pigmented tannin: structural elucidation by a complimentary ......anthocyanins and their variation...
TRANSCRIPT
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Pigmented Tannin:Structural Elucidation by a
Complimentary Suite of Mass Spectrometric Techniques
Jonathan R. CaveAndrew L. Waterhouse
Carlito B. LebrillaJames A. Kennedy
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Production
White• Vineyard• Crush• Press• Fermentation• Settling• Stabilization• Aging
– Barrel– Bottle
Red• Vineyard• Crush• Fermentation• Press• Settling• Stabilization• Aging
– Barrel– Bottle
• All Color comes from the Skin• We extract phenolics, tannin, pigments from the skins and seeds
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Control Points - Red
• Extraction/Maceration– Anthocyanins– Tannins– Cinnamates
• Retention– Oxidation– Precipitation
• Modification– Pyrano ring closures– Adducts– Bridging – Acetaldehyde and others
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Total Mass of Solutes
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Structures
• Anthocyanins
• Catechin
• Cinnamates
Ferulic Acid
Cinnamic Acid
Catechin
Malvidin Aglycon
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Compounds
+
4
OOH
OH
OH
OH
OH
4
OOH
OH
OH
OH
OH
53
O+
OH
OH
O
OH
O
OGlu
CH3
CH3
n
TA type
OOH
OH
OH
OH
OH
OOH
OH
O
OH
O
O Glu
OOH
OH
OH
OH
OH
n
A-T type
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Impact of Pigmented Tannin
Primary Quality Parameters
Flavor Aroma Texture Color
Softens astringency, alters flavor perception1
Responsible for persistent color2
Monomeric anthocyanin and copigmentation almost disappears within 2 years
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Challenges
Diversity of molecular structures3,4
Expanded Sensory Properties
Challenging Chemistry
• Hundred year problem
• Ribéreau-Gayon
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Compounds by Class
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Isomers
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Objectives• Identification of polymeric pigments from new
wine and wine during aging.
– Analysis of the components of these mixtures utilizing the mass spectrometry techniques, Q-TOF, MALDI-FT ICR, ESI-QQQ-FTICR, ESI-QQQ/DAD and HRxx
– Structural Identification by means of MS data using standard and customized de-convolution algorithms.
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Necessary MS Attributes
FT-ICR
• High Resolution
• High Mass Accuracy
• High Sensitivity
• Discrimination of Compounds
• Simultaneous analysis
QTOF
• Tandem MS
• High Sensitivity
• Collision Induced Dissociation (CID)
• High Selectivity (Q)– Mass Selectivity for fragmentation
– Eliminate coeluting ions
• Accurate Analyzer (TOF)
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Illustration of mass error
Single Quadrupole Analyzer ICR
• Absolute Error < ±0.001 m/z• Absolute Error ±0.2 m/z
Cyanidin Aglycon
Precise Mass: 287.05556307013
Example Mass Absolute Error Mass Error (ppm)
FTICR 287.0559 0.0003 1.2
Quadrupole 287.16 0.10 348.4
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Discriminating between Molecules
Accurate Mass
Absolute Mass Difference (amu)
Relative Mass Difference (ppm)
Malvidin-3-O-Glucoside acetaldehyde adduct (Vitisin B) 517.1346 0.0364 70.4 ppm
Cyanidin-3-O-Glucoside vinylformic acid adduct 517.0982
Two Pigmented Tannin molecules with the same nominal mass can be differentiated
Ion m/z = 715.0989 1 ppm 5 ppm 10 ppm
C13H22N12O23 C46H18O9 C35H18N6O12
C11H10N26O13 C17H26N6O25 C21H30O27
C28H26O22 C15H14N20O15 C19H18N14O17
C26H14N14O12 C30H18N8O14 C17H6N28O7
C41H18N2O11 C28H6N22O4 C34H22N2O16
… …
Possible Compounds 5 25 44
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Strategy
High Resolution Mass Spectrometry
Proanthocyanidin Preparation
Low Pressure Liquid Chromatography
H2O/MeOH/Acetone onSephadex LH20
Hydroxycinnamic acid/Na Matrix
MALDI-ICR
H2O/Acetone+0.1%FA
ESI-QQQ-ICR
H2O/ACN+0.1%FA
Q-TOF
w/HPLC
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FTICR Technology
• 3 Modes of Motion
• Magnetonic
• Cyclotronic
• Translational
• Fluctuation of RF Field changes Gyration radius
•
• Multiple ions in one cell• Fourier Transform Deconvolution
Lebrilla, C.B. 2013
𝑚𝑧 ∝
𝐵
𝜔
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Sample of Current ICR ConditionsCompound Exact
MassExperimental
MassMass Error
(ppm) Resolution
Malvidin-3-(6-acetyl)-monoglucoside 535.1452 535.1450 0.3 54000
Catechin-malvidin-3-glucoside (T-A
Dimer) 781.1980 781.1975 0.6 44000
Malvidin-3-glucoside-catechin (A-T
Dimer) 782.2058 782.2079 2.6 42000
Malvidin-3-glucose-4-vinyl-catechin 805.1980 805.1967 1.6 39000
Malvidin-3-(acetyl)glucose-4-vinyl-
catechin 847.2086 847.2070 1.8 38000
Malvidin-3-(p-coumaroyl)glucose-4-
vinyl-catechin 951.2348 951.2333 1.5 34000
Malvidin-3-glucoside-dicatechin (A-T
Trimer) 1070.2692 1070.2679 1.2 29000
Malvidin-3-glucose-4-vinyl-dicatechin 1093.2614 1093.2575 3.5 29000
Average 1.7 38000
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FTICR Spectra
1990 Cabernet Sauvignon
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FTICR Spectra
1990 Cabernet Sauvignon
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ICR Values
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7 Pinotin VariationsGrape Derived
1 Vanillin bridged Petunidin Cat DimerOak Derived
1.7ppm Mass Error *He et al. (3)
*
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QTOF TechnologyWhy Nano-HPLC?
