Symmetry c18 column

HPLC analysis was carried out on a Shimadzu chromatographic apparatus (Kyoto, Japan) using a Symmetry C₁₈ column. The mobile phase was selected variably at a flow rate of 1 ml min−1. Hyoscyamine and scopolamine were obtained from Sigma-Aldrich and prepared in methanol (1mg/ml). Hairy roots were extracted from in vitro raised (treated and control) samples and oven-dried at 55°C for 16 hr followed by crushing in liquid N₂. Then, 3g of the crushed sample was dissolved in 10 ml of 15:5:1 (Methanol: Ammonium Hydroxide: Chloroform), followed by extraction with CHCl₃ (10 ml per sample) was carried out and sonicated of samples for 10 minutes. The samples were kept for 1-hour at room temperature and evaporated on the rotatory evaporator till dryness. Further, 3ml of chloroform and 1.5 ml of 1NH₂SO₄ was added to each sample and then air-dried to remove the residual CHCl₃. The pellet was resuspended in 28% NH₄OH (pH = 10). The suspension was then filtered using filter paper. The filtrate is collected in a tube and the filter paper was washed with 1–2 ml CHCl₃ to purge the membrane of residual metabolites. The samples were again evaporated on the rotatory evaporator with the addition of 2 ml methanol. Further, using the corresponding standards, the samples were quantified and stored for further analysis [26 (link)].

The RP-HPLC system (LC 2030C, Shimadzu, Japan) was equipped with a reverse phase Symmetry C18 column (3.5 μm, 4.6 × 75 mm²) and an ultraviolet (UV) detector.

The release profile of SD from cubosomes was determined in terms of its accumulated mass (%) to the initial SD content in the BCM. The samples were enclosed in dialysis bags and immersed in 10 mL PBS. Aliquots of 500 μL were collected at 1, 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, and 168 h, and 500 μL of PBS were added back to replace each withdrawn aliquot. SD content was quantified using HPLC on a Prominence (series LC-20A; Shimadzu) chromatograph using a Symmetry C18 column and acetonitrile as eluent. An area (Y) versus mass (X) calibration curve (Y = 2e⁹X; R² = 0.99) was determined from the SD standard at 0.18 mg mL⁻¹ (ranging from 0.001 g to 0.006 g) prepared in PBS. Each experiment was carried out at least three times. Results are expressed as mean ± standard deviation values.
To determine the mechanism of drug release, the semi-empirical Korsmeyer-Peppas model was fitted to the release profiles. The model is given by Ref. [16 (link)]: $\text{Mt}/\mathrm{M}\infty ={\mathbf{k}}_{\text{KP}}\cdot {\mathrm{t}}^{\mathbf{n}}$
where Mt/M∞ is the fractional drug release, t is the release time, k_KP is the Korsmeyer-Peppas release kinetic constant characteristic of the drug/system (particle and matrix), and n is an exponent that characterizes the release mechanism [16 (link)]. The data were fitted using the OriginPro9 software.