Rapid liquid chromatographic method for simultaneous determination of tetracyclines antibiotics and 6-epi-doxycycline in pharmaceutical products using porous graphitic carbon column

A rapid and selective high performance liquid chromatographic (HPLC) method has been developed for the separation and determination of five commercially used tetracyclines. The chromatography was performed on a porous graphitic carbon (PGC) column, using 0.05 M potassium phosphate buffer (pH 2.0)--acetonitrile (40 + 60) as the mobile phase and ultraviolet detection at 268 nm. The method permits the simultaneous determination of oxytetracycline, metacycline, chlortetracycline and doxycycline as well as the separation of one of their common impurities (6-Epi-doxycycline) in bulk powder and pharmaceutical preparations with detection limits of 0.5-2 microg ml(-1) and recoveries of 98.9-100.5%. Correlation coefficients for calibration curves in the range of 5-50 microg ml(-1) were greater than 0.999 for all tetracyclines. The within- and between-day precision was determined for both retention times and peak area. It is suggested that the proposed HPLC-PGC method should be used for routine quality control and dosage form assay of tetracyclines in pharmaceutical preparations. The chromatographic behaviour of the five tetracyclines was examined under variable mobile phase compositions, the results revealed that elution order and selectivity were dependent on the buffer agent used. Comparison between retentions obtained with PGC and with silica-based stationary phase (ODS), showed similar variations of the capacity factors with the mobile phase composition, but with a different elution order.