Journal of Chromatography A, 1207 (2008) 101–109 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Analysis of flavonol glycoside isomers from leaves of Maytenus ilicifolia by offline and online high performance liquid chromatography–electrospray mass spectrometry Lauro M. de Souza, Thales R. Cipriani, Rodrigo V. Serrato, Denise E. da Costa, Marcello Iacomini, Philip A.J. Gorin, Guilherme L. Sassaki ∗ Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, CP 19046, CEP 81531-980, Curitiba-PR, Brazil a r t i c l e i n f o Article history: Received 16 April 2008 Received in revised form 30 July 2008 Accepted 11 August 2008 Available online 14 August 2008 Keywords: Liquid chromatography–mass spectrometry Maytenus ilicifolia Flavonol glycosides isomers Kaempferol Quercetin Radical aglycone a b s t r a c t Flavonol glycosides present in leaves of Maytenus ilicifolia, were examined after fractionation on silicagel column. Flavonol mono-, di-, tri-, and tetraglycosides, containing kaempferol, quercetin or myricetin were identified by offline electrospray mass spectrometry. Increasing the cone energy induced to adducts variation, from H+ to Na+ . Protonated ions were characteristically fragmented by sequentially removing the monosaccharide residues, whereas in the sodiated ions, the aglycone was firstly removed. Online high performance liquid chromatography–mass spectrometry, with simple gradients of water, acetonitrile and acetic acid indicated the presence of several isomers, which were further identified by gas chromatography–mass spectrometry as containing galactose or glucose. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Maytenus ilicifolia Mart. ex Reissek (Celastraceae) is an important folk medicinal plant, largely found in Paraguay, Uruguay, Argentina and Brazil. From the infusion of its leaves is made a tea, which has several benefits to human health, from gastric protection to anti-ulcerogenic and analgesic activities [1–3]. Moreover, extracts of M. ilicifolia containing phenolic compounds, particularly flavonoids, induced to vascular relaxation in rat aorta [4,5]. These compounds also have anti-oxidants, anti-allergic, anti-inflammatory, anti-microbial, and anti-cancer properties [6–13]. Flavonoids include a class of compounds widely found in higher plants, their chemical architecture is based in a three-ring structure (A, B and C). Flavonoids may exist as free aglycones, and as O- or Cglycosides, however, other substitutions or chemical groups such as acetyl or sulfate groups, may be attached to the rings [14–16]. According to their structural variability, it has been suggested that the carbohydrate moieties are important for their biological activities [17,18]. Previous investigations showed the presence of several ∗ Corresponding author. Tel.: +55 41 3361 1577; fax: +55 41 3266 2042. E-mail address: sassaki@ufpr.br (G.L. Sassaki). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.032 types of flavonol 3-O-glycosides in leaves of Maytenus spp, such as rutin, quercitrin, hyperoside, and isoquercitrin, and other structures were characterized as kaempferol and quercetin di-, tri, and tetraglycosides [19–22]. Mass spectrometry (MS) has been successfully applied on the elucidation of structures of several flavonoid glycosides [6–8,23–30]. More accurate results are usually obtained in combination with chromatographic techniques, overcoming the difficulties in identifying isomers in complex matrixes. Some techniques, such as metal complexation-mass spectrometry, have been applied to distinguish some standard of isomeric glycosides [31]. However, this offline technique is not suitable to be applied on complex mixtures, since nonspecific complexation may lead to misinterpretation. The glycosylation site of flavonols from M. ilicifolia appears exclusively at the 3-OH position. However, carbohydrate composition and interglycosidic linkages have been shown to be of great variability. Thus, we now describe the structures of flavonol glycosides, including several isomers, found in extracts of M. ilicifolia leaves, using high performance liquid chromatography (HPLC), electrospray mass ionization (ESI-MS) and gas chromatography–mass spectrometry (GC–MS). The different fragments obtained for protonated and sodiated precursor ions were the basis for monosaccharide sequencing and for structural eluci- 102 L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 dation of these flavonol glycosides, which included some previously undescribed structures. 