X bridge prep c18 5 μm obdtm

Kahle method with minor modifications was used to determined chlorogenic acid [26 (link)]. The identification and quantification of chlorogenic acid were determined through HPLC (Waters 600 system). The juice sample was filtered (0.45 μm, 13 mm Cat. # AS 021345-N Agela Technologies), and samples were fractionated through the Waters Auto Purification HPLC column (X Bridge ^TM prep C18: 5 μm OBD^TM, 150 mm × 19 mm, Waters, Ireland). The mobile phase consisted of a mixture solution of aqueous formic acid (A) with methanol (B) (0.1:99.9, v/v). We then injected 5 mL of filtrate sample into the column at a flow rate of 1 mL/minute. A linear biphasic gradient was used with 15–40% solvent B over 10 min, 40–50% over 15 min, 50–75% over 20 min, 75–90% over 25 min followed by a gradual column re-equilibration from 90–65% over 30 min, 65–40% over 35 min, 40–10% over 40 min. Samples were detected using a UV-VIS spectrophotometer at a wavelength of 320 nm, a commercially available chlorogenic acid was used as an external standard, and different concentrations of its solution (10–80 mg/mL) were used to draw a standard curve. Data were presented as mg of chlorogenic acid equivalent per milliliter of samples.

Briefly, the juice sample (25 mL) was poured into a separation funnel with an equal volume of acetone three times and filtered by a No.1 Whatman filter paper. The filter cake was re-extracted with methanol, and the extract was vigorously mixed with an equal volume of petroleum ether. The upper petroleum ether layer was dehydrated using anhydrous sodium sulfate, and after filtration, it was concentrated using a rotary evaporator at 30 °C. Then, we added 10 mL acetonitrile–methanol–acetone solution (40:40:20, v/v) and kept it at 18 °C in the dark until further analysis. The samples were filtered through a syringe filter (0.45 μm, 13 mm Cat#AS-021345 N-Agela Technologies). Filtered samples were fractionated using Waters Auto Purification high-performance liquid chromatography (HPLC) (Cat. # 1501382671, X Bridge^TM prep C18: 5 μm OBD^TM, 150 mm × 19 mm, Waters, Ireland). The mobile phase was comprised of a mixture solution of acetonitrile-acetone-methanol (40:20:40, v/v), and the flow rate was adjusted to 0.80 mL/min. Commercially available β-carotene and lutein with different concentrations were used to draw a linear regression calibration curve.

Sugar contents were determined by [27 (link)]. The identification and quantification of sugar contents were performed using a semi-preparatory high-performance liquid chromatography (HPLC) system (waters 600). The juice sample was filtered (0.45 μm, 13 mm Cat. # AS 021345-N Agela Technologies), and samples were fractionated through the Waters Auto Purification HPLC column (X Bridge ^TM prep C18: 5 μm OBD^TM, 150 mm × 19 mm, Waters, Ireland). The mobile phase consisted of acetonitrile (75:25, v/v) with a 1 mL flow rate. A Cosmosil packed column of D-sugars (4.6×250 mm) was used. Commercially available sucrose, fructose, and glucose were used as external standards. Different concentrations of each solution standard were prepared. Sucrose, fructose, and glucose were used to draw a linear regression calibration curve. Data were analyzed as g of sucrose, fructose, and glucose equivalent per liter of sample.