Liquid chromatography/mass spectrometry methods for distinguishing N-oxides from hydroxylated compounds

This study describes the application of liquid chromatography/mass spectrom etry (LC/MS) methods for distinguishing between aliphatic and aromatic hydr oxylations 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 gene rate ions from liquid chromatographic effluents. ESI-MS, ESI-MS/MS, APCI-MS , and APCI-MS/MS experiments were performed on several metabolites and deri vatives of loratadine (a long-acting and nonsedating tricyclic antihistamin e) using an ion trap mass spectrometer (LCQ) and a triple-quadrupole mass s pectrometer (TSQ), The observations are as follows: (1) LC/ESI-MS produced predominantly [M + H](+) ions with minor fragmentation. (2) LC/ESI-MS/MS da ta, however, showed a predominant loss of water from metabolites with aliph atic hydroxylation while the loss of water was not favored when hydroxylati on was phenolic, N-Oxides (aromatic and aliphatic) showed only a small amou nt of water loss in the MS/MS spectra, (3) Under LC/APCI-MS conditions, ali phatic hydroxylation could be readily distinguished from aroma;tic hydroxyl ation based on the extent of water loss. In addition, N-oxides produced dis tinct [M + H - O](+) ions. These [M + H - O](+) ions were not produced in t he APCI-IMS spectra of hydroxylated metabolites, (4) Similar to the ESI-MS/ MS spectra, the APCI-MS/MS spectra hom the (M + H)(+) ions of N-oxides yiel ded 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 me tabolites and N-oxides.