Add 1 mL 1 mol/L HCl to 10 mL spiked milk (1 µg/mL or 0.1 µg/mL). Then add 10 mL methylene chloride, vortex to mix and centrifuge for 15 min at 4500 rpm. Remove the aqueous layer, add 5 mL 0.1 mol/L HCl to the organic layer, vortex to mix for 1 min and centrifuge for 15 min at 4500 rpm. Combine the aqueous phases and add once more 5 mL 0.1 mol/L HCl to the organic layer, vortex and centrifuge. At least combine all aqueous phases for SPE.

Column conditioning: 5 mL methanol, then 5 mL water (Do not let run the column dry.)

Sample aspiration: The prepared sample is passed through the column by gravity or low vacuum.

Washing: 5 mL 0.1 mol/L HCl, then 5 mL methanol

Drying: 10 min vacuum

Elution: 2 x 2.5 mL methanol / 5 % NH3

Evaporation: Nitrogen stream

Reconstitution: With a suitable solvent for the subsequent analysis.