**3. Liquid chromatography in the analysis of flavonoid metabolism**

To show the chemical variety of flavonoids, chromatographic methods have been utilized to examine their structures. Previously, the major methods used to analyze flavonoids were paper chromatography, thin layer chromatography, column chromatography, and liquid chromatography (LC) [36]. Efficient screening of plant extracts may be accomplished using biological assays as well as chromatographic techniques such as high-performance liquid chromatography (HPLC) in conjunction with different detection modalities [37]. Because it permits systematic profiling of complex plant samples and especially focuses on their identification and consistent assessment of the found compounds, HPLC is a potent tool for the quick investigation of bioactive ingredients. Modern HPLC separation of flavonoids nearly entirely uses reversedphase liquid chromatography (RP-LC), with significant exceptions being normal phase liquid chromatography (NP-LC) for oligomeric proanthocyanins [38] and the recent rising use of hydrophilic interaction chromatography (HILIC). Other flavonoid separation methods include mixed-mode ion-exchange-reversed phase separation of anthocyanins [39–41], size exclusions chromatography (SEC) analysis of flavonol glycosides [42], and theaflavins and proanthocyanidins [43]. However, because of the infrequent usage of the later modes, this chapter will concentrate mostly on RP-LC in line with the extent and predominance of this method in flavonoid literature.

RP-LC has proved its applicability for the separation of flavonoids depending on the nature of the aglycone (including the oxidation state, substitution patterns, and stereochemistry), the type and degree of glycosylation, and the nature and degree of acylation. The vast majority of RP-LC separations are accomplished using C18

#### *Application of Liquid Chromatography in the Analysis of Flavonoid Metabolism in Plant DOI: http://dx.doi.org/10.5772/intechopen.107182*

octadecyl-silica (ODS) phases, however, C8 [44], C12 [45], phenyl or phenyl-hexyl [46–50], pentafluorophenyl (PFP) [51–53] and polar embedded RP phases [54–56] as well as polymeric RP-LC phases were still widely used [57]. Aqueous/organic phases including methanol, acetonitrile, and less commonly tetrahydrofuran [58], isopropanol [59] or ethanol [57], and acidic modifiers such as acetic acid, formic acid, ammonium acetate, or trifluoroacetic acid (TFA) [59] are typical mobile phases (phosphoric, citric, or perchloric acids have also been used in combination with UV detection, although these are not suited to hyphenation with MS). Highly acidic mobile phases (>4–10% formic acid, 0.1–0.6% TFA) [60–63] are utilized for anthocyanins to assure the presence of flavylium cationic species in solution and therefore increase chromatographic efficiency. To detect and/or identify flavonoids, a variety of detectors may and have been used in conjunction with HPLC separation. These include electrochemical detection (ED) [64, 65], fluorescence (FL) [66], UV–Vis, diode array [59, 67], NMR [68, 69], and of course MS detectors [70, 71]. The most prevalent currently are diode array and MS detectors, which will be explored briefly in this and the next sections.

#### **3.1 Liquid chromatography (LC) with ultraviolet (UV) detector**

Particularly in early flavonoid research, the conjugated aromatic nature of flavonoids proved to be a significant advantage: absorption at relatively long wavelengths increases the selectivity of qualitative and quantitative spectrophotometric methods, and the distinctive spectra of various classes of flavonoids allow differentiation between them. These qualities are similarly helpful when HPLC separation is combined with UV–Vis detection. Flavonoids exhibit two UV–Vis absorption maxima: Band II (Band II), which is attributed to the A-ring, and Band III (Band III), which is attributed to the B-ring (Band 274 I). Due to the fact that it offers more specialized information and since all flavonoids absorb between 240 and 285 nm, the latter of these is more useful. Due to the absence of conjugation between the A and B rings, flavanols, flavanones, dihydroflavonols, and isoflavones only display Band II absorption (269–279 nm). Anthocyanidins may be easily identified by their Band I absorption between 460 and 550 nm in the visible range, in contrast to flavonols and flavones, which exhibit Band I absorption between 300 and 380 nm [72]. **Figure 6** provides typical illustrations of the UV–Vis absorbance spectra of the major groups of flavonoids.

