Pyrrolizidine alkaloid detection

Hartmann T, Ober D (2000) Biosynthesis and Metabolism of Pyrrolizidine Alkaloids in Plants and Specialized Insect Herbivores. 209. 207-243 Haseley SR, Kamerling JP, Vliegenthart JFG (2002) Unravelling Carbohydrate Interactions with Biosensors Using Surface Plasmon Resonance (SPR) Detection. 218 93-114... 

Note It is reported that the use of chlorobenzene as solvent is essential when the reagent is to be used to detect aromatic amines [1]. In the case of steroids, penicillins, diuretics and alkaloids the reaction should be accelerated and intensified by spraying afterwards with dimethylsulfoxide (DMSO) or dimethylformamide (DMF), indeed this step makes it possible to detect some substances when this would not otherwise be possible [5,9-11] this latter treatment can, like heating, cause color changes [5,9]. Penicillins and diuretics only exhibit weak reactions if not treated afterwards with DMF [10, 11]. Steroids alone also yield colored derivatives with DMSO [9]. Tlreatment afterwards with diluted sulfuric acid (c = 2 mol/L) also leads to an improvement in detection sensitivity in the case of a range of alkaloids. In the case of pyrrolizidine alkaloids it is possible to use o-chloranil as an alternative detection reagent however, in this case it is recommended that the plate be treated afterwards with a solution of 2 g 4-(dimethyl-amino)-benzaldehyde and 2 ml boron trifluoride etherate in 100 ml anhydrous ethanol because otherwise the colors initially produced with o-chloranil rapidly fade [12]. 

Bober, M. A., Kurth, M. J., Milco, L. A., Roseman, D. M., Miller, R. B. and Segal, H. J. 1991. A pyrrolizidine alkaloid-enzyme-linked immunosorbent-assay detection strategy. ACS Symposium Series, 451 176-183 and, Bober, M. A., Milco, L. A., Miller, R. B., Mount, M., Wicks, B. and Kurth, M. J. 1989. A competitive enzyme-linked immunosorbent-assay (ELISA) to detect retronecine and monocrotaline in vitro. Toxicon, 27(9) 1059-1064. 

No new alkaloids have been discovered within this family. A phytochemical investigation of plants of the Boraginaceae indigenous to France has been carried out.11 Pyrrolizidine alkaloids were detected in 23 species from 14 genera, and N-oxides were present in 12 species. No definite identifications were made. 

Separation of mono- and di-ester pyrrolizidine alkaloids has been achieved by ion-pair adsorption t.l.c., using chloride (or iodide) as the counter-ion.48 Chloranil has been used to oxidize pyrrolizidine alkaloids on t.l.c. The pyrrole derivatives that were formed were then detected with Ehrlich s reagent49 or sulphuric acid.50 Mixtures of pyrrolizidine alkaloids have been separated by h.p.l.c. on a reversed-phase styrene-divinylbenzene resin.51 In a sensitive method for the detection of pyrrolizidine alkaloids, the protonated alkaloids were complexed with aqueous methyl orange. The dye was then released from the complex and estimated spectrophotometrically.52... 

Insects have evolved mechanisms to tolerate plant toxins. Such mechanisms enable them to use a plant as a food source that is avoided by other herbivores, and provides the herbivore with its own ecological niche.9 For example, insects may prevent accumulation of detrimental alkaloids in the hemolymph by efficient excretion. Larvae of Spodoptera littoralis (Lepidoptera, Noctuidae) feed freely on plants containing pyrrolizidine alkaloids that are rapidly excreted and only transiently detectable in the hemolymph. Even pyrrolizidine alkaloids directly injected into the hemolymph are efficiently excreted.48 The same has been observed... 

The presence of toxic pyrrolizidine alkaloids in honey has been known for several decades [42]. The potential health concerns associated with pyrrolizidine alkaloids in food [27] and honey [28] in particular demand a rapid, sensitive method of detection in various matrices. The application of LC-MS methods to honey samples extracted using sex SPE cartridges has facilitated the analysis of honey for the presence of pyrrolizidine alkaloids and their N-oxides [34,41]. The SPE and LC-MS analysis has shown that honey attributed to known pyrrolizidine alkaloid-producing sources can have levels in excess of 5000 jrg/kg honey. Further to this, honey attributed to non-pyrrolizidine alkaloid-producing floral sources and unspecified blended honeys can also have significant amounts of alkaloids present. 

In the West Indies especially, pyrrolizidine alkaloid toxicity is a continuing problem, as plants containing pyrrolizidine alkaloids such as Heliotropium, Senecio, and Crotolaria species are used in traditional medicine to make herbal teas. Chronic exposure to low doses of these alkaloids causes liver cirrhosis, and it is estimated that these alkaloids account for one-third of the cirrhosis cases detected at autopsy in Jamaica (see Chapter 6 for more details of these natural toxins). 

For the sensitive detection of pyrrolizidine alkaloids it is necessary to use solvent... 

Spectral analysis serves as a useful adjunct to paper and column chromatography in the fast determination of the composition of alkaloidal mixtures. A comparative study of the IR-spectra of many pyrrolizidine alkaloids, all very similar to each other, has permitted the designation of a few bands of good intensity in the low-frequency region which are characteristic of certain of the alkaloids and therefore permit the detection of small amounts of impurities in a given sample (83, 84, 120). Thus, seneciphylline has a distinctive band at 902 cm. (s) and another at 992 cm.- (m) senecionine (and its stereoisomers), a band at 757 cm.-i (m) riddelliine, 1120 cm. (m) retrorsine, 1055 cm. (m). 

The Polonovsky reaction (246) whereby a 3-pyrroline ring system is oxidized to the N-oxide and treated with acetic anhydride to give the pyrrole, has been employed by Dann (247) to detect pyrrolizidine alkaloid iV-oxides on paper chromatograms, the fluorescence of the pyrrole being observed. The method has been developed by Mattocks (248) as a spectrophotometric procedure, applicable also to biological material... 

Tic. We have used a modification of the colorimetric method to detect pyrrolizidine alkaloids on thin layer chromatograms. Alkaloid spots when sprayed with o-chloranil are converted directly to the pyrrole derivatives which then can be detected by spraying with Ehrlich s reagent (21). As expected, seco and saturated alkaloids give negative results. 

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