## 7 Analytical Techniques for the Determination of Active Compounds  
Over the last years several extraction techniques associated with different analytical methods have been reported for the chemical characterisation of _A. altissima_. For this purpose, it is crucial to consider that this type of plant possesses a complex and high number of compounds among which there are several fatty acids, volatile compounds, antioxidants, and alkaloids, among others [50; 52; 72]. These compounds were studied to check if they had any biological activity; this could help understand mechanisms of action, develop pharmacological formulations, and apply them to different research fields. Therefore, it is essential to identify and quantify the main compounds to improve this species' knowledge and possible uses. In a study carried out by [PERSON] et al. [52], compounds were extracted from the seeds in a powder format using a Soxhlet apparatus. The authors used hexane (400 mL) as a solvent and the extraction time was 8 h, after which the solvent was evaporated using a rotary evaporator set at 40 \({}^{\circ}\)C. The collected oil was analysed by (1) gas chromatography coupled with a flame ionisation detector (GC-FID) and (2) gas chromatography coupled with mass spectrometry with headspace solid-phase micro-extraction (HS-SPME-GC/MS). The GC-FID equipment was used to determine the sterols fraction and the profile of fatty acids after transformation into methyl esters by adding 1 mL of n-hexane for each 40 mL of previously extracted oil, to which was then added a solution of sodium methoxide (2M). After performing this step, the mixture was heated to 50 \({}^{\circ}\)C in a thermal bath, followed by the addition of HCl (2N). On the other hand, the analysis of volatile compounds was carried out by HS-SPME-GC/MS. The fatty acids profile revealed a predominance of linoleic and oleic acids, observing a polyunsaturated/saturated ratio of 11.86. Regarding the level of sterols, a predominant presence of \(\beta\)-sitosterol was observed, which is associated with high antioxidant activity and the control of cholesterol levels. Lastly, concerning the volatile fraction, a significant percentage of non-terpenes were observed in seed oil which could not be observed in oils from other parts of the same plant species. [PERSON] et al. [50], developed an analytical method to determine phenolic compounds with antioxidant activity by reversed-phase HPLC coupled to an ultraviolet detector (UV) using a Phenomenex Kinetex Luna 2.6 \(\upmu\)m PFF 100A reversed column, performing the monitoring of the compounds at a wavelength of 280 nm, with the extraction of the compounds of different parts of _A. altissima_ being performed with Soxhlet equipment for methanol, acetone, and ethanol extractions, with hydroalcoholic extractions carried out through refluxing, followed by evaporation under vacuum to a final volume of 150 mL. The authors verified that a greater final concentration of phenolic compounds was obtained when methanol was used for extraction, with lower concentrations of phenols observed in stems and stalks compared with extracts from leaves. In addition, hydroxcinnamic acids were revealed to be predominant, ferulic acid being the one whose percentage was higher, finding that the antioxidant activity levels were closely related to the total phenolic content, with an R2 of 0.86. [PERSON] et al. [72] developed a method to evaluate the active components of _A. altissima_ with the help of an HPLC-UV. The goal was to perform the fingerprinting and quantification from samples of different sources, proceeding to the extraction of the compounds after performing the drying and pulverization of the stem bark using ethanol (75%) for 1 kg of content, being later evaporated in a vacuum. In this study, concentrations ranged from 6.44 \(\upmu\)g/mL to 825 \(\upmu\)g/mL for ailanthone, one of the main quasinoids with anti-inflammatory, anti-microbial, and anti-allergic activity in _A. altissima_, used as quality control, with the UV detector set to a wavelength of 250 nm.