A new method for simultaneous determination of cyclic antidepressants and their metabolites in urine using enzymatic hydrolysis and fast GC-MS

Abstract

A method for the simultaneous determination of six commonly prescribed cyclic antidepressants and their major metabolites in urine is presented. This method can be used for quantitation of amitriptyline, nortriptyline, imipramine, desipramine, doxepin, desmethyldoxepin, and maprotiline in human urine, in addition to the qualitative determination of their hydroxylated metabolites. This method is suitable for confirmation of drug abuse in health care professionals and overdose cases where the identity of the abused cyclic antidepressant may not be known. Samples are spiked with internal standard and hydrolyzed with ??-glucuronidase from Escherichia coli. Hydrolysis is found to be essential to the extraction procedure as the tertiary cyclic antidepressants are found to be extensively conjugated in urine. The secondary cyclic antidepressants, on the contrary, are found to be minimally conjugated. Drugs are extracted from alkalinized urine into solvent and derivatized with MSTFA/ammonium iodide/ethanethiol reagent. This reagent produces more stable derivatives compared to reagents previously employed. Gas chromatographic (GC)-mass spectrometric analysis is performed in electron ionization mode by selective ion monitoring, using hydrogen as a carrier gas, a short narrow bore GC capillary column, and fast temperature program, allowing for a rapid analytical cycle. While maintaining specificity for these drugs, concentrations in human urine ranging from 50 to 20,000 ng/mL can be measured with intraday and interday precisions, expressed as variation coefficient, of less than 2.8% for all analytes.