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# Chiral and achiral analysis of chiral flavonoids with liquid and supercritical fluid chromatography – A review

Laurine Réset, Clément de Saint Jores, Caroline West

► To cite this version:

Laurine Réset, Clément de Saint Jores, Caroline West. Chiral and achiral analysis of chiral flavonoids with liquid and supercritical fluid chromatography – A review. Journal of Chromatography Open, 2024, 6, pp.100183. 10.1016/j.jcoa.2024.100183. hal-05422271

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Submitted on 17 Dec 2025

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# Chiral and achiral analysis of chiral flavonoids with liquid and supercritical fluid chromatography – A review

Laurine Réset, Clément De Saint Jores, Caroline West*

University of Orleans, Institute of Organic and Analytical Chemistry, CNRS UMR7311, rue de Chartres - BP 6759, 45067 Orléans Cedex 2, France.

Corresponding author:

Caroline West - orcid.org/0000-0001-7595-6777

Phone: +33 2 38 49 47 78

e-mail: caroline.west@univ-orleans.fr

<figure><img src="image_3.png" /></figure>

## Highlights

* Chiral flavonoids are analyzed with LC and SFC methods

* Achiral and chiral separations examined

* Sample complexity in natural products may require two-dimensional methods

* UV-visible detection most frequently used

* Fluorescence detection offers better sensitivity

* Mass spectrometry offers sensitivity and structure information
**Abstract**

Flavonoids have been the topic of numerous studies for decades because they possess varied interesting bioactivities including e.g. antioxidant and anticancer properties. In particular, some flavonoids possess chiral features, due to the presence of one or two chiral carbon atoms on their aglycone root. They include flavans, isoflavans, flavanones, flavanonols, flavanols and auronols. In addition, other chirality centres may be observed on the carbohydrate groups attached to the aglycone root, but these will not be considered in the present paper. However, as stereoisomers usually have different bioactivity, analysis methods focusing on these specific flavonoid classes should be of interest to better understand their mode of action and their biosynthetic pathways. In the present review, we have examined chromatographic analyses in the liquid phase (LC) and the supercritical phase (SFC) in achiral and chiral modes to analyse chiral flavonoids. One- and two-dimensional methods are reported, and the different detection modes are also examined.
Note that this review did not aim at being comprehensive, but rather at displaying the diversity and main features of current methods.

**Keywords:** chirality; enantioresolution; flavonoids; natural products
Table of content

1. Introduction to chiral flavonoids

1.1. Flavonoid properties

1.2. General structure of chiral flavonoids

1.3. Significance and difficulties of chirality

1.4. Stability of chiral flavonoids

1.4.1. Racemisation

1.4.2. Epimerisation

1.4.3. Enantiomerisation

2. Achiral analysis of chiral flavonoids

2.1. Liquid chromatography

2.2. Supercritical fluid chromatography

3. Chiral analysis

3.1. Chiral stationary phases

3.1.1. Polysaccharides

3.1.2. Cyclodextrins

3.2. Mobile phase composition

3.3. Operating parameters

3.3.1. Oven temperature

3.3.2. Pressure in compressible fluids

4. Two-dimensional chromatographic analyses

5. Detectors

5.1. UV-visible single-wavelength and diode-array detectors

5.2. Fluorescence detection

5.3. Mass spectrometry

5.4. Circular dichroism

5.5. Sensitivity

6. Conclusion
# 1. Introduction to chiral flavonoids

Flavonoids belong to the polyphenol family. They are widely distributed in nature and form an integral part of our daily lives. They are found in many food products such as fruits, vegetables, cereals, herbs and spices. As a result, flavonoids are also present in drinks such as tea, red wine and fruit juice. They contribute to the colour of certain foods and flowers, but they also play a crucial role in the survival, growth, reproduction and defence of plants. In addition to their presence in the plant kingdom, flavonoids are present in certain marine organisms [1], but their function in marine biology is not yet fully understood. Nevertheless, they also appear to be important compounds for the survival and adaptation of marine organisms in their environment.

In 1994, the number of flavonoids identified was estimated at around 4,000 [2], then increased over the years to reach 7,000 in 2005 [3]. To date, 9,000 flavonoids have been identified, which make up 60% of the known phenolic compounds [4]. This makes them one of the most important and diverse families of natural compounds yet identified. The major reason for this intensive research is that flavonoids have been associated with a number of beneficial properties to be exploited in a variety of fields, including pharmaceuticals, cosmetics and the food industry. In addition, some flavonoids have chiral features, which should contribute to differentiated properties between stereoisomers. Therefore, there is a great interest in developing analysis methods for flavonoids, and among them, for chiral flavonoids. Chromatography is one such method, on which the present review will particularly focus.

## 1.1. Flavonoid properties

Flavonoids have antioxidant properties that help protect body cells from damage caused by free radicals, which are responsible for diseases such as cancer [5-7] or cardiovascular disease [8-10], but also contributing to ageing [11]. They also have anti-inflammatory effects and have been used in traditional medicine to treat bacterial and fungal infections [12]. In 1936, Rusznyak and Szent-Gyorgyi demonstrated that flavonoids have the ability to strengthen the resistance of blood vessels [13]. They improve the elasticity and tightness of blood vessels, reducing the risk of heart disease by lowering cholesterol levels and regulating blood pressure. In addition, they have promising potential in the fight against cancer by inducing programmed cell death and inhibiting the angiogenesis feeding cancer cells [14,15]. Finally, flavonoids appear to improve cognitive function by improving blood flow to the brain, which helps protecting against oxidative damage and inflammation, and may help prevent neurodegenerative diseases such as Alzheimer's [16].

Working with flavonoids is rendered difficult since they are produced in small quantities by living organisms, and are divided into so many different structures. Although flavonoids are
widely distributed in nature, their availability in food and their bioavailability in the human body vary considerably [17]. This variation is due to numerous factors such as plant variety, growing conditions, maturity and processing method. Their complex chemical structures (see next section) mean that they cannot always be easily produced by conventional chemical synthesis. In fact, their physicochemical properties, such as sensitivity to pH, temperature [18] and light, or limited solubility [19], complicate their use and analysis.

## 1.2. General structure of chiral flavonoids

The biological properties of flavonoids stem from their chemical structure (Figure 1). The core structure consists of two rings, A and B, which may be linked through a carbon chain (in chalcones) or through a third ring (ring C).

<figure><img src="image_4.png" /><figcaption>**Figure 1.** General structures of chiral flavonoids</figcaption></figure>

Chemical modifications on this backbone notably include methoxyl groups and hydroxyl groups [20], as can be observed for example on Hesperetin in Figure 2. Hydroxyl groups are also possibly modified with carbohydrate groups, as appears in Hesperidin (Figure 2), which is the glycosylated form of Hesperetin, or in Naringin that is the glycosylated form of
Naringenin. Hydroxyl groups may also be acylated with different acids. For instance, acetylation may be obtained with gallic acid, as appears in Epigallocatechin gallate (Figure 2), which is an acetylated form of Epigallocatechin. The general classification of flavonoids into sub-families is based on the oxidation state and degree of unsaturation of the central heterocycle (ring C in Figure 1). In other words, flavonoid classification depends on the presence or absence of a double bond between C2 and C3, the position of the bond between the B and C rings, the opening or closing of the C ring, and the nature and position of the substituents in the C ring [21].

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Examples of chiral flavonoids mentioned in this paper.</figcaption></figure>
Moreover, when no double bond is present between C2 and C3, a chiral centre is present at the C2 and/or C3 positions. This is the case for the flavan, isoflavan, flavanone, isoflavanone, flavanonol and flavanol families (Figure 1). For instance, Hesperetin and Naringenin, together with their glycosylated forms Hesperidin and Naringin, all belong to the group of flavanones. Catechin and Epicatechin belong to the group of flavan-3-ols, while Silymarin is a flavanonol. Moreover, flavans, isoflavans and flavanols may bear a third chiral centre on C4 when a hydroxyl group or a larger group is present in this position, as is the case in procyanidin oligomers (Figure 2). Finally, the C ring is most often a 6-membered ring (including one oxygen atom and 5 carbon atoms), but also exists as a 5-membered ring in the auronol family, which is less frequently encountered. In auronols, a single chiral centre exists on the C2 carbon atom (Figure 1).

In this review article, we will focus solely on flavonoids bearing one or more chiral centres on the root structure (not taking account of carbohydrate chirality).

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Relation between molecular weight (MW) and hydrophobicity (octanol-water partition coefficient) in chiral flavonoids cited in this review (Tables 1, 2, 3). Bubble size is related to the topological polar surface area (TPSA). All molecular descriptors were computed with MolDesc (icoa.fr)</figcaption></figure>

To better understand chromatographic methods applied to these compounds, it is also useful to examine the variations of size and polarity within these families. Figure 3 represents the variation of log P (octanol-water partition coefficient) depending on molecular weight. The figure was drawn based on molecules found in the literature cited in Tables 1, 2 and 3 and using the same computation tool for all of them (moldesc.icoa.fr) because literature data were not available for all molecules. First, it can be easily noticed that no general trend can
be observed between molecular weight and the hydrophobicity expressed by log P when all
molecules are considered. However, a tendency can be observed with the white bubble
points on the left side of the figure (low molecular weight) where increasing numbers of
hydroxyl groups cause both an increase in molecular weight and decreasing hydrophobicity
(decreasing log P). Another trend can be seen with increasing size of glycosylated groups:
on the left are aglycon molecules, while the introduction of a monosaccharide unit or a
disaccharide unit is causing again increased molecular weight and decreased
hydrophobicity. Other points fall out of these trends: they are gallate acetylated molecules
and dimers (e.g. procyanidin dimers). For such variations, the increase in molecular weight is
not causing any significant change in log P values.

A better relation between molecular weight and polarity can be found when polarity is expressed by the topological polar surface area (TPSA), with a logarithmic tendency fitting all molecules (Figure S1). On Figure 3, TPSA is represented by the bubble size. Quite logically, all points on the right size of the figure (large molecular weight) have large TPSA values, due to the presence of numerous oxygen atoms, although they exhibit a variety of log P values as the hydrophilicity of oxygen atoms is counterbalanced by the hydrophobicity of carbon atoms. Understanding these structural variations will be useful in the next sections when discussing the choice of chromatographic methods and elution orders. Typically, log P values are useful to explain elution orders in reversed-phase liquid chromatography, where hydrophobic interactions dominate the retention mechanism, while TPSA values are useful to explain elution order in normal-phase elution modes where hydrogen bonding is most significant to explain retention.

**1.3. Significance and difficulties of chirality**

Chirality is a fundamental characteristic of nature [22] that is widely encountered in many
classes of natural compounds as carbohydrates, amino acids, lipids, and of course
flavonoids, to name a few. Chirality is well known to have a significant impact on bioactivity
as stereoisomers (enantiomers and diastereomers) usually have different biological activity
and toxicity [23]. Furthermore, they are usually metabolized at a different rate, or degrade at
a different rate in the environment. As a result, while the chirality of natural products is not
frequently considered, chiral separation is a primary concern in the pharmaceutical industry,
where enantioseparation is considered crucial. Considering their different features, the
resolution of chiral active compounds should progressively become a concern to other
application fields such as cosmetics and the food industry. The purpose of separating
stereoisomers may be to control their proportions (analytical scale) or to purify single
stereoisomers for activity testing (preparative scale). Chromatographic techniques are well
suited to both purposes.
Regarding the specific case of chiral flavonoids, in recent years most chromatographic studies were related to two specific families of chiral flavonoids, the flavanones and flavan-3-ols. These studies focused on their quantification in natural products, as well as their enantioresolution on different chiral stationary phases [24].

Chiral separations are always challenging because they are difficult to predict. Furthermore, molecules with several chiral centres cause additional complications, because the number of stereoisomers increases with the number of chiral centres: with n chiral centres, there are a maximum of 2ⁿ stereoisomers. In the case of chiral flavonoids, if one excludes the chirality on possible carbohydrate moieties that are generally enantiopure, there would most often be one or two chiral centres (on C2 and/or C3), resulting in two to four stereoisomers. A third chiral centre on C4 would induce eight stereoisomers.

Examining the conformation of chiral flavonoids is therefore important to understand their biosynthetic pathways, their interactions with other biological molecules, as well as their bioavailability and activities. Molecular modelling methods have been used to predict the physicochemical and biological properties of flavonoid enantiomers, providing a deeper understanding of their biological activity, but analytical methods are also useful in this respect, as will be further discussed in this review.

## 1.4. Stability of chiral flavonoids

Some chiral flavonoids are more stable than others, and this stability depends on the conditions to which they are exposed. Before and during chromatographic analysis, flavonoids can be subjected to various conditions such as temperature, pH, light and solvents [25]. These conditions can affect certain flavonoids, which have specific functional groups on their structure. This can lead to their degradation or transformation into other isomers, causing processes such as racemisation, epimerisation and enantiomerisation. It is important to identify such behaviours, as molecules that are sensitive to such changes of configuration should typically not be used in drug formulations. Thermodynamic studies and molecular modelling can be helpful to understand the stability of chiral flavonoids and their degradation and conversion processes [26]. The most stable isomeric forms generally have a lower free energy, indicating greater thermodynamic stability.

### 1.4.1. Racemisation

Racemisation occurs when a single enantiomer or scalemic mixture (non-equimolar mixture of the enantiomers) is transformed into a racemic mixture, i.e. an equimolar mixture of the two enantiomers. When only one chiral centre is present, it means that this chiral centre takes the opposite configuration. When more than one chiral centre is present, all of them take the opposite configuration to obtain the opposite enantiomer. In the case of chiral
flavonoids, racemisation can only concern aglycon forms, since the carbohydrate groups are stable in this respect. Several factors favour the racemisation process, such as temperature and pH. The solvent can strongly influence racemisation, affecting the chiral purity of a mixture. During acid or enzymatic hydrolysis, an inappropriate solvent can accelerate the conversion of enantiomers between them, thus reducing the enantiomeric excess and the chiral purity of the final product. In addition, it seems that flavanones substituted with hydroxyl groups are more susceptible to racemisation than those substituted with methoxyl groups [27].

### 1.4.2. Epimerisation

Epimerisation is the process of reversible conversion of an enantiomer comprising more than one chiral centre into a diastereomer, thus impacting only one chiral centre. This can again occur under the influence of factors such as exposure to light, pH variations or chemical rearrangements. Many chiral flavonoids may be subject to this effect: it may occur in aglycon molecules, where more than one chiral centre is present, as is the case in flavanols and flavanonols, or in glycosylated forms, related to the configuration change of the C2, C3 or C4 chiral atom.

For instance, in 2005, Nahoko Uchiyama et al. showed that when a sample of commercial Hesperidin (Figure 2), a glycosylated flavanone bearing the S configuration on C2, was kept in boiling water, the relative amount of Hesperidin decreased and the amount of epimer (2R) increased as a function of time [28].

In the case of aglycon flavanols, the catechin family (Figure 2) was examined by different authors and for different purposes. As appears in Tables 1, 2 and 3 (-)-Epicatechin is present in many plants. However, it is more bioactive than (+)-Catechin [29,30] and therefore more desirable. Studies have shown that it is possible to obtain the (+) enantiomer of Epicatechin from (+)-Catechin using a catalysis reaction [30]. However, Catechin may degrade during the manufacturing process [29]. For instance, it is present in cocoa beans but can no longer be found after transformation to chocolate.

Hiroyuki Terashima et al. studied the epimerisation of Catechin and Epicatechin [31]. Thanks to a chiral chromatographic method (see section 3), it was found that the epimerisation of (+)-Catechin and (+)-Epicatechin could be suppressed by adding D-fructose, but not D-glucose and D-sucrose. Conversely, this epimerisation reaction is sometimes deliberately provoked. In nature, the (+)-Epicatechin enantiomer is not present or is present in too small quantities; the predominant form is 93% (-)-Epicatechin [29]. Using a native *C. sinensis* anthocyanidin reductase (CsANR), Yumei Qian et al. succeeded in producing (-)-Epicatechin and (+)-Epicatechin using a recombinant CsANR [30]. This provides a better understanding of the biosynthesis of these molecules.
In a different study, Boulbekhari et al. examined epimerisation of naringin and hesperidin were analysed through a chiral chromatography method. Such methods will be further explained in section 3.3 below.

#### 1.4.3. Enantiomerisation

Enantiomerisation refers to the reversible conversion of one enantiomer into another with an opposite spatial configuration. This process can be catalyzed by certain chemical agents or by specific reactions.

