In Situ Determination of Fructose Isomer Concentrations in Wine Using

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In situ Determination of Fructose-Isomer Concentrations in Wine Using Quantitative 13C NMR Spectroscopy Cinzia Colombo, Clara Aupic, Andrew R. Lewis, and B. Mario Pinto J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03641 • Publication Date (Web): 09 Sep 2015 Downloaded from http://pubs.acs.org on September 13, 2015

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Journal of Agricultural and Food Chemistry

In situ Determination of Fructose-Isomer Concentrations in Wine Using Quantitative 13C NMR Spectroscopy

Cinzia Colombo,*,† Clara Aupic,† Andrew R. Lewis, † B. Mario Pinto*,†

†

Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British

Columbia, V5A 1S6, Canada.

*

Address correspondence to C. Colombo or B. Mario Pinto, Department of Chemistry, Simon

Fraser University, Burnaby, British Columbia, Canada V5A 1S6. Tel: 778-782-5650; Fax: 778782-3765; E-mail: [email protected] or [email protected].

1 ACS Paragon Plus Environment


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ABSTRACT: A practical method for simultaneously quantifying fructose and ethanol contents

2

in wines using

3

the method leaves an unmodified sample available for subsequent testing or additional

4

analyses. The relative ratios of the five known fructose isomers in ethanolic solutions at

5

different pH and their variations with temperature are also reported. The data are correlated

6

with the sweetness of wines. The technique was applied to commercially available wines and

7

the results are compared with other methods. Sugar levels above 0.6 g/L can also be

8

measured. A simple adaptation of the method permits measurement of different

9

carbohydrates using integration of single peaks for each compound, in combination with an

13

C-qNMR spectroscopy is reported. Less than 0.6 mL of wine is needed, and

13

10

external reference

C-qNMR spectrum of a sample with known concentration. The method

11

can be applied at all stages of wine production, including grape must, during fermentation,

12

before and after bottling.

13 14

KEYWORDS:

15

content.

13

C NMR spectroscopy, fructose isomers, qNMR, wine analysis, carbohydrate


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INTRODUCTION

17

Fructose occurs naturally in grape berries and is one of the principal components of sweet

18

white wines, which are composed predominantly of water, ethanol and carbohydrates (mainly

19

fructose and glucose). Grape vines produce sucrose by photosynthesis, which is moved into

20

the berries during ripening where it is hydrolyzed by the enzyme invertase into glucose and

21

fructose for storage.1 By the time of harvest, between 15 and 25% of the grape will be

22

composed of simple sugars, mainly six-carbon sugars like glucose and fructose but also small

23

amounts of three-, four-, five- and seven-carbon sugars.2 During the fermentation process that

24

transforms grapes into wine, yeast consumes the natural sugars in ripe grapes and

25

transforms them into alcohol and carbon dioxide. Hence, most wines are “dry” (meaning, no

26

apparent sweetness or residual sugar) or almost dry.3 However, many sweet wines or dessert

27

wines taste sweet because of the residual sugars they contain. Naturally sweet wines are

28

those in which the alcohol and sweetness are exclusively from grapes, which originally

29

contain high concentrations of sugars, usually achieved by leaving the grapes on the vines for

30

as long as possible, intentionally drying the grapes after harvest, or harvesting berries that

31

have frozen (i.e. eiswein or ice wine).4 Back blending is the process of sweetening wine after

32

fermentation. The most common ways of back sweetening are by adding sugar or sterilized

33

unfermented grape juice or sweet reserve to a finished wine, although in most jurisdictions

34

(including California but not for other US states), the addition of sugar rather than some form

35

of grape juice is forbidden. Typically, dry and sparkling wines contain less than 5 g/L of total

36

residual sugars, medium and sweet white wines may have up to 40 g/L, fortified wines (e.g.

37

sherry or port etc.) have as much as 150 g/L, and late harvest and ice wines may contain up

38

to 200 g/L.5



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Several methods of analysis for carbohydrate chemical composition in complex natural

40

mixtures have been developed for use in the food and beverages industries, and interest has

41

grown with the current concerns about diet composition6 and fructose-related diseases such

42

as diabetes,7

43

sources and principal sweeteners.8 Furthermore, there has been a growing interest in high-

44

quality dessert wines, which recently earned a higher degree of respect in the court of wine

45

consumer opinion.3

46

dependent, i.e., the perceived sweetness in a food or beverage system depends on several

47

factors, including temperature, pH, solids content, and the presence of other sweeteners.9-10

reflecting the predominance of fructose and sucrose as the main energy

Although sweetness is an intrinsic chemical property, it is system

48 49

Fructose is recognized as the sugar responsible for the major differences in sweetness

50

occurring with changes in temperature, and it served as a model for the development of a

51

general theory of sweetness.15 After over 40 years, there is still controversy over the possible

52

biochemical explanation (multiple receptors and multiple transduction mechanisms) for the

53

sweet taste response.11 At least two different sweet taste transduction systems have been

54

proposed to explain the relationship between sweet and bitter taste,12 and thermal sensitivity

