RP-HPLC BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF METFORMIN AND LEVOTHYROXINE IN HUMAN PLASMA- DRUG INTERACTION STUDIES

A simple, sensitive, robust and specific high performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous determination metformin and levothyroxine in human plasma. In the current study, the analysis was performed on Phenomenex Luna 5µ C18 100A (250 x 4.6mm, 5 micron) column using acetonitrile: methanol: 0.03M phosphate buffer (pH 2- 3.5) 50:10:40 v/v/v as mobile phase at flow rate 1.0 mL/min. The analytes were monitored with UV-PDA detector at 251nm. In this developed method Metformin and Levothyroxine elutes at a retention time of 2.698 and 5.929 min respectively. The proposed method is having linearity in the concentration range from 5 to 50μg/mL of Metformin and Levothyroxine. The current method was validated with respect to linearity; precision, lowest limit of detection (LOD) and lowest limit of quantification (LOQ), accuracy and recovery according to the USP guidelines. The system consisted of a pump (Shimadzu, prominence, HPLC), with 20µl sample injector, along with a PDA detector at a wavelength of 251nm. Data was compiled using Shimadzu LC Solution software. A good linear relationship over the concentration range of 5-50µg/ml was shown. Validation of the method was carried out as per the USFDA. The method developed was found to be precise, accurate, specific and selective. Statistical analysis shows that the method is reproducible and selective for the estimation of Metformin and Levothyroxine in dosage form.