# Optimization of an Extraction Protocol for Untargeted Metabolomics of Vitis vinifera L. Leaves

## TL;DR

This study optimizes a method to extract metabolites from grapevine leaves to better understand their responses to stress.

## Contribution

A reproducible extraction protocol for untargeted metabolomics in grapevine leaves is developed and validated.

## Key findings

- Optimal extraction used 750 mg leaf powder, 100 mg/mL sample-to-solvent ratio, and 80% methanol with 0.1% formic acid.
- UHPLC-QTOF-MS analysis identified discriminant metabolites using PCA and OPLS-DA.
- The protocol maximizes recovery of secondary metabolites relevant to grapevine stress responses.

## Abstract

Viticulture faces increasing challenges due to the susceptibility of Vitis vinifera L. to biotic and abiotic stresses, which trigger defense responses involving the synthesis of secondary metabolites. Untargeted metabolomics has become a powerful tool to explore these metabolic changes; however, the efficiency and reproducibility of metabolomic studies strongly depend on the extraction protocol used. Current literature shows variability in sample handling, solvent composition, and extraction conditions. This study aimed to optimize an extraction protocol for secondary metabolites in grapevine leaves to ensure high recovery of compounds relevant to untargeted metabolomics. Leaves of Vitis vinifera L. cv. Tempranillo were collected, pooled, frozen, and ground under liquid nitrogen. A factorial design was used to evaluate the effects of sample mass, sample-to-solvent ratio, and solvent type on extraction efficiency. Extracts were analyzed using UHPLC-QTOF-MS in both positive and negative ionization modes, and multivariate statistical tools (PCA and OPLS-DA) were used to identify discriminant metabolites. Optimal extraction was achieved using 750 mg of leaf powder, a sample-to-solvent ratio of 100 mg/Ml, and methanol 80% acidified with 0.1% of formic acid. This protocol maximizes the recovery of metabolites and provides a robust basis for future untargeted metabolomics studies of grapevine responses.