# 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
The LC \(\times\) LC strategy, combining different stationary phases, namely RP (or modified RP), HILIC, and, less commonly, size-exclusion chromatography (SEC), has shown to be promising for the determination of polyphenols in food products [76,77]. LC \(\times\) LC, operating in the off-line or on-line modes, offers improved separations taking advantage of the orthogonality between the first and second dimensions. Consequently, sample clean-up procedures can be simplified by reducing the total analysis time. In this regard, an RP-LC \(\times\) RP-LC method, combining a micro-bore ES-CN column (1D) with a C18 column (2D), was successfully applied to the determination of 51 polyphenolic compounds in pistachio extracts [78]. Another study performed by [PERSON] et al. for the determination of polyphenols in apple extracts samples combined HILIC \(\times\) RP in the two-dimensional LC [79]. In this case, higher peak capacities and sensitivities were obtained with the optimized online HILIC \(\times\) RP-UHPLC-MS method concerning previously 1D reported methods. Regarding detection, apart from the widespread UV-vis spectroscopy, fluorescence and mass spectrometry have been utilized to increase the sensitivity and selectivity of the methods. In UV-Vis, current multi-way spectrophotometers such as diode-array detectors allow the simultaneous detection of the different phenolic families. For instance, the range 270-280 nm is used to monitor benzoic acids, flavanols, flavanones, and other species containing phenyl moieties, 310-330 nm is used for hydroxycinnamic acids and stilbenes, 370-380 nm is used for flavones, isoflav, and flavonols, 420-430 nm is used for curcuminoids, and 450-550 nm is used for anthocyanins. As most of the polyphenolic compounds have fluorescent moieties in their structures, fluorometric detection is an excellent alternative to the UV counterpart, thus providing enhanced performance from a proper selection of the excitation and emission wavelengths. For instance, flavanols are detected at \(\lambda_{\text{ex}}\) 280 nm and \(\lambda_{\text{em}}\) 320 nm with negligible interferences from other phenolic families; \(\lambda_{\text{ex}}\) 325 nm and \(\lambda_{\text{em}}\) 375 nm are good choices for the determination of hydroxycinnamic acids; \(\lambda_{\text{ex}}\) 375 nm and \(\lambda_{\text{em}}\) 430 nm have been recommended for flavonoids, flavones, and isoflav. Currently, MS is the most powerful detector for qualitative and quantitative determinations of phenolic compounds. In our opinion, the hyphenation of MS with liquid chromatography deserves a more exhaustive development. Hence, the state of the art of application of HPLC-MS to polyphenol characterization is given below. Figure 4: Representative chromatograms of various nutraceutical samples obtained by HILIC-FLD. Sample assignation: a = antioxidant extract; b = cranberry; c = cranberry (combined); d = artichoke; e = red grape (peel and seeds); f = raspberry; g = grapevine. Compound assignation: 1 = catechin; 2 = epicatechin; 3 = procyanidin A2; 4 = procyanidin B2; 5 = trimer 1; 6 = procyanidin C1; 7 = tetramer 1; 8 = tetramer 2; 9 = pentamers; 10 = hexamers (adapted from reference [75]). Liquid Chromatography Coupled to Mass Spectrometry  
HPLC-MS techniques, and more recently, the UHPLC-MS counterparts, have been increasingly used in nutraceutical characterization, especially for bioanalytical applications dealing with pharmacokinetics and metabolism studies. LC-MS has been widely exploited for the identification and structural elucidation of known and unknown bioactive molecules, especially from LC-MS/MS experiments [80; 81]. Furthermore, high-resolution mass spectrometers (HRMS) coupled to the LC systems result in exceptional tools for food analysis. Obtaining accurate mass measurements is the major advantage of HRMS to provide almost unambiguous identifications and quantifications. For instance, the presence of curcumin and other curcuminoids in turmeric-based extracts was confirmed using this strategy.