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A HPLC fluorescence-based method for glutathione derivatives quantification in must and wine
Analytica Chimica Acta 660 (2010) 158–163
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A HPLC fluorescence-based method for glutathione derivatives quantification in must and wine Stéphanie Marchand ∗ , Gilles de Revel UMR 1219 Œnologie, ISVV, Université de Bordeaux, 210, Chemin de Leysotte, CS 50008, 33 882 Villenave d’Ornon Cedex, France
a r t i c l e
i n f o
Article history: Received 4 August 2009 Received in revised form 23 September 2009 Accepted 26 September 2009 Available online 2 October 2009 Keywords: Oxidized and reduced glutathione ␥-glutamylcysteine g-Wine Quantification Liquid Chromatography-fluorimetry
a b s t r a c t A simple and automated high performance liquid chromatography (HPLC) method for the separation and quantitative determination of ␥-glutamylcysteine and reduced glutathione (GSH) in wines is reported. This technique involves the use of a pre-column derivatization with 2,3-naphthalenedialdehyde (NDA), an isocratic separation in presence of -cyclodextrine and a fluorimetric detection. The quantification of oxidized glutathione (GSSG) has also been studied, for the first time in wines, using an additive prederivatization step for reduction using glutathione reductase. The method has been designed for use in laboratories with limited equipment. The assay has been optimized and presents very good performances in terms of sensitivity and selectivity. Then, it has been validated for linearity, LOD, LOQ, precision and accuracy. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Glutathione (␥-l-glutamyl-l-cysteinylglycine; GSH) is the principal non-protein thiol ubiquitous in animals, plants and even in microorganisms. Its monitoring is of paramount importance in studies related to oxidative stress, detoxification processes and human diseases. Because of the key role and widespread presence of GSH in biochemical systems, separative detection and quantification methods for GSH have been published from laboratories mostly linked with medical research. However, glutathione seems to be also implicated in wine aroma either as disulfur form with odorous thiols [1–4], or as a storage form of cysteine moieties [5] itself supposed to be implicated in aroma production [6]. Park et al. [7] pointed out the importance of GSH in yeast metabolism. Lavigne et al. [8] studied changes of GSH levels during alcoholic fermentation using capillary electrophoresis method. These studies and quantification methods were significant advances in understanding the role of GSH levels in must and wines. But the lack of a simple and rapid quantification method has prevented a comprehensive evaluation of GSH levels in musts and wines. GSH and ␥-GC do not have unique spectral characteristics that permit easy quantification; instead, they are derivatized with specific probes. Because of its higher sensitivity, fluorime-
∗ Corresponding author. Tel.: +33 557 575841. E-mail address: [email protected] (S. Marchand). 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.09.042
try is preferred to spectrophotometry for GSH detection, but both techniques can be used with most commercial probes. Two of the best derivatizing agents are aromatic dialdehydes, i.e. ophthaldialdehyde (OPA) and 2,3-naphthalenedialdehyde (NDA), now widely used to label nucleophilic primary amino compounds, especially amino acids and peptides [9–11]. NDA–GSH adduct has several advantages; one of them is the presence of an excitation wavelength in the visible range very close to the emission line of the argon laser, which is usually used in confocal microscopy, flow cytometry, and capillary electrophoresis. NDA is also a promising probe for in situ labelling of GSH, and the main advantage of NDA, is the capacity of NDA–aminothiol adducts to be included in the -cyclodextrine (-CD) cavity [12]. The main consequence of the inclusion is an enhancement of the fluorescence ratio. The addition of -CD to the mobile phase of a high performance liquid chromatography (HPLC) method allows better fluorescence detection capacities, better resolution and lower retention times. The present work is directed to develop a very simple and rapid technique for quantifications of ␥-GC, GSH and GSSG in wines, exploiting chemical selective steps and the high sensitivity of the fluorimetric detection. The previously presented arguments have directed our choices to develop a GSH quantification method using derivatization by NDA, and separation by HPLC with -CD. Moreover GSH, because of its very low standard redox potential (E◦ = −240 mV for thiol/disulfide exchange) can be present in large quantities under the disulfide form (GSSG). Even, if wine pH is in favor of the reduced form (GSH), the presence of GSSG in wines
S. Marchand, G. de Revel / Analytica Chimica Acta 660 (2010) 158–163
must be studied and taken into consideration. GSSG has never been quantified in wines. The strategy we chose is the enzymatic reduction of GSSG by glutathione reductase [13] and quantification of the GSH generated.The quantification methods, including reduction step, have been fully optimized and validated considering white and red wine matrixes. 2. Experimental 2.1. Chemicals and reagents GSH, GSSG, ␥-GC, glutathione reductase from baker yeast (E1.6.4.2.), NDA, ethanethiol (EtSH), boric acid (H3 BO4 ), phosphoric acid (H3 PO4 ), sodium hydroxide (NaOH), hydrochloric acid (HCl), ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA), ethanol, methanol, -cyclodextrine, phosphoric acid salt (NaH2 PO4 ·12H2 O), phosphate buffer saline (PBS) were purchased from Sigma–Aldrich (St. Quentin-Fallavier, France). All aqueous solutions were made using highly purified water produced by a Millipore Milli Q system (St. Quentin-en-Yvelines, France). GSH, ␥-GC and GSSG standard solutions were prepared by dissolving the peptides in HCl 0.1 M aqueous solutions supplemented with EDTA (2 mM) (HCl·EDTA). GSH, ␥-GC and GSSG standard solutions (1 mg mL−1 ) were stored at −18 ◦ C before use. Diluted standard solutions were done in HCl·EDTA extemporaneously. 2.2. Wine samples Red and white Bordeaux AOC wines were used to optimize and validate the quantification method. Samples of wine were spiked at several concentrations for each analysis. The wine is conserved in small scale tanks in the cellar of the institute. After validation, several wines from different regions of France were analyzed following the validated methodology. They were commercial wines or wines offered by producers. To study of GSH and GSSG levels in wine with or without malolactic fermentation (MLF), five Merlot wines have been sampled from five tanks in two wineries from the Bordeaux area, just before lactic bacteria inoculation and at the end of MLF. 2.3. GSSG quantification Oxidized glutathione (GSSG) has to be reduced before quantification. The reduction is performed using glutathione reductase. To conserve the enzyme activity, 5 L of glutathione reductase and 50 L of sample are dissolved in 950 L of phosphate buffer saline solution pH 7.4 containing 1% ethanethiol, before incubation during 5 min at 20 ◦ C. The GSSG level is obtained from total GSH level minus reduced GSH level then dividing this difference by 2. 2.4. Derivatization procedure A 1 mg mL−1 NDA solution was prepared in ethanol and stored at −18 ◦ C for up to one month without loss of activity. The derivatization procedure was performed by an autosampler as follows: to 4 L of sample were added 5 L of borate buffer and automatically mixed, then 1 L of NDA solution was added, and the resulting 10 L were mixed during 4 min before injection in the HPLC system. A borate buffer was prepared from 0.2 M H3 BO4 and 1% (v/v) ethanethiol in highly purified water and aqueous NaOH was added to adjust the pH to 9.2. NDA solution was composed of 2,3-naphthalenedialdehyde 5 mM in ethanol. Borate buffer and NDA solutions were poured separately in ready-to-use vials and stored at −18 ◦ C before use.
