Research article Received: 14 March 2014
Revised: 30 June 2014
Accepted: 25 September 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3516
MALDI-MS of flavonoids: a systematic investigation of ionization and in-source dissociation mechanisms Denise B. Silva and Norberto P. Lopes* Matrix assisted laser desorption ionization (MALDI) is a technique widely employed in the analysis of proteins and peptides, and nowadays it has also been applied to small molecules. There is little significant information regarding the in-source dissociation processes on MALDI for natural products. Twenty-six flavonoids (flavanones, flavones and flavonols) were analyzed by MALDI using different methods (with different matrices) and without matrix to comprehend the in-source reactions and establish good analysis methods for these compounds. Depending on the class, structure and the laser intensity applied, methoxylated flavonoid aglycones can eliminate methyl radicals (˙CH3) in the source, such as flavonols, but lithium 2,4-dihydroxybenzoate matrix suppresses the ˙CH3 eliminations and retro-Diels–Alder cleavages in the source. All of the flavonoid O-glycosides evaluated herein eliminated the sugar in source, even in the presence of the matrix, and its product radical ions ([M-H-sugar] ˙) were observed in the negative mode. The flavone C-glycosides suffered intense dissociation, which was reduced by the addition of a matrix and the application of low laser intensity, mainly in the negative mode. Depending on the hydroxyl substituents, the [M-H-H] ˙ ion was observed with variable relative intensity in the spectra. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: MALDI; flavone; flavonol; dissociation; flavanones
Introduction
182
Flavonoids are a group of substances with variable phenolic structures that are widely distributed in plants. These substances represent one of the most important classes of secondary metabolites present in the human diet as a result of their essential biological benefits.[1] More than 4000 flavonoids have been identified, existing as free aglycones, O-glycosides and C-glycosides in plants. In addition to the different physiological functions in plants, such as antioxidant effects and ultraviolet tissue protection, flavonoids can also be an important stimulus for industrial application. Several biological properties have been reported for flavonoids, including antioxidant,[2] antidepressant,[3] anti-inflammatory, cardiovascular and anticancer activities.[4] In a recent review, a compilation of information from epidemiological and meta-analysis studies of flavonoids showed that an inverse relationship exists between the consumption of flavonoid-rich diets and the development of many aging-associated diseases, including cancers, cardiovascular disease, diabetes, osteoporosis and neurodegenerative disorders.[5] The mechanism of action for flavonoids is mainly a result of its capacity to inhibit oxidative stress, but other pathways also contribute to the final effects.[6] In addition, Perez-Viscaino and coworkers brought attention to the flavonoid paradox, in which flavonoids, like quercetin, showed systemic biological effects but were not found in plasma after oral administration.[7] The authors suggested that quercetin is extensively metabolized into conjugated metabolites, which circulate in the plasma in this form, and through a reversible reaction can deliver the free aglycones to the tissues, exhibiting the biological effect.[7] The analysis of flavonoids in plasma samples, in addition to other biological matrix-containing polyphenol compounds, are mainly
J. Mass Spectrom. 2015, 50, 182–190
performed using liquid chromatography hyphenated to mass spectrometry (MS).[8] There are also some approaches that do not utilize prior chromatographic separation, such as direct infusion mass spectrometry, which includes electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI).[9,10] MALDI-MS is an extremely powerful technique for metabolite analysis studies because of its high sensitivity, high-speed data acquisition (≈60 s per sample) and reduced influence of contamination upon ionization.[11] Ionization by MALDI can be explained by thermodynamic and physicochemical processes, for which there are several different models that attempt to describe it.[12] Basically, the ionization process has two steps: (1) primary ion formation (matrix) and (2) secondary ion formation in the MALDI plume (analyte). The analyte is mixed with a matrix (small compounds and normally with high conjugation) to produce a ‘solid solution’, which is then subjected to a laser beam. The matrix molecules absorb laser photons resulting in ionization of the matrix molecules (primary ion formation) and desorption, producing a cloud (plume). Subsequently, the charge is transferred to the analyte molecules via proton or cation transfer from the photo-activated matrix molecules, leading to the formation of the characteristic ions ([M + H]+, [M-H] and M+˙).[12–14] The analysis of small compounds (25% for analyses without matrix) (Fig. 3C and Supporting Information Fig. 2S). The flavonols quercetin (16) and morin (19), with two hydroxyls in the B ring, however, suffered higher fragmentation compared to 18 and 17 (Supporting Information Fig. 2S), which could be related to the absorbed energy from laser. Some product ions were observed, such as the ions resulting from RDA fragmentation (the most intense fragment) in the C ring mainly involving a 1,3 scission and losses of CH2O and CO (Supporting Information Figs. 1S and 2S); both of these were already reported from ESI-MS/MS.[25] Thus, the product ion at m/z 151 was visualized from both compounds in the mass spectra acquired in the negative mode (Fig. 8), but LiDHB matrix suppressed this fragmentation by RDA, as well as the eliminations of CO, CH2O and others (Supporting Information Figs. 105S and 106S). Another important result is related to the ions formed by hydrogen radical (˙H) elimination in source. The flavonoids with no hydroxyl substituents in the B ring showed the [M-H-H] ˙ ion with very low relative intensity in the mass spectra (negative mode). The compound 16 exhibited this ion with higher intensity than 18, showing the highest relative intensities among flavonols analyzed (Fig. 5) and in analyses performed by high laser intensity (Fig. 3C). These observations may be useful in identifying the structures of flavonoids by MALDI; in addition, they can suggest the extent of conjugation and perhaps the antioxidant capacity of the flavonoids.
