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Direct and fast analysis of free and sulfite-bound carbonyl compounds in wine by two-dimensional quantitative proton (1H) and carbon (13C) nuclear magnetic resonance spectroscopy (2D q NMR) Maria Nikolantonaki, Prokopios Magiatis, and Andrew L. Waterhouse Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01682 • Publication Date (Web): 08 Sep 2015 Downloaded from http://pubs.acs.org on September 13, 2015
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Analytical Chemistry
Direct and rapid analysis of free and sulfite-bound carbonyl compounds in wine by twodimensional quantitative proton (1H) and carbon (13C) nuclear magnetic resonance spectroscopy (2D q NMR)
Maria Nikolantonaki,1,3 Prokopios Magiatis,2 and Andrew L. Waterhouse 1*
1
Department of Viticulture and Enology, University of California, Davis, California
95616, United States 2
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,
University of Athens, Panepistimioupolis Zografou 15 771, Athens, Greece 3
Current address: Institut Universitaire de la Vigne et du Vin, Jules Guyot, UMR A
02.102 PAM AgroSup Dijon/Université de Bourgogne, Rue Claude Ladrey, BP 27877, 21078 Dijon Cedex, France
*
To whom correspondence should be addressed
Telephone: +1-530-752-4777 Fax: +1-530-752-0382 E-mail:
[email protected]
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Abstract
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Recent developments that have accelerated 2D NMR methods and improved quantitation
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have made these methods accessible analytical procedures, and the large signal dispersion allows
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for the analysis of complex samples. Few natural samples are as complex as wine, so the
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application to challenges in wine analysis look promising. The analysis of carbonyl compounds
6
in wine, key oxidation products, is complicated by a multitude of kinetically reversible adducts,
7
such as acetals and sulfonates, so that sample preparation steps can generate complex
8
interferences. These challenges could be overcome if the compounds could be quantified in situ.
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Here, two-dimensional (1H-1H) homonuclear and heteronuclear (13C-1H) single quantum
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correlations (COSY and HSQC) nuclear magnetic resonance spectra of undiluted wine samples
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were observed at natural abundance. These techniques achieve simultaneous direct identification
12
and quantitation of acetaldehyde, pyruvic acid, acetoin, methylglyoxal and α-ketoglutaric acid, in
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wine with only a small addition of D2O. It was also possible to observe and sometimes quantify,
14
the sulfite, hydrate, and acetal forms of the carbonyl compounds. The accuracy of the method
15
was tested in wine samples by spiking with a mixture of all analytes at different concentrations.
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The method was applied to 15 wine samples of various vintages and grape varieties. The
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application of this method could provide a powerful tool to better understand the development,
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evolution, and perception of wine oxidation, and insight into the impact of these sulfite bound
19
carbonyls on antimicrobial and antioxidant action by SO2.
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Analytical Chemistry
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Introduction
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Wine-relevant carbonyl compounds, formed as natural fermentation or chemical oxidation
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products, including acetaldehyde (1), acetoin (2), α-ketoglutaric acid (3), pyruvic acid (4), and
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methylglyoxal (5), have been proposed as markers of wine aging. The concentration of these
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substances is tied to winemaking and storage conditions, varies significantly,1 and is especially
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high in sweet wines made from Botrytis cinerea infected grapes probably due to sugar oxidation.2
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Analyzing carbonyl compounds in such a complex matrix as wine is very daunting
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because of their chemical reactivity and instability. First, carbonyls are known to take part in the
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formation of stable colored, structures arising from the reaction between anthocyanins and 1,
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glyoxylic acid, and 4.3-5 These pigments have strong bonds between the carbonyl and flavonoid
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moiety, and are slow to release the carbonyl unit for potential analysis, but one method has been
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described.6 Second, aldehydes and ketones also react with alcohols to form acetals.7, 8 Some of
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the nitrogen and sulfur containing compounds, such as the amino acids, proteins and amines can
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also react with carbonyls by nucleophilic attack and form adducts.9 In these reactions, it is not
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always clear if the formation of these products is reversible under wine-like conditions. In
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addition, in grape juice and wine, carbonyl compounds react with sulfur dioxide yielding
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hydroxysulfonates.10, 11 The sulfite reactions are reversible and dissociation constants for sulfur-
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bound carbonyl compounds like 1, 3, L-xylosone, 5-keto-D-fluctose, 4 and galacturonic acid
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have been determined under wine-like acidic conditions by Burroughs and Sparks.10-12
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Free sulfur dioxide is a key to protecting wine from microbes and oxidation, but an
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accurate measure of free SO2 is complicated due to these many binding reactions. All of the
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carbonyl compounds listed above can bind to sulfur dioxide, but the amount depends on their
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concentrations and dissociation constants of each. And the converse is also true: the influence on 3
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the sulfite binding on the concentration of free carbonyl compounds as measured by many
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conventional methods is poorly described, except in a few cases.13 It would be most useful to
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have a method that does not initiate the dissociation of compounds, particularly where the
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dissociation constants and rates are unknown or poorly described.
