Comparative Study of High Performance Liquid Chromatographic Methods for the Determination of Tetracycline Antibiotics

Abstract

This work reviewed all the past HPLC methods utilized for the detection of tetracyclines and created a model to study the most promising ones. Three general mobile phases were investigated. They included Mobile Phase I (0.05 M buffer (A)-acetonitrile (B) programmed from 85A:15B to 40A:60B), Mobile Phase II (0.05 M buffer (A)-acetonitrile (B)-methanol (C) programmed from 80A:0B:20C to 30A:50B:20C), and Mobile Phase III (0.05 M buffer (A)-acetonitrile (B)-dimethylformamide (C) programmed from 80A:OB:20C to 30A:50B:20C). Buffers made from citrate, oxalate, and phosphate, each prepared at pH 2.0, 4.5, and 7.0, were used with each mobile phase. Ethylenediaminetetraacetic acid (EDTA) at 0.005 M was added to the pH 4.5 and pH 7.0 buffers (it would not dissolve in pH 2.0 buffers). Tetramethylammonium chloride (TMA) was also added at 0.01 M to all of the buffers at each pH. The columns chosen for this study were a CIS bonded to silica (Supelco LC-18), a “deactivated” C18 bonded column (Supelco LC-18-DB), and a polymeric styrenedivinylbenzene copolymer column (Polymer Laboratories PLRP-S). The columns and mobile phases were evaluated using a mixed standard of oxytetracycline, tetracycline, and chlortetracycline. Comparisons were based on peak shape, separations, and comparative recoveries (integrated areas) of standards. The polymeric column was markedly superior in all respects. Generally optimum results were obtained with Mobile Phase I in pH 2.0 buffers. The oxalate buffer was best with the bonded columns while little difference was noted with the polymeric column. TMA markedly improved peak shape on the bonded columns. EDTA was of little benefit. Recoveries, especially of chlortetracycline, were higher from the polymeric column.