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Table 1 Comparison of calibration curve slopes for optimization borate buffer/spiked white wine ratio for ␥-GC and GSH derivation by NDA during 4 min at 20 ◦ C. Sample
1 2 3 4 5 6 7
Slopes (A.U. M−1 )
Volumes (L) Borate buffer
NDA solution
GSH
8 7 6 5 4 3 2
1 1 1 1 1 1 1
0.8 1.3 2.1 2.7 3.3 1.8
␥-GC 0.6 1.0 1.6 2.1 2.6 1.3 No peaks
2.5. Instrumentation and operating conditions A HP 1100 HPLC instrument from Agilent Technologies (Massy, France) was used throughout this study. It comprised a binary pump, an autosampler and a fluorimetric detector. The excitation and emission wavelengths were 467 and 525 nm respectively [11]. Chromatographic parameters (peak retention times and areas) were calculated via the Chemstation® software. A Purospher STAR® RP-18 (5 m) HPLC cartridge (250 × 4.6 mm i.d.) associated with a Purospher STAR® guard column (4 mm × 4.6 mm i.d.) from Merck (Darmstadt, Germany) were used for separation of GSH and ␥-GC. This material can tolerate alkaline mobile phases. The mobile phase was composed of methanol and phosphate buffer. The ratio have been optimized (Table 2) and the chosen one is methanol:buffer 15:85 (v:v). The phosphate buffer was composed of NaH2 PO4 ·12H2 O (10 mM) and -CD (optimized at 3 mM) in highly purified water; pH is adjusted to 8.5 using NaOH solutions. The mobile phase was filtered using a vacuum filtration system through 0.45 m membrane filter. 3. Results and discussion 3.1. Method optimization 3.1.1. Optimization of derivatization parameters Several parameters of the automatic derivatization procedure were optimized to minimize the quantification limit by comparison of calibration curves slopes. A white wine was spiked with five levels of both GSH and ␥-GC (5, 12, 15, 18 and 30 M for GSH and 10, 19, 24, 29, 48 M for ␥-GC). The two sets of five samples were injected varying derivatization parameters. Peak areas were plotted versus concentrations, and they presented linear relationships. The calibration curve slopes were then plotted versus the variations of derivatization parameters and the best value was determined. As previously reported the condensation between NDA and either glutathione or ␥-GC is highly pH dependent [11]. As a result, wines were mixed with borate buffer pH 9.2 before NDA addition. The buffer/sample ratio was optimized using a 4 min derivatization time. The calibration curve slopes were proportional to sample volume if this latter is between 1 and 5 L; derivatization decreased if sample volume was above 5 L (Table 1). This result agrees with pH monitoring by adding borate buffer to white wine. The mixed pH must be greater than 8.5 to permit good derivatization. To be sure the pH conditions are optimized even with very acidic must and wines, the conditions selected were 4 L sample mixed with 5 L borate buffer and 1 L NDA solution in ethanol. The derivatization step time was optimized with variation of mixing time. Calibration curve slopes are plotted versus derivatization time (Fig. 1). Four minutes seems to be a good compromise for derivatization time.
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Fig. 2. Typical chromatogram obtained for the optimized ␥-GC and GSH quantification method.
Fig. 1. Comparison of calibration curve slopes for optimization NDA–aminothiols derivatization time in white wine. The derivatization is performed at 20 ◦ C by mixing 4 L sample, 5 L borate buffer and 1 L NDA solution.
Table 2 Influence of (a) -CD concentration in HPLC aqueous mobile phase and of (b) methanol/phosphate buffer ratio, on retention times and (c) resolution. Mobile phaseb
10:90 15:85 20:80 25:75
[-CD] = 3 mMa
[-CD] = 4 mM
trGSH (min)
tr␥-GC (min)
Rc
trGSH (min)
tr␥-GC (min)
R
25.5 15.0 10.5 8.0
20.6 12.8 9.7 8.0
6.3 4.0 2.2 0.0
13.1 10.0 6.5 5.5
11.4 8.0 6.5 5.5
3.5 2.6 0.0 0.0
3.1.2. Optimization of mobile phase Two parameters for the mobile phase can be optimized. The first one is the -CD concentration in phosphate buffer. -CD presents two main advantages: increasing fluorescence of NDA–aminothiol adducts and decreasing retention times without any loss of resolution [13]. The second one is the percentage of methanol. The tested [-CD] concentrations were 3 and 4 mM. For each concentration the effect of the methanol/phosphate buffer ratio on retention time of the analytes and on resolution (R) was studied using an aqueous solution, containing HCl and EDTA at pH 3.5, spiked with glutathione and ␥-GC at around 300 M each. Results are summarized in Table 2. A good compromise for mobile phase composition is [CD] = 3 mM and the MeOH percentage at 15%. It should be noted that in the preparation of the mobile phase the -CD mass must be precisely determined using a scale. An example of a chromatogram obtained with the optimized analysis parameters is presented in Fig. 2.