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Conventional quantitation methods of free carbonyl compounds in wine include non-
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specific methods such as distillation or reaction with bisulfite, low sensitivity methods based on
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colorimetric procedures, and single-compound quantitative enzymatic methods.11, 14 The former
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methods do not provide compound specific data, and applying the latter methods to
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simultaneously survey all major wine carbonyls would be time consuming. To date, the most
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effective alternatives for the analysis of carbonyl compounds are GC-NPD/MS and HPLC-UV. In
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both cases, sample preparation yields derivatives, such as the o-(2,3,4,5,6-pentafluorobenzyl)-
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hydroxylamine2, a cysteamine (2-aminoethanthiol)15 or the 2,4-dinitrophenylhydrazone13,
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derivatives before analysis. The effects of the derivatization steps on the various carbonyl
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equilibria are known for a few specific equilibria, but otherwise are poorly documented.
16-18
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Current quantitation methods of bound forms include a sulfonate hydrolysis step under
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strong alkaline conditions (pH = 11) followed by derivatization after acidification14, 19 or direct
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acid hydrolysis.13 Thus, levels of bound SO2-bound carbonyl compounds are calculated by the
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difference between two results: a free carbonyl level measured without hydrolysis and a total
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carbonyls result that includes hydrolysis.18 However, the derivatization conditions may promote
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the oxidation of the sample, resulting in the formation of de novo interfering carbonyl
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compounds.20, 21 In addition the application of the preparation steps may well release carbonyls
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from the bound forms. Ethylidine bridged flavonoids are reported to release acetaldehyde in acid
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at 50°C in 20 minutes, and acetals can be hydrolyzed under milder conditions.22 Thus, a method
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that can simultaneously quantitate free and sulfonate-bound carbonyl compounds without
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hydrolysis could provide insight into the chemical reactivity of these compounds, and could
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possibly be extended to other carbonyl products.
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While 2D NMR has been widely used for assignments and identification of various
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compounds, it has only recently been utilized for the quantitation of metabolites in biological
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samples.23, 24 This development has been facilitated by accelerated data acquisition and greater
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instrument stability that has improved quantitative reproducibility.25
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Our effort was aimed at investigating NMR spectroscopy as a tool for the direct analysis
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of free and sulfite-bound carbonyls in wine. Its implementation should eliminate the sample
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preparation steps and resulting artifacts present in existing analytical methods. 1H NMR spectra
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of wine contains thousands of individual resonances of varying intensities, and dispersion of
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those signals into two-dimensions may provide an opportunity to measure some signals
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quantitatively. Here we have evaluated the use of 2D 1H-13C HSQC and 1H-1H COSY spectra for
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the analysis of multiple free and sulfite-bound carbonyl compounds directly in wine.
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EXPERIMENTAL SECTION
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Chemicals. Acetaldehyde, 1, pyruvate, 4, acetoin, 2, methylglyoxal, 5, α-ketoglutarate, 3,
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pyromellitic acid, L-tartaric acid and deuterium oxide were purchased from Sigma-Aldrich, Inc.
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(St. Louis, MO). Sodium bisulfite, and ethanol were purchased from Acros Organics (Morris
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Plains, NJ). Water was purified using a Milli-Q system (Millipore, Billerica, MA). All chemicals
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were of analytical grade or of the highest available purity.
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NMR Spectroscopy Experiments. NMR experiments were performed on a Bruker 600
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MHz spectrometer using a cryoprobe at 289 K. 2D acquisition parameters for the 1H–13C HSQC
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experiment were as follows: The hsqcetgpsisp2.2 pulseprog was used, with a spectral width of
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8417 Hz in the F2 dimension and 24147 Hz in the F1 dimension, acquisition time was 0.061 s
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(F2) and 0.005 (F1), relaxation delay was 1.5 s, 1K (t2) x 256 (t1) data points, 8 scans per t1
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increment. Processing: Zero filling and FT to 2K x 1K data points after multiplication with qsine
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filter using SSB=2.2D. Acquisition parameters for the COSY experiment were as follows: The
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cosygpprqfpulseprog was used, with a spectral width of 9615 Hz, acquisition time was 0.10 s,
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relaxation delay was 1.5 s, 2K(t2) x 256 (t1) data points, 8 scans per t1 increment. Processing:
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Zero filling and FT to1K x 1K data points after multiplication with sine filter. All NMR data
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were processed using Topspin 2.1 NMR (Bruker) software.
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To test the accuracy of the 2D method for wine samples, spiking experiments were
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performed in a wine sample with the addition of increasing concentrations 10 to 200 mg L-1 of 2,
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3, 4, 5 and 1 to 50 mg L-1of 1. A correlation analysis was made between the observed and the
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added concentration values.
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NMR description of studied compounds.
