High-performance liquid chromatographic determination of phenylephrine and its conjugates in human plasma using solid-phase extraction and electrochemical detection.

An HPLC method for the determination of phenylephrine and its conjugates in human plasma was developed and validated. The method for quantitation involved extraction of diluted plasma (subject to hydrolysis with beta-glucuronidase for 30 min with 500 units of enzyme per 0.1 ml of plasma at 37 degrees C for the conjugates) on solid-phase weak cation-exchange cartridges followed by elution of the analyte and the internal standard (ethylnorphenylephrine) with 5% triethylamine in methanol. Analysis was carried out on a 15 cm ODS stationary phase using ion-pair reversed-phase chromatography. An electrochemical detector operated at + 1.15 V vs. Ag/AgCl was employed for detection. The standard curves were linear in the range 1.0-50.0 ng ml-1 for phenylephrine and 25.0-500.0 ng ml-1 for phenylephrine obtained from its conjugates. The limit of quantitation was 2.0 ng ml-1 (RSD = 17%) and 25.0 ng ml-1 (RSD = 18%), respectively. Acceptable accuracy and precision were obtained during intra- and inter-batch analyses for both the assays.