HPLC Quantification of Drug Dissolution

Each suppository (n = 3) was dissolved in 100 mL of 0.1 M phosphate buffer (pH 8.0) at 37 ± 0.5 °C under continuous magnetic stirring at 100 rpm until complete dissolution was achieved. 1 mL aliquots were withdrawn and diluted by half using absolute ethanol. Samples were then filtered via a 0.22 μm syringe filter (Sigma Aldrich, UK). The total amount of drug in each sample was quantified using a Hewlett Packard 1260II Series HPLC system (Agilent Technologies, UK). The assay involved injecting 20 μL of each sample through an Eclipse plus C18 column, 150 mm × 4.6 mm (Zorbax, Agilent Technologies, UK) at 40 °C. The mobile phase was comprised of A) methanol and B) purified water and was pumped at a flow rate of 1 mL/min. The gradient elution was as follows: 50% A from 0 to 4 min, followed by 50–80% A from 4 to 4.10 min, then kept at 80% A from 4.10 to 8 min and finally 80–50% A from 8 to 12 min. The eluents were screened at a wavelength of 254 nm.