3.1.3. Optimization of conditions for GSSG reduction The equilibrium of the GSH/GSSG redox couple could be an interesting point to study in order to collect pertinent data on GSH in wines. For the measurement, GSSG is usually reduced to GSH, and then the reduced/oxidized ratio can be determined, or the total GSH pool is calculated. Many ways for the GSSG reduction have been reported [14]. Because of the specificity of glutathione reductase, the enzymatic method is optimized for GSSG quantification in wines. The GSSG standard solution (160 M) is used to evaluate the best conditions for the enzyme performance and GSH stabilization. 5 L of glutathione reductase suspension and 50 L of sample are dissolved in 950 L of solvent during 1, 5, 7 or 10 min and at the temperatures of 4, 20 or 30 ◦ C. The tested parameters were solvent composition and pH, time and temperature of reduction. The ratio [GSH measured]/[2 × GSSG in solution] (%) is used to compare parameters. The first parameter which was optimized was the solvent. Time and temperature were arbitrarily fixed to 5 min and 4 ◦ C. Four different solvents were tested: PBS at pH 7.4, PBS with ethanethiol 1% (v/v) pH 7.3, HCl·EDTA at pH 1.45 and HCl·EDTA with ethanethiol 1% (v/v) at pH 1.43. These four solvents were selected to provide neutral medium (optimal for glutathione reductase activity) and acidic medium (optimal for GSH conservation). Ethanethiol solution was added as an antioxidant in order to enhance the stabilization of GSH after reduction. Recovery values presented in Table 3 show that PBS with ethanethiol is the only solvent in which the GSSG is correct. These results allow to discard all tested solvents except PBS with ethanethiol. The second optimized parameter was the influence of the pH on GSH recovery. Time and temperature were also arbitrary fixed to 5 min and 4 ◦ C, and the solvent was the previously selected one. Addition of either NaOH or H3 PO4 solutions was employed to change pH values. Recovery values are presented in Fig. 3 and show an optimum for pH 8.5. Because recovery values between pH 7.5 and 8.5 increase only slightly, the pure PBS with EtSH 1% at pH 7.3 was chosen for further study. The third optimized parameter was the influence of the time of reduction. Temperature was also arbitrarily fixed to 20 ◦ C, and the solvent was the previously selected one. Recovery values presented in Table 3 show that the differences between time ranges
Table 3 Optimization of solvent composition, time and temperature for GSSG reduction by glutathione reductase (n = 3). T = 4 ◦ C, t = 5 min
T = 20 ◦ C in PBS + EtSH
t = 5 min in PBS + EtSH ◦
Solvents
Recoveries (%)
Time (min)
Recoveries (%)
T ( C)
Recoveries (%)
PBS PBS + EtSH HCl·EDTA HCL·EDTA + EtSH
0 68 ± 5 0 14 ± 1
1 5 7 10
73 ± 12 79 ± 11 78 ± 11 70 ± 10
4 20 30
52 ± 9 78 ± 10 73 ± 10
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Table 4 Detection and quantification limits, repeatability, reproducibility and linearity (n = 3) of the method in red and white wine matrixes. In white wine matrix
In red wine matrix
GSH
GSSG
␥-GC
GSH
GSSG
␥-GC
LOD (M) LOQ (M) Repeatability Reproducibility
0.1 0.3 5% 5%
4 13 10% 15%
0.6 2.0 6% 13%
0.1 0.4 3% 12%
5 15 8% 16%
0.8 2.5 7% 18%
Linearity r2 Slope (M) % slope var.
0.988 2.6 9%
0.994 0.25 4%
0.990 2.1 9%
0.989 3.0 5%
0.996 0.3 5%
0.975 2.2 5%
GSH quantification linearity test, taking into account the dilution in PBS and the stoichiometry of GSSG reduction.
Fig. 3. Optimization of solvent pH (PBS + EtSH 1%, v/v) for GSSG reduction by glutathione reductase (n = 3).
are slightly noticeable. Nevertheless, running the reaction from 5 to 7 min, improves the recovery. Five minutes reduction time was selected for the further experiments. The last optimized parameter was the influence of temperature. Ambient temperature facilitates enzyme activity, but cooling facilitates GSH conservation. The tested temperatures were 4, 20 and 30 ◦ C. Recovery values presented in Table 3 reveal that the best recovery is obtained at 20 ◦ C. For GSSG quantification in wines, the reduction is performed by mixing 5 L of glutathione reductase suspension and 50 L of sample dissolved in 950 L of PBS with EtSH 1% (v/v), before incubation during 5 min at 20 ◦ C. These conditions are very easily applicable in laboratories. The GSSG quantification method was then validated in red and white wines matrixes. 3.2. Validation of the HPLC assay 3.2.1. Linearity Linearity was evaluated in both red and white wine matrixes by thiol addition (six points between 1 and 20 M; six other points between 20 and 100 M). Linearity of GSSG quantification was evaluated in both red and white wine matrixes by GSSG addition (seven points between 6.5 and 65 M). In the whole tested range the responses were linear when peak area were used for signal evaluation. Each concentration level was prepared in triplicate. A correction was applied by subtracting the peak area of the non-spiked red or white wine samples (references) from the spiked wines samples. Data are summarized in Table 4. The slopes obtained for GSSG calibration curves agree with values obtained for
