{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T20:35:25Z","timestamp":1788554125757,"version":"build-2803163510"},"reference-count":30,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2025,7,26]],"date-time":"2025-07-26T00:00:00Z","timestamp":1753488000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004166","name":"Medical University of Warsaw","doi-asserted-by":"publisher","award":["1\/F\/MB\/N\/24"],"award-info":[{"award-number":["1\/F\/MB\/N\/24"]}],"id":[{"id":"10.13039\/501100004166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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\u2122 (volumetric absorptive microsampling, VAMS) and Capitainer\u00ae (quantitative dried blood spot, qDBS). The validation followed EMA and IATDMCT guidelines, assessing linearity (1.27\u201364.80 ng\/mL for WB and 0.50\u201360 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\u2013Bablok regression and Bland\u2013Altman 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.<\/jats:p>","DOI":"10.3390\/molecules30153139","type":"journal-article","created":{"date-parts":[[2025,7,28]],"date-time":"2025-07-28T08:51:33Z","timestamp":1753692693000},"page":"3139","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Therapeutic Drug Monitoring of Everolimus Using Volumetric Absorptive Microsampling and Quantitative Dried Blood Spot Methods with LC-MS\/MS in Adult Solid Organ Transplant Recipients: An Analytical and Clinical Comparative Study"],"prefix":"10.3390","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4833-8532","authenticated-orcid":false,"given":"Arkadiusz","family":"Kocur","sequence":"first","affiliation":[{"name":"Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-8668-1036","authenticated-orcid":false,"given":"Bartosz","family":"Olkowski","sequence":"additional","affiliation":[{"name":"Students\u2019 Scientific Club by the Department of Transplantation, Immunology, Nephrology and Internal Medicine, Medical University of Warsaw, 02-006 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mateusz","family":"Moczulski","sequence":"additional","affiliation":[{"name":"Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorota","family":"Miszewska-Szyszkowska","sequence":"additional","affiliation":[{"name":"Department of Transplantation, Immunology, Nephrology and Internal Medicine, Medical University of Warsaw, 02-006 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1652-6616","authenticated-orcid":false,"given":"Olga Maria","family":"Rostkowska","sequence":"additional","affiliation":[{"name":"Department of Transplantation, Immunology, Nephrology and Internal Medicine, Medical University of Warsaw, 02-006 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna","family":"Polak","sequence":"additional","affiliation":[{"name":"Students\u2019 Scientific Club by the Department of Transplantation, Immunology, Nephrology and Internal Medicine, Medical University of Warsaw, 02-006 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna","family":"Korniluk","sequence":"additional","affiliation":[{"name":"Metabolism Disorders and Therapeutic Drug Monitoring Laboratory, Medical University of Warsaw Clinical Center, \u017bwirki i Wigury 63A, 02-091 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2956-3056","authenticated-orcid":false,"given":"Teresa","family":"B\u0105czkowska","sequence":"additional","affiliation":[{"name":"Department of Transplantation, Immunology, Nephrology and Internal Medicine, Medical University of Warsaw, 02-006 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4798-1497","authenticated-orcid":false,"given":"Magdalena","family":"Durlik","sequence":"additional","affiliation":[{"name":"Department of Transplantation, Immunology, Nephrology and Internal Medicine, Medical University of Warsaw, 02-006 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9110-4312","authenticated-orcid":false,"given":"Tomasz","family":"Pawi\u0144ski","sequence":"additional","affiliation":[{"name":"Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1097\/FTD.0000000000001250","article-title":"Everolimus Personalized Therapy: Second Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology","volume":"47","author":"Masuda","year":"2025","journal-title":"Ther. Drug Monit."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1097\/FTD.0000000000000269","article-title":"Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee","volume":"38","author":"Seger","year":"2016","journal-title":"Ther. Drug Monit."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1080\/17576180.2025.2477976","article-title":"Microsampling techniques and patient-centric therapeutic drug monitoring of immunosuppressants","volume":"17","author":"Kocur","year":"2025","journal-title":"Bioanalysis"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kocur, A., and Pawi\u0144ski, T. (2022). Volumetric Absorptive Microsampling in Therapeutic Drug Monitoring of Immunosuppressive Drugs-From Sampling and Analytical Issues to Clinical Application. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24010681"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1097\/FTD.0000000000000643","article-title":"Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring","volume":"41","author":"Capiau","year":"2019","journal-title":"Ther. Drug Monit."},{"key":"ref_6","unstructured":"Capitainer AB (2025, June 24). Capitainer\u00ae\u2014Volumetric Dried Blood Spot Sampling. Available online: https:\/\/www.capitainer.com."},{"key":"ref_7","unstructured":"Neoteryx (2025, June 24). Volumetric Absorptive Microsampling (VAMS\u2122) Technology. Available online: https:\/\/www.neoteryx.com."},{"key":"ref_8","unstructured":"European Medicines Agency (EMA) (2025, June 24). ICH Guideline M10 on Bioanalytical Method Validation. Available online: https:\/\/www.ema.europa.eu\/en\/documents\/scientific-guideline\/ich-guideline-m10-bioanalytical-method-validation-step-5_en.pdf."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1097\/FTD.0000000000001281","article-title":"Implementation of Volumetric Finger-Prick Self-Sampling for Therapeutic Drug Monitoring of Immunosuppressants After Kidney Transplantation: Lessons Learned From the Practice","volume":"47","author":"Vethe","year":"2025","journal-title":"Ther. Drug Monit."