d by HPLCMS/MS on the rat liver microsomal fraction, incubated with every single compound, respectively. The separation was performed with an Agilent 1100 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA), such as a vacuum degasser, a binary pump and an autosampler. The liquid chromatograph was equipped having a Merck LiChroCART–C18 (5 ) 150 mm 4.six mm column along with a Phenomenex SecurityGuard four.0 mm two.0 mm precolumn. The chromatographic run was carried out by a binary mobile phase of water and acetonitrile, employing 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 have been set as HIV-1 Activator Storage & Stability follows: curtain gas: 20 psi; source gas GS1: 30 psi; source gas GS2: 30 psi; probe Histamine Receptor Modulator list temperature: 350 C; gas for collisional activation: N2 at three psi; ion spray voltage: +5000 V. SRM evaluation. The mass spectrometric signal was optimized for all investigated substances upon their synthesis as analytical standards. Setup was performed by infusion in the analyte solutions in acetonitrile at ten /mL concentration. The Selected Reaction Monitoring (SRM) process was built making use of at least two transitions from the analytes protonated molecular ion to the corresponding fragment ions (Table 1). Then, the rat liver microsomal fraction, incubated with compound 5 or 7, respectively, was analyzed with all the identical SRM approach. The analyses were executed at time t = 0 and at time t = two h. Item ion scan mode analysis. The search for probable metabolites was also conducted together with the same chromatographic program but operating within the solution ion scan mode, i.e., the protonated molecular ion of your predicted metabolites was selected using the initial quadrupole (Q1), then fragmented inside the intermediate cell upon collisional activation with helium molecules (Q2) and the generated item 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 many reaction monitoring operating mode. Compd four 5 Precurson Ion (m/z) 258.2 272.1 Declustering Possible (V) 30 29 Entrance Potential (V) 4 8 Item Ions 258.two 212.two 258.two 168.2 272.1 226.2 272.1 182.2 272.1 211.1 319.three 273.1 319.three 167.0 319.three 194.8 333.two 181.0 333.two 167.1 333.two 223.2 Collision Power (V) 12 22 13 20 22 ten 18 19 18 34 17 Collision Cell Exit Possible (V) 15 15 18 14 20 18 16 25 15 30319.333.3. Benefits 3.1. Vasodilating Activity 3.1.1. In Vitro Experiments Due to the fact NO predominantly modulates the tone of huge conduit vessels [29,30], the vasodilator activities with the nitrooxyphenylalkyl derivatives 4, too as those of GTN, taken as a reference, had been assessed on rat aorta strips precontracted with 1 L-phenylephrine. The endothelium was removed so as to study the vasodilation effects only on account of the direct action of NO-donor organic nitrates. Each of the solutions had been capable to dilate the strips in a concentration-dependent manner. Their potencies as vasodilators, expressed as pEC50 , are collected in Table 2. Inhibitors of ALDH-2 (chloral hydrate and benomyl) shifted the concentration esponse curves of all nitrooxy derivatives to