3.2.2. Memory, limits of detection and quantification Memory of the automatic sampler was tested by injection of the sample matrix right after the last sample of the calibration curve. The values obtained were close to the intercept of calibration lines. The limits of detection (LODs) (concentration for signal/noise = 3) and the limits of quantification (LOQs) (concentration for signal/noise = 10) were calculated according to the OIV recommendation and repeated 10 times. This method allowed the determination of the LOD with a risk of error lower than 0.13% and a LOQ with a risk of error lower than 0.05%. Averages of the 10 calculations for each limit and each wine color associated are synthesized in Table 4. 3.2.3. Precision To evaluate the repeatability of the determination, ten identical samples of spiked red and white wines were prepared and analyzed on the same day. The concentrations are lower than the expected concentrations in wines but higher than quantification limits [GSH] = 2.99 M ⇔ 0.9 mg L−1 ; [␥-GC] = 2.57 M ⇔ 0.8 mg L−1 ). The results are presented in Table 4. The repeatability is very good. Reproducibility is calculated from 10 analysis of the same wine than for repeatability determination, frozen at −18 ◦ C in vials ready to be injected and analyzed twice a week, during one month using the strictly identical mobile phase. The results are also in Table 4 and the values are in most cases better than 15% except for ␥-GC in red wine (18%). 3.2.4. Recovery To calculate the recovery, the standard addition technique was applied to white and red wines. Both GSH and ␥-GC were spiked in wines at two different concentration levels. The recoveries are around 100% for GSH and ␥-GC for both matrixes. For GSSG, the
Table 5 Recovery capacities of the method in red and white wine matrixes (n = 3). Red wine
Initial measured concentration (M)
Low spiking level
High spiking level
Spiked concentration (M)
Measured concentration after spiking (M)
Recovery (%)
Spiked concentration (M)
Measured concentration after spiking (M)
Recovery (%)
GSH ␥-GC GSSG
12 ± 1 2.5 ± 0.6 0
6.3 11.8 13.0
18.8 ± 0.4 13 ± 2 12.5 ± 0.7
112 92 96
52.4 39.2 52.1
60 ± 4 41 ± 4 50 ± 2
93 99 96
White wine GSH ␥-GC GSSG
9±1 0.4 ± 0.2 0
6.3 11.8 13.0
15 ± 2 12 ± 1 11 ± 2
104 96 88
52.4 67.1 52.1
60 ± 2 69 ± 2 47 ± 3
98 103 90
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Table 6 Examples of GSH and ␥-GC quantification in various red and white wines (n = 3). Main variety
Vintage
[GSH] ± SD (n = 3) M
French dry white wines Chardonnay Chardonnay Chardonnay Chardonnay Chardonnay Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc
2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008
Bordeaux red wines (mainly Merlot and C. sauvignon) Blended 2007 Blended 2007 Blended 2007 Blended 2007 Blended 2005 Blended 2003 Blended 1999
[␥-GC] ± SD (n = 3) mg L
−1
M
mg L−1
5.9 7.3 5.5 5.9 9.1 13.3 23 8 15 15 19 21 12 8.4
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.6 0.3 0.3 0.6 0.6 0.9 2 1 2 2 3 2 2 0.3
1.8 2.26 1.7 1.8 2.8 4.1 7.1 2.4 4.7 4.7 6 6.6 3.7 2.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2 0.08 0.1 0.2 0.2 0.3 0.6 0.4 0.7 0.8 1 0.6 0.7 0.1
1.6 2.3 1.9 1.6 1.9 2.3 0.97
± ± ± ± ± ± ±
0.1 0.3 0.1 0.2 0.2 0.1 0.06
0.50 0.7 0.60 0.5 0.6 0.70 0.30
± ± ± ± ± ± ±
0.04 0.1 0.05 0.1 0.1 0.05 0.02
recoveries are around 100% for red wine and around 90% for white wine. Data are summarized in Table 5. 3.3. Analysis of wines samples GSH and ␥-GC were quantified in a large diversity of wines. Table 6 presents results obtained for a selection of 15 “2008 primeur” dry white wines from Chardonnay and Sauvignon blanc varieties, and a selection of red wines from Bordeaux area. The results are coherent compared to previously published ones. Our examples of Sauvignon blanc wines are richer than Chardonnay wines for GSH and ␥-GC levels. Table 6 presents also the results obtained from a selection of commercial red wines from the Bordeaux area. Perhaps because of their ages (vintages 1999–2007), they contain very small GSH amounts (and no ␥-GC), even though the method has high selectivity and sensitivity. No enological conclusion can be deduced from these results, but for the first time a glutathione quantification method is applied to old wines and is sensitive to enable quantification. For these practical examples, the quantification of ␥-GC does not seem to be really important. The quantities are small and the presence of this peptide is rare. This result agrees with previously published ones [8] where ␥-GC quantification data are not presented even if the method is able to detect it. So, for further experiment, an estimation of ␥-GC contents using calibration curves made only with GSH can be enough. This possibility decreases the method cost (␥-GC standard is expensive) and handling time. Nevertheless, sacrificing resolution between the ␥-GC and GSH peaks to reduce elution time must be avoided because of the possible presence of fluorescent endogenous compounds of wine eluted shortly after glutathione on chromatograms. In another application example, both GSH and GSSG were quantified in five Merlot wines before and after MLF. The levels of GSH, GSSG and also the total GSH ([total GSH] = [GSH] + 2 × [GSSG]) are presented at Fig. 4. Data show that no tendency can be really deducted from individual GSH or GSSG quantifications. On the other hand, a clear trend is observed for the total GSH. Total GSH levels are close for the five examples before MLF (between 116 and 151 M); and are similar after MLF (between 86 and 104 M). The decrease
Fig. 4. Reduced, oxidized and total glutathione quantification in five Merlot wines before and after MLF.
S. Marchand, G. de Revel / Analytica Chimica Acta 660 (2010) 158–163
of total GSH levels during MLF is also apparent (between 21% and 36%) for the five samples. For this example, total GSH measurement seems to be the only one good indicator of GSH contained in wines. This conclusion is not surprising because the same one is proposed the field of medical and biological analytical chemistry [14,15]. It must be considered to understand the variations of GSH levels during winemaking and ageing.
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tion of the presented work and will be used in our laboratory for further experiments. Acknowledgements The authors are grateful to Tomasz Lipiec and Aleksandra Kuchta for technical support and to Dr. Céline Franc, Dr. Olivier Leynaud and Pr. John Almy for English language editing.
