HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR THE QUANTITATION OF NADOLOL IN HUMAN PLASMA USING FLUORESCENCE DETECTION

A rapid and sensitive HPLC-fluorescence assay was developed and validated for the determination of nadolol, a beta-blocker, in human plasma, Nadolol and the internal standard (desmethyl nadolol) were extracted from alkalinized plasma into methyl-tert.-butyl ether, The organic solvent was evaporated under nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected on to a C-18 silica column (25 cm x 4.6 mm i.d.) at a flow rate of 1.4 mL/min. The mobile phase was 0.05 M monobasic ammonium phosphate (pH 4.2) and acetonitrile (84:16, v/v), Fluorimetric detection was performed at excitation 230 nm and emission 330 nm, The nominal retention times were 3.3 and 4.3 min for the internal standard and nadolol, respectively, The lower limit of quantitation was 5 ng/mL and linearity (R(2) greater than or equal to 0.994) of the standard curve was demonstrated between 5 and 500 ng/mL, The analysis of quality control (QC) samples at 60, 200 and 400 ng/mL resulted in precision estimates less than or equal to 7.0% relative standard deviation (RSD) for the inter-assay and less than or equal to 6.3% RSD for intra-assay, The predicted concentrations of the QC samples deviated < 10% from the nominal values. The extraction recovery of nadolol from human plasma was 64%, Nadolol was stable in human plasma at -20 degrees C for at least 5 months and for at least three freeze-thaw cycles, Nadolol and the internal standard were stable in the autosampler at 5 degrees C for at least 40 h, Overall, the assay was accurate, precise, sensitive, specific and reproducible for the analysis of nadolol in plasma, The validated assay procedure was applied to study the pharmacokinetics of nadolol after administration of single and multiple doses to human subjects.