Development of a Precolumn Derivatization Procedure Prior to Ultrasound-Assisted Cloud Point Extraction for Sensitive Determination of Fluoroquinolones in Eggs by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

In this work, a 4-chloro-7-nitrobenzodioxazole (NBD-Cl) derivatization coupled with the ultrasound-assisted cloud point extraction (UACPE) method prior to high-performance liquid chromatography with fluorescence detection (HPLC-FLD) analysis was developed for the determination of four widely used fluoroquinolones (FQs) including norfloxacin (NOR), ciprofloxacin (CIP), sarafloxacin (SAR), and gatifloxacin (GAT) in eggs. The derivatives of FQs with NBD-Cl were extracted into the Triton X-114 surfactant-rich phase, which was analyzed by reversed-phase HPLC-FLD. Ultrasound was applied to accelerate the phase separation in extraction and enhance the extraction efficiency of target analytes. Variable parameters affecting the derivatization and UACPE procedure were systematically evaluated and optimized. Under the optimum conditions, four FQs were successfully separated within 30 min through an Agilent TC-C₁₈ column. Good recoveries of 86.2–103.5 %, which were calculated using a range of spiked samples at three concentrations, were obtained by UACPE. The calibration graphs were linear over the range of 1.2–73.0 μg kg⁻¹ for four FQs with correlation coefficients (R) no less than 0.9957. The limits of detection were 0.2, 0.5, 0.3, and 0.4 μg kg⁻¹ for NOR, CIP, SAR, and GAT, respectively. The precisions indicated by relative standard deviations ranged from 0.6 to 4.3 % for both intraday and interday analysis. The proposed method proved to be a selective, sensitive, and eco-friendly approach which was successfully applied to analyze FQs in eggs at the local farmers market, and none of the target analytes were detected in these samples.