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…

1H-NMR-based Metabolomics to assess the impact of soil type on the chemical composition of Mediterranean red wines

The aim of this study was to evaluate the effects of different soil types on the chemical composition of Mediterranean red wines, through untargeted and targeted 1H-NMR metabolomics. One milliliter of raw wine was analyzed by means of a Bruker Avance II 400 spectrometer operating at 400.15 MHz. The spectra were recorded by applying the NOESYGPPS1D pulse sequency, to achieve water and ethanol signals suppression. No modification of the pH was performed to avoid any chemical alteration of the matrix. The generation of input variables for untargeted analysis was done via bucketing the spectra. The resulting dataset was preprocessed prior to perform unsupervised PCA, by means of MetaboAnalyst web-based tool suite. The identification of compounds for the targeted analysis was performed by comparison to pure compounds spectra by means of SMA plug-in of MNova 14.2.3 software. The dataset containing the concentrations (%) of identified compounds was subjected to one-way analysis of variance (ANOVA) to highlight significant differences among the wines. The untargeted analysis, carried out through the PCA, revealed a clear differentiation among the wines. The fragments of the spectra contributing mostly to the separation were attributed to flavonoids, aroma compounds and amino acids. The targeted analysis leaded to the identification of 68 compounds, whose concentrations were significant different among the wines. The results were related to soils physical-chemical analysis and showed that: 1) high concentrations of flavan-3-ols and flavonols are correlated with high clay content in soils; 2) high concentrations of anthocyanins, amino acids, and aroma compounds are correlated with neutral and moderately alkaline soil pH; 3) low concentrations of flavonoids and aroma compounds are correlated with high soil organic matter content and acidic pH. The 1H-NMR metabolomic analysis proved to be an excellent tool to discriminate between wines originating from grapes grown on different soil types and revealed that soils in the Mediterranean area exert a strong impact on the chemical composition of the wines.

Climate change impacts on Douro Region viticulture and adaptation measures

Climate has a significant impact in the success of any agricultural system, with a direct influence on the crops suitability to a given region, interfering on yield and quality and also with the economic sustainability of the productive activity. In the Douro Demarcated Region (RDD), as in most regions of the Mediterranean climate, the scarce precipitation (33% has less than 600 mm per year), and your high variability, associated with high rates of evapotranspiration during the summer, is usually one of the fundamental factors that limit the grapevine development, as well as the production and quality of the harvest. Thus, facing the scenario in temperature changes for the next decades (1.5-2.5°C) and confirming the predictions of precipitation decreases and/or great variability in the occurrence of heat waves and intense rainfall, the consequences for slope stability in mountain viticulture and sustainability of all operations involved, are risks to be taken into account. In this way, a deepest and sustained knowledge regarding the adaptation measures to adverse environmental conditions is of a crucial importance, enabling a more efficient adaptation of plant growth conditions and the optimization of production and quality of the grapevines. The development of this work, carried out in two commercial vineyards, one located in Soutelo do Douro, São João da Pesqueira, Cima Corgo sub-region, and another located in Numão, Vila Nova de Foz Côa, Douro Superior sub-region, it seeks to establish a relationship between climatic elements and physiological, productive and qualitative parameters, as well as to evaluate the effectiveness of adaptation measures, including different types of deficit irrigation (2002-2019) and the application of shading nets (2019-2020) in the physiological, viticultural and oenological behavior in the Touriga Nacional and Moscatel Galego Branco varieties, respectively. The results showed that the application of deficit irrigation allowed to significantly reduce the impact of the adverse weather conditions at key moments in the development of the grapevine, particularly in the period immediately before veráison and maturation, reducing the negative effects on the physiological processes and productivity, without compromise the must quality parameters. On the other hand, the application of shading nets significantly reduced de leaves temperature, allowing to increase the water potential, stomatal conductance and photosynthetic rate of grapes, which was reflected in the yield increase in the 2nd year of the study. For the maturation indicators, higher levels of total acidity, malic acid and assimilable nitrogen were obtained. The last measure presents a huge potential, being essential to carry out more years of trials to obtain stronger conclusions in terms of production parameters, but also in characteristics as important as the grape ripening components and the organoleptic characteristics of wines.