Regardless of the degradation process, the change in configuration can lead to a loss of biological activity or specific flavonoid properties. These phenomena can also affect the relative quantity of enantiomers in a final product, which can have consequences for the efficacy and safety of the product. It is important to note that these degradation and conversion processes may be reversible under certain circumstances. Moderate processing conditions or adjustments to environmental parameters can restore the original chirality of flavonoids. In order to minimize exposure of chiral flavonoids to conditions likely to degrade them or convert them into isomers, it is essential to use mild storage and analytical conditions, control temperature and pH, and protect samples from light. For instance, Roberto Cirilli et al. demonstrated the enantiomerisation of flavanone on a Chiralpak IA column in the presence of 2'-hydroxychalcone [25]. In their study, changing the pH of the borate buffer in the mobile phase (ethanol-borate) resulted in the disappearance of the interconversion zone, which appears as a plateau above the baseline between the peaks of the enantiomers (so-called "batpeaks"). In addition, increasing the percentage of aqueous solution in the mobile phase caused the order of elution to reverse.

## 2. Achiral analysis of chiral flavonoids

As mentioned above, over the last few decades, research into flavonoids has continued to grow due to their numerous bioactivities. As a result, the chromatographic methods to analyse flavonoids, and among them chiral flavonoids, have evolved to become more selective and sensitive. Due to the limited volatility of most flavonoids, their analysis with gas chromatography (GC) would require derivatization procedures [32]. In the case of polyphenolic analytes, derivatization is often causing issues because multiple derivatives may be produced from a single analyte. GC will therefore not be discussed in the following, as we will focus on direct methods. Liquid chromatography (LC), whether as high-performance (HPLC) or ultra-high-performance (UHPLC), is the most widely used to separate and quantify flavonoids. The largest proportion of research papers is concerned
with achiral analysis only (this section), while a smaller proportion of research papers is
related to chiral analysis, i.e. the separation of stereoisomers (see section 3). In achiral and
chiral LC methods, different elution modes may be applied, as further discussed below
 subsection 2.1).

Recently, other techniques have been developed to improve selectivity in the analysis of
chiral flavonoids, notably supercritical fluid chromatography (SFC, section 2.2) and two-
dimensional (2D) chromatography (in the liquid or supercritical phase, section 4). SFC offers
advantages such as highly-efficient fast separations and a complementary selectivity to the
most used reversed-phase LC methods, while 2D methods improve peak capacity with
complementary selectivity [33,34].

LC and SFC methods are both mainly used with UV detection at a single wavelength, or a
diode-array detector (DAD) allowing to record the whole UV-visible spectrum of each analyte
(see section 5.1). Alternately, higher sensitivity and some more selectivity may be obtained
with fluorescence detection (FLD, see section 5.2). When a better selectivity and sensitivity is
required, the chromatographic method is preferably hyphenated to mass spectrometry (MS).
Furthermore, tandem mass spectrometry (MS/MS) provides more sensitive detection, and a
chance for more detailed structural information (see section 5.3). Other, more specific
detectors like circular dichroism (CD) may be useful when it is necessary to differentiate
between two enantiomers (section 5.4).

**2.1. Liquid chromatography**

LC is by far the most common technique to analyse flavonoids. Achiral analysis of chiral
flavonoids can be useful in many instances. In 2016, André de Villiers et al. highlighted the
progress made in previous years in the achiral analysis of all flavonoids (not only the chiral
ones) [35]. In many cases, it is necessary to rapidly assess the purity and composition of
flavonoid samples, whether it be during (hemi)synthesis, following extraction [36,37] or
monitoring of plant profiles during harvesting [38,39]. In such cases, extensive chiral
separation is not always useful or necessary, and achiral analysis of flavonoids can provide
sufficient information. For example, in oenology, it is essential to analyse different phenolic
compound profiles to select by-products and the appropriate grape variety depending on the
phenolic compounds desired [40]. Furthermore, when the aglycon root is carrying a chiral
centre, but also a carbohydrate moiety, because the carbohydrate group is usually
enantiopure, the two opposite configurations of the chiral centre mean that the molecules
obtained are diastereomers, thus may be resolved in an achiral environment. For instance,
this would be the case for the (2S) and (2R) isomers of Hesperidin (Figure 2). Also, when two
chiral centres are present on the aglycon core structure, two pairs of enantiomers yield
diastereomeric pairs. The best example of such case is that of (+)-Catechin and (-)-
Epicatechin (see Figure 2), which are diastereomers and therefore may be resolved in an achiral environment.

A summary table of the different stationary and mobile phases used in the recent literature on achiral analysis of chiral flavonoids is presented in Table 1. As it will appear there, mostly flavanones and flavan-3-ols were reported.

<table><caption>**Table 1** - Achiral LC and SFC analyses of chiral flavonoids in the recent literature (from 2015). See main text for abbreviations of techniques, detectors, solvents and additives.</caption><thead><tr><th>Sample</th><th>Flavonoid classes</th><th>Specific flavonoids examined</th><th>Technique</th><th>Detector(s)</th><th>Stationary phase and column dimensions</th><th>Mobile phases</th><th>Flow rate (ml/min)</th><th>Temperature / Pressure</th><th>Gradient</th><th>References</th></tr></thead><tbody><tr><td>zhitzy Da Huang (Ginsengoside Ellis, Crouanthium, B.palmatum)</td><td>Flavan-3-ols Flavanones</td><td>Catechin Epicatechin Naringenin Naringutin Naringin (Neo)hesperidin</td><td>UHPLC</td><td>MS</td><td>Kinetex C18 (100 x 4.6 mm – 2.6 µm)</td><td>A) H₂O + 0.1% FA<br>B) ACN</td><td>0.8</td><td>35°C</td><td>5-95% B (55 min)</td><td>26</td></tr><tr><td>Herbal dietary supplements</td><td>Flavan-3-ols Flavanones</td><td>Hesperetin (+/-)catechin, (neo)hesperetin (-)-epicatechin (-)-epicatechin gallate</td><td>UHPLC</td><td>UV MS/MS</td><td>Chromolith RP18 (50 x 2.0 mm)</td><td>A) H₂O + 0.05% TFA<br>B) ACN</td><td>1.2</td><td>20°C</td><td>8-80% B (5 min)</td><td>35</td></tr><tr><td>Commercial chocolates</td><td>Flavan-3-ols</td><td>(+/-)-Catechin (+/-)-epicatechin</td><td>HPLC</td><td>DAD</td><td>Nova pak C18 (250 x 4.6 mm – 4 µm)</td><td>A) H₂O/ THF/TFA (92:2:0.1)<br>B) ACN + 0.1% TFA</td><td>0.7</td><td>30°C</td><td>15-100% B (24 min)</td><td>31</td></tr><tr><td>Grape pomace</td><td>Flavan-3-ols</td><td>(-)-Epicatechin (-)-galocatechin (-)-epigallocatechin (+)-catechin (-)-galocatechingallate</td><td>UHPLC</td><td>DAD</td><td>Kinetex C18 (100 x 3.0 mm – 2.6 µm)</td><td>A) H₂O + 0.1% FA<br>B) ACN</td><td>0.8</td><td>35°C</td><td>5-95% B (11.5 min)</td><td>30</td></tr><tr><td>Saraca asoca</td><td>Flavan-3-ols</td><td>Epicatechin Catechin Epicatechin gallate Epigallocatechin gallate Procyanidin</td><td>UHPLC</td><td>DAD</td><td>Acquity UPLC HSS T3 (50 x 2.1 mm – 1.8 µm)</td><td>A) H₂O + 0.1% H₂PO₄<br>B) ACN</td><td>0.75</td><td>37°C</td><td>5-80% B (9 min)</td><td>27</td></tr><tr><td>Sphaerophyso salsugae</td><td>Flavonoid glycosides</td><td></td><td>HPLC</td><td>UV</td><td>Unitary C18 (250 x 4.6 mm – 5 µm)</td><td>A) H₂O + 0.2% FA<br>B) ACN</td><td>1.0</td><td>30°C</td><td>5-45% B (40 min)</td><td>40</td></tr><tr><td>Blackcurrant</td><td>Flavan-3-ols Flavanones</td><td>Fusitin Catechin Epicatechin</td><td>HPLC</td><td>DAD MS</td><td>Synergi C18 Hydro-RP 80A (150 x 2.0 mm – 4 µm)</td><td>A) H₂O + 0.1% FA<br>B) ACN + 0.1% FA</td><td>0.3</td><td>30°C</td><td>2-100% B (35 min)</td><td>29</td></tr><tr><td>Pera Rio orange juice</td><td>Flavanones</td><td>Hesperidin Naringutin Eriocitrin</td><td>HPLC</td><td>DAD MS</td><td>BEH XBridge C18 (250 x 4.6 mm – 5 µm)</td><td>A) H₂O + 0.1% FA<br>B) ACN</td><td>1.0</td><td>-</td><td>6-100% B (43 min)</td><td>28</td></tr><tr><td>Cramberry-based pharmaceuticals</td><td>Flavan-3-ols</td><td>Catechin Epicatechin Procyanidin</td><td>UHPLC</td><td>FLD</td><td>Kinetex C18 (100 x 4.6 mm – 2.6 µm)</td><td>A) H₂O + 0.1% FA<br>B) MeOH</td><td>0.4</td><td>-</td><td>12-95% B (33min)</td><td>41</td></tr><tr><td>Tea Benifuuki (Comellio sinensis)</td><td>Flavanones</td><td>3'-O-methylated epigallocatechin gallate</td><td>HPLC</td><td>UV</td><td>XSelect CSH Phenyl hexyl XP (100 x 3.0 mm – 2.5 µm)</td><td>A) H₂O + 0.1% FA<br>B) ACN + 0.1% FA</td><td>0.7</td><td>40°C</td><td>6-95% A</td><td>42</td></tr><tr><td>Propolis</td><td>Flavanones</td><td>Ringstrobin</td><td>HPLC</td><td>MS/MS FLD</td><td>Not mentioned</td><td>MeOH / H₂O (25/75) + 1.48 g KH₂PO₃, pH = 7.1</td><td>1.0</td><td>-</td><td>-</td><td>43</td></tr><tr><td>Cito reticulatae Pericarpium</td><td>Flavanones</td><td>Didymin Hesperidin</td><td>SFC</td><td>DAD</td><td>Zorbax RX-SIL (150 x 2.1 mm – 5 µm)</td><td>A) CO₂<br>B) MeOH</td><td>0.8</td><td>45°C / 95 bar</td><td>8-36% B (11 min)</td><td>38</td></tr><tr><td>Synthetic compounds for pharmaceutical and cosmetic applications</td><td>Flavanones Flavan-3-ols</td><td>Hesperetin Naringenin Catechin Epigallocatechin</td><td>UC</td><td>DAD MS</td><td>Acquity Torus DEA (100 x 3.0 mm – 1.7 µm)</td><td>A) CO₂<br>B) MeOH + 0.1% MSA</td><td>2.0-0.7</td><td>25°C / 150-110 bar</td><td>20-100% B (12.5 min)</td><td>37</td></tr><tr><td>Bee pollen</td><td>Flavanones</td><td>Ringstrobin 2'-hydroxyflavanone Flavanone 4-hydroxyflavanon 6-hydroxyflavanone 7-hydroxyflavanone Naringin Naringenin Hesperetin Taxifolin</td><td>SFC</td><td>DAD CD</td><td>Lichrospher 100 Dijol (250 x 4.6 mm – 5 µm)</td><td>A) CO₂<br>B) ¡PROH</td><td>3.0</td><td>35°C / 150 bar</td><td>5-50% B (20 min)</td><td>39</td></tr><tr><td>Citrus</td><td>Flavanones</td><td>Hesperetin Naringenin</td><td>UHPSFC</td><td>DAD</td><td>Viridis BEH (100 x 3.0 mm -1.7µm)</td><td>A) CO₂<br>B) MeOH + 1% FA</td><td>1.0</td><td>40°C / 140 bar</td><td>3-30% B (20 min)</td><td>44</td></tr></tbody></table>

Due to their structural similarities, the achiral separation of flavonoids can however be complex. In this context, an appropriate LC mode must first be selected. As is also the case for many applications, due to its separation capability and widespread availability, reversed-phase liquid chromatography (RPLC) is the most widely used for flavonoid analysis [37,41–
43]. The reported RPLC methods ensured effective analysis of flavonoids in a variety of applications, such as the pharmaceutical industry, life science and foodomics.

In RPLC, a hydrophobic stationary phase is used, most often octadecyl-siloxane-bonded silica, although other non-polar stationary phases have been reported like phenyl-hexyl-siloxane-bonded silica. The mobile phase, on the other hand, is polar, mainly composed of a mixture of water or buffer (solvent A) and an organic solvent (solvent B), which is most often acetonitrile (ACN), or sometimes methanol (MeOH). Acidic additives such as formic acid (FA), trifluoroacetic acid (TFA) or phosphoric acid ($H_3PO_4$) are commonly introduced in the mobile phase to stabilize some of the flavonoids that are prone to degrade in aqueous solution, and therefore improve their elution with good peak shapes to favour resolution.

However, the strongest acids are inappropriate for hyphenation to mass spectrometry.

RPLC is mostly appropriate for hydrophobic and moderately hydrophilic analytes. Judging from Figure 3, this should be well applicable to flavonoids, since most of them have positive log P values. Only the glycosylated molecules are somewhat hydrophilic.

The elution order of flavonoids in RPLC depends on the position and number of hydroxyl groups on their structure. In general, increased hydrophilicity through increased number of hydroxyl groups or glycosylation logically causes earlier elution. On the contrary, increasing the number of methoxyl groups causes increased retention. The position of the hydroxyl groups also matters. For instance, hydroxyl groups located at the ends of the molecule (as positions 3', 4', and 5' of B-ring) are more accessible for polar interactions (e.g. solvation by the polar mobile phase) than those located closer to the C ring, so some resolution of isomeric species is possible. In addition, intra-molecular hydrogen bonding would also occur between proximal polar functions, which may again cause different solvation by the polar mobile phase and/or different insertion in the non-polar stationary phase, resulting in different chromatographic retention.

As is also the case for other applications fields, a trend towards ultra-high efficiency was observed in the recent applications to flavonoids analysis [40,44]. Ultra-high-performance LC (UHPLC) is best obtained with modern instruments offering reduced dead volumes combined with columns packed with sub-2 µm fully porous particles, or sub-3 µm superficially porous particles. However, many recent flavonoid analyses in RPLC were still based on larger particles (4-5 µm) and are thus still relevant of HPLC.

Ionic liquids are liquid salts with unique properties, including high thermal stability and the ability to form hydrogen bond. Ionic liquids were very trendy some 10-15 years ago, whether to be introduced in the mobile phase or to be bonded on the stationary phase. They are now less frequently observed in RPLC chromatographic methods [45]. However, the introduction of an ionic liquid in the mobile phase can improve the resolution of flavonoids: the selectivity
of the separation depends on the hydrogen bonds formed between the ionic liquid and the
hydroxyl groups present on the flavonoid structure.

Another LC mode used for flavonoid analysis is hydrophilic interaction liquid chromatography (HILIC). HILIC relies on a polar stationary phase (like bare silica gel, or polar-bonded silica), and a polar mobile phase comprising a major portion of ACN and a minor portion of aqueous buffer. Therefore, it is appropriate to retain the most polar flavonoids, *i.e.* those with glycosylated groups or flavonoid-derived polar metabolites. It was mostly employed to resolve non-chiral flavonoids that co-eluted in RPLC [43] and has been widely described for use in 2D-LC methods [44,46–48] (see section 4). A significant advantage of HILIC over RPLC is that it offers increased MS sensitivity with electrospray ionization mode (ESI), because the acetonitrile-rich mobile phase is easier to evaporate than the water-rich mobile phases of RPLC mode.

The choice between RPLC and HILIC therefore depends on the specific characteristics of the samples to be analysed (their polarity and the expected differences within the sample) and the analytical objectives.

## 2.2. Supercritical fluid chromatography

Over the last few decades, supercritical phase chromatography (SFC) has emerged as a possible alternative to LC to separate flavonoids. The specific properties of the mobile phase, mainly composed of pressurized CO2, give SFC some advantages over LC. The most obvious is the significantly reduced consumption of organic solvents, making it more environmentally-friendly, especially when preparative-scale separations are desired. In addition, supercritical fluids offer high efficiency at high flow rate, which is often resulting in better resolution per unit time. Although known for over 60 years, in the past decade the manufacturing of new-generation SFC instruments has led to a renewed interest in the technique. Indeed, these modern instruments are more robust and provide higher efficiencies than older instruments [33], notably through reduced dead volumes, thereby favouring the use of small-particle stationary phases to provide ultra-high-performance SFC (UHPSFC). In addition, hyphenation to mass spectrometry is facilitated. All of this has led to an increase in the number of application articles published in the recent years, and flavonoids analysis is one example among them.

