higher costs and difficulties in detecting underfilled samples. Overall, VAMS stands out as a microsampling technique with the potential to enhance patient compliance and operational efficiency, positioning itself as a viable tool for toxicological analysis in both clinical and forensic contexts. Full article

Our aim was to compare the performance of complementary clinical laboratory approaches to monitoring exposure to apixaban and rivaroxaban, the most prescribed direct-acting oral anticoagulants (DOAC’s): an automated commercial anti-Xa chromogenic assay suitable for emergency and pre-surgery testing and a laboratory-developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employed for non-emergency analysis in plasma and in dried blood volumetric absorptive microsamples (VAMS) collectible by the patients in their homes. The full validation of the LC-MS/MS method was performed. Cross-validation of the methodologies was accomplished by processing 60 specimens collected for whole blood count and DOAC monitoring in a central clinical laboratory. For VAMS samples, dried plasma and whole blood calibrators were found to be suitable, and a cycle run for seven days could be implemented for rational and economic sample processing. The anti-Xa chromogrenic assay and the LC-MS/MS method delivered discordant plasma analyte concentrations. Moreover, the lack of agreement between plasma and VAMS concentrations was observed. Clinical laboratories must be aware of the differences between the performance of apixaban and rivaroxaban LC-MS/MS and anti-Xa assays. Hematocrit must always be measured along with VAMS samples to obtain accurate results. Full article

Ganciclovir (GCV) and its prodrug valganciclovir (VGCV) are antiviral medications primarily used to treat infections caused by cytomegalovirus (CMV), particularly in immunocompromised individuals such as solid organ transplant (SOT) recipients. Therapy with GCV is associated with significant side effects, including bone marrow suppression. Therefore, therapeutic drug monitoring (TDM) is mandatory for an appropriate balance between subtherapeutic and toxic drug levels. This study aimed to develop and validate three novel methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for GCV determination in serum (reference methodology), dried serum spots (DSS), and VAMS-Mitra™ devices. The methods were optimized and validated in the 0.1–25 mg/L calibration range. The obtained results fulfilled the EMA acceptance criteria for bioanalytical method validation. Assessment of DSS and VAMS techniques extended GCV stability to serum for up to a minimum of 49 days (at room temperature, with desiccant). Developed methods were effectively evaluated using 80 clinical serum samples from pediatric renal transplant recipients. Obtained samples were used for DSS, and dried serum VAMS samples were manually generated in the laboratory. The results of GCV determination using serum-, DSS- and VAMS-LC-MS/MS methods were compared using regression analysis and bias evaluation. The conducted statistical analysis confirmed the interchangeability between developed assays. The DSS and VAMS samples are more accessible and stable during storage, transport and shipment than classic serum samples. Full article

Fibromyalgia (FM) is a chronic central sensitivity syndrome characterized by augmented pain processing at diffuse body sites and presents as a multimorbid clinical condition. Long COVID (LC) is a heterogenous clinical syndrome that affects 10–20% of individuals following COVID-19 infection. FM and LC share similarities with regard to the pain and other clinical symptoms experienced, thereby posing a challenge for accurate diagnosis. This research explores the feasibility of using surface-enhanced Raman spectroscopy (SERS) combined with soft independent modelling of class analogies (SIMCAs) to develop classification models differentiating LC and FM. Venous blood samples were collected using two supports, dried bloodspot cards (DBS, n = 48 FM and n = 46 LC) and volumetric absorptive micro-sampling tips (VAMS, n = 39 FM and n = 39 LC). A semi-permeable membrane (10 kDa) was used to extract low molecular fraction (LMF) from the blood samples, and Raman spectra were acquired using SERS with gold nanoparticles (AuNPs). Soft independent modelling of class analogy (SIMCA) models developed with spectral data of blood samples collected in VAMS tips showed superior performance with a validation performance of 100% accuracy, sensitivity, and specificity, achieving an excellent classification accuracy of 0.86 area under the curve (AUC). Amide groups, aromatic and acidic amino acids were responsible for the discrimination patterns among FM and LC syndromes, emphasizing the findings from our previous studies. Overall, our results demonstrate the ability of AuNP SERS to identify unique metabolites that can be potentially used as spectral biomarkers to differentiate FM and LC. Full article

