A sensitive validated method has been developed for the quantification of Nadolol in rat plasma by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) using deuterated Nadolol (Nadolol D9) as internal standard (IS). The liquid-liquid extraction method using ethyl acetate was employed for the sample pretreatment. The separation was achieved on the Agilent Zorbax XDB C18 column (150 mm × 4.6 mm ID., 3.5 μm). The column temperature was controlled at 30°C. The components were eluted by using mobile phase A (10 mM ammonium formate) and mobile phase B (acetonitrile) in the ratio of 20:80 v/v with a flow rate of 0.5 mL/min. And 15 μL aliquot was injected in an isocratic elution mode with a total run time of 2.5 min. The multiple reactions monitoring transitions, m/z 310.20/254.10 for Nadolol and IS 319.20/255.00 were selected to achieve high selective analysis. The method exhibited great selectivity and linearity over the concentration range of 6 to 3000 ng/mL. The lower limit of quantification was found to be 6 ng/mL. The developed method proved acceptable results on selectivity, sensitivity, precision, accuracy, and stability studies as per Food and Drug Administration guidelines. This HPLC-MS/MS assay was successfully applied to get the pharmacokinetics parameters in rat plasma.
Keywords: HPLC-MS/MS; deuterated internal standard; liquid–liquid extraction; pharmacokinetic analysis; rat plasma; validation.