Exilate is definitely an oral prodrug that is definitely hydrolysed towards the direct thrombin inhibitor dabigatran. The concentration of dabigatran was assessed in mouse plasma samples using a UPLC-MS/MS approach. A plasma volume of 50 was spiked with 5 of internal standard (dabigatran-13 C6 ; TRC, Toronto, Canada) at a concentration of 1 /mL. Right after gently shaking, 150 of 0.1 M HCl in MeOH (WITKO Group, Lodz, Poland) was added, mixed for ten min, and chilled at 4 C for the next 10 min. The supernatant collected after sample centrifugation (16,600g, 15 min, four C) was straight injected into an UltiMate 3000 UPLC technique (Thermo Fisher Scientific, Waltham, MA, USA) combined having a TSQ Quantum Ultra triple TLR7 Agonist Purity & Documentation quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic evaluation was performed applying an Acquity UPLC BEH C18 (3.0 one hundred mm2 , 1.7 ; Waters, Milford, MD, USA) analyticalInt. J. Mol. Sci. 2021, 22,12 ofcolumn and applying 0.1 formic acid (FA; Thermo Fisher Scientific, Waltham, MA, USA) in ACN (Sigma Aldrich, St. Louis, MO, USA) (A) and 0.1 FA in H2 O (B) as mobile phases delivered in the following gradient elution program: 95 B hold for 1 min, 95 B for 3 min, 55 B for 0.5 min, and 95 B for 2.five min for column equilibration. The mass spectrometric detection was performed in an electrospray constructive ionisation mode, and chosen ion transitions have been used for quantification: 472.4172.0 (CE = 39 V) and 478.3172.1 (CE = 39 V) for dabigatran and dabigatran-13 C6 , respectively. The mass spectrometry operating parameters had been as follows: spray voltage = 5000 V, vaporiser temperature = 300 C, auxiliary gas = 25, and sheath gas = 30. 4.three. Assessment of In Vivo Endothelial Function by Magnetic Resonance Imaging (MRI) The in vivo strategy for endothelial function assessment applying a magnetic resonance imaging (MRI) method was developed, effectively applied, and previously described by Bar et al. [31]. MRI experiments on mice have been performed working with a 9.4T scanner (BioSpec 94/20 USR, Bruker, BioSpin GmbH, Germany) under isoflurane anaesthesia (Baxter Polska Sp. z o.o., Warszawa, Poland; 1.5 vol ) in oxygen and air (1:2) mixture and continuous physique temperature maintained at 37 C employing a circulating warm water program. The heart activity, respiration, and physique temperature were monitored by applying a Monitoring and Gating System (SA Instruments Inc., Stony Brook, NY, USA). The endothelial function was evaluated in response to reactive hyperaemia applying a flow-mediated vasodilation (FMD) technique at the same time as in response to acetylcholine (Ach; Sigma Aldrich, St. Louis, MO, USA) administration. The home-made equipment for FMD measurements in mice allowed to get a short-term occlusion (five min) of a mouse femoral artery (FA) and examination of volume adjustments in the FA in response to occluder release and enhanced blood flow. Three-dimensional (3D) pictures of FA were recorded around the coronal view on the mice (on their left hind limb). The S1PR5 Agonist web vessel response to acetylcholine administration (Ach, i.p, 16.6 mg/kg b.w) was assessed inside the thoracic (ThA) and abdominal (AbA) aorta. Vasomotor response was evaluated by comparing two time-resolved 3D pictures in the vessels before and 25 min just after Ach injection. Three-dimensional (3D) pictures on the ThA and AbA had been acquired on the sagittal view on the mice, approximately 5 mm below the heart. All images had been registered utilizing the cine IntraGateTM FLASH 3D sequence and reconstructed using the IntraGate 1.two.b.