title: Determination of the natural abundance delta15N of nortropane alkaloids by gas chromatography-isotope ratio mass spectrometry of their ethylcarbamate esters.
abstract: An important route for the detoxification of tropane alkaloids involves N-demethylation to the nor-compounds followed by further degradation. In order to study the mechanisms of the pertinent reactions, a suitable means to determine the isotope ratios of the substrates and products is required. However, the polarity and functionality of the nortropane compounds makes their analysis as free bases difficult. A method is described which allows both the quantification of nortropane alkaloids and the determination of their natural abundance delta(15)N values. The protocol exploits the derivatisation of the alkaloids by reaction with ethyl chloroformate in aqueous medium and the quantitative extraction of the ensuing ethylcarbamate esters. The improved chromatographic properties of these derivatives gives ample separation of the isomeric nortropine and norpseudotropine for measurement of their delta(15)N (per thousand) values by isotope ratio mass spectrometry interfaced to gas chromatography. Adequate separation could not be achieved with the underivatised compounds. Repeatability and precision are sufficient to allow differences in the delta(15)N values (Deltadelta(15)N) > 0.8 per thousand to be measured, with a standard deviation routinely approximately 0.3 per thousand. The methodology has been tested by determining the changes in the delta(15)N values of nortropine and norpseudotropine during degradation by cell suspension cultures of a Pseudomonas strain expressing a specific capacity for tropine catabolism. The precision and reproducibility are shown sufficient to allow the evolution of the delta(15)N values to be followed during the fermentation.
authors.affiliation: Elucidation of Biosynthesis by Isotopic Spectrometry Group, Unit for Interdisciplinary Chemistry: Synthesis-Analysis-Modelling (CEISAM), University of Nantes-CNRS UMR6230, 2, rue de la Houssinière, 44322 Nantes, France.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kosieradzka
authors.forename: Illa
authors.initials: I
authors.lastname: Tea
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Gentil
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Robins
mesh_terms: D000470:Alkaloids / Q000032:analysis*; D002219:Carbamates / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D004980:Ethane / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D007554:Isotopes / Q000032:analysis*; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D009662:Nortropanes / Q000032:analysis*; D011549:Pseudomonas; D015203:Reproducibility of Results
chemical_list: D000470:Alkaloids; D002219:Carbamates; D004952:Esters; D007554:Isotopes; D009587:Nitrogen Isotopes; D009662:Nortropanes; D004980:Ethane