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Acetaldehyde (1): 1H NMR (600 MHz, H2O+D2O) δ 9.665 (1H, q, 2.3 Hz, H-1), 2.225
105 106 107
(3H, d, 2.3 Hz, H-2). 13C NMR (151 MHz, H2O+D2O) δ 207.19 (C-1), 30.74 (C-2) Acetaldehyde hydrated (6): 1H NMR (600 MHz, H2O+D2O) δ 5.235 (1H, q, 5.16 Hz, H1), 1.312 (3H, d, 5.16 Hz, H-2). 13C NMR (151 MHz, H2O+D2O) δ 88.80 (C-1), 23.85 (C-2)
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Acetaldehyde (ethyl hemiacetal)(7): 1H NMR (600 MHz, H2O+D2O) δ 4.94 (1H, q, 5.2
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Hz, H-1), 3.77 (1H, dt, Ha-CH2). 3.51 (1H, dt, Hb-CH2), 1.17 (3H, CH3), 1.303 (3H, d, 5.2 Hz,
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H-2). 13C NMR (151 MHz, H2O+D2O) δ 94.69 (C-1), 63.31 (CH2), 22.62 (C-2), 14.87 (CH3)
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Acetaldehyde-bisulfite adduct (8): 1H NMR (600 MHz, H2O+D2O) δ 4.54 (1H, q, 6.45
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Hz, H-1, overlapped), 1.46 (3H, d, 6.45 Hz, H-2). 13C NMR (151 MHz, H2O+D2O) δ 80.99 (C-
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1), 17.47 (C-2)
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Acetoin (2): 1H NMR (600 MHz, H2O+D2O) δ 4.40 (1H, q, 7.15 Hz, H-3), 2.21 (3H, s,
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H-1), 1.365 (3H, d, 7.15 Hz, H-4). 13C NMR (151 MHz, H2O+D2O) δ 215.94 (C-2), 73.60 (C-3),
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25.55 (C-1), 18.90 (C-4)
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Acetoin-bisulfiteadduct (isomer I) (9a,b): 1H NMR (600 MHz, H2O+D2O) δ 4.165 (1H,
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q, 6.54 Hz, H-3), 1.43 (3H, s, H-1), 1.24 (3H, d, 6.54 Hz, H-4). 13C NMR (151 MHz, H2O+D2O)
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δ 89.75 (C-2), 70.06 (C-3), 17.87 (C-4), 17.26 (C-1)
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Acetoin-bisulfiteadduct (isomer II) (9c,d): 1H NMR (600 MHz, H2O+D2O) δ 4.165 (1H,
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q, 6.54 Hz, H-3), 1.48 (3H, s, H-1), 1.24 (3H, d, 6.54 Hz, H-4). 13C NMR (151 MHz, H2O+D2O)
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δ 89.62 (C-2), 69.98 (C-3), 16.98 (C-4), 16.81 (C-1)
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Ketoglutaric acid (hydrated) (10a): 2.43 (2H, t, 7.5 Hz, CH2), 2.11 (2H, t, 7.5 Hz, CH2). 13
C NMR (151 MHz, H2O+D2O) δ 29.29, 34.08 Ketoglutaric acid (cyclic) (10b): 2.98 (2H, br, CH2), 2.66 (2H, t, 6.7 Hz, CH2). 13C NMR
(151 MHz, H2O+D2O) δ 34.56, 28.31
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Ketoglutaric acid-bisulfite adduct (11b): 1H NMR (600 MHz, H2O+D2O) δ 2.56 (1H, m,
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CH), 2.49 (1H, m, CH), 2.34 (1H, m, CH), 2.25 (1H, m, CH). 13C NMR (151 MHz, H2O+D2O) δ
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177.85 (CO), 172.65 (CO), 90.66 (C-2), 29.53, (CH2), 29.12 (CH2).
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Pyruvic acid (carbonyl form) (4): 1H NMR (600 MHz, H2O+D2O) δ 2.42 (3H, s, CH3).
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C NMR (151 MHz, H2O+D2O) δ 200.16 (C-2), 174.83 (CO), 26.56 (CH3). Pyruvic acid (hydrated form)(12): 1H NMR (600 MHz, H2O+D2O) δ 1.57 (3H, s, CH3).
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C NMR (151 MHz, H2O+D2O) δ 175.60 (CO), 93.30 (C-2), 25.77 (CH3). Pyruvic acid-bisulfite adduct (13): 1H NMR (600 MHz, H2O+D2O) δ 1.73 (3H, s, CH3).
134 135
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C NMR (151 MHz, H2O+D2O) δ 173.52 (CO), 88.56 (C-2), 21.37(CH3). Methylglyoxal hydrated form I(14a): 1H NMR (600 MHz, H2O+D2O) δ 5.26 (1H, s, H-
136 137
3), 2.29 (3H, s, CH3).
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(CH3).
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3), 1.36 (3H, s, CH3).
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(CH3).
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C NMR (151 MHz, H2O+D2O) δ 209.50 (CO), 90.34 (C-3), 25.01
Methylglyoxal hydrated form II (14b): 1H NMR (600 MHz, H2O+D2O) δ 5.07 (1H, s, H-
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142
13
13
C NMR (151 MHz, H2O+D2O) δ 95.76 (C-2), 92.55 (C-3), 22.07
Methylglyoxal-bisulfite adduct (15): 1H NMR (600 MHz, H2O+D2O) δ 5.20 (1H, s, H-3), 2.43 (3H, s, CH3). 13C NMR (151 MHz, H2O+D2O) δ 206.15 (CO), 88.59 (C-3), 27.76(CH3).
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Sample preparation for NMR analysis. To 0.45 ml of wine or standard solution 50µL of
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a solution containing 0.78 mM pyromellitic acid in D2O was added in a 5 mm NMR tube. The
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signal from the pyromellitic acid served as an internal standard (IS). After thorough mixing, 0.5
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mL of each sample was transferred to a 5 mm-o.d Wilmad® NMR tube for analysis.
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Quantitation. Standard addition procedure. The concentrations of free and sulfite-bound
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carbonyls were calculated using standard addition procedure. A model mixture for standard
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additions was prepared with five targeted wine-relevant free carbonyls (1, 2, 3, 4, 5), dissolved in
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1 ml H2O, each at 1 gm-L-1. This stock solution was added to a model wine (12% ethanol, pH =
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3.5, 5 gL-1 tartaric acid) to obtain a range of concentrations varying between 10 and200 mgL-1 for
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2, 3, 4, 5, and 1 to 50 mgL-1 for 1. Each standard solution was measured five times as described
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above.
