Method 1 Method 2 Method 3 Method 4 Method 5
Conditioning of cartridges 2×4 mL methanol
2×4 mL water
5 mL methanol
5 mL water
2×2.5 mL acetone
2×2.5 mL methanol
2×5 mL water
3×2 mL hexane
2 mL acetone
3×2 mL methanol
3 mL methanol
3 mL water
pH of estrogens
 standard solutions
3 2 5 3 2
Washing step 2×5 mL methanol:water (1:1)
2×5 mL acetone:water (1:2)
5 mL water 25 mL hexane and water (1:4) 5×2 mL water 2×10 mL water
  Elution 2×4.5 mL methanol 10 mL acetonitrile 2×10 mL acetone 4 x1 mL acetone 20 mL methanol
Complete dryness of solvents Vacuum Vacuum Vacuum Vacuum Vacuum
Redissolution of estrogens 2 mL methanol 2 mL acetonitrile 2 mL methanol 2 mL methanol 2 mL methanol
Table 1: SPE methods for concentrating estrogens using standards of 20.00 μg L-1 of each compound.