d by HPLCMS/MS on the rat liver microsomal fraction, incubated with each and every compound, respectively. The separation was performed with an Agilent 1100 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA), including a vacuum degasser, a binary pump and an autosampler. The liquid chromatograph was equipped having a Merck LiChroCART–C18 (five ) 150 mm four.6 mm column and also a Phenomenex SecurityGuard 4.0 mm two.0 mm precolumn. The chromatographic run was carried out by a binary mobile phase of water and acetonitrile, using isocratic conditions with acetonitrile/water 0.1 formic acid (50/50) for 14 min. The flow-rate was 1 mL/min. The LC was interfaced to an Applied Biosystems API 3200 triple uadrupole mass spectrometer (Applied Biosystems Sciex, Ontario, Canada), operating in electro spray ionization (ESI)–positive ion mode. The other MS parameters were set as follows: curtain gas: 20 psi; source gas GS1: 30 psi; supply gas GS2: 30 psi; probe temperature: 350 C; gas for collisional activation: N2 at three psi; ion spray voltage: +5000 V. SRM analysis. The mass spectrometric signal was optimized for all investigated substances upon their synthesis as analytical standards. Setup was performed by infusion from the analyte options in acetonitrile at ten /mL concentration. The Selected Reaction Monitoring (SRM) technique was constructed applying a minimum of two transitions from the analytes protonated molecular ion to the corresponding fragment ions (Table 1). Then, the rat liver microsomal fraction, incubated with compound five or 7, respectively, was analyzed BRD2 Inhibitor manufacturer together with the similar SRM system. The analyses have been executed at time t = 0 and at time t = two h. Solution ion scan mode evaluation. The search for doable metabolites was also carried out together with the very same chromatographic system but operating inside the product ion scan mode, i.e., the protonated molecular ion with the predicted metabolites was selected using the initially quadrupole (Q1), then fragmented inside the intermediate cell upon collisional activation with helium molecules (Q2) and the generated solution ions have been analyzed by the third quadrupole (Q3) under continuous scanning conditions. The analyses have been executed around the rat liver microsomal fraction at time t = 0 and at time t = two h.Antioxidants 2022, 11,six ofTable 1. Mass spectrometric acquisition parameters for the multiple reaction monitoring operating mode. Compd 4 five Precurson Ion (m/z) 258.2 272.1 Declustering Possible (V) 30 29 Entrance Potential (V) four eight Product Ions 258.two 212.two 258.two 168.2 272.1 226.2 272.1 182.2 272.1 211.1 319.3 273.1 319.3 167.0 319.3 194.8 333.two 181.0 333.two 167.1 333.two 223.2 Collision Energy (V) 12 22 13 20 22 ten 18 19 18 34 17 Collision Cell Exit Potential (V) 15 15 18 14 20 18 16 25 15 30319.333.three. Results three.1. Vasodilating Activity 3.1.1. In Vitro Experiments Because NO predominantly modulates the tone of massive conduit vessels [29,30], the COX-2 Activator Accession vasodilator activities with the nitrooxyphenylalkyl derivatives four, as well as those of GTN, taken as a reference, were assessed on rat aorta strips precontracted with 1 L-phenylephrine. The endothelium was removed in order to study the vasodilation effects only because of the direct action of NO-donor organic nitrates. Each of the merchandise have been in a position to dilate the strips in a concentration-dependent manner. Their potencies as vasodilators, expressed as pEC50 , are collected in Table two. Inhibitors of ALDH-2 (chloral hydrate and benomyl) shifted the concentration esponse curves of all nitrooxy derivatives to
