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…

De novo Vitis champinii whole genome assembly allows rootstock-specific identification of potential candidate genes for drought and salt tolerance

Vitis champinii cultivars Ramsey and Dog-ridge are main choices for rootstocks to adapt viticulture in semi-arid and arid regions thanks to their distinctive tolerance to drought and salinity. However, genetic studies on non-vinifera rootstocks have heavily relied on the grapevine (Vitis vinifera) reference genome, which difficulted the assessment of the genetic variation between rootstock species and grapevines. In the present study, this limitation is addressed by introducing a novo phased genome assembly and annotation of Vitis champinii. This new Vitis champinii genome was employed as reference for mapping RNA-seq reads from the same species under drought and salt stresses, and for comparison the same reads were also mapped to the Vitis vinifera PN40024.V4 reference genome. A significant increase in alignment rate was gained when mapping Vitis champinii RNA-seq reads to its own genome, compared to the Vitis vinifera PN40024.V4 reference genome, thus revealing the expression levels of genes specific to Vitis champinii. Moreover, differences in coding sequences were observed in ortholog genes between Vitis champinii and Vitis vinifera, which therefore challenges previous differential expression analyses performed between contrasting Vitis genotypes on the same gene from the Vitis vinifera genome. Genes with possible implications in drought and salt tolerance have been identified across the genome of Vitis champinii, and the same genomic data can potentially guide the discovery of candidate genes specific from Vitis champinii for other traits of interest, therefore becoming a valuable resource for rootstock breeding designs, specially towards increased drought and salinity due to climate change.

VineyardFACE: Investigation of a moderate (+20%) increase of ambient CO2 level on berry ripening dynamics and fruit composition

Climate change and rising atmospheric carbon dioxide concentration is a concern for agriculture, including viticulture. Studies on elevated carbon dioxide have already been on grapevines, mainly taking place in greenhouses using potted plants or using field grown vines under higher CO2 enrichment, i.e. >650 ppm. The VineyardFACE, located at Hochschule Geisenheim University, is an open field Free Air CO2 Enrichment (FACE) experimental set-up designed to study the effects of elevated carbon dioxide using field grown vines (Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon). As the carbon dioxide fumigation started in 2014, the long term effects of elevated carbon dioxide treatment can be investigated on berry ripening parameters and fruit metabolic composition.
The present study aims to investigate the effect on fruit composition under a moderate increase (+20%; eCO2) of carbon dioxide concentration, as predicted for 2050 on both Riesling and Cabernet Sauvignon. Berry composition was determined for primary (sugars, organic acids, amino acids) and secondary metabolites (anthocyanins). Special focus was given on monitoring of berry diameter and ripening rates throughout three growing seasons. Compared to previous results of the early adaptative phase of the vines [1], our results show little effect of eCO2 treatment on primary metabolites composition in berries. However, total anthocyanins concentration in berry skin was lower for eCO2 treatment in 2020, although the ratio between anthocyanins derivatives did not differ.
[1] Wohlfahrt Y., Tittmann S., Schmidt D., Rauhut D., Honermeier B., Stoll M. (2020) The effect of elevated CO2 on berry development and bunch structure of Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon. Applied Science Basel 10: 2486

20-Year-Old data set: scion x rootstock x climate, relationships. Effects on phenology and sugar dynamics

Global warming is one of the biggest environmental, social, and economic threats. In the Douro Valley, change to the climate are expected in the coming years, namely an increase in average temperature and a decrease in annual precipitation. Since vine cultivation is extremely vulnerable and influenced by the climate, these changes are likely to have negative effects on the production and quality of wine.
Adaptation is a major challenge facing the viticulture sector where the choice of plant material plays an important role, particularly the rootstock as it is a driver for adaptation with a wide range of effects, the most important being phylloxera, nematode and salt, tolerance to drought and a complex set of interactions in the grafted plant.
In an experimental vineyard, established in the Douro Region in 1997, with four randomized blocs, with five varieties, Touriga Nacional, Tinta Barroca, Touriga Franca and Tinta Roriz, grafted in four rootstocks, Rupestris du Lot, R110, 196-17C, R99 and 1103P, data was collected consecutively over 20 years (2001-2020). Phenological observations were made two to three times a week, following established criteria, to determine the average dates of budbreak, flowering and veraison. During maturation, weekly berry samples were taken to study the dynamics of sugar accumulation, amongst other parameters. Climate data was collected from a weather station located near the vineyard parcel, with data classified through several climatic indices.
The results achieved show a very low coefficient of variations in the average date of the phenophases and an important contribution from the rootstock in the dynamic of the phenology, allowing a delay in the cycle of up to10-12 days for the different combinations. The Principal Component Analysis performed, evaluating trends in the physical-chemical parameters, highlighted the effect of the climate and rootstock on fruit quality by grape varieties.

