Pyrrolizidine N-oxides

Smecio jacobaea, unlike S. ovatus, has been extensively studied around the world owing to its weedlike propensity and the toxic effect on livestock. The HPLC-ESI-ion trap MS profiles for extracts of both plants (Figure 13.14) showed the presence of pyrrolizidine-N-oxides (Colegate ef al., unpublished). 

It has been demonstrated that, when many samples are to be analyzed in automatically injected sequences, the APCI mode is more stable than the ESI mode for quantitation [32]. However, the ESI mode is more sensitive for the detection ofthe pyrrolizidine- N-oxides. 

Figure 4. Mono- and diester pyrrolizidine alkaloids and a pyrrolizidine N-oxide.

Additional studies have shown that the oxidation of alkaloid to its N-oxide occurs and competes with pyrrole formation. N-oxides when administered intravenously to rats or when treated with liver microsomal enzymes are not converted to pyrroles. Hence they are non-toxic in the liver, and, by their highly hydrophilic nature, provide a pathway for detoxification. When administered orally to rats and sheep however, N-oxides are converted in the rumen (sheep) or gut (rats) to the parent alkaloids which are then metabolized in the liver causing the normal toxic sequelae. The toxicity (or lack thereof) of the pyrrolizidine N-oxides depends on whether or not they are reduced vivo before reaching the liver (61). After intravenous administration of indicine N-oxide to human cancer patients, free alkaloid has been detected in the blood and subsequent liver damage has been observed (55). 

Cells of Senecio vulgaris in tissue culture lack the ability to synthesize pyrrolizidine alkaloids, but retain the ability to take up and accumulate the corresponding A -oxides. The translocation of exogenous A -oxides into the vacuole by carrier-mediated transport has been demonstrated cells of nonalkaloid-producing plants do not take up pyrrolizidine N-oxides (Hartmann, 1991). 

The N-oxide of indicine (49) exhibits anti-tumour activity in experimental tumour systems, without some of the toxic effects associated with other pyrrolizidine alkaloids. The N-oxides of echinatine and europine show similar anti-tumour activity against P 388 lymphocytic leukaemia tumours.23 Indicine N-oxide is metabolized to the free base in rabbits and humans,62 although the N-oxide is the more active anti-tumour agent. It has been suggested that the conversion of indicine N-oxide into indicine is not essential for its anti-tumour activity.63 Indicine N-oxide is the first pyrrolizidine alkaloid to be tested as an anti-tumour agent in humans. The toxicity and pharmacokinetics of this compound have been studied in 29 patients with advanced cancers.64 The major toxic effect was myelosuppression, but acute liver damage was not observed. 

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. 

LINDIGKEIT, R., BILLER, A., BUCH, M., SCH1EBEL, H.M., BOPPRE, M., HARTMANN, T., The two faces of pyrrolizidine alkaloids The role of the tertiary amine and its N-oxide in chemical defense of insects with acquired plant alkaloids. Eur. J. Biochem., 1997,245,626-636. 

HARTMANN, T., EHMKE, A., EILERT, U., VON BORSTEL, K., THEURING, C., Sites of synthesis, translocation and accumulation of pyrrolizidine alkaloid N-oxides in Senecio vulgaris L Planta, 1989,177,98-107. 

Whilst the N-oxidation of pyrrolizidine alkaloids is a recognized detoxifying mechanism in mammals [26], it has also been shown that ingested N-oxides are indeed toxic, presumably via in vivo reduction to the parent pyrrolizidine alkaloids... 

Fig. 13.8 General structures of some pyrrolizidine alkaloids. A Pyrrolizidine alkaloid esters, B 1,2-dehydro pyrrolizidine alkaloid esters, C the N-oxide of 1,2-dehydro pyrrolizidine alkaloid esters, and D an otonecine-based alkaloid. Ri and R2 can be H or esters of carboxylic acids that may or may not be cyclized forming a macrocyclic diester.

Gas chromatography coupled to mass spectrometry is a widely used analytical method [31] but does require prior reduction of the involatile N-oxides. Additionally, there are problems associated with some alkaloids that require prederivatization to enhance the GC characteristics. These requirements for prederivatization can adversely affect the GG-MS interpretation, especially at trace levels of alkaloids [32]. Gombined SPE-LC-MS approaches have provided methods for qualitative profiling and quantitative analysis of pyrrolizidine alkaloids and their N-oxides. 

Extraction of the pyrrolizidine alkaloids and their N-oxides from plant sources is a critical first step in LG-MS analysis. Some methods of extraction already described (Section 13.2) consist simply of treatment with aqueous methanol and, following microfiltration (0.45 p.m), immediate LC-MS analysis with no further treatment. However, the aqueous methanol treatment will extract more than just the alkaloids. 

Fig. 13.10 The reversed phase HPLC-ESI-MS base ion (mjz 200-500) chromatogram of some authenticated standard pyrrolizidine alkaloids and N-oxides. A Monocrotaline,...

Careful analysis of M S and the MS/MS data for the major components of S. ovatus identified sarradne-related alkaloids. For example, the molecular ion data for peak 7 (Figure 13.14) (MH m/z354) potentially corresponded to the N-oxides of several 1,2-saturated, macrocyclic or open chain diester pyrrolizidine alkaloids previously... 

Because of the diversity of structures, including stereochemical orientations, in the pyrrolizidine alkaloids extracted from a plant, it is not usually possible to have authenticated standards for every alkaloid extracted. An alternative approach is to generate calibration curves using standard pyrrolizidine alkaloids that best approximate those extracted from the plant [35,41]. Thus, considerations such as N-oxidation, mono- or diesterification, open chain or cyclic diesterification, and the character of the esterifying acids are important in deciding which standards to use. The analytes are then described in terms of equivalents of the standard alkaloid. At a later stage, if it becomes important, specific authenticated alkaloids can be used... 

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. 

Tab. 13.1 Quantities of pyrrolizidine alkaloids and their N-oxides extracted from E. vulgare-derived honey.

The total pyrroUzidine alkaloid content was 2850 143 xg/kg honey (ppb) expressed as equivalents oflasiocarpine-N-oxide forthe N-oxides present and equivalents of lasiocarpine for the tertiary base pyrrolizidine alkaloids present. The assignment of the minor alkaloids leptanthine-N-oxide and echimiplatine-N-oxide may be reversed... 

Figure 2.4 Biosynthesis of the pyrrolizidine alkaloid. senecionine-N-oxide. ODC, ornithine decarboxylase ADC, arginine decarboxylase SPDS, spermidine synthase HHS, homospermidine synthase. (See Plate 9 in colour plate section.)...

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