Elsevier

Journal of Chromatography A

Volume 1139, Issue 1, 12 January 2007, Pages 130-135
Journal of Chromatography A

Assay of glutathione in must and wines using capillary electrophoresis and laser-induced fluorescence detection: Changes in concentration in dry white wines during alcoholic fermentation and aging

https://doi.org/10.1016/j.chroma.2006.10.083Get rights and content

Abstract

Glutathione (GSH) was assayed in must and wine using capillary electrophoresis coupled with laser-induced fluorescence (LIF) detection. Sample preparation involved conjugating thiols with monobromobimane (MBB) in a 2-(N-cyclohexylamino)ethanesulfonic acid [CHES] buffer (179 mM). The electrophoretic conditions were 30 kV with a capillary length of 105 cm from the inlet to the detector (120 cm total length) and a 50 μm inner diameter. Under these conditions, the complete separation from the other main non-volatile thiols took less than 20 min. We also described the optimum conditions for derivatizing wine samples with MBB to increase eletrophoretic sensitivity. The detection limit for glutathione assay is 65 nmol/L. This simple, sensitive method provides a specific assay of glutathione in reduced form, as the sample preparation technique does not modify the balance of oxidized and reduced forms. We used this method to monitor changes in the reduced glutathione content of a white wine during alcoholic fermentation and barrel aging.

Introduction

Glutathione (GSH) is a major natural component of many plants and foods [1], [2], [3]. The many properties of this sulfur tripeptide, including preventing the formation free radicals and detoxifying cells, as well as inhibiting enzymatic and non-enzymatic mechanisms involved in browning fruit juice and other foods [1], [4], [5], [6] have certainly been responsible for the large body of literature on assays of compound.

Although glutathione was first discovered in grapes as early as 1989 [7], little has been written about assaying this compound in must and wine [8], [9]. Moreover, the function of this peptide in wine is not well understood. The ability of glutathione to react with certain quinones is now well documented. GSH level is important for preventing the oxidation of phenolic compounds in wine. However, GSH's potentiality to play a major role in protecting volatile thiols during the aging of bottled white wines [10] needs to be explained. It is essential to analyze GSH quickly and efficiently to have a better understanding of the protective capacity of glutathione in wine.

The first glutathione assay described in the literature involved enzymatic methods [11], [12], [13]. The methods developed later, mainly suited to assaying glutathione in blood and plasma, used high-performance liquid chromatography (HPLC) coupled with either fluorescence [2], [9], [14], [15], [16] or electrochemical detection [17], [18]. Several high-performance capillary electrophoresis (HPCE) methods have traditionally used UV detection [19], [20]. Recently, various studies related to plasma thiols using capillary electrophoresis and laser-induced fluorescence (LIF) detection, provided more sensitive detection than UV systems [21], [22], [23], [24], [25], [26], [27]. These methods were reviewed by Bayle and Couderc [22]. Many dyes used for CE-UV detection are also used for CE-LIF detection. Because the most popular laser in LIF detection is the argon-ion laser emitting light at 488 nm, fluoresceinebased dyes are best suited [28], [29], [30], [31]. Monobromobimane (MBB) is one of the most commonly used compounds for labeling thiols. However, at the present time, monobromobimane is practically no longer [23], [32] used in studying CE-LIF, probably due to a lack of lasers with appropriate excitation wavelength.

Monobromobimane is a specific reagent for the thiol functional group, forming MBB-thiol adducts with relatively high fluorescence emission, thereby enabling the detection of analytes even at low concentration (2 × 10−8 mol/L) [32]. Drawbacks of the MBB method include the fact that MBB is self-fluorescent and undergoes photodegradation, yielding fluorescent products that interfere with thiol determination. Furthermore, MBB is not selective for GSH because other thiols can react and, typically, unknown peaks are observed on the chromatogram.

The main methods described in the literature for GSH determination are summarized in Table 1. The only analytical method appropriately validated for the determination of GSH and Cys in wine was developed by Park et al. [9]. These authors found a 3.3 nmol/L detection limit for GSH assay with o-phthalaldehyde (OPA) derivatization of wine samples.

To our knowledge, there is no existing study dealing with glutathione determination by HPCE in wine. This article presents a method for assaying glutathione and other sulfur amino acids (cysteine and N-acetyl-cysteine), in must and wine. This method of assaying non-volatile thiols in poplar leaves [2], adapted from that described by Noctor and Foyer in 1998, uses capillary electrophoresis coupled with fluorimetric detection (LIF). This is expected to provide greater sensitivity, while requiring shorter analysis time and smaller sample volume.

Section snippets

Reagents

l-Homocysteine (Hcy, >95%), l-cysteine (Cys, >99.5%), N-acetyl-l-cysteine (NA-Cys, >99%), l-glutathione (GSH, >98.0%), γ-glutamylcysteine (γ–Glu-Cys, 99%), cysteine-glycine (Cys-Gly, 99%), d-l-dithiothreitol (DTT, >99%), oxidized glutathione (GSSG), monosodium phosphate (NaH2PO4, >99%), disodium phosphate (Na2HPO4, >99%), residual 2-(N-cyclohexylamino)ethanesulfonic acid (CHES, >98%), monobromobimane (MBB, 97%), sodium ethylendiaminetetraacetic acid salt (EDTA, 100%), and NaOH, all were from

Optimization of sample derivatization time

Derivatization of thiols by MBB is a very fast process. Several time incubations were assayed in this study for testing the optimal GSH derivatization procedure (Fig. 1). Twenty minutes of derivatization were necessary to detect the maximum amount of GSH in a wine sample. Beyond this limit, the fluorescence yield of GSH-MBB adduct decreases drastically. After 30 min of derivatization, the loss is about 50%. We raised the possibility that the loss in GSH-MBB fluorescence occurring 30 min after the

Application to glutathione assays in must and wine

Large quantities of glutathione were found in grapes by Cheynier et al. and Liyanage et al. [7], [38]. The very strong reactivity of this compound with both oxygen and oxidized phenolic compounds probably explains why little research has been done into assaying glutathione in must and wine.

The formation of 2-S-glutathionyl caffeoyl tartaric acid (GRP), when glutathione reacts with certain quinines, during extraction of the must has been clearly described [39], [40], [41]. Glutathione is also

Conclusion

The assays presented in this work use capillary electrophoresis coupled with laser induction fluorescence detection. The resolution quality obtained with this method makes it possible to assay various amino acids and sulfur peptides in media as complex as must or wine, quickly, reproducibly, and with good sensitivity. The most interesting results were obtained in assays for glutathione in its reduced form, as preparation of the samples did not cause any modification in the balance between

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