Development of a simple HPLC method for separation of doxycycline and its degradation products

A simple HPLC method for the separation of doxycycline and its degradation products 6-epidoxycycline and metacycline was developed. Numerous HPLC conditions were tested for the qualitative determination of doxycycline and its degradation products. The best result was achieved by using Phenomenex® Luna 5 μm C8 250×4.6 mm column with a Phenomenex® C8 4×10 mm I.D. guard column, and a mobile phase consisting of acetonitrile:water:perchloric acid (HClO4) (26:74:0.25) adjusted to pH 2.5 with 5 M sodium hydroxide, a flow-rate of 1.0 ml/min and ultraviolet detection at 350 nm. Correlation coefficients for calibration curves within the detection range of 3–60 μl/ml were 0.9990 for doxycycline and 1.000 and 0.9994 for 6-epidoxycycline and metacycline, respectively (within the range 0.5–7 μl/ml). The resolution between metacycline and 6-epidoxycycline was 1.2 and between 6-epidoxycycline and doxycycline it was 1.9 which fulfils European Pharmacopoeia requirements. The within- and between-day precision was determined for both retention time and peak area. Preliminary results indicate that this method can also be applied for separating other tetracyclines such as minocycline, chlortetracycline, tetracycline and demeclocycline.