# Analytical Methods for Exploring Nutraceuticalticals Based on Phenolic Acids and Polypphenols
## 4 Analytical Methods for the Determination of Active Compounds in Nutraceutical Products
### Chromatographic Methods
Several polyphenols such as 3-caffeoylquinic acid, methyl 4-caffeoylquinate, kaempfer-acetyl-glycoside, quercetin 3-rutinoside, quercetin-acetyl-glycoside, kaempfer 3-\(O\)-glucoside, and quercetin 3-\(O\)-glucose were identified. In another work, a Q-Orbitrap HRMS instrument was used to assess a comprehensive investigation of the polyphenolic constituents of fennel waste extract, showing that chlorogenic acids were the most abundant compounds [102]. Some applications based on MS instruments with an even higher resolution than an Orbitrap analyzer, such as Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), have also been described in the literature. For example, [PERSON] et al. [104] evaluated _Vitis vinifera_ \"Pinot nor\" leaves as a source of bioactive nutraceutical compounds using an FT-ICR-MS instrument, identifying the presence of numerous compounds that are known to possess diverse nutritional and pharmacological properties, such as caffe acid, catechhin, kaempfer and quercetin, several phytosterols, and fatty acids. The use of ambient mass spectrometry (AMS) techniques has also been described for nutraceutical characterization. AMS involves the direct analysis of compounds from sample surfaces, allowing a straightforward study of unconventional bulk samples, such as whole tablets, plant parts, or tissue sections. The analysis takes place at ambient pressure outside the MS instrument, which speeds up the sampling process and without any sample preparation (or minimal sample manipulation). For example, [PERSON] et al. described the use of direct analysis in real time (DART) for the rapid identification of Salvia species by chemometric processing [105]. The authors analyzed plant leaves in their native form by DART-HRMS without any sample preparation step, obtaining a derived chemical fingerprinting of both fresh and dried saliva material. The presence of essential oil biomarkers such as 3-carene, \(\alpha\)-pinene, \(\beta\)-pinene, \(\beta\)-thujone, \(\beta\)-caryophyllene, camphor, and bornel was confirmed. In another work, [PERSON] et al. revealed the important nutraceutical properties of Mauritius fruit (_Ananas comosus_) against diabetes [106]. In this case, some active compounds identified in ethyl acetate and methanolic extracts of the fruit using DART-HRMS were sinapic acid, daucosterol, 2-methylpropanoate, 2,3-dimethyl-4-hydroxy-4(2H)-furanone, methyl 2-methylbutanoate, and triterpen ergosterol. Desorption electrospray ionization (DESI) is another AMS technique allowing the direct identification and determination of bioactive substances deposited on surfaces and from thin biological tissue sections with minimal (or without) sample treatment. In this case, a charged spray of solvent droplets is directed toward the surface to desorb and ionize the analytes, which are directed to the MS inlet under vacuum conditions. The investigation of phytochemicals from thin-layer chromatography (TLC) imprints or direct plant tissues is well described in the literature. As an example, the chemical profiling and separation of bioactive secondary metabolites in maca (_Lepidium peruvianum_) by normal- and reversed-phase TLC coupled to DESI-HRMS was proposed by [PERSON] et al. [107]. The authors reported for the first time the identification of six potential plant antibiotics, phytoanticipins, glycosylated ascorbagens, and dihydroxascorbagens from Maca seeds. Matrix-assisted laser desorption ionization (MALDI)\(-\)TOF-MS is another excellent technique for the deeper characterization of polyphenols, especially for dealing with polymeric compounds such as PACs [108], especially for the elucidation of intermolecular link positions. The polyphenol profiling of chestnut pericarp, integument, and curing water extracts to qualify these food by-products as a source of bioactive antioxidant compounds for nutraceutical applications was also described by MALDI-TOF-MS [109]. This method allowed the identification of condensed and hydrolyzable tannins such as procyanidins and prodelphinidins.