4. Conclusion References In response to the increasing interest for GSH in grape and wine, and its newly described impact on wine aroma, a new automated, simple and quite fast method for the quantification of ␥-GC and both GSH and GSSG was developed, optimized and fully validated. The validated method shows good linearity, repeatability and reproducibility, and also high selectivity and high sensitivity. The analysis requires a very small amount of wine sample (around 1 mL) and the sample preparation is fully automated for ␥-GC and reduced GSH quantification and partially automated for GSSG quantification (in that case preparation needs less than 1 min handling and 5 min waiting time). The method requires no special equipment except for an HPLC fluorimetric detection system; this element could be replaced by an UV detector but with a significant loss of sensitivity. The method can be easily modified for UPLC systems. This allows the method to be adopted in research laboratories as well as control laboratories. Using our method as an example tends to show that GSH is not a pertinent indicator of glutathione contents in wines; total glutathione is a better one. That is why the proposed method for GSSG quantification in wines must be considered as the main contribu-
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A HPLC fluorescence-based method for glutathione derivatives quantification in must and wine Stéphanie Marchand ∗ , Gilles de Revel UMR 1219 Œnologie, ISVV, Université de Bordeaux, 210, Chemin de Leysotte, CS 50008, 33 882 Villenave d’Ornon Cedex, France
a r t i c l e
i n f o
Article history: Received 4 August 2009 Received in revised form 23 September 2009 Accepted 26 September 2009 Available online 2 October 2009 Keywords: Oxidized and reduced glutathione ␥-glutamylcysteine g-Wine Quantification Liquid Chromatography-fluorimetry
a b s t r a c t A simple and automated high performance liquid chromatography (HPLC) method for the separation and quantitative determination of ␥-glutamylcysteine and reduced glutathione (GSH) in wines is reported. This technique involves the use of a pre-column derivatization with 2,3-naphthalenedialdehyde (NDA), an isocratic separation in presence of -cyclodextrine and a fluorimetric detection. The quantification of oxidized glutathione (GSSG) has also been studied, for the first time in wines, using an additive prederivatization step for reduction using glutathione reductase. The method has been designed for use in laboratories with limited equipment. The assay has been optimized and presents very good performances in terms of sensitivity and selectivity. Then, it has been validated for linearity, LOD, LOQ, precision and accuracy. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Glutathione (␥-l-glutamyl-l-cysteinylglycine; GSH) is the principal non-protein thiol ubiquitous in animals, plants and even in microorganisms. Its monitoring is of paramount importance in studies related to oxidative stress, detoxification processes and human diseases. Because of the key role and widespread presence of GSH in biochemical systems, separative detection and quantification methods for GSH have been published from laboratories mostly linked with medical research. However, glutathione seems to be also implicated in wine aroma either as disulfur form with odorous thiols [1–4], or as a storage form of cysteine moieties [5] itself supposed to be implicated in aroma production [6]. Park et al. [7] pointed out the importance of GSH in yeast metabolism. Lavigne et al. [8] studied changes of GSH levels during alcoholic fermentation using capillary electrophoresis method. These studies and quantification methods were significant advances in understanding the role of GSH levels in must and wines. But the lack of a simple and rapid quantification method has prevented a comprehensive evaluation of GSH levels in musts and wines. GSH and ␥-GC do not have unique spectral characteristics that permit easy quantification; instead, they are derivatized with specific probes. Because of its higher sensitivity, fluorime-
∗ Corresponding author. Tel.: +33 557 575841. E-mail address: [email protected] (S. Marchand). 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.09.042
try is preferred to spectrophotometry for GSH detection, but both techniques can be used with most commercial probes. Two of the best derivatizing agents are aromatic dialdehydes, i.e. ophthaldialdehyde (OPA) and 2,3-naphthalenedialdehyde (NDA), now widely used to label nucleophilic primary amino compounds, especially amino acids and peptides [9–11]. NDA–GSH adduct has several advantages; one of them is the presence of an excitation wavelength in the visible range very close to the emission line of the argon laser, which is usually used in confocal microscopy, flow cytometry, and capillary electrophoresis. NDA is also a promising probe for in situ labelling of GSH, and the main advantage of NDA, is the capacity of NDA–aminothiol adducts to be included in the -cyclodextrine (-CD) cavity [12]. The main consequence of the inclusion is an enhancement of the fluorescence ratio. The addition of -CD to the mobile phase of a high performance liquid chromatography (HPLC) method allows better fluorescence detection capacities, better resolution and lower retention times. The present work is directed to develop a very simple and rapid technique for quantifications of ␥-GC, GSH and GSSG in wines, exploiting chemical selective steps and the high sensitivity of the fluorimetric detection. The previously presented arguments have directed our choices to develop a GSH quantification method using derivatization by NDA, and separation by HPLC with -CD. Moreover GSH, because of its very low standard redox potential (E◦ = −240 mV for thiol/disulfide exchange) can be present in large quantities under the disulfide form (GSSG). Even, if wine pH is in favor of the reduced form (GSH), the presence of GSSG in wines
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must be studied and taken into consideration. GSSG has never been quantified in wines. The strategy we chose is the enzymatic reduction of GSSG by glutathione reductase [13] and quantification of the GSH generated.The quantification methods, including reduction step, have been fully optimized and validated considering white and red wine matrixes. 2. Experimental 2.1. Chemicals and reagents GSH, GSSG, ␥-GC, glutathione reductase from baker yeast (E1.6.4.2.), NDA, ethanethiol (EtSH), boric acid (H3 BO4 ), phosphoric acid (H3 PO4 ), sodium hydroxide (NaOH), hydrochloric acid (HCl), ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA), ethanol, methanol, -cyclodextrine, phosphoric acid salt (NaH2 PO4 ·12H2 O), phosphate buffer saline (PBS) were purchased from Sigma–Aldrich (St. Quentin-Fallavier, France). All aqueous solutions were made using highly purified water produced by a Millipore Milli Q system (St. Quentin-en-Yvelines, France). GSH, ␥-GC and GSSG standard solutions were prepared by dissolving the peptides in HCl 0.1 M aqueous solutions supplemented with EDTA (2 mM) (HCl·EDTA). GSH, ␥-GC and GSSG standard solutions (1 mg mL−1 ) were stored at −18 ◦ C before use. Diluted standard solutions were done in HCl·EDTA extemporaneously. 2.2. Wine samples Red and white Bordeaux AOC wines were used to optimize and validate the quantification method. Samples of wine were spiked at several concentrations for each analysis. The wine is conserved in small scale tanks in the cellar of the institute. After validation, several wines from different regions of France were analyzed following the validated methodology. They were commercial wines or wines offered by producers. To study of GSH and GSSG levels in wine with or without malolactic fermentation (MLF), five Merlot wines have been sampled from five tanks in two wineries from the Bordeaux area, just before lactic bacteria inoculation and at the end of MLF. 2.3. GSSG quantification Oxidized glutathione (GSSG) has to be reduced before quantification. The reduction is performed using glutathione reductase. To conserve the enzyme activity, 5 L of glutathione reductase and 50 L of sample are dissolved in 950 L of phosphate buffer saline solution pH 7.4 containing 1% ethanethiol, before incubation during 5 min at 20 ◦ C. The GSSG level is obtained from total GSH level minus reduced GSH level then dividing this difference by 2. 2.4. Derivatization procedure A 1 mg mL−1 NDA solution was prepared in ethanol and stored at −18 ◦ C for up to one month without loss of activity. The derivatization procedure was performed by an autosampler as follows: to 4 L of sample were added 5 L of borate buffer and automatically mixed, then 1 L of NDA solution was added, and the resulting 10 L were mixed during 4 min before injection in the HPLC system. A borate buffer was prepared from 0.2 M H3 BO4 and 1% (v/v) ethanethiol in highly purified water and aqueous NaOH was added to adjust the pH to 9.2. NDA solution was composed of 2,3-naphthalenedialdehyde 5 mM in ethanol. Borate buffer and NDA solutions were poured separately in ready-to-use vials and stored at −18 ◦ C before use.
