2.5. Sample Preparation

1 g of sample (±0.0001 g) was weighed, and it was mixed with 10 mL of cold water acidified with HCl (pH 1.0). The mixture was stirred for 15 min at room temperature. Later, 0.1 mL of Carrez I and 0.1 mL of Carrez II were added to the mixture, the sample was shaken for 5 min and then it was centrifuged at 6000 rpm for 10 min using a Rotofix 32A centrifuge (Hettich, Tuttlingen, Germany). The supernatant was separated from the precipitate and frozen for one hour. After this time, the frozen supernatant was again centrifuged at 6000 rpm for 10 min and filtered through a 0.45 µm nylon filter. Finally, 1 mL of sample extract was purified by SPE (Figure 1).

Schematic diagram of sample preparation: solid-liquid extraction (SLE) step and solid-phase extraction (SPE) step.

To carry out the SPE procedure, 100 mg of the SBA-15-LP-NH₂ was packed in empty SPE cartridges of 3 mL capacity. For this, the material was plugged with frits of polyethylene at both ends and a nylon membrane (0.45 µm) on the lower frit was used to avoid the loss of material. After the cartridges were packed, the SPE procedure was carried out using a Supelco Visiprep SPE vacuum manifold 12 port model (Sigma Aldrich, St. Louis, MO, USA) connected to a vacuum pump at 7.6 psi. In the first place, 2 mL of acidified water (pH 1.0, HCl) conditioned the cartridge and later, 1 mL of sample extract was loaded and passed through the cartridge. Afterwards, the cartridge was washed with 2 mL of Hex to eliminate interferences and, finally, the analytes were eluted with 0.2 mL of MeOH and later with 1.8 mL of water (Figure 1). The extract was filtered through a 0.45 µm nylon filter and it was injected into the HPLC-QqQ-MS/MS. All solvents used in the SPE procedure were previously cooled in a fridge at a temperature of between 0–5 °C.