Determination of Diltiazem HCL by Reverse Phase High Performance Liquid Chromatography in Rabbit Plasma

Background: A precise, accurate, rapid and sensitive reverse phase liquid chromatographic (RP-HPLC) method has been developed in rabbit plasma spiked with Diltiazem hydrochloride (DTZ). Methods: The chromatographic method was standardized using an Agilent 5 TC- C18 column (2) 250 mm x 4.6 mm with UV detection at 237 nm and flow rate of 1 ml/min. The mobile phase consisted of methanol: water (90:10: v/v). The retention time for the Diltiazem hydrochloride was found to be 4.2 min. In spiked rabbit plasma, drug was extracted by using acetonitrile as disperser solvent for protein precipitation and the supernatant was mixed with100 mL dichloromethane as an extraction solvent. After centrifugation, this demented phase containing drug was collected and evaporated to dryness. The residue was re-dissolved in50 uL of mobile phase and injected into the HPLC system for analysis. Good linearity was obtained for the calibration curve of Diltiazem hydrochloride was found to be R2= 0.9999. Results: The intra- and inter-days precision and accuracy of three selected concentrations (25, 50, 100ng ml-1) showed % RSD within the range and excellent % recovery. The limit of detection (LOD) and limit of Quantification (LOQ) values were 1.325ng ml-1 and 4.02ng ml-1, respectively. Conclusion: The developed HPLC method for the estimation of Diltiazem HCL in rabbit plasma would be more accurate, precise, sensitive and selective.