D. B. Silva and N. P. Lopes The flavonols 3-O-methylquercetin (20) (Fig. 9) and 7-Omethylgalangin (21) eliminated methyl radical in the source, resulting in the product ions at m/z 300 and 268, which were more intensive for 20 (Fig. 3A), and probably it can be related to absorbed energy from laser, as 20 has two additional OH in the B ring (catechol). These fragment ions [M-H-˙CH3] ˙ exhibited higher relative intensities in the spectra acquired with higher laser intensities (Fig. 3A). In addition, 20 also eliminated CH4 in the source, which has already been reported for flavonoids by ESI-MS/ MS (CID), but they were only observed at higher collision energies and competed with ˙CH3 elimination for hesperetin.[32] Additionally, it is important to highlight that LiDHB matrix avoided ˙CH3 and CH4 elimination in the source from methylated flavonols, as observed in the spectra illustrated in Fig. 9. In the negative ionization mode, all of the flavonol O-glycosides produced the radical product ion [M-H-sugar] ˙ (the highest relative intensity) and non-radical product in source formed from homolytic and heterolytic cleavages, respectively, and their relative intensities were more intense in the spectra, increasing laser
188
Figure 9. Mass spectra of 3-O-methylquercetin 20 acquired without matrix in the negative mode (A) and with the matrices CHCA (B) and LiDHB (C) both in positive mode.
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intensity (Fig. 3D). The fragment ions from RDA reactions were also observed in the spectra, which were inhibited by LiDHB matrix. Moreover, all O-glycosides did not show ions formed by the hydrogen radical loss (˙H) from deprotonated ions, as was observed for flavonol aglycones. In the positive mode, it was possible to confirm, beyond the loss of sugar, other dissociations in the source, such as CO, but the use of a matrix protected these compounds and reduced the in-source fragmentation in all the analyses. Not even the LiDHB matrix prevented the cleavage of the ether bond between the sugars and the aglycones, so this in-source dissociation cannot be avoided. Thus, flavonol aglycones showed lower in-source dissociation, even in the presence of a matrix. The [M-H-H] ˙ ion can be easily observed in the negative mode, but its relative intensity is proportional to the number of hydroxyl substituents present in the substances, mainly in relation to free 4′-OH, as well as to the laser intensity. The methoxylated flavonol showed fragmentation in the source, including ˙CH3 elimination, but the LiDHB matrix prevented these dissociations and the RDA reactions observed. Finally, the use of a matrix in the negative and positive modes reduced the fragmentation of flavonol O-glycosides, but it did not completely avoid sugar elimination. The same results were observed for all flavonol O-glycosides evaluated where the dissociations did not depend on the sugar. In addition, glycosylated flavonoids with more than one sugar showed only cationizated ions, mainly with sodium [M + Na]+, so the protonated ions [M + H]+ were not observed in their mass spectra acquired in the positive mode. After the addition of sodium, the cationizated ions [M + Na]+ revealed higher relative intensity when compared with the samples without sodium addition. This strategy can be applied to increase the intensity of these ions, reducing the dissociation in the source from these flavonoids. On the basis of these data, it is possible to observe that flavonoids can react in a MALDI source giving in-source dissociation products by two competitive pathways starting with homolytic and heterolytic bond cleavage. Natural products are currently analyzed by ESI-MS, which was developed for the generation of protonated ([M + H]+) and cationized species (i.e., [M + Na]+) in the positive mode and deprotonated molecules ([M-H] ) in the negative mode. A review of the radical ion formation in ESI compiles several examples showing the formation of radical ions (molecular ions) by removal (M+˙) or addition (M ˙) of one or more electrons via redox reactions in the ESI source, which may compete with acid/base reactions and coordination with ionic species. The extent to which these processes occur depends on a combination of factors, such as proton affinity and redox potential, as well as experimental parameters.[33] A comparative analysis of homologous antioxidant molecules, such as carotenoids, xanthophylls and retinoids (which have similar proton affinities), shows that the increase of conjugation extension is an important factor for the loss of an electron in the ESI source by radical stabilization. However, compounds such as β-carotene (that have no heteroatoms) can exhibit sequential in-source homolytic cleavage, affording several fragmentation products and resulting in complex spectra.[23,34] In this case, reduction of the energies allowed for the observation of the molecular ion, a phenomenon that can also be observed in the MALDI source.[23] Other antioxidant agents like quinones can show radical ion formation by removal (M+˙) or addition (M ˙) of an electron, in addition to cross insource reactions with acetonitrile, and furnish unexpected
