# Recovery of Polyphenols from Agri-Food By-Products: The Olive Oil and Winery Industries Cases
## 4 Polyphenol Analysis: From Quantification to Antioxidant Capacity
### Chromatographic Techniques  
High-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC) is frequently used to analyze polyphenolic compounds. There are many studies of identification and quantification of polyphenols in olive oil [44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54] and wine by-products [55; 56; 57; 58; 59; 60; 61; 62] by chromatographic techniques.  
Commonly, separation is performed in the reverse phase mode, using C18 columns, and with gradient elution using mobile phases based on methanol:water or acetonitrile:water containing formic acid [63]. Different gradient programs are proposed, depending on the polyphenolic profile of the samples. The most frequently used detectors are ultraviolet (UV)-diode array (DAD) and mass spectrometry (MS) detectors, but fluorescence and electrochemical detectors are also applied. Figure 5 shows the HPLC-UV chromatogram of a wine lees extract.  
It is well known that MS allows the confirmation of the identity of the polyphenols. In this sense, high-resolution mass spectrometry (HRMS) is especially suited when dealing with complex samples, such as extracts from vegetal origin products. Thus, the HRMS spectrum and the retention time are used for confirmatory analysis [63; 64]. It must be said that low-resolution MS/MS systems are also applicable. Both low- and high-resolution MS are suited for quantitative analysis, providing excellent selectivity as well as sensitivity, but it must be kept in mind that, to obtain reliable quantitative results, calibration should account for potential matrix effects, which are quite common in liquid chromatography LC-MS.  
UV detection is simpler than MS detection, but the UV spectrum provides useful information which, combined with retention time, allows for tentative identification of compounds. As a matter of example, [PERSON] et al. [54] identified luteolin, hydroxytyrosol,  
Figure 5: HPLC-UV chromatogram at 280 nm of a wine lees extract. Peak assignment: 1 Gallic acid; 2 catechin; 3 cis-couratric acid; 4 trans-coutatric acid; 5 caffe acid; 6 p-coumaric acid; 7 astilbin.  
trans-ferulic acid, rutin hydrate, tyrosol, apigenin, and caffeic acid in olive pomace samples by HPLC-DAD-UV. [PERSON] et al. [48], also using HPLC-DAD-UV, reported the presence of hydroxytyrosol-4-glucose, hydroxytyrosol, dialdehyde form of decarboxymethyl elenolic acid linked to hydroxytyrosol (HyEDA), verbascoside, tyrosol, and salidroside in olive pomace samples. Moreover, [PERSON] et al. [60] identified quercetin, ellagic acid, gallic acid, caffeic acid, p-coumaric acid, chlorogenic acid, and kaempfer in wine lees samples by HPLC-UV. If the chromatographic peaks are well resolved, UV detection can be used for quantitative analysis of target compounds [63]. HPLC-UV quantitative analysis is simpler than in the case of MS since there are no matrix effects.  
A different approach is related to the analysis of total polyphenol content (TPC). It is also based on the use of UV detection. The chromatogram is acquired at a wavelength where polyphenols absorb (e.g., 280 nm or 320 nm), and the total area of the peaks eluting in the time window, where elution of polyphenols occurs, is calculated. This area is related to TPC, which is estimated using a calibration curve obtained with a standard (e.g., gallic acid). The TPC is expressed in terms of the equivalent concentration of the standard used in calibration [65].