🍄 Mycotoxins — aflatoxins, ochratoxin A
Determination of mycotoxins (aflatoxins B1, B2, G1, G2, and ochratoxin A) in food by immunoaffinity column (IAC) cleanup with HPLC-FLD detection.
Overview
Mycotoxins are toxic secondary metabolites of molds (Aspergillus, Penicillium, Fusarium) posing a serious threat to human and animal health. Aflatoxin B1 is the most potent known natural carcinogen (IARC Group 1), and ochratoxin A is nephrotoxic and probably carcinogenic (Group 2B). EU regulations (EC Reg. 1881/2006, EU Reg. 2023/915) establish strict limits in food: aflatoxin B1 2–12 µg/kg, sum of aflatoxins 4–15 µg/kg, ochratoxin A 3–10 µg/kg.
The analytical method based on EN 14123 (aflatoxins) and EN 15851 (ochratoxin A) standards involves sample extraction with acetonitrile/water (or methanol/water) mixture, cleanup on immunoaffinity columns (IAC), and determination by HPLC with fluorescence detection (FLD). IAC columns contain monoclonal antibodies specifically binding target mycotoxins, ensuring high cleanup selectivity.
Aflatoxins B1 and G1 require post-column fluorescence derivatization (bromination with iodine or cobalt, photochemical UV, or electrochemical KOBRA Cell) to enhance fluorescence signal. Aflatoxins B2 and G2 fluoresce natively. Ochratoxin A has strong native fluorescence and does not require derivatization.
Alternatively, LC-MS/MS detection (without derivatization) is used, allowing simultaneous determination of multiple mycotoxins (aflatoxins, ochratoxin, deoxynivalenol, zearalenone, fumonisins, T-2/HT-2) in one test (multi-mycotoxin method). VICAM Myco6in1+ or R-Biopharm EASI-EXTRACT columns enable simultaneous cleanup of multiple mycotoxins.
Method principle
Extraction: Sample (25–50 g) is extracted with acetonitrile/water (80:20 v/v) or methanol/water (80:20) mixture with NaCl. IAC cleanup: Diluted extract is passed through immunoaffinity column (e.g., VICAM AflaOchra, R-Biopharm EASI-EXTRACT). Mycotoxins selectively bind to antibodies on column, while matrix contaminants are washed with water. Mycotoxins are eluted with methanol (1–3 mL). HPLC-FLD detection: Eluate is injected onto C18 column. Aflatoxins are derivatized (KOBRA Cell — electrochemical bromination). Fluorescence detection: λex 365 nm / λem 435 nm (aflatoxins), λex 333 nm / λem 460 nm (ochratoxin A). Quantification from calibration curves.
Applications
- Official control of aflatoxins in nuts, cereals, spices (EU Reg. 2023/915)
- Ochratoxin A determination in cereals, coffee, wine, cocoa
- Feed and feed material control (aflatoxin B1)
- Infant food testing (lowest limits: 0.10 µg/kg)
- Milk monitoring — aflatoxin M1 (limit 0.050 µg/kg)
- Dried fruit control (figs, raisins, apricots)
- BRC/IFS certification of imported raw materials
Key parameters
| Parameter | Value |
|---|---|
| Analytes | Aflatoxins B1, B2, G1, G2; Ochratoxin A |
| LOQ aflatoxin B1 | 0.05–0.50 µg/kg |
| LOQ ochratoxin A | 0.10–1.0 µg/kg |
| Recovery | 70–110% (depending on matrix and analyte) |
| Detection | HPLC-FLD (fluorescence) or LC-MS/MS |
| EU limits (afla B1) | 2–12 µg/kg (depending on product) |
Standard
- Standard number
- PN-EN 14123:2008 / PN-EN 15851:2010
- Title (PL)
- Żywność — Oznaczanie aflatoksyn B1, B2, G1, G2 i ochratoksyny A z zastosowaniem kolumnek immunopowinowactwa i HPLC
- Title (EN)
- Foodstuffs — Determination of aflatoxins B1, B2, G1, G2 and ochratoxin A using immunoaffinity column clean-up and HPLC
Step-by-step procedure
1. Sample preparation
Grind/homogenize sample (min. 100 g). Weigh 25 g into conical flask.
