## 7 Analytical Techniques for the Determination of Active Compounds
[40] studied the composition of essential oils extracted from _A. altissima_ leaves from three different regions of Tunisia. The dried material was extracted by hydrodistillation of Quickfit apparatus for 3 h, in which the distillate obtained was extracted with \(n\)-pentane (twice) and the organic layer was concentrated and consequently analysed by GC-FID and GC-MS. The three essential oils were characterised as containing a high percentage of non-terpenic compounds and some of the sesquiterpene hydrocarbons and oxygenated monoterpenes. The majority of 139 compounds identified were tetradecanol, heneicosane, tricosane, docosane, \(\alpha\)-curcumcene, \(\alpha\)-gurjunene, methyl decanoate, \(\alpha\)-terpinen-7-al, geranial, \(\alpha\)-guaiene,\(\alpha\)-humulene, and (E)-\(\beta\)-farnesene. In the same study, methanolic extracts of dried and ground leaves and the residual leaves (remained after hydrodistillation) were further analysed for their content of phenolic and flavonoid compounds. The total phenolic content was performed by the Folin-Ciocalteu method, and these were characterised by high-performance liquid chromatography with a photodiode array detector and tandem mass spectrometry (HPLC-PDA-MS/MS). Total flavonoid content was performed with the AICl\({}_{3}\) colorimetric method. Comparing the two methods of total content determination, it was possible to verify a higher content of phenols in the dried leaves than the hydrodistilled residue and a higher content of flavonoids in the hydrodistilled residue compared with the dried leaves. Furthermore, from the methanolic extracts antioxidant, antimicrobial, and phytotoxic activities were analysed. To quantify the phenolic compounds, [PERSON] et al. [78] incubated the leaves in methanol with 0.1% chloride acid for 50 min. After this period, water and chloroform were added to the resulting supernatant and the sample was stirred for 45 min at 4 \({}^{\circ}\)C, followed by centrifugation and separation of the liquid phase. This process was repeated without the addition of chloroform. After extraction, the compounds were analysed by HPLC-DAD using wavelengths of 520 nm for anthocyanins, 340 nm for flavones and flavanones, 320 nm for resveratrol, coniferyl alcohol, hydroxycinamic acid and its respective derivatives, 360 nm for flavonoids and ellagic acid, and 280 nm for isoflav, catechins, benzoic acids, and the respective derivatives. The evaluation of the phenolic compounds' content, flavonoids, and non-flavonoids was also performed by [PERSON] et al. [79], using a new HPLC procedure equipped with a C\({}_{18}\) column using wavelengths of 360 nm and 310 nm to identify flavonoids and phenolic acids, respectively, which was faster (26 min) and used fewer solvents than other works. The extractions in deionised water were performed after maceration and respective extraction for 48 h, followed by filtration. The extractions in methanol were performed for a period of 72 h without maceration. The supernatants were evaporated to 140 mL, reconstituted with water to a final volume of 500 mL, and then stored at 4 \({}^{\circ}\)C until analysis. No flavonoid content was obtained from fresh leaves when water extraction was performed, while the content of both groups of compounds was higher when methanol was used for extraction.