Isomer differentiation of hydroxypyridine N-oxides using metal complexation and electrospray ionization mass spectrometry

BUTLER M.; ARROYO MAÑEZ P.; CABRERA G. M.

Salt Lake City, Utah

Conferencia; 58th ASMS Conference on Mass Spectrometry and Allied Topics; 2010

American Society for Mass Spectrometry

Novel Aspect: A new and efficient method for the rapid analysis and detection focusing on the differentiation of isomeric metabolites was developed. Introduction: The potential of electrospray ionization (ESI) for obtaining and studying gas-phase metal complexes was realized soon after the method had been introduced as a soft-ionization technique for polar involatile molecules. For example, the use of metal ions produces very stable metal coordination complexes, and therefore alkaline, alkaline earth and transition metal ions have been extensively used in ESI for many types of compounds. Metal ions have also been successfully used for stereochemical differentiation by mass spectrometry. Differentiation by mass spectrometry of heterocyclic N-oxides and their isomeric hydroxylated metabolites formed during metabolic biotransformation of many drugs is relevant since their product ion spectra are usually very similar, particularly when N-oxidation or hydroxylation occurs on the same aromatic ring. Methods: Mass spectrometric analyses were performed using a Bruker micrOTOF-Q II mass spectrometer equipped with ESI and Agilent 1200 Series LC was used for HPLC studies. The analyte solutions of 2-hydroxypyridine and 3-hydroxypyridine N-oxides, each at a concentration of 1 mg/mL, were prepared using methanol. The metal ion stock solutions, each at a concentration of 10 mM, were prepared from the metal salts of Mg (II), Al (III), Ca (II), VO (IV), Fe (III), Co (II), Ni (II), Cu (II), Zn (II) and Ga (III) in the form of chlorides, sulphates or oxides. A small excess of HCl was added to the stock solutions of the trivalent ions. The metal solutions were used in excess relative to the sample solutions. Preliminary Data Protonated cations as well as cationized complexes of the isomeric hydroxypyridine N-oxides were produced in the gas phase from the corresponding neutral molecules using ESI. Mass spectra obtained with the different metal ions were recorded for each isomer. While complex formation was successful for 2-hydroxypyridine N-oxide with trivalent ions, in the case of 3-hydroxypyridine N-oxide, only peaks related to the protonated cation were present. On the other hand, divalent cations formed specific species for each isomer, giving characteristic spectra in every case. For instance, the main complexes observed for 2-hydroxypyridine N-oxide with calcium were mono charged cations with up to three ligands and one water molecule whereas doubly charged ions or mono charged cations with more than one water molecule were important in the case of 3-hydroxypyridine N-oxide. A similar behavior was also observed for the other divalent metals. These differences may be explained considering the ability of 2-hydroxypyridine N-oxide to form stable chelates acting as a bidentate ligand that limits the coordination of surrounding water molecules in contrast to its isomer. Because metal complexation was promising in differentiating both isomers, its practical application in LC/ESI-MS as a detection method for distinguishing them was tested. For example, addition of the metal solution at the interface between the LC column and the source provided an easy way to obtain similar spectra to the previously recorded by direct infusion. Thus, with a simple LC/ESI-MS experiment, significant structural information might be derived from the spectra observed according to the metal utilized.