COMPARATIVE-STUDY OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF TETRACYCLINE ANTIBIOTICS

This work reviewed all the past HPLC methods utilized for the detectio n of tetracyclines and created a model to study the most promising one s. Three general mobile phases were investigated. They included Mobile Phase I (0.05 M buffer (A)-acetonitrile (B) programmed from 85A:15B t o 40A:60B), Mobile Phase II (0.05 M buffer (A)-acetonitrile (B)-methan ol (C) programmed from 80A:OB:20C to 30A:50B:20C), and Mobile Phase II I (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 e ach mobile phase. Ethylenediaminetetraacetic acid (EDTA) at 0.005 M wa s 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 0.0 1 M to all of the buffers at each pH. The columns chosen for this stud y were a C18 bonded to silica (Supelco LC-18), a ''deactivated'' C18 b onded column (Supelco LC-18-DB), and a polymeric styrenedivinylbenzene copolymer column (Polymer Laboratories PLRP-S). The columns and mobil e phases were evaluated using a mixed standard of oxytetracycline, tet racycline, and chlortetracycline. Comparisons were based on peak shape , separations, and comparative recoveries (integrated areas) of standa rds. The polymeric column was markedly superior in all respects. Gener ally optimum results were obtained with Mobile Phase I in pH 2.0 buffe rs. The oxalate buffer was best with the bonded columns while little d ifference was noted with the polymeric column. TMA markedly improved p eak shape on the bonded columns. EDTA was of little benefit. Recoverie s, especially of chlortetracycline, were higher from the polymeric col umn.