3.3. Chromatography Analysis and the Evaluation of the Results

Commercially available standards of phloridzin (≥99%), phloretin (≥99%), chlorogenic acid (≥95%), rutin (≥94%), HPLC gradient grade acetonitrile and phosphoric acid were purchased from Merck Sigma-Aldrich (St. Louis, MO, USA, Merck KGaA, Darmstadt, Germany).

A Shimadzu LC-10AD (Shimadzu Corporation, Kyoto, Japan) HPLC system equipped with an LC-10AD binary solvent delivery module, with an SIL-HTA autosampler, a DGU-14A on-line degasser, an SPD-M10A diode array detector, and a CTO-10AC column oven (Shimadzu Co, Kyoto, Japan) was used in this study. The data evaluation and data acquisition were performed using the Shimadzu “LC Lab-Solution” software (Shimadzu Corporation, Kyoto, Japan).

The separation was performed on an YMC-Triart C18 ExRS (150 × 4.6 mm) chromatography column (YMS Co, Ltd., Japan, Kyoto) with a particle size of 5 µm (pore size 8 nm) at a 30 °C temperature. The mobile phase consisted of a water solution of 0.1% phosphoric acid (A) and acetonitrile (B) at a flow rate 1.0 mL/min according to the following elution gradient program: 0.01–10 min 10% mobile phase B, 10–10.2 min 50% mobile phase B, 10.2–12.5 min 10% mobile phase B. An injection volume of 1 µL was used. The peaks of separated compounds were detected at the wavelength of 280 nm for phloridzin and phloretin, 327 nm for chlorogenic acid, and 354 nm for rutin. All analysed compounds were successfully separated within 13 min.

The identification of the peaks was achieved by comparing their retention times and UV spectra with reference standards. The quantitative evaluation of the peaks was calculated using the method of the external standard:

where c_x is the concentration of the analyte in a sample (mg L)⁻¹, c_ST is the concentration of the corresponding standard (mg L)⁻¹, A_x is the peak area of the analyte in a sample, and A_ST is the peak area of the standard.

The concentration of a determined compound was converted to the amount of extraction solvent used, followed by the conversion to the real sample weight with the correction to the purity of the used standard. The total amount of individual phenolic compounds was expressed in mg g⁻¹ of dried plant material, or in µg g⁻¹ of fresh fruit material. The basic validation parameters of the chromatography method and the chromatographic patterns of real extracts are shown in the Supplementary Materials, Section 1.