Copyright © 2015 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2015, 50, 182–190
In-source dissociations of flavonoids in MALDI species.[35] Flavonoids also have low oxidation potential, but the same phenomena observed in ESI were not observed, and independent of the ESI source conditions, the ionization occurred as the expected acid/base or cationization mechanisms.[8]
Conclusions As we report in the present paper, under well-defined laser energies, flavonoids can exhibit easily radical in-source reactions as opposed to the ESI in-source behavior. We propose that the absorption of the laser energy by the conjugated system increases the internal ion energy to a level that may initiate dissociation reactions. All of the present data suggest that the development of a MALDI protocol for the analysis of each flavonoid or of classes with similar structures is necessary to define the behavior of the in-source decomposition reactions. In summary, aglycones of flavone, flavonone and flavonols can have radical in-source eliminations that can be significant for flavonols and may induce spectral interpretation problems. Additionally, the three classes exhibit RDA dissociation, but in very low amounts, and flavanones resulted in the formation of the most stable ions, confirming the influence of the conjugation system for the energy absorption and increase of the internal ion energy. So methoxylated flavanones did not lose methyl radical in source, differently from that observed for flavonols, as these dissociations can be avoided when LiDHB is used as matrix, an uncommon matrix. The radical hydrogen elimination was observed in negative mode, mainly for some aglycones, which is directly correlated to the number of hydroxyl groups on the B ring; however, it is a low-intensity fragment ion. All O-glycosylated flavonoids suffered intense dissociation, producing fragment ions by the loss of sugars in source, which were reduced after the addition of a matrix. The in-source dissociation of C-glycosides was also reduced after the addition of a matrix, but in the same way it cannot be annulled. Finally, the glycosides are more prone to exhibiting in-source dissociations and must be verified in more detail before beginning any MALDI analysis. Acknowledgements The authors thank FAPESP (Process: 2009/54098-6, 2012/18031-7) and CGEN/CNPq.
References
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Copyright © 2015 John Wiley & Sons, Ltd.
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189
[1] L. P. Taylor, E. Grotewold. Flavonoids as developmental regulators. Curr. Opin. Plant Biol. 2005, 8, 317. [2] S. Burda, W. Oleszek. Antioxidant and antiradical activities of flavonoids. J. Agric. Food Chem. 2001, 49, 2774. [3] V. Butterweck, G. Jürgenliemk, A. Nahrstedt, H. Winterhoff. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test. Planta Med. 2000, 66, 3. [4] O. Benavente-Garcia, J. Castillo. Update on uses and properties of Citrus flavonoids: new findings in anticancer, cardiovascular, and antiinflammatory activity. J. Agric. Food Chem. 2008, 56, 6185. [5] V. P. A. Babu, D. Liu, E. R. Gilbert. Recent advances in understanding the anti-diabetic actions of dietary flavonoids. J. Nutr. Biochem. 2013, 24, 1777. [6] A. Borah, R. Paul, S. Choudhury, A. Choudhury, B. Bhuyan, A. Talukdar, M. D. Choudhury, K. P. Mohanakumar. Neuroprotective Potential of silymarin against CNS disorders: insight into the pathways and molecular mechanisms of action. CNS Neurosci. Ther. 2013, 19, 847.