2. Experimental 2.1. Plant material Leaves of M. ilicifolia were collected in Curitiba (Southern Brazil), and donated by the “Central de Produção e Comercialização de Plantas Medicinais, Aromáticas e Condimentares do Paraná Ltda”. The plant was identified by Prof. Olavo Guimarães, Botany Department, Federal University of Paraná (UFPR), Curitiba, Brazil and it is deposited in the Herbarium of UFPR as voucher no. 30842. 2.2. Extraction and chromatographic fractionation of components from M. ilicifolia Defatted leaves (100 g) of M. ilicifolia were extracted thrice with water (500 ml) under reflux (100 ◦ C), for 3 h. The extracts were combined, concentrated to lower volume (200 ml), and then added to cold ethanol (3 vol.) in order to precipitate macromolecules, which were removed by centrifugation (10,000 rpm). The ethanol-soluble compounds (ESC) were concentrated and then freeze-dried. A fraction of ESC (500 mg) was applied to a column (50 cm × 5 cm) packed with silica-gel 60 G (Merck), and eluted using a series of mixtures of CHCl3 –MeOH (ranging from 95:5, v/v to 100% of MeOH). Each collected fraction (10 ml) was evaporated under nitrogen stream at room temperature (∼25 ◦ C) and analyzed on thin layer chromatography (TLC) with silica-gel 60 G (Merck), using n-PrOH–H2 O (7:3, v/v) as solvent. The plates were general stained by 10% H2 SO4 in ethanol (v/v) at 100 ◦ C, or with orcinol–H2 SO4 at 100 ◦ C, in order to identify carbohydratecontaining compounds [32]. Similar fractions were combined to give rise to 10 further fractions, namely F1–F10. 2.3. HPLC conditions HPLC analysis was performed in a Shimadzu apparatus, equipped with an SPA-10 UV/Vis detector, the samples were detected at 360 nm, or by online HPLC–ESI-MS. The oven temperature was held at 60 ◦ C, and the separation was carried out at a flow rate of 1 ml min−1 on a Supelco LC-18 column, 250 mm × 4.6 mm (4 ␮m, particle size) as analytical and semi-preparative modes. Two mobile phases using HPLC grade solvents (Tedia-USA) prepared in deionized water, were: solvent A 1% of acetic acid in water (v/v), and solvent B H2 O:CH3 CN:HOAc (49:50:1, v/v). The gradient I (used on F3) was: 10–30% B in 5 min, 30–50% B in 25 min held for 5 min, back to 10% B in 35 min held for more 10 min. The gradient II (used on F4 and F5) was: 10–40% B in 15 min, held for 2 min, back to 10% in 20 min held for more 10 min. The samples were dissolved in MeOH:H2 O (1:1, v/v), the concentration of the analytical run being 1 mg/ml, with 10 ␮l injected. For purification purposes, semi-preparative runs were carried out using concentration of 10 mg/ml, with 50 ␮l of injection volume. Non-resolved peaks were collected together for further purifying by preparative TLC, using n-PrOH–H2 O (7:3, v/v) as solvent, and being detected at UV 366 nm. 2.4. Mass spectrometry Mass spectrometry (MS) analysis was acquired in an ESI-MS source, with a triple quadrupole, Quattro LC (Micromass-Waters), at atmospheric pressure ionization (API). For detecting the negative ions, the energy setup was: cone 25 V (low), 50 V (medium) and 100 V (high) and capillary 2.73 kV, and for positive ions was: cone 35 V (low), 63 V (medium), 85 V (high), and the capillary at 2.6 kV. Nitrogen was used as nebuliser and desolvation gas, the source block temperature was held at 80 ◦ C and desolvation gas at 200 ◦ C. For tandem-MS experiments, each precursor ion was selected by MS1 and fragmented by collision-induced dissociation (CID) with argon as collision gas. The CID-energies were 20 eV (low), 40 eV (medium) and 60–80 eV (high). The samples (200 ␮g/ml) were solubilized in MeOH–H2 O (1:1, v/v), or supplemented with 5 mM of HCl or NaCl. Offline analysis was performed by direct injection into the ESI-MS source with a syringe-infusion pump (KDScientific) at a constant flow rate of 10 ␮l min−1 . Online analysis was carried out by splitting the HPLC flux at the column exit, into ESI-MS. Data acquisition and processing were performed using MassLynx 3.5 software, and all scans were obtained in the continuum mode. The glycosides fragments were analyzed based on the nomenclature proposed by Domon and Costello [33], adapted to denote the observed product ions (Fig. 1). Considering that the fragmentation occurred mainly in the glycosidic linkages, those ions containing the aglycone moiety were designated as Yj , where j is the number of glycosidic residues remaining linked to aglycone. Y0 meaning that no sugar remained linked to the aglycone and Y* meaning the rearrangement of monosaccharide linked to the aglycone. Product ions containing only the carbohydrate moiety were designated as Bi , where i indicates the number of monosaccharide units, counting from the non-reducing end. Bx indicates the product ion from a monosaccharide residue linked to the aglycone. The charge of each ion is shown as superscripts (Fig. 1). Fig. 1. Fragmentation nomenclature for flavonoid glycosides. L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 103 Table 1 Flavonol glycosides detected in offline negative ESI-MS/MS MS1 MS2 [M−H] [Y0 −H] 417 433 447 463 579 593 593 609 625 625 739 755 771 771 871 887 901 284:285 300:301 284:285 300:301 300:301 284:285 300:301 300:301 300:301 316:317 284:285 300:301 300:301 316:317 284:285 300:301 284:285 − •− :Y0 − NL Main fraction 132a 132a 162a 162a 278b 308b 292b 308b 324b 308b 454c 454c 470c 454c 586d 586d 616d F2 F2, F3 F2, F3 F2, F3 F2, F3 F2, F3 F2, F3 F2, F3 F3, F4 F3, F4 F3, F4 F4, F5 F4, F5 F4, F5 F4, F5 F5 F5 R – 3.9 4.1 4.5 4.3 3.9 3.3 4.2 3.8 – 3.8 3.6 3.5 3.3 4.1 4.3 4.2 255 271 255 271 271 255 271 271 271 – 255 271 271 – 255 271 255 227 243 227 243 243 227 243 243 243 – 227 243 243 – 227 243 227 163 163 163 163 163 163 163 163 163 163 163 163 163 163 163 163 163 179 179 179 179 179 179 179 179 179 179 179 179 179 179 179 179 179 151 151 151 151 151 151 151 151 151 151 151 151 151 151 151 151 151 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 135 133 149 133 149 149 133 149 149 149 165 133 149 149 165 133 149 133 121 137 121 137 137 121 137 137 137 153 121 137 137 153 121 137 121 93 108 93 108 108 93 108 108 108 125 93 108 108 125 93 108 93 R: ratio between [Y0 −H]• − and Y0 − obtained in 40 eV CID-MS; NL: neutral losses—consistent with: a monosaccharide; b disaccharide; c trisaccharide; and d tetrasaccharide. 2.5. Derivatization and GC–MS analysis The samples (∼500 ␮g) were hydrolyzed in 1 M TFA (200 ␮l) at 100 ◦ C for 14 h, and then dried under a nitrogen stream. The resulting monosaccharides were reduced to alditols with NaBH4 (Sigma), and acetylated in 200 ␮l of pyridine and acetic anhydride (1:1, v/v) (Merck), at 100 ◦ C for 2 h. Ice was then added and the alditol acetates formed were extracted with CHCl3 (Merck), and analyzed by GC–MS on a capillary column DB-225MS (J&W-USA). The injector temperature was 250 ◦ C, with the column being initiated at 50 ◦ C, heated at 40 ◦ C min−1 , and held at 220 ◦ C for 25 min. For methylation analysis, the samples were dissolved in 200 ␮l DMSO (Merck), powdered NaOH (5 mg) and 200 ␮l CH3 I (Merck) [34]. The suspension was vigorously stirred for 30 min and left overnight at room temperature. The O-methylated derivatives were extracted with CHCl3 . The extract was washed with water several times, until neutral pH, and then it was dried under nitrogen stream. The resulting per-O-methylated compounds were hydrolyzed in 45% formic acid (v/v) at 100 ◦ C for 14 h. The samples were reduced using