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3690","DOI":"10.1111\/bcp.15870","article-title":"Clinical performance of volumetric finger-prick sampling for the monitoring of tacrolimus, creatinine and haemoglobin in kidney transplant recipients","volume":"89","author":"Vethe","year":"2023","journal-title":"Br. J. Clin. Pharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"340797","DOI":"10.1016\/j.aca.2023.340797","article-title":"Liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants and creatinine from a single dried blood spot using the Capitainer\u00ae qDBS device","volume":"1242","author":"Deprez","year":"2023","journal-title":"Anal. Chim. Acta."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"12893","DOI":"10.1021\/acs.analchem.8b03512","article-title":"Evaluation of the Capitainer-B Microfluidic Device as a New Hematocrit-Independent Alternative for Dried Blood Spot Collection","volume":"90","author":"Velghe","year":"2018","journal-title":"Anal. Chem."},{"key":"ref_13","unstructured":"U.S. Food and Drug Administration (FDA) (2025, June 24). Process Validation: General Principles and Practices, Available online: https:\/\/www.fda.gov\/files\/drugs\/published\/Process-Validation--General-Principles-and-Practices.pdf."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1097\/FTD.0000000000000205","article-title":"Impact of Laboratory Practices on Interlaboratory Variability in Therapeutic Drug Monitoring of Immunosuppressive Drugs","volume":"37","author":"Christians","year":"2015","journal-title":"Ther. Drug Monit."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1016\/j.clinbiochem.2004.11.007","article-title":"Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry","volume":"38","author":"Taylor","year":"2005","journal-title":"Clin. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3019","DOI":"10.1021\/ac020361s","article-title":"Strategies for the Assessment of Matrix Effect in Quantitative Bioanalytical Methods Based on HPLC-MS\/MS","volume":"75","author":"Matuszewski","year":"2003","journal-title":"Anal. Chem."},{"key":"ref_17","unstructured":"Chromsystems Instruments & Chemicals GmbH (MassTox\u00ae Immunosuppressants in Whole Blood\u2014Instruction Manual for LC-MS\/MS Analysis, 2023). MassTox\u00ae Immunosuppressants in Whole Blood\u2014Instruction Manual for LC-MS\/MS Analysis, Version 1.0 IVDR."},{"key":"ref_18","unstructured":"RECIPE Chemicals + Instruments GmbH (ClinMass\u00ae TDM Kit System: Immunosuppressants in Whole Blood\u2014Instruction Manual for LC-MS\/MS Analysis, 2024). ClinMass\u00ae TDM Kit System: Immunosuppressants in Whole Blood\u2014Instruction Manual for LC-MS\/MS Analysis, Version 1.1 IVDD."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"141","DOI":"10.11613\/BM.2015.015","article-title":"Understanding Bland Altman analysis","volume":"25","author":"Giavarina","year":"2015","journal-title":"Biochem. Med."},{"key":"ref_20","first-page":"49","article-title":"Comparison of methods: Passing and Bablok regression","volume":"21","year":"2011","journal-title":"Biochem. Med."},{"key":"ref_21","unstructured":"Clinical and Laboratory Standards Institute (CLSI) (2013). Measurement Procedure Comparison and Bias Estimation Using Patient Samples. Approved Guideline, CLSI. [3rd ed.]. CLSI Document EP09-A3."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1097\/FTD.0000000000001273","article-title":"A Narrative Review of Chromatographic Bioanalytical Methods for Quantifying Everolimus in Therapeutic Drug Monitoring Applications","volume":"47","author":"Nascimento","year":"2025","journal-title":"Ther. Drug Monit."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/j.jchromb.2018.11.030","article-title":"Validation and clinical application of an LC-MS\/MS method for the quantification of everolimus using volumetric absorptive microsampling","volume":"1104","author":"Verheijen","year":"2018","journal-title":"J. Chromatogr. B."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"339889","DOI":"10.1016\/j.aca.2022.339889","article-title":"Quantification of immunosuppressants from one 3.2 mm dried blood spot by a novel cold-induced phase separation based LC-MS\/MS method","volume":"1210","author":"Le","year":"2022","journal-title":"Anal. Chim. Acta."},{"key":"ref_25","first-page":"1039","article-title":"Results from a proficiency testing pilot for microsampling in therapeutic drug monitoring","volume":"60","author":"Veenhof","year":"2022","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Yoo, S., Kim, G., Kim, S., Ha, J., Cho, B.S., Joo, D.J., and Lee, J.I. (Ther. Drug Monit., 2022). Volumetric Absorptive Microsampling for the Therapeutic Drug Monitoring of Everolimus in Patients who have Undergone Liver Transplant, Ther. Drug Monit., ahead of print.","DOI":"10.1097\/FTD.0000000000001033"},{"key":"ref_27","unstructured":"Paniagua-Gonz\u00e1lez, L., D\u00edaz-Louzao, C., and \u00c1lvarez-Fern\u00e1ndez, M. (2020). Volumetric absorptive microsampling (VAMS) for assaying immunosuppressant drugs in capillary blood. J. Pharm. Biomed. Anal., 178."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1373\/jalm.2018.028126","article-title":"Feasibility of Immunosuppressant Drug Monitoring by a Microsampling Device","volume":"4","author":"Gruzdys","year":"2019","journal-title":"J. Appl. Lab. Med."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1208\/s12248-024-00947-1","article-title":"Self-sampling by adolescents at home: Assessment of the feasibility to successfully collect blood microsamples by inexperienced individuals","volume":"26","author":"Boffel","year":"2024","journal-title":"AAPS J."},{"key":"ref_30","unstructured":"Cayman Chemical (2025, June 24). Hemoglobin Colorimetric Assay Kit (Item No. 700540). Available online: https:\/\/www.caymanchem.com\/product\/700540\/hemoglobin-colorimetric-assay-kit."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/15\/3139\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:16:37Z","timestamp":1760033797000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/15\/3139"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,26]]},"references-count":30,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["molecules30153139"],"URL":"https:\/\/doi.org\/10.3390\/molecules30153139","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,26]]}}}