55

has been considered to have a role in taste-perceived intensity.13 Although the interesting

56

question about sweet receptors and transductions remains unanswered, this temperature

57

effect has some important consequences for foods that are consumed hot, ice-cold, or at

58

room temperature. For example, the suggested temperature for optimal taste (and

59

sweetness) in the case of white wines is usually between 8 and 12°C. In this temperature

60

range, the most pleasant or favorable balance of the complex, highly inter-related

61

organoleptic properties including sweetness, volatility of chemicals responsible for flavors and

62

fragrances, bitterness, acidity, viscosity and mouth-feel, etc. is reached. 4 ACS Paragon Plus Environment

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Fructose is a reducing sugar that exists as a mixture of 5 isomers, when dissolved in water.

65

The different isomers in the form of β-pyranose, α-pyranose, β-furanose, α-furanose and

66

ketohexose (Figure 1) are distinct compounds, differing from each other in their chemical,

67

physical, and biological properties.14 These forms can be observed by NMR spectroscopy and

68

their proportion in the equilibrium mixture can be measured.

69

sensitivity of

70

mainly in pure water solutions. The sweetness arising from fructose is the most variable

71

among the naturally occurring sugars (this variation is directly related to the mutarotational

72

behavior), and it is well known that the sweetness of aqueous solutions containing fructose

73

markedly decreases as the temperature increases.15 The β-pyranose isomer is reported as

74

the major form in aqueous solution and as the isomer responsible for sweetness, while β-

75

furanose and α-furanose are nearly void of taste; the decrease in sweetness with increasing

76

temperature is attributed to the lesser proportion of the β-pyranose isomer.15

77

techniques including gas chromatography (GC), high-performance liquid chromatography

78

(HPLC),16-17-18-19-20 solid-phase microextraction (SPME)21-22 and spectroscopic techniques

79

such as infrared (IR)23 have been applied to the detection and measurement of fructose and

80

other carbohydrates in wines. However, some of these analytical procedures are somewhat

81

restricted and usually require time-consuming sample preparation procedures.

82

spectroscopy has been used extensively for identifying carbohydrates and other compounds

83

in wines, but its application is limited in that the spectral region where the carbohydrate

84

signals are observed is highly congested due to the presence of overlapping resonances,

85

requiring multidimensional NMR approaches for deconvolution. Consistent analyses of 1H

86

NMR spectra of wine is further complicated by the large number of compounds present and

However, due to the low

13

C NMR, the majority of the studies have been performed with 1H NMR and


Analytical

1

H NMR


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1

87

also by the variations in

H chemical shifts that can occur due to changes in pH,

88

concentration, and the presence of other compounds. For this reason, proper analysis

89

typically requires sophisticated data treatment techniques or alternatively, samples need to be

90

pH-buffered, and ideally have accurate pH adjustments made to minimize chemical shift

91

variations so that reliable assignment of specific resonances to individual compounds is

92

possible.24

93

alignment and fine adjustments are often still required before spectral analyses can be

94

reliably undertaken.25 Although the qualitative and quantitative measurement of sugars in

95

wine based on 1H NMR spectra is challenging, advances have been made possible by the

96

availability of innovative statistical analysis methods25-26 and modern chemometric tools.27

97

There have been multi-site demonstrations for the methodology of the WineScreener,28 which

98

showed the use of identical (400 MHz) NMR instrumentation, in conjunction with

99

standardized, optimized sample preparation, data acquisition, data processing and data

Even with these sample manipulations prior to spectral acquisition, peak

1

100

analysis protocols for chemical analysis of wines and fruit juices based on

H NMR

101

spectra.24,29

102

Although

103

complex mixtures, relatively few papers have described its use for quantitative analysis of

104

carbohydrates in natural mixtures.30-31 Analytical evaluation of honey by

105

revealing its origin and possible adulteration, which utilized a cryogenically-cooled NMR probe

106

to achieve higher sensitivity, has been reported.32-33 Duquesnoy et al. developed a procedure

107

using 13C-qNMR spectroscopy, which allowed the direct identification and quantification of the

108

carbohydrates in ethanolic extracts of two conifers, using an internal standard method.34

109

Concerning wine analysis, a first quantitative analysis of contaminants (diethylene glycol and

110

natural compounds such as sugars, sugar alcohols, glycerol, and sugar acids) by

13

C NMR has been used for the identification of individual components within


13

C-qNMR, aimed at

13

C NMR

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111

was reported in the mid-1980s.35 Most recently, with the introduction of advanced

112

technologies for direct sample analysis, full bottle

113

of acetic acid content in wine.36 Although the method was not practical for the quantification of

114

wine components like tannins, flavonoids, phenols, and aldehydes, it was perfectly suitable

115

for the study of the specific enological problem of acetic acid spoilage, and the quality of fine

116

wines.

