High Performance Liquid Chromatography

A modified liquid- liquid extraction based reverse phase high performance liquid chromatographic method has been developed and validated for the determination of Furosemide in human plasma. The chromatographic separation was achieved with Pinnacle C18 column (250 x 4.60 mm, 5μm i.d.) and a mobile phase comprising of a mixture of Methanol: Water: Triethylamine (70:30:0.1v/v/v), pH adjusted to 3.2 with orthophosphoric acid was used with UV detection at 274nm. The internal standard spironolactone structurally related to Furosemide was used. The retention time for Furosemide and spironolactone was found to be 3.75min and 8.12min respectively.  A calibration curve was linear in the concentration range of 200-2200ng/ml for Furosemide in blank human plasma. The % recovery from human plasma was found to be in the range of 89.41 to 93.91 for Furosemide. The lower limit of quantitaion was found to be 200ng/ml and no interference was found from endogenous compound. The specificity, linearity, accuracy, precision and stability of the method were evaluated from spiked human plasma sample as per EMA (European Medicine Agency) guideline. The method can be used for supporting therapeutic drug monitoring and pharmacokinetic studies.