MS n Identification and Structural Characterization of Phenolic Compounds

The complete and unequivocal identification of each phenolic compound found in a plant extract can only be performed using NMR spectroscopy isolated and/or combined with other

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analytical techniques. This fact comes from the existence for a wide range number of phenolic compounds with positional isomers or chiral carbons.

A large number of phenolic compounds have been studied directly or extracted from plants and characterized by ¹³C and ¹H-NMR experiments.

There are recent studies using HPLC for separation of components from crude extracts and the eluent is split between MS and NMR (March and Brodbelt, 2008), for simultaneous HPLC-MS and HPLC-NMR analysis.

Nevertheless, the use of HPLC coupled to mass spectrometry, mostly ESI-MS has been widely used for structural identification of phenolic compounds present in several natural samples (Ablajan et al., 2006; Cuyckens and Claeys, 2004; Fabre et al., 2001; de Rijke et al., 2003; Ye et al., 2007). FAB was also used for identification of phenolic compounds after HPLC separation (Edenharder et al., 2001; Sano et al., 1999).

A review on the application of MS techniques for the determination of flavonoids in biological samples was reported by Praisan et al. (2004).

7.1. Flavonoids

Cuyckens and Claeys (2004) found that in the structure analysis of flavonoids by HPLC/ESI-MS/UV-DAD, the negative-ion mode is more sensitive and the fragmentation behavior is different, giving additional and complementary information, then the positive mode. Depending on the structure, flavonoid O-glycosides undergo collision-induced cleavage of the O-glycosidic bonds producing the free deprotonated aglycone.

In order to help the analysis of mass fragmentation of flavonoid compounds, either as free aglycones and/or O-glycosilated aglycones. Ma et al. (1997) proposed a nomenclature for the main fragment ions obtained (Figure 9) (Cuyckens and Claeys, 2004).

In the negative mode for free aglycones, the ^, A and ^, B labels correspond to ions containing intact A- and B-rings, respectively, in which i and j indicate the C-ring bonds that have been broken. For conjugated aglycones, Y is used to refer to the aglycone fragment [M–H–glycoside]^-. When positive mode is used, the ions are denotade ^, A and ^, B , respectively.

Figure 9. Ion nomenclature used for flavonoid glycosides (illustrated on apigenin 7-O-rutinoside).

Adaptaded from(Cuyckens and Claeys, 2004).

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Phenolic Compounds and Antioxidant Capacity of Medicinal Plants 23

The most useful fragmentations for the identification of flavonoid aglycones are those that require cleavage of two C-C bond of the C-ring, resulting in structurally informative ^, A and ^, B ions. These ions are obtained by specific retro Diels-Alder (RDA) reactions and give information on the number and type of substituent in the A- and B-rings (Cuyckens and Claeys, 2004). RDA reactions occur in six-membered cyclic structures containing a double bond and involve the relocation of three pairs of electrons in the cyclic ring. As a result, the cleavage of two -bonds and the formation of two π-bonds take place; for example, cyclohexene will fragment into butadiene and ethylene (de Rijke et al., 2006).

The MSⁿ analysis and main fragment ions of several flavonoid aglycones in the negative mode were reported by Fabre et al. (2001).

The RDA C-ring cleavage of the 1,3 bonds giving ^, A and ^, B fragment ions appears as the main fragments in the negative ion mode, as it is also true for the positive mode.

The ^, B ion is the major peak and it is characteristic for isoflavones (daidzein and genistein) (de Rijke et al., 2006). ^, A and ^, B fragments are reported at low intensity for some members of the main types of flavonoids.

3’,4’-dihydroxyflavonol (quercetin and fisetin) give characteristic 1,2 C-ring cleavage with ^, A ions as more abundant, rather than the ^, B fragment ions; this type of cleavage is not observed for other flavonols (Fabre et al., 2001). ^, A ions have also been detected from the fragmentation of two isoflavones (formononetin and biochanin A) (Aramendia et al., 1995; de Rijke et al., 2003).

The number of hydroxyl groups in the B-ring is clearly observed in the fragmentation pattern. Flavonols with two or more hydroxyl groups in the B-ring display ^, A and ^, B . In some cases, a direct cleavage of the bond between the B- and C-rings, resulting in an [M-B-ring-H]^- fragment ion, can be observed (Cuyckens and Claeys, 2004).

In addition to RDA reaction fragment ions, loss of small groups, such as H₂O (18 Da), CO (28 Da), CO₂ (44 Da) and C₂H₂O (42 Da), are commonly detected in negative and positive ion mode. These fragments are helpful in the identification of those specific functional groups. Compounds presenting methoxyl groups have a typical loss of 15 Da resulting in a [M − H − CH ]^. radical ion (Cuyckens and Claeys, 2004).

Flavonoids are found in nature often conjugated with sugar units. Glucose is the most commonly found sugar moiety followed by galactose, rhamnose, xylose and arabinose.

Fragment ions from glyconjugate flavonoids are labelled based on the nomenclature introduced by Domon and Costello (Cuyckens and Claeys, 2004) represented in Figure 9. Y represents the diglycoside unit, with fragments that contain the aglycone part being denominated Y₁ (loss of one sugar unit) and Y₀ (loss of two sugar units); the relative sugar fragments are labeled B₁ and B₀. Ions formed due to the cleavage of the sugar ring, and which contain the aglycone, are designated ^,X , where j is the number of the interglycosidic bonds broken, counting from the aglycone; the superscripts k and l indicate the interglycosidic bonds, with the glycosidic bond linking the glycose part to the aglycone being numbered 0 (de Rijke et al., 2006).

