A One-Step Polyphenol Removal Approach for Detection of Multiple Phytohormones from Grape Berry

Phytohormones play an important role in regulating the maturation process and the quality-related metabolite accumulation of fruits, and their concentration variation has always been concerned during fruit development. However, berry fruits, such as grape berries, are rich in a large number of secondary metabolites, which brings great challenges to the isolation and determination of hormones. In this work, we used grapes as experimental materials and proposed a solid-phase extraction (SPE) protocol to efficiently isolate multiple hormones from phenol-rich matrix using a mixture of dichloromethane, methanol and formic acid as eluent. A highly sensitive method based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to quantify a total of 11 plant growth regulators, including the recognized phytohormones, in grape pericarp and seed. The established method showed satisfactory precision (RSD < 11.3%) and linearity (R-2 > 0.9980). The limits of detection (LOD) and the limit of quantification (LOQ) were 0.001-0.75 ng/mL and 0.004-2.5 ng/mL, respectively. The recovery for the three levels of analytes spiked ranged from 63% to 118%, and the matrix effect was between 73% and 119%. Finally, the proposed method was applied to investigate the dynamic hormone concentration in Vitis vinifera L. cv. Cabernet Sauvignon berries from different vineyards, and assess the changes in endogenous hormones in grapes after treatment with exogenous growth regulators. We found that the contents of IP, ABA and IAA in pericarp and IP, IAA, IBA and SA in seed were significantly down-regulated after 10 days of treatment with NAA concentrations of 10 mg/L and 40 mg/L. In conclusion, this method helps to elucidate the role played by phytohormones in the maturation process and the accumulation of quality-related metabolites in phenol-rich fruits.

Automated summary

This study proposed a method for efficient isolation and determination of hormones in grape berries using solid-phase extraction and UHPLC-MS/MS. The method showed satisfactory precision and linearity, with low limits of detection and quantification. The method was applied to investigate the changes in hormone concentration in grape berries and the results showed significant down-regulation of certain hormones after treatment with growth regulators.