Development of a rapid extraction and high-performance liquid chromatographic separation for amitriptyline and six biological metabolites.
The development of a rapid high-performance liquid chromatographic method for the determination of amitriptyline, amitriptyline-N-oxide, 10-hydroxyamitriptyline, 10-hydroxynortriptyline (E and Z isomers), nortriptyline and desmethylnortriptyline in plasma and liver tissue is described. A liquid--liquid extraction with hexane--butanol and back-extraction into phosphoric acid provides efficient extraction of amitriptyline-N-oxide along with amitriptyline and the other metabolites. A Supelcosil C8 reversed-phase column with 5-micron packing and a methanol--sodium phosphate buffer--amine modifier mobile phase was used. The combination of mobile phase pH and amine modifier concentration for the best separation within a reasonable analysis time for all seven solutes plus an internal standard was determined using a factorial design coupled with a multi-factor window diagram technique. Ultraviolet detection at 214 nm provided limits of detection of approximately 1 ng/ml.
D000639:Amitriptyline / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D051381:Rats; D013056:Spectrophotometry, Ultraviolet
D000639:Amitriptyline