Chromatography, Reversed-Phase Liquid

Berry skins were freeze-dried (Cold Trap 7385020, Labconco, Kansas City, MO, United States). Dried tissues were ground with a tissue lyser (MM400, Retsch, Germany). Fifty mg of the powder were extracted with methanol: water: 7 M hydrochloric acid (70:29:1, V:V:V) to determine flavonol concentration and profile. Extracts were filtered (0.45 μm, Thermo Fisher Scientific, San Jose, CA, United States) and analyzed using reversed-phase high performance liquid chromatography (HPLC) coupled to a diode array detector (DAD). The HPLC system was an Agilent 1260 series (Agilent, Santa Clara, CA, United States) with a reversed-phase C₁₈ column LiChrospher^® 100, 250 mm × 4 mm with a 5 μm particle size and a 4 mm guard column of the same material. Anthocyanins may interfere significantly with the quantification of flavonols. Anthocyanin removal through solid phase extraction using a cationic exchange resin (e.g., Dowex 50X4-400, Acros Organics, Fair Lawn, NJ, United States) has been proposed for the determination of flavonols (Hilbert et al., 2015 (link)). However, the determination of flavonols is also possible avoiding co-elution between anthocyanins and flavonols (Downey and Rochfort, 2008 (link)). As Downey and Rochfort (2008) (link) method was not possible to implement directly on our HPLC system, the method was fine-tunned for our instruments. Flow was set to 0.5 ml per minute and temperature was set to 25°C. Two mobile phases were designed to always maintain the following proportions (V/V) of acetonitrile, 0–8 min 8%, at 25 min 12.2%, at 35 min 16.9%, at 70 min 35.7%, 70–75 min 65%, and 80–90 min 8%. This acetonitrile gradient and different isocratic concentrations of formic acid (HCOOH) from 1.8 to 10% were tested by adjusting the gradients and concentrations of two mobile phases (aqueous HCOOH and HCOOH in acetonitrile) as in Supplementary Information 3. A concentration 5% of HCOOH was the only one, avoiding coelution and allowing the simultaneous quantification (Figure 2 and Supplementary Information 4). The remaining volume up to 100% was achieved with purified water. For our HPLC system and column, a 5% HCOOH helped to avoid co-elution, separation of individual flavonols and a high degree of peak sharpness in both anthocyanins and flavonols.
For the identification of flavonols, standards of myricetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside and syringetin-3-O-glucoside (Extrasynthese, Genay, France) were used. Flavonols were quantified determining the peak area of the absorbance at 365 nm. Quercetin-3-O-glucoside was used as a quantitative standard for all the flavonols. It must be noted that each individual anthocyanin and flavonol have a different molar relative response factors (e.g., absorbance per M unit) and even though calculating a response factors for each flavonol would have been possible using commercial standards, this is not the standard practice in the literature and would make comparisons of flavonol profiles harder.

Hippocampus, striatum and cerebellum were dissected from one hemisphere under microscope between 2 and 4 h after the last cycle of alcohol exposure, weighed and snap frozen at −80°C. Hippocampus and striatum samples were each homogenized in 250 μL 0.1 M perchloric acid using an immersion hand disperser (Polytron PT 1200 E, Kinematica Inc., Keyland Court Bohemia, NY, USA). Cerebellum samples were homogenized with the same method in 500 μL milliQ water. Samples were then centrifuged at 14000 rpm at 4°C for 20 min and then supernatant collected using a 0.22 μm filter (Advantec, Sierra Court, CA, USA, 13CP020AS).
The concentration of the cations sodium (Na⁺), magnesium (Mg⁺⁺), potassium (K⁺), and calcium (Ca⁺⁺) in the cerebellum were determined by ion chromatography (IC, Dionex Aquion 1100, Thermo Fisher Scientific, Waltham, MA, USA). The cation chromatography consisted of a CS12A 4 mm analytical and a Dionex IonPac CG12A 4 mm guard column set. 20 mmol/L methanesulfonic acid was the eluent and was sonicated for 20 min followed by degassing with nitrogen for an additional 10 min prior to IC. 10 μl of sample were injected, was eluted for 20 min with isocratic 20 mmol/L methanesulfonic acid. Chromeleon Chromatography Data System software (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze and calculate the peaks.
The concentrations of norepinephrine (NE), 3,4-dihydroxy- phenylacetic acid (DOPAC), dopamine (DA), 5-hydroxy- indoleacetic acid (5-HIAA), homovanillic Acid (HVA) and serotonin (5-HT) in the striatum and hippocampus were determined by HPLC with electrochemical detection. The monoamines were separated by reverse-phase liquid chromatography with a Prodigy C18 column (DA 2 × 100 mm, particle size 3 μm, Phenomenex, YMC Europe, Schermbeck, Germany). The mobile phase (55 mM sodium acetate, 1 mM octanesulfonic acid, 0.1 mM Na2EDTA and 8% acetonitrile, adjusted to pH 3.7 with 0.1 M acetic acid) was de-gassed with an online de-gasser. 10 μl of sample were injected with a flow rate of 0.15 mL/min. Electrochemical detection was accomplished using an amperometric detector Antec Decade (Antec Scientific, Alphen aan den Rijn, Netherlands) with a glassy carbon electrode set at 0.8 V and an Ag/AgCl reference electrode. The output was recorded, and peak areas were calculated by LC solution software (Shimadzu, Kyoto, Japan).

Reverse-phase liquid chromatography was carried out. The mobile phase consisted of a solution containing acetonitrile (56%), methanol (37%), and water (7%) in an isocratic process. The stationary phase consisted of a C-18 precolumn and a C-18 column, respectively, a Supelguard reverse-phase chromatographic column C-18 (2 cm long, 4.6 mm internal diameter, and 5 µm average particle diameter) and a C-18 Supelcosil reverse-phase chromatographic column (25 cm long, 4.6 mm internal diameter, and 5 µm average particle diameter).
All HPLC analyses were conducted at room temperature (20°C). Before injecting the samples, the system was equilibrated for 30 minutes under the conditions described above. The flow was set at 1.2 mL/min, and the wavelength at 245 nm. A volume of 100 µL was injected for each sample run.
The ivermectin standard (0.05 µg/mL), the internal standard (0.05 µg/mL), the blank (Figure 1), and the fortified controls (Figure 2) were analyzed first. Subsequently, all samples were run and the standard injections were repeated every three samples.
The analysis was continuously monitored by Accredia, the Italian accreditation body, which certifies the quality of laboratory practices and conformity of the results obtained.