[7] F. Perez-Vizcaino, J. Duarte, C. Santos-Buelga. The flavonoid paradox: conjugation and deconjugation as key steps for the biological activity of flavonoids. J. Sci. Food Agric. 2012, 92, 1822. [8] D. Steinmann, M. Ganzera. Recent advances on HPLC/MS in medicinal plant analysis. J. Pharm. Biomed. Anal. 2011, 55, 744. [9] K. Dettmer, P. A. Aronov, B. D. Hammock. Mass spectrometry-based metabolomics. Mass Spectrom. Rev. 2007, 26, 51. [10] L. Lin, Q. Yu, X. Yan, W. Hang, J. Zheng, J. Xing, B. Huang. Direct infusion mass spectrometry or liquid chromatography mass spectrometry for human metabonomics? A serum metabonomic study of kidney cancer. Analyst 2010, 135, 2970. [11] D. Miura, Y. Fujimura, H. Tachibana, H. Wariishi. Highly sensitive matrixassisted laser desorption ionization-mass spectrometry for highthroughput metabolic profiling. Anal. Chem. 2010, 82, 498. [12] R. Zenobi, R. Knochenmuss. Ion formation in MALDI mass spectrometry. Mass Spectrom. Rev. 1998, 17, 337. [13] M. Karas, U. Bahr, U. Giessmann. Matrix-assisted laser desorption ionization mass spectrometry. Mass Spectrom. Rev. 1991, 10, 335. [14] U. Bahr, M. Karas, F. Hillenkamp. Analysis of biopolymers by matrixassisted laser desorption/ionization (MALDI) mass spectrometry. Fresenius J. Anal. Chem. 1994, 348, 783. [15] L. H. Cohen, A. I. Gusev. Small molecule analysis by MALDI mass spectrometry. Anal. Bioanal. Chem. 2002, 373, 571. [16] S. Moco, B. Schneider, J. Vervoort. Plant micrometabolomics: the analysis of endogenous metabolites present in a plant cell or tissue. J. Proteome Res. 2009, 8, 1694. [17] Y. Li, B. Shrestha, A. Vertes. Atmospheric pressure infrared MALDI imaging mass spectrometry for plant metabolomics. Anal. Chem. 2008, 80, 407. [18] D. B. Silva, I. C. C. Turati, D. R. Gouveia, M. Ernst, S. P. Teixeira, N. P. Lopes. Mass spectrometry of flavonoid vicenin-2, based sunlight barriers in Lychnophora species. Sci. Rep. 2014, 4, 1, DOI: 10.1038/srep04309. [19] P. M. Angel, R. M. Caprioli. Matrix-assisted laser desorption ionization imaging mass spectrometry: in situ molecular mapping. Biochemistry 2013, 52, 3818. [20] M. S. Soares, D. F. Silva, M. R. Forim, M. F. G. F. Silva, J. B. Fernandes, P. C. Vieira, D. B. Silva, N. P. Lopes, S. A. Carvalho, A. A. Souza, M. A. Machado. Phytochemistry 2014, submitted. [21] X. Wang, J. Han, A. Chou, J. Yang, J. Pan, C. H. Borchers. Hydroxyflavones as a new family of matrices for MALDI tissue imaging. Anal. Chem. 2013, 85, 7566. [22] R. E. March, H. Li, O. Belgacem, D. Papanastasiou. High-energy and lowenergy collision-induced dissociation of protonated flavonoids generated by MALDI and by electrospray ionization. Int. J. Mass Spectrom. 2007, 262, 51. [23] T. Guaratini, R. L. Vessecchi, F. C. Lavarda, P. M. B. G. M. Campos, Z. Naal, P. J. Gates, N. P. Lopes. New chemical evidence for the ability to generate radical molecular ions of polyenes from ESI and HR-MALDI mass spectrometry. Analyst 2004, 129, 1223. [24] J. Cvacka, A. Svatos. Matrix-assisted laser desorption/ionization analysis of lipids and high molecular weight hydrocarbons with lithium 2,5dihydroxybenzoate matrix. Rapid Commun. Mass Spectrom. 2003, 17, 2203. [25] N. Fabre, I. Rustan, E. Hoffmann, J. Quetin-Leclercq. Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 707. [26] P. J. A. Madeira, M. H. Florêncio. Flavonoid–matrix cluster ions in MALDI mass spectrometry. J. Mass Spectrom. 2009, 44, 1105. [27] R. Vessecchi, G. J. Zocolo, D. R. Gouvea, F. Hübner, B. Cramer, M. R. R. Marchi, H. U. Humpf, N. P. Lopes. Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies. Rapid Commun. Mass Spectrom. 2011, 25, 2020. [28] A. Arora, M. G. Nair, G. M. Strasburg. Structure–activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radic. Biol. Med. 1998, 24, 1355. [29] C. A. Rice-Evans, N. J. Miller, G. Paganga. Structure–antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933. [30] E. Hvattum, D. Ekeberg. Study of the collision-induced radical cleavage of flavonoid glycosides using negative electrospray ionization tandem quadrupole mass spectrometry. J. Mass Spectrom. 2003, 38, 43.
D. B. Silva and N. P. Lopes [31] R. E. March, E. G. Lewars, C. J. Stadey, X. S. Miao, X. Zhao, C. D. Metcalfe. A comparison of flavonoid glycosides by electrospray tandem mass spectrometry. Int. J. Mass Spectrom. 2006, 248, 61. [32] P. J. Gates, N. P. Lopes. Characterisation of flavonoid aglycones by negative ion chip-based nanospray tandem mass spectrometry. Int. J. Anal. Chem. 2012, 2012, 1. [33] R.Vessecchi,A.E.M.Crotti,T.Guaratini,P.Colepicolo,S.E.Galembeck,N.P.Lopes. Radical ion generation processes of organic compounds in electrospray ionizationmassspectrometry.Mini-Rev.Org.Chem.2007,4,75. [34] T. Guaratini, R. Vessecchi, E. Pinto, P. Colepicolo, N. P. Lopes. Balance of xanthophylls molecular and protonated molecular ions in electrospray ionization. J. Mass Spectrom. 2005, 40, 963.