#### **Figure 6.** *UV–vis absorbance spectra of the principal classes of flavonoids: (a) Luteolin [73]; (b) quercetin [74].*

**Figure 7.** *HPLC–UV at 260 nm (a), 1 and 2 represent UV spectra of peak 7 (hispidol 40-O-glucoside) and peak 34 (afrormosin), respectively [75].*

In research by Mohamed A. Farag et al., an integrated approach utilizing HPLC– UV was used for the large-scale and systematic identification of polyphenols in *Medicago truncatula* root and cell culture. UV spectra (200–600 nm) were recorded for different flavonoid sub-classes including 26 isoflavones (peaks 1–6, 8, 10, 12, 14, 19–29, and 31–35), 4 flavones (peaks 9, 13, 18, and 31), 2 flavanones (peaks 21 and 30), 2 aurones (peaks 7 and 11), and a chalcone (peak 32) with isoflavone representing the major sub-class. For example, isoflavones typically have a maximum absorbance near 255 nm with a second maximum between 300 and 330 nm (peak 34 in **Figure 7**), whereas aurones have the first maximum near 250 nm and the second peak around 390 nm (peak 7 in **Figure 7**) [75].

Kim-Ngan Huynh Nguyen et al.'s study quantified seven major compounds, including phenolic acids (chlorogenic acid, caffeic acid, and p-coumaric acid) and flavonoids (rutin, quercitrin, quercetin, and kaempferol) in three aerial parts of *Physalis angulata*, that is, leaves, calyces, and fruits. Chromatographic separation was carried out on a Kromasil C18 column (150 mm × 4.6 mm i.d., 5 μm) with a gradient elution of 0.1% formic acid in acetonitrile, 0.2% ammonium acetate/0.1% formic acid in water and methanol at a flow rate of 1.0 mL/min; detection was at 250 and 300 nm. The applications of liquid chromatography (LC) with ultraviolet (UV) for the analysis of flavonoid metabolism of plants that show in **Table 1** (**Figure 8**) [81].

#### **3.2 Liquid chromatography (LC) with mass spectrometry (MS)**

In contrast to 30 years ago, routine separation and preliminary identification of complex mixtures of flavonoids ranging over many orders of magnitude in concentration are now achievable because of the combination of chromatographic resolution offered by HPLC and structural data offered by MS. Furthermore, during the past 10 years, significant advancements in LC technology have been realized. UHPLC (ultra-high pressure liquid chromatography), alternative stationary phases including monoliths and superficially porous phases, high-temperature HPLC [82, 83], and multidimensional HPLC [84–87] are a few noteworthy advancements.

Cheminformatics methods combined with LC-MS/MS provide a potent tool for high-throughput surveys of flavonoid variety [88, 89]. Utilizing straightforward solvent combinations and LC columns, glycosylated, acylated, and prenylated flavonoid molecules and their aglycones may be separated. For MS/MS analysis, the isolated


*Application of Liquid Chromatography in the Analysis of Flavonoid Metabolism in Plant DOI: http://dx.doi.org/10.5772/intechopen.107182*

#### **Table 1.**

*Applications of liquid chromatography (LC) with ultraviolet (UV) for the analysis of flavonoid metabolism.*

molecules are ionized next. In order to analyze flavonoids, LC-tandem mass spectrometry (LC-MS/MS) has emerged as the method of choice. Algae had previously been thought to have no flavonoids. But using an LC-MS/MS technique, flavonoids were identified as intermediates and end products, demonstrating the occurrence of flavonoid production in microalgae [90]. It implies that the undiscovered flavonoids in every plant species can be discovered using cutting-edge metabolomics technology.

