Polyphenolic Compounds in Hops and Analytical Methods: A Survey. Oregon State University Mass Spectrometry Laboratory

Transcription

1 Polyphenolic Compounds in ops and Analytical Methods: A Survey regon State University Mass Spectrometry Laboratory

2 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

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

4 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)

5 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

6 Parent Ion Scan of Quercetin-3-rutinoside (Rutin) m/z C m/z MW = 162 m/z 465

7 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

8 Flavonol Glycosides in Commercial eer Samples eer 1 eer 2 eer 3 eer 4 eer 5

9 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 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

10 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 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

11 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

12 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

13 Long-range -C correlations by MC-NMR 1" 3' A 6' 1' 2' C 3 1 4

14 Multiple Reaction Monitoring for Prenyl flavonoids Positive Ion APCI-CAD (collision energy = 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

15 LC-MRM for Prenylflavonoids in eer isoxanthohumol xanthohumol 2, 4-dihydroxychalcone 8-prenylnarigenin 6-prenylnarigenin No. 6 = 8-geranylnarigenin No. 7 = 6-geranylnarigenin

16 Prenylflavonoids in eer eer (µg/l) b Xanthohumol Isoxanthohumol 8-Prenylnaringenin US major brand Lager/pilsner Lager/pilsner Lager/pilsner Lager/pilsner Northwest/US microbrews American porter Amer. hefeweizen Strong ale India pale ale Imported beers European stout European lager European pilsner European pilsner ther Non-alcohol beer

17 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 Amount in rew (mg)

18 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 Amount in rew (mg)

19 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)

20 Catechins Epicatechin-catechin- 1 Epicatechin-epicatechin- 2 Catechin- (4α 6)-gallocatechin Gallocatechin-(4α 8)-catechin -type Gallocatechin-(4α 8)-catechin A-type

21 ESI-Mass Spectrum of a hop extract Relative Abundance (A) monomer dimer trimer 867 tetramer pentamer hexamer 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

22 Chromatographic Purification of ops PAs for Analysis Me 2 Sephadex L-20 column PLC chromatogram of hops extract Semi-preparative PLC mm Econosil C18 column Analytical PLC: mm Synergi C18 column Me 2 PLC chromatogram of hops extract

23 Fragmentation Pathways for Catechin + m /z F F -122 Da + m /z m /z A C m/z 291 RDA Da + m/z 139 RF + m/z Da + m/z 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 (epi)catechin (epi)gallocatechin

24 Characteristic Fragmentation Pathways of -Type Dimer RF F Da E m/z 165 A m/z 563 C + E D F RF C 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 RDA F Da A m/z 411 C D FF C Da m/z 287

25 Proanthocyanidin trimer identification by ESI- MS/MS MS/MS A 593, m/z ΟΗ ΟΗ 595, 593, 305 C 595, 577, 307 ΟΗ

26 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%).

27 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

28 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

29 Quantification of Proanthocyanidins in ops regon Willamette ops PA Extract No. R t (min) (M) + Comp Catechin Epicatechin Epicatechin-catechin Catechin-catechin Gallocatechin-catechin Epicatechin-(catechin) (Gallocatechin) 2 -catechin PLC by 250 mm x 4.6 Synergi 4 µm ydro-rp-80a. 5-50% methanol in 1% formic acid. Extinction 280 nm Monomer = 3975 Dimer = 6725 Trimer = Molar response ratios 1: 1.69: The chemical composition of hops is qualitatively similar, but quantitatively different. 2. The chemical composition of a given cultivar varies quantitatively with geographic location.

30 Acknowledgements Dr. ui-jing Li Mr. Alan W. Taylor Dr. J. Fred Stevens Dr. Martin Sägesser Anheuser usch Cos. op Research Council