Benzophenone 3

Tazarotene (ethyl 6-[2-(4,4-dimethyl-2,3-dihydrothiochromen-6-yl)ethynyl]pyridine-3-carboxylate) was purchased from Sigma-Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC)-grade methanol, acetonitrile, and formic acid (98%) were purchased from J.T. Baker (Phillipsburg, NJ, USA). Ethanol absolute for HPLC ≥99.8% was obtained from POCH (Gliwice, Poland). Water (quadruple-distilled) with a conductivity of less than 1 μS cm⁻¹ was prepared using the S2-97A2 distillation apparatus (ChemLand, Stargard Szczecin, Poland). Zinc oxide (nanopowder <100 nm particle size) and titanium (IV) oxide (anatase, nanopowder < 25 nm particle size, 99.7% trace metals basis) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The UV absorbers—benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4, avobenzone, and octocrylene—were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The following reagents were used: formic acid and ammonium acetate from Panreac (Barcelona, Spain); C18 disperse sorbent from Scharlab (Barcelona, Spain); HPLC-grade methanol, and water from Romil (Barcelona, Spain); Standards of benzophenone-1 (BP-1, purity: 99%), benzophene-2 (BP-2, purity: 97%), benzophenone-3 (BP-3, purity: 98%), benzophenone-8 (BP-8, purity: ≥98%) from Sigma-Aldrich (Steinheim, Germany). Isotopically labelled BP used as internal standard (BP-d₁₀) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Individual stock standard solutions were prepared at 1000 mg/L in methanol and kept at -18°C. Working solutions were made up by dilution of the stock standard solution in methanol.

The chemical components and the preparation of the tested ointments were as follows: almond oil (All Organic Treasures, Wiggensbach, Germany) (30 wt.%) was melted, and glyceryl stearate (SE, Caesar & Loretz, Hilden, Germany) (5 wt.%), cetearyl alcohol (Aliacura, Scheinfeld, Germany) (1 wt.%), glyceryl caprylate (Herbana, Ljubljana, Slovenia) (0.5 wt.%), and benzophenone-3 (Sigma-Aldrich, Darmstadt, Germany) (5 wt.%) were added to the oil in a water bath. The antioxidants β-carotene (Sigma-Aldrich, Darmstadt, Germany) (1 wt.%), or trans-resveratrol (Sigma-Aldrich, Acros Organics) (1 wt.%) were then added slowly to ensure that the lipid phase was completely molten and homogeneous in appearance. Demineralized water (58 wt.%) was heated to the same temperature (60–65 °C) and added to the lipid phase during homogenization at the fastest speed. Homogenization took place for another 3 to 5 min in a cold bath until the ointment was cooled to about body temperature. Xanthan (Organic Factory, Vailate, Italy) (0.3 wt.%) was suspended in glycerol (All Organic Treasures, Wiggensbach, Germany)) (5 wt.%) and mixed thoroughly. Next, a preservative, dehydroacetic acid (Sigma-Aldrich, Darmstadt, Germany) (8 wt.%) in benzyl alcohol (Sigma-Aldrich) was added, mixed thoroughly, and acidified to pH 5 to 5.5 with lactic acid (90%, Caesar & Loretz Hilden, Germany).