Analysis of Daphnane Orthoesters in Poisonous Australian Pimelea Species by Liquid Chromatography-Tandem Mass Spectrometry.

Cattle grazing in arid rangelands of Australia suffer periodic extensive and serious poisoning by the plant species Pimelea trichostachya, P. simplex, and P. elongata. Pimelea poisoning (also known as St. George disease and Marree disease) has been attributed to the presence of the diterpenoid orthoester simplexin in these species. However, literature relating to previous studies is complicated by taxonomic revisions, and the presence of simplexin has not previously been verified in all currently recognized taxa capable of inducing pimelea poisoning syndrome, with no previous chemical studies of P. trichostachya (as currently classified) or P. simplex subsp. continua. We report here the isolation of simplexin from P. trichostachya and the development of a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to measure simplexin concentrations in pimelea plant material. Simplexin was quantified by positive-ion atmospheric pressure chemical ionization (APCI) LC-MS/MS with selected reaction monitoring (SRM) of the m/z 533.3 > 253.3 transition. LC-MS/MS analysis of the four poisonous taxa P. trichostachya, P. elongata, P. simplex subsp. continua, and P. simplex subsp. simplex showed similar profiles with simplexin as the major diterpenoid ester component in all four taxa accompanied by varying amounts of related orthoesters. Similar analyses of P. decora, P. haematostachya, and P. microcephala also demonstrated the presence of simplexin in these species but at far lower concentrations, consistent with the limited reports of stock poisoning associated with these species. The less common, shrubby species P. penicillaris contained simplexin at up to 55 mg/kg dry weight and would be expected to cause poisoning if animals consumed sufficient plant material.

LC/MS/MS analysis of the daphnane orthoester simplexin in poisonous Pimelea species of Australian rangelands.

Liquid chromatography/mass spectrometry (MS)/MS was used to analyse toxins in P. trichostachia, P. simplex subsp. continua, P. simplex subsp. continua and P. elongata samples (flowers, seeds, branches, main stem, leaves and roots) collected from various locations in Queensland, Saskatchewan and New South Wales, Australia. Simplexin was the major analyte in all taxa, with varying minor levels of huratoxin. Simplexin levels in P. trichostachia and P. elongata were higher (580 and 540 mg/kg in flowering foliage, respectively) than in P. simplex (255 mg/kg). Levels of huratoxin were higher in P. simplex (relative to simplexin) than in P. trichostachia or P. elongata. P. simplex flower heads and roots contained similar simplexin levels, with very small amounts of toxins detected in branches, stems and leaves. In P. trichostachia, simplexin levels were high in flower heads but low in the the other plant parts. The simplexin levels in aerial parts were generally higher from the pre-flowering to the flowering stage, decreasing towards the post-flowering stage; similar trends were recorded for P.elongata samples collected from a site near Bollon and P. trichostachia samples collected from a site near Jericho (both sites in Queensland). The simplexin concentration in roots was much less variable. Flowers and seeds had much higher simplexin levels than the foliage. The breakdown of the toxin in litter was more rapid compared to seeds under the same weathering conditions. Unlike the results from the litter samples, no significant decrease occurred in seed samples after 18 months of exposure.

Seasonal and Species Variation of the Hepatotoxin Indospicine in Australian Indigofera Legumes as Measured by UPLC−MS/MS

Livestock industries have maintained a keen interest in pasture legumes because of the high protein content and nutritive value. Leguminous Indigofera plant species have been considered as having high feeding values to be utilized as pasture, but the occurrence of the toxic constituent indospicine in some species has restricted this utility. Indospicine has caused both primary and secondary hepatotoxicosis and also reproductive losses, but has only previously been determined in a small number of Indigofera species. This paper validates a high throughput ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method to determine indospicine content of various Indigofera species found in Australian pasture. Twelve species of Indigofera together with Indigastrum parviflorum plants were collected and analysed. Out of the 84 samples analyzed, *I. spicata contained the highest indospicine level (1003 ± 328 mg/kg DM, n = 4) followed by I. linnaei (755 ± 490 mg/kg DM, n = 51). Indospicine was not detected in 9 of the remaining 11 species, and at only low levels (<10 mg/kg DM) in 2 out of 8 I. colutea specimens and in 1 out of 5 I. linifolia specimens. Indospicine concentrations were below quantitation levels for other Indigofera spp. (I. adesmiifolia, I. georgei, I. hirsuta, I. leucotricha,* I. oblongifolia, I. australis and I. trita) and Indigastrum parviflorum. One of the more significant findings to emerge from this study is that the indospicine content of I. linnaei is highly variable (159 to 2128 mg/kg DM, n = 51), and differs across both regions and seasons. Its first re-growth after spring rain has a higher (p < 0.01) indospicine content than growth following more substantial summer rain. The species collected include the predominant Indigofera in Australia pasture, and of these, only *I. spicata and I. linnaei contain high enough levels of indospicine to pose a potential toxic threat to grazing herbivores.

