Determination of Doxycycline, 4-epidoxycycline, and 6-epidoxycycline in Aquatic Animal Muscle Tissue by an Optimized Extraction Protocol and Ultra-performance Performance Liquid Chromatography with Ultraviolet Detection

Abstract A fast ultra-performance liquid chromatography method was developed for the separation and quantification of doxycycline, 4-epidoxycycline, and 6-epidoxycycline in the muscles of channel catfish, shrimp, eel and turtle. The analytes were separated by comprehensive optimization of the chromatographic conditions. The extraction method was performed using a modified QuEChERS methodology. 2 g of muscle sample were mixed with 5 mL Mcllvaine buffer (pH 4), and 4 g of sodium sulfate and 2 g of sodium chloride were added for liquid–liquid partitioning. The mixture was agitated for 30 s and extracted with 10 mL of acidified acetonitrile. The resulting extract was purified using 200 mg of a C18-silica adsorbent. The samples were analyzed using ultra-performance liquid chromatography with ultraviolet detection using a BEH C18 column (2.1 mm ×100 mm, 1.7 μm). The limits of detection were less than 25 μg/kg and the limits of quantitation were less than 50 μg/kg for all analytes. The recoveries of the analytes were from 62.3% to 89.0% with relative standard derivations below 10.0%. The method provided efficient extraction and purification that enabled rugged and low-cost determination of the analytes.