Relative energy deficiency in sport (RED-S) is a condition that arises from persistent low energy availability (LEA), which affects the hypothalamic–pituitary axis and results in alterations of several hormones in both male and female athletes. As frequent blood hormone status determinations using venipuncture are rare in sports practice, microsampling offers promising possibilities for preventing and assessing RED-S. Therefore, this study aimed at developing a liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS/MS) method for quantifying relevant steroids and thyroid hormones in 30 μL of capillary blood obtained using Mitra^® devices with volumetric absorptive microsampling technology (VAMS^®). The results of the study showed that all validation criteria were met, including a storage stability of more than 28 days in a frozen state (−18 °C) and 14 days at room temperature (20 °C). The validated assay provided precise (<12%) and accurate (<13%) results for all the target analytes. Furthermore, as a proof of concept, autonomously collected VAMS^® samples from 50 female and male, healthy, active adults were analyzed. The sensitivity of all analytes was adequate to quantify the decreased hormone concentrations in the RED-S state, as all authentic samples could be measured accordingly. These findings suggest that self-collected VAMS^® samples offer a practical opportunity for regular hormone measurements in athletes and can be used for early RED-S assessment and progress monitoring during RED-S recovery. Full article

The diagnostic criteria for fibromyalgia (FM) have relied heavily on subjective reports of experienced symptoms coupled with examination-based evidence of diffuse tenderness due to the lack of reliable biomarkers. Rheumatic disorders that are common causes of chronic pain such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and chronic low back pain are frequently found to be comorbid with FM. As a result, this can make the diagnosis of FM more challenging. We aim to develop a reliable classification algorithm using unique spectral profiles of portable FT-MIR that can be used as a real-time point-of-care device for the screening of FM. A novel volumetric absorptive microsampling (VAMS) technique ensured sample volume accuracies and minimized the variation introduced due to hematocrit-based bias. Blood samples from 337 subjects with different disorders (179 FM, 158 non-FM) collected with VAMS were analyzed. A semi-permeable membrane filtration approach was used to extract the blood samples, and spectral data were collected using a portable FT-MIR spectrometer. The OPLS-DA algorithm enabled the classification of the spectra into their corresponding classes with 84% accuracy, 83% sensitivity, and 85% specificity. The OPLS-DA regression plot indicated that spectral regions associated with amide bands and amino acids were responsible for discrimination patterns and can be potentially used as spectral biomarkers to differentiate FM and other rheumatic diseases. Full article

Volumetric absorptive microsampling (VAMS) has arisen as a relevant tool in biological analysis, offering simplified sampling procedures and enhanced stability. Most of the attention VAMS has received in the past decade has been from pharmaceutical research, with most of the published work employing VAMS targeting drugs or other exogenous compounds, such as toxins and pollutants. However, biomarker analysis by employing blood microsampling has high promise. Herein, a comprehensive review on the applicability of VAMS devices for the analysis of endogenous metabolites/biomarkers was performed. The study presents a full overview of the analysis process, incorporating all the steps in sample treatment and validation parameters. Overall, VAMS devices have proven to be reliable tools for the analysis of endogenous analytes with biological importance, often offering improved analyte stability in comparison with blood under ambient conditions as well as a convenient and straightforward sample acquisition model. Full article

Volumetric absorptive microsampling (VAMS) is the newest and most promising sample-collection technique for quantitatively analyzing drugs, especially for routine therapeutic drug monitoring (TDM) and pharmacokinetic studies. This technique uses an absorbent white tip to absorb a fixed volume of a sample (10–50 µL) within a few seconds (2–4 s), is more flexible, practical, and more straightforward to be applied in the field, and is probably more cost-effective than conventional venous sampling (CVS). After optimization and validation of an analytical method of a drug taken by VAMS, a clinical validation study is needed to show that the results by VAMS can substitute what is gained from CVS and to justify implementation in routine practice. This narrative review aimed to assess and present studies about optimization and analytical validation of assays for drugs taken by VAMS, considering their physicochemical drug properties, extraction conditions, validation results, and studies on clinical validation of VAMS compared to CVS. The review revealed that the bio-analysis of many drugs taken with the VAMS technique was optimized and validated. However, only a few clinical validation studies have been performed so far. All drugs that underwent a clinical validation study demonstrated good agreement between the two techniques (VAMS and CVS), but only by Bland–Altman analysis. Only for tacrolimus and mycophenolic acid were three measurements of agreement evaluated. Therefore, VAMS can be considered an alternative to CVS in routine practice, especially for tacrolimus and mycophenolic acid. Still, more extensive clinical validation studies need to be performed for other drugs. Full article

