Development and validation of LC-MS/MS method for the simultaneous estimation of tenofovir disoproxil fumarate and pioglitazone HCL in human plasma

For the simultaneous detection of tenofovir and pioglitazone in a human plasma LC-MS/MS technique was utilized. API-4000 mass spectrometer was used. It should be operated in positive ion mode and contains ion source of turbo spray. Inverted C18 column was used which produces good resolution and symmetrical peaks. Metoprolol is used as internal standard. It requires acidic PH for elution but  Tenofovir disoproxil fumarate and Pioglitazone requires neutral PH after extraction which is equity diluted with H2O for supernate. Finally, it provides good resolution and elution. By using Acetonitrile 100mg of Tenofovir Disoproxil fumarate and Pioglitazone HCL were dissolved and the final volume is made with H2O and Acetonitrile in 1:1 ratio to give 1mg/ml. standard stock solution are stored at -2to ± 20 C till analysis is completed. The guidelines were provided by the FDA to fulfill the essentials requires for the technique. To regulate the concentration the procedure starts with standard curve which is built form the quantitative analysis standard ranges form 1-1000ng/mL. To determine the stability freeze-thaw technique were used. To determine the stability the analyte is kept at room temperature for 24 hours. The correlation coefficients for all calibration curves were more than 0.99.