*3.5. Sample Preparation*

In the present study, defatted bovine milk was used and the proteins' precipitation was achieved by adding 3 mL of ACN in 1.5 g of milk. The pH was adjusted to 5.0 by using 0.5 mL of buffer solution (70% CH3COONa 0.2 M/30% CH3COOH 0.2 M). The sponge was initially placed in a vial containing 1.5 g of milk and the system was centrifuged at low rpm for 15 min. The material was rinsed with deionized water and then squeezed to wash the water away. Subsequently, 1.5 mL of 1% CH3COOH/ACN/MeOH solution (50:40:10 *v*/*v*/*v*) was added to the sponge and the analytes were eluted by centrifugation at low rpm for 15 min. The eluent was filtered and injected in the HPLC column.

In the case of fat containing milk samples, centrifugation was applied for fat removal prior to deproteinization. Moreover, sample preconcentration was applied by evaporation of elution solvent prior to HPLC analysis and reconstitution to 100 μL when necessary and in order to reach the legislation demands.

## *3.6. Standard Solution Preparation*

For the chromatographic analysis, stock standard solutions of each analyte were prepared at a concentration of 100 ng μL−<sup>1</sup> using a solvent with the same composition as the mobile phase. Stock standard solutions were stable for six months at 4 ◦C, while working standards were prepared on a daily basis. The calibration curves were constructed by the use of solutions being prepared at concentrations of 0.5–10 ng μL−1.
