A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass

Abstract

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography–mass spectrometry (LC–MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC–MS (for peramine). This work provides the first highly sensitive quantitative LC–MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Ergot alkaloid mycotoxins: physiological effects, metabolism and distribution of the residual toxin in mice

“…All animal studies were approved by the La Trobe University Animal Ethics Committee (Protocol number 18-21) and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes set out by the National Health and Medical Research Council of Australia. The mice were housed in groups of two to four during the experimental period in individually-ventilated cages (Tecniplast, Buguggiate, Italy) with standard pellet food and water available ad libitum 42 . Ambient temperature of housing and testing rooms was 21 ± 2 °C and mice were housed under a 12-h light-dark cycle (lights on at 7am) 42 .…”

“…The mice were housed in groups of two to four during the experimental period in individually-ventilated cages (Tecniplast, Buguggiate, Italy) with standard pellet food and water available ad libitum 42 . Ambient temperature of housing and testing rooms was 21 ± 2 °C and mice were housed under a 12-h light-dark cycle (lights on at 7am) 42 . A total of 48 male 8-12-week-old C57Bl/6 J mice were sourced from a breeding colony at the Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria.…”

“…A total of 48 male 8-12-week-old C57Bl/6 J mice were sourced from a breeding colony at the Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria. Animals were allowed to acclimatise to the facility conditions for a period of one week prior to behavioural testing 42 . All animals underwent rotarod testing to examine effects of intoxication on movement and tail plethysmography to observe vasomotor effects.…”

“…Sample tubes were placed into pre-frozen 24 well cryo-blocks on the Geno/Grinder 2010 (SPEX Sample Prep, Metuchen, NJ, USA) and the tissues were homogenised at 1,700 rpm for 1-5 min. The fine powder was stored at −80 °C until weighed 42 .…”

Alkaloid Concentrations of Lolium perenne Infected with Epichloë festucae var. lolii with Different Detection Methods—A Re-Evaluation of Intoxication Risk in Germany?

“…Many of the published toxicity thresholds for peramine, lolitrem B, and ergovaline are based on analyses of plant dry weight. In best practice, new methods need to be validated, compared to other licensed methods, and tested for their robustness [32,43], before they can be licensed for the quantitation of alkaloids. However, there can be essential differences in methods, which might lead to different concentrations.…”