title: Investigation of low-abundant in vitro metabolites of stable isotope-labelled BAL4815 by accurate mass capillary-LC-ESI-qTof-MS and MS/MS.
abstract: The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.
authors.affiliation: Analytics, Basilea Pharmaceutica Ltd, Basel, Switzerland. mathias.wind@basilea.com
authors.forename: Mathias
authors.initials: M
authors.lastname: Wind
authors.forename: Jochen
authors.initials: J
authors.lastname: Spickermann
authors.forename: Michael
authors.initials: M
authors.lastname: Schleimer
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Donzelli
authors.forename: Klaus
authors.initials: K
authors.lastname: Gebhardt
authors.forename: Rima
authors.initials: R
authors.lastname: Sturm-Haurany
authors.forename: Dominique
authors.initials: D
authors.lastname: Klauer
authors.forename: Pascal
authors.initials: P
authors.lastname: Fullhardt
authors.forename: Anne
authors.initials: A
authors.lastname: Schmitt-Hoffmann
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000737:chemistry* / Q000302:isolation & purification; D002197:Capillary Action; D002853:Chromatography, Liquid / Q000379:methods; D003903:Deuterium; D022781:Hepatocytes / Q000378:metabolism*; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D009570:Nitriles / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014230:Triazoles / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011725:Pyridines
chemical_list: D000935:Antifungal Agents; D003903:Deuterium; D009570:Nitriles; D014230:Triazoles; C508735:isavuconazole; D011725:Pyridines