Detection of High Levels of Pyrrolizidine-N-oxides in the Endangered Plant Cryptantha crassipes (Terlingua Creek Cat's-eye) Using HPLC-ESI-MS
Department of Natural Resource Management, Sul Ross State University, Texas, USA. Phytochemical Analysis
(Impact Factor: 2.34).
11/2011; 22(6):532-40. DOI: 10.1002/pca.1314
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.
Available from: Paula N Brown
- "This will improve global acceptance of analytical methods, enhance the confidence in the validity and relevance of any obtained results and likely reduce the amount of inhouse validations required by users. The HPLC-MS method described for the quantification of four PAs and one PANO in PA-containing plants and honey was optimised from previously published methods to provide an improved method for evaluating plants and honey for PAs (Betteridge et al. 2005; Williams et al. 2011). Plants and honeys were collected in the British Columbia region and analysed with the optimised method during the validation procedures according to AOAC International guidelines (AOAC 2013). "
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ABSTRACT: Pyrrolizidine alkaloids (PAs) are a class of naturally occurring compounds produced by many flowering plants around the World. Their presence as contaminants in food systems has become a significant concern in recent years. For example, PAs are often found as contaminants in honey through pollen transfer. A validated method was developed for the quantification of four pyrrolizidine alkaloids and one pyrrolizidine alkaloid N-oxide in plants and honey grown and produced in British Columbia. The method was optimised for extraction efficiency from the plant materials and then subjected to a single-laboratory validation to assess repeatability, accuracy, selectivity, LOD, LOQ and method linearity. The PA content in plants ranged from1.0 to 307.8 µg/g with repeatability precision between 3.8 and 20.8% RSD. HorRat values were within acceptable limits and ranged from 0.62 to 1.63 for plant material and 0.56-1.82 for honey samples. Method accuracy was determined through spike studies with recoveries ranging from 84.6 to 108.2% from the raw material negative control and from 82.1-106.0 % for the pyrrolizidine alkaloids in corn syrup. Based on the findings in this single-laboratory validation, this method is suitable for the quantitation of lycopsamine, senecionine, senecionine N-oxide, heliosupine and echimidine in common comfrey (Symphytum officinale), tansy ragwort (Senecio jacobaea), blueweed (Echium vulgare) and hound's tongue (Cynoglossum officinale) and for PA quantitation in honey and found that PA contaminants were present at low levels in BC honey.
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ABSTRACT: Pyrrolizidine alkaloid (PA)-containing plants are widely distributed in the world. PAs are hepatotoxic, affecting livestock and humans. PA N-oxides are often present together with PAs in plants and also exhibit hepatotoxicity but with less potency. HPLC-MS is generally used to analyze PA-containing herbs, although PA references are unavailable in most cases. However, to date, without reference standards, HPLC-MS methodology cannot distinguish PA N-oxides from PAs because they both produce the same characteristic ions in mass spectra. In the present study, the mass spectra of 10 PA N-oxides and the corresponding PAs were systemically investigated using HPLC-MS to define the characteristic mass fragment ions specific to PAs and PA N-oxides. Mass spectra of toxic retronecine-type PA N-oxides exhibited two characteristic ion clusters at m/z 118-120 and 136-138. These ion clusters were produced by three unique fragmentation pathways of PA N-oxides and were not found in their corresponding PAs. Similarly, the nontoxic platynecine-type PA N-oxides also fragmented via three similar pathways to form two characteristic ion clusters at m/z 120-122 and 138-140. Further application of using these characteristic ion clusters allowed successful and rapid identification of PAs and PA N-oxides in two PA-containing herbal plants. Our results demonstrated, for the first time, that these characteristic ion clusters are unique determinants to discriminate PA N-oxides from PAs even without the availability of reference samples. Our findings provide a novel and specific method to differentiate PA N-oxides from PAs in PA-containing natural products, which is crucial for the assessment of their intoxication.
Available from: Joseph M Betz
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ABSTRACT: Cultivation of Crotalaria juncea L. (Sunn Hemp cv. 'Tropic Sun') is recommended as a green manure crop in a rotation cycle to improve soil condition, help control erosion, suppress weeds, and reduce soil nematodes. Because C. juncea belongs to a genus that is known for the production of toxic dehydropyrrolizidine alkaloids, extracts of the roots, stems, leaves, and seeds of 'Tropic Sun' were analyzed for their presence using HPLC-ESI/MS. Qualitative analysis identified previously unknown alkaloids as major components along with the expected macrocyclic dehydropyrrolizidine alkaloid diesters, junceine and trichodesmine. The dehydropyrrolizidine alkaloids occurred mainly as the N-oxides in the roots, stems, and, to a lesser extent, leaves, but mainly as the free bases in the seeds. Comprehensive spectrometric and spectroscopic analysis enabled elucidation of the unknown alkaloids as diastereoisomers of isohemijunceine, a monoester of retronecine with an unusual necic acid. The dehydropyrrolizidine alkaloid contents of the roots, stems, and leaves of immature plants were estimated to be 0.05, 0.12, and 0.01% w/w, respectively, whereas seeds were estimated to contain 0.15% w/w.
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