Development and validation of UV-Spectrophotometric and RP-HPLC method for simultaneous estimation of Metformin and Doxycycline in bulk and synthetic mixture

A simple, rapid, sensitive, accurate and precise UV spectrophotometric and isocratic RP-HPLC method have been developed for simultaneous estimation of Metformin and Doxycycline in bulk and synthetic mixture. Spectrophotometric estimation was done by two methods. First method was Q-absorbance ratio method, where two wavelengths 236 nm (λmax of Metformin) and 248 nm (Iso-absorptive point) were used. The second method was first derivative method. In this method the zero-crossing point of Metformin was selected at 282 nm and for Doxycycline, it was 232 nm. The solvent used was methanol in both the above UV-spectrophotometric methods. Metformin and Doxycycline showed good linearity in the series of 1-9 µg/ml and 2-20 µg/ml respectively by both the two methods with an excellent correlation coefficient (r2≥0.998). In RP-HPLC method, the chromatographic separation was achieved on Luna Phenomenex C18 (150 mmХ6 mm, 5 µm) analytical column. A mixture of Acetonitrile: Phosphate buffer (50 mM): Triethylamine (TEA): Tetrahydrofuran (THF) (30:66:2:2) pH adjusted to 2.1 with orthophosphoric acid was used as the mobile phase, at a flow rate of 1 ml/min and at detector wavelength 248 nm. The retention time of Metformin and Doxycycline was found to be 3.561±0.0017 and 5.574±0.0131 minutes respectively. A linear response was observed over the concentration range 4-64 µg/ml of Metformin and 5-80 µg/ml of Doxycycline. All the three methods were validated in accordance to ICH guidelines for linearity, accuracy, precision, LOD and LOQ. The proposed methods were effectively utilized for the concurrent estimation of Metformin and Doxycycline in synthetic mixture. Keywords: Metformin, Doxycycline, Q-absorbance ratio method, First derivative method, RP-HPLC