Developing and optimizing an SFC method involves various parameters, starting with the selection of stationary phase, which influence on the separation is paramount, followed by the nature and percentage of co-solvent in the CO2-based mobile phase, to ensure the elution of all target analytes. The back-pressure applied after the separation and UV-visible detection to ensure that the fluid is maintained above its critical pressure, the oven temperature and the flow rate are fine optimization parameters. All of them influence the
density of the mobile phase in SFC, making it possible to tune the method to the specific flavonoid separation requirements.

Methanol is by far the most employed co-solvent in SFC. In the case of flavonoid analysis, isopropanol was also reported (see Table 1). In most cases, the percentage of co-solvent introduced in pressurized CO₂ remains below 50%, but the most polar flavonoids may require larger proportions of co-solvent at the end of an elution gradient. There is actually no limit to this proportion of co-solvent, although the fluid state may not be supercritical anymore when such large proportions are used. Similar to LC modes, additives are often introduced in the SFC mobile phase, especially to facilitate the elution of ionisable analytes. In the case of flavonoids, acidic additives like FA or TFA are preferred, for the same reasons mentioned in the previous section. A less common acid, methanesulfonic acid (MSA) was found to greatly enhance the peak shapes and resolution of flavonoids [46].

One of the special features of SFC is its ability to analyse a wide range of flavonoids, covering different polarities. A study carried out in 2020 [46] employed a broad gradient, covering a wide range of compositions in the mobile phase starting with CO₂-rich mobile phase and ending in pure liquid. Such wide gradients, covering SFC and LC conditions, are usually called “unified chromatography” analyses (UC). This approach offers great flexibility to analyse complex samples containing flavonoids with a wide range of polarities more completely and accurately.

Similar to LC, there are several retention modes in SFC, mostly depending on the stationary phase polarity, as the mobile phase is always composed of CO₂ and co-solvent. SFC can be similar to either the normal phase mode (NP), where a polar stationary phase retains polar compounds preferentially. In that case, similar elution orders to HILIC may be expected. Alternately, SFC can be similar to the reversed-phase mode (RP), where a non-polar stationary phase favours the retention of non-polar analytes. However, RPSFC may yield different elution orders from RPLC because the absence of water in the SFC mobile phase significantly changes the selectivity as no hydrophobic interactions occur. For instance, in one particular study, it was observed that using C18 columns in SFC yielded undesirable co-elution of some analytes and generally poor selectivity [47]. In the recent literature, flavonoids were mostly analysed on polar stationary phases, comprising a variety of chemistries (bare silica gel, diethyl-aminopropyl-bonded silica, propanediol-bonded silica or 2-ethylpyridine-bonded silica), thus these separations were all NPSFC. For instance, Ana M. Ares *et al*. were able to separate 10 flavonoids in a single analysis on a 5-µm propanediol-bonded silica stationary phase in 20 min [48].

Whatever the chromatographic technique (LC or SFC) or mode (RPLC, NPLC or HILIC) employed, it should be noted that a single achiral chromatographic method is not always
sufficient, and that it may be necessary to use complementary approaches such as off-line or on-line two-dimensional chromatography to obtain a more complete and accurate resolution of complex flavonoid samples, which will be discussed in section 4.

### 3. Chiral analysis

As explained in section 1, chiral analysis is often necessary for in-depth characterization of a sample. This is particularly true when stereoselectivity of a newly found molecule must be determined, or when biological activities must be assessed.

The separation of enantiomers can be achieved with different chromatographic techniques.

For a long time, the preferred method was the chemical derivatization with a chiral and enantiomerically pure agent, to form diastereomers that could be resolved in an achiral environment. A major advantage is the possibility to use classical achiral chromatographic methods like RPLC on a C18-bonded stationary phase. However, it requires highly pure derivatization agents and involves chemical reactions to produce the diastereomers [49], then possibly reversed reactions to recover the initial products when purification is desired, which can be lengthy and costly [44]. As a result, this method is no longer a favourite.

A second possible method is the use of chiral additives in the mobile phase, to form transient diastereomers. With this method, an achiral stationary phase can still be used. However, the consumption of chiral additive can be costly, unless some effort is done to recycle it. As a result, a single reference of such method was observed in the recent literature [31], employing cyclodextrins as mobile phase additives together with an achiral phenyl-bonded stationary phase to achieve the enantioresolution of Catechin and Epicatechin enantiomers in green tea. Cyclodextrins as chiral additives are also often used in capillary electrophoresis [50], as the quantities required are much lower than in chromatography, but this is outside the scope of the present paper.

Finally, the last and preferred method nowadays is the use of chiral stationary phases (CSPs), where a chiral selector is deposited, immobilized or bonded on a silica support, to form transient diastereomers with the chiral analytes. From the years 1980s', a variety of CSPs have been developed to achieve enantioseparations in the liquid phase, including brush-type phases, ligand and inclusion complexes (e.g. cyclodextrins), biomolecules (like proteins and macrocyclic glycopeptides) and synthetic or natural polymeric phases, with the most famous and versatile being polysaccharides. Depending on the choice of CSP, the same chromatographic modes as achiral chromatography may be used (NPLC, RPLC, HILIC, SFC, etc.).

Thanks to the wide possibilities offered by modern CSPs, chiral flavonoids are now commonly resolved on CSPs with LC methods [51]. As for SFC, although it is usually recognized as an excellent technique for chiral separations [52], making use of the same
CSPs as enantioselective LC, its specific application to the enantioresolution of flavonoids was only little explored to this date, with only a few papers relating it.

Unlike achiral chromatography, where separation is based on differences in hydrophobicity or polarity, chiral chromatography exploits the specific interactions between the chiral selector of the CSP and the stereoisomers. Also, steric parameters are significant, as the chiral analyte must access the enantiorecognition sites of the chiral selector. As a result, the choice of CSP is highly significant to the success of a chiral separation. In addition, the choice of mobile phase composition (solvents and additives) and temperature conditions also have a significant influence on the enantioseparation and must be carefully optimized. The literature related to enantioseparations of flavonoids in the liquid and supercritical phase is presented in Table 2.

<table><caption>**Table 2 - Chiral LC and SFC analyses of flavonoids**</caption><thead><tr><th>Sample</th><th>Types of flavonoids</th><th>Flavonoids</th><th>Mode</th><th>Detector(s)</th><th>Stationary phase (column dimensions)</th><th>Mobile phases</th><th>Flow rate</th><th>Temperature / Pressure</th><th>Gradient</th><th>MS mode</th><th>References</th></tr></thead><tbody><tr><td>Standards</td><td>Flavanones</td><td>Flavanone</td><td>HPLC</td><td>UV CD</td><td>Chiralpak IA (250 x 4.6 mm – 5 µm)</td><td>A) MeOH<br>B) H<sub>2</sub>O + 10 mM Na<sub>2</sub>HPO<sub>4</sub> (pH=9)</td><td>1.0</td><td>25°C</td><td>-</td><td>-</td><td>71</td></tr><tr><td>Serum and Yerba Santa</td><td>Flavanones</td><td>Homoeriodictyol</td><td>HPLC</td><td>UV</td><td>Chiralcel OJ-RH (150 x 4.6 mm – 5 µm)</td><td>ACN/H<sub>2</sub>O/H<sub>2</sub>PO<sub>4</sub> (22/78/0.5)</td><td>1.0</td><td>25°C</td><td>80 min</td><td>-</td><td>89</td></tr><tr><td>Biologic fluids (serum)<br>Tufu ling (Rhizoma Similae glabrosæ)<br>Apple (Molus domesticæ)</td><td>Flavan-3-ols</td><td>Taxifolin</td><td>HPLC</td><td>CD UV MS</td><td>Chiralcel OJ-RH (150 x 4.6 mm – 5 µm)</td><td>ACN/H<sub>2</sub>O/H<sub>2</sub>PO<sub>4</sub> (15/85/0.5)</td><td>0.35</td><td>25°C</td><td>140 min</td><td>ESI(-)</td><td>90</td></tr><tr><td>Launce<br>arborescence</td><td>Flavanones</td><td>Naringin<br>Hesperidin</td><td>HPLC</td><td>UV Polarisomé</td><td>Chiralpak IC (250 x 4.6 mm – 3 µm)</td><td>A) Hexane<br>B) EtOH (70/30)</td><td>1.0</td><td>40°C</td><td>-</td><td>-</td><td>91</td></tr><tr><td>Albedo of pomelo and sour orange</td><td>Flavanones</td><td>Naringin</td><td>HPLC</td><td>CD</td><td>Chiralpak IB (250 x 4.6 mm – 5 µm)</td><td>A) n-hexane<br>B) EtOH + 0.5% TFA (60/40)</td><td>1.2</td><td>-</td><td>-</td><td>-</td><td>92</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Flavanone</td><td>HPLC</td><td>UV</td><td>B-CD (250 x 4.6 mm)</td><td>A) MeOH<br>B) H<sub>2</sub>O + 1% TEAA pH=5.5 (35/65)</td><td>0.5</td><td>-</td><td>-</td><td>-</td><td>84</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Naringenin</td><td>HPLC</td><td>UV</td><td>Chiralpak AD-H (150 x 4.6 mm – 5 µm)</td><td>MeOH</td><td>1.0</td><td>30°C</td><td>-</td><td>-</td><td>66</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Naringenin<br>Eriodictyol</td><td>HPLC</td><td>CD</td><td>Chiralpak AD-RH (150 x 4.6 mm – 5 µm)</td><td>A) MeOH<br>B) H<sub>2</sub>O + 0.1%TFA</td><td>0.4</td><td>30°C</td><td>33-100%B (8min)</td><td>ESI(-), MRM</td><td>67</td></tr><tr><td>human plasma and urine</td><td>Flavanones</td><td>Hesperetin<br>Homoeriodictyol</td><td>HPLC</td><td>CD MS/MS</td><td>Chiralpak IA (150 x 2 mm – 3 µm)</td><td>A) H<sub>2</sub>O + 0.1% FA<br>B) ACN</td><td>0.4</td><td>30°C</td><td>33-100%B (8min)</td><td>ESI(-), MRM</td><td>73</td></tr><tr><td>rat serum</td><td>Flavanones</td><td>Liquiritigenin</td><td>HPLC</td><td>UV</td><td>Chiralpak AD-RH (150 x 4.6 mm – 5 µm)</td><td>ACN/H<sub>2</sub>O/acetic acid (50:50:0.05)</td><td>0.6</td><td>25 °C</td><td>-</td><td>-</td><td>63</td></tr><tr><td>Canadian natural health product</td><td>Flavanones</td><td>Liquiritigenin<br>Pinocembrin<br>Pinostrobin</td><td>HPLC</td><td>MS</td><td>Chiralpak AD-RH (150 x 4.6 mm – 5 µm)</td><td>1) ACN/H<sub>2</sub>O/FA (50/50/0.01)<br>2) MeOH + 0.1% AcNH<sub>4</sub><br>3) ACN/H<sub>2</sub>O/FA (80/20/0.01)</td><td>1) 0.6<br>2) 0.5<br>3) 1.0</td><td>-</td><td>-</td><td>-</td><td>ESI</td><td>65</td></tr><tr><td>Cocoa-Based Ingredients</td><td>Flavan-3-ols</td><td>(*/-)-Catechin<br>(*/-)-epicatechin</td><td>HPLC</td><td>Fluorescence</td><td>Astec Cyclobond I-2000 RSP) + B-CD (250 x 4.6 mm – 5 µm)</td><td>A) 20 mM AcNH<sub>4</sub> buffer (pH=4)<br>B) MeOH (70/30)</td><td>1.0</td><td>35°C</td><td>-</td><td>-</td><td>83</td></tr></tbody></table>
<table><tbody><tr><td>Tea (Camellia sinensis)</td><td>Flavan-3-ols</td><td>Epicatechin</td><td>HPLC</td><td>DAD</td><td>Chiralpak IA (250 x 4.6 mm - 5 µm)</td><td>A) 0.5% acetic acid + hexane<br>B) 0.5% acetic acid + EtOH</td><td>1.0</td><td>-</td><td>15-50%B (45min)</td><td>-</td><td>32</td></tr><tr><td>Commercial chocolates</td><td>Flavan-3-ols</td><td>(+/-)Catechin<br>(+/-)epicatechin</td><td>HPLC</td><td>DAD</td><td>Cyclobond I 2000 RSP (β-CD)<br>(250 x 4.6 mm - 5 µm)</td><td>A) H₂O + 50mM NaH₂PO₄ (pH=3)<br>B) ACN/H₂O + 30mM NaH₂PO₄ (pH=3)<br>80/20</td><td>1.0</td><td>30°C</td><td>10-45%B (70min)</td><td>-</td><td>31</td></tr><tr><td>spices, roots, plants</td><td>Flavanones</td><td>Naringenin, Hesperitin</td><td>HPLC</td><td>MS/MS</td><td>Chiralpak AD-3R (150 x 2.1 mm - 3 µm)</td><td>MeOH</td><td>0.5</td><td>40°C</td><td>-</td><td>ESI+ (MRM)<br>TQ</td><td>64</td></tr><tr><td>Bottled orange juice</td><td>Flavanones</td><td>Hesperetin, Naringenin, Flavanone</td><td>HPLC</td><td>CD DAD MS/MS</td><td>Chiralpak AD-3R (150 x 2.1 mm - 3 µm)</td><td>MeOH</td><td>0.5</td><td>40°C</td><td>-</td><td>API QTRAP ESI</td><td>68</td></tr><tr><td>Standards</td><td>Flavanones</td><td>6-methoxyflavanone<br>7-methoxyflavanone<br>2'-hydroxyflavanone<br>4'-hydroxyflavanone<br>6-hydroxyflavanone<br>7-hydroxyflavanone</td><td>HPLC</td><td>UV MS</td><td>N-benzyl-<br>phenethylamino-β-<br>cyclodextrin-bonded<br>ordered mesoporous<br>SBA-15 (82CDP)<br>(150 x 4.6 mm)</td><td>A) H₂O + 1% TEAA<br>B) MeOH (70/30) pH = 5</td><td>1.0</td><td>-</td><td>-</td><td>ESI</td><td>87</td></tr><tr><td>Synthetic compounds</td><td>Flavanones</td><td>4-iminoringenin derivatives 2-6</td><td>HPLC</td><td>UV</td><td>Chiralpak IB (100 x 4.6 mm - 5 µm)</td><td>βPrOH</td><td>0.2</td><td>25°C</td><td>-</td><td>-</td><td>80</td></tr><tr><td>Pericarp</td><td>Flavanones</td><td>Naringin<br>Flavanone-7-O-diglycosides</td><td>HPLC</td><td>CD</td><td>Chiralcel OD-H (250 x 4.6 mm - 5 µm)</td><td>A) n-Hexane<br>B) EtOH (70/30)</td><td>1.0</td><td>30°C</td><td>-</td><td>-</td><td>78</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Flavanone<br>6-methoxyflavanone<br>2-hydroxyflavanone<br>4-hydroxyflavanone<br>6-hydroxyflavanone<br>Naringin<br>Hesperidin<br>Silymarin</td><td>HPLC</td><td>MS</td><td>bridged bis(β-<br>cyclodextrin)<br>(150 x 4.6 mm)</td><td>ACN/H₂O +0.1%FA (15/85)</td><td>0.8</td><td>30°C</td><td>-</td><td>ESI(+)</td><td>88</td></tr><tr><td>Citrus pulp and peel</td><td>Flavanones</td><td>Naringenin</td><td>HPLC</td><td>CD MS/MS</td><td>Chiralpak IG-3 (150 x 2.0 mm - 3 µm)</td><td>A) MeOH<br>B) H₂O +0.1% FA (85/15)</td><td>0.5</td><td>40°C</td><td>-</td><td>-</td><td>85</td></tr><tr><td>Urine</td><td>Flavanones</td><td>Flavanone, Hesperetin</td><td>HPLC</td><td>DAD</td><td>Chiralcel OD-H (250 x 4.6 mm - 5 µm)</td><td>A) hexane<br>B) βPrOH (70/30)</td><td>0.3</td><td>-</td><td>-</td><td>-</td><td>75</td></tr></tbody></table>

<table><tbody><tr><td>Green tea</td><td>Flavan-3-ols</td><td>Catechin Epicatechin</td><td>HPLC</td><td>DAD Fluorescence</td><td>Inert sustain phenyl (150 x 4.6 mm)</td><td>H₂O + 1M NaCl + 0.1% H₂PO₄ (0.05%-0.6% B-CD)</td><td>1.0</td><td>35°C</td><td>-</td><td>-</td><td>93</td></tr><tr><td rowspan="2">Standards</td><td rowspan="2">Flavanones</td><td>Naringin<br>Naringenin<br>Hesperetin<br>Hesperidin</td><td rowspan="2">nano LC</td><td rowspan="2">UV</td><td>capillary column packed with phenyl carbamate 2-propyl-CD (100 µm x 220 mm)</td><td>EtOH/hexane/TFA (60/40/0.5)</td><td>240 nL/min</td><td>-</td><td>-</td><td>-</td><td>85</td></tr><tr><td>4'-methoxyflavanone<br>6-methoxyflavanone<br>7-methoxyflavanone<br>2'-hydroxyflavanone<br>4'-hydroxyflavanone<br>6-hydroxyflavanone<br>7-hydroxyflavanone</td><td>Segapak 1, 2, 3 (100 µm id. 357 µm od)</td><td>A) MeOH<br>B) βPrOH (20/80)</td><td>240 nL/min</td><td>-</td><td>-</td><td>81</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Flavanone</td><td>SFC</td><td>UV</td><td>capillary column packed with mesoporous silica 2-propyl-CD (100 µm x 220 mm)</td><td>ACN<br>B) H₂O + 10 mM ammonium acetate pH=4.5 (40/60)</td><td>281 nL/min</td><td>-</td><td>-</td><td>-</td><td>86</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Naringenin</td><td>SFC</td><td>UV</td><td>RegisCell (250 x 4.6 mm - 5 µm)</td><td>CO₂/MeOH with 25 mM isobutylamine</td><td>2.0</td><td>35°C/200 bar</td><td>4-40% B (14min))</td><td>-</td><td>95</td></tr><tr><td>Standards</td><td>Flavanones</td><td>Pinostrobin<br>2'-hydroxyflavanone<br>Flavanone</td><td>SFC</td><td>UV</td><td>RegisPack (250 x 4.6 mm)</td><td>CO₂/MeOH (75/25)</td><td>4.0</td><td>30°C</td><td>-</td><td>-</td><td>66</td></tr><tr><td>Bee pollen</td><td>Flavanones</td><td>4-hydroxyflavanone<br>6-hydroxyflavanone<br>7-hydroxyflavanone<br>Naringin<br>Naringenin<br>Hesperetin, taxifolin</td><td>SFC</td><td>CD DAD</td><td>Chiralpak AD (250 x 4.6 mm - 10 µm)</td><td>A) CO₂<br>B) EtOH/MeOH (80/20) +0.1%TFA</td><td>3.0</td><td>30°C / 150 bar</td><td>15-50%B (40min)</td><td>-</td><td>39</td></tr></tbody></table>

### 3.1. Chiral stationary phases

In recent years, the development and marketing of new CSPs has continued apace.