[35] R. Vessecchi, Z. Naal, J. N. C. Lopes, S. E. Galembeck, N. P. Lopes. Generation of naphthoquinone radical anions by electrospray ionization: solution, gas-phase, and computational chemistry studies. J. Phys. Chem. A 2011, 115, 5453.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
190 wileyonlinelibrary.com/journal/jms
Copyright © 2015 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2015, 50, 182–190
Revised: 30 June 2014
Accepted: 25 September 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3516
MALDI-MS of flavonoids: a systematic investigation of ionization and in-source dissociation mechanisms Denise B. Silva and Norberto P. Lopes* Matrix assisted laser desorption ionization (MALDI) is a technique widely employed in the analysis of proteins and peptides, and nowadays it has also been applied to small molecules. There is little significant information regarding the in-source dissociation processes on MALDI for natural products. Twenty-six flavonoids (flavanones, flavones and flavonols) were analyzed by MALDI using different methods (with different matrices) and without matrix to comprehend the in-source reactions and establish good analysis methods for these compounds. Depending on the class, structure and the laser intensity applied, methoxylated flavonoid aglycones can eliminate methyl radicals (˙CH3) in the source, such as flavonols, but lithium 2,4-dihydroxybenzoate matrix suppresses the ˙CH3 eliminations and retro-Diels–Alder cleavages in the source. All of the flavonoid O-glycosides evaluated herein eliminated the sugar in source, even in the presence of the matrix, and its product radical ions ([M-H-sugar] ˙) were observed in the negative mode. The flavone C-glycosides suffered intense dissociation, which was reduced by the addition of a matrix and the application of low laser intensity, mainly in the negative mode. Depending on the hydroxyl substituents, the [M-H-H] ˙ ion was observed with variable relative intensity in the spectra. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: MALDI; flavone; flavonol; dissociation; flavanones
Introduction
182
Flavonoids are a group of substances with variable phenolic structures that are widely distributed in plants. These substances represent one of the most important classes of secondary metabolites present in the human diet as a result of their essential biological benefits.[1] More than 4000 flavonoids have been identified, existing as free aglycones, O-glycosides and C-glycosides in plants. In addition to the different physiological functions in plants, such as antioxidant effects and ultraviolet tissue protection, flavonoids can also be an important stimulus for industrial application. Several biological properties have been reported for flavonoids, including antioxidant,[2] antidepressant,[3] anti-inflammatory, cardiovascular and anticancer activities.[4] In a recent review, a compilation of information from epidemiological and meta-analysis studies of flavonoids showed that an inverse relationship exists between the consumption of flavonoid-rich diets and the development of many aging-associated diseases, including cancers, cardiovascular disease, diabetes, osteoporosis and neurodegenerative disorders.[5] The mechanism of action for flavonoids is mainly a result of its capacity to inhibit oxidative stress, but other pathways also contribute to the final effects.[6] In addition, Perez-Viscaino and coworkers brought attention to the flavonoid paradox, in which flavonoids, like quercetin, showed systemic biological effects but were not found in plasma after oral administration.[7] The authors suggested that quercetin is extensively metabolized into conjugated metabolites, which circulate in the plasma in this form, and through a reversible reaction can deliver the free aglycones to the tissues, exhibiting the biological effect.[7] The analysis of flavonoids in plasma samples, in addition to other biological matrix-containing polyphenol compounds, are mainly
J. Mass Spectrom. 2015, 50, 182–190
performed using liquid chromatography hyphenated to mass spectrometry (MS).[8] There are also some approaches that do not utilize prior chromatographic separation, such as direct infusion mass spectrometry, which includes electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI).[9,10] MALDI-MS is an extremely powerful technique for metabolite analysis studies because of its high sensitivity, high-speed data acquisition (≈60 s per sample) and reduced influence of contamination upon ionization.[11] Ionization by MALDI can be explained by thermodynamic and physicochemical processes, for which there are several different models that attempt to describe it.[12] Basically, the ionization process has two steps: (1) primary ion formation (matrix) and (2) secondary ion formation in the MALDI plume (analyte). The analyte is mixed with a matrix (small compounds and normally with high conjugation) to produce a ‘solid solution’, which is then subjected to a laser beam. The matrix molecules absorb laser photons resulting in ionization of the matrix molecules (primary ion formation) and desorption, producing a cloud (plume). Subsequently, the charge is transferred to the analyte molecules via proton or cation transfer from the photo-activated matrix molecules, leading to the formation of the characteristic ions ([M + H]+, [M-H] and M+˙).[12–14] The analysis of small compounds (25% for analyses without matrix) (Fig. 3C and Supporting Information Fig. 2S). The flavonols quercetin (16) and morin (19), with two hydroxyls in the B ring, however, suffered higher fragmentation compared to 18 and 17 (Supporting Information Fig. 2S), which could be related to the absorbed energy from laser. Some product ions were observed, such as the ions resulting from RDA fragmentation (the most intense fragment) in the C ring mainly involving a 1,3 scission and losses of CH2O and CO (Supporting Information Figs. 1S and 2S); both of these were already reported from ESI-MS/MS.[25] Thus, the product ion at m/z 151 was visualized from both compounds in the mass spectra acquired in the negative mode (Fig. 8), but LiDHB matrix suppressed this fragmentation by RDA, as well as the eliminations of CO, CH2O and others (Supporting Information Figs. 105S and 106S). Another important result is related to the ions formed by hydrogen radical (˙H) elimination in source. The flavonoids with no hydroxyl substituents in the B ring showed the [M-H-H] ˙ ion with very low relative intensity in the mass spectra (negative mode). The compound 16 exhibited this ion with higher intensity than 18, showing the highest relative intensities among flavonols analyzed (Fig. 5) and in analyses performed by high laser intensity (Fig. 3C). These observations may be useful in identifying the structures of flavonoids by MALDI; in addition, they can suggest the extent of conjugation and perhaps the antioxidant capacity of the flavonoids.
