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Levosimendan is a positive inotrope and vasodilator used in patients with acute and chronic decompensated heart failure. It is metabolized into OR-1855 (inactive metabolite), which is further acetylated into OR-1896 (active metabolite having a prolonged half-life, hence a sustained effect). Levosimendan represents a valuable alternative to traditional inotropes with broad clinical applications in critically ill patients with cardiogenic shock, advanced heart failure and post-cardiac surgery. However, while levosimendan demonstrates dose-dependent hemodynamic effects, its pharmacokinetics has not yet been investigated in adult critically ill patients, a vulnerable population characterized by complex and unstable conditions that may significantly alter drug disposition. Therefore, pharmacokinetics studies of levosimendan and metabolites in critically ill patients require a reliable and sensitive quantification method. We developed and validated a highly sensitive method using ultra-high-performance liquid-chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the quantification of levosimendan, OR-1855 and OR-1896 in human plasma. To achieve the required analytical sensitivity, plasma sample preparation included protein precipitation with acetonitrile, subsequent supernatant's evaporation to dryness under nitrogen, and reconstitution of the solid residues with a solution of H 2 O:MeOH 4:1, followed by a 40 µL-aliquot injection into the LC column. Chromatographic separation of the three analytes was achieved in a 6-minute run in gradient mode, using an Acquity UPLC BEH C18 1.7 µm, 2.1 × 150 mm column. The method was extensively validated according to international bioanalytical assay guidelines, on a clinically relevant concentration range of 0.1-100 ng/mL, for each analyte. Considering these very low concentrations, the assay showed excellent performances in terms of trueness (94.3-105.3 %), repeatability (1.9-7.2 %) and intermediate fidelity (2.3-9.7 %). Of note, during our ex vivo studies on whole blood samples stability, acetylation of the metabolite OR-1855 into the active OR-1896 metabolite was observed in the presence of red blood cells. The UHPLC method is being applied for a prospective observational pharmacokinetics study of levosimendan in patients undergoing extracorporeal membrane oxygenation support. The benefit of levosimendan therapeutic drug monitoring in intensive care patients remains to be assessed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Janus kinase inhibitors (JAKi) are oral small molecules used in the treatment of a broad spectrum of autoimmune and myeloproliferative diseases. JAKi exhibit significant intra- and inter-individual pharmacokinetic variabilities, due to fluctuations in compliance with oral treatments and their metabolism essentially driven by cytochrome P450 enzymes. Intrinsically, JAKi have dose-response relationship and narrow therapeutic index: therapeutic drug monitoring (TDM) is expected to optimize and adapt their dosage regimen in order to resolve problems of efficacy and tolerance linked to dose and safety. A sensitive analytical method using multiplex high-performance liquid-chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the simultaneous quantification in plasma of the 6 major currently used JAKi, namely abrocitinib, baricitinib, fedratinib, ruxolitinib, tofacitinib, and upadacitinib. Plasma samples are subjected to protein precipitation with MeOH, using stable isotopically labelled internal standards. The separation of JAKi in supernatants diluted 1:1 with ultrapure H 2 O was performed using a C 18 column Xselect HSS T3 2.5 µm, 2.1x150 mm using a mobile phase composed of formic acid (FA) 0.2% and acetonitrile (+FA 0.1%) in gradient mode. The analytical run time for the multiplex assay was 7 min. JAKi drugs were monitored by electrospray ionization in the positive mode followed by triple-stage quadrupole MS/MS analysis. The method was validated according to SFSTP and ICH guidelines over the clinically relevant concentration ranges (0.5-200 ng/mL for abrocitinib, baricitinib and upadacitinib; 1-400 ng/mL for tofacitinib; 0.5-400 ng/mL for ruxolitinib, and 10-800 ng/mL for fedratinib). This multiplex HPLC-MS/MS assay achieved good performances in term of trueness (91.1-113.5%), repeatability (3.0-9.9%), and intermediate precision (4.5-11.3%). We developed and validated a highly sensitive method for the multiplex quantification of the JAKi abrocitinib, baricitinib, fedratinib, ruxolitinib, tofacitinib, and upadacitinib in human plasma. The method will be applied for prospective clinical pharmacokinetic studies to determine whether TDM programs for JAKi based on residual drug concentrations can be recommended using disease-specific therapeutic ranges., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)