Development of an HPLC-PDA Method for the Simultaneous Estimation of Three Antibiotics in Pharmaceutical Formulations and Bovine Milk and Health Risk Assessment

A simple new, rapid, sensitive and cost-effective HPLC–PDA method was developed and validated for the determination of tetracycline (TC), oxytetracycline (OTC) and ciprofloxacin (CIP) simultaneously. Chromatographic separations were carried out using a reversed-phase Shim-pack GIS C18 column (4.60 × 250.00 mm; 5.00 µm) at 30°C. Oxalic acid (0.05 M), acetonitrile and methanol were used as mobile phase under gradient elution conditions at the flow rate 1.50 mL min−1. Detection wavelength was set at 330 nm. Aliquot of 20.00 µL solution was injected and three drugs were eluted within 7.39±0.05 min. As per ICH guidelines linearity, recovery, accuracy, precision, selectivity, specificity, sensitivity, stability, column efficiency, system suitability and robustness were determined for the validation of the proposed method. Calibration curves were linear over a studied concentration range of 8.00 µg mL-1 with correlation coefficient (r) 0.999 for all drugs. Relative standard deviation (RSD) for intra and inter day precision was found less than 2.87% and 3.22%, respectively indicating method to be reproducible. The proposed method has been suitably applied for the estimation of TC, OTC and CIP in pharmaceutical formulation and milk samples collected from local market in Bangladesh. Among fifteen milk samples analyzed, most of the cases (more than 50%) TC, OTC and CIP were detected above maximum residue level (MRLs) though no significant toxicological effect on the health of consumers in the study area was identified.