D. B. Silva and N. P. Lopes The flavonols 3-O-methylquercetin (20) (Fig. 9) and 7-Omethylgalangin (21) eliminated methyl radical in the source, resulting in the product ions at m/z 300 and 268, which were more intensive for 20 (Fig. 3A), and probably it can be related to absorbed energy from laser, as 20 has two additional OH in the B ring (catechol). These fragment ions [M-H-˙CH3] ˙ exhibited higher relative intensities in the spectra acquired with higher laser intensities (Fig. 3A). In addition, 20 also eliminated CH4 in the source, which has already been reported for flavonoids by ESI-MS/ MS (CID), but they were only observed at higher collision energies and competed with ˙CH3 elimination for hesperetin.[32] Additionally, it is important to highlight that LiDHB matrix avoided ˙CH3 and CH4 elimination in the source from methylated flavonols, as observed in the spectra illustrated in Fig. 9. In the negative ionization mode, all of the flavonol O-glycosides produced the radical product ion [M-H-sugar] ˙ (the highest relative intensity) and non-radical product in source formed from homolytic and heterolytic cleavages, respectively, and their relative intensities were more intense in the spectra, increasing laser
188
Figure 9. Mass spectra of 3-O-methylquercetin 20 acquired without matrix in the negative mode (A) and with the matrices CHCA (B) and LiDHB (C) both in positive mode.
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intensity (Fig. 3D). The fragment ions from RDA reactions were also observed in the spectra, which were inhibited by LiDHB matrix. Moreover, all O-glycosides did not show ions formed by the hydrogen radical loss (˙H) from deprotonated ions, as was observed for flavonol aglycones. In the positive mode, it was possible to confirm, beyond the loss of sugar, other dissociations in the source, such as CO, but the use of a matrix protected these compounds and reduced the in-source fragmentation in all the analyses. Not even the LiDHB matrix prevented the cleavage of the ether bond between the sugars and the aglycones, so this in-source dissociation cannot be avoided. Thus, flavonol aglycones showed lower in-source dissociation, even in the presence of a matrix. The [M-H-H] ˙ ion can be easily observed in the negative mode, but its relative intensity is proportional to the number of hydroxyl substituents present in the substances, mainly in relation to free 4′-OH, as well as to the laser intensity. The methoxylated flavonol showed fragmentation in the source, including ˙CH3 elimination, but the LiDHB matrix prevented these dissociations and the RDA reactions observed. Finally, the use of a matrix in the negative and positive modes reduced the fragmentation of flavonol O-glycosides, but it did not completely avoid sugar elimination. The same results were observed for all flavonol O-glycosides evaluated where the dissociations did not depend on the sugar. In addition, glycosylated flavonoids with more than one sugar showed only cationizated ions, mainly with sodium [M + Na]+, so the protonated ions [M + H]+ were not observed in their mass spectra acquired in the positive mode. After the addition of sodium, the cationizated ions [M + Na]+ revealed higher relative intensity when compared with the samples without sodium addition. This strategy can be applied to increase the intensity of these ions, reducing the dissociation in the source from these flavonoids. On the basis of these data, it is possible to observe that flavonoids can react in a MALDI source giving in-source dissociation products by two competitive pathways starting with homolytic and heterolytic bond cleavage. Natural products are currently analyzed by ESI-MS, which was developed for the generation of protonated ([M + H]+) and cationized species (i.e., [M + Na]+) in the positive mode and deprotonated molecules ([M-H] ) in the negative mode. A review of the radical ion formation in ESI compiles several examples showing the formation of radical ions (molecular ions) by removal (M+˙) or addition (M ˙) of one or more electrons via redox reactions in the ESI source, which may compete with acid/base reactions and coordination with ionic species. The extent to which these processes occur depends on a combination of factors, such as proton affinity and redox potential, as well as experimental parameters.[33] A comparative analysis of homologous antioxidant molecules, such as carotenoids, xanthophylls and retinoids (which have similar proton affinities), shows that the increase of conjugation extension is an important factor for the loss of an electron in the ESI source by radical stabilization. However, compounds such as β-carotene (that have no heteroatoms) can exhibit sequential in-source homolytic cleavage, affording several fragmentation products and resulting in complex spectra.[23,34] In this case, reduction of the energies allowed for the observation of the molecular ion, a phenomenon that can also be observed in the MALDI source.[23] Other antioxidant agents like quinones can show radical ion formation by removal (M+˙) or addition (M ˙) of an electron, in addition to cross insource reactions with acetonitrile, and furnish unexpected
