Apigenin 7 o glucoside

Choline chloride, L-(+)-lactic acid, glycerol, 1, 2-propanediol were purchased from Sigma-Aldrich (Steinheim, Germany). Citric acid and L-proline were purchased from Merck (Darmstadt, Germany). Verbascoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside and gallic acid commercial standards of HPLC grade (>99%) (Extrasynthese, Genay, France) were used as references for identification. Methanol, ethanol, chloroform, ethyl acetate, n-butanol, methyl tert-butyl ether and acetonitrile were of HPLC quality. Formic acid was of analytical grade (>99%).

All reagents and solvents for extraction, as well as UPLC and HPLC-MS analyses (methanol, acetonitrile, formic acid, dimethylsulfoxide, polyamide and chloroform), were from Sigma-Aldrich (Poznań, Poland), and ultrapure water was obtained from a Millipore Direct Q3 device. Standards of flavonoids (apigenin, apigenin-7-O-glucoside, apigenin-6-C-glucoside, apigenin-8-C-glucoside, isorhamnetin, luteolin, luteolin 7-O-glucoside, luteolin 4′-O-glucoside, luteolin-8-C-glucoside, luteolin-6-C-glucoside, quercetin 3-O-galactoside, quercetin, rutin, isorhamnetin, isorhamnetin 7-O-glucoside) were purchased from Extrasynthese (Genay, France).
Extracts were prepared according to [21 (link)]. Weighted samples of frozen leaves (about 100 mg) were placed in Eppendorf tubes with 1.4 mL of 80% methanol and the internal standard, homogenized using a ball mill (MM 400, Retsch, Haan, Germany) and subsequently placed in an ultrasonic bath for 30 min and centrifuged (11,000× g) for 10 min. apigenin (5 µL of 1 mg/mL solution in dimethylsulfoxide) was used as the internal standard in extracts prepared for the UPLC analyses. Samples of the secondary metabolite extract were directly subjected to HPLC-MS analysis and, prior to the UPLC analysis, 200 µL were diluted with 80% methanol (800 µL).

Methanol (MeOH) and acetonitrile (AcN) were purchased from Merck (Darmstadt, Germany) and phosphoric acid from Sigma-Aldrich (St. Louis, MO, USA). Standards of apigenin, apigenin-7-O-glucoside, catechin, hydroxytyrosol, luteolin, luteolin-7-O-glucoside, oleuropein, rutin, tyrosol, and verbascoside were purchased from Extrasynthese (Genay, France). Deionized water was obtained by Siemens UltraClear (Siemens AG, München, Germany).
Multi-element standard solution from Perkin Elmer (NexION Setup Solution, Waltham, MA, USA) was used. Argon used to form plasma for the inductively coupled plasma mass spectrometer (ICP-MS) analysis was of purity 6.0 (Messer, Austria). Acetylene was supplied by Messer (Messer Croatia Plin d.o.o., Zaprešić, Croatia).
Deionized water was obtained by Siemens UltraClear (Siemens AG, München, Germany).