2. Extraction
Add 5 g NaCl and 100 mL acetonitrile/water (80:20). Shake vigorously 30 min (orbital shaker).
3. Filtration and dilution
Filter through folded filter. Take aliquot (e.g., 10 mL) and dilute with PBS to 50 mL (reduce MeCN concentration < 25%).
4. IAC cleanup
Pass diluted extract through IAC column (flow 1–2 drops/s). Mycotoxins bind to antibodies.
5. Washing
Wash column with 20 mL deionized water (removing matrix contaminants). Apply vacuum 30 s.
6. Elution
Elute mycotoxins with 1.5 mL methanol (flow 1 drop/2 s). Collect into HPLC vial. Evaporate to dryness under N₂ and reconstitute in mobile phase.
7. HPLC injection
Inject 50–100 µL onto C18 column. Mobile phase: H₂O/MeCN/MeOH with acetic acid. Gradient or isocratic.
8. FLD detection
Fluorescence detection with KOBRA cell: aflatoxins λex 365 / λem 435 nm, ochratoxin A λex 333 / λem 460 nm.
9. Quantification
Calculate concentrations from calibration curves (5–7 points). Include dilution factor and sample mass.
10. Quality control
Every series: blank, fortified sample, CRM (FAPAS/BIPEA). Aflatoxin recovery 70–110%, ochratoxin 70–110%. PT participation.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| HPLC system with FLD detector | Shimadzu Nexera + RF-20Axs, Agilent 1260 Infinity II + FLD, Waters Alliance + FLR | 150 000–350 000 PLN |
| LC-MS/MS system (alternative) | Shimadzu LCMS-8060NX, Waters Xevo TQ-XS | 600 000–1 200 000 PLN |
| KOBRA derivatization cell | R-Biopharm KOBRA Cell (electrochemical bromination) | 15 000–25 000 PLN |
| Immunoaffinity columns (IAC) | VICAM AflaTest / OchraTest / AflaOchra, pack of 25 pcs. | 2 500–5 000 PLN / 25 pcs. |
| HPLC C18 column | Waters Symmetry C18 (150 × 4.6 mm, 5 µm), Phenomenex Kinetex | 1 500–3 000 PLN |
| Vacuum system for IAC | 12/24-position vacuum manifold, SUPELCO Visiprep | 3 000–8 000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Acetonitrile HPLC grade | 75-05-8 | HPLC mobile phase and extraction, 2.5 L |
| Methanol HPLC grade | 67-56-1 | IAC elution and mobile phase, 2.5 L |
| Aflatoxin B1, B2, G1, G2 standards | — | Certified CRM (Romer Labs, Trilogy), solutions in MeCN, 5 mL |
| Ochratoxin A standard | 303-47-9 | Certified CRM, solution in MeCN or toluene, 1 mL |
| Potassium bromide KBr (KOBRA Cell) | 7758-02-3 | Salt for electrochemical cell, in-situ Br⁻ generation |
| Nitric acid HNO₃ 4 mol/L (KOBRA Cell) | 7697-37-2 | Electrolyte for KOBRA cell |
| PBS (phosphate buffer pH 7.4) | — | For diluting extract before IAC, tablets or ready-made |
| Sodium chloride NaCl | 7647-14-5 | Extraction additive (improving phase separation) |
Health and safety (OHS)
- Aflatoxins — highly carcinogenic (IARC Group 1). Use double gloves, work in dedicated area.
- Mycotoxin standards — toxic. Store in freezer (-20°C). Prepare solutions under fume hood.
- Acetonitrile — flammable and toxic (H225, H302, H312, H332). Work under fume hood.
- Methanol — flammable and toxic (H225, H301, H311, H331). Toxic by inhalation and ingestion.
- Aflatoxin decontamination — sodium hypochlorite solution (5% NaOCl) neutralizes aflatoxins on surfaces.
- Mycotoxin-containing waste — collect separately and chemically neutralize before disposal.