Copyright © 2015 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2015, 50, 182–190
In-source dissociations of flavonoids in MALDI species.[35] Flavonoids also have low oxidation potential, but the same phenomena observed in ESI were not observed, and independent of the ESI source conditions, the ionization occurred as the expected acid/base or cationization mechanisms.[8]
Conclusions As we report in the present paper, under well-defined laser energies, flavonoids can exhibit easily radical in-source reactions as opposed to the ESI in-source behavior. We propose that the absorption of the laser energy by the conjugated system increases the internal ion energy to a level that may initiate dissociation reactions. All of the present data suggest that the development of a MALDI protocol for the analysis of each flavonoid or of classes with similar structures is necessary to define the behavior of the in-source decomposition reactions. In summary, aglycones of flavone, flavonone and flavonols can have radical in-source eliminations that can be significant for flavonols and may induce spectral interpretation problems. Additionally, the three classes exhibit RDA dissociation, but in very low amounts, and flavanones resulted in the formation of the most stable ions, confirming the influence of the conjugation system for the energy absorption and increase of the internal ion energy. So methoxylated flavanones did not lose methyl radical in source, differently from that observed for flavonols, as these dissociations can be avoided when LiDHB is used as matrix, an uncommon matrix. The radical hydrogen elimination was observed in negative mode, mainly for some aglycones, which is directly correlated to the number of hydroxyl groups on the B ring; however, it is a low-intensity fragment ion. All O-glycosylated flavonoids suffered intense dissociation, producing fragment ions by the loss of sugars in source, which were reduced after the addition of a matrix. The in-source dissociation of C-glycosides was also reduced after the addition of a matrix, but in the same way it cannot be annulled. Finally, the glycosides are more prone to exhibiting in-source dissociations and must be verified in more detail before beginning any MALDI analysis. Acknowledgements The authors thank FAPESP (Process: 2009/54098-6, 2012/18031-7) and CGEN/CNPq.
References
J. Mass Spectrom. 2015, 50, 182–190
Copyright © 2015 John Wiley & Sons, Ltd.
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[1] L. P. Taylor, E. Grotewold. Flavonoids as developmental regulators. Curr. Opin. Plant Biol. 2005, 8, 317. [2] S. Burda, W. Oleszek. Antioxidant and antiradical activities of flavonoids. J. Agric. Food Chem. 2001, 49, 2774. [3] V. Butterweck, G. Jürgenliemk, A. Nahrstedt, H. Winterhoff. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test. Planta Med. 2000, 66, 3. [4] O. Benavente-Garcia, J. Castillo. Update on uses and properties of Citrus flavonoids: new findings in anticancer, cardiovascular, and antiinflammatory activity. J. Agric. Food Chem. 2008, 56, 6185. [5] V. P. A. Babu, D. Liu, E. R. Gilbert. Recent advances in understanding the anti-diabetic actions of dietary flavonoids. J. Nutr. Biochem. 2013, 24, 1777. [6] A. Borah, R. Paul, S. Choudhury, A. Choudhury, B. Bhuyan, A. Talukdar, M. D. Choudhury, K. P. Mohanakumar. Neuroprotective Potential of silymarin against CNS disorders: insight into the pathways and molecular mechanisms of action. CNS Neurosci. Ther. 2013, 19, 847.