13

C NMR was applied for the measurement

117 13

118

We describe herein the use of

C-qNMR for the quantitative determination of the relative

119

fructose-isomer composition and the total fructose content in wines. The method is also

120

applicable to the detection and quantification of other compounds including ethanol, other

121

sugars like glucose and sucrose, or additional chemicals as long as they have at least one

122

13

C signal which does not overlap with resonances from other compounds present.

123 124 125

MATERIALS AND METHODS

126

NMR Sample Preparation. Fructose, sucrose and ethanol-OD (CH3CH2OD, 99.95% pure)

127

were purchased from Sigma Aldrich. D-[UL-13C6] fructose was purchased from Omicron

128

Biochemicals. White wines produced from different grape varieties including Baccus Weiss

129

(2012 vintage), Pinot Gris (2013), Chardonnay Blanc (2013), and a late harvest Riesling

130

(2012) were purchased from wine stores in British Columbia. A sweet Moscato wine (2013)

131

purchased from an Italian winery. 600 MHz-grade 5 mm NMR tubes (from Bruker) were filled

132

with 550 µL of the wine or solution to be analyzed.

133

13

134

operating at 600.33 MHz for 1H and 150.90 MHz for

C NMR Spectra. Spectra were acquired on a Bruker AVANCE II digital spectrometer 13

C equipped with a Bruker 5 mm QNP



13

C has cold (~20 K)

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13

C, 1H and 2H coils, and the

13

135

cryoprobe. This direct-detect probe for

C

136

and

137

maintained at the desired temperature (283, 293 and 310 K) using a Bruker BVT-3000 and a

138

Bruker BCU-05 air controller to regulate the sample temperature by actively controlling the

139

temperature of flow of heated air over the NMR tubes. The air flows ranged from 670 L/h for

140

293 K to 800 L/h for 310 K. These higher than normal flows compared to those used on

141

conventional NMR probes are necessary to compensate for the cooling effect of the cold but

142

vacuum-insulated NMR r.f. coils surrounding the sample. The sample temperature was

143

calibrated using 99.9% methanol37 and was found to show a variation of less than 0.1 K from

144

the displayed value. An inversion recovery pulse sequence was used to measure

145

the carbons of interest with a recycle delay of 120 s and an array of 12 different recovery

146

delays covering the range from 100 ms to 120 s. Relaxation data were fit using the T1/T2

147

analysis routines in the Topspin software. T1 relaxation times for the C-2 carbons of fructose

148

in 15 vol. % ethanol at 298 K were 11.3 s for the keto form, 8.9 s (α-furanose), 8.2 s (β-

149

furanose), 8.5 s (β-pyranose) and 10.2 s (α-pyranose). Quantitative proton-decoupled

150

NMR spectra were acquired using an inverse-gated pulse sequence to eliminate NOE

151

enhancements.38 The

152

ppm was used, and 32 scans, each consisting of 131072 complex points, were accumulated

153

with a recycle delay of 120 s between scans (65 min per spectrum). Proton decoupling was

154

performed using the WALTZ-64 decoupling sequence (1H pulses of 100 µs, 1H transmitter set

155

to 4.00 ppm).39 Five to twelve spectra were recorded for each sample.

156

Dependence of the Equilibrium Composition of Fructose in Wine on Temperature.

157

Fructose was dissolved in 15% ethanol-OD, 85% H2O (fructose concentration 1.11 M). Upon

158

complete dissolution, the samples were transferred to 5 mm NMR tubes and equilibrated at

1

H preamplifier channels are both cooled to 77 K. The sample temperature was

13

C T1's for

13

C

13

C transmitter frequency was set to 100 ppm, a spectral width of 250


Page 9 of 33


13

159

three different temperatures (283, 293 and 310 K).

C-qNMR experiments of 32 scans (65

160

min for each spectrum) were recorded and 6 experiments were performed for each

161

temperature. The effect of pH on the isomeric composition was evaluated by dissolving

162

fructose (fructose concentration 1.11 M) in 15% ethanol-OD, 85% H2O solution, addition of

163

diluted HCl or NaOH to the desired pH (pH 2.0 and pH 6.2, respectively).

164

of 32 scans were recorded for each sample and 6 experiments were performed for each

165

temperature (283, 293 and 310 K). For direct wine analysis, NMR tubes containing 550 µL of

166

pure Riesling and Moscato were were prepared, and five

167

were acquired for each wine at each temperature.

168

Equilibrium Composition of Fructose at 293 K (. Samples at different pH were prepared by

169

dissolving fructose (1.11M) in 15% ethanol-OD, 85% H2O, previously acidified with diluted

170

HCl and NaOH to the desired pH (pH 2.0, 3.0, 5.0, 6.2, respectively). Samples containing

171

different volume percentages of ethanol were prepared by dissolving fructose (1.11M) in 5%,

172

10%, 20%, 30% ethanol-OD respectively. Six 13C-qNMR spectra (32 scans) were recorded for

173

each sample at 293 K.

174

Quantitative Determination using PULCON method. An aqueous solution of sucrose of

175

accurately known concentration (0.293M) was prepared in 15 vol. % ethanol-OD for the

176

external reference sample and

177

respectively. For ethanol quantification, a solution of ethanol-OD (99.95% pure) was used as

178

an external reference and

179

unaltered Baccus Weiss, Pinot Gris, and Chardonnay Blanc wines were added to separate

180

NMR tubes and five successive

181

293 K.

13

C-qNMR spectra

13

C-qNMR spectra (32 scans each)

13

C-qNMR spectra were recorded at 283, 293 and 310 K,

13

C-qNMR spectra were recorded at 293 K. Samples of 550 µL of

13

C-qNMR spectra (32 scans) were recorded of each wine at



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13

C NMR Spectra. Fourier transformation of the FIDs was performed with two-

182

Analysis of

183

fold zero-filling and application of an exponential apodization (line broadening of 2 Hz).

184

Spectra were manually phased and baseline corrected using Bruker Topspin software. The

185

resulting series of quantitative

186

in a Mathematica 5.1 notebook written by Dr. Darren Brouwer.38 Since

187

may be comparable with resonances of low-concentration components, deconvolution was

188

applied and the contribution of satellites was taken into account (see Supporting Information).

189

An initial, automated deconvolution over the chemical shift range of interest for the carbon

190

signals of fructose isomers was performed by reading in the phased, baseline-corrected

191

spectrum. Experimental spectra were fit using the least-squares "nonlinearRegress" function

192

in Mathematica. Starting from the initial parameters, the peak positions, the peak widths at

193

half-height and optimal combination of Lorentzian and Gaussian peaks were allowed to vary,

194

to reach the best matching for the specific sample's resonances.

13

C NMR spectra were deconvoluted using procedures coded 13

C satellites peaks

195 196

RESULTS AND DISCUSSION

197

13

198

fructose in water containing up to 15% ethanol by volume in order to accurately determine the

199

relative isomer ratios. High resolution spectra and accurate quantification using peak area

200

integration and peak deconvolution analysis of these data permitted accurate measurements

201

of relative ratios of the fructose isomers.

202

carried out (Figure 2). Late harvest and dessert wines are made with naturally dehydrated

203

grapes and are very concentrated in sugars. A few hours of data acquisition using a

204

cryoprobe provided

205

quantification and less than 1 mL aliquot of wine was required to perform the analysis. Here

C-qNMR experiments were initially carried out on wine-like mixtures made by dissolving

13

C-qNMR analysis of wines at 293 K was then

13

C NMR spectra with signal to noise ratios suitable for reliable


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206

we describe the application of the PULCON method40 to

13

207

measuring fructose concentrations in wines sampled directly from the bottle and added to a

208

NMR tube by using a sucrose solution as the external quantification standard. PULCON is a

209

method that correlates the absolute intensities of two spectra measured in different solution

210

conditions.40 It overcomes the need to add an additional reference compound (internal

211

standard) of known concentration to the sample being analyzed. PULCON therefore permits

212

direct comparisons of integrals from different spectra and thus also different samples.41 The

213

use of

214

are present in the highest concentrations after water (which has no 13C signal and is therefore

215

not detected) and ethanol (whose

216

Other organic compounds are present at much lower concentrations and do not interfere with

217

the identification of the sugar resonances or peak area determinations of these signals. The

218

protocol used in the present study only requires that an accurately measured volume of wine

219

be transferred directly to the NMR tube. No solvents, standards, or buffer solutions are added,

220

and no pH adjustments, chemical separations, derivatizations, or other pre-treatments are

221

carried out. The high spectral dispersion and narrow peak widths in

222

coupled with the field stability and slow drift rates of the main magnet field available on most

223

modern, shielded superconducting magnets also makes it possible to turn off the usual 2H-

224

based field-locking if desired. This approach eliminates the need to add a deuterated solvent,

225

which means the wine can be tested and analyzed in its native condition.

226

The limit of detection (LOD) for

227

acquisition on the 600 MHz QNP cryoprobe is 0.6 g/L. LOD was measured with samples of

228

fructose of known concentration in wine-like solution. By comparison using 1H NMR, the

229

reported limits of quantification (LOQ) for the Winescreener are 0.2 g/L for sucrose, and 0.5

C-qNMR spectra of wines for

13

C NMR is inherently selective for the fructose isomers in wine because these sugars

13

C signals are well resolved from those of the sugars).

13

C NMR spectra, when

13

C-qNMR, assuming signal to noise of 3:1 for a 1 hour



Page 12 of 33

230

g/L for glucose and fructose. Assuming the LOQ is based on a 10:1 signal to noise ratio, we

231

can estimate that Winescreener LODs are approximately one-third of the LOQ values. The

232

13

233

straightforward, alternative analytical technique for analyzing sugars in wine (and other

234

beverages).

235

(which 1H-NMR based WineScreener requires), eliminates the need for pH adjustments (as

236

13

237

require a specific brand of NMR spectrometer or type of NMR probe, and can be carried out

238

at any magnetic field strength, making it possible to implement it on virtually all NMR

239

spectrometers designed for analyzing liquid samples. Furthermore, in the current

240

method, 550 µL of intact, unmodified wine is simply added directly to an NMR tube, there is

241

no extra time, special equipment or chemical addition required. The WineScreener protocol

242

requires several pre-treatment steps including adding buffer, and adjusting the pH.

243

Equilibrium Composition of Fructose in Wine - Variation with Temperature. Previous

244

investigations aimed at determining fructose isomeric composition resulted sometimes in

245

inconsistent data, due essentially to the complexity of the mutarotation phenomenon and the

246

limitations of the different methods employed.42-43 Isomeric composition studies have been

247

performed in different solvents, suggesting different relative compositions but also giving rise

248

to confusing and incorrect interpretations.44-45

249

unsuitable for determination of the isomeric ratios of fructose because of its low sensitivity

250

and the difficulties in detecting minor isomers like α-pyranose and keto forms.46 However, in

251

our cryprobe-enhanced experiments, 13C signals for all five isomers of fructose (Figure 1) at a

252

reasonable concentration (about 1 M) were observed. The analyses were performed on the

253

resonances corresponding to the C-2 carbon of each isomer: β-pyranose (98.0 ppm), α-

C NMR–based methodology presented in the current work provides a useful,

13

C NMR removes the need to suppress the strong water and ethanol signals

C chemical shifts do not vary much for the range of pH that most wines have), does not

13

C NMR

13

C-qNMR spectroscopy has been considered


Page 13 of 33


254

pyranose (97.8 ppm), β-furanose (101.5 ppm), α-furanose (104.3 ppm), and ketohexose

255

(213.4 ppm). These signals were chosen since they generally do not overlap with other

256

signals in wine samples. In order to guarantee the quantitative reliability of

257

it is necessary to set an interscan delay at least five times as long as the longest T1 value,

258

before applying another pulse, combined with a 90° pulse, to allow complete relaxation of

259

each carbon of the wine compounds to be analyzed. Quantitative spectra can also be

260

obtained using a fast train of short pulses, which leads to a minor change in the steady-state

261

magnetization as T1 is varied. The integration of these two approaches usually provides

262

excellent results.34 We measured the T1 values of all the carbons for pure fructose in ethanol

263

solution 15%, finding T1 values ranging from 0.2 s to 11.3 s. The highest T1s were observed

264

for carbon 2 (11.3 s for the keto form). Based on the largest T1 value, the interscan delay was

265

fixed at 120 s (10 times the highest T1) to ensure accurate signal intensities. WALTZ-16

266

composite pulse decoupling was used to minimize any r.f.-induced heating of the samples.

267

The main objective of this set of experiments was to confirm the general trend observed for

268

changes in the relative ratios of the fructose isomers in wines as the temperature of the

269

samples was altered.

270

temperatures in Moscato and late harvest Riesling is shown in Figure 3 and is very similar to

271

wine-like solutions at different pH (See Supporting Information).

272

The relative amounts of the various isomers of fructose, including the contribution of the keto

273

form, was recently reported by Barclay et al. based on 1H NMR spectroscopic measurements

274

of pure fructose in D2O.47 An accurate quantitative analysis of isomeric forms by

275

spectroscopy was reported by Mega et al. who used

276

(30% D2O, 70% H2O).48

13

C spectroscopy,

The dependence of the individual components with increasing

13

13

C-qNMR

C-enriched fructose in water solution



Page 14 of 33

277

To the best of our knowledge, the current analysis of the relative ratios of the various fructose

278

isomers is the first using real wine samples. These results provide useful information about

279

the actual isomeric composition in aqueous-ethanolic solutions and wines at different

280

temperatures and help resolve the previous inconsistencies in the literature regarding the

281

equilibrium fructose concentrations in ethanolic solutions.45 Our results correlate with those

282

reported by Barclay et al.47 and Mega et al.,48 suggesting no significant variation in the

283

equilibrium isomeric ratios with either the pH, solvent composition (pure water, aqueous

284

solution containing 15% ethanol by volume, or wines) or a combination of these

285

parameters.47-48

286

were apparent in our measurements when comparing the ratios obtained at different pH or

287

fructose concentrations in the presence of ethanol or in wines. The absolutely critical

288

parameter to fix a priori for a quantitative evaluation of fructose is the temperature, so for all

289

other evaluations of the fructose content, the temperature was held constant at 293 K . The

290

dependence of fructose isomeric composition in wines upon temperature variation gives rise

291

to fascinating speculations about the suggested serving temperatures for white wines.

292

Although there are a number of factors responsible for the complexity of wine taste,

293

temperature should be taken into consideration for its contribution to sweetness, which is

294

determined primarily by fructose-isomer proportions.

295

Equilibrium Composition of Fructose at Constant Temperature. Equilibrium compositions

296

of fructose at 293 K of wine and wine-like samples at different pH are reported in Table 1.

