A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass
The rapid identification and quantitation of alkaloids produced by <i>Epichloë</i> 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 l...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2019-11-01
|
| Series: | Toxins |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2072-6651/11/11/649 |
| _version_ | 1798039336321548288 |
|---|---|
| author | Simone Vassiliadis Aaron C. Elkins Priyanka Reddy Kathryn M. Guthridge German C. Spangenberg Simone J. Rochfort |
| author_facet | Simone Vassiliadis Aaron C. Elkins Priyanka Reddy Kathryn M. Guthridge German C. Spangenberg Simone J. Rochfort |
| author_sort | Simone Vassiliadis |
| collection | DOAJ |
| description | The rapid identification and quantitation of alkaloids produced by <i>Epichloë</i> 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 <i>Epichloë</i>-infected perennial ryegrass (<i>Lolium perenne</i> 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. |
| first_indexed | 2024-04-11T21:52:29Z |
| format | Article |
| id | doaj.art-bef22f03fdfa4174a283e55bed32083a |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-04-11T21:52:29Z |
| publishDate | 2019-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-bef22f03fdfa4174a283e55bed32083a2022-12-22T04:01:12ZengMDPI AGToxins2072-66512019-11-01111164910.3390/toxins11110649toxins11110649A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial RyegrassSimone Vassiliadis0Aaron C. Elkins1Priyanka Reddy2Kathryn M. Guthridge3German C. Spangenberg4Simone J. Rochfort5Agriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaAgriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, VIC 3083, AustraliaThe rapid identification and quantitation of alkaloids produced by <i>Epichloë</i> 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 <i>Epichloë</i>-infected perennial ryegrass (<i>Lolium perenne</i> 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.https://www.mdpi.com/2072-6651/11/11/649<i>epichloë endophyte</i><i>lolium perenne</i> l.ryegrassalkaloidmycotoxinsquantitationliquid chromatography–mass spectrometry |
| spellingShingle | Simone Vassiliadis Aaron C. Elkins Priyanka Reddy Kathryn M. Guthridge German C. Spangenberg Simone J. Rochfort A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass Toxins <i>epichloë endophyte</i> <i>lolium perenne</i> l. ryegrass alkaloid mycotoxins quantitation liquid chromatography–mass spectrometry |
| title | A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass |
| title_full | A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass |
| title_fullStr | A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass |
| title_full_unstemmed | A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass |
| title_short | A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass |
| title_sort | simple lc ms method for the quantitation of alkaloids in endophyte infected perennial ryegrass |
| topic | <i>epichloë endophyte</i> <i>lolium perenne</i> l. ryegrass alkaloid mycotoxins quantitation liquid chromatography–mass spectrometry |
| url | https://www.mdpi.com/2072-6651/11/11/649 |
| work_keys_str_mv | AT simonevassiliadis asimplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT aaroncelkins asimplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT priyankareddy asimplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT kathrynmguthridge asimplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT germancspangenberg asimplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT simonejrochfort asimplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT simonevassiliadis simplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT aaroncelkins simplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT priyankareddy simplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT kathrynmguthridge simplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT germancspangenberg simplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass AT simonejrochfort simplelcmsmethodforthequantitationofalkaloidsinendophyteinfectedperennialryegrass |