NaBD4 (Sigma) and acetylated in 200 ␮l of pyridine and acetic anhydride (1:1, v/v). The resulting partially methylated alditol acetates (PMAAs) were extracted in CHCl3 and analyzed by GC–MS (Varian-USA, model Saturn 2000R), using a capillary column DB-225MS. The injector temperature was 250 ◦ C, with the column being initiated at 50 ◦ C, heated at 40 ◦ C min−1 , and held at 210 ◦ C for 30 min. The EI-MS spectra were compared with those obtained from the library for O-glycosidic linkage analysis [35]. arising from isotopes of Cl− adducts, were observed. By increasing the cone voltage to 50 V, both ions were formed, at m/z 755 [M−H]− and at m/z 791 [M+Cl]− , with similar intensities, and with the cone energy at 100 V the main ion observed was the deprotonated form, at m/z 755. Each fraction was analyzed by CID-MS, and the resulting fragments showed the formation of the deprotonated regular aglycone ion [Y0 ]− , as well as the formation of the deprotonated radical aglycone ion ([Y0 −H]•− ) which is obtained by a hemolytic cleavage of the glycosidic bond, between the aglycone and the monosaccharide [26,36]. The main fragment ions observed for deprotonated radical 3. Results and discussion 3.1. Offline negative ESI-MS analysis Fractions were collected from the silica-gel column and those reactive to orcinol–H2 SO4 (F2–F5) were submitted to negative ESI detection, which provided mixtures of flavonol glycosides, according to the pseudo-molecular ions observed (Fig. 2A–D, Table 1). Fraction F5, which contained mainly compound with m/z 755 [M−H]− , was solubilized in MeOH–H2 O (1:1, v/v), and MeOH–H2 O with 5 mM HCl or 5 mM NaCl, and analyzed in the negative ESI mode using a capillary energy at 2.73 kV. It was observed that changes on the cone energy affected the relative abundances of resulting ions. Thus, at low cone energy (25 V) two main ions at m/z 791 and 793, Fig. 2. Offline negative ESI-MS, in scan mode, of fractions F2–F5. 104 L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 Fig. 3. Formation of radical aglycone ([Y0 −H]− • ) and regular aglycone (Y0 − ) ions in negative ESI mode (A), and negative CID-MS of flavonol triglycosides, showing the formation of radical from kaempferol, quercetin and myricetin, respectively (B–D). aglycones and regular deprotonated aglycone were m/z 284–285, consistent with kaempferol, and m/z 300–301, consistent with quercetin (Fig. 3A–C). These two aglycones had been previously found in M. ilicifolia leaves [21,22]. However, at least 2 precursor ions gave fragments at m/z 316 and 317, indicating that flavonol glycosides from M. ilicifolia can contain myricetin as aglycone (Fig. 3A and D). The formation of the [Y0 −H]•− ion is dependent on the glycosylation sites, and it is directly affected by the energy used in CID-MS. Thus, different flavonoid glycosides may form both deprotonated radical aglycone and deprotonated regular aglycone ions, with different ratios, providing useful information about the glycosylation sites [26]. However, no significant variation in the ratio between [Y0 −H]•− :Y0 − was observed, and both CID-energies used (20 and 40 eV) favored the formation of the radical aglycone. Although the glycosylation site could not be inferred only by negative CID Fig. 4. Influence of the cone energy on ionization of a flavonol triglycoside from F5. Positive ionization with cone energy at: (A) 35, (B) 63 and (C) 85 V. examination, Cuyckens and Claeys [26] observed that under certain energy conditions, the radical aglycone had been formed in high abundance, especially for flavonol 3-O-glycosides. Thus the pronounced [Y0 −H]•− ion observed in all CID-fragments (Fig. 3A–D) suggests a possible glycosylation site on 3-OH. The other compounds exhibited similar results (Table 1, Figs. S-1–S-5). In the negative ESI detection, the pseudo-molecular ions in MS1 , and the neutral losses (NL) observed in MS2 , showed that the flavonols present in F2–F5 are mainly linked to mono-, di-, tri-, and tetrasaccharides, respectively (Table 1). This separation according to molecular weight is due to increments of MeOH in the solvent used on silica-gel column fractionation, i.e. the higher the polarity of the solvent, the better its ability to remove the glycosides with higher degree of polymerization. 