Isoquercetin

Extract analyses were performed using reversed-phase chromatography, as described previously^{16 (link)} using a Ultimate 3000 HPLC system comprised of a LPG-3400SD pump with built-in degasser, a WPS-3000 U(T)SL cooled autosampler, a temperature-controlled column compartment and an DAD-3000(RS) diode array detector equipped with the Chromeleon software (all Thermo Scientific, Dreieich, Germany). Analyte separation was performed on an Accucore C18 column (150 mm × 3.0 mm × 2.6 μm; Thermo Scientific). Column temperature was 40 °C and the injection volume 1 μL. Detection wavelength was 260 nm. Analytes were separated via gradient elution with mobile phase A containing 0.1% formic acid (FA) in water and mobile phase B containing 0.1% FA in acetonitrile at a flow rate of 0.5 mL/min. The elution gradient starting conditions were 95% A and 5% B. After 5 min of equilibration time, the proportion of B was increased to 20% at 8 min and to 40% at 12 min, followed by 60% B at 15 min and 80% B at 17 min for 3 min. B was reduced to 5% at 20 min until 25 min.
Chlorogenic acid (3-CQA, Sigma Aldrich), neochlorogenic acid (5-CQA, Sigma Aldrich), rutin (Sigma-Aldrich) and isoquercetin (Extrasynthese, Genay, France) were used as standards. 4-CQA was assigned tentatively.

The solvents used for extraction (methanol, acetonitrile and formic acid) were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). A Milli-Q purification system (Millipore, Bedford, MA, USA) was used to prepare high-performance liquid chromatography (HPLC) grade water. Standards of gallic acid, caffeic acid, p-coumaric acid, ellagic acid, ferulic acid, sinapic acid, quinic acid, syringic acid, chlorogenic acid, phloridzin, kaempferol, luteolin, quercetin, isorhamnetin, myricetin, apigenin, epicatechin and catechin were purchased from Sigma-Aldrich. Standards of quercetin rhamnoside, isoquercetin, rutin, robinin, isorhamnetin rutinoside, neohesperidin, quercetin galactoside, myricitrin, myricetin galactoside, epigallocatechin, epigallocatechin gallate, procyanidin B1, procyanidin B2, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, cyanin, delphinidin-3-O-glucoside, malvidin-3-O-glucoside, and pelargonidin-3-O-glucoside chloride were purchased from Extrasynthese (Genay, France).
The reference standard mixture of 37 FAME (FAME mix 37) was acquired from Supelco (Sigma-Aldrich, Bellefonte, PA, USA); other reference standards were purchased from Matreya Inc. (Pleasant Gap, PA, USA): PUFA-2, a nonconjugated 18:2 isomer mixture of individual PUFA, eicosapentaenoic acid, (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), C18:3 cis-6,9,12, and C18:3 cis-9,12,15.