# Chemical Characterization of Cider Produced in Hardanger--From Juice to Finished Cider
## 2 Materials and Methods
### HPLC-UV and HPLC-MS Analysis of Phenolic Compounds and Total Phenol Using Folin-Ciocalteu Method  
Total phenols were measured spectrophotometrically using the Folin-Ciocalteu method following the protocol of [PERSON] [6]. The juice and cider samples were filtered using syringe filters (MACHAREY-NAGEL GmbH & Co. KG, Duren, Germany), Folin-Ciocalteu reagent (Sigma-Aldrich, Steinheim, Germany) was used in accordance with [PERSON] et al. [12], and a gallic acid standard (Merck KGaA, Darmstadt, Germany) was used for calibration. All results are expressed as gallic acid equivalents (GAE mg L\({}^{-1}\)). Total phenol content was determined using a VWR-UV 3100 PC spectrophotometer at 765 nm (VWR, Leuven, Belgium).  
Quantitative and qualitative analyses of individual phenolic compounds in juice and cider samples were carried out using the HPLC-UV method, employing an Agilent 1100 series HPLC system (described in Section 2.2) at a wavelength of 285 nm for identification (comparisons to the standard compound) and quantification with external curves, prepared with HPLC grade standards from Extransynthese (Genay, France), as listed in Table S1.  
Briefly, the studied compounds were separated on a Phenomenex Luna C18(2) HPLC column (150 mm \(\times\) 2 mm i.d., 3 um) protected by a guard column. The mobile phase consisted of solvent A, 0.5% acetic acid, and solvent B, HPLC-grade methanol, and the following solvent gradient was applied: 100% A, 0-6 min; 90% A, 6-7 min; 85% A, 7-35 min; 70% A, for 35-53 min; 55% A, 53-68 min; 100% B for, 68.1-77 min; 100% A, and 78-90 min. The flow rate was set to 0.3 mL min\({}^{-1}\), the tray and column oven temperatures were maintained at 13 \({}^{\circ}\)C and 30 \({}^{\circ}\)C, respectively, and 10 \(\upmu\)L injections were made for each analysis. Samples were thawed and filtered through a 0.45 \(\upmu\)m polyvinylidene difluoride (PVDF) syringe membrane filter prior to analysis. The data from the UV detector were acquired at 285 nm.  
Qualitative analysis of phenolic compounds in cider samples was performed using HPLC-UV-MS\({}^{2}\). The analytical system consisted of an Acela 1250 UHPLC system (Thermo Scientific, Waltham, MA, USA) coupled with an LTQ Velos MS system (Thermo Scientific, Waltham, MA, USA). This method was adopted according to [PERSON] et al. [13] with some modifications. The studied compounds were separated on the same column as for HPLC-UV analysis, the Luna C18(2) HPLC column from Phenomenex (150 \(\times\) 2 mm i.d., 3 um), which was protected by a guard column. The mobile phase consisted of solvent A, 0.5% acetic acid, and solvent B, methanol, and the following solvent gradient was applied: 0-6 min (0% B), 6-7 min (0-10% B), 7-35 min (10-35% B), 35-53 min (35-45% B), 53-68 min (45-70% B), 68-68.1 min (70-0% B), and 68.1-88 min (0% B). The flow rate was set to 0.3 mL/min, tray and column oven temperatures were maintained at 13 \({}^{\circ}\)C and 38 \({}^{\circ}\)C, respectively, and 5 \(\upmu\)L injections were used for each analysis. Samples were thawed and filtered through a 0.45 \(\upmu\)m PVDF syringe membrane filter prior to analysis. Data were acquired with the PDA detector at 280 nm, and UV-Vis spectra were recorded in the range of 230-600 nm. Ionization of the compounds was achieved with ESI operating in negative mode. The settings for the MS system were as follows: source voltage, 3 kV; source temperature, 325 \({}^{\circ}\)C; capillary temperature, 350 \({}^{\circ}\)C; sheath gas, 60 arbitrary units(a.u.); auxiliary gas, 10 a.u. The presence of the analytes in the samples was confirmed by comparison of the retention time, targeted MS\({}^{2}\) spectra, and UV-Visible (Vis) spectra (where possible) with those of the reference standard materials (Table S2).