HPLC‐MS detection of pyrrolizidine alkaloids and their N‐oxides in herbarium specimens dating back to the 1850s

Abstract

Premise of the StudyUnderstanding the phylogenetic distribution of defensive plant secondary metabolites is essential to the macroevolutionary study of chemically mediated plant–animal interactions. The chemical ecology of pyrrolizidine alkaloids (PAs) has been extensively studied in a number of plant–herbivore systems, including Apocynaceae (the milkweed and dogbane family) and Danainae (the milkweed and clearwing butterflies). A systematic survey is necessary to establish a detailed understanding of their occurrence across Apocynaceae. A survey of this species‐rich, mainly tropical and subtropical family will rely heavily on small tissue samples removed from herbarium specimens, some of which will be very old and/or preserved with alcohols or mercuric chloride.MethodsWe optimized PA extraction methods from small leaf fragments of recently collected silica‐dried leaves of the PA‐positive Echites umbellatus, varying crushing and extraction time. We then applied our optimized method to leaf fragments from 70–167‐year‐old herbarium specimens of E. umbellatus. To simulate the effect of alcohol treatment on PA detectability in herbarium specimens, we incubated freshly collected leaves of the PA‐positive Parsonsia alboflavescens in three different alcohols before drying and compared PA recovery to freshly dried controls. PAs were quantified using high‐performance liquid chromatography–mass spectrometry analysis. X‐ray fluorescence was used to identify mercury‐containing specimens.ResultsFifteen seconds of leaf crushing followed by 24 h of extraction were optimal for PA free‐base and N‐oxide recovery. This method yielded ~50‐fold greater PA recovery than prior methods. Half of the herbarium specimens (13 of 23), including the oldest, tested positive for PAs; leaf age did not correlate with success in PA extraction. Treatment of fresh leaves with alcohol before drying did not diminish PA recovery; mercury was observed in both PA‐positive and PA‐negative specimens.Conclusions PAs can be reliably detected in small tissue samples from herbarium specimens up to 167 years old, including specimens that had been treated with alcohol or mercury salts. The variability of PA presence among herbarium specimens of E. umbellatus indicates that multiple specimens will need to be tested before a particular species is determined to lack PAs.

Chemotaxonomic Investigation of Apocynaceae for Retronecine-Type Pyrrolizidine Alkaloids Using HPLC-MS/MS

“…We report the largest survey to date of PA occurrence in Apocynaceae, leveraging the long-term stability of PAs in dried leaves (Tasca et al, 2018), the taxonomically diverse collection of leaf samples that one of us (T.L.) has assembled for phylogenetic analysis, and advances in PA identification using HPLC-MS/MS to overcome the barriers that had previously precluded systematic study of their occurrence.…”

“…has assembled for phylogenetic analysis, and advances in PA identification using HPLC-MS/MS to overcome the barriers that had previously precluded systematic study of their occurrence. We also follow up on preliminary evidence of infraspecific polymorphism of PA presence in Echites umbellatus (Tasca et al, 2018) Apocynum cannabinum L. was used as a negative control because it is a chemically well-studied species from which PAs have not been reported (Abe and Yamauchi, 1994), and because it has a homospermidine synthase pseudogene and hence should not be able to produce PAs (unpublished data). Subjecting the negative control leaf sample to the four PREC scans resulted in a TIC with a signal to noise ratio lower than three to one (<3:1 S/N).…”

“…Most of these specimens had been dried at ca. 30 °C when collected from the living plant, potentially with alcohol and/or mercuric chloride treatment, and stored at room temperature (Tasca et al, 2018).…”

“…The published literature on natural products contains multiple biases, with the absence of a class of metabolites in a species being particularly hard to infer from studies that have not specifically targeted a compound class. Systematic surveys for chemicals of interest are often also impeded by availability of tissues across a breadth of species; this can be compounded by the lack of high-throughput, sensitive and selective analytical methods to maximize compound identification from small quantities of test material (Tasca et al, 2018). Infraspecific variation, a ubiquitous feature of plant specialized metabolism (Hartmann, 1996;Moore et al, 2014), can further introduce confusion about the distribution of a metabolite via contradictory reports in the literature.…”

“…Woodson (Burzynski et al, 2015), and variation in PA detectability among herbarium specimens of Echites umbellatus Jacq. (Tasca et al, 2018), indicate the potential for infra-specific variation in PA presence.…”