High-pressure liquid chromatographic determination of amitriptyline and its major metabolites in human whole blood.

A sensitive, specific, high-pressure liquid chromatographic method using an internal standard was developed for the determination of amitriptyline and its major metabolites in whole blood. Analysis was carried out on a microparticulate silica column with a mobile phase consisting of acetonitrile-methanol-aqueous ammonium hydroxide (93:7:0.4). Linear calibration curves ranging to 250 ng/ml were obtained for all compounds using UV absorbance detection at 220 nm. The lower limit of detection was 2 ng/ml for amitriptyline and 10-hydroxyamitriptyline, and 6 and 16 ng/ml for nortriptyline and its 10-hydroxylated metabolite, respectively. Human whole blood samples collected after single intravenous and single oral doses can be analyzed using this procedure.