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…

The rootstock, the neglected player in the scion transpiration even during the night

Water is the main limiting factor for yield in viticulture. Improving drought adaptation in viticulture will be an increasingly important issue under climate change. Genetic variability of water deficit responses in grapevine partly results from the rootstocks, making them an attractive and relevant mean to achieve adaptation without changing the scion genotype. The objective of this work was to characterize the rootstock effect on the diurnal regulation of scion transpiration. A large panel of 55 commercial genotypes were grafted onto Cabernet Sauvignon. Three biological repetitions per genotype were analyzed. Potted plants were phenotyped on a greenhouse balance platform capable of assessing real-time water use and maintaining a targeted water deficit intensity. After a 10 days well-watered baseline period, an increasing water deficit was applied for 10 days, followed by a stable water deficit stress for 7 days. Pruning weight, root and aerial dry weight and transpiration were recorded and the experiment was repeated during two years. Transpiration efficiency (ratio between aerial biomass and transpiration) was calculated and δ13C was measured in leaves for the baseline and stable water deficit periods. A large genetic variability was observed within the panel. The rootstock had a significant impact on nocturnal transpiration which was also strongly and positively correlated with maximum daytime transpiration. The correlations with growth and water use efficiency related traits will be discussed. Transpiration data were also related with VPD and soil water content demonstrating the influence of environmental conditions on transpiration. These results highlighted the role of the rootstock in modulating water deficit responses and give insights for rootstock breeding programs aimed at identifying drought tolerant rootstocks. It was also helpful to better define the mechanisms on which the drought tolerance in grapevine rootstocks is based on.

Mobile device to induce heat-stress on grapevine berries

Studying heat stress response of grapevine berries in the field often relies on weather conditions during the growing season. We constructed a mobile heating device, able to induce controlled heat stress on grapes in vineyards. The heater consisted of six 150 W infrared lamps mounted in a profile frame. Heating power of the lamps could be controlled individually by a control unit consisting of a single board computer and six temperature sensors to reach a pre-set temperature. The heat energy applied to individual berries within a cluster decreases by the squared distance to the heat source, enabling the establishment of temperature profiles within individual clusters. These profiles can be measured by infrared thermography once a steady state has been reached. Radiant flux density received by a berry depending on the distance was calculated based on a view factor and measured lamp surface temperature and resulted to 665 Wm-2 at 7cm. Infrared thermography of the fruit surface was in good agreement with measurements conducted with a thermocouple inserted at epidermis level. In combination with infrared thermography, the presented device offers possibilities for a wide range of applications like phenotyping for heat tolerance in the field to proceed in the understanding of the complex response of plants to heat stress. Sunburn necrosis symptoms were artificially induced with the aid of the device for cv. Bacchus and cv. Sylvaner in the 2020 and 2021 growing season. Threshold temperatures for sunburn induction (LT5030min) were derived from temperature data of single berries and visual sunburn assessment, applying logistic regression. A comparison of threshold temperatures for the occurrence of sunburn necrosis confirmed the higher susceptibility of cv. Bacchus. The lower susceptibility of cv. Sylvaner did not seem to be related to its phenolic composition, rendering a thermoprotective role of berry phenolic compounds unlikely.

Ecophysiological performance of Vitis rootstocks under water stress

The use of rootstocks tolerant to soil water deficit is an interesting strategy to cope with limited water availability. Currently, several nurseries are breeding new genotypes, but the physiological basis of its responses under water stress are largely unknown. To this end, an ecophysiological assessment of the conventional 110-Richter (110R) and SO4, and the new M1 and M4 rootstocks was carried out in potted ungrafted plants. During one season, these Vitis genotypes were grown under greenhouse conditions and subjected to two water regimes, well-watered and water deficit. Water potentials of plants under water deficit down to < -1.4 MPa, and net photosynthesis (AN) <5 μmol m-2 s-1 did not cause leaf oxidative stress damage compared to well-watered conditions in any of the genotypes. The antioxidant capacity was sufficient to neutralize the mild oxidative stress suffered. Under both treatments, gravimetric differences in daily water use were observed among genotypes, leading to differences in the biomass of root, shoot and leaf. Under well-watered conditions, SO4 and 110R were the most vigorous and M1 and M4 the least. However, under water stress, SO4 exhibited the greatest reduction in biomass while M4 showed the lowest. Remarkably, under these conditions, SO4 reached the least negative stem water potential (Ψstem), while M1 reduced stomatal conductance (gs) and AN the most. In addition, SO4 and M1 genotypes also showed the highest and lowest hydraulic conductance values, respectively. Our results suggest that there are differences in water use regulation among genotypes, not only attributed to differences in stomatal regulation or intrinsic water use efficiency at the leaf level. Therefore, because no differences in canopy-to-root ratio were achieved, it is hypothesized that xylem vessel anatomical differences may be driving the reported differences among rootstocks performance. Results demonstrate that each Vitis rootstock differs in its ecophysiological responses under water stress.