The combined effects of climate, soils, and deficit irrigation on yield and quality of Touriga Nacional under high atmospheric demand in the Douro Region

Global warming is one of the biggest environmental, social and economic threats in several viticultural regions. In the Douro Valley, changes are expected in the coming years, namely an increase in temperature and a decrease in precipitation. These changes are likely to have consequences for the production and quality of wine.
The aim of this study was to explore the effects of different soil characteristics combined with several deficit irrigation strategies, managed throughout ETc references and predawn leaf water potentials thresholds, on physiology, yield, and qualitative attributes on the Touriga Nacional variety under years of mild to severe water and heat stress.
The studies were conducted over seven years (2015 to 2021) in two plots of a commercial vineyard located at Quinta do Ataíde (Symington Family Estates) planted in 2011 and 2014 at 170 meters elevation, growing under three water regimes: non-irrigated (NI) and two deficit irrigation strategies (30% and 60% ETc) assessed weekly by Ψpd. The site has an annual rainfall below 500 mm, with high atmospheric demand. Climate data was collected from a weather station, located on site. Berry ripening was followed weekly for fruit analysis. At harvest, yield, vigour and pruning weight per vine were determined from 90 vines by treatment. Each season at veraison the NDVI Index was accessed by a drone. The soils physic-chemistry in the experimental blocs were analysed and grouped by SWHC. Delta C-13 analyses were also performed per treatment in two years.Irrigation had a positive effect on yield per vine, mostly due to an increase in berry and cluster weight, and fertility index through the years. A significant increase in sugar content, colour and phenols was observed with deficit irrigation in some years, but vine vigour related to soil characteristics had by far the greatest impact on quality.

Climate projections over France wine-growing region and its potential impact on phenology

Climate change represents a major challenge for the French wine industry. Climatic conditions in French vineyards have already changed and will continue to evolve. One of the notable effects on grapevine is the advancing growing season. The aim of this study is to characterise the evolution of agroclimatic indicators (Huglin index, number of hot days, mean temperature, cumulative rainfall and number of rainy days during the growing season) at French wine-growing regions scale between 1980 and 2019 using gridded data (8 km resolution, SAFRAN) and for the middle of the 21th century (2046-2065) with 21 GCMs statistically debiased and downscaled at 8 km. A set of three phenological models were used to simulate the budburst (BRIN, Smoothed-Utah), flowering, veraison and theoretical maturity (GFV and GSR) stages for two grape varieties (Chardonnay and Cabernet-Sauvignon) over the whole period studied. All the French wine-growing regions show an increase in both temperatures during the growing season and Huglin index. This increase is accompanied by an advance in the simulated flowering (+3 to +9 days), veraison (+6 to +13 days) and theoretical maturity (+6 to +16 days) stages, which are more noticeable in the north-eastern part of France. The climate projections unanimously show, for all the GCMs considered, a clear increase in the Huglin index (+662 to 771 °C.days compared to the 1980-1999 period) and in the number of hot days (+5.6 to 22.6 days) in all the wine regions studied. Regarding rainfall, the expected evolution remains very uncertain due to the heterogeneity of the climates simulated by the 21 models. Only 4 regions out of 21 have a significant decrease in the number of rainy days during the growing season. The two budburst models show a strong divergence in the evolution of this stage with an average difference of 18 days between the two models on all grapevine regions. The theoretical maturity is the most impacted stage with a potential advance between 40 and 23 days according to wine-growing regions.