Detection of High Levels of Pyrrolizidine-N-oxides in the Endangered Plant Cryptantha crassipes (Terlingua Creek Cat's-eye) Using HPLC-ESI-MS
ABSTRACT A previous investigation of pyrrolizidine alkaloids produced by nine species of Cryptantha identified at least two chemotypes within the genus. Other research has postulated that pyrrolizidine-N-oxide concentrations increase as the growing conditions become harsher, particularly with respect to water availability. Cryptantha crassipes is an endangered plant with a very limited distribution range within a dry, harsh Texan ecosystem.
To determine the pyrrolizidine alkaloid (and their N-oxides) profile and concentrations in Cryptantha crassipes.
Methanolic extracts of Cryptantha crassipes were partitioned into dilute sulphuric acid and the alkaloids concentrated using strong cation exchange, solid-phase extraction columns. Extracts were analysed using reversed-phase high-pressure liquid chromatography coupled to electrospray ionisation ion trap mass spectrometry.
The N-oxides of lycopsamine and intermedine were the major pyrrolizidine alkaloids detected in Cryptantha crassipes. Smaller to trace amounts of other pyrrolizidine alkaloids observed were: the 7- and 3'-acetylated derivatives and the 1,2-dihydro analogs of lycopsamine-N-oxide and/or intermedine-N-oxide; a pair of unidentified N-oxides, isobaric with lycopsamine-N-oxide; and the N-oxides of leptanthine, echimiplatine, amabiline, echiumine and dihydroechiumine. Only trace amounts, if any, of the parent free base pyrrolizidine alkaloids were detected. The concentration of pyrrolizidine alkaloids was estimated to be 3-5% of the dry weight of milled leaves, or 10-50 times the levels previously reported for similar chemotypes.
The high levels of the N-oxides of lycopsamine and intermedine establish the genus chemotype of the endangered Cryptantha crassipes and support earlier data linking high levels of N-oxides to dry, harsh growing conditions.
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ABSTRACT: Four Greek endemic Boraginaceae plants, Onosma erecta Sibth. & Sm., Onosma kaheirei Teppner, Onosma leptantha Heldr., and Cynoglossum columnae L. (aerial parts), were screened for their content of pyrrolizidine alkaloids (PAs). TLC with the Mattocks-Molyneux visualization reagent was used as a preliminary qualitative test for PA or PA N-oxide detection. The extracts of the species found to contain PAs and their N-oxides were further analyzed by GC/MS, so as to identify their structures by means of the mass spectra and retention index values of known PAs already published in the literature. Twenty-three PAs were identified. For additional peaks, recognized as possible PAs by their MS pattern, no exact structures were tentatively suggested, as a result of lack of matching literature data. Furthermore, a quantitative PA profile of the species was obtained.Journal of AOAC International 10/2014; 97(5). DOI:10.5740/jaoacint.SGEDamianakos · 1.39 Impact Factor
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ABSTRACT: Species of the Amsinckia genus (Boraginaceae) are known to produce potentially hepato-, pneumo- and/or geno-toxic dehydropyrrolizidine alkaloids. However, the taxonomic differentiation of Amsinckia species can be very subtle and there seems to be marked differences in toxicity towards grazing livestock. Methanol extracts of mass-limited leaf samples from herbarium specimens (collected from 1899 to 2013) of 10 Amsinckia species and one variety were analyzed using HPLC-esi(+)MS and MS/MS for the presence of potentially toxic dehydropyrrolizidine alkaloids and/or their N-oxides. Dehydropyrrolizidine alkaloids were detected in all specimens examined ranging from about one to 4000 g/g of plant. Usually occurring mainly as their N¬-oxides, the predominant alkaloids were the epimeric lycopsamine and intermedine. Also sometimes observed in higher concentrations were the 3'- and 7-acetyl derivatives of lycopsamine/intermedine and their N-oxides. Within a designated species, an inconsistent profile was often observed that may be due to natural variation, taxonomic misassignment or non-uniform degradation due to plant collection and storage differences.Journal of Agricultural and Food Chemistry 03/2014; DOI:10.1021/jf500425v · 3.11 Impact Factor
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ABSTRACT: IntroductionThe diversity of structure and, particularly, stereochemical variation of the dehydropyrrolizidine alkaloids can present challenges for analysis and the isolation of pure compounds for the preparation of analytical standards and for toxicology studies.Objective To investigate methods for the separation of gram-scale quantities of the epimeric dehydropyrrolizidine alkaloids lycopsamine and intermedine and to compare their NMR spectroscopic data with those of their heliotridine-based analogues echinatine and rinderine.Methods Lycopsamine and intermedine were extracted, predominantly as their N-oxides and along with their acetylated derivatives, from commercial samples of comfrey (Symphytum officinale) root. Alkaloid enrichment involved liquid–liquid partitioning of the crude methanol extract between dilute aqueous acid and n-butanol, reduction of N-oxides and subsequent continuous liquid–liquid extraction of free base alkaloids into CHCl3. The alkaloid-rich fraction was further subjected to semi-automated flash chromatography using boronated soda glass beads or boronated quartz sand.ResultsBoronated soda glass beads (or quartz sand) chromatography adapted to a Biotage Isolera Flash Chromatography System enabled large-scale separation (at least up to 1–2 g quantities) of lycopsamine and intermedine. The structures were confirmed using one- and two-dimensional 1H- and 13C-NMR spectroscopy. Examination of the NMR data for lycopsamine, intermedine and their heliotridine-based analogues echinatine and rinderine allowed for some amendments of literature data and provided useful comparisons for determining relative configurations in monoester dehydropyrrolizidine alkaloids. A similar NMR comparison of lycopsamine and intermedine with their N-oxides showed the effects of N-oxidation on some key chemical shifts. A levorotatory shift in specific rotation from +3.29° to −1.5° was observed for lycopsamine when dissolved in ethanol or methanol respectively.ConclusionA semi-automated flash chromatographic process using boronated soda glass beads was standardised and confirmed as a useful, larger scale preparative approach for separating the epimers lycopsamine and intermedine. The useful NMR correlations to stereochemical arrangements within this specific class of dehydropyrrolizidine alkaloid cannot be confidently extrapolated to other similar dehydropyrrolizidine alkaloids. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.Phytochemical Analysis 09/2014; 25(5). DOI:10.1002/pca.2511 · 2.45 Impact Factor