Assays on the simultaneous determination and elimination of chloroanisoles and chlorophenols from contaminated cork samples

Journal of Chromatography A, 1122 (2006) 215–221

Assays on the simultaneous determination and elimination of chloroanisoles and chlorophenols from contaminated cork samples夽 Sara Insa, Vict`oria Salvad´o, Enriqueta Antic´o ∗ Department of Chemistry, University of Girona, Campus Montilivi, 17071 Girona, Spain Received 15 December 2005; received in revised form 10 April 2006; accepted 11 April 2006 Available online 6 May 2006

Abstract A method for the simultaneous determination of the chloroanisoles and chlorophenols in cork samples with gas chromatography has been evaluated in view to its application. All the stages of the suggested procedure have been submitted to an in-depth examination using spiked ground corks. The recoveries of the method, which involves a simultaneous extraction with n-pentane followed by a second extraction using an aqueous basic solution where the phenolic derivates are transferred and, subsequently, derivatised, have been satisfactory for the all analytes at the studied spiking concentration levels. Good precision data and limits of detection between 1 ng/g and 2 ng/g were obtained for almost all compounds. As real samples, naturally contaminated cork slabs taken from different sources have been analysed, showing the presence of 2,4,6-trichloroanisole (TCA) and, in lesser extent, its direct precursor, 2,4,6-trichlorophenol (TCP). Removal studies have been performed by washing these tainted cork slabs with different solutions: Milli-Q water, sodium hydroxide and commercial products. Sodium hydroxide solutions have led to better analyte elimination, and the complete removal of TCP from the cork has been accomplished together with 72% of TCA reduction has been achieved. © 2006 Elsevier B.V. All rights reserved. Keywords: Cork taint; Chloroanisoles; Chlorophenols; Cork; Gas chromatography/mass spectrometry (GC/MS); Washing tests

1. Introduction The unique physical properties attributed to cork have encouraged its use for sealing wine bottles, especially high-quality wines and those stored for long periods of time. Although other materials have been tested as alternative closures [1], cork is probably the best choice to guarantee an effective seal as well as to ensure the proper maturation of the wine. Nevertheless, there have occasionally been problems resulting from defective cork stoppers, including leakages, cork-derived deposits and the appearance of sensorial alterations, which have led to the implantation of rigorous quality controls in the cork industry. Among the unpleasant aromas detected in bottled wines, cork taint still constitutes a major concern because of its frequent occurrence in spoiled wines, which represent a significant source of economical losses worldwide [2]. According to the

夽 Presented at the 11th Meeting on Instrumental Analysis, Barcelona, 15–17 November 2005. ∗ Corresponding author. Tel.: +34 972418276; fax: +34 972418150. E-mail address: [email protected] (E. Antic´o).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.04.022

European QUERCUS project, at least 80% of spoiled wines analysed exhibited the presence of 2,4,6-trichloroanisole (TCA) [3]. This off-flavour, described as mouldy/musty, is commonly related to the chloroanisole family of compounds, especially TCA and, to a lesser extent, 2,3,4,6-tetrachloroanisole (TeCA) and pentachloroanisole (PCA). However, the contribution of other chemical substances to cork taint has been reported in the current literature [4–6]. The mechanisms leading to the appearance of TCA in wines have been discussed by several authors [7]. There is still some disagreement about the origin of chloroanisoles because certain processes performed in either the cork or wine industries can also lead to TCA formation. In any case, it can be assumed that the direct precursors of chloroanisoles are chlorophenols, 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP), which are converted into less toxic compounds through Omethylation reactions mediated by different microbial species. Therefore, chlorophenolic products used in cellars or during the manufacture of cork stoppers can be potential sources of cork taint. As a result of the need to preserve the quality of cork, factories involved in cork stopper production have been required to ensure

