Systematic evaluation of sample preparation for fractionation of phytohormone salicylic acid in fresh leaves

Autor(es)

Marques, Thiago L. ^[1] ; Moraes, Liz Mary B. ^[1] ; Rocha, Fabio R. P. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Ctr Nucl Energy Agr, POB 96, BR-13400970 Piracicaba, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Talanta; v. 208, FEB 1 2020.
• Citações Web of Science: 2

Resumo

The determination of salicylic acid (SA), an important phytohormone responsible for stress signaling in plants, is of great importance in agricultural studies. However, a critical evaluation of the procedures for the extraction of the analytes and hydrolysis of the conjugated forms of SA is lacking in the literature and the available alternatives are complex, time-consuming, and laborious. In this study, the sample preparation methods for SA fractionation were critically evaluated to develop a simpler and faster alternative procedure. Microwave-assisted extractions were carried out with 2.0 g of fresh leaves and 8.0 mL of a 75% v/v ethanol:water solution at 40 degrees C for 10 min, followed by alkaline hydrolysis using 100 mu L of 0.1 mol L-1 NaOH at 80 degrees C for 60 min. Free and total SA were determined in crude and hydrolyzed extracts, respectively, by fluorimetry after chromatographic separation of the sample matrix under isocratic elution (25% v/v acetonitrile/phosphate buffer) using a C18 column. Recovery experiments using methyl salicylate and acetylsalicylic acid model compounds demonstrated that the soft microwave-assisted extraction did not decompose the SA derivatives and that alkaline hydrolysis was quantitative. The proposed procedure was successfully applied for fractionation of SA in sugarcane, corn, and soybean leaves with extraction and hydrolysis yields up to 70 and 20% higher than those achieved in previously proposed approaches, respectively. The developed procedure is a simple, fast, and reliable alternative for SA fractionation in crude extracts without sample clean-up, and utilizes dilute reagents and green solvents. (AU)

• Processo FAPESP: 16/25333-0 - Desenvolvimento de sistemas de preparo de amostras em fluxo aplicados a estudos agronômicos: determinação de sinalizadores de estresse e fracionamento de nitrogênio em tecidos foliares
• Beneficiário: Thiago Linhares Marques
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado