## 3 Primary Metabolites: Extraction and Analysis
### Sugars  
For many fruits and vegetables, sweetness is an important parameter influencing their quality and is determined by the level of soluble sugars. Therefore, the determination of sugars and sweetness are of great importance in many fields of plant-food-science research [4]. In apples, sweetness is one of the most important quality parameters that determines the overall acceptability of the fruit, and it is affected mainly by fructose, glucose, sucrose and sorbitiol concentrations [31]. Sorbitol and sucrose are biosynthetized in the leaves, and then they are translocated into the fruits, where they can be converted into fructose, glucose, malic acid and starch depending on the stage of fruit development. Sorbitol and sucrose represent almost the total translocated carbohydrates [31]. Although the individual sugar content may vary greatly between and within apple cultivars, during fruit storage, the concentrations of sucrose tend to decrease due to the conversion to fructose and glucose [31]. Sugars quantification in apples is preceded by their extraction from the fruit tissues. The extraction of sugars is usually performed by using water as an extraction solvent, since it yields more reliable results. Indeed, [PERSON] et al. [32] found that, with alcoholic extraction, some sugars may not adequately dissolve in proportion to their true concen  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{2}{c}{**Extraction and**} & \multicolumn{2}{c}{**Recommendations**} \\  
tration because of solvent vaporization, even at low temperatures. However, in general, a preliminary immersion in methanol/water solution of the fruit tissues before the extraction, is important to inhibit the invertase activity, thus avoiding the hydrolysis of sucrose into free sugars (glucose and fructose) [3]. As traditional colorimetric and iodometric methods are unable to quantitate sugars individually, non-structural carbohydrates in apples are analyzed by HPLC coupled with suitable columns and detectors, including refractive index detector (RI), evaporative light scattering detector (ELSD) and pulsed amperometric detector (PAD) [32; 33; 34]. HPLC coupled to diode-array detector is quite limited, as sugars do not absorb UV light at a wavelength longer than 200 nm [32]. Although less common, GC-MS-based methods for sugars' analysis have also been proposed. In these cases, preliminary derivatization stages with methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) are needed [35]. One of the most used methods for the quantification of soluble carbohydrates, is HPLC-RI, being simple, rapid and economical [34]. In fact, an accurate HPLC-RI method for the simultaneous determination of glucose, fructose, sucrose and sorbitol in leaf and/or fruit peel from different apple cultivars was developed and validated by [PERSON] et al. [34]. This method was found to be reproducible and sensitive. The HPLC-RI method was successfully applied to qualitative and quantitative establish the sugars of seven apple cultivars and two rootstocks originating from a germplasm collection [34]. Instead, [PERSON] et al. [33] developed an HPLC-ELSD method to analyze sugars without derivatizations, with short analysis times (25 min) and good chromatographic separations. Recently, [PERSON] et al. [36] also used the same method to quantify glucose, fructose, sucrose and sorbitol in \"Orin\" apples, founding that their content generally increased during the fruit-ripening periods and reached the maximum at full-ripening stage of 180 days after full-bloom stage [36]. ELSD does not suffer of limitations such as composition, flow rate of mobile phase and temperature. In this case, the detection is based on the ability of particles to cause photon scattering, thus with ELSD most compounds less volatile than the mobile phase could be detected, regardless the optical properties of the analytes. The methods discussed above require considerable financial investment, advanced analytical skills and time.