Improvement of chemical analysis of antibiotics : X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Abstract

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform—methanol—5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol—acetonitrile—0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C 8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 μg using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C 8 column was possible using methanol—acetonitrile—0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracyline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50°C.