HPLC-Fluorescence Detection Method for Quantitative Determination of Tetracycline Antibiotic Residues in Honey

Abstract

A high performance liquid chromatography method utilizing fluorescence detectionwas optimized and validated to determine tetracycline residues in honey. The separation ofthree tetracycline residues; oxytetracycline, tetracycline and chlortetracycline was carried outon a reverse???phase C8 column with a gradient elution. A mobile phase system consisted of50% (v/v) methanol and 25 mM sodium acetate buffer (containing disodium ethylenediaminetetraaceticacid and calcium chloride, pH 8.10). Fluorescence detection was observed at 518nm (excitation wavelength at 393 nm) with 20 minute analysis time. The extraction withdisodium ethylenediaminetetraacetic acid (Na2EDTA)-McIlvaine buffer pH 4 was performedand followed by HLB cartridge clean up step. Calibration curves for oxytetracycline,tetracycline and chlortetracycline showed good linearities (r2 > 0.998) at concentrationsranged from 5 to 1000 ng/mL. The limits of detection (LODs) and quantifications (LOQs) foroxytetracycline, tetracycline and chlortetracycline were found to be 3.40, 0.29, 4.69 ??g/kg,and 11.35, 0.96, 15.62 ??g/kg, respectively. The recoveries of oxytetracycline, tetracycline andchlortetracycline, at 50, 100 and 200 ??g/kg spiked samples were higher than 80% for allcompounds. The analytical method was successfully applied to honey samples collected fromnorthern part of Thailand.

Keywords: tetracyclines, honey, fluorescence detection, HPLC