However, for flavonoid analysis, only two types of CSP stand out: polysaccharide-based stationary phases and CSPs based on native or modified cyclodextrins (Figure 4). Note that other types of enantioselective stationary phases are employed in LC and SFC, like brush-type phases, macrocyclic glycopeptides, cyclofructans, calixarenes and others, but none of them were reported in the chiral analysis of flavonoids.
<figure><img src="image_7.png" /><figcaption>**Figure 4.** General structure of *tris*(3,5-dimethylphenylcarbamate) polysaccharides and β-Cyclodextrin (β-CD) chiral stationary phases.</figcaption></figure>

**3.1.1. Polysaccharides**

CSPs based on modified polysaccharides are by far the most widespread [53]. A first reason is their high success rate in a variety of applications. Indeed, polysaccharides include a large number of chiral centres and chiral cavities to interact with the analytes, and therefore have a high probability of interacting specifically with the analytes, thereby providing better chances to separate stereoisomers. Interactions between enantiomeric compounds and the modified polysaccharides occur mainly *via* hydrogen bonds and π-π interactions [54], which is perfectly adequate for flavonoids, as they contain both hydrogen-bonding groups (carbohydrate moieties, hydroxyl and methoxyl groups) and aromatic rings. Secondly, polysaccharide CSPs also have a high compatibility with a wide range of solvents, especially for the most recent immobilized versions. Actually, the initial versions of polysaccharide CSPs developed by Okamoto and co-workers [55] were simply coated on modified silica, which offered limited possibilities to optimize mobile phase composition, as strong solvents would have dissolved the polymer. Modern versions based on the works of Francotte and co-workers may be immobilized on silica through thermal or photochemical cross-linking of the polymer [56,57], offering the possibility to use a wider range of solvents to elute difficult compounds or adjust enantioselectivity. Coated phases are generally considered to be more versatile, while immobilized phases are more stable and can provide a higher loading
capacity [58], which is a useful feature when preparative-scale separations are desired. Also, in terms of silica support, some of these phases are now available in sub-2 µm particles, making it possible to reach high-efficiency enantioresolutions [59], although this possibility has not been reported yet for flavonoids. Some studies have also been achieved with nano LC, as it offers excellent resolution and sensitivity, thanks to the use of small columns and high-quality packing of the particles. This enabled efficient separation of flavonoid enantiomers, even at low concentrations [60]. This work also included chlorinated polysaccharide CSPs.

Summarizing Table 2, it can be seen that the majority of recent separations were achieved on the older coated polysaccharides (68%), and a minority (32%) on immobilized phases. We also note that two CSPs appear most frequently: amylose and cellulose modified with tris(3,5-dimethylphenylcarbamate) ligands (Figure 4). As they are among the most versatile, they offer great flavonoid separation capacity. They are available in coated or immobilized versions from different manufacturers. In Table 2, we can see the coated Chiralcel AD [27,48,61–66] and RegisPack [63], and the immobilized Chiralpak IA [25,28,30,67,68] for the amylose phase, and the coated Chiralcel OD [67,69–74] and Sepapak 1 [60], and the immobilized Chiralpak IB [49] for the cellulose phase.

Polysaccharide CSPs have the advantage of being usable under normal-phase (NPLC), reversed-phase (RPLC) or polar organic (PO) LC conditions, as will be further discussed in section 3.2. The same stationary phases can also be used in SFC. However, as mentioned above, SFC has been little used to date in the enantioresolution of flavonoids. Enantiomeric separations of flavonoid samples were described in a single paper. In 2022, Ana M. Ares et al. examined the chiral separation of flavanones from a sample of bee pollen [48]. They found that retention on the Chiralpak AD column was higher than on achiral (diol) stationary phases, possibly resulting from the specific interactions in chiral binding sites, together with the added π-π interactions. On this column, they achieved the simultaneous enantioresolution of 10 pairs of flavanone enantiomers (Figure 5). In this study, the 2R enantiomer eluted first for most analytes, except for Naringenin, 2-hydroxyflavanone and Taxifolin, for which the 2S enantiomer eluted first.
<figure><img src="image_8.png" /><figcaption>**Figure 5.** Chiral separation using the Chiralpak AD column. Chromatographic conditions 30 °C, 3mL/min, 150 bar. Gradient of ethanol/methanol (80:20): from 0.0 to 10.0 min it was held at 15%, from 10.0 to 23.0 min it was increased to 28% and from 23.0 to 40.0 min (A: without TFA and B: with 0.1% TFA) [48]. Reprinted with permission from Elsevier.</figcaption></figure>

**3.1.2. Cyclodextrins**

Cyclodextrins are inclusion-type chiral selectors that can be used in different separation techniques, such as LC, SFC or GC, but also in capillary electrophoresis or in asymmetric synthesis [75]. Cyclodextrin-bonded stationary phases were often employed for the enantioresolution of flavonoids, as they offer an effective and selective method for these separations [51]. Cyclodextrins are macrocycles made of glucose units connected by glycosidic links, and they come in different sizes, with the most common being α-cyclodextrin (6 glucose units), β-cyclodextrin (7 glucose units) and γ-cyclodextrin (8 glucose units). This structure forms a basket-shaped hydrophobic cavity (Figure 4) in which molecules can be inserted according to their size and shape. The analytes may also interact with the secondary hydroxyl groups at the cyclodextrin “entrance”. These hydroxyl groups may also be modified to vary the possible interactions with analytes [70,76]. In the case of chiral flavonoid separations, native and modified β-cyclodextrins (β-CD) have been used [29,77,78].

Over the years, despite the strong interest in polysaccharide stationary phases, new β-CD-based stationary phases continue to be created [78–82]. Recently, Ning Zhang *et al*.
prepared a new bridged bis(β-cyclodextrin) stationary phase that they compared with a
native β-CD stationary phase [82]. This new phase showed a higher separation capacity than
the native phase. In fact, with the chemical modification the new phase had two cavities
instead of one in the native phase, which made it possible to accommodate larger chiral
analytes, thereby improving selectivity.

As described above for polysaccharide stationary phases, nano-LC was also described with cyclodextrin stationary phases [79,80]. In 2017, Mariana Silva et al. used a β-CD-bonded stationary phase to achieve enantioseparation of 12 flavanones and flavanone glycosides by nano-LC with high efficiency [80].

**3.2. Mobile phase composition**

In addition to the choice of stationary phase, the selection of the mobile phase is of
paramount importance in chiral analysis, as it can have a considerable impact on
chromatographic enantioresolution. Firstly, as is also the case in achiral separations, the
choice of mobile phase composition affects the possible interactions between the
enantiomers and the chiral selector in the CSP as the chromatographic process relies on a
balance of interactions between analyte and stationary phase on the one hand, analyte and
mobile phase on the other hand. Secondly, and more specific to chiral separations, some
stationary phases with flexible chiral selectors (typically, polysaccharide-based CSPs) may
somewhat change their three-dimensional structure depending on the choice of solvent,
opening some chiral cavities or, on the contrary, limiting access to them. Finally, additives
like acids, bases or salts can also contribute to the enantiorecognition process in different
ways: limiting non-enantioselective interactions with the supporting phase, forming ion pairs
with ionic analytes, etc. Thus, by modifying the composition of the mobile phase, it is
possible to radically alter the separation performance and optimize chromatographic
selectivity.

Keeping these elements in mind, the mobile phase can be adjusted by using different
solvents and additives, or by modulating the pH in order to optimize the specific interactions
between the enantiomers and the stationary phase. We will first discuss the choices of
solvents, then the additives usually employed for flavonoids.

As appears in Table 2, most flavonoid enantioseparations reported in the recent years were
carried out in RPLC mode, where the interaction between the stationary phase and the target
compounds is largely driven by hydrophobicity. Similar to achiral RPLC, a mobile phase
composed of an alcohol and an aqueous buffer is used to achieve the separation. Widely
used in chiral chromatography, the NPLC mode also remains a means of achieving
flavonoids enantioresolution. In this mode, the mobile phase usually contains a non-polar
solvent as a major component (typically hexane) and a polar co-solvent to adjust retention
and enantioresolution [28,30,69–73,79,83,84]. While this type of solvent is convenient for preparative scale as they can be evaporated rather easily, they are however not environmentally friendly, especially as hexane is rather toxic. In this context, SFC is of great interest [52] as CO₂ would replace hexane [48,63,85–87]. As the eluent strength of CO₂ was often compared to that of alkanes, replacing hexane by CO₂ often enables enantioseparations to be carried out with comparable selectivity, although higher flow rates can be employed without damage to efficiency, so resolution per unit time is generally improved. The addition of an alcohol-type co-solvent increases the polarity of the mobile phase, as described above for achiral separations. Among the alcohols used in chiral analyses in RPLC, NPLC and SFC are isopropanol (iPrOH), EtOH and MeOH (Table 2). All separations reported on β-CD-based stationary phases employed H₂O / ACN [29,80] or H₂O / MeOH [77,81] mobile phases. For instance, Ning Zhang *et al.* studied the influence of the percentage of MeOH on resolution and retention [82] (Figure 6). In this study, enantioseparations were examined on a modified β-CD-based stationary phase with a H₂O / MeOH mobile phase comprising 0.1% formic acid. They showed that, the higher the percentage of MeOH, the lower the retention and resolution, which corresponds to RPLC behaviour. A balance therefore needed to be found between analysis time and resolution.

<figure><img src="image_9.png" /><figcaption>**Figure 6.** Effect of operating parameters on the chromatographic separation of flavanone enantiomers on a cyclodextrin-type stationary phase. (a) Effect of methanol content in the aqueous mobile phase on the retention factors and resolution of 6-methoxyflavanone enantiomers; (b) effect of column temperature on the retention factors, separation factor and resolution of 2-hydroxyflavanone enantiomers; (c) effect of the concentration of formic acid in the mobile phase on retention factors, separation factor and resolution of 4-hydroxyflavanone enantiomers [82]. Reprinted with permission from RSC publishing.</figcaption></figure>
As mentioned above, additives such as acids, bases or salts are also frequently added to the mobile phase to modify the interactions between the enantiomers and the stationary phase, which can impact the selectivity and resolution of the separation (Figures 5 and 6). In aqueous compositions, additives can also modify the pH of the mobile phase, thereby modifying the ionization state of acidic and basic analytes, or the ionization state of the stationary phase (silanol groups, ionizable bonded ligands). Flavonoids have alcohol and phenol functional groups that can be ionized at different pH levels, with pKₐ values typically varying between 6 and 13 [88]. By adjusting the mobile phase pH, the ionization state of flavonoids varies and, consequently, their interactions with the chiral selectors also vary: when the alcohol and phenol functions remain protonated, hydrogen-bonding with the chiral selector is favoured, which should be favourable to enantiorecognition. This can lead to significant changes in their retention and enantioseparation. However, as mentioned before, flavonoids have better stability in acidic conditions, so this parameter cannot be varied in a large range. In consequence, acidic additives are generally used in the mobile phase (Table 2). These include formic acid [31,62,68,81,82,89], acetic acid [61], phosphoric acid [31,66,74,90,91] and trifluoroacetic acid [28,48,64,69,72,73,79,84]. In SFC as well, Ana M. Ares *et al.* as previously reported, carried out the separation of 10 flavanone enantiomeric pairs [48] (Figure 5). Their work showed that the addition of an acidic additive, in this case 0.1% TFA, improved the symmetry of the peaks, particularly that of taxifolin, but also modified enantioresolution for several pairs of enantiomers.

When using β-CD as the stationary phase, a basic additive or a salt can be added (Table 2) to promote the formation of an inclusion complex [75]. These additives can be ammonium acetate [77,80], triethylamine acetate [78,82], isobutylamine [86], or monosodium phosphate [25,29]. However, as mentioned earlier, basic conditions may cause analyte instability. None of the above references mentioned any such issue.

## 3.3. Operating parameters

### 3.3.1. Oven temperature

Temperature conditions can also have an impact on enantiomer separation through several modes of action. In liquid mobile phases, an increase in temperature reduces solvent viscosity, which is favourable to increased column efficiency, thereby possibly improving resolution. However, following van't Hoff law, increasing temperature in liquid phases usually yields lower retention, which is not so favourable to enantioresolution. In addition, lowering temperature is causing the analytes to be more rigid, which should be favourable to the enantioresolution of flexible molecules. But it may also cause the chiral selector to be more
rigid, which can affect the enantiorecognition process in an unpredictable, positive or negative way. In this respect, it is frequent to observe that the effect of temperature on enantioresolution is not monotonous, possibly with different trends in different ranges of temperature. In supercritical fluids, the situation is even more complicated [48,63,85,86] as the fluid density decreases with an increase of temperature, which is changing the mobile phase elution strength and has several other consequences [92].

An example of the effect of temperature in LC mode can be seen in Figure 6, where Ning Zhang et al. monitored retention, selectivity and resolution between flavanone enantiomers, when increasing temperature on a CD-bonded stationary phase [81,82]. In that case, and in the range of temperature explored, retention and resolution decreased as temperature increased, while selectivity remained mostly unchanged.

We may note that the range of temperatures explored is usually rather small because the stability of most chiral stationary phases does not allow for temperatures higher than 40-45°C. In the lower range, reducing temperature below room temperature cannot always be achieved, depending on the type of oven available in the chromatographic system.

In addition to being an optimization parameter, temperature variation may also give access to thermodynamic parameters and is useful to understand enantiorecognition mechanisms [83,85]. Indeed, according to Van't Hoff law, the change in free energy (ΔG) accompanying the chromatographic separation of two enantiomers is directly related to the enantioseparation and is given by Eq. (1):

$$ (\Delta G) = -RT \ln \alpha \qquad (1) $$

where R is the gas constant, T is the absolute temperature and α is the enantioseparation factor. Plotting retention and separation factors according to the reversed temperature is thereby providing access to enthalpy and entropy changes. For the reasons explained above and unlike achiral separations, chiral separations often yield non-linear trends as the retention and enantioseparation mechanism are likely to vary over the range of temperatures examined.

For instance, in 2022, Ali et al. demonstrated a correlation between the free energy change, that is to say between the enantioseparation factor observed on a Chiralcel®OD-H column, and the carbon chain length of bis-asymmetric compounds derived from flavanes [70] (Figure S2). It was observed that higher molar mass molecules (long carbon chain lengths) had stronger interactions with the stationary phase, leading to increased retention and, in that case, to increased enantioseparation. However, it should be noted that the correlation between molecular weight and retention and/or separation can vary depending on the specific nature of the interactions and the chemical properties of the flavonoids studied. In other cases, increased molecular weight would cause reduced penetration of the analytes in
the chiral cavities, thereby reducing enantioresolution, so no general conclusion should be drawn from this observation.

