A method for the determination of total phenylephrine (free plus conjugated) in human serum was developed using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. After serum was deproteinized with acetonitrile, the conjugated phenylephrine was hydrolyzed with diluted hydrochloric acid. The solution was evaporated to dryness. The reconstituted residue was analyzed with HPLC using a heart-cut technique. Good recoveries of the analytes from spiked human serum samples were obtained with small coefficients of variation. A good linear response was obtained for the concentration range 5-500 ng/ml. The lower limit of quantitation for phenylephrine in human serum was 5 ng/ml. The method was applied to the determination of phenylephrine in human serum after oral administration of phenylephrine hydrochloride.
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