Chemicals and reagents
Capsaicin, pharmaceutical grade, was purchased from Formosa Labs (Taoyean, Taiwan) and stored at -20°C until use. 190 proof ethyl alcohol (95% EtOH) was purchased from Decon Labs (King of Prussia, PA). HPLC grade water was purchased from Fischer-Scientific (Fair Lawn, NJ). Tween-20 was purchased from MP Biomedicals (Solon, OH). All solutions were stored at room temperature, unless otherwise stated.
Prior to each study, a stock solution of 25 mM CAP was prepared. Initially, the CAP powder was dissolved in a solution made-up of 80:10:10 H2O:Tween-20:95% EtOH (hereafter referred to as "Tween solution"), but this method did not allow sufficient solubility of high concentrations of CAP. Instead, the stock solution was prepared by dissolving 76 mg (250 μmol) of solid CAP in 10 mL of 95% EtOH. Preparing such a high concentration of CAP for the stock solution allowed any error associated with variation of volume to be considered negligible. In the research presented, solutions with EtOH levels at 5, 10, 15, 20, 25, 35, 50, 65, 75, and 95% EtOH in H2O were prepared. Additionally, solutions of CAP were prepared at concentrations of 0, 200, 350, and 500 μM for all percentages of EtOH mentioned. Finally, a much broader range of concentrations (0, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 μM) were prepared in 10% EtOH for more in-depth analysis. All samples were diluted to a total volume of 5 mL in triplicate.
To prepare the Tween solutions, a solution of 80:10:10 of H2O:Tween-20:95% EtOH (v/v/v) was initially prepared. To this Tween solution, a specified volume of 25 mM stock CAP was added to achieve the desired concentration of CAP. Solutions of CAP were prepared at concentrations of 0, 50, 150, 250, 350, and 450 μM, with a total volume of 5 mL. Similar to the CAP solutions in EtOH, the Tween solutions were prepared in triplicate.
To study the stability of CAP in the optimized solvent system, solutions at concentrations of 0, 200, 350, and 500 μM CAP in 10% EtOH/H2O (without Tween-20) were prepared, and left in one of four different environments: 1) room temperature and exposed to light, 2) room temperature and protected from light, 3) approximately 3°C and protected from light, and 4) approximately -20°C and protected from light. All solutions were kept in glass vials, and placed in a cardboard box to shelter them from light, except one set of vials which was directly exposed to room light for the purposes of the study. For solutions stored below room temperature (3°C and -20°C), the solutions were allowed to warm up to room temperature for two hours and then vortexed, before analysis. Visual inspections of the solutions prior to analysis indicated no turbidity. All samples were prepared in triplicate to a final volume of 5 mL.