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The calibration curves for the sulfite-carbonyl adducts were prepared using 1 gmL-1stock
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solution as above, diluted into model wine, to obtain concentrations between 10 to 200 mgL-1for
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all compounds. To five tubes, each containing 500 µL model wine spiked with carbonyls, 25 µL
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of sulfur dioxide (10 mM) was added. The mixture was mixed for 10s and incubated for 1h to
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achieve equilibrium. For each sample, the sulfite-adduct was analyzed as described above. It is
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presumed that all the added carbonyl would be in the sulfite adduct form under these conditions.
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For each free and sulfite-bound carbonyl compound, a standard addition curve was fitted
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by the linear regression equation: V = a[c] + b, where V represents the 2D peak volume
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integration ratio between pyromellitic acid (IS) and each free and/or sulfite-bound carbonyl
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compound, and [c] the concentration (mgL-1) in wine. Table 1 and Table 2 show the number of
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signals that were used for compound identification. However, due to overlap of signals even in
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the 2D spectra, only a subset of these signals could be used for quantitation. The observed
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concentration of each compound was calculated by a b/a ratio, where a is the slope and b the y-
168
intercept of the linear regression curve.
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Analytical evaluation. The standard addition curve linearity was evaluated by its
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regression coefficient r2, Table 3.To test the accuracy of the 2D method for wine samples, spike
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experiments were performed using a wine sample with the addition of two different
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concentrations of each compound (15, 50 mgL-1for 2, 3, 4, 5 and 3, 10 mgL-1for 1). The accuracy
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was expressed as the percent recovery (%R) calculated for each compound using: %R = (Cs-
174
Cu)/Csa, where Cs: the measured concentration in spiked aliquot; Cu: the measured concentration
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in unspiked aliquot and Csa: the concentration of spike added. The precision of NMR
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measurement was determined on five successive analysis of the same sample at two
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concentrations (Table 4) and evaluated via the percentage of the coefficient variation (CV% =
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SD 100/AV), where SD is the standard deviation and AV the average value of replicates. The
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limit of detection (LOD) and quantitation (LOQ) of each analyte were estimated based on the
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standard deviation of the response and the slope (a) of the calibration curve according to the
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formulas: LOD = 3.3 (SD/a) and LOQ = 10 (SD/a). Because of the reactivity and instability of
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the carbonyl compounds in wine, method validation was performed using a wine sample
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containing low levels of targeted compounds.
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RESULTS AND DISCUSSION
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Assignments of free and sulfite-bound carbonyl signals. NMR spectra (1D and 2D) of
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model wine solutions containing 1 mM carbonyl compounds (1, 2, 3, 4, and 5), with and without
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6 mM (384 mg/L) sulfur dioxide revealed the presence of the free forms, hydrates, diethyl acetals
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and sulfonate adducts, but not all for every compound. The progress of the conversions was
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monitored by 1H NMR. In all reaction mixtures, the equilibria were reached within a few
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minutes.
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Identification of free and sulfite-bound acetaldehyde. The 1H-NMR of acetaldehyde, 1, in
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model wine exhibited three sets of peaks; one corresponding to the carbonyl form (1), one
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corresponding to the hydrate form (6) and one corresponding to the ethyl hemiacetal (7). The
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expected resonances of 1 consisted of a highly deshielded quartet at 9.66 ppm (aldehydic proton)
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and a doublet at 2.22 ppm corresponding to the methyl group. The aldehydic carbon was
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observed at 207.19 ppm. The hydrate 6 revealed a proton at 5.23 ppm (quartet) coupled with the
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methyl group at 1.31 ppm (doublet). The dihydroxy carbon was observed at 88.8 ppm confirming
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the presence of two oxygen atoms. Compound 7 was a hemiacetal (4.94 ppm/94.69 ppm) also
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containing an ethyl group identified in the carbon spectrum with two peaks at 63.31 and 14.87
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ppm. The two protons of the CH2 group were observed at 3.77 and 3.51 ppm as two doublets of
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triplets. The methyl group overlapped with the ethanol peak. After addition of sodium
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metabisulfite a new set of peaks appeared, corresponding to the bisulfite adduct 8. The methyl
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group of the adduct was observed as a doublet at 1.46 ppm while the methine proton overlapped
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with the peak of tartaric acid at 4.54, as clarified by the COSY spectrum. In the HSQC spectrum
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the methine proton was correlated with a carbon at 80.99 as expected for a bisulfite adduct.
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Identification of free and sulfite-bound acetoin. In contrast to acetaldehyde, racemic 2 in
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model wine was observed as a single compound in the free carbonyl form, with no hydrate or
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acetals present. The 1H-NMR spectrum showed one methyl adjacent to a carbonyl at 2.21 ppm,
209
one methyl at 1.365 ppm, and one methine on the oxygenated carbon at 4.40 ppm. The carbonyl
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of 2 was observed at 215.94 ppm in the 13C spectrum. Addition of sodium metabisulfite led to the
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appearance of two new compounds. Indeed the addition of sulfite group at position 2 of racemic 2
212
leads to 2 diasteriomers, as two pairs of enantiomers (9 a-d). Each diastereomer gave four distinct
213
signals in the carbon spectrum, and in the 1H-NMR two distinct signals for the methyl groups of
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position 1 (1.43 and 1.48 ppm) and two over-lapping signals for positions 3 and 4 at 4.165 and
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1.24 ppm respectively.
