IVAS 2022 banner
IVES 9 IVES Conference Series 9 IVAS 9 IVAS 2022 9 Screening of hydroxytyrosol and tyrosine related metabolites in commercial wines by an UHPLC/MS validated method.

Screening of hydroxytyrosol and tyrosine related metabolites in commercial wines by an UHPLC/MS validated method.

Abstract

Hydroxytyrosol (HT) is a bioactive phenolic compound with antioxidant activity. Yeast synthetise tyrosol from tyrosine by the Ehrlich pathway which is subsequently hydroxylated to HT. The aim of the present work is to develop and validate an UHPLC–HRMS method to assess the metabolites involved in this pathway as well as to screen Spanish commercial wines for HT bioactive compound.

A total of 100 samples of commercial wines were analysed including 57 red wines and 43 white. The analysis was carried out in a Waters Acquity UHPLC (Milford, Massachusetts, USA) coupled to a Waters Xevo TQ (Milford, Massachusetts, USA) triple quadrupole mass spectrometer. The MassLynx MS software was used. The column used was an Acquity UPLC BEH C18. The chromatographic conditions consisted of two mobile phases, water with 0.2% acetic acid (A) and acetonitrile (B), with a gradient elution programmed.

This analytical method was validated following AOAC instructions (AOAC 2012). Linearity, LOD, LOQ, intermediate accuracy, repeatability and matrix effects were the parameters assessed.  Calibration standards were prepared for each analytical batch and three replicates were determined at different concentrations for each compound with 7 degrees of linearity.

Linearity values were calculated through the correlation coefficient (R2) of the curves obtained for each compound. The detection limits were calculated based on the standard deviation of the response and the slope (Ich, 2005).

The intermediate precision was calculated measuring standard deviation (RSD) in a set of three concentrations (LOQ, 10x LOQ and 100x LOQ ng mL−1) for 5 days with 6 replicates per concentration. Repeatability was assessed in a single day-long work session, with six replicates of each concentration.

The matrix effect was tested in a wine synthetic matrix by spiking with the same standard
solution as described above. The slopes resulting from the spiked matrix and calibration solutions (acetonitrile 10% v/v) in the linear range were used to evaluate the matrix effect.

In order to elucidate the effect that filtration caused on the compounds, most usual filters such as nylon (NY), polytetrafluoroethylene (PTFE) and cellulose acetate (CA) were tested. In the case of hydroxytyrosol the LOD was 0.052 ng mL−1 and LOQ 0.157 ng mL-1. For tyrosol, LOD 13,020 and LOQ 39,455 ng mL -1. Tyrosine, LOD 1,567 and LOQ 4,748 ng mL−1 and hydroxyphenylpyruvic acid, LOD 6,795 and LOQ 20,591 ng mL-1. All the values had an R2 between 0.9991 and 0.9999, showing quite good linearity. As we know, this is the first study available in which all the compound of the formation route for hydroxytyrosol has been identified and quantified. This could be accomplished thanks to a validated HRM method developed specifically to diminish LOD and LOQ. Furthermore, we ascertained the differences in the content of hydroxytyrosol in a great range of Spanish wines.

References

AOAC (2012) Appendix F: guidelines for Standard Method Performance
Requirements (SMPR). AOAC Official methods of analysis.
Ich (2005). ICH Topic Q2 (R1) Validation of analytical procedures: Text and methodology. International Conference on Harmonization, 1994 (November 1996), 17.

DOI:

Publication date: June 24, 2022

Issue: IVAS 2022

Type: Poster

Authors

González-Ramírez Marina1, Valero Eva2, Cerezo Ana B.1, Troncoso Ana M.1 and Garcia-Parrilla M. Carmen1

1Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla
2Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de Olavide, Sevilla, Spain

Contact the author

Keywords

hydroxytyrosol, wine, UHPLC, mass spectrometry, tyrosine.

