## 4 Secondary Metabolites
### Carotenoids
Compounds are identified using mass spectral libraries and linear retention indices, calculated from a series of n-alkanes from C\({}_{6}\) to C\({}_{30}\). Such an approach was used by [PERSON] et al. [57] to identify several terpen occurring in different apple varieties of _Malus domestica_ Borkh from different geographic regions at Madeira Islands, including farnesol and \(\alpha\)-farnesene. The authors found that for the whole fruit, terpen accounted for 3.10% of the total GC peak area of the chromatograms. In order to increase the selectivity and sensitivity of the analytical method, [PERSON] et al. [148] used tandem mass spectrometry (QqQ) for metabolite profiling of volatile compounds, including terpen, in apples. The developed analytical method allowed the separation of co-eluted terpen, with the same nominal masses, which could be not identified by conventional detectors such as FID and/or qMS. In this work, the triple quadrupole mass spectrometer operated in multiple selected reaction monitoring mode (MRM) and at the optimal collision energy for a given compound, the most intense fragmentation ion was chosen as a quantifier (Q) and the second most intense as a qualifier (q). The confirmation of molecules identity was achieved by comparing the q/Q ratio of samples and those of reference standards. The choose of two selected ions transition, one for quantification and one for confirmation, during MRM, ensured high selectivity and sensitivity, using the q/Q ratio as a confirmatory parameter. As triterpenoids such as betulinic acid (BA) and unsolic acid (UA) are polar metabolites, they are not efficiently volatilized for analysis using GC. Thus, LC-MS is more suitable for their analysis [59; 149; 150; 151]. LC coupled with ultraviolet (UV) or diode array detectors found a limited application in the study of apples triterpenes, as these compounds absorb UV radiation weakly and only at wavelength of 200 nm. Thus, only few studies reported the use of LC-DAD for triterpenoids analysis in apple fruits [152]. On the contrary, LC-MS/MS has been widely preferred, as it allows compounds unequivocal identification by examining their fragment ions, obtained through MS/MS experiments [153]. [PERSON] and colleagues [59] investigated the fragmentation pathways of apple triterpenes, i.e., pomaceic, annurcoic, euscaphic, pomolic, corosolic, maslinic, betulinic, unsolic and oleanolic acid by LC-APCI-MS/MS, in order to allow their fast identification without the use of reference standards. Negative ion mode was the polarity chosen for the analytes. The main ions observed in ion trap and Q-TOF measurements for the considered triterpene acids are summarized in Table 6. The application of LC-MS/MS for triterpenoids analysis also ensures more sensitive quantification. [PERSON] et al. [149] developed a practical and reliable analytical method for the extraction, identification and quantification of UA and BA in apple peel extracts, by using LC coupled to MS with positive electrospray ionization mode (ESI+). An isocratic run, consisting of 80% acetonitrile and 20% ammonium acetate 10 mM pH 6.0, allowed the separation of the analytes. The developed method was validated in terms of sensibility, precision and accuracy. The LOD and LOQ values were 0.087 and 0.266 \(\upmu\)g/mL for BA and 0.398 and 2.117 \(\upmu\)g/mL for UA. For betulinic acid, LOD and LOQ values were lower compared to those obtained with the LC-DAD method developed by [PERSON]\(\rm\ddot{a}\)t\(\rm\ddot{e}\) et al. [152] (LOD: 0.15 \(\upmu\)g/mL and LOQ: 0.45 \(\upmu\)g/mL). \begin{tabular}{l l l l l l l l l} \multicolumn{10}{c}{Table 6. Cont.} \\ \hline
**Ion** & \begin{tabular}{l} **Ponacetic** \\ **Acid** \\ \end{tabular}  & \begin{tabular}{l} **Annurocic** \\ **Acid** \\ \end{tabular}  & \begin{tabular}{l} **Escapolic** \\ **Acid** \\ \end{tabular}  & \begin{tabular}{l} **Poncoli Acid** \\ \end{tabular}  & \begin{tabular}{l} **Consocioci** \\ **Acid** \\ \end{tabular}  & \begin{tabular}{l} **Masilic Acid** \\ \end{tabular}  &