Method development and validation of Metformin HCL in human plasma using LC-ESI-MS/MS
Student name: Mr Harsh Pratap Singh
Guide: Dr Chaitanya Madhurantakam
Year of completion: 2016
Host Organisation: Cadila pharmaceuticals, Ahmedabad
Supervisor (Host Organisation): Mr Niraj Patel
Abstract: A simple, rapid & sensitive liquid chromatography tandem MS (MS/MS) assay method has been developed and fully validated for the simultaneous quantification of metformin(anti diabetic drug) in human plasma.
metformin D8 was used as internal standard. A complete and stepwise trials were done for the selection of different solution(diluents, buffer ,mobile phase ,seal wash solution, weak wash solution, precipitating agent and strong wash solution) column and different chromatographic condition(run time, flow rate, cone temperature ,auto sampler temperature ,column temperature, cone gas flow, collision gas flow T-wave parameters etc.
after reviewing previous study we used protein-protein precipitation method as extraction method and found result in acceptance criteria.
The analytes were extracted from 200 micro liter aliquots of human plasma via protein-protein precipitation method using acetonitrile as precipitating agent. The reconstituted samples were chromatographed on a Luna 5 micron CN 100 A(100*4.6MM) by using a 50:50 ration(2mM ammonium formate in water : acetonitrile) as the mobile phase at a flow rate of 0.600ml/min and pH was set to 3.5 .
The calibration curve were linear (r2>=0.99) over the concentration range of (12.14-1207ng/ml ) for metformin.
The waters acuity LC-MS/MS in multi reaction monitoring (MRM) mode was used for the detection. volume of injection was 8micro liter, mass detector we used, ion source was electron spray ionization (ESI).
The results of the intra and inter day precision and accuracy studies were well within the acceptable limits. all the analytes were found to be stable in a battery of stable studies.
The method is precise & sensitive enough for intended purpose. a run time of 6min for each sample.The main objective we achieved that we developed and validated analytical method for the estimation of Metformin in human plasma with due consideration of rapidity ,economy ,selectivity, according to USFDA guideline which can detect the concentration of drug up to Nano gram level.
