mass spectroscopy of flavonoid

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fragmentation pattern of flavonoids - mass spectroscopy

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APPLICATION OF MASS SPECTROSCOPY IN STRUCTURE ELUCIDATION OF FLAVONOIDS Submitted to: Submitted by: Dr. Hardik Bodiwala Satyender Kumar 1

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Flavonoids (or bioflavonoids) (from the Latin word flavus meaning yellow, their colour in nature), are a class of plant secondary metabolites. 2

The flavonoids are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain. The skeleton above, can be represented as the C6 - C3 - C6 system:

The flavonoids are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain. The skeleton above, can be represented as the C 6 - C 3 - C 6 system 3

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A bicyclic heterocycle in which a benzene ring is fused to one of dihydro-pyran. The chemical structure of flavonoids are based on a C 15 skeleton with a Chromane ring bearing a second aromatic ring B in position 2, 3 or 4. Chromane ring 4

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Most of these (flavanones, flavones, flavonols, and anthocyanins) bear ring B in position 2 of the heterocyclic ring. In isoflavonoids, ring B occupies position 3. 5

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Flavonoids are often hydroxylated in positions 3, 5, 7, 3’, 4’ and/or 5’ Frequently, one or more of these hydroxyl groups are methylated, acetylated, prenylated or sulphated In plants, flavonoids are often present as O - or C- glycosides O bonding in flavonoids occurs far more frequently than C bonding The O -glycosides have sugar substituent's bound to a hydroxyl group of the aglycone, usually located at position 3 or 7, whereas the C -glycosides have sugar groups bound to a carbon of the aglycone, usually 6-C or 8-C The most common carbohydrates are rhamnose, glucose, galactose and arabinose Flavonoid -diglycosides are also frequently found 7

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LC-MS (LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY ) In flavonoid analysis: LC-MS : state - of - the - art detection technique in LC-MS Tandem MS : identification of unknowns, (MS/MS or MS n ) better selectivity, wider ranging information 8

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LC-MS (LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY ) In LC-MS of flavonoids : APCI and ESI are used almost ESI is more frequently used in flavonoid analysis NI mode provide more sensitivity for both ESI and APCI Other techniques which are next to ESI and APCI: EI : Electrochemical ionization CI : Chemical ionization FAB : Fast atom bombardment MALDI : Molecular assisted laser desorption ionization MALDI-TOF MS : MALDI-Time of Flight MS The composition of the LC (gradient) eluent, its pH and the nature of buffer composition can have distinct influence Most commonly used additives : Acetic acid, Formic acid, Ammonium-acetate, Ammonium- formate 9

FRAGMENTATION PATHWAYS FOR FLAVONOIDS:

FRAGMENTATION PATHWAYS FOR FLAVONOIDS The most important fragmentation Retro Dials-alder reaction of flavonoids reaction (RDA) RDA fragments are especially important for the structural characterization of aglycone and the aglycone part of flavonoids conjugates. RDA occur in six membered cyclic structures containing a double bond and involves the relocalization of three pairs of electrons in the cyclic ring. Net result is cleavage of σ bonds and formation of two π bonds 10

FRAGMENTATION PATHWAYS FOR FLAVONOIDS:

FRAGMENTATION PATHWAYS FOR FLAVONOIDS E.g.: C- ring cleavage of Apigenin and Luteolin can be used to determine the number and nature of the substituent's on A and B rings Apegenin (R2=H) Luteolin (R2=OH) 11 m/z-153 m/z-135

FRAGMENTATION PATHWAYS FOR FLAVONOIDS:

Fragmentation pathways are largely independent of the ionization mode (ESI and APCI) and the type of instrument (triple quadruple or ion trap) Significant difference do occur as regards the relative abundance of the various fragments ions FRAGMENTATION PATHWAYS FOR FLAVONOIDS 12

RDA - FRAGMENTATION:

RDA - FRAGMENTATION In the PI mode, the ions that are formed after the cleavage of two bonds in the C-ring, are denoted i,j A + and i,j B + with ion A containing the A-ring and ion B, the B-ring. When the NI mode is used, the ions are denoted i,j A - and i,j B - , respectively. Ions derived from the fragment ions by the loss of a fragment X, are denoted [ i,j A ± −X] and [ i,j B ± −X], respectively. The indices i and j represent the C-ring bonds which are broken. NI = Negative ionization PI = Positive ionization 13

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Figure1: The structures of the flavonoids: Quercetin (molecular weight = 302); Myricetin (molecular weight = 318); Apigenin (molecular weight = 270); Luteolin (molecular weight = 286); Narigenin (molecular weight = 272); Hesperetin (molecular weight = 302); Catechin (molecular weight = 290) ; Epigallocatechin (molecular weight = 306). 14

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Figure2: Fragmentation pathways for flavonoids by cleavage of c- ring bonds; (A) 1 and 3, (A2) 0 and 4; (B) 0 and 2, (B2) 1 and 4 and (C1) 0 and 3, (C2) 1 and 2, (C3) 1 and 4, (C4) 2 and 4 15

Table1: Fragments ions for the selected flavonoids classes in the PI mode (1):

Table1: Fragments ions for the selected flavonoids classes in the PI mode (1) 16

FRAGMENTATION IN PI MODE:

FRAGMENTATION IN PI MODE RDA cleavage, which generates 1,3 A + and 1,3 B + fragments ions, is the most important fragmentation pathways for flavonones , flavones an flavonols Cleavage of the 1,3 bonds : 1,3 A + ion as the most prominent ion with the: Flavonones : Naringenin Flavones : Luteolin, Apegenin Flavonols : Kaempferol 1,3 B + ion is also observed even proposed as a diagnostic ion for flavones 17

FRAGMENTATION IN PI MODE:

FRAGMENTATION IN PI MODE Cleavage of the 0,2 bonds: 0,2 A + ion may be used to distinguish flavonols since it does not occur in the spectra of other classes of flavonoids 0,2 B + ion with relative abundances ranging from 1 to 90 % as a diagnostic ion for flavones e.g.: Kaempferol , Quercetin, Myricetin, Isorhamnetin, Apigenin, Luteolin, Acacetin 18

FRAGMENTATION IN PI MODE:

FRAGMENTATION IN PI MODE The 0,4 C-ring cleavage: Only protonated flavones fragment via this pathway under low energy FAB-CID conditions 0,4 B + ions can be diagnostic for flavones aglycone after 0,4 B + ion after loss of water FAB: Fast atom bombardment CID: Collision induced dissociation 19

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Fragments common to flavonoids that arising from: H 2 O (18 Da ), CO (28 Da ), [M+H-42] + C 2 H 2 O (42 Da ), and [M+H-46] + successive loss of H 2 O and CO (46 Da ), do not show in the spectra of isoflavonoids. [M+H-42] + (loss of 2×CO) indicate presence of isoflavones [M+H-15] +• formed by loss of methyl radical, is predominantly in O -methylated isoflavones, flavones and flavonols 20

FRAGMENTATION IN NI MODE:

FRAGMENTATION IN NI MODE Cleavage of 1,3 bonds : The most important fragmentation Pathways in NI mode as well as in PI mode 1,3 A - and 1,3 B - ions are reported for many flavonoids 1,3 A - ions are most abundantly with 1,3 B - ions Cleavage of 0,3 bonds: 1,3 A - and 0,3 B - fragments were observed only for the isoflavones daidzein and genistein in a study However, the same fragment was observed for a flavone 21

FRAGMENTATION IN NI MODE:

FRAGMENTATION IN NI MODE Cleavage of 0,4 bonds: 0,4 A - and 0,4 B - fragments are observed for at relatively low abundance for at least some members of all classes of flavonoids In a study of isoflavones and flavones, fragmentation of isoflavones mainly resulted in 0,4 B - ions whereas for flavones 0,4 A - ions was prominent (1) 22

FRAGMENTATION IN NI MODE:

FRAGMENTATION IN NI MODE Cleavage of 1,2 bonds: 1,2 A - the main ion of the main ion of flavonols quercetin ( m/z 171) But in a study no peak was observed in the spectra of two isoflavones, formononetin and biochenin A (1) Cleavage of 1,4 bonds: Proposed to explain the formation of m/z 149 [ 1,4 B+H] - in the mass spectrum of apigenin formation of m/z 149 ion is either 1,4 B - or 1,3 B - m/z 149 ion has also found in spectrum of luteolin . However, is probably not equivalent to apigenin , due to hydroxyl substituent on the B-ring higher mass than epigenin 23

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES:

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES Flavonoids commonly occur as flavonoid- O- glycosides; 3- and 7- hydroxyl groups are the typical glycosylation sites Flavonoids-diglycosides are also found in nature with rutinose ( rhamnosyl –(1→6)-glucose) and neohesperidose ( rhamnosyl -(1→2)-glucose) being the most common sugar moities Flavonoid- C- glycosides in which the sugar is directly linked to aglyone by a C-C bond comprise flavonoid-mono and di -C-glycosides and O-C -glycosides To date, C -glycosylation has only been found at the C-6 and C-8 positions of the flavonoid aglycone 24

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES:

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES Examples: Naringenin-7- O- neohesperidose (A) and naringenin-7- O- glucoside (B) Y represents: Diglycosides Y 1 represents: fragment contain aglycone part with loss of one glycoside Y 0 represents: fragment contain aglycone with loss of two glycosides B 1 represents: glycose fragments B 0 represents: glycose fragments 25

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES:

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES In Fegure 3 Y 1 ion is formed after loss of a rhamnose unit (146 Da ), and the Y 0 ion after further loss of a glucose unit (162 Da ) The interglycosydic linking can be determined on the basis of the ratio Y 1 ⁻/ Y 0 ⁻ When [M+H-146]⁺ (Y 0 ⁻)˃ [M+H-(146+162)]⁺ (Y 1 ⁻) indicates a 1→6 linkage in Naringenin-7 -O -rutinose While, [M+H-146]⁺˂[M+H-(146+162)]⁺ indicates a 1→2 linkage in Naringenin-7- O- neohesperidose The type of O - glycosidic linkage determined by Y* ion which corresponds to [M+H-162]⁺ ion i.e. removal of a glucose. This ion only observed in 7- O -glycosides not in 3- O- glycosides Naringenin-7- O -rutinoside 26

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES:

FRAGMENTATION PATHWAY IN FLAVONOID-(DI)-GLYCOSIDES MS/MS of flavonoid-( di )-glycosides is useful to: Differentiate b/w 1→2 and 1→6 glucose linking types of diglycosides To distinguish b/w Naringenin-7- O- neohesperidose and naringenin-7- O glycoside Figure 3 : MS/MS spectra obtained for [M+H] + ions of Naringenin-7- O- neohesperidose (A) and naringenin-7-O-rutinside (B), using LC-ESI(+)-MS/MS 27

Characterization of flavonoids aglycones by Negative Ion Chip-Based Nanospray Tandem Mass Spectrometry :

Characterization of flavonoids aglycones by Negative Ion Chip-Based Nanospray Tandem Mass Spectrometry Instrumentation : QStar-XL quadruple-time-of-flight Instrument hybrid (Applied Biosystems, Warrington, UK) Analyst QS version 1.1 software Instrument control, (Applied Biosystems, Warrington, UK) data acquisition and data processing Nanomate HD automatic chip-based nanospray system (Advion Biosciences, Norwich, UK) 28

Characterization of flavonoids aglycones by Negative Ion Chip-Based Nanospray Tandem Mass Spectrometry (2):

Characterization of flavonoids aglycones by Negative Ion Chip-Based Nanospray Tandem Mass Spectrometry (2) Nanospray ionization is an improvement over traditional ESI for the analysis of low volume, low concentration samples In ‘chip-based’ nanospray the analytic solution is sprayed from a conductive pipette tip pressed against the rear of the chip using a small gas pressure and low voltage to create the spray Each nanospray needle in the array is used only once to avoid contamination 29

Characterization of flavonoids aglycones by Negative Ion Chip-Based Nanospray Tandem Mass Spectrometry :

Characterization of flavonoids aglycones by Negative Ion Chip-Based Nanospray Tandem Mass Spectrometry In CID a collision energy of around -35eV was determined to result in the ‘best’ product ion spectra The main neutral molecules lost from the [M-H]⁻ ions consisted of a combination of H 2 O, CO, CO 2 and/or H 2 CCO A detailed analysis of all the spectra indicates that a combination of a specific order of neutral eliminations occurs along with the presence of a series of diagnostic low-mass product ions for each of the flavonoid classes analyzed All of the flavonoids (except the flavanols ) have the previously described ions at m/z 151 and 107, whereas the flavanols catechin and epigallocatechin (with no oxidation at carbon 3, but following a similar ring contraction mechanism) result in the product ions at m/z 137 and 109. Also, all of the flavonoids except the flavones have an ion at m/z 125, the flavones have an ion at m/z 121. 30

