Rapid-resolution liquid chromatography/mass spectrometry for determination and quantitation of polyphenols in grape berries.

A rapid-resolution liquid chromatography/mass spectrometric (RRLC/MS) method for detection and quantitation of polyphenols in grape berry skins and seeds has been developed. Pulp-free berry skins were treated with liquid nitrogen and ground; seeds were also ground. Then, 3 g of samples were extracted with 30 mL of a mixture of methanol/water/formic acid 70:30:1 (v/v/v) under sonication and 1 microL of the final extract was injected into two 100 x 2.1 mm i.d., 1.8 microm Zorbax Eclipse plus C18 columns connected in series. Compounds were fractionated using a gradient elution of acidified acetonitrile/methanol 50:50 (v/v)/water. Columns were thermostatted at 70 degrees C. MS was carried out on an Agilent 6410 QqQ instrument equipped with an electrospray ionization source. Positive and negative MS/MS product ion scans were used for compound identification, whereas positive full scan MS in the m/z range 200-1400 was used for quantitation. By means of mass spectra comparison, various flavonols, flavan-3-ols, anthocyanins and stilbenes were identified. Quantitation was performed by external calibration, and concentration values were corrected for matrix effect that was evaluated in separate experiments. Semi-quantitative estimation was performed for compounds for which standards were not commercially available. Recoveries ranged from 90-102% with relative standard deviation (RSD) <5%, whereas the between samples RSD was in the range 4-12%. Two surrogate standards were used for quality control. The developed method was applied to analyze the polyphenol content of three Vitis vinifera table cultivars at physiological maturity and after proper preservation for 6 weeks. Results demonstrated that during preservation about half of the polyphenol content was lost.