### 3.3.2. Pressure in compressible fluids

Unlike conventional liquid chromatography, SFC uses a compressible fluid as the mobile phase. Thus, pressure plays a key role in modifying the physicochemical properties of the mobile phase, as increasing pressure causes increased density, thereby increasing eluting strength, which can influence the retention, separation and efficiency, all impacting enantioresolution [48,63,85,86]. This provides additional flexibility for adjusting separation conditions to achieve optimum results in chiral SFC.

## 4. Two-dimensional chromatographic analyses

When further increases in column efficiency are not possible and a single chromatographic analysis cannot achieve the full resolution of a sample, an extra dimension [93,94] should be added. Two-dimensional liquid chromatography [34] (2D-LC) is an advanced technique that combines two complementary separation dimensions to improve the resolution of compounds in a complex sample, thereby providing more detailed information about the composition of samples, and to identify compounds that would be difficult to separate using a single chromatographic dimension. However, certain combinations, although orthogonal, are not feasible due to the immiscibility of solvents (e.g. an aqueous fraction to be transferred to a hexane-based mobile phase) and incompatibility between the mobile and stationary phases (e.g. a strong solvent from the first dimension could not be introduced in a second dimension operating under HILIC mode). Consequently, particular care is required in the design of 2D systems, bearing in mind that the first-dimension eluent is also the second-dimension injection solvent. Recent literature on this topic is reported in Table 3.
<table><caption>**Table 3** - 2D analyses involving chiral flavonoids</caption><thead><tr><th>Sample</th><th>Types of flavonoids</th><th>Flavonoids</th><th>Mode</th><th>Detector(s)</th><th>Dim</th><th>Stationary phase (column dimensions)</th><th>Mobile phases</th><th>Flow rate (ml/min)</th><th>Temperature / Pressure</th><th>Gradient</th><th>MS</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Camellia sinensis in green and black tea</td><td rowspan="2">Flavan-3-ols</td><td rowspan="2">(+/-)Catechin<br>(+/-)galocatechin<br>(+/-)galocatechin gallate<br>(+/-)catechin gallate</td><td rowspan="2">SEC x RPLC</td><td>UV</td><td>1D</td><td>Ultra hydrogel-120<br>(300 x 7.8 mm - 5.10<sup>3</sup> Da)</td><td>A) H<sub>2</sub>O<br>B) ACN</td><td>1.0</td><td>60°C</td><td>0-50% B</td><td>ESI (+/-) - TQ</td><td rowspan="2">42</td></tr><tr><td>MS</td><td>ELSD</td><td>2D</td><td>BEH-C18<br>(50 x 2.1 mm - 1.7 µm)</td><td>A) H<sub>2</sub>O + 0.1% FA<br>B) MeOH</td><td>1.0</td><td>80°C</td><td>0-100% B</td><td></td></tr><tr><td rowspan="3">Denzhan Shenmai</td><td rowspan="3">Flavanones</td><td rowspan="3">Eriodictyol<br>Naringin-7-O-glucuronide<br>Homoisoflavonone</td><td rowspan="3">RPLC x RPLC</td><td rowspan="3">MS</td><td>1D</td><td>C8 XTerra MS<br>(100 x 2.1 mm - 1.7 µm)</td><td>A) H<sub>2</sub>O + 0.1% FA<br>B) MeOH</td><td>0.1</td><td>40°C</td><td>10-100% B</td><td>QTOF</td><td rowspan="3">45</td></tr><tr><td>2D</td><td>Eclipse Plus C18<br>(50 x 4.6 mm - 1.8 µm)</td><td>A) H<sub>2</sub>O + 0.1% FA<br>B) MeOH</td><td>2.0</td><td>40°C</td><td></td><td></td></tr><tr><td>2D<br>chiral</td><td>Chiralpak IC-3<br>(30 x 4.6 mm - 2.7 µm)</td><td>A) H<sub>2</sub>O + 0.1% FA<br>B) MeOH</td><td>1.0</td><td>40°C</td><td></td><td></td></tr><tr><td rowspan="2">Red wine</td><td rowspan="2">Flavan-3-ols</td><td rowspan="2">Catechin<br>Epicatechin</td><td rowspan="2">RPLC x RPLC</td><td rowspan="2">UV</td><td>1D</td><td>RP Amide<br>(250 x 1.0 mm - 5 µm)</td><td>A) H<sub>2</sub>O + FA (pH=3)<br>B) ACN + FA (pH=3)</td><td>0.1</td><td>25°C</td><td>20-90% B<br>(70 min)</td><td>-</td><td rowspan="2">46</td></tr><tr><td>2D</td><td>Ascentis Express<br>(30 x 4.6 mm - 2.7 µm)</td><td>A) H<sub>2</sub>O + FA (pH=3)<br>B) ACN + FA (pH=3)</td><td>4.0</td><td>40°C</td><td>0-100% B<br>(1.7 min)</td><td>-</td></tr><tr><td rowspan="2">Citrus lemon peels</td><td rowspan="2">Flavanones</td><td rowspan="2">(Neo)hesperidin<br>(iso)naringin<br>Neoerioctirin</td><td rowspan="2">CCC x CCC</td><td rowspan="2">MS/MS</td><td>1D</td><td>n hexane/n butanol/MeOH/H<sub>2</sub>O<br>(0.23/1.0/0.25/1.13)</td><td></td><td>2.0</td><td>-</td><td>ESI (-)</td><td rowspan="2">47</td></tr><tr><td>2D</td><td>n hexane/n butanol/MeOH/H<sub>2</sub>O<br>(0.47/1.0/0.38/1.46)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Green tea</td><td rowspan="2">Flavan-3-ols</td><td rowspan="2">(Epi)galocatechin gallate<br>(Epi)galocatechin<br>(Epi)catechin</td><td rowspan="2">HILIC x RPLC</td><td>PDA</td><td>1D</td><td>Nomura Chemical Deyelosil<br>Diol-100<br>(250 x 1.0 mm - 5 µm)</td><td>A) ACN + Acetic acid<br>(99:1)<br>B) MeOH/H<sub>2</sub>O/Acetic acid<br>(94.05/4.95/1)</td><td>0.05</td><td></td><td>4-40% B<br>(55 min)</td><td>ESI (-)</td><td rowspan="2">48</td></tr><tr><td>MS</td><td>Fluorescence</td><td>2D</td><td>2x Zorbax SB-C18<br>(50 x 4.6 mm - 1.8 µm)</td><td>A) H<sub>2</sub>O + 0.1% FA<br>B) ACN</td><td>0.8</td><td>50°C</td><td>2-100%<br>(22 min)</td><td></td></tr><tr><td rowspan="2">Rooibos<br>(Aspalathus<br>linearis)</td><td rowspan="2">Flavanones</td><td rowspan="2">Hesperidin<br>Eriodictyol<br>flavanone-O-hexoside<br>flavanone-O-<br>malonylhexoside</td><td rowspan="2">NP-<br>HPCC<br>x RP-<br>UHPLC<br>off line</td><td>UV</td><td>1D</td><td>Et<sub>3</sub>OAc/H<sub>2</sub>O (1/1) (A)</td><td>n-BuOH/H<sub>2</sub>O (1/1) (B)</td><td>1.5</td><td>-</td><td>100-50% B<br>(47 min)</td><td>QTOF. ESI</td><td rowspan="2">49</td></tr><tr><td>MS</td><td>2D</td><td>Poroshell 120 SB-C18<br>(100 x 2.1 mm - 1.8 µm)</td><td>A) H<sub>2</sub>O + acetic acid 2%<br>B) ACN</td><td>0.8</td><td>44.5°C</td><td>10-100% B<br>(17 min)</td><td></td></tr><tr><td rowspan="2">Saherophyso<br>galoside</td><td rowspan="2">Flavanoids<br>glycosides</td><td rowspan="2">-</td><td rowspan="2">RPLC x<br>HILIC<br>off line</td><td>UV</td><td>1D</td><td>Xagua C18 prep<br>(250 x 20 mm - 10 µm)</td><td>A) H<sub>2</sub>O + 0.2% FA<br>B) ACN</td><td>15</td><td>-</td><td>5-30% B<br>(55 min)</td><td>-</td><td rowspan="2">40</td></tr><tr><td>MS</td><td>2D</td><td>Xamide prep.<br>(250 x 20 mm - 10 µm)</td><td>A) H<sub>2</sub>O + 0.2% FA<br>B) ACN</td><td>15</td><td>-</td><td>3-95% A<br>(30 min)</td><td>-</td></tr><tr><td>Grapevine canes<br>(Vitis vinifera L.)</td><td>Flavan-3-ols<br>Flavanones</td><td>Catechin<br>Epicatechin</td><td>HILIC x RPLC</td><td>DAD</td><td>1D</td><td>Uchropeperiol<br>(150 x 1.0 mm - 5 µm)</td><td>A) ACN + 1% FA</td><td>0.18</td><td>25°C</td><td>2-40% B<br>(80 min)</td><td></td><td>50</td></tr></tbody></table>

Complementarity may be achieved through different means. The simplest way is to maintain the same chromatographic mode in the two dimensions but changing the composition of the mobile phase [95]. For instance, RPLC in acidic conditions may be hyphenated to RPLC in basic conditions, ensuring facilitated transfer between the first and second dimensions as there would be no significant incompatibility of mobile phases. However, this solution is unfit for flavonoids analysis as they require acidic analytical conditions to ensure their stability. Hence another simple way to achieve complementarity is to use different stationary phases in the same chromatographic mode. For instance, both dimensions could use RPLC mode, but different stationary phases like octadecyl-bonded silica and phenyl-bonded silica, to obtain different selectivity. Here again, mobile phase compatibility remains excellent, but it is difficult to obtain high orthogonality with such strategies. To improve this point, different chromatographic modes (e.g. RPLC and HILIC) can be selected in the two dimensions [96–99]. The compatibility between RPLC and HILIC remains reasonably good as they may both employ mixtures of aqueous buffers and ACN. Compatibility would however be much worse when RPLC is hyphenated to NPLC, which operates in purely organic solvents.
As a general rule, the second dimension is used to resolve co-elutions observed in the first dimension. The flavonoids that pose the greatest difficulty in terms of separation are the glycosylated forms because, as pointed out in section 1.2 and Figure 3, they are larger and more polar than non-glycosylated forms. Increasing the size of the glycosylated group (monosaccharide, disaccharide...) further increases both molecular weight and polarity. Therefore, two different analyte features may be focused on: size and polarity. In this context, keeping RPLC as one of the two dimensions and introducing a different chromatographic mode in the other dimension seems a logical approach.

Considering the size feature, Scoparo *et al.* reported a 2D-LC method combining size exclusion chromatography (SEC) and RPLC in a comprehensive fashion (SECxRPLC), to analyse the flavonoids of tea leaves extracts [100]. While SECxRPLC is commonly used in the analysis of biopharmaceuticals, it was rarely described for natural products. This paper was the first to cover such a broad spectrum of compound polarity on a single plant extract [100].

Considering the polarity feature, there has been an ongoing development in the use of HILIC in 2D-LC, either in the first or second dimension. The high orthogonality between HILIC and RPLC, estimated around 80% in this case, shows the strong potential of this coupling [97,101]. Often used in off-line mode, this coupling has already proven to be very effective, both for analytical and preparative purposes [102].

Another complementarity that may be sought in the specific case of chiral flavonoids is that of stereoisomers. Réset *et al.* recently proposed to combine an achiral SFC method in the first dimension to a chiral SFC method in the second dimension in a multiple-heart cutting 2D-SFC system [103]. This allowed retrieving pairs of stereoisomers from the first dimension to be resolved in the second dimension. As both dimensions employed the same fluid (carbon dioxide and methanol co-solvent with acidic additive), no compatibility issues were encountered. In addition, the compressible nature of the supercritical fluid proved to be a great advantage in re-focusing the peaks upon entering the second dimension, which was favourable to resolution and sensitivity.

Besides complementarity of the two separation dimensions, in on-line analysis the way to transfer fractions from the first dimension to the second, and the number of fractions to be transferred may vary. For instance, a single fraction from a single peak can be transferred (heart-cut mode), or several separated fractions from different peaks (multiple heart-cut mode), or several fractions from a single peak (selective comprehensive mode), or all fractions from the first dimension (fully comprehensive mode). Not all transfer modes have been applied to the analysis of flavonoids. In the multiple heart-cut approach to 2D, compounds are separated primarily in one dimension, with selective and targeted cutting of
specific fractions in the first dimension, transferred to the second dimension for further analysis. This approach reduces sample complexity and focuses on specific analytes, while minimizing interference from other compounds [104,105]. In contrast, the comprehensive LCxLC approach transfers all fractions from the first dimension to the second dimension and offers a most complete view of the sample. To achieve this, it is necessary to have a fast analysis speed in the second dimension, which means using a shorter column, preferably packed with small particles to retain sufficient efficiency [93,99,101].

The purpose of 2D-chromatography is to increase peak capacities compared to one-dimensional analysis and thereby obtain a more complete view of a complex sample, which is generally the case. Off-line 2D methods usually pose few difficulties since the fractions obtained from the first dimension may be evaporated to dryness and reconstituted in an appropriate dilution solvent, but the situation is different for on-line coupling. In that case, the volume and mobile phase composition transferred from the first to the second dimension should be strictly controlled to avoid degrading the resolution or distorting the peaks [98,106]. When the first dimension is operated in gradient elution, shifting gradients can be used in the second dimension to progressively adapt the elution conditions in second dimension to the transferred fraction. Additionally, sensitivity may be poorer than in one-dimensional LC due to dilution caused by high flow rates in the second dimension [76]. Thus, it is important to take into account all the interfering parameters and optimize them. The group of André de Villiers is largely involved in the development of 2D-LC methods, particularly combining RPLC and HILIC. In 2022, they showed the limits of this coupling. In their approach, a solution proposed was to switch from one dimension to the other by stopping the first-dimension flow in multiple-heart-cut mode [99].

Ning Sheng et al. in 2017 also addressed the difficulties of 2D-LC to analyse flavonoids in a mixture of medicinal herbs [107]. By testing different gradient modes, they showed that adding a lag time before starting the second-dimension gradient was beneficial. As shown on Figure 7 (Mode C) this strategy offered a good distribution of peaks in the two-dimensional space compared with conventional or segmented gradient modes (A and B modes). This served both for achiral-achiral and achiral-chiral analysis.
<figure><img src="image_10.png" /><figcaption>**Figure 7.** Comparison of contour plots from LC × LC analysis of a mixture of medicinal herbs under three types of gradient modes (contour plots with absorbance at 280 nm are shown on the left and the corresponding gradient program is shown on the right [107]. Reprinted with permission from Elsevier.</figcaption></figure>

Two-dimensional methods may also be used at the preparative scale for the purpose of (i) purifying single flavonoids for accurate structure determination and/or activity testing or (ii) recovering flavonoid fractions to be further analysed with a high-resolution method like UHPLC-MS/MS. Counter-current chromatography (CCC) was reported for both purposes [108,109]. CCC is a liquid-liquid chromatographic technique relying on the partition of the target compounds between two non-miscible liquid phases. Although on-line hyphenation of CCC to HPLC is possible [110], in the case of flavonoids only off-line methods were reported. Using this technique, in 2017 Walters *et al*. demonstrated for the first time the presence of Hesperidin and 18 other compounds in Rooibos [109].

**5. Detectors**

Detection is essential for chromatographic monitoring and to characterize chiral flavonoids. In recent years, detection techniques have been improved to ensure greater sensitivity and selectivity of compounds.

Regardless of whether the chromatography is chiral or achiral, the detectors used are mostly the same. They include UV-visible single-wavelength or diode-array detectors (DAD), fluorescence detectors (FLD) and MS detectors. In addition, circular dichroism detector is
useful when enantioseparation is achieved, or with achiral analysis when a single enantiomer is expected to be present in the sample. By far the most commonly used detectors are DAD and MS detectors (Figure 8).

<figure><img src="image_11.png" /><figcaption>**Figure 8.** Comparison of the different detectors listed in Tables 1, 2 and 3 for the analysis of chiral flavonoids.</figcaption></figure>

**5.1. UV-visible single-wavelength and diode-array detectors**

Single-wavelength UV-visible detector is widely used to detect and quantify chiral flavonoids in LC and SFC, as can be seen in Tables 1, 2, and 3. Although improved by electronic filters, sensitivity is still moderate, but linearity range is rather good and repeatability is usually excellent. Currently, it can accurately detect quantities of flavonoids ranging from a few nanograms to several milligrams. A major limitation is however its specificity, as it cannot differentiate between the stereoisomers of chiral flavonoids that possess similar UV absorption properties [48,64,65].