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Table 1 Comparison of calibration curve slopes for optimization borate buffer/spiked white wine ratio for ␥-GC and GSH derivation by NDA during 4 min at 20 ◦ C. Sample
1 2 3 4 5 6 7
Slopes (A.U. M−1 )
Volumes (L) Borate buffer
NDA solution
GSH
8 7 6 5 4 3 2
1 1 1 1 1 1 1
0.8 1.3 2.1 2.7 3.3 1.8
␥-GC 0.6 1.0 1.6 2.1 2.6 1.3 No peaks
2.5. Instrumentation and operating conditions A HP 1100 HPLC instrument from Agilent Technologies (Massy, France) was used throughout this study. It comprised a binary pump, an autosampler and a fluorimetric detector. The excitation and emission wavelengths were 467 and 525 nm respectively [11]. Chromatographic parameters (peak retention times and areas) were calculated via the Chemstation® software. A Purospher STAR® RP-18 (5 m) HPLC cartridge (250 × 4.6 mm i.d.) associated with a Purospher STAR® guard column (4 mm × 4.6 mm i.d.) from Merck (Darmstadt, Germany) were used for separation of GSH and ␥-GC. This material can tolerate alkaline mobile phases. The mobile phase was composed of methanol and phosphate buffer. The ratio have been optimized (Table 2) and the chosen one is methanol:buffer 15:85 (v:v). The phosphate buffer was composed of NaH2 PO4 ·12H2 O (10 mM) and -CD (optimized at 3 mM) in highly purified water; pH is adjusted to 8.5 using NaOH solutions. The mobile phase was filtered using a vacuum filtration system through 0.45 m membrane filter. 3. Results and discussion 3.1. Method optimization 3.1.1. Optimization of derivatization parameters Several parameters of the automatic derivatization procedure were optimized to minimize the quantification limit by comparison of calibration curves slopes. A white wine was spiked with five levels of both GSH and ␥-GC (5, 12, 15, 18 and 30 M for GSH and 10, 19, 24, 29, 48 M for ␥-GC). The two sets of five samples were injected varying derivatization parameters. Peak areas were plotted versus concentrations, and they presented linear relationships. The calibration curve slopes were then plotted versus the variations of derivatization parameters and the best value was determined. As previously reported the condensation between NDA and either glutathione or ␥-GC is highly pH dependent [11]. As a result, wines were mixed with borate buffer pH 9.2 before NDA addition. The buffer/sample ratio was optimized using a 4 min derivatization time. The calibration curve slopes were proportional to sample volume if this latter is between 1 and 5 L; derivatization decreased if sample volume was above 5 L (Table 1). This result agrees with pH monitoring by adding borate buffer to white wine. The mixed pH must be greater than 8.5 to permit good derivatization. To be sure the pH conditions are optimized even with very acidic must and wines, the conditions selected were 4 L sample mixed with 5 L borate buffer and 1 L NDA solution in ethanol. The derivatization step time was optimized with variation of mixing time. Calibration curve slopes are plotted versus derivatization time (Fig. 1). Four minutes seems to be a good compromise for derivatization time.
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Fig. 2. Typical chromatogram obtained for the optimized ␥-GC and GSH quantification method.
Fig. 1. Comparison of calibration curve slopes for optimization NDA–aminothiols derivatization time in white wine. The derivatization is performed at 20 ◦ C by mixing 4 L sample, 5 L borate buffer and 1 L NDA solution.
Table 2 Influence of (a) -CD concentration in HPLC aqueous mobile phase and of (b) methanol/phosphate buffer ratio, on retention times and (c) resolution. Mobile phaseb
10:90 15:85 20:80 25:75
[-CD] = 3 mMa
[-CD] = 4 mM
trGSH (min)
tr␥-GC (min)
Rc
trGSH (min)
tr␥-GC (min)
R
25.5 15.0 10.5 8.0
20.6 12.8 9.7 8.0
6.3 4.0 2.2 0.0
13.1 10.0 6.5 5.5
11.4 8.0 6.5 5.5
3.5 2.6 0.0 0.0
3.1.2. Optimization of mobile phase Two parameters for the mobile phase can be optimized. The first one is the -CD concentration in phosphate buffer. -CD presents two main advantages: increasing fluorescence of NDA–aminothiol adducts and decreasing retention times without any loss of resolution [13]. The second one is the percentage of methanol. The tested [-CD] concentrations were 3 and 4 mM. For each concentration the effect of the methanol/phosphate buffer ratio on retention time of the analytes and on resolution (R) was studied using an aqueous solution, containing HCl and EDTA at pH 3.5, spiked with glutathione and ␥-GC at around 300 M each. Results are summarized in Table 2. A good compromise for mobile phase composition is [CD] = 3 mM and the MeOH percentage at 15%. It should be noted that in the preparation of the mobile phase the -CD mass must be precisely determined using a scale. An example of a chromatogram obtained with the optimized analysis parameters is presented in Fig. 2.
