## 4 Secondary Metabolites
### Phenolics and Organic Acids  
The most common method for the separation and analysis of polyphenols and organic acids occurring in apple fruits is the high-performance liquid chromatography (HPLC) due to its high-resolution, efficiency, reproducibility and relatively short analysis time, without derivatization and no restriction on sample volatility [97; 98; 99; 100; 101; 102]. Recent advances in apple phytochemicals analysis show a tendency for the application of environmentally friendly and faster techniques. This is evidenced, for example by the recently developed separative techniques, such as Ultrahigh-Performance Liquid Chromatography (UHPLC) and Ultra-Fast Liquid Chromatography that came from the evolution of packing materials used to improve resolution, and also contributed to such advances. Recently, UHPLC has been proposed for the analysis of polyphenols [103; 104; 105; 106; 107; 108]. In general, peak efficiency and chromatographic resolution provided in UHPLC are higher than conventional HPLC. In addition, UHPLC methods can be considered more cost-effective because they typically need 80% less organic solvents than conventional HPLC methods [91]. Both HPLC and UHPLC can be easily coupled to a variety of detectors for polyphenols detection, including UV-Vis and MS.  
To date, no single chromatographic methods capable of separating the different types of phenolic compounds, occurring in apples, are available. It is necessary to optimize the stationary phase, mobile phase, gradient elution, temperature and flow rate for each group of compounds [109]. Moreover, polyphenols stereochemistry, molecular weight, polarity and degree of polymerization could influence compounds retention. However, the reported methods for the separation of phenolics, as well as their glycosides, have been carried out mainly by reverse phase liquid chromatography (RPLC), on silica-based C18-bonded phase columns [71; 106; 110; 111]. The average particle diameter of HPLC packings is typically 3-10 \(\upmu\)m. With columns of smaller particle size, a larger number of plates per unit time is provided, with respect to columns with larger particle size [112]. As mobile phase, binary mixtures of aqueous formic acid or acetic acid and acetonitrile (ACN) or methanol (MeOH) as organic modifiers have been employed [71; 106; 110; 111]. Typically, gradient elutions have been preferred, since multiple-step gradients are more suitable for complex mixtures, such as apple extracts. Although RPLC has been mostly chosen for apple polyphenols separation, [PERSON] et al. [113] used hydrophilic interaction chromatography (HILIC) to develop a robust and reliable analytical method for the extraction, separation and identification of monomeric and oligomeric procyanidins in apple extracts. HILIC separation mechanism is opposite to that of RP systems: polar stationary phase retains polar analytes, which are eluted by mixture of organic solvent (usually acetonitrile) and water [114]. Due to the complexity of procyanidins oligomers structures in the apple extract, normal phase silica columns were found to be not suitable for quantification purposes, particularly at a higher degree of polymerization. Instead, HILIC column ensured a better resolution of the chromatographic peaks [113]. As for phenolic compounds, reverse phase liquid chromatography is used also for the detection of organic acids by C18-bonded phase column. However, a method based on the use of an Aminex HPX cation-exchange column and an elution solvent consistingof sulfuric acid in bi-distilled water has been also reported for the quantification of malic acid [115]. The Aminex HPX-series of strong cation-exchange resins are prepared from a sulfonated polystyrene-divinylbenzene copolymer and are available in prepacked columns. One of the major problems experienced with the use of these columns are related to the co-elution of non-acid components and the poor resolution of the chromatographic peaks, thus limiting their use.