Vardenafil hydrochloride

All chemicals used were of reagent grade. Synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), and cholesterol (Chol) were obtained from Avanti^® Polar Lipids (Alabaster, Alabama, AL, USA). Nonactin, di-8-ANEPPS, KCl, HEPES, KOH, DMSO, gramicidin A (GrA), nystatin (Nys), sildenafil citrate, vardenafil hydrochloride, and tadalafil were purchased from Sigma-Aldrich^® (Merck KGaA, Darmstadt, Germany). The chemical structures of the tested PDE-5 inhibitors are presented in Table 1.
KCl solutions (0.1 M or 2.0 M) were buffered using 5 mM HEPES–KOH at pH 7.4. All experiments were performed at room temperature (25 ± 2 °C).

Sildenafil citrate (CAS No. 171599-83-0), Tadalafil (CAS No. 171596-29-5), and Vardenafil hydrochloride (CAS No. 330808-88-3) standards were prepared with certified reference materials, which were purchased from the Sigma-Aldrich (St. Louis, MO, USA) and the United States Pharmacopeia (Rockville, MD, USA). Sildenafil-d₃, widely used as a surrogate standard, was obtained from the Toronto Research Chemicals (North York, ON, Canada). Methanol, acetonitrile, water, and acetic acid (MS grade) were obtained from the Honeywell (Muskegon, MI, USA) and Sigma-Aldrich. Disodium ethylene diamine tetra acetic acid (Na₂EDTA, ACS reagent grade) was also purchased from the Sigma-Aldrich.
Individual stock solutions (1 mg mL⁻¹) were prepared by dissolving in Methanol and then stored at less than 4 °C. Supplementary Table S2 presents the chemical structures and properties of each target compound. The mixtures of working solutions for the calibration were prepared by diluting the stock solution to desirable concentrations in a mixture of 0.1% acetic acid, 10% Methanol, and 400 mg L⁻¹ Na₂EDTA.