# 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
As can be seen in Figure 5, the accurate mass spectra obtained by LC-HRMS with an orbitrap analyzer gave the authors reliable information for the identification of curcumin and other major components in the studied samples [82]. By far, ESI is the most generalized ionization source, although atmospheric-pressure chemical ionization (APCI) has also been proposed, especially for those compounds exhibiting low ionization efficiencies under ESI conditions [83; 84; 85]. This is the case, for instance, of the identification and determination of triterpenes and phenolic acids from ancient apples of Friuli Venezia Giulia as nutraceutical ingredients [85]. While ESI resulted in the best option for the determination of phenolic acids, APCI in negative ion mode was selected as the ion source of terpen due to the opportunity to obtain the pseudomolecular [M-H]\({}^{-}\) ions with higher intensity than with ESI [86]. Carotenoids are also a typical family of compounds exhibiting better ionization behavior under APCI conditions in comparison to ESI [87; 88; 84]. For example, [PERSON] et al. [84] described the determination of carotenoids from Araza (_Etugenia stipinate_), an Amazonian fruit with potential value as a nutraceutical source. Recently, UHPLC-APCI-TOF/MS was also proposed for the screening of genistein from selected seeds of Apiacea [89]. To date, dozens of applications of (U)HPLC-ESI-MS/(MS) have been published. Low-resolution mass spectrometers based on triple quadrupole and ion trap analyzers are typically proposed for the sensitive determination of well-known bioactive substances, as well as for identification purposes when working in tandem mass spectrometry [90; 91; 92; 93; 94]. For example, [PERSON] et al. carried out the quantification of 30 bioactive compounds in coffee silverskin extracts by HPLC-MS/MS using a triple quadrupole analyzer [94]. Eighteen phenolic compounds were detected and quantified, with caffeoylquinic acids being the most abundant ones. In contrast, HPLC-DAD-MS using an ion-trap analyzer was employed to address a comprehensive characterization of the phytochemicals present in an Italian ancient apple \"Mela Rosa dei Monti Sibillini\" [93]. Extracts from lyophilized material were richer in polyphenolic compounds than the dried counterparts. These findings suggested that this ancient apple variety was an excellent source of active components for nutraceuticalals. HRMS based on time-of-flight (TOF) and Orbitrap analyzers is the technique of choice for a comprehensive characterization of plant-based extracts due to the higher-resolution  
Figure 5: Reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) total ion chromatogram (**a**) and extracted ion chromatogram of curcumin (_m/z_ 367.1187) (**b**). MS spectrum of peak at retention time of 13.52 min (**c**). Adapted from reference [82]. capabilities and the accurate mass measurements (with errors below 2 ppm depending on the instrument). In these cases, hybrid configurations combining quadrupole and ion-trap analyzers coupled with HRMS instruments such as Q-TOF [95; 96; 97; 98; 99; 100], IT-TOF [101], and Q-Orbitrap [102; 103] were preferred to take advantage of the high sensitivity of these platforms under full-scan acquisition mode. Furthermore, fragmentations can be employed for identification purposes, especially when profiling strategies are applied. For instance, [PERSON] et al. evaluated the potential nutraceutical properties of leaves from several commonly cultivated plants such as lemon (_Ctrus limon_), red silk-cotton (_Bombax ceiba_), henna (_Lawsonia inernis_), eucalyptus (_Eucalyptus globulus_), basil (_Ocimum basilicum_), Mandarin orange (_Citrus reticulata_), and spearmint (_Mentha spicata_) [95]. RP HPLC coupled to a Q-TOF instrument working in negative ion electrospray mode was proposed as the instrumental technique.