A Simple Thin-Layer Chromatographic Method for the Detection of Ergovaline in Leaf Sheaths of Tall Fescue (Festuca Arundinacea) Infected with Neotyphodium Coenophialum

Abstract

Abstract.A relatively simple and inexpensive thin-layer chromatographic (TLC) method is described for the detection and semiquantitative measurement of ergovaline in leaf sheaths of tall fescue (Festuca arundinacea). Samples were finely ground and extracted with methanol. The extracts were filtered and the methanol was evaporated. The aqueous residue was extracted with hexane, followed by chloroform at pH 9. The chloroform extract was concentrated and further purified on a preparative silica gel TLC plate, developed with toluene/ethyl acetate/acetonitrile (50:10:40). The ergovaline band was scraped and eluted with methanol. The eluant was concentrated and an aliquot was applied to a silica gel TLC plate. The plate was developed successively with chloroform/acetone/acetic acid (90:10:5) and chloroform/ethanol (9:1). Ergovaline was visualized with p-dimethylaminobenzaldehyde and sulfuric acid. Semiquantitation of ergovaline was achieved by comparison with a known standard of ergotamine, which was shown to have the same Rf as ergovaline in this system. Spike recovery of ergotamine averaged 60%, with a limit of detection of 200 g/kg of dry tall fescue leaf sheaths. The method was applied to 15 tall fescue samples with varying degrees of fungal infection, and ergovaline was identified in all contaminated samples with endophyte infection above 15%. Thin-layer chromatography may be also applicable for tall fescue seed, where the ergovaline content is usually higher and the amount of interfering pigments is much lower.Tall fescue (Festuca arundinacea) is one of the most widely used forage pastures in many countries due to its excellent agronomic properties. However, tall fescue pastures may be toxic, giving way to a disease that may have serious consequences for livestock. 4,17 The symptoms may be variable, depending on the climatic season. In the summer, the most frequent alterations consist of severe discomfort, with pronounced hyperthermia, accelerated respiratory rate, excessive salivation, rough hair coat, lowered milk production, and decreased forage intake, whereas in very cold winter seasons, peripheral circulatory collapse may develop. 7 Generally, the toxicity does not produce serious impairment or death of the animals, but they may suffer a host of subclinical problems, such as low weight gain, reduced reproductive efficiency, reduced milk production, decreased growth rate, etc. These symptoms are reminiscent of the syndrome known as ergotism, which is caused by consumption of several fungal toxins collectively known as ergot alkaloids. 3 In 1977, it was reported that tall fescue toxicity is associated with the infection of plants and seeds with an endophytic fungus, formerly known as Acremonium coenophialum and now renamed Neotyphodium coenophialum. 5,6 This organism was able to synthesize several ergot alkaloids in in vitro cultures. 12 These alkaloids were later detected in the tall fescue plants infected with the fungus. 18 Noninfected plants did not produce ergot alkaloids nor cause animal toxicity. Take...

Development and validation of a new LC–MS/MS method for the simultaneous determination of six major ergot alkaloids and their corresponding epimers. Application to some food and feed commodities

“…It is therefore important to consider both epimers when the ergot alkaloid contamination level of a cereal or cereal product has to be determined. Different methods have been reported for the analysis of ergot alkaloids, including thin layer chromatography (TLC) (Salvat & Godoy, 2001), capillary electrophoresis (CE) (Franch & Blaschke, 1998), enzyme linked immunosorbent assay (ELISA) (Hill et al, 2001), gas chromatography (GC) with electron capture detection (ECD) (Barrow & Quigley, 1975), liquid chromatography (LC) with ultraviolet (Veress, 1993), fluorescence (Komarova & Tolkachev, 2001;Storm et al, 2008) or mass spectrometric (MS) (Burk et al, 2006;Kokkonen & Jestoi, 2010;Krska, Stubbings, Macarthur, & Crews, 2008;Mohamed, Gremaud, Rychoz-Payot, Tabet, & Guy, 2006) detection. In recent years, LC-MS has become the method of choice for mycotoxin determination (Shephard et al, 2011;Spanjer, 2010), and has provided an unequivocal identification of ergot alkaloids in various matrices (Friedrich et al, 2004;Lehner, Craig, Fannin, Bush, & Tobin, 2005;Mohamed et al, 2006).…”

Mass spectral characterization of ergot alkaloids by electrospray ionization, hydrogen/deuterium exchange, and multiple stage mass spectrometry: usefulness of precursor ion scan experiments

“…Several analytical techniques have been used to monitor the presence of such contamination in food products. Thin‐layer chromatography (TLC),14, 15 high‐performance liquid chromatography (HPLC) with ultraviolet16, 17 or fluorimetric18, 19 detection, a gas liquid chromatography system (GLC) with electron capture detection,20, 21 and gas chromatography coupled to mass spectrometry (GC/MS)22, 23 have been reported for the determination of ergot alkaloids and lysergic acid derivatives. Several matrices were studied such as cereals and cereal products22, 24, 25 and human and animal biological fluids 15, 16, 21, 23, 26.…”

An integrated targeted and untargeted approach for the analysis of ergot alkaloids in cereals using UHPLC – hybrid quadrupole time-of-flight mass spectrometry

“…Evaluation of the exposure and actual risk due to consumption of contaminated food and feedstuffs requires suitable analytical strategies. Analytical methods for the determination of ergot alkaloids include thin layer chromatography (TLC) (Salvat and Godoy, 2001), capillary electrophoresis (CE) (Franch and Blaschke, 1998), enzyme linked immunosorbent assay (ELISA) (Hill et al, 2001), gas chromatography (GC) with electron capture detection (ECD) (Barrow and Quigley, 1975), liquid chromatography (LC) with ultraviolet (Veress, 1993), fluorescence (Komarova and Tolkachev, 2001a;Storm et al, 2008) or mass spectrometric (MS) (Diana di Mavungu et al, 2012;Burk et al, 2006;Kokkonen and Jestoi, 2010;Krska et al, 2008a;Mohamed et al, 2006b) detection. Over the last decade, LC-MS has become a dominant tool for mycotoxin determination, and has provided an unequivocal identification of ergot alkaloids in various matrices (Diana di Mavungu et al, 2012;Friedrich et al, 2004;Kokkonen and Jestoi, 2009;Krska et al, 2008;Lehner et al, 2005;Mohamed et al, 2006a;Mohamed et al, 2006b).…”