## 4 Secondary Metabolites
### Carotenoids  
The diversified structural characteristic of apple carotenoids requires accurate methods for their separation and identification. To date, many chromatographic techniques are available for the determination of carotenoids in plant-based foods; however, their characterization is more challenging due to similar molecular mass (structural isomers or geometrical isomers) and other derivatives [139]. Routinely, HPLC with UV-Vis, are used for the separation and quantification of carotenoids, occurring in apples [11; 140; 141], as these compounds show absorption in the visible region (400 and 500 nm), due to the long-conjugated double-bond system [142]. However, the \(\uplambda_{\max}\) of individual carotenoids can vary depending on functional groups (see Table 5). To confirm the occurrence of unknown carotenoids in a given sample, single run by HPLC (normal or reversed-phase) systems with isocratic or gradient elution are used. Typically, chromatographic separation of carotenoids is based on HPLC analysis using C18 and C30 columns. In general, C30 and C18 stationary phases are extremely employed for the separation of geometrical isomers. As regard the composition of the mobile phase, [PERSON] et al. [11] quantified carotenoids in apples with a HPLC-UV-Vis (DAD) method, which involved the use of methanol/MTBE (2-methoxy-2 methylpropane)/water (95:3:2, \(v/v\), with 1.5% ammonium acetate in water) and methanol/MTBE/water (8:90:2, \(v/v\), with 1.0% ammonium acetate in water). Instead, a binary-gradient elution with acetone and deionized water was used by [PERSON] et al. [140] for the characterization and the quantification of chlorophyll and carotenoid pigments in the peel and flesh of thirteen commercial apple cultivars, including red-skinned varieties (Ariane, Fuji (I) from Italy, Fuji (F) from France, Pink Lady, Royal Gala and Starking Red Chief), green-skinned varieties (Granny Smith, Green Doncella, Green Golden Delicious and Reina de Reineta), yellow-skinned and yellow-green and varieties (Golden Montana, Golden Delicious and Golden Rosett). It should be noted that, for more reliable results, the identification of the pigment profile is often performed not only by comparing the UV-visible spectra and retention time, but also by comparing mass spectra with those of standard pigments and data in the literature [11; 141]. Indeed, closely related carotenoids and their metabolites could co-elute in various analytical methods. Thus, although spectral confirmation of these carotenoids is based on characteristic UV-Vis spectra, obtained by PDA/DAD, additional characterization is required by MS analysis [139]. Remarkable milestones are provided by the development of analytical methodsfor qualitative and quantitative analysis of carotenoids, through liquid chromatography coupled to mass spectrometry; first of all, the possibility to confirm their structures through tandem mass spectrometric experiments. MS/MS is used in order to find out the characteristic/typical fragmentation pattern of carotenoids, thus allowing their identification. The fragmentation patterns of the main carotenoids occurring in apples are reported in Table 5. Most of the MS analysis of carotenoids is performed, by using positive ion mode. APCI (atmospheric pressure chemical ionization) has become the most widely used ionization technique for various carotenoids in apples, because of high sensitivity [139]. Recently, also ultrahigh-performance liquid chromatography (UHPLC) technology has been used for the analysis of carotenoids in _Malus_ fruits. As the UHPLC C30 stationary phase columns are not commercially available, C18 columns have been used [143]. Terpenes are secondary metabolites obtained by the combinations of several isoprene units (C\({}_{5}\)) and they can be grouped, based on the number of carbon atoms, in monoterpenes (C\({}_{10}\)), sesquiterpenenes (C\({}_{15}\)), diterpenenes (C\({}_{20}\)) and triterpenenes s(C\({}_{30}\)).