*4.5. Compared Strategies for Sample Preparation and Clean-Up*

In addition to IACs, several other techniques of sample preparation have been tested: SLE, QuEChERS, SPE with OASIS HLB cartridges and Mycosep 225 Trich column (contain a mixture of adsorbent materials). The protocol for sample preparation for SLE and QuEChERS was based on our previously described methods [41,42].

The OASIS HLB column was tested according to the manufacturer's protocol. Briefly, 1 g of sample was weighed into a 50 mL plastic tube and extracted for 30 min with 8 mL of H2O. Next, the sample was centrifuged and 2 mL of extract was transferred into OASIS HLB cartridges previously conditioned with 3 mL of MeOH and 3 mL of H2O. Subsequently, the column was washed with 5 mL of 5% MeOH and eluted with 3 mL of MeOH. All sample were collected and evaporated to dryness under a gentle nitrogen stream at 40 ◦C. The residue was dissolved in 200 µL of 0.2% CH3COOH and transferred to an autosampler vial.

For IAC DONTEST 1 g of sample was weighed into a 50 mL plastic tube and extracted for 30 min with 8 mL of H2O. Next, the sample was centrifuged and 2 mL of extract was passed onto DONTEST. Subsequently, the column was washed with 10 mL water and finally eluted with 2 mL methanol and collected to glass tube. After evaporation (N2, 40 ◦C) sample was dissolved in 200 µL of 0.2% CH3COOH and transferred to an autosampler vial.

For the sample preparation using for clean-up Mycosep 225 Trich column (based on the manufacturer's recommendation with slight modification) 1 g of previously milled sample was weighed into a 50 mL centrifuge tube. Next, 8 mL of ACN:H2O (84:16; *v*/*v*) mixture was added and was extracted in a rotary shaker for 30 min, followed by centrifugation for 15 min at 4000 rpm. Subsequently, 6 mL of supernatant was transferred to a glass tube and pushed through a Mycosep 225 Trich column. Purified extract (2 mL) was collected, and the sample was evaporated to dryness in a gentle nitrogen stream at 40 ◦C. The dry residue was reconstituted in 200 µL of 0.2% CH3COOH and transferred to an autosampler vial. For final optimized sample preparation additionally, 10 µL of MIX IS was added before sample evaporation. Extraction recovery (ER) and matrix effect (ME) were calculated to find an appropriate method for sample preparation. In this case, ER was calculated as the ratio of the area of the analyte(s) recorded for the sample spiked with the target compound(s) before extraction to the area for the spiked sample after extraction.
