Liquid-chromatographic determination of amitriptyline and its metabolites in serum, with adsorption onto glass minimized

Abstract

To study correlations between the concentrations, in serum, of amitriptyline and its most important metabolites with clinical response in patients, we developed a 'high-performance' liquid-chromatographic method for routine determination of amitriptyline, nortriptyline, total 20-hydroxy-amitriptyline, desmethylnortriptyline, and E(trans)- and Z(cis)-10-hydroxynortriptyline. These compounds are extracted from 1 mL of alkalinized serum to hexane/isoamyl alcohol (99/1 by vol). Perazine is the internal standard. To minimize irreversible adsorption of the drugs onto the glassware, 5 μg of maprotiline is added to the organic phase just before evaporation. After a 10-min resolution on a silica column eluted with acetonitrile/methanol NH4OH (1 mol/L), absorbance is measured at 240 nm. Only chlorimipramine, doxepin, procainamide, and N-acetylprocainamide may interfere with assay of the compounds that probably are therapeutically relevant: amitriptyline, nortriptyline, and E-10-hydroxynortriptyline. Uremia, lipemia, and icterus also do not affect the analysis.