# 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  
Beyond the spectroscopic and voltammetric methods that provide information on the overall content of polyphenols, separation methods offer excellent opportunities for profiling. Thus, chromatographic techniques result in the best option for the simultaneous quantification of several bioactive components occurring in the samples. Among the arsenal of techniques available in our current analytical laboratories, liquid chromatography seems to be the system of choice. Capillary electrophoresis (CE) can also be used owing to the ionizable nature of polyphenols. For instance, the phenolic content of herbal, fruit, or vegetable extracts has been determined using different CE modes such as capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary isotachophoresis (CITP), and capillary electrochromatography (CEC) [61; 62; 63; 64; 65]. The polar and thermolabile nature of polyphenols hinders the use of gas chromatography (GC) for their direct determination. Hence, GC methods for the determination of polyphenols rely on derivatization procedures to reduce the polarity of analytes. In this regard, silylation has been successfully applied to the identification and quantification of phenolic compounds in plant and fruit extracts [66; 67; 68; 69]. As mentioned, liquid chromatography (LC) is the best option for the determination of polyphenols in nutraceutical products and in plant extracts. In general, phenolic acids, stilbenes, and flavonoids can be separated successfully by reversed-phase (RP) mode, typically with C18 or C8 columns. Beyond conventional High Performance LC (HPLC), the newest and most powerful instrumentation based on ultra-high-performance liquid chromatography (UHPLC) and core-shell column technology can be used. Mobile phases in LC are prepared with MeOH or ACN as organic solvents and acid aqueous solutions (e.g., using formic or acetic acids). The high complexity of the nutraceutical samples often requires customized elution gradients to achieve a good resolution of components. For instance, up to 15 polyphenols were determined in Taurisolo(r) (grape pomace extract supplement, MBMed, Turin, Italy) using a polar RP-C18 HPLC column and an elution gradient generated with 0.1% formic acid in water (\(v/v\)) and acetonitrile [70]. [PERSON] et al. determined more than 30 polyphenol-related compounds in nutraceutical samples (tablets and capsules) derived from green tea and grapes. The UHPLC separation was accomplished with sub-2 \(\upmu\)m particle-size C18 columns together with mass spectrometry (MS) detection [71; 72]. To avoid expensive instrumental platforms, core-shell columns appeared as a good alternative to achieve excellent efficacies. In this regard, [PERSON] et al. used a Kinetex C18 reversed-phase (100 mm \(\times\) 4.6 mm ID, 2.6-\(\upmu\)m particle size) column for the determination of flavanols in dietary products and pharmaceuticals [73]. In another example, sub-2 \(\upmu\)m and porous-shell columns were compared for the determination of 29 polyphenols in cranberry-based pharmaceuticals obtaining, in that case, better performances using the UHPLC option [74]. As an alternative to the reverse-phase mode, the hydrophilic interaction chromatography (HILIC) has opened up great possibilities, especially for dealing with the most polar analytes that are weakly retained in RP columns. HILIC was used, for example, for the separation of flavanols and related compounds in dietary supplements and nutraceuticals from berries, grapes, or artichoke extracts [75] (see Figure 4). The mobile phase consistedof acidified acetonitrile and methanol solutions, applying an elution gradient based on increasing the percentage of methanol. In this study, the authors highlighted the improved separation of flavanol oligomers, as well as the great potential to separate small phenolic acid molecules.