3.2. Offline positive ESI-MS analysis At low cone energy, the main ions observed were those corresponding to protonated compounds, whereas at high energies the sodiated compounds were predominant when the fractions were dissolved in MeOH:H2 O (1:1, v/v). This typical behavior was also tested with the fraction F5 dissolved in MeOH:H2 O (1:1, v/v) containing 5 mM of HCl or NaCl. With HCl, the ionization followed a similar behavior, alternating adducts with changes on the cone voltage. Setting the cone energy at 35 V, the main ion was at m/z 757 [M+H]+ , increasing the cone voltage to 63 V both species were observed, at m/z 757 and 779 [M+Na]+ , and finally at 85 V the main ion observed was the more stable form at m/z 779. In all cases the capillary energy was 2.6 V (Fig. 4A–C). As expected, the solution containing NaCl gave only Na+ adducts, with the main ion formed at m/z 779, even changing the cone voltage. L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 105 Fig. 5. (A) Positive CID-MS of protonated ions of the major flavonol triglycosides present in F4. (B) Precursor ion at m/z 741 [M+H]+ and (C) at m/z 757. 3.3. Tandem-MS analysis of protonated compounds The protonated ions were obtained using the cone energy at 35 V. The CID-fragmentation profiles fallowed the nomenclature proposed by Domon and Costello [33] by the formation of Yj + fragments, which were important to determine the sequence of monosaccharide units linked to flavonols. For example, the fraction F4, containing mainly the flavonol triglycosides with m/z 741 and 757 [M+H]+ , produced CIDfragments correspondent to the sequential monosaccharide losses (Fig. 5A–C). Thus, the precursor ion at m/z 741 gave rise to fragments Y2 + at m/z 595 [M−146]+ , Y1 + at m/z 449 [M−292]+ , and Y0 + at m/z 287 [M−454]+ , which is compatible with the structure previously found, kaempferol-3-O-˛-l-rhamnopyranosyl-(1 → 6)- Fig. 6. (A) CID-MS of flavonol triglycosides from F4 as Na+ adducts, showing the Bi -type fragments of precursor ion at m/z 763 (B) and 779 (C). Its worth noting that the formation of radical aglycone ([Y0 −H]+ • ) was intensified by increasing the CID energy. 106 L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 Table 2 Offline positive ESI-MS/MS detection of sodiated flavonol glycosides MS1 MS2 [M+Na] Y0 • + :Y0 + *R BX + B1 + (␣) B1 + ␤ B2 + (␣) B2 + ␤ B3 + (␣) B3 + ␤ B4 + 441 457 471 487 603 617 617 633 649 649 763 779 795 795 895 895 911 911 925 308:309 324:325 308:309 324:325 324:325 308:309 324:325 324:325 324:325 340:341 308:309 324:325 324:325 340:341 308:309 308:309 324:325 324:325 308:309 – 5.1 4.9 4.5 4.8 4.5 4.9 4.8 5.2 – 3.8 3.4 3.6 3.8 3.4 3.4 3.3 3.3 3.4 155 155 185 185 169 185 169 185 185 185 185 185 185 185 185 169 185 169 185 – – – – 155 169 169 169 185 169 169 169 169 169 169 169 169 169 163 – – – – – – – – – – 169 169 185 169 155 185 155 185 185 – – – – 301 331 315 331 347 331 331 331 331 331 331 315 331 315 331 – – – – – – – – – – 331 331 347 331 301 317 301 317 331 – – – – – – – – – – 477 477 493 477 477 447 477 447 477 – – – – – – – – – – – – – – 463 463 463 463 493 – – – – – – – – – – 609 609 609 609 639 + R: ratio between [Y0 +Na]• + and [Y0 +Na]+ , *CID energy 60–80 eV. O-[-˛-l-rhamnopyranosyl-(1 → 2)]-O-ˇ-d-galactopyranoside [19,20]. The precursor ion at m/z 757 [M+H]+ gave rise to fragments Y2 + at m/z 611 [M−146]+ , Y1 + at m/z 465 [M−292]+ , and Y0 + at m/z 303 [M−454]+ , compatible with the quercetin-3-O-˛-l-rhamnopyranosyl-(1 → 6)-O-[-˛-l-rhamnopyranosyl-(1 → 2)]-O-ˇ-d-galactopyranoside [20,21]. Additionally, both precursor ions had similar neutral losses corresponding to sugar moieties, confirming that both structures have similar