2.2.5. Dissolution Studies

Dissolution studies under sink conditions were performed using the United States Pharmacopeia (USP) paddle method at 37.0 ± 0.5 °C (apparatus 2) in Erweka DT 80 (Erweka GmbH; Langen, Germany). Amounts equivalent to 10 mg of EZ or ATV were used in the dissolution vessels.

The ezetimibe dissolution study was performed at 50 rpm with 500 mL of dissolution medium containing 0.45% of sodium lauryl sulphate in 0.05 M of sodium acetate buffer, adjusted to pH 4.5. The use of a medium with sodium lauryl sulphate is necessary to increase the low solubility of the active ingredient, this dissolution test method of ezetimibe is established by the United States Pharmacopeia (USP42-NF37, 2019). At different times, the samples were filtered by 0.45 μm (Acrodisc, NY, USA). The quantity of EZ was determined at 233 nm using a UV–VIS Jasco V-730 spectrophotometer (Tokyo, Japan) by the following calibration curve y = 0.0394x (μg/mL) − 0.0089 (r² = 0.9996) across a range of 1–15 μg/mL.

The atorvastatin dissolution study was performed at 75 rpm with 900 mL of dissolution medium containing 0.05 M phosphate buffer, adjusted to pH 6.8. This dissolution test method of atorvastatin is established by the United States Pharmacopeia (USP42-NF37, 2019). Samples were collected periodically and filtered through 0.45 μm; ATV was then determined at 241 nm by the following calibration curve: y = 0.0404x (μg/mL) − 0.0165 (r² = 0.9994) with a range of 2–20 μg/mL. Both dissolution methods were validated according to ICH Q2 (R1) (CPMP/ICH/381/95). Each determination was performed in triplicate and the error bars on the graphs represent the standard deviation.

Ezetimibe and atorvastatin were evaluated in a biorelevant media under non-sink conditions. The dissolution study of the different formulations was performed at 100 rpm and 37.0 ± 0.5 °C using a magnetic stirrer thermostatic bath (FisherbrandTM; Milan, Italy). Amounts equivalent to 50 mg of EZ and ATV were added in 50 mL of pH 6.5 FaSSIF (fasted-state simulated intestinal fluid) biorelevant media purchased from biorelevant.com (London, UK). At different times, 0.5 mL samples were withdrawn and filtered through 0.45 μm (Acrodisc, NY, USA), and 0.3 mL was discarded before collecting the final 0.2 mL of filtrate. The filtered samples were immediately diluted in methanol and analyzed [16,17].

The sample quantification was carried out by HPLC (Agilent 1100 series FLD G1321A) equipped with a 50 µL loop. Samples were separated using a Zorbax SB C-8 column (4.6 × 250 mm, 5 µm). The mobile phase consisted of acetate buffer pH 4.0 (40%) and HPLC-grade acetonitrile (60%), with a flow rate of 1 mL/min. Ezetimibe was analyzed with a UV–VIS detector set at a wavelength of 233 nm and atorvastatin at 241 nm.