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Identification of free and sulfite-bound α-ketoglutaric acid. Ketoglutaric acid was more
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complex than the others because in addition to the free form (3) and the hydrated form (10a), a
218
cyclic form could also be observed (10b). The equilibrium was dependent on the pH of the
219
solution and also on the concentration of the compound. However at the pH of the model wine,
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and the concentration range used for the calibration curve, the dominant form was the cyclic 10b,
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but with some contribution from the free 3 via rapid (on the NMR timescale) interconversion. It
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was observed as two peaks (one triplet and one distorted broad peak) corresponding to two
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methylenes. The formation of the SO2 adduct would lead to either 11a or 11b. There were four
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distinct multiplets corresponding to the four protons of the two methylenes, and two methylenes
225
in the 13C spectrum plus an oxysulfated quaternary carbon and the two carbonyls. A large
226
difference in the chemical shift of the two carbonyls suggests that one correspond to a free
227
carboxylic acid and one to a lactone, leading to the conclusion that the observed form was 11b.
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Identification of free and sulfite-bound pyruvate. Pyruvic acid in model wine was
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observed as a mixture of the carbonyl form (4) and the hydrate form (12). Each form gave a
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distinct peak for the methyl group, 4 at 2.42 ppm and 12 at 1.57 ppm, assigned using HSQC and
231
13C NMR . The carbonyl form was identified by the signal at 200.16 ppm while the hydrate by
232
the oxygenated carbon at 93.3 ppm. As expected, the methyl of the carbonyl form was deshielded
233
compared to the methyl of the hydrated form. The addition of the sulfite group led to the
234
appearance of a new methyl signal at 1.73 ppm corresponding to compound 13.
235
Identification of free and sulfite-bound methylglyoxal. Compound 5 in model wine was
236
observed as a mixture of the two hydrated forms (14a, 14b) while the carbonyl form 5 was not
237
observed. The major hydrate form 14a corresponded to the compound with one hydrated
238
aldehyde while the minor compound 14b corresponded to the case where both carbonyls were
239
hydrated. In the first there was one carbonyl at 209.5 ppm and a doubly oxygenated carbon at
240
90.3 ppm. In the second case the molecule showed two doubly oxygenated carbons at 92.5 and
241
95.7 ppm. The addition of sodium metabisulfite led to only one observable adduct, 15, where the
242
sulfite group had been added to the aldehydic carbon. The adduct showed one carbonyl at 206.1
243
ppm, one oxysulfated methine carbon and one methyl group.
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Quantitative 2D NMR analysis.
245
Calibration of 2D NMR results. Our strategy to quantify free and sulfur-bound carbonyls
246
compounds directly in wine was to apply 2D 1H-1H COSY and 1H-13C HSQC techniques
247
(Figure2) which have not been used previously in wine quantitation. For the quantitative 2D
248
NMR experiments, 10% D2O was added to wine samples for locking.
249
Since these techniques had not been applied to quantitation of wine components, it was
250
important to verify whether volumes of 2D signals could be proportional to the concentration of
251
the corresponding analyte. For quantitation, the non-sulfonate forms of each compound (formed
252
instantly) were summed and expressed in carbonyl form equivalents. So the concentration of 1, 4,
253
and 5 were expressed as the sum of 1,6, plus 7; 4, plus 12;and 14a, plus 14b, respectively.
254
Method Validation. The method showed satisfactory linearity for all free carbonyl
255
compounds within the range of 1.2-10 mgL-1 to 200 mgL-1 for 2, 3, 4, 5 (see Table 3), and 0.7
256
mgL-1 to 50 mgL-1 for 1, with the corresponding regression correlation coefficients of (r2) above
257
0.990. Moreover, calibration curves showed strong linear (r2 > 0.989) responses for the sulfite-
258
bound carbonyls, 8, 13, and 15, within the ranges of 2.5-600,12.3-432 and 8.6-435 mg L-1,
259
respectively. Calibration curves of all free and sulfite-bound carbonyls showed the same order of
260
magnitude for their slope value, suggesting a similar sensitivity of the analytical method for all
261
the assessed compounds. LOQ were observed for 1/8, 2, 3, 4/13 and 5, at 1.1/7, 13.2, 16.8,
262
3.6/10.3, and 12.5/19.1 mg L-1, respectively. However, because of the apparent high dissociation
263
constant, and consequently low sulfite binding power of 2, none of its sulfite adduct, 9abcd,
264
could be detected in wine-like conditions containing a 4-fold excess of sulfur dioxide (relative to
265
the concentration of 2). In addition to that, severe peak overlap in HSQC spectra was encountered
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266
on the assigned 11b in wine samples. As a consequence of these problems, neither the 9abcd nor
267
the 11b species could be observed in the course of wine analysis.
268
The accuracy and precision of the method were assessed using low (15 mg L-1 for 2, 3,
269
4/13, 5/15, 8, and 3 mg L-1 for 1) and high (50 mg L-1 for 2, 3, 4/13, 5/15, 8, and 10 mg L-1 for 1)
270
concentrations in triplicate. All samples were assessed over two days and this stability assay
271
showed no significant reduction of analyte concentrations in the wine matrix at either low or high
272
concentrations. The accuracy of the method was calculated as the ratio of the mean observed
273
increased concentration and the known spiked amount added to the wine matrix and expressed as
274
the percent recovery for the three samples (Table 4). Percent recovery (IS-normalized) results in
275
wine for 2, 3, 4/13, 5/15 and 8 were within 92 and 116 %, highlighting the accuracy of the
276
quantitation in wine. The coefficient of variation ranged from 0.5% to 7.2%, demonstrating good
277
precision for an NMR based method.
278
Sensitivity and limitations of the method. The proposed method is based on the fact that
279
cross-peak volumes observed in the 2D 1H-1H COSY and 1H-13C HSQC NMR spectrum can be
280
correlated with the concentration of the analytes. The lower limit of detection of the proposed
281
method will depend upon several experimental parameters which govern cross-peak volumes in
282
the COSY and HSQC spectra. With the present experimental results we have been able to
283
measure the concentration of free and sulfite bound low molecular weight carbonyl compounds
284
directly in wine to within a few tens of milligrams per liter range. In our experiments, performed
285
in a 600 MHz NMR magnet equipped with a cryo-probe, we have used approximately 1h for
286
recording the COSY and HSQC spectra of the wine samples. We have found (data not shown)
287
that by increasing to 9 h the signal averaging recording time the LOD of all tested analytes can be
288
lowered. Nevertheless, considering that the quantitation limits of all analytes were adequately
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below the reported mean levels of the tested carbonyls in wines when they were measured with 1
290
h recording time, these experimental parameters were set as the default in order to avoid
291
excessive analysis time.
292
Method application in wine. Several California wines (n = 15) from different vintages
293
(1974 - 2012) were analyzed for 1/8, 2, 3, 4/13, and 4/15 using the above described method. The
294
observed concentrations and standard deviations are shown in Figure 3. The mean concentrations
295
of 1, 2, 3, 4 and 5 were found to be 17.5 ± 32.6, 12.7 ± 7.8, 106.5 ± 56.7,36 ± 27.8 and 6.4 ± 2.8
296
mg L-1 respectively. The observed concentrations of these carbonyl compounds from 2D COSY
297
and HSQC spectra are well within the range of those reported previously.1,
298
survey of targeted carbonyl-bisulfite adducts, reported for the first time, show distinct differences
299
and variation in their concentration between the 8, 13 and 15 bisulfites. In the wines tested, 8 and
300
13 were found at higher concentrations, 28.2 ± 60.2 and 110 ± 106.2 mgL-1, respectively,
301
followed by 15 (7.8 ± 8.8 mgL-1). Not surprisingly, acetaldehyde and pyruvate bisulfite adducts
302
have some of the lowest reported dissociation constants of quantitatively important carbonyls,11
303
whereas the significance of the methyl glyoxal-bisulfite adducts, 14 and 15 has not been clarified
304
in dry wines. However, the identification of these bisulfites at significant concentrations in wines
305
older than one year (data not shown) indicates stability and apparently slow reaction of the free
306
carbonyl with other substrates under wine aging conditions. In addition, the distribution of these
307
bisulfites in the assessed wines showed no correlation with the vintage or the grape variety,
308
suggesting a that other factors, perhaps fermentation conditions or oxidation during processing
309
contribute to the free and sulfite bound carbonyls levels in wine.
16
Moreover, the
310
Influence of sulfur dioxide concentration at bottling on the levels of free and sulfite
311
bound carbonyls in wines. The development of free and sulfite bound carbonyls during bottle
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312
aging in the presence of different levels of sulfur dioxide was studied by the screening of these
313
compounds in a wine (Cabernet sauvignon, Oakville, Napa, 1997) bottled with 30 and 120 mgL-
314
1
315
sulfur dioxide addition generally resulted in both treatments having little accumulation of 1,
316
indicating the control of chemical oxidation even at the low sulfur dioxide concentration (30
317
mgL-1), or a reaction pathway comparable to the reaction rate.
sulfur dioxide (Figure 4). The analysis of the wines at 15 years of bottle aging showed that the
318
High sulfur dioxide containing treatment (120 mg L-1) increased 8, 13 and 15 bisulfite
319
adducts formation by 18%, >100% and >100%, respectively, indicating that hydroxysulfonate
320
adducts have substantial stability under wine aging conditions. Interestingly, in the case of high
321
sulfur dioxide treatment, between 1, 4 and 5, only 4 was found present in free form. This suggests
322
that, under wine aging conditions, 4 is a weaker sulfur dioxide binder compared to 1 and 5.
323
However, sulfur dioxide excess at bottling had not affected 10b concentration in wines,
324
indicating weak sulfite binding by 3. Moreover, 1 showed the highest binding with sulfur dioxide,
325
since even in the low sulfur dioxide treatment (30 mg L-1) only its hydroxysulfonate adduct, 8,
326
was detected. This observation is supportive of the low dissociation constant (Ks) reported for 8
327
in model wine solution.2,
328
resulted in much higher levels of both 5 and its hydroxysulfonate, 15.
12
One mystery is why the elevated level of sulfites during aging
329
CONCLUSIONS
330
2D NMR can be used for the accurate quantitation of carbonyl compounds and their
331
sulfite bound forms in wine. This analytical capacity paves the way toward a number of studies to
332
better understand how wine carbonyls could be formed or react with other wine constituents.
333
Such studies could provide insight into yeast metabolism during fermentation, when a large
334
fraction of the carbonyls are formed, or as a result of oxidation, such as during micro-
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335
oxygenation treatments. While the hydroxysulfonate of acetaldehyde is often attributed to the
336
“bound” sulfites observed in wine, this data clearly shows there are several additional carbonyls
337
that are found in sulfite-bound forms in substantial amounts, adding up to some tens of
338
milligrams per liter. The data provided here leads to the question of whether or not these
339
hydroxysulfonates can contribute to, or subtract from, microbial stability or antioxidant
340
protection of wines provided by SO2, particularly as the wine ages and sulfites level decline.
