Solid-phase extraction and HPLC-MS profiling of pyrrolizidine alkaloids and theirN-oxides: a case study ofEchium plantagineum

Abstract

Pyrrolizidine alkaloids and their N-oxides can be extracted from the dried methanolic extracts of plant material using dilute aqueous acid. The subsequent integration of solid-phase extraction (with a strong cation exchanger) of the alkaloids and N-oxides from the aqueous acid solution, together with analysis using HPLC-ESI/MS, provides a method for the simultaneous profiling of the pyrrolizidine alkaloids and their N-oxides in plant samples and the collection of useful structural data as an aid in their identification. The N-oxide character of the analytes may be confirmed by treating analytical samples with a redox resin and observing the formation of the corresponding parent pyrrolizidine alkaloids. The present case study of Echium plantagineum highlighted a higher ratio of N-oxides to the parent tertiary bases than has been previously reported. Furthermore, a higher proportion of acetylated pyrrolizidine-N-oxides was observed in the flower heads relative to the leaves. Six pyrrolizidine alkaloids or pyrrolizidine-N-oxides, not previously reported from E. plantagineum, were tentatively identified on the basis of MS and biogenetic considerations. Three of these, 3'-O-acetylintermedine/lycopsamine, leptanthine-N-oxide and 9-O-angelylretronecine-N-oxide, have been reported elsewhere, whilst three others, 3'-O-acetylechiumine-N-oxide, echimiplatine-N-oxide and echiuplatine-N-oxide, appear unreported from any other source.

Diversity of Pyrrolizidine Alkaloids in the Boraginaceae Structures, Distribution, and Biological Properties

“…Several saturated and 1,2-unsaturated PAs (mainly as N-oxides) were detected using HPLC-ESI-MS in fresh pollen collected from flowers of the PAs producing plants Echium vulgare and E. plantagineum and /or from bee pollen from bees that had foraged on PA plants [12]. The same method has also been used to determine the PA profile in E. plantagineum [93], Cryptantha crassipes [64], and Anchusa strigosa [50]. PAs (N-oxides and free bases) occurring in small amounts could be efficiently analyzed using an RP-HPLC ion trap MS method with an atmospheric pressure chemical ionization (APCA) interface [167].…”

“…For analytical purposes high-resolution capillary GLC alone or in combination with mass spectrometry is the method of choice for free PA bases [40,59,67,70,90,91,96,163,[191][192][193][194][195]. HPLC and HPLC-MS are also helpful but less sensitive than GLC and GLC-MS [60,93,[196][197][198][199]. In addition, the resolution is much lower than for GLC but in HPLC also PA-N-Oxides can be directly analysed.…”

Effects of supplementing a mixed diet with echium (<i>Echium plantagineum</i>) oil on methanogenesis in a rumen simulation system

“…It is difficult to distinguish whether the effects of oil supplementation in methane mitigation are due to secondary metabolites, a particular fatty acid, or mixtures of fatty acids. Secondary compounds, like the pyrrolizidine alkaloids echimidine and echiumine, known to contribute to liver damage and photosensitivity in grazing livestock (Weston et al, 2013), are found in greatest quantity in E. plantagineum (Colegate et al, 2005). There are marked differences in the sensitivity of domestic species to echium toxicosis, with cattle and horses showing the highest levels of sensitivity (Cheeke, 1984).…”

Identification of a Second Site of Pyrrolizidine Alkaloid Biosynthesis in Comfrey to Boost Plant Defense in Floral Stage,

“…After centrifugation (10 min at 5,000g), zinc dust was added in excess to the supernatants, which were then stirred for 3 h for the complete reduction of alkaloid N-oxides. As SCX-SPE purification has been shown to be suitable for the enrichment of PAs (Colegate et al, 2005), the samples were further centrifuged (10 min at 5,000g) and then applied to SCX-SPE cartridges (Phenomenex) equilibrated with 6 mL of methanol and 6 mL of 0.05 M H 2 SO 4 . After each column had been washed with 12 mL of water and 12 mL of methanol, the alkaloids were eluted by applying 3 3 6 mL of methanol containing 5% (v/v) NH 4 OH.…”