Inertsil ods 4 column

To select appropriate components for the development of the SMEDDS formulation, solubility studies were conducted for various oils and surfactants.18 (link) Excess amounts of MTX (approximately 50 mg) was transferred to 15 mL conical tube (SPL #50,015) containing 3 mL of pure oils or 10% (w/v) aqueous surfactant solutions. Then, the mixture was vortexed and kept for 7 days at 25 °C in a shaking water-bath to facilitate the solubilization. Subsequently, 1 mL of samples were centrifuged at 10,000 g for 10 min (Hanil Science Industrial Co., South Korea) to separate the undissolved MTX. The supernatant was taken and diluted with mobile phase for high performance liquid chromatography (HPLC) analysis for the quantification of MTX. The concentration of MTX in the sample was quantified by the HPLC system (Agilent 1260 Infinity, Agilent Technologies, USA) consisting of the Chem Station software, G1311C 1260 Quat Pump, and G1314B 1260 VWD VL detector. The Inertsil ODS-4 column (GL Sciences, Japan, 4.6 mmI.D. x 250 mm, 5 μm) was used, and the column temperature was maintained at 25 °C. The mobile phase consisted of 0.1 M dibasic phosphate (pH was adjusted to 3.0 with hydrochloride solution) and methanol at the volume ratio of 74/26, respectively. The mobile phase was eluted at a flow rate of 1 mL/min. The eluent was monitored by the detector at 303 nm.

The methods described in Jin et al. (2019 (link)) and Li, Liu, Peng, et al. (2020 ) were followed for the extraction and determination of GABA with some modifications. Briefly, 0.1 g of fresh sample was homogenized with 1 mL of 50% ethanol solution (containing 0.1 M HCl), then centrifuged for 15 min at 13,500 × g, filtered through a 0.22‐μm filter, and finally diluted 10 fold with methanol (Sigma‐Aldrich) for detection by LC–MS (LC: AC, ExionLC; MS: Q‐trap5500, AB Sciex Pret. Ltd). Separation used an Inertsil ODS‐4 column (3 μm particle size, 3 × 150 mm; GL Sciences Inc.). The mobile phases A and B were 0.1% methanoic acid and acetonitrile, respectively. The GABA standard (A2192) was purchased from Sigma‐Aldrich. The GABA retention time was 1.29 min.

The experiment was performed on a Thermo Ultimate 3000 HPLC system coupled with an Ultimate 3000 diode array detector (Thermo Fisher Scientific, USA). The chromatographic separation was performed on an Inertsil ODS-4 column (4.6 mm × 250 mm × 5 μm; GL-science Inc., Tokyo, Japan), using ultrapure water–0.1% formic acid solution (eluent A) and methanol (eluent B) as mobile phases at a flow rate of 0.8 mL min⁻¹. The column temperature was maintained at 30 °C. Two mobile phases were programmed as follows: 0–5 min, 5–22% B; 5–20 min, 22% B; 20–35 min, 22–24% B; 35–45 min, 24–25% B; 45–50 min, 25–40% B; 50–60 min, 40–45% B. The injection volume was 10 μL. Chromeleon Chromatography Data System software 7.0 was used for instrument control, data acquisition, and data analysis. The chromatographic peaks of these nine compounds (GA, C, EC, EGC, ECG, EGCG, theobromine, theophylline, and caffeine) were confirmed by comparing their retention time and UV-Vis absorbance spectra with those of the authentic standards. Quantification of these compounds was conducted by the integration of the peak using the external standard method. Calibration curves of these nine compounds were obtained at a detection wavelength of 280 nm over different concentrations, of which the calibration curves were shown in Fig. S1 (ESI†).