High-performance liquid-chromatographic simultaneous determination of commonly used tricyclic antidepressants

Abstract

We report a method for simultaneous measurement of five commonly used tricyclic antidepressant drugs (doxepin, desipramine, nortriptyline, imipramine, and amitriptyline) in serum by paired-ion high-performance liquid chromatography, with use of a reversed-phase column and ultraviolet detection at 254 nm. The drugs are extracted from 2 ml of serum at pH 14 into hexane/isoamyl alcohol (99/1 by vol) and re-extracted into 200 microliter of 0.1 mol/liter HCI. An aliquot of the aqueous acid phase is chromatographed with use of a methanol/acetonitrile/water (41/15/44) solvent system, containing 5 mmol of pentanesulfonic acid per liter of phosphate buffer (0.1 mol/liter, pH 6.5), at a flow rate of 1,5 ml/min. Analytical recoveries of the drugs from serum increase with increasing concentration, from 62% at 25 microgram/liter to 93% at 300 microgram/liter. Linear response is observed for drug concentrations up to 1500 microgram/liter and the detection limit is 2-3 microgram/liter. Within-run precision ranges from 1.4 to 2.9% and day-to-day precision from 1.7 to 7%, depending on the specific drug. The entire procedure can be completed within 45 min and is well adapted to the routine clinical laboratory. Of 48 common basic and several neutral drugs tested for possible interferences, only three benzodiazepines, three phenothiazines, and three antihistamines interfere with the assay of doxepin, desipramine, and nortriptyline, respectively.