Quantitative Determination of Ochratoxin A
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Variable analysis
- Addition of 20 μl of internal standard (13C-OTA)
- Addition of 20 μl of β-glucuronidase
- Incubation for 18 hours at 37°C
- Addition of 1 cm3 of MeOH and 1.96 cm3 of ACN
- Vortexing for 2 minutes
- Centrifugation for 10 minutes at 7,000 rpm
- Evaporation of a 3 ml portion of the supernatant in a stream of nitrogen
- Dissolution of the samples in 1 cm3 of MeOH
- Addition of 25 cm3 of PBS
- Quantitative transfer to an OchraPrep immunoaffinity column
- Washing the column with 20 cm3 of distilled water
- Elution of OTA with 1.5 cm3 of MeOH:CH3COOH (98:2)
- Evaporation of the eluted sample in a stream of nitrogen at 40°C
- Dissolution of the sample in 150 μl of H2O:MeOH (7:3) mixture
- Concentration of OTA in the serum sample
- Volume of serum (1 cm3)
- Temperature of incubation (37°C)
- Centrifugation speed (7,000 rpm)
- Composition of the elution solvent (MeOH:CH3COOH, 98:2)
- Addition of 13C-OTA as an internal standard
- No negative controls are explicitly mentioned in the protocol
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