Reliable analysis of volatile compounds from small samples of Eucalyptus magnificata (Myrtaceae)

Abstract

Phytochemistry is a source of data for plant systematics. This tool has much more value if herbarium specimens can be used without major damage and if results are comparable with fresh samples. A modified method for the solvent extraction of eucalypt leaf oils for phytochemical analysis and chemotaxonomy studies, including historical herbarium samples by gas chromatography–mass spectrometry (GC–MS), has been statistically assessed using Eucalyptus magnificata L.A.S.Johnson & K.D.Hill leaves. Leaf sample size was reduced by a factor of 250 to minimise damage to herbarium specimens, reduce solvent volume and simplify preparation of solvent extract before analysis. Leaf sampling treatments assessed the effects of the number of leaves and post-harvest air-drying on variation in components in the solvent extract. The results showed no statistically significant effect of leaf mass or the number of leaves used in GC–MS analyses on the precision of the measurements, but a significant difference among treatments for some oil constituents, particularly 1,8-cineole. Most differences in terpenoid concentration were due to variation among plants rather than extraction treatments. Extracts from air-dried herbarium leaves up to 44 years old were directly comparable with those from fresh leaves. Solvent extraction in 2 mL GC–MS vials of ~0.5 cm² (16 mg) of leaf material, using fragments of fresh or air-dried leaves, drastically reduced sample and solvent volumes and showed that sampling from E. magnificata herbarium specimens for chemotaxonomy and chemotyping is a valid method, enabling broader sampling with much lower costs than for traditional fieldwork collections.