The objective of this study was to validate a novel assay using the volumetric absorptive microsampling (VAMS) technique combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the determination of the antiseizure medication perampanel in saliva and its clinical applicability in patients with epilepsy. VAMS tips were loaded with 30 μL of saliva and dried for 60 min. Analytes were extracted with methanol. The supernatant was evaporated under a gentle stream of nitrogen and reconstituted with 60 μL of methanol. Separation and quantification were achieved on a monolithic column connected to a mass spectrometer. Calibration curves were linear between 0.5 and 300 ng/mL. Intra- and inter-day accuracy was within 85.6–103.2% and intra-day and inter-day precision did not exceed 12.1%. Perampanel was stable in samples collected by VAMS and stored under different storage conditions. The VAMS-LC-MS/MS method was validated according to internationally accepted criteria and tested in patients with epilepsy who were receiving a combination of perampanel and other antiseizure medications. The method showed adequate bioanalytical performances, holding great potential as an alternative strategy to support domiciliary TDM in patients with epilepsy treated with perampanel according to the simplicity of sample collection. Full article

Therapeutic drug monitoring (TDM) is a specialized area of laboratory medicine which involves the measurement of drug concentrations in biological fluids with the aim of optimizing efficacy and reducing side effects, possibly modifying the drug dose to keep the plasma concentration within the therapeutic range. Plasma and/or whole blood, usually obtained by venipuncture, are the “gold standard” matrices for TDM. Microsampling, commonly used for newborn screening, could also be a convenient alternative to traditional sampling techniques for pharmacokinetics (PK) studies and TDM, helping to overcome practical problems and offering less invasive options to patients. Although technical limitations have hampered the use of microsampling in these fields, innovative techniques such as 3-D dried blood spheroids, volumetric absorptive microsampling (VAMS), dried plasma spots (DPS), and various microfluidic devices (MDS) can now offer reliable alternatives to traditional samples. The application of microsampling in routine clinical pharmacology is also hampered by the need for instrumentation capable of quantifying analytes in small volumes with sufficient sensitivity. The combination of microsampling with high-sensitivity analytical techniques, such as liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), is particularly effective in ensuring high accuracy and sensitivity from very small sample volumes. This manuscript provides a critical review of the currently available microsampling devices for both whole blood and other biological fluids, such as plasma, urine, breast milk, and saliva. The purpose is to provide useful information in the scientific community to laboratory personnel, clinicians, and researchers interested in implementing the use of microsampling in their routine clinical practice. Full article

In toxicology, screenings are routinely performed using chromatographic methods coupled to detection systems such as high-resolution mass spectrometry (HR/MS). The increase in specificity and sensitivity of HRMS is responsible for the development of methods for alternative samples such as Volumetric Adsorptive Micro-Sampling. Whole blood overloaded with 90 drugs was sampled with 20 µL Mitra^TM to optimize the pre-analytical step as well as to determine the identification limits of drugs. Elution of chemicals was carried out in a solvent mixture through agitation and sonication. After dissolution, 10 μL was injected into the chromatographic system coupled to the Orbitrap^TM HR/MS. Compounds were confirmed against the laboratory library. The clinical feasibility was assessed in fifteen poisoned patients using the simultaneous sampling of plasma, whole blood and Mitra^TM. The optimized extraction procedure allowed us to confirm 87 compounds out of the 90 present in the spiked whole blood. Cannabis derivatives were not detected. For 82.2% of the investigated drugs, the identification limits were below 12.5 ng·mL⁻¹, with the extraction yields ranging from 80.6 to 108.7%. Regarding the patients’ analysis, 98% of the compounds in plasma were detected in Mitra^TM compared to whole blood, with a satisfying concordance (R² = 0.827). Our novel screening approach opens new insights into different toxicologic fields appropriate for pediatrics, forensics or to perform mass screening. Full article