3.1.3. Optimization of conditions for GSSG reduction The equilibrium of the GSH/GSSG redox couple could be an interesting point to study in order to collect pertinent data on GSH in wines. For the measurement, GSSG is usually reduced to GSH, and then the reduced/oxidized ratio can be determined, or the total GSH pool is calculated. Many ways for the GSSG reduction have been reported [14]. Because of the specificity of glutathione reductase, the enzymatic method is optimized for GSSG quantification in wines. The GSSG standard solution (160 M) is used to evaluate the best conditions for the enzyme performance and GSH stabilization. 5 L of glutathione reductase suspension and 50 L of sample are dissolved in 950 L of solvent during 1, 5, 7 or 10 min and at the temperatures of 4, 20 or 30 ◦ C. The tested parameters were solvent composition and pH, time and temperature of reduction. The ratio [GSH measured]/[2 × GSSG in solution] (%) is used to compare parameters. The first parameter which was optimized was the solvent. Time and temperature were arbitrarily fixed to 5 min and 4 ◦ C. Four different solvents were tested: PBS at pH 7.4, PBS with ethanethiol 1% (v/v) pH 7.3, HCl·EDTA at pH 1.45 and HCl·EDTA with ethanethiol 1% (v/v) at pH 1.43. These four solvents were selected to provide neutral medium (optimal for glutathione reductase activity) and acidic medium (optimal for GSH conservation). Ethanethiol solution was added as an antioxidant in order to enhance the stabilization of GSH after reduction. Recovery values presented in Table 3 show that PBS with ethanethiol is the only solvent in which the GSSG is correct. These results allow to discard all tested solvents except PBS with ethanethiol. The second optimized parameter was the influence of the pH on GSH recovery. Time and temperature were also arbitrary fixed to 5 min and 4 ◦ C, and the solvent was the previously selected one. Addition of either NaOH or H3 PO4 solutions was employed to change pH values. Recovery values are presented in Fig. 3 and show an optimum for pH 8.5. Because recovery values between pH 7.5 and 8.5 increase only slightly, the pure PBS with EtSH 1% at pH 7.3 was chosen for further study. The third optimized parameter was the influence of the time of reduction. Temperature was also arbitrarily fixed to 20 ◦ C, and the solvent was the previously selected one. Recovery values presented in Table 3 show that the differences between time ranges
Table 3 Optimization of solvent composition, time and temperature for GSSG reduction by glutathione reductase (n = 3). T = 4 ◦ C, t = 5 min
T = 20 ◦ C in PBS + EtSH
t = 5 min in PBS + EtSH ◦
Solvents
Recoveries (%)
Time (min)
Recoveries (%)
T ( C)
Recoveries (%)
PBS PBS + EtSH HCl·EDTA HCL·EDTA + EtSH
0 68 ± 5 0 14 ± 1
1 5 7 10
73 ± 12 79 ± 11 78 ± 11 70 ± 10
4 20 30
52 ± 9 78 ± 10 73 ± 10
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Table 4 Detection and quantification limits, repeatability, reproducibility and linearity (n = 3) of the method in red and white wine matrixes. In white wine matrix
In red wine matrix
GSH
GSSG
␥-GC
GSH
GSSG
␥-GC
LOD (M) LOQ (M) Repeatability Reproducibility
0.1 0.3 5% 5%
4 13 10% 15%
0.6 2.0 6% 13%
0.1 0.4 3% 12%
5 15 8% 16%
0.8 2.5 7% 18%
Linearity r2 Slope (M) % slope var.
0.988 2.6 9%
0.994 0.25 4%
0.990 2.1 9%
0.989 3.0 5%
0.996 0.3 5%
0.975 2.2 5%
GSH quantification linearity test, taking into account the dilution in PBS and the stoichiometry of GSSG reduction.
Fig. 3. Optimization of solvent pH (PBS + EtSH 1%, v/v) for GSSG reduction by glutathione reductase (n = 3).
are slightly noticeable. Nevertheless, running the reaction from 5 to 7 min, improves the recovery. Five minutes reduction time was selected for the further experiments. The last optimized parameter was the influence of temperature. Ambient temperature facilitates enzyme activity, but cooling facilitates GSH conservation. The tested temperatures were 4, 20 and 30 ◦ C. Recovery values presented in Table 3 reveal that the best recovery is obtained at 20 ◦ C. For GSSG quantification in wines, the reduction is performed by mixing 5 L of glutathione reductase suspension and 50 L of sample dissolved in 950 L of PBS with EtSH 1% (v/v), before incubation during 5 min at 20 ◦ C. These conditions are very easily applicable in laboratories. The GSSG quantification method was then validated in red and white wines matrixes. 3.2. Validation of the HPLC assay 3.2.1. Linearity Linearity was evaluated in both red and white wine matrixes by thiol addition (six points between 1 and 20 M; six other points between 20 and 100 M). Linearity of GSSG quantification was evaluated in both red and white wine matrixes by GSSG addition (seven points between 6.5 and 65 M). In the whole tested range the responses were linear when peak area were used for signal evaluation. Each concentration level was prepared in triplicate. A correction was applied by subtracting the peak area of the non-spiked red or white wine samples (references) from the spiked wines samples. Data are summarized in Table 4. The slopes obtained for GSSG calibration curves agree with values obtained for
3.2.2. Memory, limits of detection and quantification Memory of the automatic sampler was tested by injection of the sample matrix right after the last sample of the calibration curve. The values obtained were close to the intercept of calibration lines. The limits of detection (LODs) (concentration for signal/noise = 3) and the limits of quantification (LOQs) (concentration for signal/noise = 10) were calculated according to the OIV recommendation and repeated 10 times. This method allowed the determination of the LOD with a risk of error lower than 0.13% and a LOQ with a risk of error lower than 0.05%. Averages of the 10 calculations for each limit and each wine color associated are synthesized in Table 4. 3.2.3. Precision To evaluate the repeatability of the determination, ten identical samples of spiked red and white wines were prepared and analyzed on the same day. The concentrations are lower than the expected concentrations in wines but higher than quantification limits [GSH] = 2.99 M ⇔ 0.9 mg L−1 ; [␥-GC] = 2.57 M ⇔ 0.8 mg L−1 ). The results are presented in Table 4. The repeatability is very good. Reproducibility is calculated from 10 analysis of the same wine than for repeatability determination, frozen at −18 ◦ C in vials ready to be injected and analyzed twice a week, during one month using the strictly identical mobile phase. The results are also in Table 4 and the values are in most cases better than 15% except for ␥-GC in red wine (18%). 3.2.4. Recovery To calculate the recovery, the standard addition technique was applied to white and red wines. Both GSH and ␥-GC were spiked in wines at two different concentration levels. The recoveries are around 100% for GSH and ␥-GC for both matrixes. For GSSG, the
