A NOVEL, SENSITIVE, BIOANALYTICAL METHOD FOR ESTIMATION OF AMLODIPINE BESYLATE IN RAT PLASMA USING FLOURESCENCE DETECTION BY RP-HPLC

The aim of the present work was to develop and validate a novel, rapid, sensitive, accurate and specific RP-HPLC assay with fluorescence detection for the quantification of amlodipine besylate in rat plasma to be applied to preclinical studies. Flourescein was used as internal standard (IS) and the detection was carried out by an online wavelength switching which enabled detection of amlodipine & flourescein in the same method. The plasma proteins were precipitated by a single step protein precipitation extraction procedure using methanol. Mean recovery of amlodipine besylate from rat plasma was more than 85.0 % for 25 – 5000 ng/ml concentrations. The assay exhibited good linear relationship with an r² of 0.999. Lower limit of Quantification limit (LLOQ) was 1.84 µg/ml of amlodipine besylate and accuracy and precision were over the concentration range 25 – 5000 ng/ml. Thus, the bionanalytical method for quantification of amlodipine besylate is novel, since use of fluorescence detection & using flourescein as the internal standard has made the method with comparable sensitivity to LC-MS/MS methods. The proposed method is convenient for routine determination of amlodipine besylate in rat plasma and quality control laboratories that require an economic and rapid method.