Nylon filter

Two seized samples (A and B) collected during investigative operations by RIS between September 2017 and December 2018 were analyzed by LC–HRMS. Since these samples are connected to criminal activity, it is not possible to provide further information such as location and exact data collection.
Seized samples were stored at room temperature until extraction, then 1 mg of seizure was extracted with 1 mL of methanol, vortexed for 1 min, sonicated at 25°C for 10 min and finally filtered through a 0.22 μm nylon filter from Agilent (Santa Clara, CA, USA). The obtained extracts are diluted 1: 10000 and subsequently 5 μL are injected into the UHPLC system.

All solvents were purchased from Sigma-Aldrich (Taufkirchen, Germany). Water was purified by a Milli-Qplus system from Millipore (Milford, MA, USA). Sephadex LH-20 was purchased from Sigma-Aldrich. Nylon filters (0.45 µm pore size) were from Agilent (Agilent Technologies, Palo Alto, CA, USA).

The solvents used for the isolation of the flavonoids were of reagent grade, whereas the solvents used for HPLC analysis were HPLC grade. All solvents were purchased from Sigma-Aldrich (MilliporeSigma, Burlington, MA, USA). Water was purified by a Milli-Qplus system from Millipore (Milford, MA, USA). Sephadex LH-20 was purchased from Sigma-Aldrich. Nylon filters (0.45 µm pore size) were from Agilent (Agilent Technologies, Palo Alto, CA, USA). Rosmarinic acid (97% purity) was purchased from Alfaesar (Kandel, Germany). Apigenin 7-O-glucoside (≥99% purity) and luteolin 7-O-glucoside (>98% purity) were purchased from Extrasynthèse (Genay, France). A series of stock solutions were prepared and kept at −20 °C in 100% methanol. From these stock solutions, a series of fresh working solutions were prepared immediately prior to analysis.

The analysis of amino acids was carried out to demonstrate enzyme immobilization. For that purpose, the immobilized enzyme was hydrolyzed with 6 M HCl using a Milestone ETHOS D microwave oven from Gomensoro (Madrid, Spain) under argon conditions at 170 °C for 20 min. After digestion, samples were diluted up to 10 mL with Milli-Q water and filtered with nylon filters (13 mm diameter and 0.45 μm pore size) from Agilent Technologies (Palo Alto, CA, USA). Amino acids were analyzed by RP-HPLC previous derivatization with OPA using a Hypersil AA-ODS column (2.1 mm × 200 mm, 5 μm particle size) from Agilent Technologies. OPA derivatization was carried out in the injector by mixing 8 μL of OPA, 6 μL of borate buffer, 4 μL of sample, and 6 μL of borate buffer. The reaction took 2 min and immediately after, samples were analyzed by RP-HPLC. The chromatographic conditions were: mobile phase A, sodium acetate (50 mM, pH 7.2); mobile phase B, ACN; injection volume, 24 μL; flow rate, 0.5 mL/min; column temperature, 20 °C; elution gradient, 0–50% B for 40 min, 50–90% B for 5 min, and 90% B for 5 min. Fluorescence detection was recorded at a λexc of 340 nm and a λem of 450 nm. Amino acid identification was carried out by comparison with amino acid standards.

All used reagents were GC or HPLC grade. Chloroform (cat no. 34854), methanol (cat no. 1.06018), hexane (cat no. 270504), acetonitrile (cat no. 20060.320), acetic acid (cat no. 45754), boron trifluoride in methanol (cat no. B1252), NaCl (cat no. S3014) and 2,6-Di-tert-butyl-4-methylphenol (BHT) (cat no. B1378), ethyl acetate (cat no. 34858), and hydrochloric acid (cat no. H1758) were purchased from Merck KGaA (Warsaw,Poland). Double-distilled water was obtained from a Milli-Q Water System (Millipore, Billerica, MA, USA). Buffers used for HPLC analysis were filtered through 0.22 µm nylon filters (Agilent).