Pyrrolizidine alkaloids (PAs) are small organic hepatotoxins, produced by various plant species, which mediate plant-herbivore interactions. The interactions between the PA-philic milkweed butterflies (Danainae, Lepidoptera) and their larval host plant family (Apocynaceae, milkweeds), are well documented, but the phylogenetic distribution of PAs in Apocynaceae has yet to be established. A thorough survey of PAs across the ca. 4500 species of Apocynaceae must rely on minute tissue samples removed from herbarium specimens. We optimized an extraction method to maximize PA yield from small leaf fragments and used it to detect PAs in very old (70-177 years old) herbarium specimens of the PA-positive Echites umbellatus. Results showed that 15 seconds of crushing followed by 24 hours of methanol extraction were optimal for PA free-base and N-oxide recovery from recently collected dried leaf samples. This method yielded approximately 33 times greater PA signal intensity, as measured by reverse-phase LC-MS using positive electrospray ionization, than prior methods. Twelve of the 24 herbarium specimens tested positive for PAs, including the oldest, collected in 1840. A simulation of field alcohol preservation often used on herbarium specimens collected in the tropics was conducted to determine its effect on PA content in Parsonsia alboflavescens leaves. PA recovery was not affected by alcohol preservation; however, we have found that preservation with mercuric chloride might lead to false negative assessments of PA content. We demonstrate that a survey of PA occurrence in Apocynaceae can be conducted using herbarium specimens, however, since PAs were detected in only half of specimens tested, multiple specimens must be assayed before a species is determined to lack PAs.

LC-MS detection of pyrrolizidine alkaloids and their N-oxides in herbarium specimens dating back to 1840

255th National Meeting (2018)

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