O-glycosides, C-glycosides and O,C-glycosides can be distinguished based on their MSⁿ fragmentation pattern.

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Flavonoid O-glycosides can suffer both a collision-induced homolytic and heterolytic cleavage of the O-glycosidic bond producing deprotonated radical aglycone [Y − H]^. and deprotonated aglycone ion, Y (Hvattum and Ekeberg, 2003).

The radical aglycone ions are very common for deprotonated flavonol 3-O-glycosides.

The nature and position of the glycoside group on the flavonol structure plays an important role on the formation of radical aglycone ions. Hvattum and Ekeberg (2003) verified that the product ion spectrum of kaempferol-7-O-neohesperidoside showed only a minor radical aglycone product ion, as opposed to kaempferol-3-O-rutinoside.

The homolytic to heterolytic cleavage ratio increases with the increasing number of OH groups in the B-ring. There are minor differences between positional isomers: ^,B products are more easily formed for 7-O-glycosides, whereas ^,A fragments are more abundant for 4’-O-glycosides (Cuyckens and Claeys, 2005).

Flavonoid C-glycosides have the sugar moiety linked directly to the flavonoid aglycone via an acid-resistant C-C bond. Tandem MSⁿ analysis in combination with CID allows for the characterization of this type of compounds both in negative and positive ion modes.

The major fragment ions observed are related to the cross-ring cleavages of the sugar residue (Figure 10) and the loss of water molecules (Figure 11) (Cuyckens and Claeys, 2004).

Figure 10. Characteristic product ions formed by cross-ring cleavages in a pentose and hexose residue (Cuyckens and Claeys, 2004).

Figure 11. Loss of water observed for 6-C-glycosyl flavonoids involving the hydroxyl group at the 2’’-position of the sugar residue and the hydroxyl group at the 5-or 7-2’’-position of the aglycone (Cuyckens and Claeys, 2004).

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Phenolic Compounds and Antioxidant Capacity of Medicinal Plants 25

The known C-glycosilation positions are the C-6 and/or C-8 of the flavonoid nucleus.

Thus, the main goal is to differentiate 6-C- and 8-C-glycosyl flavonoids. The loss of a water molecule is observed, in positive and negative ion modes, and it is more pronounced for 6-C- than 8-C-glycosyl compounds.

In di-C-glycosides, sugar residues of different mass can be located, since the C6-sugar residue shows more extensive fragmentation than the C8-sugar residue.

Very few flavonoid glycosides are commercially available as standards, so their quantitative analysis is seldom performed. Usually, plant extracts are subject to hydrolysis of those glycosides and the released aglycones are identified and quantified.

In addition to glycosilation, several flavonoids have been described containing an acyl group linked to the sugar part. These acyl groups can be observed in mass spectrometry experiments, based on typical neutral losses. The most common acyl groups naturally occurring in flavonoids are acetyl, malonyl, benzoyl, galloyl, coumaroyl, feruloyl and sinapoyl (Cuyckens and Claeys, 2004).

The exact linkage position of acyl groups to sugar units is difficult to define through ESI/MSⁿ data, but they appear to be mainly linked at the 6-position of a hexose moiety which is confirmed when a ^, X fragment is present in the spectrum.

7.2. Non –flavonoids

Ionization of hydroxybenzoic and hydroxycinnamic acids can be performed either in the negative (deprotonation, [M-H]^-) or positive (protonation, [M+H]⁺) ion mode.

Tandem mass spectrometry in the negative-ion mode of deprotonated phenolic acids produce a common loss of 44 Da by elimination of a carboxyl group from the deprotonated molecular ions, [M-H-CO₂]^-.

As mentioned before, chlorogenic acids (CQA) are a family of esters formed between some trans-cinnamic acids (caffeic, coumaric, ferulic and tartaric) and (-)-quinic acid. This class of compounds is found in high levels in coffee, where esterification occurs at positions 3, 4 and 5 of the quinic acid structure. In addition to coffee, there are other plants rich in CQA and substitution at position C-1 has been reported in some Asteraceae, such as arnica and artichoke (Clifford et al., 2005).

Despite that this class of compounds is widely distributed in nature, only few commercial standards are available, therefore accurate identification of individual compounds in complex samples is quite difficult. The application of tandem MSⁿ fragmentation of the different isomers makes it possible to discriminate each one. Clifford and co-workers (Clifford, 2003, 2005) studied exhaustively these compounds by HPLC-ESI/MSⁿ and presented a hierarchical key for the identification of caffeoylquinic acid (mono, di and tri-isomers), coumaroylquinic acids and feruloylquinic acids. For dicaffeoylquinic acids, the caffeoyl group is more or less easily removed, depending on which position of quinic acid it is connected, in the following order: 1~5 > 3 > 4. The discrimination between the 1-CQA and 5-CQA is easy to establish on HPLC using a reverse phase column since 5-CQA is more hydrophobic and therefore elutes later (Clifford et al., 2005). This key was also used for the identification of a large number of hydroxycinnamate esters of quinic, tartaric and shikimic acid in several species of Asteraceae (Clifford et al., 2005).

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These authors found cis and trans hydroxycinnamate moieties in CQAs and were able to distinguish them based on their fragmentation patterns, relative retention times and UV irradiation response.