341 342
AKNOWLEDGMENT
343
We sincerely thank the American Vineyard Foundation (AVF) for its financial support.
344
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References
346
(1)
Jackowetz, J. N.; Mira de Orduña, R. Food Control 2013, 32, 687-692.
347 348
(2)
Barbe, J.-C.; de Revel, G.; Joyeux, A.; Lonvaud-Funel, A.; Bertrand, A. Journal of Agricultural and Food Chemistry 2000, 48, 3413-3419.
349 350
(3)
Bakker, J.; Timberlake, C. F. Journal of Agricultural and Food Chemistry 1997, 45, 3543.
351 352
(4)
Fulcrand, H.; Cheynier, V.; Oszmianski, J.; Moutounet, M. Phytochemistry 1997, 46, 223227.
353 354
(5)
Fulcrand, H.; Benabdeljalil, C.; Rigaud, J.; Cheynier, V.; Moutounet, M. Phytochem. 1998, 47, 1401-1407.
355 356
(6)
Drinkine, J.; Lopes, P.; Kennedy, J. A.; Teissedre, P. L.; Saucier, C. Journal of Agricultural and Food Chemistry 2007, 55, 1109-1116.
357 358
(7)
Lorette, N. B.; Howard, W. L.; Brown, J. H. The Journal of Organic Chemistry 1959, 24, 1731-1733.
359
(8)
Peterson, A. L.; Gambuti, A.; Waterhouse, A. L. Tetrahedron 2015, 71, 3032-3038.
360
(9)
Wlodek, L. Acta Biochim. Pol. 1988, 35, 307-317.
361 362
(10)
Burroughs, L. F.; Sparks, A. H. Journal of the Science of Food and Agriculture 1973, 24, 199-206.
363 364
(11)
Burroughs, L. F.; Sparks, A. H. Journal of the Science of Food and Agriculture 1973, 24, 207-217.
365 366
(12)
Burroughs, L. F.; Sparks, A. H. Journal of the Science of Food and Agriculture 1973, 24, 187-198.
367
(13)
Han, G.; Wang, H.; Webb, M. R.; Waterhouse, A. L. Talanta 2015, 134, 596-602.
368
(14)
Ough, C. S.; Amerine, M. A. 2d ed. Wiley & Sons, New York 1988.
369 370
(15)
Lau, M. N.; Ebeler, J. D.; Ebeler, S. E. American Journal of Enology and Viticulture 1999, 50, 324-333.
371 372
(16)
Elias, R. J.; Laurie, V. F.; Ebeler, S. E.; Wong, J. W.; Waterhouse, A. L. Anal. Chim. Acta 2008, 626, 104-110.
373
(17)
Jackowetz, J. N.; Mira de Orduña, R. Food Chemistry 2013, 139, 100-104.
374 375
(18)
de Azevedo, L. C.; Reis, M. M.; Pereira, G. E.; da Rocha, G. O.; Silva, L. A.; de Andrade, J. B. Journal of Separation Science 2009, 32, 3432-3440.
376 377
(19)
Lea, A. G. H.; Ford, G. D.; Fowler, S. International Journal of Food Science & Technology 2000, 35, 105-112.
378
(20)
Wildenradt, H. L.; Singleton, V. L. Am. J. Enol. Vitic. 1974, 25, 119-126.
379
(21)
Waterhouse, A. L.; Laurie, V. F. Am. J. Enol. Vitic. 2006, 57, 306-313.
380
(22)
Chiang, Y.; Kresge, A. J. Journal of Organic Chemistry 1985, 50, 5038-5040. 18
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381 382
(23)
Gronwald, W.; Klein, M. S.; Kaspar, H.; Fagerer, S. R.; Nürnberger, N.; Dettmer, K.; Bertsch, T.; Oefner, P. J. Analytical Chemistry 2008, 80, 9288-9297.
383
(24)
Rai, R. K.; Tripathi, P.; Sinha, N. Analytical Chemistry 2009, 81, 10232-10238.
384 385
(25)
Van, Q. N.; Issaq, H. J.; Jiang, Q. J.; Li, Q. L.; Muschik, G. M.; Waybright, T. J.; Lou, H.; Dean, M.; Uitto, J.; Veenstra, T. D. Journal of Proteome Research 2008, 7, 630-639.
386 387
(26)
Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. The Journal of Organic Chemistry 1997, 62, 7512-7515.