Nevertheless, the aromaticity of flavonoids is proving to be a real advantage for characterizing these compounds. Their absorptions at relatively long wavelengths improve the selectivity of qualitative and quantitative spectrophotometric methods, while the characteristic spectra of the different classes of flavonoids enable them to be distinguished from one another, and to be distinguished from other compounds that may be present in a complex sample of natural product. For this reason, DAD detectors are often preferred over single-wavelength detectors.

The UV spectra of flavonoids generally show two main absorption bands in the region of 240 to 400 nm. The absorption band I, attributed to ring B (Figure 1), is located between 300 and 395 nm, while absorption band II, attributed to ring A, is located between 240 and 295 nm (Table 4). Increasing the number of hydroxyl groups on the aglycone leads to a bathochromic
shift of the absorption bands (increasing wavelength at maximum absorption). On the other hand, methylation or glycosylation, particularly on the hydroxyl groups in positions 3, 5, 7 and 4', causes a hypsochromic shift in the absorption bands (decreasing wavelength at maximum absorption). It should be noted that the specific nature of the sugar moiety generally has no significant effect on these spectral shifts.

<table><caption>**Table 2** - UV absorption bands of the flavanol, flavanone and flavanonol families</caption><thead><tr><td>Structure type</td><td>Band II (nm)</td><td>Band I (nm)</td></tr></thead><tbody><tr><td>Flavonol (3-OH is substituted)</td><td>250-280</td><td>328-357</td></tr><tr><td>Flavonol (3-OH is free)</td><td>250-280</td><td>358-385</td></tr><tr><td>Flavanone and flavanonol</td><td>270-295</td><td>300-330</td></tr></tbody></table>

## 5.2. Fluorescence detection

The fluorescence detector (FLD) is a highly sensitive detector that can detect very small quantities of chiral flavonoids. Because it relies on light emission, it is typically more sensitive than the absorption-based UV detector [99,111]. For example, Inal Bakhytkyzy et al. have shown that the detection limit for FLD was 10 times better than for UV detection [111] (Figure S3). In addition, because not all molecules can emit fluorescence light, and because different fluorescent molecules would require different excitation wavelengths, FLD is also more specific than UV detectors, with fewer spurious signals. Indeed, in fluorescence there are always two wavelengths to be used: an excitation wavelength and an emission wavelength. In the case of flavanols, which were mostly studied in recent years, the excitation wavelength is between 270 and 280 nm and the emission wavelength between 315 and 347 nm (Table 5). For instance, it appears in Figure S3 that the FLD chromatogram shows a cleaner trace than the DAD chromatogram, as the former allows focusing on selected compounds through excitation and emission wavelengths. However, FLD is usually more expensive than DAD. Whether in chiral or achiral chromatography, FLD allows monitoring the separation but in no case provides information on the chiral identity of stereoisomers [31,77,97].

<table><caption>**Table 3** - Excitation and emission bands of flavanols for fluorescence detection</caption><thead><tr><th>Type of Flavonoids</th><th>Flavonoids</th><th>Excitation (nm)</th><th>Emission (nm)</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="5">Flavanols</td><td>(Epi)catechin</td><td>276</td><td>316</td><td>83</td></tr><tr><td>(Epi)catechin Procyanidin</td><td>280</td><td>347</td><td>41</td></tr><tr><td>(Epi)gallocatechin gallate, (Epi)gallocatechin, (Epi)catechin</td><td>276</td><td>316</td><td>48</td></tr><tr><td>(Epi)catechin (Epi)gallocatechin Fisetinidol Epifisetinidol</td><td>230</td><td>321</td><td>52</td></tr><tr><td>(Epi)catechin</td><td>270</td><td>315</td><td>93</td></tr></tbody></table>
### 5.3. Mass spectrometry

The mass detector is a highly specific detector that allows both detection and quantification but may also provide some structural information. It offers both high sensitivity and high specificity. It can measure the exact molecular weight of chiral flavonoid molecules [35].

However, mass analysers are usually significantly more expensive than other detectors and often require more sample preparation prior to analysis. They may also impose specific mobile phase compositions as volatile components should be used, and corrosive additives should be particularly avoided. Hyphenation to SFC must be done with a little more caution. Solubility problems can arise due to the depressurization of CO₂ when entering the mass spectrometer. In addition, as CO₂ is not easily ionized in electrospray, it is useful to introduce an extra source of protons to favour analyte ionization. Both issues can be simply solved with the introduction of a make-up fluid prior to entering the MS [46].

When all the data in the tables 1, 2 and 3 are taken together, it can be seen that many analyses using UV detection were also coupled with simple or tandem MS. In all cases, the electrospray ionization mode was used in positive or negative mode [112]. For the analysis of chiral flavonoids, there was no preferred ionization mode, with positive mode being used for 48% and negative mode for 52% of the reported publications.

Although the ionization source was common to all the analyses of chiral flavonoids listed here, analysers were more varied [35]. The accessibility of low-resolution mass in terms of price, makes it a criterion of choice. In fact, the majority (72%) of reported analyses were done with a low-resolution analyser of single- [36,38,42,46,62,113] or triple-quadrupole [27,43,44,65,89,97,114] type. The remaining 28% were analysed with high-resolution mass spectrometry (HRMS, Figure 9). HRMS is generally used for applications requiring high precision and selectivity, such as the precise identification of compounds and the analysis of complex mixtures [39,97,99,101,107,108]. Thanks to the high resolution provided by the Q-TOF analyser, Lidia Montero et al. in 2017 carried out the first determination of the presence of Hesperidin in grapevine cane [101].

In addition to identification, MS can also be used to quantify flavonoids. This quantification is possible in simple MS [36,62] or in MS/MS [27,44,89].

Finally, mass spectrometry is a powerful tool for characterizing molecules but is not always capable of differentiating between all flavonoids, as fragmentation patterns do not always allow differentiating between isomers, and especially not between stereoisomers.
<figure><img src="image_12.png" /><figcaption>**Figure 9.** Comparison of MS analysers used in low and high resolution (HRMS).</figcaption></figure>

**5.4. Circular dichroism**

Circular dichroism (CD) is often used as a detection method in enantioseparations, and in particular for chiral flavonoids (Figure 10). Widely used in biochemistry for proteins and nucleic acids [115,116], the CD detector can be used to study the structure and conformation of molecules. It is based on the difference in absorption of left and right circularly-polarized light as it passes through a solution containing a chiral molecule [117]: enantiomers have opposite CD spectra, so the chromatographic peaks seen with CD detector are either positive or negative, as appears in Figure 10. This detector is therefore specific of chiral molecules. It is very useful to detect and quantify flavonoid enantiomers, as it can distinguish between molecules with different spatial configurations [25,28,69,71,72,113].

<figure><img src="image_13.png" /><figcaption>**Figure 10.** Chromatogram of Naringenin obtained by HPLC analysis on a Chiralcel OD-H column in UV and CD [72]. Reprinted with permission from Elsevier.</figcaption></figure>

CD can provide information on the conformation of flavonoids, but it may have limited specificity for distinguishing between different types of flavonoids. Some flavonoids may have
similar CD spectra, making it difficult to distinguish them on the basis of this technique alone.
In such cases, another detector as MS or MS/MS may be required for accurate identification.
Used both in LC [25,28,49,64,65,69–73,78,113] and SFC [48,87], the CD detector is used for
a variety of purposes. It can be used to monitor enantiomer configurations as a function of
fruit maturity [69,84], to assess the optical activity of new compounds [73], or to determine
the elution order of enantiomers [64,65]. For example, in 2005, Salvatore Caccamese et al.
performed several chiral separations of flavonoids such as Naringenin (Figure 10) in HPLC
using CD detection [72]. Thanks to this detection, it is possible to determine which of the
enantiomers elutes first. In cases where a scalemic mixture would be present, it would
therefore be useful to determine which enantiomer is the most concentrated. By studying
flavonoids that are widespread in the plant kingdom, they showed the potential of this
detector and its great need for application examples such as natural products. However, at
the time, the potential of CD was underestimated: it was mostly used as an independent
spectroscopic technique to determine molecular structure, but not so much as a
chromatographic detector.

**5.5. Sensitivity**

Detector sensitivity plays a crucial role in molecular analysis, as it determines the ability to
accurately detect and measure low concentrations of target analytes. This makes the
comparison of flavonoid profiles in different matrices more complete, to identify and quantify
the different classes of flavonoids present. It also makes it possible to assess fine variations
in composition as a function of various factors, such as fruit maturity, growing conditions or
processing methods. In the case of chiral flavonoids, sensitive detectors would also provide a
better view of isomer ratios and isomerisation effects when a chiral chromatographic method
is placed in front of the detector.

It is important to note that the sensitivity of a detector can be influenced by various factors
such as background interference, sample matrix or experimental conditions. Philip Machonis
et al. determined the presence of Catechin and Epicatechin in cocoa [77]. In this matrix, they
showed the presence of the 2 enantiomers of each molecule. Measuring the limit of
quantification (LOQ) with fluorescence detection for each enantiomer, they found that the two
Catechin enantiomers had the same LOQ. However, for Epicatechin the LOQs were very
different, with 0.6 µg/mL for (+)-Epicatechin and 12.1 µg/mL for (-)-Epicatechin. It is difficult
to understand such a large difference, unless some other compound co-eluted with one of
the two enantiomers.

Regarding two-dimensional chromatography, it is important to note that the limits of detection
and quantification are often deteriorated compared to one-dimension separation, because
this small quantity is divided into several fractions in the second dimension. For instance, in
2009, Paola Dugo et al showed that adding an extra RPLC dimension increased the LOQ by around a factor of 5 [76]. However, in other cases, it could also be favourable to detectability, e.g. when a low-concentration compound co-eluted with a large-concentration compound in the first dimension.

Figure 11 shows representative values of LOQs reported in the literature with UV, FLD and MS detectors for different chiral flavonoids. Naturally, these values vary greatly from one detector to another. If we take a closer look at the Naringenin compound, we can see that the UV detector used with SFC [48,118] was not very sensitive compared with the same detector employed with an LC separation. This is a common observation, as SFC-UV is typically twice less sensitive than LC-UV, when all system dimensions are in the same scale. However, between a UV detector used in 2005 [66] and one used in 2018 [38], there was an increase in sensitivity of a factor of 5. If we now compare the difference between an HPLC-UV analysis [38] and an UHPLC-UV analysis [65], a lower limit of quantification was observed for Naringenin in UHPLC-UV than in HPLC-UV, which seems quite logical since ultra-high-performance should offer higher efficiency, thereby improving detectability.

MS detection naturally offers the best sensitivity whatever the year, compound or chromatographic mode [36,44]. The introduction of tandem MS/MS detection also increases sensitivity [27,89]. Whereas the performance of UV and FLD detectors can hardly be further increased, MS sensitivity continues to improve in new instruments.

<figure><img src="image_14.png" /><figcaption>**Figure 11.** Representation of the sensitivities (LOQ in mg/mL) of the detectors (UV, MS, FLD) used in the analysis of hesperidin (blue), hesperetin (green), epicatechin (red), and naringenin (purple).</figcaption></figure>
HPLC-UV (2018) [38]; SFC-UV (2019) [47]; UHPLC-UV (2015) [43]; HPLC-MS (2015) [36]; HPLC-MS (2013) [44]; SFC-UV 1 (2022) [118]; SFC-UV 2 (2022) [48]; HPLC-UV (2005) [66]; UHPLC-MS/MS (2016) [27]; UHPLC-UV (2016) [65]; HPLC-UV (2005) [74]; UHPLC-MS/MS (2022) [89]; UHPLC-UV (2017) [30]; HPLC-UV (2016) [40]; HPLC-UV (2009) [76]; HPLC-FLD (2014) [77]

Finally, and as is well known, the chromatographic technique used upstream of detection has a strong influence on sensitivity. Typically, HILIC mobile phases are more favourable to MS detection than RPLC, since mobile phases in HILIC contain a larger proportion of organic solvent thus are easier to evaporate. For the same reason, SFC is usually favourable to MS detection as most of the mobile phase is made of pressurized CO2. Technical developments in chromatography and detection mean that we can now work with more sensitive instruments, which also makes it easier to identify and quantify samples.

**6. Conclusions**

Advances in liquid chromatography and supercritical fluid chromatography, in one or two separation dimensions and combined with the most sensitive detectors, have improved the detection, quantification and identification of flavonoids. Chiral flavonoids set specific issues related to their limited stability. However, the latter can be improved with optimised storage conditions, advanced sample preparation and analysis techniques to reduce degradation and/or isomerisation effects. In short, these advances are enabling more accurate and reliable analysis of chiral flavonoids, opening up new prospects for research and applications.

Overall, the evolution of chromatographic methods to analyse chiral flavonoids has led to a better understanding of the importance of stereochemistry in the molecular and biological interactions of flavonoids. These advances have major implications for drug design and understanding the interactions between natural compounds and biological processes.

**CRediT Author Contributions**

**Laurine Réset:** Investigation, Methodology, Visualization, Writing – Original Draft.

**Clément de Saint Jores:** Writing – Review & Editing, Supervision.

**Caroline West:** Visualization, Writing – Original Draft, Review & Editing, Supervision, Funding acquisition.

**Conflicts of interest**

Nothing to declare.
## Acknowledgement

Région Centre Val-de-Loire is acknowledged for a PhD grant allocated to Laurine Réset. ICOA is supported by the University of Orleans, the National Centre for Scientific Research (CNRS), the projects CHemBio (FEDER-FSE 2014-2020-EX003677), Valbiocosm (FEDER-FSE 2014-2020-EX003202), Techsab (FEDER-FSE 2014-2020-EX011313), QUALICHIM (APR-IA-PF 2021-00149467), ESTIM-ICOA (CPER/FEDER-FSE+ 2021-2027-00022860), the RTR Motivhealth (2019-00131403) and the Labex programs SynOrg (ANR-11-LABX-0029) and IRON (ANR-11-LABX-0018-01).
## References

[1] B.T. Martins, M. Correia da Silva, M. Pinto, H. Cidade, A. Kijjoa, Marine natural flavonoids: chemistry and biological activities, Nat. Prod. Res. 33 (2019) 3260-3272. https://doi.org/10.1080/14786419.2018.1470514.

[2] J.B. Harborne, The Flavonoids: Advances in Research since 1980, Springer, 2013.

[3] O.M. Andersen, K.R. Markham, Flavonoids: Chemistry, Biochemistry and Applications, CRC Press, 2005.

[4] I. Erlund, Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology, Nutr. Res. 24 (2004) 851-874. https://doi.org/10.1016/j.nutres.2004.07.005.

[5] W. Ren, Z. Qiao, H. Wang, L. Zhu, L. Zhang, Flavonoids: Promising anticancer agents, Med. Res. Rev. 23 (2003) 519-534. https://doi.org/10.1002/med.10033.

[6] K.P. Devi, T. Rajavel, S.F. Nabavi, W.N. Setzer, A. Ahmadi, K. Mansouri, S.M. Nabavi, Hesperidin: A promising anticancer agent from nature, Ind. Crops Prod. 76 (2015) 582-589. https://doi.org/10.1016/j.indcrop.2015.07.051.

[7] J. Choi, D.-H. Lee, H. Jang, S.-Y. Park, J.-W. Seol, Naringenin exerts anticancer effects by inducing tumor cell death and inhibiting angiogenesis in malignant melanoma, Int. J. Med. Sci. 17 (2020) 3049-3057. https://doi.org/10.7150/ijms.44804.

[8] D. Barreca, G. Gattuso, E. Bellocco, A. Calderaro, D. Trombetta, A. Smeriglio, G. Laganà, M. Daglia, S. Meneghini, S.M. Nabavi, Flavanones: Citrus phytochemical with health-promoting properties: Citrus phytochemical with health-promoting properties, BioFactors 43 (2017) 495-506. https://doi.org/10.1002/biof.1363.

[9] L. Testai, V. Calderone, Nutraceutical Value of Citrus Flavanones and Their Implications in Cardiovascular Disease, Nutrients 9 (2017) 502. https://doi.org/10.3390/nu9050502.

[10] A. Chanet, D. Milenkovic, C. Manach, A. Mazur, C. Morand, Citrus Flavanones: What Is Their Role in Cardiovascular Protection?, J. Agric. Food Chem. 60 (2012) 8809-8822. https://doi.org/10.1021/jf300669s.

[11] S. Kumar, A.K. Pandey, Chemistry and Biological Activities of Flavonoids: An Overview, Sci. World J. 2013 (2013) 1-16. https://doi.org/10.1155/2013/162750.

