Liquid Chromatography/Mass Spectrometry Methods for Distinguishing N-Oxides from Hydroxylated Compounds

Abstract

This study describes the application of liquid chromatography/mass spectrometry (LC/MS) methods for distinguishing between aliphatic and aromatic hydroxylations and between hydroxylations and N-oxidations. Hydroxylations and N-oxidations are common biotransformation reactions of drugs. Electrospray (ESI) and atmospheric pressure chemical ionization (APCI) were used to generate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS, and APCI-MS/MS experiments were performed on several metabolites and derivatives of loratadine (a long-acting and nonsedating tricyclic antihistamine) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass spectrometer (TSQ). The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H]+ ions with minor fragmentation. (2) LC/ESI-MS/MS data, however, showed a predominant loss of water from metabolites with aliphatic hydroxylation while the loss of water was not favored when hydroxylation was phenolic. N-Oxides (aromatic and aliphatic) showed only a small amount of water loss in the MS/MS spectra. (3) Under LC/APCI-MS conditions, aliphatic hydroxylation could be readily distinguished from aromatic hydroxylation based on the extent of water loss. In addition, N-oxides produced distinct [M + H - O]+ ions. These [M + H - O]+ ions were not produced in the APCI-MS spectra of hydroxylated metabolites. (4) Similar to the ESI-MS/MS spectra, the APCI-MS/MS spectra from the (M + H)+ ions of N-oxides yielded a small amount of water loss but no [M + H - O]+ ions. These results indicate that LC/APCI-MS can be used to distinguish between hydroxylated metabolites and N-oxides.

Flavin-Containing Monooxygenase Activity in Hepatocytes and Microsomes: In Vitro Characterization and In Vivo Scaling of Benzydamine Clearance

“…2). Whereas other metabolites were apparently formed from P450 and UGT activity, it is clear that It is well known that N-oxide metabolites can undergo in-source breakdown to the parent amine under some mass spectrometry conditions (Ramanathan et al, 2000;Tong et al, 2001). If this occurred with benzydamine N-oxide, it would complicate the determination of benzydamine lability and its quantitation in various matrices.…”

“…1) It is unknown if enterohepatic recirculation of benzydamine occurs in humans, although N-oxides and glucuronides are primary candidates for recirculation due to enzymes in gut flora with potent reductase and glucuronidase activity, respectively (Rowland and Tozer, 1995;Cashman, 1999). 2) The mass spectral source-induced and/or thermal deoxygenation of N-oxides is well documented and has recently been studied as a diagnostic tool for N-oxide structural assignment (Ramanathan et al, 2000;Tong et al, 2001). Mass spectral source-induced decomposition could easily result in misquantitation of N-oxide metabolite as unchanged parent compound in pharmacokinetic studies and thus an artificially increased area under the curve for parent and reduced clearance value.…”

Persistence of the tricyclic antidepressant drugs amitriptyline and nortriptyline in agriculture soils

“…The E-2-OH-nortriptyline follows E-10-OH-nortriptyline in abundance as a nortriptyline metabolite in rat bile and also occurs in rabbit urine [17]. There is a predominant loss of water from metabolites with aliphatic hydroxylation, while the loss of water was not favored when hydroxylation was phenolic [27]. Aliphatic hydroxylation could be distinguished readily from aromatic hydroxylation based on the extent of water loss [27].…”

Metabolism and Excretion of Capravirine, a New Non-Nucleoside Reverse Transcriptase Inhibitor, Alone and in Combination With Ritonavir in Healthy Volunteers

“…ϩ under atmospheric pressure chemical ionization (APCI) conditions (Ramanathan et al, 2000;Tong et al, 2001). This methodology was used to identify N-oxide metabolites of capravirine in humans.…”