Otimização da extração e condições cromatográficas para quantificação de ácido abscísico por HPLC em diferentes espécies vegetais

Abstract

Population growth has increased the global demand for food, agricultural inputs, and sustainable solutions to ensure food security. One of the major challenges in modern agriculture is the difficulty plants face in developing and surviving under abiotic stresses, especially soil salinity, which is exacerbated by inadequate irrigation practices. To maintain homeostasis and adapt to these conditions, plants activate specific signaling pathways, including the modulation of phytohormones, with abscisic acid (ABA) being a key player. Given its importance in stress responses, quantification of ABA is an essential tool in physiological studies; however, traditional methodologies still face several limitations. In this context, high-performance liquid chromatography (HPLC) stands out as an available technique, offering high sensitivity, good resolution, and compatibility with thermolabile compounds. Therefore, the present study aimed to optimize and validate a method for ABA quantification by HPLC using small amounts of leaf tissue from different plant species. Plants (Solanum lycopersicum, Vigna unguiculata, and Zea mays) were grown in a greenhouse and subjected to saline stress with 100 mM NaCl for seven days. Leaves were harvested, and different ABA extraction methods—including solvents and resuspension strategies—were evaluated. Extracts were analyzed by HPLC using different mobile phases and flow rates. Maize yielded the highest ABA content and was selected for further analyses, with four replicates per treatment. HPLC analyses revealed species-specific differences in ABA detection. The method showed strong linearity (R² = 0.9818) for the standard curve. ABA levels tended to increase under saline stress in maize leaves. However, the t-test indicated a p-value > 0.05, showing no statistically significant difference. Overall, solvent choice, mobile phase composition and flow rate, as well as chromatographic conditions and sample age, were crucial for method performance. Maize leaves exhibited the most consistent results, indicating a trend toward increased ABA levels under saline stress. However, further analyses are necessary to enhance reproducibility and reduce variability among replicates.