216

S. Insa et al. / J. Chromatogr. A 1122 (2006) 215–221

the absence of undesirable substances in their final products before they are introduced into the market. Although nowadays all the steps involved in the cork manufacturing process have been improved, residual chloroanisoles and chlorophenols can remain in the finished corks and contribute to spoilage in bottled wine. Diverse strategies have been proposed to remove contaminants from cork: sterilisation of corks to remove the microflora which, under favourable conditions, can produce the metabolites responsible for disagreeable aromas [8], electron beam irradiation [9], different improved washing procedures [10], and extractions using supercritical carbon dioxide [11,12] or ultrasounds [12]. Rocha et al. [8] found a decrease in the presence of all volatile compounds in the cork after the autoclaving process. As far as electron beam irradiation is concerned, the authors demonstrate that degradation of TCA occurs, thus obtaining its radiolytic products, mono- and di-chloroanisole [9]. The other strategies, assayed in an industrial scale and already patented, also seem to produce satisfactory results according to the promoters. Even though odour thresholds of chloroanisoles and chlorophenols in cork samples are usually not determined, the presence of these compounds becomes a potential risk even at low concentration level. Then, a reliable analytical method is needed which allows to verify their absence. These two groups of compounds exhibit different chemical behaviours (due to the phenolic group in chlorophenols) and for this reason, the majority of studies have been focused on the analysis of chloroanisoles or chlorophenols separately. Determining concentrations of chloroanisoles, mainly TCA, in cork samples has been widely described [11,13–20]. However, few papers deal with the analysis of chlorophenolic compounds in this matrix [21,22]. Methods including a joint determination of the target substances in corks have hardly been reported in the literature. Most of them are based on solvent extraction using n-hexane [23,24] or dichloromethane [25] as solvents, and then analysis of the resulting organic cork extracts by gas chromatography (GC). In addition, solid-phase extraction (SPE) [26] and solid-phase microextraction (SPME) methods [27] have also been reported for the analysis of these analytes in corks, which were previously extracted with hydroalcoholic solutions. In this work, we propose a simple methodology capable of detecting chloroanisoles and chlorophenols in cork samples by gas chromatography with electron capture detection (ECD) and a mass spectrometric detector (MS) operating in MS–MS mode. The procedure is based on extraction according to the method developed by Juanola et al. [15] for the determination of TCA. Taking into account the ability of organic solvents to recover chlorophenols from a cork matrix [23–25], we propose herein the simultaneous extraction of both chloroanisoles and chlorophenols followed by the acetylation of the chlorophenol compounds. The results obtained are discussed in terms of recovery, repeatability and limit of detection. Finally, the developed approach has been applied to a naturally contaminated cork bark which has been submitted to various types of washing treatments. To our knowledge this is the first time that such washing treatments, which can be easily implemented in the cork manufacturing

industry, are examined in-depth and the percentage of elimination is quantified. 2. Experimental 2.1. Chemicals 2,4,6-Trichloroanisole was purchased from Sigma–Aldrich Qu´ımica (Madrid, Spain), 2,3,4,6-tetrachloroanisole from Ultra Scientific (North Kingstown, RI, USA) and pentachloroanisole from Chem Service (West Chester, PA, USA). All compounds had a purity of over 95%. 2,6-Dichloroanisole (DCA) (Sigma–Aldrich Qu´ımica) was used as an internal standard for the analysis of chloroanisoles. The stock solution containing 400 ␮g/mL of each analyte was prepared in n-hexane (Panreac, Barcelona, Spain). Calibration solutions (1–30 ng/mL) were made by diluting the concentrated stock solution with hexane and adding the internal standard to a final concentration of 198 ng/mL. 2,4,6-Trichlorophenol (Fluka, Vienna, Austria), 2,3,4,6tetrachlorophenol (North Kingstown) and pentachlorophenol (Sigma–Aldrich Qu´ımica) had a purity of at least 97% and were used in this study to prepare standard solutions. As an internal standard, 2,4,6-tribromophenol (TBP) (Supelco, Bellefonte, PA, USA) was employed. A stock chlorophenolic solution was prepared in methanol (Carlo Erba Reagenti, Milan, Italy) at a concentration level of 400 ␮g/mL. Calibration solutions (1–30 ng/mL) were prepared daily by careful dilution of the concentrated solution with methanol. TBP was added to the calibration set, the final concentration being 20 ng/mL. All stock solutions were stored in a refrigerated environment at 4 ◦ C and kept in darkness before their use. Anhydrous sodium sulfate, acetic anhydride and K2 CO3 were of analytical-reagent grade. High purity water was taken from a Milli-Q plus water purification system. The washing reagents were: Milli-Q water, sodium hydroxide solutions (NaOH 0.1 M and 0.01 M) and two commercial cork disinfectants based on acid composition (product 1: peracetic and product 2: sulfamic) which were kindly supplied by AECORK (Associaci´o d’Empresaris Surers de Catalunya, Palafrugell, Spain). These commercial products were diluted according to the supplier’s recommendations. 2.2. Acetylation of chlorophenolic standards To avoid adsorption problems and tailed peaks, chlorophenols were derivatised using the procedure described by Insa et al. [22] prior to separation and quantification by gas chromatography. 2.3. Cork samples To develop the method we employed natural cork stoppers which were kindly supplied by AECORK. The absence of chloroanisoles and chlorophenols had been previously checked. Additionally, naturally contaminated cork slabs provided by a cork manufacturer (Francisco Oller S.A.) were used as real

