Enantioselective HPLC Determination of R and S Trimipramine in Human Serum Using an Octyldecylsilane Column with β-Cyclodextrin as Mobile Phase Additive and Solid Phase Extraction

Abstract

A stereospecific HPLC method was developed for the analysis of the enantiomers of trimipramine in human serum. The assay uses amitriptyline as the internal standard and a C18 solid phase extraction column for serum sample clean-up. It is free of interference from desmethyl-trimipramine, 2-hydroxy-desmethyl-trimipramine and 2-hydroxy-trimipramine, the three major metabolites of trimipramine. Recoveries of 98.8% and 97.5% were obtained for the R and S enantiomers of trimipramine, respectively. Resolution of the enantiomers was obtained using an octyldecylsilane column with β-cyclodextrin as the mobile phase additive. The composition of the mobile phase was 80:20 v/v aqueous 10mM ammonium acetate buffer pH 4 (adjusted with acetic acid)- absolute ethanol containing 20mM β-cyclodextrin at a flow rate of 0.7mL/min. Linear calibration curves were obtained in the 25-400ng/mL range for each enantiomer in serum. The detection limit based on a S/N = 3 was 10ng/mL for each enantiomer in serum with UV detection at 220nm. The limit of quantitation for each enantiomer was 25ng/mL. Precision calculated as %RSD and accuracy calculated as % error were in the range 0.7–4.5% and 0.9–3.1%, respectively, for the R enantiomer and 0.7–5.1% and 0.4–4.4% respectively, for the S enantiomer. Separation of the three major metabolites of trimipramine was also investigated.