## 4 Secondary Metabolites
### Carotenoids
Terpenes with 15 carbons or less are typically volatile compounds, due to their small size and low polarity, and they represent in apples a minor component of total volatile organic compounds (VOCs), responsible for the fruits' aroma [56]. Some interesting triterpenoids, occurring in _Malus_ fruits, are more polar and less volatile molecules, because they are linked to one or multiple polar groups: among them, oleanolic, annurcoic, unsolic and betulinic acid are the most important polar triterpenoids [144]. The detection and quantification of volatile terpen in apples are widely performed through the use of the GC coupled to mass spectrometry (MS). Few works were based on GC coupled to flame ionization detector (FID) method [145; 146]; however, GC-FID is one of the most used detectors in the flavor and fragrance field, due to its low cost and simplicity. More commonly, the nature of terpen is investigated by using single quadrupole MS detectors [57; 81; 147]. The easy electron impact (EI) ionization ensures high reliability in compounds detection. However, problems in identification of monoterpenes and sesquiterpenes could be found, due to their structural similarities, which result in \begin{table}
\begin{tabular}{c c c c} \hline
**Pigment** & \(\mathbf{\lambda_{\text{max}}}\) **(nm)** & \multicolumn{2}{c}{**HPLC/MS (APCI+) Fragmentation Pattern \(\mathbf{m/z}\)**} \\ \cline{3-4}  & **[M + H]\({}^{+}\)** & **Characteristic Fragments** \\ \hline all-_trans_-Neoxanthin & 419, 444, 472 & 601 & 583 ([M + H\(-\)18 \({}^{+}\)), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)), 547 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ \(9^{\prime}\)-_cis_-Neoxanthin & 415, 439, 468 & 601 & 583 ([M + H\(-\)18 \({}^{+}\)), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)), 547 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ Neochrome & 401, 424, 452 & 601 & 583 ([M + H\(-\)18 \({}^{+}\), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)), 547 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ all-_trans_-Violaxanthin & 418, 443, 472 & 601 & 583 ([M + H\(-\)18 \({}^{+}\)) \({}^{+}\), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\
9-_cis_-Vioxanthin & 411, 436, 468 & 601 & 583 ([M + H\(-\)18 \({}^{+}\)) \({}^{+}\), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\
13-_cis_-Vioxanthin & 410, 435, 466 & 601 & 583 ([M + H\(-\)18 \({}^{+}\)), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ Luteoxanthin & 400, 424, 451 & 601 & 583 ([M + H\(-\)18 \({}^{+}\)) \({}^{+}\), 565 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ all-_trans_- & 424, 448, 476 & 585 & 505 ([M + H\(-\)80 \({}^{+}\)) \({}^{+}\)) \\ all-_trans_-Zeaxanthin & 428, 455, 481 & 569 & 551 ([M + H\(-\)18 \({}^{+}\)),533 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ all-_trans_-Lutein & 428, 448, 476 & 569 & 551 ([M + H\(-\)18 \({}^{+}\)),533 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\
9-_cis_-Lutein & 330, 420, 444, 472 & 569 & 551 ([M + H\(-\)18 \({}^{+}\)) \({}^{+}\)),533 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\
13-_cis_-lutein & 334, 418, 441, 470 & 569 & 551 ([M + H\(-\)18 \({}^{+}\)),533 ([M + H\(-\)18\({}^{-}\)18 \({}^{+}\)) \({}^{+}\)) \\ Chlorophyll b & 457, 646 & 907 & 629 ([M + H\(-\)278 \({}^{+}\)) \({}^{+}\)) \\ Chlorophyll b\({}^{\prime}\) & 457, 646 & 907 & 629 ([M + H\(-\)278 \({}^{+}\)) \({}^{+}\)) \\ Chlorophyll a & 430, 662 & 893 & 615 ([M + H\(-\)278 \({}^{+}\)) \({}^{+}\)) \\ Chlorophyll a\({}^{\prime}\) & 430, 662 & 893 & 629 ([M + H\(-\)278 \({}^{+}\)) \({}^{+}\)) \\ all-_trans_-\(\beta\)-Carotene & 427, 454, 479 & 537 & 445 ([M + H\(-\)92] \({}^{+}\)) \\ \hline \end{tabular}
\end{table} 
Table 5: Wavelengths of maximum absorption \(\lambda_{\text{max}}\) and characteristic fragmentation pattern of the main carotenoids occurring in apples. Reproduced with the permission of Elsevier [140]. similar MS spectra. To overcome these problems retention index values (RI) and MS spectrum interpretation should be used for a univocal peak assignment [92].