Accumulation, persistence and effects of indospicine residues in camels fed Indigofera plant

Indospicine (L-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as I. linnaei and I. spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution and histopathological effects of dietary indospicine on camels. Six young camels (2 – 4 year old), weighing 270 − 390 kg were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw); followed by the muscle, heart and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining 3 camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated mono-exponential decreases.

Volatile components associated with bacterial spoilage of tropical prawns

Analysis of headspace volatiles by gas chromatography/mass spectrometry from king (Penaeus plebejus), banana (P. merguiensis), tiger (P. esculentus/semisulcatus) and greasy (Metapenaeus bennettae) prawns stored in ice or ice slurry, which is effectively an environment of low oxygen tension, indicated the presence of amines at the early stages of storage (less than 8 days) irrespective of the nature of the storage media. Esters were more prevalent in prawns stored on ice (normal oxygen conditions) at the latter stages of storage (more than 8 days) and were only produced by Pseudomonas fragi, whereas sulphides and amines occurred whether the predominant spoilage organism was Ps.fragi or Shewanella putrefaciens. The free amino acid profiles of banana and king prawns were high in arginine (12–14%) and low in cysteine (0.1–0.17%) and methionine (0.1–0.2%). Filter sterilised raw banana prawn broth inoculated with a total of 15 cultures of Ps. fragi and S. putrefaciens and incubated for two weeks at 5°C, showed the presence of 17 major compounds in the headspace volatiles analysed using gas chromatography/mass spectrometry (GC/MS). These were mainly amines, sulphides, ketones and esters. Principal Component Analysis of the results for the comparative levels of the volatiles produced by pure cultures, inoculated into sterile prawn broth, indicated three subgroupings of the organisms; I, Ps. fragi from a particular geographic location; II, S. putrefaciens from another geographic location; and III, a mixture of Ps. fragi and S. putrefaciens from different geographic locations. The sensory impression created by the cultures was strongly related to the chemical profile as determined by GC/MS. Organisms, even within the same subgrouping classified as identical by the usual tests, produced a different range of volatiles in the same uniform substrate.

Host recognition by a polyphagous lepidopteran (Helicoverpa armigera): Primary host plants, host produced volatiles and neurosensory stimulation

An important question in the host-finding behaviour of a polyphagous insect is whether the insect recognizes a suite or template of chemicals that are common to many plants? To answer this question, headspace volatiles of a subset of commonly used host plants (pigeon pea, tobacco, cotton and bean) and nonhost plants (lantana and oleander) of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) are screened by gas chromatography (GC) linked to a mated female H. armigera electroantennograph (EAG). In the present study, pigeon pea is postulated to be a primary host plant of the insect, for comparison of the EAG responses across the test plants. EAG responses for pigeon pea volatiles are also compared between females of different physiological status (virgin and mated females) and the sexes. Eight electrophysiologically active compounds in pigeon pea headspace are identified in relatively high concentrations using GC linked to mass spectrometry (GC-MS). These comprised three green leaf volatiles [(2E)-hexenal, (3Z)-hexenylacetate and (3Z)-hexenyl-2-methylbutyrate] and five monoterpenes (α-pinene, β-myrcene, limonene, E-β-ocimene and linalool). Other tested host plants have a smaller subset of these electrophysiologically active compounds and even the nonhost plants contain some of these compounds, all at relatively lower concentrations than pigeon pea. The physiological status or sex of the moths has no effect on the responses for these identified compounds. The present study demonstrates how some host plants can be primary targets for moths that are searching for hosts whereas the other host plants are incidental or secondary targets.

