Determination Of The Natural Abundance Delta N-15 Of Nortropane Alkaloids By Gas Chromatography-Isotope Ratio Mass Spectrometry Of Their Ethylcarbamate Esters

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 N-15 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 nor-tropine and norpseudotropine for measurement of their delta N-15 (parts 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 N-15 values (Delta delta N-15)>0.8 parts per thousand to be measured, with a standard deviation routinely similar to 0.3 parts per thousand. The methodology has been tested by determining the changes in the delta N-15 values of nor-tropine 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 N-15 values to be followed during the fermentation.