A column chromatographic method for the determination of demethylchlortetracycline (DMCTC), tetracycline (TC), and chlortetracycline (CTC) was developed. The chromatogram involved the separation of these tetracyclines from their related compounds on a column of acid-washed diatomaceous earth treated with a buffer consisting of 0.1 M ethylenediaminetetraacetic acid disodium salt (EDTA) at pH 6.0 and pH 7.0, glycerin, and polyethylene glycol 400 (PEG 400), followe dby a modified spectral determination of column eluates. Although only DMCTC, TC, CTC, and their related compounds were studied in the work reported here, it appears reasonable to assume that the technique described could be extended to the assay of other tetracyclines.
HE MOST widely used and official method of T analysis of tetracyclines is the microbiological assay which is based on the ability of tetracyclines to inhibit growth of certain organisms. Inhibition of growth is used as a direct measure of activity and/or potency of these tetracyclines. However, the microbiological assay is subject to inherent sources of error which limits its precision to =tl5%. Specificity is also limited by the fact that several tetracycline-related compounds exhibit activity toward the test organism similar to the parent compound.
The methods currently inuse for the determination of tetracyclines and related compounds are based on spectral behavior in the ultraviolet. The procedure of Woolford and Chiccarelli ( l ) , which determined T C in CTC, is based upon the change in spectrophotometrk absorption in acid and basic aqueous media. A second paper by Chiccarelli et al. ( 2 ) described an additional assay method for T C and CTC based on the formation of anhydro compounds, and the fact that CTC heated a t pH 7.5 is cleaved to isochlortetracycline. whereas T C under the same conditions is relatively stable. Doskocil and Vondracek (3) determined T C and CTC with a method based on the different rates at which tetracyclines are inactivated in alkaline medium. A differential spectrophotometric method has been used in these laboratories for determination of related compounds.
Various chromatographic methods-paper, column, and thin-layer-are being used for the quantitation and investigation of tetracyclines. Selzer and Wright (4) described paper chromatog-