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Development of an Efficient HPLC Fluorescence Detection Method for Brassinolide by Ultrasonic-Assisted Dispersive Liquid-Liquid Microextraction Coupled with Derivatization
Chromatographia Chromatographia : An International Journal for Rapid Communication in Chromatography, Electrophoresis and Associated Techniques
Format: Journal Article
Publication Year: 2014
Pages: 1653 - 1660
Source ID: shanti-sources-105056
Abstract: A novel hyphenated technique based on ultrasonic-assisted dispersive liquid-liquid microextraction (UA-DLLME) coupled with derivatization has been established for the determination of brassinolide (BL, a representative of brassinosteroids) by HPLC fluorescence detection. 9-Phenanthreneboronic acid is used as labeling reagent of BL. UA-DLLME parameters containing type and volume of extraction and disperser solvent, pH and ultrasonication time are optimized. Derivatization parameters are optimized included amount of 9-phenanthreneboronic acid, volume ratio of pyridine, derivatization time and temperature. Under optimal conditions, quantitative linear range of BL is 50-1,000 ng L−1 and excellent linear response is observed with correlation coefficient of 0.9996. Limit of detection and limit of quantification are calculated as 8.0 and 25.0 ng L−1, respectively. RSDs of retention time and peak area are in the range of 0.68-0.97 % and 4.61-6.54 % for intra-day precision, 1.32-1.94 % and 7.28-9.75 % for inter-day precision, respectively. Accuracy is satisfactory in the range of 82.3-125.1 %. RSDs’ values of repeatability are in the range of 0.82-1.79 and 3.95-8.53 % for retention time and peak area, respectively. Enrichment factor for BL is 189. The results of recovery and matrix effect are in the range of 82.0-108.6 and 90.0-115.3 %, respectively. The proposed method has been applied for the determination of BL in Arabidopsis thaliana, Daucus carota and Brassica campestris L. leaves with much higher sensitivity than many other methods.