Modified Method for Determining Tetracycline, 4-Epitetracycline, and Anhydrotetracyclines in Tetracycline Base or Hydrochloride

A partition column chromatographic procedure was developed for the assay of tetracycline samples for tetracycline (I), 4-epitetracycline (II), anhydrotetracycline (III), and 4-epianhydrotetracycline (IV). Two separate portions of a sample are used, one for the assay of I and II and the other, after a concentration step, for the assay of III and IV. Both portions are assayed with the same chromatographic system. The compounds are eluted from a column of diatomaceous earth coated with a pH 7.0 buffer containing disodium ethylenediaminetetraacetate, glycerin, and polyethylene glycol 400. The individual eluates are assayed by absorption spectroscopy. The precision of the assay for I, SD ± 0.5%, suggests that a chemical method such as this should be considered as the official assay method for tetracycline samples.