Table 5 Recovery capacities of the method in red and white wine matrixes (n = 3). Red wine
Initial measured concentration (M)
Low spiking level
High spiking level
Spiked concentration (M)
Measured concentration after spiking (M)
Recovery (%)
Spiked concentration (M)
Measured concentration after spiking (M)
Recovery (%)
GSH ␥-GC GSSG
12 ± 1 2.5 ± 0.6 0
6.3 11.8 13.0
18.8 ± 0.4 13 ± 2 12.5 ± 0.7
112 92 96
52.4 39.2 52.1
60 ± 4 41 ± 4 50 ± 2
93 99 96
White wine GSH ␥-GC GSSG
9±1 0.4 ± 0.2 0
6.3 11.8 13.0
15 ± 2 12 ± 1 11 ± 2
104 96 88
52.4 67.1 52.1
60 ± 2 69 ± 2 47 ± 3
98 103 90
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Table 6 Examples of GSH and ␥-GC quantification in various red and white wines (n = 3). Main variety
Vintage
[GSH] ± SD (n = 3) M
French dry white wines Chardonnay Chardonnay Chardonnay Chardonnay Chardonnay Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc Sauvignon blanc
2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008
Bordeaux red wines (mainly Merlot and C. sauvignon) Blended 2007 Blended 2007 Blended 2007 Blended 2007 Blended 2005 Blended 2003 Blended 1999
[␥-GC] ± SD (n = 3) mg L
−1
M
mg L−1
5.9 7.3 5.5 5.9 9.1 13.3 23 8 15 15 19 21 12 8.4
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.6 0.3 0.3 0.6 0.6 0.9 2 1 2 2 3 2 2 0.3
1.8 2.26 1.7 1.8 2.8 4.1 7.1 2.4 4.7 4.7 6 6.6 3.7 2.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2 0.08 0.1 0.2 0.2 0.3 0.6 0.4 0.7 0.8 1 0.6 0.7 0.1
1.6 2.3 1.9 1.6 1.9 2.3 0.97
± ± ± ± ± ± ±
0.1 0.3 0.1 0.2 0.2 0.1 0.06
0.50 0.7 0.60 0.5 0.6 0.70 0.30
± ± ± ± ± ± ±
0.04 0.1 0.05 0.1 0.1 0.05 0.02
recoveries are around 100% for red wine and around 90% for white wine. Data are summarized in Table 5. 3.3. Analysis of wines samples GSH and ␥-GC were quantified in a large diversity of wines. Table 6 presents results obtained for a selection of 15 “2008 primeur” dry white wines from Chardonnay and Sauvignon blanc varieties, and a selection of red wines from Bordeaux area. The results are coherent compared to previously published ones. Our examples of Sauvignon blanc wines are richer than Chardonnay wines for GSH and ␥-GC levels. Table 6 presents also the results obtained from a selection of commercial red wines from the Bordeaux area. Perhaps because of their ages (vintages 1999–2007), they contain very small GSH amounts (and no ␥-GC), even though the method has high selectivity and sensitivity. No enological conclusion can be deduced from these results, but for the first time a glutathione quantification method is applied to old wines and is sensitive to enable quantification. For these practical examples, the quantification of ␥-GC does not seem to be really important. The quantities are small and the presence of this peptide is rare. This result agrees with previously published ones [8] where ␥-GC quantification data are not presented even if the method is able to detect it. So, for further experiment, an estimation of ␥-GC contents using calibration curves made only with GSH can be enough. This possibility decreases the method cost (␥-GC standard is expensive) and handling time. Nevertheless, sacrificing resolution between the ␥-GC and GSH peaks to reduce elution time must be avoided because of the possible presence of fluorescent endogenous compounds of wine eluted shortly after glutathione on chromatograms. In another application example, both GSH and GSSG were quantified in five Merlot wines before and after MLF. The levels of GSH, GSSG and also the total GSH ([total GSH] = [GSH] + 2 × [GSSG]) are presented at Fig. 4. Data show that no tendency can be really deducted from individual GSH or GSSG quantifications. On the other hand, a clear trend is observed for the total GSH. Total GSH levels are close for the five examples before MLF (between 116 and 151 M); and are similar after MLF (between 86 and 104 M). The decrease
Fig. 4. Reduced, oxidized and total glutathione quantification in five Merlot wines before and after MLF.
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of total GSH levels during MLF is also apparent (between 21% and 36%) for the five samples. For this example, total GSH measurement seems to be the only one good indicator of GSH contained in wines. This conclusion is not surprising because the same one is proposed the field of medical and biological analytical chemistry [14,15]. It must be considered to understand the variations of GSH levels during winemaking and ageing.
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tion of the presented work and will be used in our laboratory for further experiments. Acknowledgements The authors are grateful to Tomasz Lipiec and Aleksandra Kuchta for technical support and to Dr. Céline Franc, Dr. Olivier Leynaud and Pr. John Almy for English language editing.
4. Conclusion References In response to the increasing interest for GSH in grape and wine, and its newly described impact on wine aroma, a new automated, simple and quite fast method for the quantification of ␥-GC and both GSH and GSSG was developed, optimized and fully validated. The validated method shows good linearity, repeatability and reproducibility, and also high selectivity and high sensitivity. The analysis requires a very small amount of wine sample (around 1 mL) and the sample preparation is fully automated for ␥-GC and reduced GSH quantification and partially automated for GSSG quantification (in that case preparation needs less than 1 min handling and 5 min waiting time). The method requires no special equipment except for an HPLC fluorimetric detection system; this element could be replaced by an UV detector but with a significant loss of sensitivity. The method can be easily modified for UPLC systems. This allows the method to be adopted in research laboratories as well as control laboratories. Using our method as an example tends to show that GSH is not a pertinent indicator of glutathione contents in wines; total glutathione is a better one. That is why the proposed method for GSSG quantification in wines must be considered as the main contribu-
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