297

The negligible variability of peak chemical shifts and invariability of fructose-isomer peak

298

intensities from spectra recorded on different samples is shown in Figure 4. Table wines

299

generally have a pH between 2.5 and 4.5. However, considering that late harvest Riesling

300

and Moscato wine have pH 3.0 and pH 3.9, respectively, a range between 2.0 and 6.2 was

No significant differences in the relative amounts of the various isomers


Page 15 of 33


301

examined in this study. Table 1 shows the variations among the set of ethanolic solution at

302

different pH, revealing a slight percentage variation in the wide range considered. The values

303

reported by Barclay et al.47 and Mega et al.48 are included for comparison. Although the pH

304

has been reported to have an influence on the rate of the mutarotation of fructose48 (which is

305

often approximated by using a simple first-order process representing just the conversion of

306

β-pyranose to the furanose forms), it was previously also reported that the isomeric

307

composition at equilibrium was not significantly affected by variations in pH,

308

corroborated by our data. In order to find a mean percentage value reflecting the distribution

309

of the isomeric forms at constant temperature, we combined data generated from samples at

310

different pH and selected ethanol contents ranging from 5 to 30 % vol ethanol (without pH

311

buffering). As a consequence, the small variations observed are considered to derive from

312

random effects and thus mimic the case of directly analyzing wines without pretreatment, and

313

without knowledge of the actual pH or ethanol concentration.

48

as

314 315

Artificial mixtures of fructose were investigated, and mean values of the molar % ratios were

316

obtained and compared with the values measured using different methods of analyzing the

317

13

318

mean values determined using integration of the peaks for all of the carbons, including the

319

contributions from the associated

320

obtained by peak deconvolutions of signals from all carbons, including the

321

and 3) values calculated using peak areas from peak fitting (deconvolution) using the main

322

resonance and the associated satellite peaks for the signals related to only carbon 2 for each

323

isomer (Figure 6). These satellite peaks arise from the coupling of the observed

324

an adjacent (directly bonded)

C-qNMR spectra (see Supporting Information). Three different techniques were used: 1)

13

C satellite peaks; 2) mean values from peak areas

13

13

C satellite peaks

13

C nuclei to

C at natural abundance and have separations of 30-70 Hz as



Page 16 of 33

325

expected for 1JC-C couplings, and are not due to incomplete proton decoupling. In the latter

326

case, C-2 was selected because it exhibited a well-resolved, reasonably strong peak for all

327

five fructose isomers. Histograms describing the isomeric composition depending on the

328

different analysis methods employed are reported in Figure 5. The largest standard deviations

329

were found when the integration method was used. The smallest spread about the average

330

was achieved using deconvolution analysis for all signals, even though deconvolution applied

331

to just carbon-2 of each isomer also yielded a smaller standard deviation than integration.

332

However, the data obtained from deconvolutions just at carbon-2 of each isomer (Table 2)

333

represent a good compromise between speed and accuracy and were thus considered more

334

appropriate for further evaluation of fructose content in wine. The mean values also match

335

perfectly with the isomeric ratios obtained from a fully

336

ethanolic solution (Table 2).

337

Fructose Quantification in Wine. Concentration measurements with the PULCON method

338

use the principle of reciprocity which indicates that the lengths of a 90° or 360° pulse are

339

inversely proportional to the NMR signal intensity.49-50 When the concentration of external

340

reference sample is known precisely, the unknown concentrations of different samples can be

341

obtained using equation (1), where U and R indices stand for unknown and reference,

342

respectively (fructose and sucrose, respectively).

13

C-enriched fructose sample in 15 %

∙ ∙ ∙

= ∙ ∙ ∙ ∙

343

(1)

344

C is the concentration, T is the temperature, θ90 is the 90° pulse length, n is the number of

345

transients used for the experiments, and k is a correction factor taking into account the use of

346

different receiver gains for measurement of the reference and of the unknown samples, or

347

incomplete relaxation. In our experiments T and n have the same values for both the 16 ACS Paragon Plus Environment

Page 17 of 33


348

reference and the unknown sample, so the formula could be further simplified. The reference

349

sucrose spectra were measured five times and areas were calculated for all the carbon

350

signals, averaged and used in the PULCON equation (AR, eq 1). We investigated the

351

reliability of the PULCON method for the determination of fructose content with wines already

352

analyzed, comparing our results with those from a WineScreener analysis carried out on

353

different bottles of the (supposedly) identical wines.

354

Pinot Gris and Chardonnay Blanc wines were measured. The most important cause of

355

different signal strength (peak areas) between spectra derives from differences in

356

conductivities of the samples, which results in a variation of 90° r.f. pulse length. However, as

357

long as the probe is properly tuned and matched for each sample, this effect can be

358

compensated for by applying the principle of reciprocity.49 For each sample, the magnetic field

359

was shimmed to provide symmetrical peak shapes and the actual duration required for a true

360

90° r.f. pulse was determined, keeping the power of the r.f. pulse fixed. All other acquisition

361

and processing parameters remained unaltered. The quantitative reliability of the NMR peak

362

area measurements can suffer if the delay between successive r.f. pulses scans is too short

363

to allow full recovery of the magnetization. An inter-scan delay of 120 s was employed, which