Effect of Packaging Materials and Storage on Major Volatile Compounds in Three Australian Native Herbs

Lemon myrtle, anise myrtle, and Tasmanian pepper leaf are commercial Australian native herbs with a high volatile or essential oil content. Packaging of the herbs in high- or low-density polyethylene (HDPE and LDPE) has proven to be ineffective in preventing a significant loss of volatile components on storage. This study investigates and compares the effectiveness of alternate high-barrier property packaging materials, namely, polyvinylidene chloride coated polyethylene terephthalate/casted polypropylene (PVDC coated PET/CPP) and polyethylene terephthalate/polyethylene terephthalate/aluminum foil/linear low-density polyethylene (PET/PET/Foil/LLDPE), in prevention of volatile compound loss from the three native herbs stored at ambient temperature for 6 months. Concentrations of major volatiles were monitored using gas chromatography?mass spectrometry (GC-MS) techniques. After 6 months of storage, the greatest loss of volatiles from lemon myrtle was observed in traditional LDPE packaging (87% loss) followed by storage in PVDC coated PET/CPP (58% loss) and PET/PET/Foil/LLDPE (loss of 23%). The volatile loss from anise myrtle and Tasmanian pepper leaf stored in PVDC coated PET/CPP and PET/PET/Foil/LLDPE packaging was <30%. This study clearly indicates the importance of selecting the correct packaging material to retain the quality of herbs with high volatile content.

Earthy-muddy tainting of cultured barramundi linked to geosmin in tropical northern Australia

Tainting of outdoor pond-reared barramundi Lates calcarifer by muddy-earthy off-flavours is frequently reported across tropical Australia. To investigate the possible causes and effects of off-flavour tainting, we analysed water samples from outdoor rearing ponds for the presence of geosmin (GSM) and 2-methylisoborneol (2-MIB), 2 microbial metabolites often associated with tainting episodes. We then conducted controlled dose-effect experiments which measured the accumulation of tainting metabolites in the flesh, and the impact tainting had on taste and flavour attributes. GSM was deemed to be the compound most likely responsible for off-flavour tainting, persisting at moderate (similar to 1.00 mu g l(-1)) to extreme levels (similar to 14.36 mu g l(-1)), while 2-MIB was never detected during the study. Controlled experiments revealed that the accumulation of GSM in the flesh of market-sized barramundi was directly related to GSM levels of the holding water (0 to similar to 4 mu g l(-1)), with higher levels resulting in significant increases in undesirable taste and flavour attributes, particularly muddy-earthy flavour and weedy aftertaste. We identified the sensory detection threshold for GSM in farmed barramundi to be <= 0.74 mu g kg(-1), similar to estimates for GSM detection in rainbow trout Oncorhynchus mykiss (similar to 0.9 mu g kg(-1)) and for 2-MIB in channel catfish Ictalurus punctatus (0.7 mu g kg(-1)). Quantitative estimation of flesh-bound GSM using gas chromatography-mass spectrometry (GC-MS) agreed well with human sensory assessment scores and highlights the reliability of chemical analysis of GSM in barramundi flesh while also indicating the value of GC-MS analysis in predicting the impact of GSM on the sensory properties of farmed barramundi.

Impact of low temperature storage on active and storage forms of folate in choy sum (Brassica rapa subsp parachinensis)

Choy sum (Brassica rapa subsp. parachinensis) is a dark green leafy vegetable that contains high folate (vitamin B9) levels comparable to spinach. Folate is essential for the maintenance of human health and is obtained solely through dietary means. Analysis of the edible portion of choy sum by both microbiological assay and LC-MS/MS indicated that total folate activity remained significantly unchanged over 3 weeks storage at 4 degrees C. Inedible fractions consisted primarily of outer leaves, which showed signs of rotting after 14d, and a combination of rotting and yellowing after 21 d, contributing to 20% and 40% of product removal, respectively. Following deconjugation of the folate present in choy sum to monoglutamate and diglutamate derivatives, the principal forms (vitamers) of folate detected in choy sum were 5-methyltetrahydrofolate and 5-formyl tetrahydrofolate, followed by tetrahydrofolate (THF), 5,10-methenyl-THF, and 10-formyl folic acid. During storage, a significant decline in 5-formyl-THF was observed, with a slight but not significant increase in the combined 5-methyl-THF derivatives. The decline in 5-formyl-THF in relation to the other folate vitamers present may indicate that 5-formyl-THF is being utilised as a folate storage reserve, being interconverted to more metabolically active forms of folate, such as 5-methyl-THF. Although folate vitamer profile changed over the storage period, total folate activity did not significantly change. From a human nutritional perspective this is important, as while particular folate vitamers (e.g. 5-methyl-THF) are necessary for maintaining vital aspects of plant metabolism, it is less important to the human diet, as humans can absorb and interconvert multiple forms of folate. The current trial indicates that it is possible to store choy sum for up to 3 weeks at 4 degrees C without significantly affecting total folate concentration of the edible portion. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