[12] D. Tungmunnithum, A. Thongboonyou, A. Pholboon, A. Yangsabai, Flavonoids and Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical Aspects: An Overview, Medicines 5 (2018) 93. https://doi.org/10.3390/medicines5030093.

[13] St. Rusznyák, A. Szent-Györgyi, Vitamin P: Flavonols as Vitamins, Nature 138 (1936) 27-27. https://doi.org/10.1038/138027a0.

[14] R.E. Mutha, A.U. Tatiya, S.J. Surana, Flavonoids as natural phenolic compounds and their role in therapeutics: an overview, Future J. Pharm. Sci. 7 (2021) 25. https://doi.org/10.1186/s43094-020-00161-8.

[15] Z.N. Md Nesran, N.H. Shafie, S.F. Md Tohid, M.E. Norhaizan, A. Ismail, Iron Chelation Properties of Green Tea Epigallocatechin-3-Gallate (EGCG) in Colorectal Cancer Cells: Analysis on Tfr/Fth Regulations and Molecular Docking, Evid. Based Complement. Alternat. Med. 2020 (2020) 1-8. https://doi.org/10.1155/2020/7958041.

[16] A. Calderaro, G.T. Patanè, E. Tellone, D. Barreca, S. Ficarra, F. Misiti, G. Laganà, The Neuroprotective Potentiality of Flavonoids on Alzheimer's Disease, Int. J. Mol. Sci. 23 (2022) 14835. https://doi.org/10.3390/ijms232314835.

[17] C. Di Lorenzo, F. Colombo, S. Biella, C. Stockley, P. Restani, Polyphenols and Human Health: The Role of Bioavailability, Nutrients 13 (2021) 273. https://doi.org/10.3390/nu13010273.

[18] K. Chen, Y. Hu, L. Chen, J. Zhang, H. Qiao, W. Li, K. Zhang, Y. Fang, Role of dehydration temperature on flavonoids composition and free-form volatile profile of raisins during the drying process, Food Chem. 374 (2022) 131747. https://doi.org/10.1016/j.foodchem.2021.131747.
[19]C.M.M. Gachon, M. Langlois-Meurinne, P. Saindrenan, Plant secondary metabolism glycosyltransferases: the emerging functional analysis, Trends Plant Sci. 10 (2005) 542–549. https://doi.org/10.1016/j.tplants.2005.09.007.

[20]T. Iwashina, The Structure and Distribution of the Flavonoids in Plants, J. Plant Res. 113 (2000) 287–299. https://doi.org/10.1007/PL00013940.

[21]F. Ren, Y. Nian, C.A. Perussello, Effect of storage, food processing and novel extraction technologies on onions flavonoid content: A review, Food Res. Int. 132 (2020) 108953. https://doi.org/10.1016/j.foodres.2019.108953.

[22]S. Mason, The origin of chirality in nature, Trends Pharmacol. Sci. 7 (1986) 20–23. https://doi.org/10.1016/0165-6147(86)90235-X.

[23]M. Goel, E. Larson, C.J. Venkatramani, M.A. Al-Sayah, Optimization of a two-dimensional liquid chromatography-supercritical fluid chromatography-mass spectrometry (2D-LC-SFS-MS) system to assess “in-vivo” inter-conversion of chiral drug molecules, J. Chromatogr. B 1084 (2018) 89–95. https://doi.org/10.1016/j.jchromb.2018.03.029.

[24]M.K. Khan, Zill-E-Huma, O. Dangles, A comprehensive review on flavanones, the major citrus polyphenols, J. Food Compos. Anal. 33 (2014) 85–104. https://doi.org/10.1016/j.jfca.2013.11.004.

[25]R. Cirilli, R. Ferretti, E. De Santis, B. Gallinella, L. Zanitti, F. La Torre, High-performance liquid chromatography separation of enantiomers of flavanone and 2'-hydroxychalcone under reversed-phase conditions, J. Chromatogr. A 1190 (2008) 95–101. https://doi.org/10.1016/j.chroma.2008.02.101.

[26]Z.A. ALOthman, A.Y. Badjah, K.M. Alsheetan, Mohd. Suhail, I. Ali, Enantiomeric resolution of quinolones on crown ether CSP: Thermodynamics, chiral discrimination mechanism and application in biological samples, J. Chromatogr. B 1166 (2021) 122550. https://doi.org/10.1016/j.jchromb.2021.122550.

[27]I. Baranowska, J. Hejniak, S. Magiera, Development and validation of a RP-UHPLC-ESI-MS/MS method for the chiral separation and determination of flavanone, naringenin and hesperetin enantiomers, Talanta 159 (2016) 181–188. https://doi.org/10.1016/j.talanta.2016.06.020.

[28]N. Uchiyama, I.H. Kim, N. Kawahara, Y. Goda, HPLC separation of hesperidin and the C-2 epimer in commercial hesperidin samples and herbal medicines, Chirality 17 (2005) 373–377. https://doi.org/10.1002/chir.20175.

[29]M.E. Alañón, S.M. Castle, P.J. Siswanto, T. Cifuentes-Gómez, J.P.E. Spencer, Assessment of flavanol stereoisomers and caffeine and theobromine content in commercial chocolates, Food Chem. 208 (2016) 177–184. https://doi.org/10.1016/j.foodchem.2016.03.116.

[30]Y. Qian, X. Zhao, L. Zhao, L. Cui, L. Liu, X. Jiang, Y. Liu, L. Gao, T. Xia, Analysis of stereochemistry and biosynthesis of epicatechin in tea plants by chiral phase high performance liquid chromatography, J. Chromatogr. B 1006 (2015) 1–7. https://doi.org/10.1016/j.jchromb.2015.10.024.

[31]H. Terashima, M. Seki, S. Watanabe, A. Yamamoto, S. Aizawa, A. Taga, I. Mikami, S. Kodama, Chiral separation of catechin and epicatechin by reversed phase high-performance liquid chromatography with β-cyclodextrin stepwise and linear gradient elution modes, J. Chromatogr. A 1673 (2022) 463029. https://doi.org/10.1016/j.chroma.2022.463029.

[32]E. De Rijke, P. Out, W.M.A. Niessen, F. Ariese, C. Gooijer, U.A.Th. Brinkman, Analytical separation and detection methods for flavonoids, J. Chromatogr. A 1112 (2006) 31–63. https://doi.org/10.1016/j.chroma.2006.01.019.

[33]C. West, Current trends in supercritical fluid chromatography, Anal. Bioanal. Chem. 410 (2018) 6441–6457. https://doi.org/10.1007/s00216-018-1267-4.

[34]R.S. van den Hurk, M. Pursch, D.R. Stoll, B.W.J. Pirok, Recent trends in two-dimensional liquid chromatography, TrAC Trends Anal. Chem. 166 (2023) 117166. https://doi.org/10.1016/j.trac.2023.117166.
[35] A. de Villiers, P. Venter, H. Pasch, Recent advances and trends in the liquid-chromatography-mass spectrometry analysis of flavonoids, J. Chromatogr. A 1430 (2016) 16-78. https://doi.org/10.1016/j.chroma.2015.11.077.

[36] Z. Tang, R. Yin, K. Bi, H. Zhu, F. Han, K. Chen, F. Wang, Simultaneous quantitative determination of 20 active components in the traditional Chinese medicine formula Zhi-Zi-Da-Huang decoction by liquid chromatography coupled with mass spectrometry: application to study the chemical composition variations in different combinations, Biomed. Chromatogr. 29 (2015) 1406-1414. https://doi.org/10.1002/bmc.3438.

[37] T. Dhanani, R. Singh, S. Kumar, Extraction optimization of gallic acid, (+)-catechin, procyanidin-B2, (-)-epicatechin, (-)-epigallocatechin gallate, and (-)-epicatechin gallate: their simultaneous identification and quantification in Saraca asoca, J. Food Drug Anal. 25 (2017) 691-698. https://doi.org/10.1016/j.jfda.2016.08.004.

[38] E. Mesquita, M. Monteiro, Simultaneous HPLC determination of flavonoids and phenolic acids profile in Pêra-Rio orange juice, Food Res. Int. 106 (2018) 54-63. https://doi.org/10.1016/j.foodres.2017.12.025.

[39] J.W. Allwood, T.L. Woznicki, Y. Xu, A. Foito, K. Aaby, J. Sungurtas, S. Freitag, R. Goodacre, D. Stewart, S.F. Remberg, O.M. Heide, A. Sønsteby, Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit, Metabolomics 15 (2019) 12. https://doi.org/10.1007/s11306-018-1462-5.

[40] A.R. Fontana, A. Antoniolli, R. Bottini, Development of a high-performance liquid chromatography method based on a core-shell column approach for the rapid determination of multiclass polyphenols in grape pomaces, Food Chem. 192 (2016) 1-8. https://doi.org/10.1016/j.foodchem.2015.06.101.

[41] T. Paquignon, M. Scholz, B.F. Zimmermann, Can home-brewed Benifuuki green tea deliver health-relevant amounts of 3"-O-methyl epigallocatechin gallate?, NFS J. 24 (2021) 8-14. https://doi.org/10.1016/j.nfs.2021.05.004.

[42] M. Delourdesmatabilbao, C. Andreslacueva, O. Jauregui, R. Lamuelaraventos, Determination of flavonoids in a Citrus fruit extract by LC-DAD and LC-MS, Food Chem. 101 (2007) 1742-1747. https://doi.org/10.1016/j.foodchem.2006.01.032.

[43] S. Magiera, I. Baranowska, A. Lautenszleger, UHPLC-UV method for the determination of flavonoids in dietary supplements and for evaluation of their antioxidant activities, J. Pharm. Biomed. Anal. 102 (2015) 468-475. https://doi.org/10.1016/j.jpba.2014.10.004.

[44] L. Di Donna, D. Taverna, F. Mazzotti, H. Benabdelkamel, M. Attya, A. Napoli, G. Sindona, Comprehensive assay of flavanones in citrus juices and beverages by UHPLC-ESI-MS/MS and derivatization chemistry, Food Chem. 141 (2013) 2328-2333. https://doi.org/10.1016/j.foodchem.2013.05.034.

[45] S. Li, M. Tian, K.H. Row, Effect of Mobile Phase Additives on the Resolution of Four Bioactive Compounds by RP-HPLC, Int. J. Mol. Sci. 11 (2010) 2229-2240. https://doi.org/10.3390/ijms11052229.

[46] J. Molineau, M. Meunier, A. Noireau, F. Laëtitia, A.-M. Petit, C. West, Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry—method development and application to compounds of pharmaceutical and cosmetic interest, Anal. Bioanal. Chem. 412 (2020) 6595-6609. https://doi.org/10.1007/s00216-020-02798-z.

[47] Z.-M. Jiang, L.-J. wang, W.-J. Liu, H.-Y. Wang, P.-T. Xiao, P. Zhou, Z.-M. Bi, E.-H. Liu, Development and validation of a supercritical fluid chromatography method for fast analysis of six flavonoids in Citri Reticulatae Pericarpium, J. Chromatogr. B 1133 (2019) 121845. https://doi.org/10.1016/j.jchromb.2019.121845.

[48] A.M. Ares, J. Bernal, A. Janvier, L. Toribio, Chiral and achiral separation of ten flavanones using supercritical fluid chromatography. Application to bee pollen analysis, J. Chromatogr. A 1685 (2022) 463633. https://doi.org/10.1016/j.chroma.2022.463633.

[49] M. Bouanini, N. Belboukhari, J.C. Menéndez, K. Sekkoum, A. Cheriti, H.Y. Aboul-Enein, Chiral separation of novel imininaringenin derivatives, Chirality 30 (2018) 484-490. https://doi.org/10.1002/chir.22812.
[50]Y. Jeon, C. Kwon, E. Cho, S. Jung, Carboxymethylated cyclosophoraose as a novel chiral additive for the stereoisomeric separation of some flavonoids by capillary electrophoresis, Carbohydr. Res. 345 (2010) 2408–2412.
https://doi.org/10.1016/j.carres.2010.08.009.

[51]J.A. Yáñez, P.K. Andrews, N.M. Davies, Methods of analysis and separation of chiral flavonoids, J. Chromatogr. B 848 (2007) 159–181.
https://doi.org/10.1016/j.jchromb.2006.10.052.

[52]S. Felletti, O.H. Ismail, C. De Luca, V. Costa, F. Gasparrini, L. Pasti, N. Marchetti, A. Cavazzini, M. Catani, Recent Achievements and Future Challenges in Supercritical Fluid Chromatography for the Enantioselective Separation of Chiral Pharmaceuticals, Chromatographia 82 (2019) 65–75.
https://doi.org/10.1007/s10337-018-3606-1.

[53]D. Mangelings, S. Eeltink, Y. Vander Heyden, Recent developments in liquid and supercritical fluid chromatographic enantioseparations, in: Handb. Anal. Sep., Elsevier, 2020: pp. 453–521.
https://doi.org/10.1016/B978-0-444-64070-3-00009-6.

[54]S. Khater, M.-A. Lozac'h, I. Adam, E. Francotte, C. West, Comparison of liquid and supercritical fluid chromatography mobile phases for enantioselective separations on polysaccharide stationary phases, J. Chromatogr. A 1467 (2016) 463–472.
https://doi.org/10.1016/j.chroma.2016.06.060.

[55]E. Yashima, Y. Okamoto, Chiral Discrimination on Polysaccharides Derivatives, Bull. Chem. Soc. Jpn. 68 (1995) 3289–3307.
https://doi.org/10.1246/bcsj.68.3289.

[56]E. Francotte, D. Huynh, Immobilized halogenophenylcarbamate derivatives of cellulose as novel stationary phases for enantioselective drug analysis, J. Pharm. Biomed. Anal. 27 (2002) 421–429.
https://doi.org/10.1016/S0731-7085(01)00568-4.

[57]E. Francotte, D. Huynh, Immobilization of 3,5-dimethylphenyl carbamate of cellulose and amylose on silica by photochemical and thermal radical processes, Chirality 34 (2022) 711–731.
https://doi.org/10.1002/chir.23426.

[58]B. Chankvetadze, Recent developments on polysaccharide-based chiral stationary phases for liquid-phase separation of enantiomers, J. Chromatogr. A 1269 (2012) 26–51.
https://doi.org/10.1016/j.chroma.2012.10.033.

[59]T.A. Berger, Preliminary kinetic evaluation of an immobilized polysaccharide sub-2 µm column using a low dispersion supercritical fluid chromatograph, J. Chromatogr. A 1510 (2017) 82–88.
https://doi.org/10.1016/j.chroma.2017.06.021.

[60]K. Si-Ahmed, Z. Aturki, B. Chankvetadze, S. Fanali, Evaluation of novel amylose and cellulose-based chiral stationary phases for the stereoisomer separation of flavanones by means of nano-liquid chromatography, Anal. Chim. Acta 738 (2012) 85–94.
https://doi.org/10.1016/j.aca.2012.05.055.

[61]C.L. Sayre, M. Hopkins, J.K. Takemoto, N.M. Davies, Chiral analytical method development of liquiritigenin with application to a pharmacokinetic study: Chiral separation of liquiritigenin with pharmacokinetics, Biomed. Chromatogr. (2012) n/a-n/a.
https://doi.org/10.1002/bmc.2787.

[62]C. Sayre, N. Davies, Quantification of Three Chiral Flavonoids with Reported Bioactivity in Selected Licensed Canadian Natural Health Products and US Marketed Dietary Supplements, J. Pharm. Pharm. Sci. 16 (2013) 272.
https://doi.org/10.18433/J3X01Q.

[63]R. Gaggeri, D. Rossi, S. Collina, B. Mannucci, M. Baierl, M. Juza, Quick development of an analytical enantioselective high performance liquid chromatography separation and preparative scale-up for the flavonoid Naringenin, J. Chromatogr. A 1218 (2011) 5414–5422.
https://doi.org/10.1016/j.chroma.2011.02.038.

[64]Guo, Separation of the enantiomers of naringenin and eriodictyol by amylosebased chiral reversed-phase high-performance liquid chromatography, Drug Discov. Ther. (2012).
https://doi.org/10.5582/ddt.2012.v6.6.321.

[65]I. Baranowska, J. Hejniak, S. Magiera, Simultaneous Chiral Separation of Flavanone, Naringenin, and Hesperetin Enantiomers by RP-UHPLC-DAD: Chiral Separation of Flavanone, Naringenin, and Hesperetin, Chirality 28 (2016) 147–152.
https://doi.org/10.1002/chir.22559.
[66]J.A. Yáñez, X.W. Teng, K.A. Roupe, N.M. Davies, Stereospecific high-performance liquid chromatographic analysis of hesperetin in biological matrices, J. Pharm. Biomed. Anal. 37 (2005) 591–595. https://doi.org/10.1016/j.jpba.2004.10.028.