Tags

IVAS 2022 | IVES Conference Series

Citation

Related articles…

Anthocyanin profile is differentially affected by high temperature, elevated CO2 and water deficit in Tempranillo (Vitis vinifera L.) clones

Anthocyanin potential of grape berries is an important quality factor in wine production. Anthocyanin concentration and profile differ among varieties but it also depends on the environmental conditions, which are expected to be greatly modified by climate change in the future. These modifications may significantly modify the biochemical composition of berries at harvest, and thus wine typicity. Among the diverse approaches proposed to reduce the potential negative effects that climate change may have on grape quality, genetic diversity among clones can represent a source of potential candidates to select better adapted plant material for future climatic conditions. The effects of individual and combined factors associated to climate change (increase of temperature, rise of air CO2 concentration and water deficit) on the anthocyanin profile of different clones of Tempranillo that differ in the length of their reproductive cycle were studied. The aim was to highlight those clones more adapted to maintain specific Tempranillo typicity in the future. Fruit-bearing cuttings were grown in controlled conditions under two temperatures (ambient temperature versus ambient temperature + 4ºC), two CO2 levels (400 ppm versus 700 ppm) and two water regimes (well-watered versus water deficit), both in combination or independently, in order to simulate future climate change scenarios. Elevated temperature increased anthocyanin acylation, whereas elevated CO2 and water deficit favoured the accumulation of malvidin derivatives, as well as the acylation and tri-hydroxylation level of anthocyanins. Although the changes in anthocyanin profile observed followed a common pattern among clones, such impact of environmental conditions was especially noticeable in one of the most widely distributed Tempranillo clones, the accession RJ43.

Aromatic maturity is a cornerstone of terroir expression in red wine

Harvesting grapes at adequate maturity is key to the production of high-quality red wines. Enologists and wine makers define several types of maturity, including technical maturity, phenolic maturity and aromatic maturity. Technical maturity and phenolic maturity are relatively well documented in the scientific literature, while articles on aromatic maturity are scarcer. This is surprising, because aromatic maturity is, without a doubt, the most important of the three in determining wine quality and typicity (including terroir expression). Optimal terroir expression can be obtained when the different types of maturity are reached at the same time, or within a short time frame. This is more likely to occur when the ripening takes place under mild temperatures, neither too cool, nor too hot. Aromatic expression in wine can be driven, from low to high maturity, by green, herbal, fresh fruit, ripe fruit, jammy fruit, candied fruit or cooked fruit aromas. Green and cooked fruit aromas are not desirable in red wines, while the levels of other aromatic compounds contribute to the typicity of the wine in relation to its origin. Wines produced in cool climates, or on cool soils in temperate climates, are likely to express herbal or fresh fruit aromas; while wines produced under warm climates, or on warm soils in temperate climates, may express ripe fruit, jammy fruit or candied fruit aromas. Growers can optimize terroir expression through their choice of grapevine variety. Early ripening varieties perform better in cool climates and late ripening varieties in warm climates. Additionally, maturity can be advanced or delayed by different canopy management practices or training systems.

The effects of alternative herbicide free cover cropping systems on soil health, vine performance, berry quality and vineyard biodiversity in a climate change scenario in Switzerland

There is an urgent need in viticulture to adopt alternative herbicide-free soil management strategies to mitigate climate change, increase biodiversity, reduce plant protection products and improve soil quality while minimizing detrimental effects on grapevine’s stress tolerance and fruit quality. To propose sustainable solutions, adapted to different pedoclimatic conditions in Switzerland, we developed a multidisciplinary 4-year project, started in 2020. Objectives of the project are to a) evaluate the impact of green covers (spontaneous flora, winter cover crop and permanent ground cover) on environmental and agronomic parameters and b) develop subsequently innovative strategies for different viticultural contexts of Switzerland. The project is divided into 3 phases: 1) diagnosis, 2) on-farm and 3) on-station experiments. Phase 1) consisted in an assessment of 30 commercial vineyards all over Switzerland, where growers already use different herbicide-free soil management strategies. The most promising practices identified in this exploratory phase will be replicated in commercial vineyards across Switzerland (“on-farm”) as well as in a classical randomized block design in an experimental plot (“on-station”). For phase 1), measurements consisted in evaluation of soil status (compaction, structure, roots development), soil microbial diversity (metagenomics), plant diversity and biomass, vine physiology (water stress, vigor, leaf nitrogen) and berry quality (acidity, sugar, available nitrogen). Interestingly, the permanent ground cover resulted in a higher Shannon index thus a higher biodiversity as compared to the other itineraries. The winter cover crop increased vine nitrogen and vigor while deteriorating soil quality, leaving the soil more exposed and compacted likely due to more frequent tillage. The spontaneous flora led to higher berry sugar accumulation, less nitrogen and higher malic acid concentration putatively due to a higher water retention of the flora in a particularly wet vintage. Phases 2) and 3) are required to confirm those tendencies, over the 3 next vintages and different climatic conditions.

