Development of novel methods for tannin quantification in grapes and wine

[摘要] ENGLISH ABSTRACT: Phenolic compounds, and condensed tannins in particular, are of utmost importance in red grapes and wine due to their contribution to the sensory properties and potential health benefits. However, the detailed analysis of these compounds is hampered by their complexity and the lack of reliable quantitative analytical methods. In this study, the analysis of wine tannins using different chromatographic methods was evaluated in order to develop an improved methodology for their accurate characterisation and quantification.Standard compounds for use in calibration were isolated from cocoa using semi-preparative high performance liquid chromatography or purchased commercially. Calibration curves were constructed and relative response factors based on degree of polymerisation (DP), class of compound and mobile phase composition were determined. Response factors were found to vary as a function of DP and class, indicating the errors associated with quantification as (epi)-catechin equivalents as is often done due to the lack of standards.Both hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RP-LC) methods for tannin analysis were developed. For HILIC, an amide column was used, which provided separation according to DP as well as a separation of isomers within specific elution windows. In RP-LC compounds were separated based on hydrophobicity, resulting in separation of isomers, with compounds of various DPs overlapping. In both separation modes, three detectors were connected in series: a photodiode array ultraviolet (UV) detector, a fluorescence detector (FLD) and a quadrupole-time-of-flight mass spectrometer (Q-TOF-MS). FLD was found to be the most sensitive for procyanidins (PCs), while UV demonstrated the best sensitivity toward gallated PCs. Negative electrospray ionisation (ESI)-Q-TOF-MS proved essential in identifying 161 tannin species based on accurate mass data, and was the most selective of the detectors when using extracted ion chromatograms.Quantification of tannins in 9 red wine samples and a grape seed extracts indicated that each of the detectors was useful for particular compounds. Co-elution caused overestimation of some compounds by UV and occasionally by FLD as well. Nevertheless, there was good agreement between the HILIC and RPLC methods, as well as between the various detectors in each mode. Quantitative data for the red wine and seed samples were in agreement with those obtained in previous studies. The total number of compounds identified (161) and quantified (74 and 41 in HILIC and RP-LC, respectively), was greater than could previously be obtained. Both methods were shown to be viable options for the analysis of condensed tannins in grape and wine samples. HILIC was found to be more sensitive, and therefore HILIC-UV-FLD-Q-TOF-MS is recommended as the method of choice for detailed quantitative condensed tannin analysis.

[发布机构] Stellenbosch University