Phenolic composition of Tempranillo Blanco grapes changes after foliar application of urea

Our research aimed to determine the effect and efficiency of foliar application of urea on the phenolic composition of Tempranillo Blanco grapes. The field experiment was carried out in 2019 and 2020 seasons and the plot was located in D.O.Ca Rioja (North of Spain). The vineyard was Vitis vinifera L. Tempranillo Blanco and grafted on Richter-110 rootstock. The treatments were control (C), whose plants were sprayed with water and three doses of urea: plants were sprayed with urea 3 kg N/ha (U3), 6 kg N/ha (U6) and 9 kg N/ha (U9). The applications were performed in two phenological stages, pre-veraison (Pre) and veraison (Ver). Also, each of the treatments was repeated one week later. Control and treatments were performed in triplicate and arranged in a randomised block design. Grapes were harvested at optimum ripening stage. High-performance liquid chromatography was used to analyse the phenolic composition of the grapes. Finally, the results obtained from the analytical determinations – flavonols, flavanols and non-flavonoid (hydroxybenzoic acids, hydroxycinnamic acids and stilbenes) – were studied statistically by analysis of variance. The results showed that, in 2019, U6-Pre and U9-Pre treatments increased the hydroxybenzoic acid content in grapes, and also all foliar treatments applied at Pre enhanced the stilbene concentration. Moreover, U3-Ver was the only treatment that rose flavonol and stilbene contents in the Tempranillo Blanco grapes. In 2020, all treatments applied at Pre enhanced the flavonol concentration in grapes. Furthermore, U3-Pre and U9-Pre treatments increased stilbene content in grapes. Nevertheless, the hydroxybenzoic acid content was improved by U6-Ver and U9-Ver and besides, hydroxycinnamic acid concentration in grapes was increased by all treatments applied at Ver. In conclusion, the lower and highest dose of urea (U3 and U9), applied at pre-veraison, were the best treatments to improve the Tempranillo Blanco grape phenolic composition.

The plantation frame as a measure of adaptation to climate change

The mechanization of vineyard work originally led to a reduction in planting densities due to the lack of machinery adapted to the vineyard. The current availability of specific machinery makes it possible to establish higher planting densities. In this work, three planting densities (1.40×0.80 m, 1.80×1 m and 2.20×1.20 m, corresponding to 8928, 5555 and 3787 plants/ha respectively) were studied with four varieties autochthonous of Galicia (northwestern Spain): Albariño and Treixadura (white), Sousón and Mencía (red). The vines were trained in a vertical shoot positioning system using a single Royat cordon, and pruned to spurs with two buds each. Agronomic data (yield, pruning wood weight, Ravaz index) and oenological data in must were collected. The higher planting density (1.40×0.80 m) had no significant effect on grape yield per vine in white varieties, although production per hectare was much higher due to the greater number of plants. In red varieties, this planting density resulted in a significantly lower production per vine, compensated by the greater number of plants. In addition, it significantly reduced the Brix degree in the must of the Albariño, Treixadura and Sousón varieties, and increased the total acidity in the latter two and Mencía. It also caused an increase in extractable and total anthocyanins and IPT in red grapes. The effects of high planting density on grapes are of great interest for the adaptation of varieties in the context of climate change. In the future, it could be advisable to modify the limits imposed by the appellations of origin on the planting density of these varieties in order to obtain more balanced wines.