oligosaccharides. It is worth noting that in both compounds, the fragment-ion Y* was formed, at m/z 433 (Fig. 5 B) and m/z 449 (Fig. 5C). Y* is an irregular ion that corresponds to the loss of the inner hexosyl residue, and a rearrangement with a rhamnose residue, usually observed at lower collision energies. Although the Y* ion may yield false monosaccharide sequence, with appropriate instrumentation, the ratio Y1 /Y* can provide useful information on the type of flavonoid, and can also be used to infer differentiation between neohesperidose and rutinose, two common disaccharides found attached to flavonoids [24]. 3.4. Tandem-MS analysis of sodiated compounds When a higher energy was applied to the cone (above 85 V), the formation of more stable ions from flavonoid glycosides was favored, which were found as Na+ adducts. Since the sodiated compounds are more stable, they need more CID energy to produce fragments, rather than protonated compounds. Interestingly, the sodiated flavonol glycosides gave rise to fragment-ions different from those obtained in the protonated form, yielding mainly the Bi + -type fragments, evidencing the glycan moieties, and confirming those neutral losses observed in negative detection. Typically, the Bi + fragments are formed as a consequence of a breakdown on glycosidic linkages, referred to as A1 -type cleavage, that generates an oxonium ion, due to the formation of a third bond to the oxygen from the glycosidic ring. On the other hand, in the formation of Yj + fragments, the positive charge is retained in the portion of the molecule that contains the aglycone group, releasing the adjacent sugar residues as neutral species, with a double bond between C-1 and C-2 (see Fig. 1), being referred to as ˇ-cleavage [33,37]. Interestingly, in our examinations these neutral carbohydrate-species, produced by ˇ-cleavage, were observed as sodiated ions. For example, fraction F4 gave rise to 2 main ions at m/z 763 [M+Na]+ and m/z 779 [M+Na]+ , both producing similar CID-fragments, characteristic of glycosyl moieties at m/z 477 [B3 +Na]+ , confirming that these flavonols are attached by a trisaccharide group and not by monosaccharides at different sites on the aglycone. It were also observed fragments at m/z 331 [B2 +Na]+ , and others at m/z 169 [B1 +Na]+ , and 185 [Bx +Na]+ (Fig. 6A–C, see Figs. S-1–S-8 for other compounds). Table 3 GC–MS analysis of flavonol glycosides as PMAAs derivatives PMAAs 2,3-Me2 -Ara 2,3,4-Me3 -Ara 2,4-Me2 -Rha 2,3,4-Me3 -Rha 3,4-Me2 -Glc 2,3,4-Me3 -Glc 3,4,6-Me3 -Glc 2,3,4,6-Me4 -Glc 3,4-Me2 -Gal 2,3,4-Me3 -Gal 3,4,6-Me3 -Gal 2,3,4,6-Me4 -Gal Peaks 2 3 4 5 6 7 8 9 11 12 13 14 15 17/19 18/20 23/25 27 – 1 – – – – – – – – – – – – – – – – – – – – – 1 – – – – – – – 1 – – – – – – – – – – – – – – – 1 – – – – – – – 1 – – – – 1 – – 1 – – – – – – – – 1 – – 1 – – – – – – – – – – – 2 – – 1 – – – 1 – – – – 1 – – – – – – 1 – – – – 1 – – – – – 1 – – – – – 1 – – 1 – – – – – – – – 1 – 1 – – – – – – – – – – – 1 1 4 – 1 1 – – – – 2 – – – – 1 – – – – – – 2 1 – – – – – – – – 1 1 1 – – – – 1 – – – – – 1 1 – – – 1 1 – – – The values are indicated as molar ratios. The peaks 1, 10, 16, 21, 22, 24, 26, 28 were not detected in GC–MS analysis. L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 107 Fig. 7. HPLC-UV at  360 nm of fractions F3 (A1), F4 (B1), F5 (B2), and HPLC–ESI-MS of selected ions from F3 (A2–A8) and from F5 (B3–B8). As observed for deprotonated compounds on CID-MS, the Na+ adducts also produced the fragments of regular aglycone and radical aglycone ions, and the ratio of [Y0 −H]•+ /Y0 + was dependent of the energies used. Cuyckens and Claeys [26] discussed its application to determine the glycosylation sites of flavonoid monoglycosides, employing the ratio of [Y0 −H]•+ /Y0 + ions, observed at high CID-energy. In our examination, the collision energy at 40 eV produced similar ratios between [Y0 −H]•+ and Y0 + . On the other hand, using CID at 60–80 eV the formation of radical aglycone was ∼3–4-fold