Determination of the natural abundance δ15N of nortropane alkaloids by gas chromatography–isotope ratio mass spectrometry of their ethylcarbamate esters
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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 δ15N 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 δ15N (‰) 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 δ15N values (∆δ15N) > 0.8‰ to be measured, with a standard deviation routinely ∼0.3‰. The methodology has been tested by determining the changes in the δ15N 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 δ15N values to be followed during the fermentation.
KeywordsDerivatisation Isotope ratio measurement by mass spectrometry Nortropine Reaction mechanism Tropane alkaloids
K Kosieradzka thanks the French Ministry of Science and Education for a doctoral bursary. We thank Boehringer Ingelheim (Ingelheim, Germany) for their generous gift of nortropinone·HCl. We are most grateful to our colleagues Nadia Guignard and Isabelle Louvet for their assistance with the irm-MS analyses. We thank Dr P W Trudgill (Department of Biochemistry, University of Wales, Aberystwyth, UK) for supplying us with the Pseudomonas culture.
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