The impact of sustainable management regimes on amino acid profiles in grape juice, grape skin flavonoids, and hydroxycinnamic acids

One of the biggest challenges of agriculture today is maintaining food safety and food quality while providing ecosystem services such as biodiversity conservation, pest and disease control, ensuring water quality and supply, and climate regulation. Organic farming was shown to promote biodiversity and carbon sequestration, and is therefore seen as one possibility of environmentally friendly production. Consumers expect organically grown crops to be free from chemical pesticides and mineral fertilizers and often presume that the quality of organically grown crops is different or higher compared to conventionally grown crops. Integrated, organic, and biodynamic viticulture were compared in a replicated field trial in Geisenheim, Germany (Vitis vinifera L. cv. Riesling). Amino acid profiles in juice, grape skin flavonoids, and hydroxycinnamic acids were monitored over three consecutive seasons beginning 7 years after conversion to organic and biodynamic viticulture, respectively. In addition, parameters such as soil nutrient status, yield, vigor, canopy temperature, and water stress were monitored to draw conclusions on reasons for the observed changes. Results revealed that the different sustainable management regimes highly differed in their amino acid profiles in juice and also in their skin flavonol content, whereas differences in the flavanol and hydroxycinnamic acid content were less pronounced. It is very likely that differences in nutrient status and yield determined amino acid profiles in juice, although all three systems showed similar amounts of mineralized nitrogen in the soil. Canopy structure and temperature in the bunch zone did not differ among treatments and therefore cannot account for the observed differences in favonols. A different light exposure of the bunches in the respective systems due to differences in vigor together with differences in berry size and a different water status of the vines might rather be responsible for the increase in flavonol content under organic and biodynamic viticulture.

austrianvineyards.com: online viewer of all designations of Austrian wine

To digitally record and present all the origins of Austrian wines in the same perfect and clear way was the motivation for the Austrian Wine Marketing Board (Austrian Wine) to start with the project in 2018. In June 2021 the results were presented to the public in an online viewer showing all the designations of Austrian wine, available at https://austrianvineyards.com in a largely barrier-free manner. The online viewer provides tailored individual maps fitted to the respective zoom level. The smallest unit of wine-origins in Austria is called Ried and is displayed in a plot-specific manner highlighting areas under vine. Information on the Ried include administrative district, winegrowing municipality, cadastral municipality, large collective vineyard site, specific winegrowing region, generic winegrowing region, winegrowing area and, in many cases, an illustrative picture. Complementary data on the size, elevation (minimum-maximum), orientation (in 8 sectors plus flat) and gradient (minimum, maximum, average) are based on the area under vine according to the EU’s Integrated Administration and Control System. Additional information covers climate data. The diagrams are taken from the monthly breakdown of data in the annals of the Central Institute for Meteorology and Geodynamics, Austria provide a display of values for air temperature, precipitation, and sunshine hours for the reference year and the long-term average. Seasonal aggregated data on temperature, precipitation, and sunshine hours complete the display. Short descriptions with emphasis on geology and soil, field name in historical maps, etymology of the denomination, and main planted variety complements the available information for the main designations in the online viewer. These descriptions are compiled by winegrowers, geologists, historians, and journalists. All the information and data can be extracted to a pdf-file. Printed vineyard maps are also available. Missing content regarding wine origins in Styria will be completed in winter 2021/22.