# From Vine to Wine: Coloured Phenolics as Fingerprints
## 2 Materials and Methods
### Analytical Methods  
HPLC was utilized to separate phenolic compounds using a Waters 2690 Separation Module equipped with a Waters 996 DAD and interfaced with a Waters data-processing station running Millennium32 software (version 3.0.1). Chromatograms were recorded at 520 nm wavelength.  
Red wine samples were previously filtered through a 0.45 \(\upmu\)m membrane filter, and then 15 \(\upmu\)L aliquots were injected onto a Nova-Pak C18 reversed-phase column (3.9 \(\times\) 150 mm, 4 \(\upmu\)m particle size; Waters) maintained at 30 \({}^{\circ}\)C. The chromatographic conditions were adapted from [PERSON] et al. (2018). Solvent A was Milli-Q grade water, and solvent B was gradient-grade acetonitrile (Sigma-Aldrich, St. Louis, MO, USA); both solvents were acidified with 0.2% trifluoroacetic acid (spectrophotometric grade, \(\geq\)99%, Sigma-Aldrich). The flow rate was set at 1.5 mL/min. The linear gradient for solvent B was programmed as follows: 0 min, 0%; 2 min, 2%; 8 min, 8%; 15 min, 15%; 20 min, 23%; and returning to 0% at 25 min.  
Detection was performed using the DAD, with peak identification based on their retention times and ultraviolet and visible spectra. Anthocyanin compounds were identified by direct comparison with commercial standards when they were available. Additional confirmation was achieved by spiking red wine samples with the available phenolic standards. Retention times obtained with the chromatographic conditions used by us are detailed in Table S1 from Supplementary Materials. Other compounds were tentatively identified by comparing their relative retention times and spectral data with those published under similar chromatographic conditions [23; 24; 25].  
Anthocyanins were detected at 520 nm and quantified using oenin as the standard (Sigma-Aldrich), expressed as mg of oenin equivalents/L. The calibration curve was constructed over the concentration range observed in the samples and was generated by plotting peak area (absorbance) vs. concentration (mg/L). Standards had a linear response within the studied concentration ranges, with correlation coefficient (r) always \(\geq\)0.999. The anthocyanin compounds identified (abbreviated name was included between brackets) were the following: Delphinidin-3-glucoside (D-3-glc); Cyanidin-3-glucose (C-3-glc); Petunidin-3-glucose (Pt-3-glc); Peonidin-3-glcoside (Pe-3-glc); Malvidin-3-glucoside (M-3-glc, oenin); Cyanidin-(6-acetyl)-3-glucose (C-6-ac-3-glc); Petunidin-(6-acetyl)-3-glucose (Pe-6-ac-3-glc); Malvidin-(6-acetyl)-3-glucose (M-6-ac-3-glc); Peonidin-(6-counaroyl)-3-glucose (Pe-6-co-3-glc); Malvidin-(6-coumaroyl)-3-glucose (M-6-co-3-glc).  
Using these eleven individual anthocyanin compounds, several groups with different anthocyanin derivatives were calculated as follows:  
Glucosides without oenin (_Glc/entin_) = D-3-glc + C-3-glc + Pt-3-glc + Pe-3-glc (1)  
Glucosides with oenin (_Glc_) = _Glc/entin_ + M-3-glc (_oenin_) (2)  
Acetylated Anthocyanins (_Acet_) = C-6-ac-3-glc + Pt-6-ac-3-glc + Pe-6-ac-3-glc + M-6-ac-3-glc (3)  
Counaroyled Anthocyanins (_Cou_) = Pe-6-co-3-glc + M-6-co-3-glc (4)  
Total Anthocyanins (_Tot_) = _Glc_ + _Acet_ + _Cou_  
Anthocyanin glucosides were grouped in two distinct ways, as described by two different equations: (1) Excluding malvidin-3-O-glucose (oenin), the most abundant anthocyanin in wine; this approach minimizes the effect of its predominance overshadowing observations related to less abundant pigments; and (2) including all individual glucosides in order to know the behaviour of the overall glucosides together.