## 3 Primary Metabolites: Extraction and Analysis
### Fatty Acids  
The mesocarp, or pulp, of apples generally contains very low levels of lipid material (0.2-0.6%), and therefore it is not an important source of edible or industrial fats and oils [39]. Saturated and unsaturated fatty acids are, in fact, degraded to precursor molecules for straight-chain esters. However, despite their low levels of concentration, fatty acids (FAs) and lipids are important structural and metabolic constituents [40]. Indeed, alterations of the lipids constituting the cellular membranes, could cause serious problems to the cell's adaptability to stress conditions, thus resulting in fruit storage disorders. Moreover, FAs and lipids often play a crucial role as precursors of important volatile aroma and regulatory compounds cells [41]. Although there are differences among varieties and ripening stage, the content of saturated fatty acids is generally higher than that of the unsaturated ones in each apple variety. The optimal value of the ratio between the content of the latter and the former is around 30/70 or less [42]. Among the saturated and unsaturated FAs classes, palmitic acid (C16:0) and linoleic acid (C18:2) are the dominant compounds, respectively [42]. In addition to palmitic and linoleic acid, there are C18:0 (stearic acid), C18:1(oleic acid) and C18:3 (linolenic acid), whose concentration levels dynamically fluctuate during fruit growth and development. Other FAs, such as C20:0 (arachidonic acid), show only small variations, which may indicate that they are constituents of the fruit cuticle, rather than participants in actual fruit metabolism [41].  
Gas chromatography (GC) coupled to flame ionization detector (FID) or mass spectrometric detector (MS) is the most commonly used technology for the detection of fatty acids in apples. The analysis of fatty acids by GC-FID and GC-MS-based analytical methods involve three main steps, i.e., their extraction from the sample matrix, their derivatization, and the GC-FID or GC-MS analysis [43]. Lipids extraction from apple tissues is performed with hexane, and it is often followed by filtration of the supernatant through anhydrous sodium sulfate [44; 45]. Soxhlet extraction with petroleum ether has also been used [45; 46]. The derivatization is requested, because most fatty acids have high boiling points, thus being difficult to evaporate, and have a low FID response [47]. Fatty acids in apples are usually analyzed by GC-FID and GC-MS as methyl ester derivatives. The derivatization is typically made by treating apple extract with methanol and sulfuric acid under a stream of nitrogen, before the GC-MS analysis. Five fatty acids were identified as their methyl esters with this approach by [PERSON] et al. [45] in apple fruits, i.e., oleic acid, linoleic acid, palmitic acid, stearic acid and arachidonic acids. Since mass spectra of derivatized fatty acids rarely contain ions indicative of structural features (the positions of double bonds in the aliphatic chain, for example, cannot be determined), retention indices (RI) of the compounds relative to a mixture of n-alkanes are calculated and are typically used for their identification, alongside with a comparison of their mass spectral data with Wiley, NIST, NBS library and the literature data [45].