Exilate is definitely an oral prodrug which is hydrolysed to the direct thrombin inhibitor dabigatran. The concentration of dabigatran was assessed in mouse plasma samples employing a UPLC-MS/MS method. A plasma volume of 50 was spiked with 5 of internal typical (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 10 min, and chilled at 4 C for the next 10 min. The supernatant collected after sample centrifugation (16,600g, 15 min, 4 C) was straight injected into an UltiMate 3000 UPLC program (Thermo Fisher Scientific, Waltham, MA, USA) combined with a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic evaluation was carried out using an Acquity UPLC BEH C18 (three.0 100 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 inside the following gradient elution system: 95 B hold for 1 min, 95 B for 3 min, 55 B for 0.5 min, and 95 B for 2.5 min for column equilibration. The mass spectrometric detection was performed in an electrospray good ionisation mode, and chosen ion transitions were utilised 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 have been as follows: spray voltage = 5000 V, vaporiser temperature = 300 C, auxiliary gas = 25, and sheath gas = 30. four.three. Assessment of In Vivo Endothelial Function by Magnetic Resonance Imaging (MRI) The in vivo strategy for endothelial function assessment utilizing a magnetic resonance imaging (MRI) approach was created, effectively applied, and previously described by Bar et al. [31]. MRI experiments on mice were carried out employing a 9.4T scanner (BioSpec 94/20 USR, Bruker, BioSpin GmbH, Germany) beneath isoflurane anaesthesia (Baxter Polska Sp. z o.o., Warszawa, Poland; 1.5 vol ) in oxygen and air (1:two) mixture and continual physique temperature maintained at 37 C employing a circulating warm water system. The heart activity, respiration, and body temperature had been monitored by applying a Monitoring and Gating Method (SA Instruments Inc., Stony Brook, NY, USA). The endothelial function was evaluated in response to reactive hyperaemia applying a flow-mediated vasodilation (FMD) approach as well as in response to acetylcholine (Ach; Sigma Aldrich, St. Louis, MO, USA) administration. The home-made equipment for FMD measurements in mice allowed for any short-term occlusion (5 min) of a mouse femoral artery (FA) and MMP-10 Inhibitor Purity & Documentation examination of volume changes from the FA in response to occluder release and elevated blood flow. Three-dimensional (3D) pictures of FA were recorded around the coronal view of the mice (on their left hind limb). The β-lactam Inhibitor web vessel response to acetylcholine administration (Ach, i.p, 16.six mg/kg b.w) was assessed within the thoracic (ThA) and abdominal (AbA) aorta. Vasomotor response was evaluated by comparing two time-resolved 3D images from the vessels before and 25 min following Ach injection. Three-dimensional (3D) pictures on the ThA and AbA have been acquired on the sagittal view in the mice, approximately five mm below the heart. All photos have been registered making use of the cine IntraGateTM FLASH 3D sequence and reconstructed with the IntraGate 1.2.b.
