A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass
"> Figure 1
<p>Extracted ion chromatogram (EIC) of peramine, ergovaline, ergotamine and lolitrem B in perennial ryegrass with standard toxic endophyte (E+). Ergotamine was used as an internal standard (IS) for ergovaline.</p> "> Figure 2
<p>Comparison of mean alkaloid concentrations (parts per million, ppm; mg/kg) in eight different glasshouse-grown ryegrass–endophyte associations using the Thermo Q Exactive Plus (QE) and Agilent 6460C Triple Quadruple (QQQ) mass spectrometers, and the Agilent 1100 fluorescence detector (FLD): (<b>a</b>) peramine, (<b>b</b>) ergovaline and (<b>c</b>) lolitrem B. No alkaloids were detected in Trojan-WE (omitted). Bars represent the standard error of the mean (SEM). Bars with different letters are significantly different when compared to the QE (<span class="html-italic">t</span>-test, <span class="html-italic">p</span> < 0.05).</p> ">
Abstract
:1. Introduction
2. Results
2.1. Method Performance
2.2. LOD, LOQ and Linearity
2.3. Accuracy (Bias), Precision and Selectivity
2.4. Extraction of Alkaloids
2.5. Matrix Effect and Recovery
2.6. Method Robustness and Application
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Chemicals
5.2. Plant Material
5.3. Sample Preparation
5.4. Sample Extraction
5.5. Method Validation
5.6. Analytical Instruments and Conditions
5.7. Method Application
5.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Compound | RT | Ion Mass | LOD | LOQ | Standard Concentrations (ng/mL) | Equation | R2 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
(min) | (m/z) | (ng/mL) | (ng/mL) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |||
peramine | 3.59 | 248.1501 | 0.2 | 0.8 | 0.8 | 8.0 | 19.9 | 39.8 | 79.7 | 199.2 | 398.5 | 796.9 | 1593.9 | y = 217004x | 0.9912 |
ergovaline/ergotamine 1 | 5.34/5.85 | 534.2709/582.2701 | 0.2 | 0.8 | 0.8 | 8.4 | 21.0 | 42.1 | 84.2 | 210.5 | 421.0 | 842.0 | 1684.0 | y = 54031x | 0.9998 |
lolitrem B | 11.07 | 686.4037 | 1.8 | 6.0 | 6.0 | 12.0 | 30.0 | 60.0 | 120.0 | 300.0 | 600.0 | 1200.0 | 2400.0 | y = 12113x | 0.9970 |
Standard | Actual Concentration (ng/mL) | Measured Concentration (ng/mL) | Accuracy (bias) (%) | Precision (% RSD) | |
---|---|---|---|---|---|
peramine | Low | 8.0 | 7.9 | 100.2 | 3.5 |
Med | 79.7 | 79.2 | 99.7 | 3.4 | |
High | 796.9 | 792.3 | 99.6 | 1.0 | |
ergotamine | Low | 8.4 | 7.9 | 99.1 | 1.4 |
Med | 84.2 | 83.2 | 99.4 | 2.4 | |
High | 842.0 | 843.9 | 99.9 | 2.6 | |
lolitrem B | Low | 12.0 | 11.9 | 99.3 | 2.3 |
Med | 120.0 | 119.3 | 99.5 | 3.1 | |
High | 1200.0 | 1194.8 | 100.0 | 2.8 |
Peramine | Ergovaline | Lolitrem B | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
Extract | Mean ± SD | RSD (%) | Σ | Mean Conc. (ng/mL) | RSD (%) | Σ | Mean Conc. (ng/mL) | RSD (%) | Σ | |
Method 1 | 1 | 32.2 ± 0.1 | 0.4 | 39.5 | 0.1 ± 0.01 | 2.2 | 0.1 | 13.6 ± 0.2 | 1.2 | 15.3 |
2 | 7.3 ± 0.2 | 3.3 | - | - | - | - | 1.7 ± 0.1 * | 4.2 | - | |
3 | 2.8 ± 0.1 * | 5.0 | - | - | - | - | 0.2 ± 0.02 * | 7.5 | - | |
4 | 1.2 ± 0.1 * | 5.6 | - | - | - | - | - | - | - | |
5 | 0.6 ± 0.1 * | 8.3 | - | - | - | - | - | - | - | |
Method 2 | 1 & 2 | 33.2 ± 0.1 | 0.4 | - | 0.1 ± 0.003 | 2.0 | - | 14.3 ± 0.8 | 5.4 | - |
QC Sample | Spike 1 | Peramine | Ergotamine | Lolitrem B | |||
---|---|---|---|---|---|---|---|
RE | ME | RE | ME | RE | ME | ||
E− | low | 77 | 66 | 85 | 76 | 87 | 84 |
med | 79 | 70 | 87 | 77 | 86 | 80 | |
high | 81 | 81 | 85 | 76 | 80 | 71 | |
E+ | low | 88 | 61 | 85 | 74 | 84 | 88 |
med | 85 | 67 | 87 | 74 | 90 | 78 | |
high | 83 | 67 | 86 | 73 | 86 | 67 |
Cultivar/Variety | Endophyte Strain | Qualitative Alkaloid Profile | Supplier |
---|---|---|---|
Alto | SE | P, E, L | Barenbrug Agriseeds |
Alto | NEA3 | P, E | Barenbrug Agriseeds |
Trojan | WE 1 | nil | Barenbrug Agriseeds |
Trojan | NEA6 | P, E | Barenbrug Agriseeds |
Trojan | NEA10 | P, E | Barenbrug Agriseeds |
Trojan | NEA11 | P, E | Barenbrug Agriseeds |
Trojan | NEA47 | P, E | Barenbrug Agriseeds |
LP534 | NEA21 | P, L | Barenbrug Agriseeds |
Shogun 2 | NEA2 | P, E, L (low) | Barenbrug Agriseeds |
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Vassiliadis, S.; Elkins, A.C.; Reddy, P.; Guthridge, K.M.; Spangenberg, G.C.; Rochfort, S.J. A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass. Toxins 2019, 11, 649. https://doi.org/10.3390/toxins11110649
Vassiliadis S, Elkins AC, Reddy P, Guthridge KM, Spangenberg GC, Rochfort SJ. A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass. Toxins. 2019; 11(11):649. https://doi.org/10.3390/toxins11110649
Chicago/Turabian StyleVassiliadis, Simone, Aaron C. Elkins, Priyanka Reddy, Kathryn M. Guthridge, German C. Spangenberg, and Simone J. Rochfort. 2019. "A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass" Toxins 11, no. 11: 649. https://doi.org/10.3390/toxins11110649