A quantitative determination of penbutolol and its metabolite in plasma by liquid chromatography tandem mass and its application to pharmacokinetic studies

Abstract

A Simple sensitive and specific tandem mass spectrometric (LC-MS/MS) method for the determination of penbutolol (PEN) and its metabolite 4-Hydroxy penbutolol (4HPEN) in human plasma was developed and validated. The detection of the analytes was achieved in positive ion in selected reaction monitoring (SRM) mode. The deuterated compounds of the analytes were used as internal standards (IS). The extraction procedure involved solid phase extraction of PEN, 4HPEN and IS from plasma by using Strata-X cartridges. The chromatographic separation of PEN, 4HPEN and IS was carried out on a Chromatopak C₁₈ column with 5mM ammonium acetate (pH 4.5) buffer and acetonitrile (15:85, v/v) as the mobile phase under isocratic conditions at a flow rate of 0.6 mL/min. The nominal retention times obtained for PEN, 4HPEN, DPEN and D4HPEN were 2.42, 1.98, 2.40 and 2.00 minutes respectively. The lower limit of quantification for PEN and 4HPEN were 0.2 and 0.1 ng/mL respectively. The standard curves were linear (r²>0.99) over the concentration range of 0.2-302.7 ng/mL for PEN and 0.1-30.0 ng/mL for HPEN. Method validation was performed as per FDA guidelines and the obtained results met the acceptance criteria. The proposed method was found to be acceptable to a pharmacokinetic study in human volunteers.