## 4 Secondary Metabolites
### Phenolics and Organic Acids
Moreover, the ion exchange's separation mechanism implies that organic acids should be in their ionic form, so that a severe control of pH is required [116]. As regard detection, the most commonly used detector for HPLC is a variable-wavelength UV or UV-Vis detector, because both phenolics and organic acids absorb very well in the UV region [21, 28, 117, 118, 119, 120, 121]. The use of low UV detection wavelength, which ranged between 185 nm and 254 nm, allows to achieve high sensitivities in the determination of organic acids [122]. Moreover, for phenol compounds, HPLC-DAD provides extensive information; however, no single wavelength is ideal for monitoring all classes of phenolics, because they display absorbency maxima at different wavelengths (Table 4). For comprehensive and simultaneous monitoring of different groups of polyphenols, [PERSON] et al. [21] set the detector at 254, 280 and 320 nm to identify and quantify, into fifteen different apple cultivars, twenty polyphenolic compounds belonging to flavanols, phenolics acids, dihydrohalcones and flavonols. Among them, quercetin glycosides were found to be the main polyphenols in the peel (203 \(\pm\) 108 mg/100 g) and phenolic acids (10 \(\pm\) 5 mg/100 g) in the flesh. Instead, by [PERSON] et al. [117] flavonols were quantitated at 360 nm. The method implemented by these authors allowed the identification of 11 analytes: proxyanidin B1, (+)-catechin, chlorogenic acid, procyanidin B2, (\(-\))-epicatechin, rutin, hyperoside, isoquercitrin, avicularin, quercitin and phloridzin. The elution order of quercetin-3-\(O\)-glycos was as follows: quercetin-3-\(O\)-rutinoside (rutin), quercetin-3-\(O\)-galactoside (hyperoside), quercetin-3-\(O\)-glucose (iso-quercitin), quercetin-3-\(O\)-arabinoside (avicularin) and quercetin-3-\(O\)-rhammoside (quercitin), mainly according to other studies aimed at the evaluation of the quercetin derivatives profile in food extracts [92]. \begin{table}
\begin{tabular}{l c c} \hline \hline
**Group of Polyphenols** & **Phenolic Compound** & \(\mathbf{\lambda_{\text{max}}}\) **(nm)** \\ \hline \multirow{8}{*}{Flavanols} & Procyanidin B1 & 281 \\  & (+)-Catechin & 281 \\  & Procyanidin B2 & 281 \\  & Procyanidin C1 & 275 \\  & (\(-\))-Epicatechin & 280 \\  & Procyanidin A2 & 280 \\ \hline \multirow{8}{*}{Phenolic acids} & Gallic acid & 272 \\  & Protocatechuic acid & 261, 298 \\  & 5-O-Caffeoylquinic acid & 326 \\ \cline{1-1}  & Caffe acid & 324 \\ \cline{1-1}  & p-Coumaric acid & 310 \\ \cline{1-1}  & Ferulic acid & 324 \\ \hline \multirow{8}{*}{Flavonols} & Phloretin-2-\(O\)-\(\beta\)-glucose & 287 \\ \cline{1-1}  & Quercitin-3-\(O\)-galactoside & 259, 348 \\ \cline{1-1}  & Quercitin-3-\(O\)-glucose & 259, 351 \\ \cline{1-1}  & Quercitin-3-\(O\)-rutinoside & 259, 348 \\ \cline{1-1}  & Quercitin-3-\(O\)-xyloside & 260, 348 \\ \cline{1-1}  & Quercitin-3-\(O\)-arabinoside & 260, 347 \\ \cline{1-1}  & Quercitin-3-\(O\)-rhammoside & 260, 347 \\ \cline{1-1}  & Quercitin & 256, 372 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Absorption maximum (\(\lambda_{\text{max}}\)) of polyphenols occurring in apples and belonging to flavanols, phenolics acids, dihydrohalcones and flavonols classes. Reproduced under the terms of the Creative Common CC BY license which permits reuse in any medium, provided the original open access work is properly cited [21]. HPLC-UV for the quantification of polyphenols on _Malus_ extracts allowed to reach limits of detection (LOD) and limits of quantification (LOQ) in the range 0.2-5.8 \(\upmu\)g/mL and 0.1-7.1 \(\upmu\)g/mL, respectively. Fluoresce detector (FL) in some cases offers higher selectivity and sensitivity compared to UV-Vis detection methods, so that it could be offered as a robust and reliable alternative or to be complementary to UV-Vis detection systems. For example, [PERSON] et al. [106] determined the polyphenolic profile in leaves and fruits of 2 cultivars of _Malus domestica_ by UPLC-PDA-FL. In this case, the identification was also achieved, by using LC-MS/MS.