L 7400

HPLC technique was used to quantify the free quercetin according to the methods of Schieber et al. (2001 (link)). UV–Vis detection system (L7400, Hitachi) with Synergi Fusion-RP 80 (250 × 4.60 mm I.D., size 4 μm, Phenomenex) and binary gradient intelligent pump (L6200A, Hitachi, Tokyo, Japan) was employed. The mobile phase consisted of 2.5% (v/v) acetic acid water solution (solvent A) and acetonitrile (solvent B). The gradient program consisted of 3% B, initially, changing to 21% B after 4 min, was maintained at 21% B until 10 min and raised to 22% B after 11 min, was maintained at 22% B until 15 min and increased to 30% B after 16 min, was increased to 50% B after 15 min and raised to 80% B after 15 min, and was maintained at 100% B until 40 min and then reduced to 3% B after 45 min. The injection volume of all the samples was 20 μL. Simultaneous monitoring was performed at 280 nm, and the flow rate was 0.8 mL/min.

We added BG puree (1 ± 0.5 g) to 10 mL of 80% methanol. After mixing, the mixture was sonicated for 30 min. The post-extraction solution was filtered through Whatman No. 4 filter paper and a 0.22 μm syringe filter. The filtered samples were analyzed by high-performance liquid chromatography (HPLC)-UV (L-7400, Hitachi, Hitachi shi, Japan). The column was a C18 column (250 mm × 4.6 mm ID, 5 μm, Nacalai Tesque Inc., Kyoto, Japan) with a flow rate of 1.0 mL/min. The mobile phase consisted of deionized water and acetonitrile (Merck & Co., Inc., Darmstadt, Germany) (88:12, v/v). The injected sample volume was 20 μL, and the detection wavelength was 284 nm [1 (link)].

The reaction mixture of octyl cinnamate was analyzed by a high-performance liquid chromatography (HPLC). The 10-fold diluted sample was injected (20 µL) into an HPLC (Hitachi L-7400; Tokyo, Japan) equipped with a UV detector and an Inertsil ODS-3 column (5 µM, 250 mm × 4.6 mm). The isocratic elution was performed with 0.1% acetic acid and methanol at a flow rate set to 1 mL/min, and octyl cinnamate was detected under UV light at 307 nm. The integrated area of octyl cinnamate and methyl cinnamate in the HPLC chromatogram was used in order to calculate the molar conversion. The molar conversion was defined as: Peak area of octyl cinnamate per peak area of methyl cinnamate and octyl cinnamate × 100%.

The extracts of Lonicera japonica were analyzed by high-performance liquid chromatography (HPLC) (Hitachi L-7400; Tokyo, Japan) according to the method described by Lin et al. [15 (link)]. Twenty μL of the extract was loaded into a Thermo C18 capillary column (5 μm, 250 × 4.6 mm, Agilent, Waltham, MA, USA) and assayed in gradient elution mode during the chromatographic analysis. Elution was carried out using 0.1% acetic acid in water and methanol at a flow rate of 1.0 mL/min. Gradient elution was performed as follows: Methanol was set 30% for the first 5 min, then the methanol was increased to 50% between 5 and 10 min, and held at 100% for the last 5 min. The UV detector was set at a wavelength of 325 nm. Calibration curves were established using CGA standards, and samples were analyzed by comparing their retention times with those of the standards. The yield of CGA from Lonicera japonica was calculated according to the following formula Equation (2):

The BG samples were ground to a mash using a mortar and homogeneously mixed. Then, a fixed weight of BG mash (1 ± 0.5 g) was added to 10 mL of distilled water. After mixing, the mixture was processed with ultrasound for 30 min. The extraction solution was filtered through Whatman No. 4 filter paper and a 0.22 μm syringe filter. The filtered samples were subjected to HPLC with an ultraviolet detector (L-7400, Hitachi, Hitachi shi, Japan). The samples were separated on a C18 column (250 mm × 4.6 mm ID, 5 μm, Waters, Milfor, MA, USA) at a flow rate of 0.6 mL/min. Distilled water and acetonitrile (88:12, v/v) were used as the mobile phase. The sample injected volume was 20 μL, and the detection wavelength was 210 nm [30 (link)].

Reversed-phase HPLC was used to assay flavonoid composition. The Agilent HPLC system used consisted of a Model 1100 pump equipped with a multisolvent delivery system, an L-7400 (Hitachi) ultraviolet (UV) detector, and fitted with an Agilent C18 (5 μm, 4.6 × 250 mm) column. The mobile phase consisted of (A) 0.03 M phosphoric acid in water and (B) methanol. The mobile phase was filtered under vacuum through a 0.45 um membrane filter before use. The flow rate was maintained at 1 mL/min and UV absorbance was measured at 260–360 nm. The operating temperature was maintained at room temperature [19 , 20 ]. Identification of the compounds was achieved by comparison of retention times with standards, UV spectra, and UV absorbance ratios after coinjection of samples and standards.