• Nano-ESI5
• Enrichment column– Concentration
– Cleanup on Loading
– Narrows injection band
Our Chip
• Custom Diol Chip
• Normal Phase Gradient
• Separation by length
http://www.kjemi.com/artikkel/1947http://www.chibi.ubc.ca/facilities-2/proteomics-core-facility-3
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Nano-HPLC QTOF Method
• Modification of Kelm et al.
• Expansion of elution gradient
• Modification of Kelm Fluorescence to MS
• FA 3.75 vs. Acetic 4.76
– Order of magnitude greater proton strength
• Proton accessibility of solvent increased
• ACN:HOAc (98:2) ACN:H2O:FA (95:3:2)
• MeOH:H2O:HOAc (95:3:2) MeOH:H2O:FA (95:3:2)
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QTOF of Tannin
QTOF fragmentation spectrum of ion 1443.3, Catechin Pentamer M+H+, from 2010 Caymus
Cabernet Sauvignon.
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Current Status
• >4,000 Compounds of Interest
• Isomeric Complications
• ~450 Distinct Signals
• ~150 signals matched to database
• Remainder could be new, fragments
• Boost QTOF Sensitivity
• Other Wine Varieties and Ages
• Fragmentation Analysis – By Hand
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Impact on Wine Production
Insight into desired control featuresMechanisms of pigmented tannin development
Which pigments are important
Refined aging conditions for desired traitsOak, Micro-ox, etc.
Enhancement of precursors
Implications for oxygen management
Basis for innovation in production techniques
Greater Stylistic Control
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With these methods in place we proceed on the following objectives.
35 Year Vertical (Observe pigmented tannin evolution throughout aging)
• Employ our method of complimentary mass spectrometric techniques for comprehensive identification of wine matrix compounds.
• Observe the changes in relative abundance, depletion and accumulation in pigmented tannin composition.
• Postulate wine pigment precursors for examination of mechanistic pathways.
• Employ standards to quantitate the classes of polymeric pigments in wine.
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Thank You
• American Vineyard Foundation
• Evan Parker
• Andres Guerrero
• Carlito Lebrilla
• James A. Kennedy
• Waterhouse Lab
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References
1) Vidal, S., L. Francis, A. Noble, M. Kwiatkowski, V. Cheynier, and E. Waters. 2004. Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine. Analytica ChimicaActa 513: 57-65.
2) Monagas, M., C. Gómez-Cordovés, and B. Bartolomé. 2005. Evolution of polyphenols in red wines from Vitis vinifera L. during aging in the bottle. European Food Research and Technology 220: 607-614.
3) He, F., N.N. Liang, L. Mu, Q.H. Pan, J. Wang, M.J. Reeves, and C.Q. Duan. 2012. Anthocyanins and their variation in red wines. II. Anthocyanin derived pigments and their color evolution. Molecules 17: 1483-519.
4) Mcrae, J.M., R.J. Falconer and J.A. Kennedy. 2010. Thermodynamics of grape and wine tannin interaction with polyproline: Implications for red wine astringency. Journal of Agricultural and Food Chemistry 58: 12510-12518.
5) Wilm, M., G. Neubauer, and M. Mann. 1996. Parent Ion Scans of Unseparated Peptide Mixtures. Analytical Chemistry 68: 527-533.