364

afforded > 99.9% recovery of magnetization for the fructose carbons having the largest

365

longitudinal relaxation times (C-2 in the keto form, T1 = 11.3 s). For fructose quantification, the

366

information obtained from relative isomer composition (Table 2) was used and equation (1)

367

was modified, leading to a simpler formula for calculating the fructose concentrations in wine

368

samples. Just considering the area of the region between 97.0 and 99.9 ppm (related to the

369

β-pyranose and α-pyranose isomers) which from reported evaluations (Table 2) accounts for

370

70% of the total concentration of fructose, fructose content in wine samples could be

13

C-qNMR spectra of the Baccus Weiss,



Page 18 of 33

371

expressed as reported in equation (2), where cR is the molar concentration of a sucrose

372

reference sample, ∗ is the area of the region 97.0-99.9 ppm.

= ∙

373

∙ ∙

∙ ∗ ∙ ∙

(2)

374

Fructose concentrations (in g/L) can be directly calculated using equation (2) by measuring

375

the peak areas related to the two isomers (13C satellite peaks included) and applying a

376

multiplication factor determined from product of the molecular weight of fructose M (180

377

g/mol) and the fractional amount of total fructose (based on a percentage estimation of the

378

isomers being used; 100/Pu, where PU is 70).

379

The values obtained compare remarkably well with those measured using the 1H NMR-based

380

WineScreener analysis (Table 3). These

381

repeated 5 times for each wine sample, and the precision of the measurements was

382

estimated using the standard deviations calculated from these separate measurements.

383

Equation (2) can be extended to measure other carbohydrates such as glucose and sucrose

384

present in wine, by measuring the peak areas related to one of the two isomers for glucose

385

(β-pyranose and α-pyranose isomers) or by measuring the peak area of one isolated signal

386

for

387

fructofuranoside). The multiplication factor M and percentage estimation of the isomers

388

should be adapted to the carbohydrate to be measured. Baccus Weiss wine contained 1.6 g/L

389

sucrose (WineScreener) which we could detect readily by 13C-qNMR peaks at 81.3 and 103.6

390

ppm (see Supporting Information). From the same spectra, ethanol concentrations could also

391

be determined. An external reference, a sample of 99.95% Ethanol-OD was used and

392

equation (3) was employed.

sucrose

(which

exists

as

a

13

C NMR-based concentration measurements were

single

isomer:

O-α-D-glucopyranosyl-(1-2)-β-D-

∙ ∙

ℎ"#$ %&$ %( = 99.95 ∙ ∙ ∙

393


(3)

Page 19 of 33


394

These results are also in close agreement with those obtained using the official WineScreener

395

analyses and printed on the bottle labels (Table 4), especially for the vol. % ethanol contents.

396

The one outlier was the Baccus Weiss wine, which yielded a significantly (more than 1% by

397

volume) higher alcohol content based on the two independent NMR-based analyses than

398

reported on the label.

399

In conclusion, a straightforward method is described for the direct determination of fructose

400

isomers and total contents in wines without chemically altering the samples.

401

analysis works essentially as a sensitivity “filter”, removing the problem of water and ethanol

402

suppression (which are the highest abundance components of wines) on the one hand and

403

avoiding the detection of minor components and low abundance substances on the other

404

hand. This type of analysis can also be readily performed on 400 MHz NMR spectrometers,

405

extending the acquisition to a simple overnight experiment (see Supporting Information). This

406

method can detect and quantify ethanol and multiple sugars (and other compounds) at all

407

stages of wine production including grape must, during fermentation, before and after bottling,

408

providing a way to monitor possible back blending practices. The method is also applicable to

409

the analysis of fruit juices (up to 100 g/L sugar), carbonated soft drinks(100-120 g/L), and

410

certain energy drinks (up to 160 g/L sugar).

13

C-qNMR

411 412

ABBREVIATIONS USED

413

GC, gas chromatography; HPLC, high-performance liquid chromatography; IR, infrared; LOD,

414

limit of detection; LOQ, limit of quantification; NMR, nuclear magnetic resonance;

415

13

416

microextraction;

13

C-qNMR,

C quantitative nuclear magnetic resonance; r.f., radio frequency; SPME, solid-phase

417 19 ACS Paragon Plus Environment


Page 20 of 33

418

ASSOCIATED CONTENT

419

Supporting Information Available: 13C-NMR spectra and tables. This material is available free

420

of charge via the Internet at http://pubs.acs.org.”

421 422

AUTHOR INFORMATION

423

Corresponding Authors :

424

[email protected];

425

FUNDING SOURCES

426

This work was supported by the Natural Sciences and Engineering Research Council of

427

Canada, through a Discovery Grant to BMP.

Cinzia Colombo [email protected] or B. Mario Pinto.

428 429

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Page 26 of 33

Page 27 of 33


Figure captions

Figure 1. Structures of fructose isomers, with chemical shift of carbon-2 in wines indicated.

Figure 2. 13C-qNMR spectra of two wines analyzed: a) Moscato; b) late harvest Riesling.

