C(18) columns for the simultaneous determination of oxytetracycline and its related substances by reversed-phase high performance liquid chromatography and UV detection.

Simultaneous determination of oxytetracycline, 4-epioxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline on C(18) columns has been accomplished using a high performance liquid chromatographic method with UV detection. Separation was achieved on a Hypersil BDS RP-C(18) column (250 mm x 4.6 mm) and on a Waters C(18) Symmetry column (150 mm x 3.9mm), 5 microm particle size each. These columns were equilibrated with mobile phases consisted of methanol-acetonitrile-0.1M phosphate buffer pH 8.0 (12.5:12.5:75, v/v/v) and (15:15:70, v/v/v), respectively. The flow rate was 1.0 ml/min and the total elution time was 15 and 5 min, respectively. Both methods were applied to oxytetracycline raw material, human and veterinary formulations, where the excipients did not interfere. External standard calibration curves were linear for 4-epioxytetracycline, oxytetracycline, alpha-apooxytetracycline, tetracycline and beta-apooxytetracycline in the concentration range of 0.27-200 microM, 0.05-200 microM, 0.03-200 microM, 0.35-200 microM and 0.20-200 microM on column A and 0.08-200 microM, 0.15-200 microM, 0.09-200 microM, 0.25-200 microM and 0.47-200 microM on column B, respectively. Day-to-day relative standard deviation of the determination for every component was less than 3%. Concerning the first column, limits of detection and quantification of the above compounds were in the concentration ranges of 10-106 nM and 30-352 nM, respectively, whereas on the second column these ranges became 27-144 nM and 81-475 nM, respectively. Recovery of the separated compounds was 95-105%.