Identification of the sex pheromone of the tree infesting Cossid Moth Coryphodema tristis (Lepidoptera: Cossidae)

The cossid moth (Coryphodema tristis) has a broad range of native tree hosts in South Africa. The moth recently moved into non-native Eucalyptus plantations in South Africa, on which it now causes significant damage. Here we investigate the chemicals involved in pheromone communication between the sexes of this moth in order to better understand its ecology, and with a view to potentially develop management tools for it. In particular, we characterize female gland extracts and headspace samples through coupled gas chromatography electro-antennographic detection (GC-EAD) and two dimensional gas chromatography mass spectrometry (GCxGC-MS). Tentative identities of the potential pheromone compounds were confirmed by comparing both retention time and mass spectra with authentic standards. Two electrophysiologically active pheromone compounds, tetradecyl acetate (14:OAc) and Z9-tetradecenyl acetate (Z9-14:OAc) were identified from pheromone gland extracts, and an additional compound (Z9-14:OH) from headspace samples. We further determined dose response curves for the identified compounds and six other structurally similar compounds that are common to the order Cossidae. Male antennae showed superior sensitivity toward Z9-14:OAc, Z7-tetradecenyl acetate (Z7-14:OAc), E9-tetradecenyl acetate (E9-14:OAc), Z9-tetradecenol (Z9-14:OH) and Z9-tetradecenal (Z9-14:Ald) when compared to female antennae. While we could show electrophysiological responses to single pheromone compounds, behavioral attraction of males was dependent on the synergistic effect of at least two of these compounds. Signal specificity is shown to be gained through pheromone blends. A field trial showed that a significant number of males were caught only in traps baited with a combination of Z9-14:OAc (circa 95 of the ratio) and Z9-14:OH. Addition of 14:OAc to this mixture also improved the number of males caught, although not significantly. This study represents a major step towards developing a useful attractant to be used in management tools for C. tristis and contributes to the understanding of chemical communication and biology of this group of insects.

Transfer of Dietary Plant Natural Flavor Compounds From Essential Oils to Maternal Fluids in Pigs

Fetal flavor conditioning during the perinatal stage could be essential at the time of the weaning to reduce the stress and improve the feed intake in pigs. The transfer of flavor compounds from maternal diet to amniotic fluid and milk has been shown in behavioral experiments, but not through analytical procedures such as gas chromatography–mass spectrometry (GC–MS). The aim of the experiment was to trace the principal essential oils compounds supplied in the diet in maternal fluids. Twenty Large White sows around their 104th gestational day were allocated to individual farrowing crates. Two groups of 10 sows were fed either a standard gestation diet or the same diet supplemented with a mix of 8 essential oils at a rate of 1kg/ton during the last 10 days of gestation. At approximately the 113th gestational day, animals were individually treated with 10mg of Lutalyse IM was to induce farrowing. Fresh amniotic fluid was collected during the farrowing in 100-mL glass bottles and immediately stored at −20 °C freezer. During the second lactation day, 10–20 IU of Oxytocin IM was administered to each sow to facilitate collection of milk samples in 20-mL glass bottles. The samples were stored at −20 °C until analyzed by GC–MS. The presence of significant amounts of principal components of all the essential oils except one were found in the milk and amniotic fluid samples of the treated sows relative to the control sows. Our data prove the transfer of selected dietary flavors to maternal fluids and sets the scenario for further trials to manipulate postweaning behavior in piglets.