Cyanide Trapping of Iminium Ion Reactive Intermediates Followed by Detection and Structure Identification Using Liquid Chromatography−Tandem Mass Spectrometry (LC-MS/MS)

Abstract

Secondary and tertiary alicyclic amines are widely found in pharmaceuticals and environmental compounds. The formation of iminium ions as reactive intermediates in the metabolic activation of alicyclic amines has previously been investigated in radiometric assays where radiolabeled cyanide is typically employed. In this paper, we report a relatively high throughput LC-MS/MS method for the detection of the nonradiolabeled cyanide adduct formed in rat or human liver microsomal incubations via constant neutral loss scan followed by structural characterization using product ion scan on a triple quadrupole mass spectrometer. A total of 14 alicyclic amine compounds were investigated with the cyanide trapping LC-MS/MS screen and also with the glutathione (GSH) trapping screen, a well-established and commonly employed technique for reactive metabolite screening. Our results are found to be in general agreement with the previous metabolism reports for these compounds, demonstrating the effectiveness, speed, and simplicity of the cyanide trapping LC-MS/MS method to study the iminium ion intermediates from alicyclic amines and its complementarities to GSH trapping method for reactive metabolite screenings.

Comparison of the Bioactivation Potential of the Antidepressant and Hepatotoxin Nefazodone with Aripiprazole, a Structural Analog and Marketed Drug

“…The 14 mass units appear to be consistent with an additional enzymatic step involving oxidation of the piperazine ring to the corresponding lactam metabolite. An identical finding was also reported by Argoti et al (2005) in their characterization of the cyanide adducts of N-dearylated nefazodone metabolites in rat liver microsomes. Thus the molecular weight of CN2 (MH ϩ ϭ 399; m/z ϭ 274, 246) was consistent with the addition of one molecule of cyanide to the N-dearylated nefazodone M3 (see Table 1) plus the 14-Da mass addition to the piperazine ring.…”

“…Our present studies on nefazodone metabolism in human liver microsomes and recombinant P4503A4 revealed additional bioactivation pathways involving ␣-carbon oxidation on the piperazine ring in nefazodone and several of its downstream metabolites, yielding reactive iminium ion intermediates. Argoti et al (2005) have also reported a similar finding following nefazodone incubations with rat liver microsomes. Overall, the observations that nefazodone bioactivation by P4503A4 results in the mechanism-based inactivation of the isozyme presumably via covalent modification of an active site nucleophile(s) by reactive quinonoid and/or iminium intermediates (Kalgutkar et al, 2005b) suggests a possibility that these metabolites also can covalently modify macromolecules other than P450, some of which may be essential for critical pathophysiology in the liver.…”

“…Additionally, reactive iminium ion intermediates arising from ␣-carbon oxidation (adjacent to the piperazine ring nitrogen) in nefazodone and its downstream metabolites also have been characterized in rat liver microsomes as the corresponding cyano conjugates ( Fig. 1) (Argoti et al, 2005).…”

“…Nefazodone was included as a positive control in these studies. Special emphasis was placed on the characterization of reactive iminium species of nefazodone in human liver microsomes, since previous information on this bioactivation pathway was limited to the rat (Argoti et al, 2005). Studies comparing the inhibition (reversible and time-dependent) of P450 enzymes involved in aripiprazole and nefazodone metabolism were also undertaken, since time-dependent, irreversible inactivation can also be caused by covalent modification of P450 enzyme(s) that catalyze bioactivation.…”

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure–activity relationship, in vivo evaluation, mechanistic and bioactivation studies

“…20 The trapping reagents used include glutathione (for soft electrophiles such as the quinone imine) as well as methoxylamine and potassium cyanide (for hard electrophiles such as the iminium ion). [21][22][23] Electrochemical oxidation online with electrospray ionization mass spectrometry (EC-ESI/MS) is a relatively new technique that avoids the complexity of working with biological matrices. Electrochemical oxidation has been found to successfully mimic CYP450 benzylic hydroxylation, hydroxylation of aromatic rings containing electron-donating groups, Ndealkylation, S-oxidation, dehydrogenation and, less efficiently, N-oxidation and Odealkylation.…”