2.7. Quantification of Residual Pluronic® F68

The residual amount of surfactant in the H-NMeds was determined by a colorimetric method [39]. Briefly, ~1 mg of a freeze-dried H-NMeds sample was solubilized in 0.5 mL of dichloromethane. Then, 10 mL of distilled water was added and the organic solvent was evaporated at r.t. under stirring for 2 h. The suspension was filtered (cellulose acetate filter, porosity 0.45 µm, Sartorius, Florence, Italy) to obtain an aqueous solution (A).

To calculate the amount of Pluronic^® F68 in the formulation, 2 mL of the aqueous solution (A) was treated with 2 mL of 0.5% (w/v) BaCl₂ in HCl 1 N and 0.5 mL of an aqueous solution of I₂/KI (0.05 M/0.15 M). The obtained solution was incubated at r.t. for exactly 10 min in the dark. Pluronic^® F68 concentration was determined measuring the absorbance at 540 nm (Model V530, Jasco, Cremella, Italy). A calibration curve was calculated using the same method on stock solutions of Pluronic^® F68, and linearity was found in the range of 2–18 µg/mL. All data are expressed as the mean of at least three determinations. The amount of surfactant in the formulation was expressed as the Pluronic^® F68 Content % (PC %) and calculated using the following equation: