Polyphenolic Compounds in ops and Analytical Methods: A Survey regon State University Mass Spectrometry Laboratory
op il Polyphenols Flavonol Glycosides R C 2 R=, kaem pferol-3- -(6"--m alonylglucoside) R=, quercitin-3- -(6"--m alonylglucoside) Me Prenylfavonoids xanthohumol Me dehydrocycloxanthohumol Condensed Tannins (Proanthocyanidins) P ro cyan id in 3
Analytical Methods for Flavonol Glycosides Flavonol Glycosides are highly soluble in aqueous systems Extraction from hops or pellets by aqueous homogenization Direct analysis of aqueous extracts by PLC or LC-MS Sugar residues on aglycones result in shorter retention times on C-18 RP columns Larger molecules have shorter retention times on C-18 columns Negative ion electrospray mass spectrometry (ESI-MS) is most sensitive Postive ion ESI-MS is structurally most informative Tandem mass spectrometry (MS/MS) yields information of aglycone and sugar units eer samples analyzed directly without prior concentration or purification R 1 R 2 R 1 = R 2 = : Kaempferol MW =287 R 1 =, R 2 = : Quercetin MW = 303
Tandem Mass Spectrometry (MS/MS) Product Ion Scanning Select ion (M+) + in MS-1 and scan MS-2 for products Parent Ion Scanning Transmit all ions in MS-1 and set MS-2 to monitor for product ion (m/z 287 or 303 for flavonol glycosides)
Tandem Mass Spectrometry of Flavonol Glycosides + Product Ions (m/z 287 and 303) arise from [M+]+ (m/z 741 and 757) and fragment ions from loss of 1, 2 and 3 sugar units CID of [M+]+ (m/z 611) produces Product fragment ions from loss of one (m/z 465) and two (m/z 303) sugar units Select m/z 303 in MS-2 Ions from m/z 611 Select m/z 287in MS-2
Parent Ion Scan of Quercetin-3-rutinoside (Rutin) m/z 611 3 C -308-146 m/z 303 + MW = 162 m/z 465
Specific detection of quercetin and kaempferol glycosides by MS/MS M K = 773, 611, 465, 303 C= 757, 611, 465, 303 F= 611, 465, 303 K= 697, 551, 303 M= 551, 465, 303 C F LN E= 741, 595, 449, 287 Simultaneous Parent Ion Scans for quercetin and kaempferol L= 449, 287 N= 535, 287 E Parent ion scans of chromatographically separated aqueous allertauer hop extract Flavonol Glycosides eer
Flavonol Glycosides in Commercial eer Samples eer 1 eer 2 eer 3 eer 4 eer 5
Chalcones in ops R 3 R 2 R 4 R 1 Me Xanthohumol (0.1-1%) R 1 * R 2 R 3 * R 4 3 C 3 C C 3 3 4 5 6 7 8 Pn Pn Gn Pn Pn Me Me Pn Pn Me Me Me *Pn = Prenyl, Gn = Geranyl At least 13 chalcones related to xanthohumol have been identified in hops
Chalcone-Flavonone Conversion 8-Prenylnarigenin is the most Potent phytoestrogen isolated Me Me Xanthohumol Isoxanthohumol Cyclization catalyzed under basic and acidic conditions, oth chalcones and flavonones t ½ = 30 min in boiling brew. have been isolated from hops, but Desmethylxanthohumol only chalcones are biosynthesized 8 6 8-Prenylnaringenin Xanthohumol and other preylflavonoids have received considerable attention as chemopreventive agents 6 8 Conversion to prenylnarigenins complete within 15 min in boiling brew. 6-Prenylnaringenin
Isolation and Identification of Prenylflavonoids Prenylflavonoids more lipophilic than flavonol glycosides Immersion of hop cones in acetone or chloroform yields phenolics and resins Lipids and resins precipitated with hot methanol Soluble fraction purified by Sephadex L-20 chromatography Phenolic component resolution by silica gel and RP-18 chromatography UV/Vis spectroscopy: chalcones (370 nm) flavonones (290 nm) Mass spectrometry: electron ionization, chemical ionization, FA-MS, APCI-MS/MS Chalcone/flavonone interconversion during mass spectral analysis [M + ] + and [M - ] - and fragmentation by CID using argon to show ring substitutions 2-D NMR spectroscopy of proton and carbon-13 resonances shows regiochemistry
Mass Spectrometry Positive Ion APCI-CAD (Ar, N gas with collision energy = 11 V) A Me A Me -C 4 8 Me RDA C Me + Negative Ion APCI-CAD Chalcone- - - - - A RDA C Me
Long-range -C correlations by MC-NMR 1" 3' A 6' 1' 2' C 3 1 4
Multiple Reaction Monitoring for Prenyl flavonoids Positive Ion APCI-CAD (collision energy = 11 30 V ) Me A m/z 355 -C 4 8 Me A Multiple reaction monitoring 1. oth analyzers static 2. First analyzer set to transmit precursor 3. Second analyzer records product Me C m/z 179 RDA n-u
LC-MRM for Prenylflavonoids in eer isoxanthohumol xanthohumol 2, 4-dihydroxychalcone 8-prenylnarigenin 6-prenylnarigenin No. 6 = 8-geranylnarigenin No. 7 = 6-geranylnarigenin
Prenylflavonoids in eer eer (µg/l) b Xanthohumol Isoxanthohumol 8-Prenylnaringenin US major brand Lager/pilsner 34 500 13 Lager/pilsner 9 680 14 Lager/pilsner 14 400 17 Lager/pilsner Northwest/US microbrews American porter 690 1330 240 Amer. hefeweizen 5 300 8 Strong ale 240 3440 110 India pale ale 160 800 39 Imported beers European stout 340 2100 69 European lager 2 40 1 European pilsner 28 570 21 European pilsner 12 1060 8 ther Non-alcohol beer 3 110 3
Fate of xanthohumol in brew XAN IS Spent hops ot trub Cold trub Yeast slug Unhopped wort ops 15 min after hops added After 30 min After 70 min After whirlpool Cold supernatant After 7 days primary fermentation After 7 days secondary fermentation Raw beer Lagered beer -200 0 200 400 600 Amount in rew (mg)
Fate of desmethylxanthohumol in brew DMX 8-PN 6-PN Spent hops ot trub Cold trub Yeast slug Unhopped wort ops 15 min after hops added After 30 min After 70 min After whirlpool Cold supernatant After 7 days primary fermentation After 7 days secondary fermentation Raw beer Lagered beer -40-20 0 20 40 60 80 100 Amount in rew (mg)
Flavan-3-ol monomers Catechin (CT) Epicatechin (ET) G allocatechin (G C) MW 290 MW 306 Present in hops MW 274 Transferred to beer uilding units of catechins Enantiomers referred to as epi, i.e. epicatechin, epigallocatechin Referred to as (epi)gallochatechin when stereochemistry is in doubt Afzelechin (AF)
Catechins Epicatechin-catechin- 1 Epicatechin-epicatechin- 2 Catechin- (4α 6)-gallocatechin Gallocatechin-(4α 8)-catechin -type Gallocatechin-(4α 8)-catechin A-type
ESI-Mass Spectrum of a hop extract Relative Abundance 80 60 40 20 (A) monomer 577 579 291 409 593 307 427 563 595 dimer trimer 867 tetramer pentamer 865 1155 881 883 1171 1443 863 895 1187 1459 899 1203 1475 hexamer 1731 1747 1881 1917 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z Distinction between groups of monomers, dimers, oligomers Mass differences of 16 Th between mass peaks signifies a difference in the number of hydroxyl groups, e.g. 867, 883, 899 Mass differences of 2 Th between mass peaks indicates the presenece of an A- or -type proanthocyanidine, e.g. 867, 865, 863
Chromatographic Purification of ops PAs for Analysis Me 2 Sephadex L-20 column PLC chromatogram of hops extract Semi-preparative PLC 250 10 mm Econosil C18 column Analytical PLC: 250 4.6 mm Synergi C18 column Me 2 PLC chromatogram of hops extract
Fragmentation Pathways for Catechin + m /z 2 9 1 F F -122 Da + m /z 1 6 9 m /z 1 2 3 + A C m/z 291 RDA - 152 Da + m/z 139 RF + m/z 291-126 Da + m/z 165-2 m/z 147 RDA = Retro Diels Alder fragmentation FF = enzofuran ring forming fragmentation RF = eterocyclic ring fragmentation Neutral Losses through Fissions from PA Subunits Cmpd RF RDA FF 2 /FF (epi)afzelechin -126-136 -106-124 (epi)catechin -126-152 -122-140 (epi)gallocatechin -126-168 -138-156
Characteristic Fragmentation Pathways of -Type Dimer RF F - 398 Da E m/z 165 A m/z 563 C + E D F RF C - 126 Da QMCD D E F m/z 437 RDA F D m/z 285 m/z 273 QM CD QM = Quinone methide fragmentation RDA F = Retro Diels Alder fission of ring F FF C = enzofuran forming fission of ring C RF C = eterocyclic ring fission of ring C A C QM CD- + QM CD + D E F m/z 291 + RDA F - 152 Da A m/z 411 C D + - 2 FF C - 124 Da m/z 287
Proanthocyanidin trimer identification by ESI- MS/MS 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 MS/MS 883.00 426.7 579.0 A 593, 305 730.7 550.8 532.7 424.9 593.0 696.5 289.1 712.8 406.9 594.5 317.0 604.5 467.0 694.7 883.0 305.1 275.1 379.0 365.1 468.7 605.3693.8 757.0 865 679.0 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z ΟΗ ΟΗ 595, 593, 305 C 595, 577, 307 ΟΗ
Proanthocyanidin profile of USA hop varieties -type Proanthocyanidins a) monomers: CT>EC>>GC. b) dimers: procyanidin 3(26.2%), procyanidin 1(25.3%), procyanidin 4(20.6%), procyanidin 2 (12.2%), others (15.7%). 7 dimers, 6 trimers, 9 tetramers, 6 pentamers, and 6 hexamers c) trimers: EC-EC-CT (49.0%) EC-CT-CT (24.5%) CT-CT-CT (20.9%) others (5.6%).
Quinone Methide Fragmentations for A- and -type Proanthocyanidins by ESI-MS/MS QM CD for A-type PA A C D R 1 R 2 R 3 E F R 4 QM 1 A C D R 1 F R 2 R 3 E R 4 QM 2 A C R 1 R 2 + (M + - x - 4) + Two quinone-methide funcitionalities D F R 3 E R 4 QM CD for -type PA A C D R 1 R 2 R 3 E F R 4 QM R 1 R 2 A C + D F R 3 E R 4 (M + - x - 2) + ne quinone-methide funcitionality
A-type Proanthocyanidin oligomers indentified in hops (epi)azelechin-a-(epi)catechin (epi)catechin-a-(epi)catechin (epi)gallocatechin-a-(epi)catechin (epi)gallocatechin-a-(epi)gallocatechin (epi)catechin-a-(epi)catechin-a-(epi)catechin (epi)catechin-a-(epi)catechin-(epi)catechin (epi)catechin-a-(epi)gallocatechin-a-(epi)catechin (epi)gallocatechin-a-(epi)gallocatechin-a-(epi)catechin (epi)gallocatechin-a-(epi)gallocatechin-a-(epi)catechin (epi)catechin-a-(epi)gallocatechin-(epi)catechin (epi)catechin-(epi)catechin-a-(epi)catechin-(epi)catechin (epi)catechin-(epi)catechin-(epi)catechin-(epi)catechin-a-(epi)catechin
Quantification of Proanthocyanidins in ops regon Willamette ops PA Extract No. R t (min) (M) + Comp 1 25.3 291 Catechin 2 32.2 291 Epicatechin 4 22.1 579 Epicatechin-catechin 6 21.0 579 Catechin-catechin 8 15.6 595 Gallocatechin-catechin 14 11.7 867 Epicatechin-(catechin) 2 17 9.6 899 (Gallocatechin) 2 -catechin PLC by 250 mm x 4.6 Synergi 4 µm ydro-rp-80a. 5-50% methanol in 1% formic acid. Extinction Coefficients @ 280 nm Monomer = 3975 Dimer = 6725 Trimer = 11360 Molar response ratios 1: 1.69: 2.87 1. The chemical composition of hops is qualitatively similar, but quantitatively different. 2. The chemical composition of a given cultivar varies quantitatively with geographic location.
Acknowledgements Dr. ui-jing Li Mr. Alan W. Taylor Dr. J. Fred Stevens Dr. Martin Sägesser Anheuser usch Cos. op Research Council