388
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Table and Figure captions Table 1:Assignment of 2D 1H-1HCOSY NMR signals of free and sulfite-bound carbonyl compounds in wine. Table 2:Assignment of 2D 1H - 13C HSQC NMR signals of free and sulfite-bound carbonyl compounds in model wine and real wine that were used for calibration and quantitation. Table 3:Calibration line parameters average concentrations, standard deviations, and ranges of selected wine free and sulfite bind carbonyls (n = 15). The compound numbering is defined in Figure 1. *1; 4; 5 concentrations are expressed are the sum of 1,6,7; 4, 12; 14a, 14b,respectively. Table 4: Method accuracy and precision estimated by performing triplicate analyses of a wine sampled spiked with two known concentrations of each free and sulfite bound carbonyl compound. The compound numbering is defined in Figure 1. *1; 4; 5 concentrations are expressed as the sum of 1,6,7; 4, 12; 14a, 14b,respectively. Figure 1: Structures of identified free and sulfite bound carbonyl compounds. Acetaldehyde (1), hydrated acetaldehyde (6), acetaldehyde ethyl hemiacetal (7), acetaldehyde bisulfide (8), acetoin (2), acetoin bisulfite (9ab, isomer I), acetoin bisulfite (9cd, isomer II), αketoglutaric acid (3), hydrated ketoglutaric acid (10a), hydrated ketoglutaric acid (10b, cyclic form), α-ketoglutaric acid bisulfite (11a), α-ketoglutaric acid bisulfite (11b, cyclic form), pyruvic acid (4), hydrated pyruvic acid (12), pyruvic acid bisulfite (13), methylglyoxal (5), hydrated methylglyoxal (14a, form I), hydrated methylglyoxal (14b, form II), and methylglyoxal bisulfite (15).
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Figure 2: Portion of the (A) 1H-13C HSQC and (B) 1H-1HCOSY 600 MHz cryo-probe spectrum of identified free and sulfite bind carbonyl compounds in wine sample. The compound numbering is defined in Figure 1. Figure 3: Average values along with the standard deviation of the concentration of free and sulfite bound carbonyl compounds of wine samples (n=15). The compound numbering is defined in Figure 1. *1; 4; 5 concentrations are expressed as the sum of 1,6,7; 4, 12; 14a, 14b respectively. Figure 4: Quantitation of free and sulfite bind carbonyls compounds in wine samples (Cabernet Sauvignon from Oakville, Napa, 199 9, spiked at bottling with 20 and 120 mg/L of sulfites. All quantitation assays were performed in April 2013.The compound numbering is defined in Figure 1. *1; 4; 5 concentrations are expressed as the sum of 1,6,7; 4, 12; 14a, 14b,respectively.
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Table 1
compound 1 2 6 7 8 9ab 9cd
chemical shift (δ) (ppm) 1 1 H H assignment 9.67 2.23 H1-H2 1.36 4.40 H3-H4 5.24 1.31 H1-H2 4.94 1.30 H1-H2 4.54 1.46 H1-H2 1.24 4.16 H3-H4 1.24 4.00 H3-H4
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Table 2 chemical shift (δ) (ppm) 1 13 compound H C assignment 2.21 25.53 H1/C1 2 2.37 26.90 H3/C3 4 4.16 70.06/69.98 H3/C3 9abcd 2.68 28.24 CH2 10b 2.56 29.53 CH2 11b 2.49 29.53 CH2 2.34 29.12 CH2 2.25 29.12 CH2 1.56 25.81 H3/C3 12 1.72 21.28 H3/C3 13 2.29 25.18 CH3 14a 1.35 22.15 CH3 14b 2.42 27.73 CH3 15
NOTE The spectra were calibrated according to the ethanol peak at 1.17 ppm (proton) and 17.47 ppm (carbon) according to 26.
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Table 3
compounds calibration curve fitted regression equation
r2
linear range (mgL-1)
validation range (mgL-1)
wine conc (mgL-1) mean ± SD
1*
y = 0.0022x + 0.008
0.997
0.7-50
1.1-50
17.5 ± 32.6
2
y = 0.0057x + 0.007
0.992
9.8-200
13.2-200
12.7 ± 7.8
4*
y = 0.0039x + 0.006
0.997
1.2-200
3.6-200
36.0 ± 27.8
5*
y = 0.0032x + 0.004
0.994
8.6-150
12.5-200
6.4 ± 2.8
8
y = 0.0052x + 0.080
0.989
2.5-600
7.0-600
28.2 ± 60.2
10b
y = 0.0045x + 0.022
0.990
10.3-200
16.8-200
106.5 ± 56.7
13
y = 0.0034x + 0.062
0.998
8.2-432
10.3-432
110.0 ± 106.2
15
y = 0.0037x + 0.001
0.994
16.2-435
19.1-435
7.8 ± 8.8
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Analytical Chemistry
Table 4
compounds
1* 2 4* 5* 8 10b 13 15 1
concentration accuracy (%R)1
precision SD
CV (%)
3
102 ± 0.01
0.00
2.5
10
98 ± 0.01
0.00
2.9
15
100 ± 0.02
0.32
7.1
50
92 ± 0.01
0.02
4.2
15
102 ± 0.10
0.06
3.4
50
99 ± 0.02
0.01
2.7
15
98 ± 0.01
0.07
4.3
50
109 ± 0.06
0.01
3.6
15
92 ± 0.07
0.06
1.2
50
96 ± 0.02
0.01
0.8
15
84 ± 0.25
0.14
5.6
50
116 ± 0.38
0.08
6.1
15
97 ± 0.01
0.21
7.2
50
113 ± 0.04
0.13
5.4
15
96 ± 0.04
0.15
4.5
50
98 ± 0.03
0.07
3.5
%R = percentage recovery
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Figure 1
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Figure 2
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Figure3
40
120
35
100
30
80
25
60
20
40
15
20
10
0
5
-20
Mean Mean±SE Mean±SD
-40
1*
2
4*
13
5*
15
28
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1
0
n
Conentration (mg/L)
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8
10b
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Figure 4
Wine-low SO2 Wine-High SO2
1*#
8#
2#
4*#
13#
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5*#
15#
10b"
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2D qNMR
HSO3-
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