Lc 2010a ht hplc system

A matcha sample was performed according to our previous experimental method [24 (link)] with some modifications. Briefly, powdered matcha green tea (10 g) was ultrasonically extracted twice with 150 mL of boiling distilled water in a boiling water bath for 30 min. The total infusion was concentrated using a vacuum rotary evaporator at 70 °C and the extract was freeze-dried using a freeze dryer (Christ, Osterode, Germany).
The total phenolic content assay was determined by the Folin–Ciocalteu method. The protein content assay was determined via the Coomassie brilliant blue method. Amino acid content assay was determined using the ninhydrin colorimetry method. The caffeine and tea catechins assay was determined using the Shimadzu LC-2010A HT HPLC system (Shimadzu Corporation, Tokyo, Japan). The above methods were implemented according to our previous report [25 (link)]. The total sugar content assay was determined by using the phonel-sulfate method according to Dubois’ report [26 (link)]. All chemical standards were of high-performance liquid chromatography grade.

High-performance liquid chromatography (HPLC) was used to quantify the amount of FP and SX in the co-SD powders. This method was performed with similar conditions reported by previously.^{40,41 (link)} Shimadzu LC-2010AHT HPLC system, with autosampler fitted to a 20 μL sampling loop and UV-Vis detector and reverse phase C₁₈ phenomenex column with 250 × 4.6 mm, 5 μm particle size was used. The mobile phase consisted of 75 : 25 v/v methanol-0.6% (w/v) aqueous ammonium acetate solution degassed and run at a flow rate of 1.0 mL min⁻¹. The column was maintained at 40 °C with a run time of 10 min. The injection volume was 20 μL. The UV detector was set to 228 nm, since both FP and SX had good absorption at this wavelength. The retention time of salmeterol was 5.7 min and fluticasone was 8.4 minutes. The retention peaks of xinafoic acid was seen around 3.09 min, but it was not used for quantification of the compounds. Different concentrations between 0.005–0.5 mg mL⁻¹ was used for calibration curves of FP and SX respectively. All co-SD powders were dissolved at a known concentration (0.5 mg mL⁻¹) and analyzed similar to the drug standards.