higher than the regular aglycone ion. The higher [Y0 –H]•+ to Y0 + ratio observed (Fig. 6 B and C) might be an evidence of flavonoid 3-O-glycosides, since it is distinct from ratios observed for flavonoid 7-O-glycosides, that exhibit a ratio value of ∼1. However, only the ratio between [Y0 −H]+• and Y0 + , did not provide enough information to differentiate between flavonoid 3O-glycosides and flavonoid 4′ -O-glycosides, but characteristically flavonoid 4′ -O-glycosides must form fragments correspondent of Bring containing the carbohydrate moieties [26], which were absent in our spectra. These results strongly suggested that the flavonols studied herein are 3-O-glycosilated. The CID-MS from other sodiated compounds are shown in Table 2. 3.5. Online HPLC–ESI-MS analysis For the online analysis, the cone energy at 50 V in the ESIMS, and the acidic solvent used in the HPLC separation, favored the protonation of compounds, observed in the positive ESI detection. Furthermore, under these conditions, the ionization promoted the in-source fragmentation, providing similar fragments to those obtained in offline CID-MS from protonated precursor ions. The insource fragmentation was useful for monosaccharide sequencing, however it could lead to a misinterpretation of the chromatograms. 108 L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 Table 4 Online positive ESI-MS detection of protonated flavonol glycosides Peak Rt MS1 [M+H]+ MS2 Y0 1a 2a 3 4 5 6 7a 8a 9 10a 11 12a 13a 14 15a 16a 17 18a 19 20a 21a 22a 23 24a 25 26a 27 28a 14.34 14.90 15.39 17.04 12.63 13.19 14.06 14.48 12.91 13.50 11.09 11.40 12.14 12.59 10.00 11.23 15.15 15.50 13.68 13.96 12.24 12.52 15.04 15.53 13.68 14.10 14.06 14.80 435 435 449 449 465 465 581 581 595 595 611 611 611 611 627 627 741 741 757 757 773 773 873 873 889 889 903 903 + 303 303 287 287 303 303 303 303 287 303 303 303 303 303 303 319 287 287 303 303 303 319 287 287 303 303 287 – Compound identification Y1 + – – – – – – 435 435 449 449 465 465 465 465 465 481 449 449 465 465 465 481 449 433 465 449 449 – Y2 + ␣ – – – – – – – – – – – – – – – – 595 595 611 611 611 627 595 565 611 581 595 – Y2 + ␤ – – – – – – – – – – – – – – – – 595 595 611 611 627 627 595 577 611 595 595 – Y3 + ␣ – – – – – – – – – – – – – – – – – – – – – – 741 711b 757 727 741b – Y3 + ␤ – – – – – – – – – – – – – – – – – – – – – – 727 727 743 743 757 – Ara-(1 → )-Quer Araf-(1 → 3)-Quer Galp-(1 → 3)-Kaemp Glcp-(1 → 3)-Kaemp Galp-(1 → 3)-Quer (hyperoside) Glcp-(1 → 3)-Quer (isoquercetrin) Rhap-(1 → 4/5)-Ara-(1 → 3)-Quer Rhap-(1 → 4/5)-Ara-(1 → 3)-Quer Rhap-(1 → 2)-Galp/Glcp-(1 → 3)-Kaemp Rhap(1 → )-Rha-(1 → 3)-Quer Rhap-(1 → 2)-Galp-(1 → 3)-Quer Rhap-(1 → 6)-Galp-(1 → 3)-Quer Rhap-(1 → 2)-Glcp-(1 → 3)-Quer Rhap-(1 → 6)-Glcp-(1 → 3)-Quer (rutin) Glcp-(1 → 2/6)-Gal/Glc-(1 → 3)-Quer Rha-(1 → )-Hex-(1 → 3)-Myr Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Kaemp Rhap-(1 → 6)-[Rhap-(1 → 2)]-Glclp-(1 → 3)-Kaemp Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Quer Rhap-(1 → 6)-[Rhap-(1 → 2)]-Glcp-(1 → 3)-Quer Hex-(1 → )-[Rha-(1 → )]-Hex-(1 → )-Quer Rhap-(1 → )-[Rhap-(1 → )]-Hex-(1 → 3)-Myr Arap-(1 → 3)-Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Kaemp Hex-(1 → )-Ara-(1 → )-[Rha-(1 → )]-Rha-(1 → 3)-Kaemp Arap-(1 → 3)-Rhap-(1 → 6)-[Rhap-(1 → 2)]-Galp-(1 → 3)-Quer Hex-(1 → )-Ara-(1 → )-[Rha-(1 → )]-Rha-(1 → 3)-Quer Glcp-(1 → 3)-Rhap-(1 → 2)-[Rha-(1 → 6)]-Gal-(1 → 3)-Kaemp – Arap: arabinopyranose, Araf: arabinofuranose, Rhap: Rhamnopyranose, Hex: hexose (galactose or glucose), Galp: galactopyranose, Glcp: glucopyranose, Quer: quercetin, Kaemp: kaempferol and Myr: myricetin. a Novel structures described in leaves of Maytnus ilicifolia. b Expected but not observed fragments. Thus, the online analysis was also carried out in the negative ESI detection, which allowed identifying the peaks without generating in-source fragmentation. Two gradients were developed in order to obtain the good peak shape, resolution, and short time running. Considering