Determination of gibberellins using HPLC coupled with fluorescence detection

Abstract

Gibberellins (GAs) are a class of carboxylic phytohormones that regulate growth and influence various developmental processes of plants. Due to the ultra trace levels of GAs in plants and the complicated matrix of samples, highly selective and sensitive methods are required for their determination, and mass spectrometry (MS) is the preferred technique. To avoid the complex sample pretreatment and requirement of expensive isotope internal standards in MS-based methods, a simple and practical approach for the determination of GAs was developed in this study as a supplementary of MS-based approaches using HPLC coupled with fluorescence detection for the daily analysis or high throughput analysis of GAs. 1,3,5,7-Tetramethyl-8-butyrethylenediamine-difluoroboradiaza-s-indacene (TMBB-EDAN), a labeling reagent for carboxylic compounds developed in our group, was applied as a pre-column derivatization reagent. TMBB-EDAN could efficiently react with GAs at 25 °C for 75 min in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (EDC) and pyridine, and then the GA derivatives could be well separated within 45 min with gradient elution on a C₁₈ column. Excellent linear responses were observed with regression coefficients >0.997, and the limits of detection were in the range of 0.081–0.22 nM (S/N = 3). Finally, the proposed method was applied to determine gibberellins in biological samples with recoveries of 91.8–109.7%.