Figure 3. Equilibrium composition of fructose isomers (β-pyranose, β-furanose, α-furanose, α-pyranose and ketohexose in order of abundance) and dependence of the individual components with increasing temperatures in wines: a) Moscato; b) late harvest Riesling.

Figure 4: Representative 13C-NMR spectra of wine-like solutions at 293 K at different pH (pH 2.0, 3.0, 5.0 and 6.2 from the top) and for wines. Wine 1: Late harvest Riesling, pH 3.0; wine 2: Moscato pH 3.9.

Figure 5. Variation in the equilibrium composition of fructose isomers (β-pyranose, βfuranose, α-furanose, α-pyranose and ketohexose in order of abundance) in wine-like solutions at 293 K . Averages values are reported in the graph; the error bars reflect the range of the standard deviation. For each isomer, the three bars reflect different methods of data analysis (in order from left to right, deconvolution of all carbon peaks, deconvolution applied to C2 peaks, and integration of all carbon peaks).

Figure 6. Portion of the

13

C-qNMR spectrum of fructose in ethanolic solvents, showing the

expansion of the area containing carbon-2 from fructose isomers. Each isomer is shown close to its corresponding C-2 peak. 27 ACS Paragon Plus Environment


Table 1.

Page 28 of 33

13

C NMR quantification of individual isomers of fructose at equilibrium, using

the relative molar ratios.

Reference

Method

pH

β-Pyr

β-Fur

α-Fur

α-Pyr

Keto

%

%

%

%

%

Solvent

Barclay et 1

- a)

D2O

68.23

22.35

6.24

2.67

0.50

13

- a)

30% D2O

69.6

21.1

5.7

3.0

0.5

13

- a)

67.22

23.96

6.18

2.01

0.63

69.70

22.35

5.62

1.84

0.48

67.58

23.49

5.91

2.15

0.87

67.86

23.48

5.95

1.98

0.72

69.30

22.71

5.73

1.70

0.57

H NMR

al.

d) 47

Mega et C NMR

al.

e) 48

This

EtOD C NMR

d)

research

15% EtOD 13

C NMR

2.0 15% EtOD

13

C NMR

3.0 15% EtOD

13

C NMR

5.0 15% EtOD

13

C NMR

6.2 15%

13

3.0

Wine 1 b)

68.72

22.68

6.26

1.90

0.44

13

3.9

Wine 2 c)

68.72

22.60

6.22

1.92

0.54

C NMR C NMR

a) Unbuffered solvent. b) Late harvest Riesling (10% EtOH), c) Moscato (15.5% EtOH), d) Measured at 293 K, e) Measured at 294 K.


Page 29 of 33


Table 2. Fructose isomer percentage determination from peak deconvolution at C-2. 13

ppm

isomers

Mean

min

max

σ (g/L)

C6-fructose

98.0

β-pyr

68.12

65.65

69.70

1.28

68.42

104.3

β-fur

23.16

22.08

24.55

0.77

22.93

101.5

α-fur

5.94

5.50

6.79

0.36

5.72

97.9

α-pyr

2.10

1.70

2.63

0.25

2.35

213.4

keto

0.68

0.44

0.97

0.15

0.59

Table 3. Fructose content determination in wine. WineScreener (g/L)

Eq 2 (g/L)

σ (g/L)a)

Baccus Weiss

8.7

8.7

1.2

Pinot Gris

5.3

5.1

1.1

Chardonnay Blanc

4.5

4.5

0.4

a) standard deviation related to the concentrations obtained from five experiments. Each experiments used a 13

C{1H} inverse gated WALTZ-64 CPD, SFU Bruker AV II 600 with QNP cryoprobe (unlocked. T = 293 K. D1 =

120 s, NS = 32, 65 min).

Table 4. Ethanol content determination in wine. Label

WineScreener (%)

Eq 3 (%)

σ (%)a)

Baccus Weiss

12.9

13.5

13.3

0.07

Pinot Gris

13.4

13.5

13.3

0.09

Chardonnay Blanc

13.8

14.0

13.5

0.05

a) ) standard deviation related to the ethanol percentage measured from five experiments.



Figure 1. OH

OH 2

2

O HO HO

O

OH OH

HO HO

β-pyr 98.0 ppm

HO

O HO HO

OH

OH

α-pyr 97.8 ppm

OH

HO

OH

O HO

2

2

OH

OH

HO

β-fur 101.5 ppm

α-fur 104.3 ppm

OH

O

HO

2

OH

OH

OH

keto 213.4 ppm

Figure 2:


Page 30 of 33

Page 31 of 33


Figure 3.

Figure 4.



Figure 5.

deconvolution

deconvolution-C2

integration

80 70

Percentage (%)

60 50 40 30 20 10 0 β-pyr

β-fur

α-fur

α-pyr

keto

Figure 6.


Page 32 of 33

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TABLE OF CONTENTS GRAPHIC


In Situ Determination of Fructose Isomer Concentrations in Wine Using

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