Miniaturisation and simplification are novel approaches in clinical bioanalysis, especially in therapeutic drug monitoring (TDM). These contemporary trends are related to the sampling, pre-treatment, and analysis of biological fluids. Currently, dried blood spot (DBS), one of the most popular microsampling techniques, is feasible and inexpensive. However, obtaining reliable results with sample homogeneity and volume variability is difficult. Volumetric Absorptive Microsampling (VAMS) has recently enabled the accurate and precise collection of a fixed blood volume. It reduced the hematocrit effect, improved volumetric accuracy, and generated results correlating with the dose and drug exposure from wet blood. This review focuses on VAMS-Mitra™ devices, which have become increasingly important since 2014, mainly for TDM and toxicology studies. First, the current literature has been reviewed based on immunosuppressants and their determination in samples obtained using Mitra™. Second, the critical points, weaknesses, and strengths have been characterized in contrast to classic venipuncture and other microsampling methods. Finally, we indicate the points of attention according to the perspective of Mitra™ as well as its usefulness in clinical practice. VAMS is currently state-of-the-art in microsampling and seems to be a good instrument for improving adherence to immunosuppressive therapy, especially in the pediatric population.

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

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

Immunosuppressive pharmacotherapy after solid organ transplantation (SOT) requires therapeutic drug monitoring (TDM) for therapy individualization. The venous whole blood is still considered as routine matrix for monitoring immunosuppressive drug concentration. On the other hand, as an alternative, capillary blood collected using noninvasive sampling is convergent with a patient-centric approach. Despite their disadvantages regarding sample homogeneity and the hematocrit effect, well-known dried blood spot techniques have shown promising results. Volumetric absorptive microsampling (VAMS) and quantitative dried blood spot (qDBS) have successfully eliminated these unfavorable biased elements. Microsampling can be used in transplant recipients' care, mainly due to long-term therapy under control drug concentrations and the long distance between the place of the patient's residence and the diagnostic laboratory in the transplant center. The study aimed to discuss the clinical consequences of implementing microsampling techniques for TDM of immunosuppressants. Additionally, we have discussed the 'hot topics' in microsampling: home-based self-sampling, adherence to therapy monitoring, and drug concentration conversion to estimated traditional matrices. Finally, based on our experience and current practice, we propose best practices for microsampling implementation from bench to bedside. Microsampling techniques can potentially revolutionise immunosuppressive pharmacotherapy by enabling patient-centric individualisation in various subpopulations, significantly improving post-transplant care.

Ciclosporin (CSA) is an immunosuppressive agent that requires therapeutic drug monitoring (TDM). High partitioning in erythrocytes indicates that whole blood (WB) is a suitable matrix for CSA determination. Alternative sampling strategies, such as volumetric absorptive microsampling (VAMS), are novel possibilities for blood collection during TDM for various analytes, including immunosuppressants. This technique is attractive for vulnerable pediatric patients, including home-based self-sampling, remote therapy, and adherence control. This study aimed to develop and validate a new method for CSA determination based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of WB and VAMS samples. Additionally, these methods were applied for CSA determination in clinical samples from pediatric transplant recipients. A strong point of this study is the assessment of an external proficiency testing scheme. Both methods were successfully validated within the 1-2000 ng/mL calibration range, with LOD 0.5 and 1 ng/mL for WB and VAMS methods, respectively. All the validation parameters fulfilled the international acceptance criteria for bioanalytical methods. Cross-validation confirmed the interchangeability of the LC-MS/MS method developed in this study. This study developed and validated novel methods for CSA determination in whole blood and VAMS using LC-MS/MS. Clinical validation and proficiency testing confirmed their utility in routine clinical practice.

Methotrexate (MTX) is an antifolic agent used in the first line of anti-inflammatory disease treatment and some oncologic issues. The metabolism of MTX is rapid, and the MTX concentration in the blood is not significant 24 h after administration. Unlike this, methotrexate polyglutamates (MTXPGs) can be informative biomarkers of drug exposure. It is widely concluded that MTXPG retention in red blood cells (RBCs) is related to appropriate efficacy, drug exposure, and toxicity during treatment. Therefore, the mentioned biomarker may be appropriately used for the PK/PD monitoring of low-dose MTX (LDMTX) treatment. The presented review study aimed to review published studies about MTX determination in clinical practice, including pre-analytical variability, bioanalytical considerations, and clinical applications of the methods for pharmacotherapy supporting target populations. In total, 14 papers from the field of bioanalytics have been included in the main review. For each phase of an analytical process, the best practises and main findings were defined as guidelines for proper analytical method optimisation, validation, and standard operation procedure implementation in clinical practice. The presented study is the first comprehensive review of MTX and its methods of metabolite determination to account for pre-analytical, analytical, and post-analytical phases concerning the TDM process.