[67]M.E.A. Zaid, N. Belboukhari, K. Sekkoum, B. Ibtissam, H.Y.A. Enein, Synthesis and Chiral Separation of Some 4-thioflavones, J. Chromatogr. Sci. 59 (2021) 856–862.
https://doi.org/10.1093/chromsci/bmab007.

[68]A. Lévèques, L. Actis-Goretta, M.J. Rein, G. Williamson, F. Dionisi, F. Giuffrida, UPLC-MS/MS quantification of total hesperetin and hesperetin enantiomers in biological matrices, J. Pharm. Biomed. Anal. 57 (2012) 1–6.
https://doi.org/10.1016/j.jpba.2011.08.031.

[69]S. Caccamese, L. Manna, G. Scivoli, Chiral HPLC separation and CD spectra of the C-2 diastereomers of naringin in grapefruit during maturation, Chirality 15 (2003) 661–667.
https://doi.org/10.1002/chir.10262.

[70]I. Ali, M. El Amin Zaid, N. Belboukhari, K. Sekkoum, W.H. Al-Qahtani, A. Mahmoud Karami, M. Locatelli, Chiral HPLC separation and simulation studies of two chiral centered bis-imino flavans (Schiff base), Microchem. J. 178 (2022) 107429.
https://doi.org/10.1016/j.microc.2022.107429.

[71]P. Ficarra, R. Ficarra, C. Bertucci, S. Tommasini, M. Calabrò, D. Costantino, M. Carulli, Direct Enantiomeric Separation of Flavanones by High Performance Liquid Chromatography using Various Chiral Stationary Phases, Planta Med. 61 (1995) 171–176.
https://doi.org/10.1055/s-2006-958041.

[72]S. Caccamese, C. Caruso, N. Parrinello, A. Savarino, High-performance liquid chromatographic separation and chiroptical properties of the enantiomers of naringenin and other flavanones, J. Chromatogr. A 1076 (2005) 155–162.
https://doi.org/10.1016/j.chroma.2005.04.024.

[73]S.-G. Ma, R.-B. Wang, W.-R. Li, Y.-B. Liu, J. Qu, Y. Li, X.-J. Wang, S. Xu, S.-S. Yu, New C-2 diastereomers of flavanone glycosides conjugated with 3-hydroxy-3-methylglutaric acid from the pericarp of Citrus grandis (L.) Osbeck, Bioorganic Chem. 80 (2018) 519–524.
https://doi.org/10.1016/j.bioorg.2018.06.024.

[74]J.A. Yáñez, N.M. Davies, Stereospecific high-performance liquid chromatographic analysis of naringenin in urine, J. Pharm. Biomed. Anal. 39 (2005) 164–169.
https://doi.org/10.1016/j.jpba.2005.02.025.

[75]G. Crini, Review: A History of Cyclodextrins, Chem. Rev. 114 (2014) 10940–10975.
https://doi.org/10.1021/cr500081p.

[76]P. Dugo, F. Cacciola, P. Donato, D. Airado-Rodríguez, M. Herrero, L. Mondello, Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines, J. Chromatogr. A 1216 (2009) 7483–7487.
https://doi.org/10.1016/j.chroma.2009.04.001.

[77]P. Machonis, M. Jones, B. Schaneberg, C. Kwik-Uribe, D. Dowell, Method for the Determination of Catechin and Epicatechin Enantiomers in Cocoa-Based Ingredients and Products by High-Performance Liquid Chromatography: First Action 2013.04, J. AOAC Int. 97 (2014) 506–509.
https://doi.org/10.5740/jaoacint.13-351.

[78]X. Lai, W. Tang, S.-C. Ng, Novel cyclodextrin chiral stationary phases for high performance liquid chromatography enantioseparation: Effect of cyclodextrin type, J. Chromatogr. A 1218 (2011) 5597–5601.
https://doi.org/10.1016/j.chroma.2011.06.020.

[79]K. Si-Ahmed, F. Tazerouti, A.Y. Badjah-Hadj-Ahmed, Z. Aturki, G. D'Orazio, A. Rocco, S. Fanali, Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-β-cyclodextrin chiral stationary phase, J. Chromatogr. A 1217 (2010) 1175–1182.
https://doi.org/10.1016/j.chroma.2009.07.053.

[80]M. Silva, D. Pérez-Quintanilla, S. Morante-Zarcero, I. Sierra, M.L. Marina, Z. Aturki, S. Fanali, Ordered mesoporous silica functionalized with β-cyclodextrin derivative for stereoisomer separation of flavanones and flavanone glycosides by nano-liquid chromatography and capillary electrochromatography, J. Chromatogr. A 1490 (2017) 166–176.
https://doi.org/10.1016/j.chroma.2017.02.012.
[81] L. Li, B. Cheng, R. Zhou, Z. Cao, C. Zeng, L. Li, Preparation and evaluation of a novel N-benzyl-phenethylamino-β-cyclodextrin-bonded chiral stationary phase for HPLC, Talanta 174 (2017) 179-191. https://doi.org/10.1016/j.talanta.2017.06.009.

[82] N. Zhang, S. Guo, B. Gong, Preparation of a novel bridged bis(β-cyclodextrin) chiral stationary phase by thiol-ene click chemistry for enhanced enantioseparation in HPLC, RSC Adv. 11 (2021) 35754-35764. https://doi.org/10.1039/D1RA04697G.

[83] N. Belboukhari, A. Cheriti, C. Roussel, N. Vanthuyne, Chiral separation of hesperidin and naringin and its analysis in a butanol extract of *Launeae arborescens*, Nat. Prod. Res. 24 (2010) 669-681. https://doi.org/10.1080/14786410903178376.

[84] S. Caccamese, R. Chillemi, Racemization at C-2 of naringin in pummelo (Citrus grandis) with increasing maturity determined by chiral high-performance liquid chromatography, J. Chromatogr. A 1217 (2010) 1089-1093. https://doi.org/10.1016/j.chroma.2009.10.073.

[85] L. Toribio, C. Alonso, M.J. del Nozal, J.L. Bernal, J.J. Jiménez, Enantiomeric separation of chiral sulfoxides by supercritical fluid chromatography, J. Sep. Sci. 29 (2006) 1363-1372.

[86] Z. Pirzada, M. Personick, M. Biba, X. Gong, L. Zhou, W. Schafer, C. Roussel, C.J. Welch, Systematic evaluation of new chiral stationary phases for supercritical fluid chromatography using a standard racemate library, J. Chromatogr. A 1217 (2010) 1134-1138. https://doi.org/10.1016/j.chroma.2009.10.004.

[87] L. Toribio, I. Magdaleno, B. Martín-Gómez, M.T. Martín, S. Valverde, A.M. Ares, Study of Different Chiral Columns for the Enantiomeric Separation of Azoles Using Supercritical Fluid Chromatography, Separations 10 (2023) 9. https://doi.org/10.3390/separations10010009.

[88] E. Fuguet, C. Ràfols, M. Mañé, R. Ruiz, E. Bosch, Acidity constants of hydroxyl groups placed in several flavonoids: Two flavanones, two flavones and five flavonols, Talanta 253 (2023) 124096. https://doi.org/10.1016/j.talanta.2022.124096.

[89] Z. Yao, S. Wu, H. Zhang, X. Feng, Z. Wang, M. Lin, Chiral Determination of Naringenin by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry and Application in Citrus Peel and Pulp, Front. Nutr. 9 (2022) 906859. https://doi.org/10.3389/fnut.2022.906859.

[90] J.A. Yáñez, N.D. Miranda, C.M. Remsberg, Y. Ohgami, N.M. Davies, Stereospecific high-performance liquid chromatographic analysis of eriodictyol in urine, J. Pharm. Biomed. Anal. 43 (2007) 255-262. https://doi.org/10.1016/j.jpba.2006.06.027.

[91] K.R. Vega-Villa, J.A. Yáñez, C.M. Remsberg, Y. Ohgami, N.M. Davies, Stereospecific high-performance liquid chromatographic validation of homoeriodictyol in serum and Yerba Santa (Eriodictyon glutinosum), J. Pharm. Biomed. Anal. 46 (2008) 971-974. https://doi.org/10.1016/j.jpba.2007.07.020.

[92] E. Lesellier, C. West, The many faces of packed column supercritical fluid chromatography – A critical review, J. Chromatogr. A 1382 (2015) 2-46. https://doi.org/10.1016/j.chroma.2014.12.083.

[93] X. Qiao, W. Song, S. Ji, Q. Wang, D. Guo, M. Ye, Separation and characterization of phenolic compounds and triterpenoid saponins in licorice (Glycyrrhiza uralensis) using mobile phase-dependent reversed-phase × reversed-phase comprehensive two-dimensional liquid chromatography coupled with mass spectrometry, J. Chromatogr. A 1402 (2015) 36-45. https://doi.org/10.1016/j.chroma.2015.05.006.

[94] F. Cacciola, P. Dugo, L. Mondello, Multidimensional liquid chromatography in food analysis, TrAC Trends Anal. Chem. 96 (2017) 116-123. https://doi.org/10.1016/j.trac.2017.06.009.

[95] M. Iguiniz, S. Heinisch, Two-dimensional liquid chromatography in pharmaceutical analysis. Instrumental aspects, trends and applications, J. Pharm. Biomed. Anal. 145 (2017) 482-503. https://doi.org/10.1016/j.jpba.2017.07.009.

[96] L. Montero, M. Herrero, E. Ibáñez, A. Cifuentes, Profiling of phenolic compounds from different apple varieties using comprehensive two-dimensional liquid chromatography, J. Chromatogr. A 1313 (2013) 275-283. https://doi.org/10.1016/j.chroma.2013.06.015.
[97] K.M. Kalili, A. de Villiers, Off-line comprehensive two-dimensional hydrophilic interaction×reversed phase liquid chromatographic analysis of green tea phenolics, J. Sep. Sci. 33 (2010) 853–863. https://doi.org/10.1002/jssc.200900673.

[98] L. Jiao, Y. Tao, W. Wang, Y. Shao, L. Mei, Q. Wang, J. Dang, Preparative isolation of flavonoid glycosides from *Sphaerophysa salsula* using hydrophilic interaction solid-phase extraction coupled with two-dimensional preparative liquid chromatography, J. Sep. Sci. 40 (2017) 3808–3816. https://doi.org/10.1002/jssc.201700675.

[99] E.H. Nell, A. de Villiers, Using multiple heart-cutting valves and stop-flow operation to enable variable second dimension gradient times in online comprehensive two-dimensional liquid chromatography, J. Chromatogr. A 1685 (2022) 463583. https://doi.org/10.1016/j.chroma.2022.463583.

[100] C.T. Scoparo, L.M. de Souza, N. Dartora, G.L. Sassaki, P.A.J. Gorin, M. Iacomini, Analysis of Camellia sinensis green and black teas via ultra high performance liquid chromatography assisted by liquid–liquid partition and two-dimensional liquid chromatography (size exclusion×reversed phase), J. Chromatogr. A 1222 (2012) 29–37. https://doi.org/10.1016/j.chroma.2011.11.038.

[101] L. Montero, V. Sáez, D. von Baer, A. Cifuentes, M. Herrero, Profiling of Vitis vinifera L. canes (poly)phenolic compounds using comprehensive two-dimensional liquid chromatography, J. Chromatogr. A 1536 (2018) 205–215. https://doi.org/10.1016/j.chroma.2017.06.013.

[102] J. Dang, L. Zhang, Q. Wang, L. Mei, H. Yue, Z. Liu, Y. Shao, Q. Gao, Y. Tao, Target separation of flavonoids from *Saxifraga tangutica* using two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography, J. Sep. Sci. 41 (2018) 4419–4429. https://doi.org/10.1002/jssc.201800534.

[103] L. Réset, C. De Saint Jores, I. François, C. West, Development of a Two-Dimensional Supercritical Fluid Chromatography System in Multiple Heart-Cutting Modes, Anal. Chem. 96 (2024) 11969–11976. https://doi.org/10.1021/acs.analchem.4c01795.

[104] D.R. Stoll, H.R. Lhotka, D.C. Harmes, B. Madigan, J.J. Hsiao, G.O. Staples, High resolution two-dimensional liquid chromatography coupled with mass spectrometry for robust and sensitive characterization of therapeutic antibodies at the peptide level, J. Chromatogr. B 1134–1135 (2019) 121832. https://doi.org/10.1016/j.jchromb.2019.121832.

[105] I. François, A. de Villiers, B. Tienpont, F. David, P. Sandra, Comprehensive two-dimensional liquid chromatography applying two parallel columns in the second dimension, J. Chromatogr. A 1178 (2008) 33–42. https://doi.org/10.1016/j.chroma.2007.11.032.

[106] K.M. Kalili, D. Cabooter, G. Desmet, A. de Villiers, Kinetic optimisation of the reversed phase liquid chromatographic separation of proanthocyanidins on sub-2µm and superficially porous phases, J. Chromatogr. A 1236 (2012) 63–76. https://doi.org/10.1016/j.chroma.2012.02.067.

[107] N. Sheng, H. Zheng, Y. Xiao, Z. Wang, M. Li, J. Zhang, Chiral separation and chemical profile of Dengzhan Shengmai by integrating comprehensive with multiple heart-cutting two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, J. Chromatogr. A 1517 (2017) 97–107. https://doi.org/10.1016/j.chroma.2017.08.037.

[108] S. Li, C. Liu, Y. Zhang, D. Shi, R. Tsao, Application of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography for continuous extraction and separation of bioactive compounds from *Citrus limon* peel, J. Sep. Sci. 43 (2020) 3793–3805. https://doi.org/10.1002/jssc.202000588.

[109] N.A. Walters, A. de Villiers, E. Joubert, D. de Beer, Phenolic profiling of rooibos using off-line comprehensive normal phase countercurrent chromatography × reversed phase liquid chromatography, J. Chromatogr. A 1490 (2017) 102–114. https://doi.org/10.1016/j.chroma.2017.02.021.

[110] T. Michel, E. Destandau, L. Fougère, C. Elfakir, New “hyphenated” CPC-HPLC-DAD-MS strategy for simultaneous isolation, analysis and identification of phytochemicals:
application to xanthones from Garcinia mangostana, Anal. Bioanal. Chem. 404 (2012) 2963-2972. https://doi.org/10.1007/s00216-012-6430-8.

[111] I. Bakhytkyzy, O. Nuñez, J. Saurina, Determination of flavanols by liquid chromatography with fluorescence detection. Application to the characterization of cranberry-based pharmaceuticals through profiling and fingerprinting approaches, J. Pharm. Biomed. Anal. 156 (2018) 206-213. https://doi.org/10.1016/j.jpba.2018.04.031.

[112] M. Commisso, A. Anesi, S. Dal Santo, F. Guzzo, Performance comparison of electrospray ionization and atmospheric pressure chemical ionization in untargeted and targeted liquid chromatography/mass spectrometry based metabolomics analysis of grapeberry metabolites: APCI well ionizes strongly polar metabolites, Rapid Commun. Mass Spectrom. 31 (2017) 292-300. https://doi.org/10.1002/rcm.7789.

[113] K.R. Vega-Villa, C.M. Remsberg, Y. Ohgami, J.A. Yáñez, J.K. Takemoto, P.K. Andrews, N.M. Davies, Stereospecific high-performance liquid chromatography of taxifolin, applications in pharmacokinetics, and determination in tu fu ling (Rhizoma smilacis glabrae) and apple (Malus × domestica), Biomed. Chromatogr. 23 (2009) 638-646. https://doi.org/10.1002/bmc.1165.

[114] Meral Kekecoglu, E. Sonmez, M.K. Acar, S.A. Karaoglu, Pollen Analysis, Chemical Composition and Antibacterial Activity of Anatolian Chestnut Propolis Collected From Yıgilca Region, Biol. Bull. 48 (2021) 721-728. https://doi.org/10.1134/S106235902106011X.

[115] A.J. Miles, B.A. Wallace, Circular dichroism spectroscopy of membrane proteins, Chem. Soc. Rev. 45 (2016) 4859-4872. https://doi.org/10.1039/C5CS00084J.

[116] S.M. Kelly, T.J. Jess, N.C. Price, How to study proteins by circular dichroism, Biochim. Biophys. Acta BBA - Proteins Proteomics 1751 (2005) 119-139. https://doi.org/10.1016/j.bkapap.2005.06.005.

[117] C. Bertucci, D. Tedesco, Advantages of electronic circular dichroism detection for the stereochemical analysis and characterization of drugs and natural products by liquid chromatography, J. Chromatogr. A 1269 (2012) 69-81. https://doi.org/10.1016/j.chroma.2012.09.046.

[118] B.Q. Li, Z.Y. Li, X.K. Li, L.F. Tan, Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of four target flavonoids components in citrus samples, Acta Chromatogr. 35 (2023) 115-122. https://doi.org/10.1556/1326.2022.01010.