S. Insa et al. / J. Chromatogr. A 1122 (2006) 215–221

samples and to test the efficiency of the different washing treatments.

217

2.6. Cork washing tests

The cork material was milled with a conventional grinder. Immediately after grinding and before its use, it was stored in darkness at −18 ◦ C in order to avoid losses of the target compounds. The detachable parts of the grinder were washed between samples using the standard procedure for glassware. The extraction of the target analytes from cork matrices was based on Juanola et al. [15]. One gram of ground cork was placed in a 100 mL glass bottle together with an appropriate volume of n-pentane. Two successive extractions were carried out by using 80 mL and 40 mL of the solvent, respectively. The combined extracts were reduced to approximately 1 mL, first with the aid of a rotary evaporator (RE100 Bibby, Staffordshire, UK) and then under a stream of nitrogen. Due to the lack of the available reference material for the validation of the method, spiked cork was prepared by adding 1 mL of n-pentane (Romil Pure Chemistry, Cambridge, UK) solution containing the six target compounds at the desired concentration to 1 g of the ground cork placed in a 100 mL glass bottle. Afterwards, the sample was allowed to dry until a constant weight (this process took approximately 1 h) and was then extracted with n-pentane according to the method described above.

The efficiency of the different washing treatments to remove chloroanisoles and chlorophenols from cork was tested by using a naturally contaminated slab, which was conveniently grinded and homogenised. The analytes present in this cork sample were identified and quantified using the developed method (see Section 3). First, 1.5 g of this ground cork slab were placed in a 100 mL glass bottle and 80 mL of the considered washing agent were added. The mixture was shaken in a rotary mixer (Dinko Instruments, Dinter, Barcelona, Spain) or immersed in an ultrasonic bath for 90 min. At the end of this stage, the cork was separated from the rinsing solution and was allowed to dry in the open air until a constant weight was achieved (approximately 3 h). Afterwards, 1 g of cork was analysed using the method developed here so as to determine the remaining content of compounds in the sample. For some experiments, the washing solution was also analysed. Standard solutions containing the six target compounds and the respective internal standards in water were prepared. Either standard or washing solutions were extracted by applying the headspace solid-phase microextraction (HS-SPME) method developed by Insa et al. [28]. We had previously checked that the HS-SPME methodology, which was applied in [28] only for chlorophenols, allowed the quantification of chloroanisoles as well when using gas chromatography with mass spectrometric detection.

