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 sa mples was developed. After being alkalinized, plasma samples (1 mL) were ex tracted 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 mo nochloroacetate (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 conce ntration range, were linear (r=0.999). Intra-assay coefficients of variatio n and accuracy for the determined concentrations were comprised within 7.6- 10.9% and 94.0-105.6%, respectively. Inter-assay coefficient of variation a nd accuracy ranges were 10.4-20.7% and 93.2-102.7%, respectively. The metho ds detection limit was 0.2 ng/mL, allowing determination of oral phentolami ne 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 use d in the treatment of erectile dysfunction.