With an emphasis on general ionization and fragmentation processes, a brief overview of the underlying knowledge pertinent to the MS detection and MS/MS structural elucidation of flavonoids will be provided in this part. Additionally, specialized research reports provide much more in-depth information on particular classes of flavonoids, including dihydroflavonols [91], isoflavones [92], flavone-di-C-glycosides [93], flavonoid-aglycones [94], flavonoid-O-glycosides [95], and flavonoid glycosides [96]. The discussion that follows will be restricted to the API sources electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure chemical ionization, which are presently the most pertinent LC-MS

#### **Figure 8.**

*HPLC chromatogram of the mixed standards solution (a), P. angulata leaves (b). P1–P13: Phenolic acids, including P4: Chlorogenic acid, P6: Caffeic acid, and P8: p-coumaric acid. F1–F9: Flavonoids, including F4: Rutin and F9: Quercetin [81].*

ionization sources (APPI). The applications of liquid chromatography (LC) with mass spectrometry (MS) to the analysis of flavonoid metabolism are shown in **Table 2**.

Shoucuang Wang et al. (2017) researched comprehensive profiling of metabolites in citrus fruits. Non-targeted high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS/MS) was used to profile the metabolites in fruit tissues. As a result, 7416 metabolic signals were detected. In addition to those reported metabolites, seven O-glycosylpolymethoxylated flavonoids were newly annotated in the study. To better characterize these flavonoids, the 3′,4′,5,6,7,8-hexamethoxyflavone standard (m70, RT 15.3 min, m/z 403.1389, error − 0.5 ppm) was analyzed first. The precursor ions of the standard compound lost one to four methyl radicals in the MS/MS spectrum to form the base peaks of [M + H - 15]+, [M + H - 30]+, [M + H - 45]+, or [M + H - 60] + (**Figure 9A**). The characteristic loss of 162 Da was observed in the MS/MS spectra corresponding to the dissociation of a hexose moiety and a series of methyl loss of the diagnostic fragments of 15 and 30 Da (**Figure 9B**–**D**) [102].

Paola Montoro et al. (2012) researched the metabolic profiles of different extracts (obtained by petals, stamens, and flowers) by LC-ESI-IT MS (liquid chromatography coupled with electrospray mass spectrometry equipped with an ion trap analyzer). MS/MS experiments were diagnostic for the identification of specific fragmentation patterns, that is, sugar loss for flavonoid O-glycosides or the loss of specific esterification units. Interpretation of the ESI-MS/MS experiment obtained by the analysis of *Crocus sativus* petals extracts allowed us to tentatively identify 31 flavon derivatives in the extracts under investigation, mainly glycosidated and metoxilated derivatives of kaempferol, quercetin, isorhamnetin, and tamaryxetin (**Figure 10**) [103].

#### **3.3 High-performance liquid chromatography in chiral flavonoid**

Enantiomer separation, resolution, and analysis have traditionally been achieved by the transitory or covalent synthesis of diastereoisomers. Diastereoisomers can be separated on an achiral chromatographic column by differential contact and retention because they have distinct physicochemical characteristics in an achiral environment. On a chemically bonded chiral stationary phase (CSP) with an achiral mobile phase,


*Application of Liquid Chromatography in the Analysis of Flavonoid Metabolism in Plant DOI: http://dx.doi.org/10.5772/intechopen.107182*

#### **Table 2.**

*Applications of liquid chromatography (LC) with mass spectrometry (MS) for the analysis of flavonoid metabolism.*

racemic flavonoid resolution has typically been achieved through chromatographic enantiospecific resolution through transient production of diastereoisomers.