Diagnosis of soil quality and evaluation of the impact of viticultural practices on soil biodiversity in a vineyard in southwestern France

Viticulture is facing two major changes – climate change and agroecological transition. In both cases, soil quality is seen as a lever to move towards a more sustainable viticulture. However, soil biological quality is little considered in the implementation of viticultural practices. Gascogn’Innov (2017-2022) is an Operational Group funded by the European Innovation Partnership for Agriculture. As such, it brings together winegrowers from the south-west of France, scientists, advisors and technicians, around a project focused on viticultural soil biological functioning and the design of technical routes more respectful toward soil heritage. To achieve this, the project aims to acquire references on the impact of viticultural practices on soil biology from a dynamic way, and to test a methodology to integrate information provided by the soil bioindicators to manage farming systems. A set of indicators of soil biological quality are evaluated in the project: microorganisms (bacteria and fungi abundance and diversity), fauna (abundance and diversity of nematodes and earthworms), physico-chemical characteristics, soil structure assessment and degradation rate of organic matter. Based on a network of 13 plots that have been subject to an initial diagnosis in 2017, several agronomical practices to restore soil fertility are experimented to redesign the cropping system (for instance plant cover, organic matter inputs, reduction of herbicides, mineral fertilizers). System redesign was made in collaboration by winegrowers and an interdisciplinary group of experts (agronomists, biologists). Several indicators are measured on vine and soil at each vintage to assess vine health and productivity. At the end of the project (2021), a final diagnosis was carried out. Gascogn’Innov allowed to create a regional database on the quality of wine-growing soils, which permitted to evaluate the effect of practices according to soil types. Especially, decreasing the intensity of tillage and increasing the duration and diversity of grass coverage tends to increase the abundance of all the organisms studied. This project confirmed the value of soil biological quality indicators to drive the sustainability of practices, but also highlighted the key-role of expertise, in both agronomy and soil biology, to help winegrowers understand and appropriate their soil quality diagnoses.

Protected Designation of Origin (D.P.O.) Valdepeñas: classification and map of soils

The objective of the work described here is the elaboration of a map of the different types of vineyard soils that to guide the famers in the choice of the most productive vine rootstocks and varieties. 90 vineyard soils profiles were analysed in the entire territory of the Origen Denominations of Valdepeñas. The sampling was carried out in 2018 (June to October) by making a sampling grid, followed by photointerpretation and control in the field. The studied soils can be grouped into 9 different soil types (according to FAO 2006 classification): Leptosols, Regosols, Fluvisols, Gleysols, Cambisols, Calcisols, Luvisols and Anthrosols. A map showing the soil distribution with different type of soils has been made with the ArcGIS program. Regarding to the choice of rootstock, Calcisoles are soils with a high active limestone content, so the rootstocks used in these soils must be resistant to this parameter; Luvisols are deep soils with high clay content, so they will support vigorous rootstocks. Because the cartographic units are composed of two or more subgroups, with are associated in variable proportions, 9 different soil associations have been established; Unit 1: Leptosols, Cambisols and Luvisols (80%, 15% and 5% respectively); Unit 2: Cambisols with Regosols and Luvisols (40%, 30% and 30% respectively); Unit 3: Cambisols and Gleysols with Regosols (40%, 40% and 20% respectively); Unit 4: Regosols with Cambisols, Leptosols and Calcisols (40%, 30%, 15% and 15% respectively); Unit 5: Cambisols, Leptosols, Calcisols and Regosols (25% each of them); Unit 6: Luvisols with Cambisol and Calcisols (80%, 10% and 10% respectively); Unit 7: Luvisols and Calcisols with Cambisols (40%, 40% and 20% respectively); Unit 8: Calcisols with, Cambisols and Luvisols (80%, 10% and 10% respectively); Unit 9: Anthrosols. These study allow to elaborate the first map of vineyard soils of this Protected Designation of Origin in Castilla-La Mancha.

VINIoT: Precision viticulture service for SMEs based on IoT sensors network

The main innovation in the VINIoT service is the joint use of two technologies that are currently used separately: vineyard monitoring using multispectral imaging and deployed terrain sensors. One part of the system is based on the development of artificial intelligence algorithms that are feed on the images of the multispectral camera and IoT sensors, high-level information on water stress, grape ripening status and the presence of diseases. In order to obtain algorithms to determine the state of ripening of the grapes and avoid losing information due to the diversity of the grape berries, it was decided to work along the first year 2020 at berry scale in the laboratory, during the second year at the cluster scale and on the last year at plot scale. Different varieties of white and red grapes were used; in the case of Galicia we worked with the white grape variety Treixadura and the red variety Mencía. During the 2020 and 2021 campaigns, multispectral images were taken in the visible and infrared range of: 1) sets of 100 grapes classifying them by means of densimetric baths, 2) individual bunches. The images taken with the laboratory analysis of the ripening stage were correlated. Technological maturity, pH, probable degree, malic acid content, tartaric acid content and parameters for assessing phenolic maturity, IPT, anthocyanin content were determined. It has been calculated for each single image the mean value of each spectral band (only taking into account the pixels of interest) and a correlation study of these values with laboratory data has been carried out. These studies are still provisional and it will be necessary to continue with them, jointly with the training of the machine learning algorithms. Processed data will allow to determine the sensitivity of the multispectral images and select bands of interest in maturation.