[7] F. Perez-Vizcaino, J. Duarte, C. Santos-Buelga. The flavonoid paradox: conjugation and deconjugation as key steps for the biological activity of flavonoids. J. Sci. Food Agric. 2012, 92, 1822. [8] D. Steinmann, M. Ganzera. Recent advances on HPLC/MS in medicinal plant analysis. J. Pharm. Biomed. Anal. 2011, 55, 744. [9] K. Dettmer, P. A. Aronov, B. D. Hammock. Mass spectrometry-based metabolomics. Mass Spectrom. Rev. 2007, 26, 51. [10] L. Lin, Q. Yu, X. Yan, W. Hang, J. Zheng, J. Xing, B. Huang. Direct infusion mass spectrometry or liquid chromatography mass spectrometry for human metabonomics? A serum metabonomic study of kidney cancer. Analyst 2010, 135, 2970. [11] D. Miura, Y. Fujimura, H. Tachibana, H. Wariishi. Highly sensitive matrixassisted laser desorption ionization-mass spectrometry for highthroughput metabolic profiling. Anal. Chem. 2010, 82, 498. [12] R. Zenobi, R. Knochenmuss. Ion formation in MALDI mass spectrometry. Mass Spectrom. Rev. 1998, 17, 337. [13] M. Karas, U. Bahr, U. Giessmann. Matrix-assisted laser desorption ionization mass spectrometry. Mass Spectrom. Rev. 1991, 10, 335. [14] U. Bahr, M. Karas, F. Hillenkamp. Analysis of biopolymers by matrixassisted laser desorption/ionization (MALDI) mass spectrometry. Fresenius J. Anal. Chem. 1994, 348, 783. [15] L. H. Cohen, A. I. Gusev. Small molecule analysis by MALDI mass spectrometry. Anal. Bioanal. Chem. 2002, 373, 571. [16] S. Moco, B. Schneider, J. Vervoort. Plant micrometabolomics: the analysis of endogenous metabolites present in a plant cell or tissue. J. Proteome Res. 2009, 8, 1694. [17] Y. Li, B. Shrestha, A. Vertes. Atmospheric pressure infrared MALDI imaging mass spectrometry for plant metabolomics. Anal. Chem. 2008, 80, 407. [18] D. B. Silva, I. C. C. Turati, D. R. Gouveia, M. Ernst, S. P. Teixeira, N. P. Lopes. Mass spectrometry of flavonoid vicenin-2, based sunlight barriers in Lychnophora species. Sci. Rep. 2014, 4, 1, DOI: 10.1038/srep04309. [19] P. M. Angel, R. M. Caprioli. Matrix-assisted laser desorption ionization imaging mass spectrometry: in situ molecular mapping. Biochemistry 2013, 52, 3818. [20] M. S. Soares, D. F. Silva, M. R. Forim, M. F. G. F. Silva, J. B. Fernandes, P. C. Vieira, D. B. Silva, N. P. Lopes, S. A. Carvalho, A. A. Souza, M. A. Machado. Phytochemistry 2014, submitted. [21] X. Wang, J. Han, A. Chou, J. Yang, J. Pan, C. H. Borchers. Hydroxyflavones as a new family of matrices for MALDI tissue imaging. Anal. Chem. 2013, 85, 7566. [22] R. E. March, H. Li, O. Belgacem, D. Papanastasiou. High-energy and lowenergy collision-induced dissociation of protonated flavonoids generated by MALDI and by electrospray ionization. Int. J. Mass Spectrom. 2007, 262, 51. [23] T. Guaratini, R. L. Vessecchi, F. C. Lavarda, P. M. B. G. M. Campos, Z. Naal, P. J. Gates, N. P. Lopes. New chemical evidence for the ability to generate radical molecular ions of polyenes from ESI and HR-MALDI mass spectrometry. Analyst 2004, 129, 1223. [24] J. Cvacka, A. Svatos. Matrix-assisted laser desorption/ionization analysis of lipids and high molecular weight hydrocarbons with lithium 2,5dihydroxybenzoate matrix. Rapid Commun. Mass Spectrom. 2003, 17, 2203. [25] N. Fabre, I. Rustan, E. Hoffmann, J. Quetin-Leclercq. Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 707. [26] P. J. A. Madeira, M. H. Florêncio. Flavonoid–matrix cluster ions in MALDI mass spectrometry. J. Mass Spectrom. 2009, 44, 1105. [27] R. Vessecchi, G. J. Zocolo, D. R. Gouvea, F. Hübner, B. Cramer, M. R. R. Marchi, H. U. Humpf, N. P. Lopes. Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies. Rapid Commun. Mass Spectrom. 2011, 25, 2020. [28] A. Arora, M. G. Nair, G. M. Strasburg. Structure–activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radic. Biol. Med. 1998, 24, 1355. [29] C. A. Rice-Evans, N. J. Miller, G. Paganga. Structure–antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20, 933. [30] E. Hvattum, D. Ekeberg. Study of the collision-induced radical cleavage of flavonoid glycosides using negative electrospray ionization tandem quadrupole mass spectrometry. J. Mass Spectrom. 2003, 38, 43.
D. B. Silva and N. P. Lopes [31] R. E. March, E. G. Lewars, C. J. Stadey, X. S. Miao, X. Zhao, C. D. Metcalfe. A comparison of flavonoid glycosides by electrospray tandem mass spectrometry. Int. J. Mass Spectrom. 2006, 248, 61. [32] P. J. Gates, N. P. Lopes. Characterisation of flavonoid aglycones by negative ion chip-based nanospray tandem mass spectrometry. Int. J. Anal. Chem. 2012, 2012, 1. [33] R.Vessecchi,A.E.M.Crotti,T.Guaratini,P.Colepicolo,S.E.Galembeck,N.P.Lopes. Radical ion generation processes of organic compounds in electrospray ionizationmassspectrometry.Mini-Rev.Org.Chem.2007,4,75. [34] T. Guaratini, R. Vessecchi, E. Pinto, P. Colepicolo, N. P. Lopes. Balance of xanthophylls molecular and protonated molecular ions in electrospray ionization. J. Mass Spectrom. 2005, 40, 963.
[35] R. Vessecchi, Z. Naal, J. N. C. Lopes, S. E. Galembeck, N. P. Lopes. Generation of naphthoquinone radical anions by electrospray ionization: solution, gas-phase, and computational chemistry studies. J. Phys. Chem. A 2011, 115, 5453.
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