In 1980, one of the earliest publications on flavanone glycoside HPLC separation appeared. Both naringin and narirutin may be acetylated using an equal mixture of pyridine and acetic anhydride and then resolved at low temperatures (between 0 and 5 OC) [104]. Prunus callus (sweet cherries), oranges, and grapefruit's prunin (naringenin-7-O-glucoside) epimers were initially separated in the middle of the 1980s using benzoylated derivatives [105]. On Cyclobond I columns, the separation of

#### **Figure 9.**

*Mass spectra and structures of polymethoxylated flavonoids glycosides in citrus. (A) 3, 4, 5, 6, 7,8-hexamethoxyflavone (m070). (B) Dihydroxy-trimethoxyflavone -O-hexoside (m117). (C) Hydroxytetramethoxyflavone-O-hexoside (m119). (D) Monohydroxy-hexamethoxyflavone-O-hexoside (m133). PMFs, DFI, diagnostic fragment ions of polymethoxylated flavonoids [102].*

prunin benzoate and naringin benzoate has also been shown. Naringenin derivatization to naringenin tribenzoate and separation on a Chiralcel OD column are also mentioned in the literature. Naringenin's hydroxyl groups may prevent chiral identification in this stationary phase as the enantiomers could not be resolved [34].

The main advantage of chiral separation methods over achiral methods is a better understanding of the pharmacokinetics of flavanones and the development of effective dosing regimens. In the case of racemic flavanones or stereochemically pure flavanones, this requires knowledge of the *in vivo* behavior of the enantiomers and epimers. During the drug development process, understanding and comprehending the conformational stability of chiral compounds may have a significant influence on the pharmacological, pharmacokinetic, and pharmacodynamic data. Using stereospecific analytical techniques, racemization and enantiomerization/epimerization may be studied. Rapid interconversion *in vivo* would eliminate any potential distinctions in the enantiomers' medicinal or harmful effects, making the synthesis of stereochemically pure enantiomers useless. Chirality must be taken into account from the beginning of the development process for stereochemically pure compounds and racemates [34].

In a study by Gaggeri R et al. (2011), the HPLC enantioselective separation of (R/S)-naringenin (**Figure 11**), a chiral flavonoid found in several fruits juices and well-known for its beneficial health-related properties, including antioxidant, anti-inflammatory, cancer chemopreventive, immunomodulating and antimicrobial *Application of Liquid Chromatography in the Analysis of Flavonoid Metabolism in Plant DOI: http://dx.doi.org/10.5772/intechopen.107182*

#### **Figure 10.**

*LC-ESI-MS. TIC and reconstructed ion chromatograms for Crocins qualitative analysis in H2O/EtOH extract of Crocus sativus petals [103].*

activities, has been performed on both analytical and (semi)-preparative scale using amylose-derived Chiralpak AD chiral stationary phase (CSP). A standard screening protocol for cellulose and amylose-based CSPs was firstly applied to analytical Chiralcel OD-H and Chiralpak AD-H, as well as to Lux Cellulose-1, Lux Cellulose-2, and Lux Amylose-2 in order to identify the best experimental condition for the subsequent scaling-up. Using Chiralpak AD-H and eluting with pure methanol (without acidic or basic additives), relatively short retention times, high enantioselectivity, and good resolution (Rs = 3.48) were observed. Therefore, these experimental conditions were properly scaled up to (semi)-preparative scale using both a prepacked Regispack column and a Chiralpak AD column packed in-house with bulk CSP [106].

### **4. Conclusion**

Many beneficial health effects have been attributed to flavonoids, which are popular in the plant. The study of metabolism and bioavailability is very important in defining the pharmacological and toxicological profile of these flavonoid compounds. Due to great structural diversity among flavonoids, these profiles differ greatly from one compound to another, so the most abundant polyphenols in our diet are not necessarily the ones that reach target tissues. Therefore, careful analysis of flavonoids and their metabolites in biological systems is critical. Several hundred papers on the HPLC of flavonoids have been published in the past 20 or so years, yet HPLC methods can detect flavonoids across one, two, or perhaps three subclasses in one run. The improvements in HPLC flavonoid analysis closely resemble and, to a certain extent, build on those in domains like proteomics and metabolomics, which are supported by important breakthroughs.
