Novel Eco-friendly HPLC Methods Using Refractive Index Detector for Analysis of Three Veterinary Antibiotics in Pharmaceutical Formulations and Rat Plasma

Antibiotic resistance increases the human mortality rate nowadays. The main purpose of the present study was to develop green reversed-phase high-performance liquid chromatography (RP-HPLC) methods with a refractive index detector for the assay of the three veterinary antibiotics (VAs), i.e., maduramicin ammonium (MA), apramycin sulfate (AS) and clarithromycin (CLA) in pharmaceutical dosage forms and spiked rat plasma. The method utilized isocratic elution using an ODP-40 C18 column, the flow rate was set at 1.0 mL/min and negative polar signals. The linearity ranges were 3.0–18.0 μg/mL for MA, 1.5–4.0 μg/mL for AS and 0.5 to 3.0 μg/mL for CLA, respectively. Liquid-liquid extraction (LLE) procedure was optimized in plasma samples. The recoveries percentages were 85.4, 81.2 and 88.8 correspondingly, in rat plasma. However, the drugs extraction by protein precipitation method yields very poor recoveries (around 50%). The new HPLC- refractive index (RI) methods are better than the previously reported HPLC-ultra violet methods in terms of greenness and simplicity of procedures. Moreover, the previously reported LC–MS methods lack the simplicity and availability of such expensive techniques in Quality control (QC) labs. The novelty of this research is the use of refractive index detector for the first time for VAs analysis.