1260 infinity 2 lc

The extraction and determination of anthocyanins from mulberry fruit were performed using a previously described method [42 ]. Anthocyanin extraction was carried out using acidified methanol (methanol and 1.0 N HCl, 85:15, v/v), assisted by ultrasonic disruption. The extracts were concentrated by evaporation at 50 °C using a rotary evaporator, and were then re-dissolved in acidified methanol. Individual anthocyanins were separated and quantified using a high-performance liquid chromatography (HPLC) system (1260 Infinity II LC, Agilent Technologies Inc., CA, USA), coupled to a dual wavelength ultraviolet–visible detector and a data acquisition system (Millennium Chromatography Manager version 2.15.01; Waters Corporation, MA, USA). A reversed-phase chromatography column (Supelcosil LC-18-dB, 25 cm × 4.6 mm i.d.; Agilent) was used at room temperature.

NMR data were acquired on a Bruker Avance III 800 MHz spectrometer equipped with a 5 mm TCI cryoprobe, with the residual solvent used as an internal standard (CDCl₃, δ_H 7.26, δ_C 77.16 ppm). High resolution (HR)TOFMS (ESI⁺) and tandem MS data were recorded on an Agilent 1260 infinity II LC coupled to a 6545 QToF MS. The mobile phase consisted of ultra-pure H₂O (A) and ACN (B) with 0.1% formic acid. A gradient method from 15% B to 90% B in 9 min at a flow rate of 0.4 mL/min was used. The column (Phenomenex Kinetex C₁₈, 2.6 μm, 100 Å, 50 mm x 2.1 mm) was re-equilibrated before each injection and the column compartment was maintained at 40 ˚C throughout each run. Semi-preparative HPLC (Phenomenex Kinetex C₁₈, 5 μm, 100 Å, 250 mm x 10 mm) utilized isocratic elution conditions or a gradient system with a flow rate of 4 mL/min on a Shimadzu LC-20AD HPLC system operating at room temperature, equipped with an SPD-M20A photodiode array detector. All samples were filtered through a 0.2 μm nylon filter or centrifuged at 14,000 rpm for 5 min before LC–MS and HPLC analysis. General reagents were from VWR International.

All experiments were performed on a 1260 infinity II LC (Agilent Technologies, Santa Clara, CA, USA). The isolated compounds and extract were dissolved in methanol at the concentration of 1 and 3 mg/mL, respectively. They were filtered through Millipore 0.20 μm filters before injection for chromatographic separation (Millex-LG, Tokyo, Japan). Then, 5 μL of each was injected and the flow rate was 0.4 mL/min. The separation was performed on YMC triart-C18 column (4.6 × 150 mm, 5 μm), (YMC Company, Kyoto, Japan) at 40 °C with the gradient system of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B) as follows 0–2 min, 20% B; 2–22 min, 20–100% B; 22–30 min, 100% B, finally the B content was reduced to the initial conditions in 5 min and the column was re-equilibrated for 5 min. The diode array detector (DAD) was measured over the rage of 200–600 nm. The evaporation temperature of the ELSD detector was set at 80 °C. The nebulizer temperature of the ELSD detector was set at 30 °C.