its complexity, the fraction F3 needed a different gradient from that used in fractions F4 and F5. The gradient I provided the best resolution for fraction F3, which contained the majority of flavonol mono-, and diglycosides. Moreover, scanning the individual pseudo-molecular ions on the chromatograms, it was observed that of at least two peaks emerged for each ion scanned (Fig. 7A1–A8), this being observed due to the presence of isomers. Accordingly, in a previous study Tiberti et al. [22] observed some flavonol glycoside isomers in M. ilicifolia. Considering that the glycosylation site is at the 3-OH position for all flavonol glycosides, the isomerism could only be in the type of sugar, or in the type of interglycosidic linkages. In fact, both possibilities were confirmed by methylation/GC–MS analysis (Table 3). Thus, it was confirmed that the most of flavonols contain galactose or glucose linked to aglycone. An example was the ion at m/z 609 [M−H]− , that showed 4 peaks on the chromatogram. The methylation analysis showed the presence of glucose and galactose, which were branched by rhamnose in 1 → 6 or 1 → 2 O-glycosidic linkages. On the other hand, in the case of peaks 1–2 and 7–8, the isomerism seems to be due to ring configuration of arabinose residues, that appears to have both, pyranosidic and furanosidic forms. Fractions F4 and F5, containing mainly flavonol tri-, and tetraglycosides, were analyzed using the gradient II, and despite of their simplicity when compared to F3, all exhibit similar behavior when scanned for individual pseudo-molecular ions, also showing the presence of two peaks for each ion scanned (Fig. 7B1–B8). The majority of flavonol triglycosides had isomerism consisting in the presence of galactose or glucose linked to aglycone, whereas in the flavonol tetraglycosides the isomerism appeared as result of differences on monosaccharide sequence, as observed in MS2 (Table 4). Due to the complexity of samples, some peak overlapping occurred, making the methylation analysis especially laborious. However the data were well consistent with the monosaccharide sequences obtained in tandem-MS analysis. The HPLC–ESI-MS results and a structure description are summarized in Table 4. 4. Conclusion A wide variety of flavonol glycosides has been shown to be produced by M. ilicifolia. The monosaccharide composition showed the presence of arabinose (Ara), rhamnose (Rha), galactose (Gal) and glucose (Glc) in all fractions studied. Furthermore, the flavonols can also contain, mainly, glucose and galactose linked to aglycone, and the glycosidic chain can be further elongated, with rhamnose, arabinose and glucose. The online analysis proved to be a powerful technique for the identification of several diastereoisomers, allowing the scan of specific and individual ions throughout the chromatograms. However, the chromatograms resulted in high complex mixtures, which would hardly be resolved without the previous fractionation on silica-gel. The compound ionization on ESI source was directly affected by the cone energies, and consequently the H+ and Na+ adducts obtained interfered with the fragmentation profiles. Thus, following the Domon and Costello nomenclature [33], the protonated ions gave rise to Yj + fragments, while the sodiated ions gave rise to Bi + fragments, which allowed the appropriate monosaccharide sequencing. Moreover, the sodiated ions at high CID energy produced abundant radical aglycone ions, which were used to identify glycosylation sites as being in 3-OH position, in accordance to pre- L.M. de Souza et al. / J. Chromatogr. A 1207 (2008) 101–109 vious investigations [19–22]. The aglycone moieties were identified by their CID-MS partners in negative and positive ESI modes, compared with literature [7,38–41]. They were confirmed as kaempferol and quercetin. However it was also found myricetin-glycosides, which had not been described in M. ilicifolia to date. 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