An improved assay by HPLC with amperometric detection for the determination of phentolamine in plasma

An improved method for the determination of phentolamine in human plasma samples was developed. After being alkalinized, plasma samples (1 mL) were extracted with diethyl ether and then back-extracted with O.1 N HCl. Analyses were carried out on a Novapak C8 column eluted with a mixture of sodium monochloroacetate (pH 3) and acetonitrile (75:25). Amperometric detection was performed by oxidation at 1000 mV, using a glassy carbon electrode against Ag/AgCl. Calibration curves, constructed over a 1 to 30 ng/mL plasma concentration range, were linear (r=0.999). Intra-assay coefficients of variation and accuracy for the determined concentrations were comprised within 7.6-10.9% and 94.0-105.6%, respectively. Inter-assay coefficient of variation and accuracy ranges were 10.4-20.7% and 93.2-102.7%, respectively. The method's detection limit was 0.2 ng/mL, allowing determination of oral phentolamine pharmacokinetics after administration of a 40 mg dose. It is concluded that the present procedure is suitable for pharmacokinetic and bioavailability studies of oral phentolamine formulations presently used in the treatment of erectile dysfunction.