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Quercetin (molecular weight = 302) Myricetin (molecular weight = 318) (PI) m/z 301) (PI) m/z 317) PI : Precursor ion Flavonols: Characteristic neutral losses: -28,-44, -18 (H 2 O, CO, CO 2 ) Characterize product ions: 151, 125, 107 31

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Apigenin (molecular weight =270) Luteolin (molecular weight =286) (PI) m/z 269) (PI) m/z 285) Flavones: Characteristic neutral losses: -28,-44, -44, -28, -42 (CO, CO 2 , H 2 CCO) Characterize product ions: 151, 121, 107 33

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Narigenin (molecular weight = 272) Hesperetin (molecular weight = 302) (PI) m/z 271) (PI) m/z 301) Flavanones : Characteristic neutral losses: -18,-44, -44, -18, -42 (H 2 O, CO 2 , H 2 CCO) Characterize product ions: 151, 125, 107 34

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Catechin (molecular weight = 290) Epigallocatechin (molecular weight=306) (PI) m/z 289) (PI) m/z 305) Flavanols : Characteristic neutral losses: -18,-44, -44, -18, -42 (H 2 O, CO 2 , H 2 CCO) Characterize product ions: 137, 125, 109 35

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Nanospray allows the easy distuishing of methoxylated flavonoids with identical molecular mass E.g., Hesperitin : has a methoxyl group substitution at the aromatic ring and showed elimination of a methyl redical (15 Da ) After increasing the collision energy elimination of 16 due to CH4 (16 Da ) involving the methoxyl group or ortho -hydroxyl group Both of these mechanisms, when taken together are very useful for di -substituted flavonoids 36

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MSn Analysis with fast polarity switching (3):

MS n Analysis with fast polarity switching (3) Early LC/MS systems could typically operate in only one mode During, analysis many compounds respond better to a particular ionization mode either positive or negative mode Analytical challenge Need an advance and fast instruments The faster a mass instrument can switch polarities during a particular run More MS and MS n scans that can be performed over a chromatographic peak, the better the data will be and the more sensitive the analysis. 38

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Figure: Comparison of positive, negative and alternating modes of detection (3) 39

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Figure: MS n analysis using positive/negative time segments versus alternating positive/negative scans (3) TIC = Total ion current 40

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Figure: Positive ion MS 4 spectra for progesterone (3) 41

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References : Rijke D.E., Out P., Niessen W.M.A., Ariese F., Gooijer C., Brinkman U.A.T. Analytical separation and detection meyhods for flavonoids. Journal of Chromatography A. 2006, 1112: 31-63. Gates P.J., Lopes N.P. Characterization of flavonoid aglycones by negative ion spray chip-based Tandem Mass Spectrometry. Miller C. MS n analysis with fast polarity switching in the agilent 1100 series LC/MSD trap SL. Application notes, Agilent Technologies. 2002. 42

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Chalcone Flavone Flavonol Flavanone Anthocyanins Isoflavanoids Most of these (flavanones, flavones, flavonols, and anthocyanins) bear ring B in position 2 of the heterocyclic ring. In isoflavonoids, ring B occupies position 3. 44

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Group Skeleton Examples Description Functional groups 3-hydroxyl 2,3-dihydro Flavone 2-phenylchromen-4-one ✗ ✗ Luteolin , Apigenin , Tangeritin Flavonol or 3-hydroxyflavone 3-hydroxy-2-phenylchromen-4-one ✓ ✗ Quercetin , Kaempferol , Myricetin , Fisetin , Isorhamnetin , Pachypodol , Rhamnazin Flavanone 2,3-dihydro-2-phenylchromen-4-one ✗ ✓ Hesperetin , Naringenin , Eriodictyol , Homoeriodictyol Flavanonol or 3-Hydroxyflavanone or 2,3-dihydroflavonol 3-hydroxy-2,3-dihydro-2-phenylchromen-4-one ✓ ✓ Taxifolin (or Dihydroquercetin ), Dihydrokaempferol 45

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