Amanitins in Wild Mushrooms: The Development of HPLC-UV-EC and HPLC-DAD-MS Methods for Food Safety Purposes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Materials
2.2. Working Solutions, Calibration Curve, and Quality Control Samples
2.3. Mushroom Collection
2.4. Sample Preparation and Extraction Procedure
2.5. Chromatographic System and Conditions
2.6. Validation of the Method
2.6.1. Selectivity
2.6.2. Linearity
2.6.3. Precision
2.6.4. Limit of Detection (LOD) and Limit of Quantification (LOQ)
2.6.5. Recovery and Matrix Effect
2.7. Method Application
3. Results and Discussion
3.1. Optimization of Electrochemical Detection
3.2. Optimization of Sample Extraction Procedures
3.3. Optimization of the Chromatographic Conditions
3.4. Validation of the Method
3.5. Method Application
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compound | Detection | Range (µg mL−1) | Linearity (R2) | Linear Regression Equation |
---|---|---|---|---|
α-amanitin | EC | 0.5–20.0 | 0.9998 | y = 621,020x + 10,701 |
UV | 0.5–20.0 | 0.9993 | y = 36,624x + 5896.6 | |
β-amanitin | EC | 0.5–20.0 | 0.9997 | y = 346,265x + 31,251 |
UV | 0.5–20.0 | 0.9996 | y = 27,942x + 1373.6 |
Matrix | Mushroom Toxin | LOD (ng g−1) | LOQ (ng g−1) | Method | Reference |
---|---|---|---|---|---|
Mushroom tissue | α-amanitin β-amanitin | EC-62 UV-55 EC-24 UV-64 | EC-189 UV-168 EC-72.6 UV-193 | HPLC-UV-EC | Present Work |
Mushroom tissue | α-amanitin β-amanitin | 20 20 | NS | HPLC-ESI-MS | [42] |
Mushroom tissue | α-amanitin β-amanitin | 30 30 | NS | HPLC-TOF-MS | [30] |
Mushroom tissue | α-amanitin β-amanitin | 230 190 | 26.8 33.3 | HPLC-TOF-MS | [27] |
Mushroom tissue | α-amanitin β-amanitin | 2 2 | NS | HPLC-TOF-MS | [52] |
Compound | Detection | Concentration (µg mL−1) | Recovery (%) (RSD) | Matrix Effect (%) (RSD) |
---|---|---|---|---|
α-amanitin | EC | 0.5 | 117 (4.78) | 96.9 (8.01) |
2.0 | 99.4 (1.37) | |||
10.0 | 96.1 (1.48) | |||
UV | 0.5 | 104 (3.72) | 95.8 (8.87) | |
2.0 | 92.5 (1.64) | |||
10.0 | 96.8 (0.34) | |||
β-amanitin | EC | 0.5 | 114 (3.25) | 95.8 (11.4) |
2.0 | 103 (2.94) | |||
10.0 | 93.6 (0.48) | |||
UV | 0.5 | 92.0 (3.9) | 97.6 (10.9) | |
2.0 | 89.0 (2.7) | |||
10.0 | 93.3 (0.6) |
Compound | Detection | Concentration (μg mL−1) | Precision (%RSD) | |
---|---|---|---|---|
Intra-day | Inter-day | |||
α-amanitin | EC | 0.5 | 5.35 | 12.95 |
2.0 | 1.05 | 7.64 | ||
10.0 | 0.41 | 9.95 | ||
UV | 0.5 | 10.23 | 3.73 | |
2.0 | 1.13 | 2.46 | ||
10.0 | 0.62 | 11.75 | ||
β-amanitin | EC | 0.5 | 4.20 | 10.38 |
2.0 | 3.34 | 5.45 | ||
10.0 | 1.22 | 3.81 | ||
UV | 0.5 | 5.33 | 5.80 | |
2.0 | 4.55 | 3.69 | ||
10.0 | 0.69 | 3.51 |
Mushroom Species | HPLC-UV-EC |
---|---|
Amanita citrine; | |
Amanita pantherine; | |
Amanita boudieri; | |
Amanita gemmate; | |
Amanita muscaria; | |
Amanita rubescens; | |
Amanita vaginatae; | ND |
Amanita var alba; | |
Boletus edulis; | |
Cantharellus cibarius; | |
Tricholoma equestre; | |
Tricholoma joachimii; | |
Tricholoma portentosum | |
Amanita phalloides | α-amanitin: (EC) < > 290 µg g−1 β-amanitin: (EC) < > 280 µg g−1 α-amanitin: (UV) < > 280 µg g−1 β-amanitin: (UV) < > 288 µg g−1 |
Compounds (Formula) | Molecular Weight | Retention Time (min) | Protonated Molecular Ions (MS1, m/z) | Major Fragment Ions (MS2, m/z) |
---|---|---|---|---|
α-amanitin (C39H54N10O14S) | 918.97 | 18.1 | [M+H]+ 919.00 | 901.14 (100%) 661.43 (44.8%) 546.71 (60%) |
β-amanitin (C39H53N9O15S) | 919.95 | 16.6 | [M+H]+ 920.35 | 902.09 (100%) 661.77 (45%) 546.64 (64.8%) |
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Barbosa, I.; Domingues, C.; Barbosa, R.M.; Ramos, F. Amanitins in Wild Mushrooms: The Development of HPLC-UV-EC and HPLC-DAD-MS Methods for Food Safety Purposes. Foods 2022, 11, 3929. https://doi.org/10.3390/foods11233929
Barbosa I, Domingues C, Barbosa RM, Ramos F. Amanitins in Wild Mushrooms: The Development of HPLC-UV-EC and HPLC-DAD-MS Methods for Food Safety Purposes. Foods. 2022; 11(23):3929. https://doi.org/10.3390/foods11233929
Chicago/Turabian StyleBarbosa, Isabel, Cátia Domingues, Rui M. Barbosa, and Fernando Ramos. 2022. "Amanitins in Wild Mushrooms: The Development of HPLC-UV-EC and HPLC-DAD-MS Methods for Food Safety Purposes" Foods 11, no. 23: 3929. https://doi.org/10.3390/foods11233929