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Abstract No.: MoP-179
Session: Metabolomics, Metabonomics
Presentation date: Mon, Aug 28, 2006
Presentation time: 09:50 – 11:20

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of γ-Dodecanolactone Metabolites

Marko Lehtonen1, Seppo Auriola1, Risto O. Juvonen2

1 University of Kuopio, Department of Pharmaceutical Chemistry, Kuopio, Finland
2 University of Kuopio, Department of Pharmacology and Toxicology, Kuopio, Finland

Correspondence address: Marko Lehtonen, University of Kuopio, Department of Pharmaceutical Chemistry, Harjulantie 1, Kuopio, FIN-70211 Finland.

Keywords: Ionization, Chemical; Ionization, Electron; Mass Spectrometry, Gas Chromatography; Metabolism, Metabolites.

Novel aspect: GC-MS method is developed to identify hydroxylated metabolites of γ-dodecanolactone. Mouse liver microsomes can oxidize γ-dodecanolactone to eight hydroxylated metabolites, and CYP2A5 is involved in these reactions.

 

Qualitative GC-MS method was developed to identify hydroxylated metabolites of γ-dodecanolactone (DDL). The other aims were to compare oxidation rate of DDL in vitro between human and pyrazole treated mouse liver microsomes, and to study if mouse CYP2A5 oxidizes DDL.
DDL (100 µM) was incubated in 100 mM Tris-HCl pH 7.4 containing 5 mM MgCl2, 200 µg microsomal protein without and with 0.5 mM NADPH at 37 °C for 10 60 min. Compounds of interest were extracted into dichloromethane. Prior to GC-MS analysis samples were silylated with BSTFA, evaporated to dryness and the residue was dissolved to hexane. Chromatographic conditions were as follows: a cross-linked 5 % phenyl methyl siloxane capillary column was used with pulsed splitless injection at 250 °C. Injection volume was 1 µl. The oven temperature increased from 50 °C by 5 °C/min to 300 °C. The carrier gas was helium. The metabolites of DDL were identified in both electron-impact (EI) mode and positive chemical ionization (PCI) with methane as a reagent gas mode. MS conditions were as follows: EI mode temperatures of MS ionization source and quadrupole were 230 °C and 150 °C, respectively. PCI mode temperatures of MS ionization source and quadrupole were 150 °C. Mass spectral data was collected with SCAN mode (EI 30 - 500 amu and PCI 100 - 400 amu). Fragmentation of metabolites in EI mass spectrum was used to identify the location of hydroxyl group in the molecule. PCI was used for determination of molecule masses.
The chromatogram showed that the size of DDL peak was decreased, and consequently six major and two minor DLL related peaks were formed during the incubation with mouse liver microsomes. The reaction was time dependent. The mass spectra indicated that the metabolites of DDL contained oxygen and/or hydroxyl group. PCI mass spectra of metabolites revealed expected [M+1]+ ion at 287 m/z and main fragment ion at 197 m/z ([M-C3H9OSi]+). According to EI mass spectral information the main metabolites were identified as 5-(1-hydroxy-octyl)-dihydro-furan-2-one, 5-(4-hydroxy-octyl)-dihydro-furan-2-one, 5-(5-hydroxy-octyl)-dihydro-furan-2-one, 5-(6-hydroxy-octyl)-dihydro-furan-2-one, 5-(7-hydroxy-octyl)-dihydro-furan-2-one, and 5-(8-hydroxy-octyl)-dihydro-furan-2-one. However, in human liver samples mainly 5-(8-hydroxy-octyl)-dihydro-furan-2-one was formed. Polyclonal antibody against CYP2A5 inhibited almost completely the formation of seven from eight DDL metabolites in mouse liver microsomes. Formation of 5-(8-hydroxy-octyl)-dihydro-furan-2-one was increased.
We concluded that developed GC-MS method is capable to determinate and to identify hydroxylated metabolites of DDL. Mouse liver microsomes can oxidize DDL to eight hydroxylated metabolites, and CYP2A5 is involved in these reactions. Oxidation rate of DDL is more rapid in mouse liver microsomes than human liver microsomes.