2.3. Characterisation of the Optimised s-SEDDS

Powder flowability was determined using Carr’s compressibility index (CI), the Hausner ratio (HR), and the angle of repose. CI and HR were derived from bulk and tapped density. The angle of repose was determined using the funnel method [45,46]. Bulk and tapped density were measured using the Digital Bulk Density Apparatus (Edutek Instrumentation, India) with a 25-mL glass measuring cylinder. The tap height was adjusted to 3 mm. Powder (5 g) was weighed accurately and poured into a 25 mL graduated measuring cylinder, and the bulk volume was recorded. The same cylinder was placed on the tapped density measuring apparatus and tapped 200 times, with 100 times increment until no further change in volume was observed. The tapped volume was recorded directly from the cylinder marks.

The s-SEDDS (100 mg) were carefully weighed out into a 50 mL centrifuge tube and added with 50 mL of buffer solution (pH 1.2, 4.5 or 6.8). The contents in the centrifuge tube were rotated for 3 h to ensure complete release, before it was centrifuged for 5 min at 450 RCF (relative centrifugal force) to precipitate the solids [25]. Supernatant was carefully collected and diluted before injecting into the HPLC. The extent of content release was calculated in terms of percentage detected in the media compared to the amount added.

The assays of tocotrienols (delta, gamma, and alpha isomers) were determined using high-performance liquid chromatography (HPLC) method as reported and validated by Yap et al. [47]. The HPLC system consisted of a Waters 2695 Alliance^® Separation Module (Waters, Milford, MA, USA) and Waters 2475 Fluorescent Detector (Waters, Milford, MA, USA). The detector operated at an excitation wavelength of 296 nm and emission wavelength of 330 nm, with sensitivity set at 10,000 EUFS. A Hichrom C18 (4 µm, 250 × 4.6 mm i.d.) analytical column (Reading, Berkshire, UK) was fitted to a refillable guard column (2 mm × 2 cm) (Upchurch Scientific, Oak Harbor, WA, USA) packed with Whatman Partisil-10 ODS-3 (Clifton, NJ, USA). The mobile phase used was pure methanol, and the system was set to operate at 25 °C. The injection volume was set at 50 µL. Analyses were carried out at a flow rate of 1.0 mL/min, and the samples were quantified using peak area. The chromatograms are available in the Supplementary Materials (Figures S1 and S2).

The physical stability of the emulsion products formed after standing for 2 h at room temperature (25 °C) was evaluated. An amount (700 mg) of each formulation was dispersed into 250 mL of distilled water and stirred using a paddle stirrer rotating at 100 rpm for 10 min to obtain an emulsion product. Then, approximately 10 mL of the emulsion formed was immediately transferred into a test tube and allowed to stand. After two hours, samples were visually inspected. The physical stability of the emulsion products based on visual observation was categorised as no separation, slight creaming, creaming, and complete separation of phases. All experiments were conducted in triplicate.

The formulations (100 mg) were weighed carefully into 50 mL centrifuge tubes and added with 50 mL of filtered (0.22-µm) distilled water. The tubes were rotated for 1 h and then centrifuged at 450 RCF for 5 min to remove the water-insoluble Neusilin [25]. The supernatant collected from each formulation was diluted (0.5 mL in 45 mL of filtered distilled water) prior to analysis. Emulsion droplet size was measured by means of photon correlation spectroscopy (PCS) using the Malvern Zetasizer 1000HS (Worcestershire, UK). For all procedures involved in the measurements of emulsion droplet size, filtered distilled water was used to ensure a count rate value of below 3 K at a 90° scattering angle. All samples were diluted to obtain a count rate of 200–300 K. Ten measurements were taken as set by the method of the software.