Extraction steps | Method 1 | Method 2 | Method 3 | Method 4 (Martins et al. (2014)) |
---|---|---|---|---|
1) Sample amount | 3g | 10g | 10g | 0.5g |
2) Type of container | 50mL Falcon tube | 2mL Eppendorf tube | ||
3) Mixed internal standard solution, 1MRL (EU2377/90) | 150μL | 150μL | 500μL | 12.5μL |
4) EDTA, 0.1M | 200μL | 200μL | 667μL | 30μL |
5) Mixture methanol/acetonitrile | 7mL | 7mL | 23mL | / |
6) Sodium sulfate anhydride | 3g | 3g | 3g | / |
7) Mixed standard solution* | 75μL | 75μL | 250μL | 12.5μL |
8) Vortexing | 1min | 10sec and equilibration for 10min from light | ||
9) Centrifugation | 10000rpm, 10min at 4°C |  | ||
10) Deprotonization |  |  |  | 0.6mL Ethanol/acetic acid (96/4) Followed by short vortexing and 30min at -18°C incubation |
11) Supernatant | 5mL evaporated to dryness at 40°C followed by addition of 300 μL of water MilliQ | / | ||
12) Vortex and centrifugation | Few seconds 12000rpm during 30min | Â | ||
13) Supernatant | All supernatant | 0.75mL with addition of 0.25mL Formic acid 0.1% in water/ formic acid in acetonitrile (98:2) | ||
14) Filtration | 0.2μm filter | / |