## 4 Secondary Metabolites
### Phenolics and Organic Acids
Although LC-UV-Vis or LC-UV-FL are cheap and robust techniques for the quantification of polyphenols, their identification could be uniquely achieved through the comparison of retention times and UV-Vis spectra with those of authentic standards. Moreover, phenolic compounds are often linked to saccharidic moieties that are not UV-active, thus preventing the correct polyphenols identification. Considering these difficulties, in many cases, it is necessary to use a more sensitive and selective detector such as a mass spectrometer to an LC system (LC-MS), as it allows unequivocal identification of the analytes thanks to the possibility to conduct MS/MS experiments [123; 124; 95; 109; 125; 126]. Liquid chromatography coupled to mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS) are among the most widely used techniques for the analysis of polyphenols occurring in apple fruits [127; 128; 129; 130; 131]. The employment of these methods is particularly helpful not only for their quantitative determination but also for their characterization and structural elucidation, especially when MS\({}^{\text{n}}\) fragmentation can be achieved [91]. For the ionization of apple polyphenols in LC-MS, electrospray ionization (ESI) in negative mode has been, by far, the most generalized interface employed [128; 130; 132]. The negative ionization mode provides the highest sensitivity and results in limited fragmentation of flavonoids. Instead, for the identification of anthocyanins, positive ionization mode is mainly chosen, as it gives the best results [133]. As an extra-certainty to the molecular mass determination, the combination of both ionization modes (positive and negative) in MS\({}^{\text{n}}\) scan could be implemented [13]. Other less common techniques used in the analysis of polyphenols areatmospheric pressure ionization techniques, such as atmospheric pressure chemical ionization (APCI). For instance, LC-APCI-MS in positive ionization mode was proposed for the characterization of apple polyphenols by [PERSON] et al. [134], who reported for the first time five isorhamnetin glycosides, two hydroxyphloretin glycosides and quercetin in apple peel. As mass analyzers, multiple types are available and have been proposed for phenols detection, among them triple quadrupole (QQQ) [135], linear ion-trap [103], time-of-flight [103], Orbitarp [105] and QTrap [136], among others. QTrap mass analyzers are hybrid instruments combining a quadrupole and a liner ion-trap in a similar configuration to a QQQ instrument and they gaining popularity for the analysis of food products. LC-MS methods offer a better selectivity compared to LC-UV methods. In this regard, [PERSON] et al. [128] developed an UHPLC-UV and UHPLC-MS/MS for the quantification of phenolic compounds in apple juices. The developed methods were validated for 15 major compounds based on linearity, limits of detection and quantification, recovery and precision tests (see Supplementary Materials Table S1). A comparison of the quantifications showed that both UHPLC-UV and UHPLC-MS/MS had an excellent correlation for major compounds, quantified in 120 different samples. However, the slope value showed an overestimation of the UV detector for chlorogenic acid, explained by the co-elution of unknown UV-absorbing minor compounds, highlighting the advantage of using MS as detector and the selected reaction monitoring (SRM) mode to quantify highly concentrated samples. LC coupled to high-resolution mass spectrometry (HRMS), which provides accurate mass measurements, has recently obtained popularity due to its ability to give more comprehensive information concerning the exact molecular mass, elemental composition and detailed molecular structure of a given compound. LC-HRMS provides data of exceptional quality regarding apple metabolites. Indeed, it is currently used to aid in the identification of a broader range of phenolic compounds.