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…

Combining effect of leaf removal and natural shading on grape ripening under two irrigation strategies in Manto negro (Vitis vinifera L.)

The increasingly frequent heat waves during grape ripening pose challenges for high quality wine grape production. Defoliation is a common practice that can improve the control of diseases in bunches, but also it increases the exposure to sunlight. Grapes exposed to solar radiation reach temperatures over the optimum for berry development and maturation. This makes the development of irrigation and canopy management techniques of great importance to maximize yield and grape quality. A field experiment was carried out during 2021 using Manto negro wine grapes to study the effect of applied irrigation and different light exposure levels on grape quality. Two irrigation treatments were imposed based on the frequency and amount of water doses in a four-block experimental vineyard at Bodega Ribas (Mallorca). Three light exposure treatments were randomly applied in each irrigation plot. The light treatments included exposed clusters from pea size, non-exposed clusters, and shaded clusters after softening. Leaf area index and canopy porosity was estimated every 2 weeks. Midday leaf water potential was measured weekly. Additionally, apparent electrical conductivity was measured between rows to estimate the soil water content variability. Light and temperature sensors were installed at the bunch level to quantify the differences in bunch temperature and light intensity among treatments. The effect of irrigation and cluster light exposure on berry weight, TSS, TA, malic acid, tartaric acid, K+, and pH were analysed at 5 moments along grape ripening. During different heat waves, the natural shading technique decreased the maximum bunch temperature around 10 °C respect to the exposed bunches in both irrigation strategies. The combination of defoliation and shading techniques after softening decreased TSS at harvest and affected most of the quality parameters during the last stages of ripening, showing an interesting technique to delay ripening in warm viticulture areas.

Comparison of imputation methods in long and varied phenological series. Application to the Conegliano dataset, including observations from 1964 over 400 grape varieties

A large varietal collection including over 1700 varieties was maintained in Conegliano, ITA, since the 1950s. Phenological data on a subset of 400 grape varieties including wine grapes, table grapes, and raisins were acquired at bud break, flowering, veraison, and ripening since 1964. Despite the efforts in maintaining and acquiring data over such an extensive collection, the data set has varying degrees of missing cases depending on the variety and the year. This is ubiquitous in phenology datasets with significant size and length. In this work, we evaluated four state-of-the-art methods to estimate missing values in this phenological series: k-Nearest Neighbour (kNN), Multivariate Imputation by Chained Equations (mice), MissForest, and Bidirectional Recurrent Imputation for Time Series (BRITS). For each phenological stage, we evaluated the performance of the methods in two ways. 1) On the full dataset, we randomly hold-out 10% of the true values for use as a test set and repeated the process 1000 times (Monte Carlo cross-validation). 2) On a reduced and almost complete subset of varieties, we varied the percentage of missing values from 10% to 70% by random deletion. In all cases, we evaluated the performance on the original values using normalized root mean squared error. For the full dataset we also obtained performance statistics by variety and by year. MissForest provided average errors of 17% (3 days) at budbreak, 14% (4 days) at flowering, 14.5% (7 days) at veraison, and 17% (3 days) at maturity. We completed the imputations of the Conegliano dataset, one of the world’s most extensive and varied phenological time series and a steppingstone for future climate change studies in grapes. The dataset is now ready for further analysis, and a rigorous evaluation of imputation errors is included.

Legacy of land-cover changes on soil erosion and microbiology in Burgundian vineyards

Soils in vineyards are recognized as complex agrosystems whose characteristics reflect complex interactions between natural factors (lithology, climate, slope, biodiversity) and human activities. To date, most of the unknown lies in an incomplete understanding of soil ecosystems, and specifically in the microbial biodiversity even though soil microbiota is involved in many key functions, such as nutrient cycling and carbon sequestration. Soil biological properties are indicative of soil quality. Therefore, understanding how soil communities are related to soil ecosystem functioning is becoming an essential issue for soil strategy conservation. Here, we propose to assess the importance of land-cover history on the present-day microbiological and physico-chemical properties. The studied area was selected in the Burgundian vineyards (Pernand-Vergelesses, Burgundy, France) where land occupation has been reconstructed over the last 40 years. Soil samples were collected in five areas reflecting various land cover history (forest, vineyards, shifting from forest to vineyards). For each area, physico-chemical parameters (pH, C, N, P, grain size) were measured and DNA was extracted to characterize the abundance and diversity of microbial communities. The obtained results show significant differences in the five areas suggesting that present-day microbial molecular biomass and bacterial taxonomic is partly inherited from past land occupation. Over longer period of time, such study of land-uses legacies may help to better assess ecosystem recovery and the impact of management practices for a better soil quality and vineyards sustainability.

Projected changes in vine phenology of two varieties with different thermal requirements cultivated in La Mancha DO (Spain) under climate change scenarios

The aim of this work was to analyze the phenology variability of Tempranillo and Chardonnay cultivars, related to the climatic characteristics in La Mancha Designation of Origin, and their potential changes under climate change scenarios. Phenological dates referred to budbreak, flowering, veraison and harvest were analyzed for the period 2000-2019. The weather conditions at daily time scale, recorded during the same period, were also evaluated. The thermal requirements to reach each of these phenological stages were calculated and expressed as the GDD accumulated from DOY=60. Changes in phenology were projected by 2050 and 2070 taking into account those values and the projected temperatures and precipitation, simulated under two Representative Concentration Pathway (RCP) scenarios –RCP4.5 and RCP8.5– using an ensemble of models. The average phenological dates during the period under study were, April 16th ± 6.6 days and April 5th ± 6.0 days for budbreak, May 31st ± 6.0 days and May 27th ± 5.3 days for flowering, July 26th ± 5.6 days and July 25th ± 5.8 days for veraison, and Ago 23rd ± 10.8 days and Ago 17th ± 9.0 days for harvest, respectively, for Tempranillo and Chardonnay. The projected changes in temperature imply an average change in the maximum growing season (April-August) temperatures of 1.2 and 1.9°C by 2050, and 1.6 and 2.6°C by 2070, under the RCP4.5 and RCP8.5 scenarios, respectively. A reduction in precipitation is predicted, which vary between 15% for 2050 under RCP4.5 scenario and up to 30% by 2070 under RCP8.5. The advance of the phenological dates for 2050, could be of 6, 7, 7, and 8 days for Tempranillo and 4, 6, 6 and 9 days for Chardonnay, respectively for budbreak, flowering, veraison and harvest under the RCP4.5 scenario. Under the RCP8.5 emission scenario, the advance could be up to 30% higher.

Effects of graft quality on growth and grapevine-water relations

Climate change is challenging viticulture worldwide compromising its sustainability due to warmer temperatures and the increased frequency of extreme events. Grafting Vitis vinifera L.