2.5. Separation of chloroanisoles and chlorophenols

2.7. Equipment and chromatographic conditions

Two milliliter of 5% K2 CO3 solution were added to the pentane extract and the mixture was shaken for 10 min using an ultrasonic bath (P Selecta, Barcelona, Spain). The aqueous layer containing the chlorophenols in the phenolate form was carefully separated from the organic fraction where the chloroanisoles remained. A second extraction with 1 mL of the 5% K2 CO3 solution was performed (5 min) to ensure the complete separation of both families of compounds. Chloroanisoles were recovered in the organic phase which was dried over anhydrous sodium sulphate and 1 mL of internal standard solution (DCA, 198 ng/mL) was added before the concentration of the extract to 1 mL under a stream of nitrogen. A 1 ␮L of this solution was injected and analysed by the GC method. Chlorophenols remaining in the aqueous phases were derivatised by applying the procedure used for methanolic standards, with only a few modifications: 1 mL of internal standard (TBP, 20 ng/mL), 200 ␮L of acetic anhydride and 1 mL of n-hexane were added to the aqueous extract. After mixing for 1 min, the organic phase containing the acetylated compounds was separated and the aqueous phase was then re-extracted with 1 mL of n-hexane for a further minute. The two organic fractions were mixed, dried over anhydrous sodium sulphate and reduced to 1 mL under a gentle stream of nitrogen. A volume of 1 ␮L of the recovered chlorophenolic derivates in n-hexane was injected into the GC system.

The experiments designed to develop the method to determine both chloroanisoles and chlorophenols were performed with a GC 8000 Series (8160) gas chromatograph equipped with an ECD 80 electron capture detector (Fisons Instruments, Milan, Italy). A DB-5 capillary column (J&W Scientific, Folsom, CA, USA) (30 m × 0.25 mm I.D., film thickness 0.25 ␮m) was used. The operating conditions were as follows: an injector temperature of 270 ◦ C; a detector temperature of 330 ◦ C; helium was the carrier gas at 30 cm/s; N2 was the make-up gas at 43 cm/s; the oven temperature programme was 2 min at 70 ◦ C, then increasing by 5 ◦ C/min up to 180 ◦ C and then by 10 ◦ C/min up to 270 ◦ C, and finally 3 min at 270 ◦ C. Splitless mode injections were carried out with the purge valve opened at 1 min. The chromatographic data were analysed using ChromCard software. To identify the target compounds, their retention times were compared with those obtained for standard solutions injected separately. Additionally, real samples were analysed by a Thermo Finnigan TRACE GC 2000 gas chromatograph coupled to a PolarisQ ion trap mass spectrometer (Milan, Italy) operating in the electron impact (EI) mode at 70 eV. Chromatographic separation was achieved using an Rtx® -5MS capillary column (30 m × 0.25 mm I.D., film thickness 0.25 ␮m) (Restek, Bellefonte, PA, USA). The operating conditions were the same as those in the GC-ECD system, except for the flow of the carrier gas which was fixed at 1 mL/min. The ion source was set at 250 ◦ C and the transfer line

2.4. Extraction of chloroanisoles and chlorophenols from cork and spiking procedure

218

S. Insa et al. / J. Chromatogr. A 1122 (2006) 215–221

Table 1 MS–MS parameters description for the analysis of chloroanisoles and chlorophenols Compound

Precursor ion (m/z)

Excitation voltage (V)

Product ion range (m/z)

Quantitative ions (m/z)

TCA TeCA PCA DCA (IS)

195 231 265 176

0.75 0.70 0.70 0

100–200 100–235 100–270 170–185

167 203 237 176

TCP TeCP PCP TBP (IS)

196 230 264 332

1.8 1.8 1.8 0

100–200 100–235 100–270 325–335

132 + 160 194 + 166 165 + 200 332

was held at 270 ◦ C. Xcalibur 1.4 software was employed for the data acquisition and parameter control. In preliminary trials, scan runs were recorded in the mass range of m/z 50–400 for qualitative purposes. Compounds were clearly identified by comparison with reference spectra (Wiley7 database). In order to improve sensitivity and selectivity in GC detection, the ion trap was operated in MS–MS mode. The excitation voltage required to fragment the precursor ion was evaluated for the studied compounds. The MS–MS parameters, including the precursor ion and product ions chosen for quantitative purposes, are described in Table 1.

Table 2 Recovery and precision data (repeatability) of the analytical method for the analysis of chloroanisoles and chlorophenols in cork samples spiked at 20 ng/g, 10 ng/g and 5 ng/g Compound

TCA TeCA PCA TCP TeCP PCP

Recovery (repeatability RSD) (%) 20 ng/g spiked (n = 9)

10 ng/g spiked (n = 5)

5 ng/g spiked (n = 5)

107.1 (8.2) 101.6 (8.0) 97.7 (9.0) 90.8 (10.1) 75.4 (4.5) 57.4 (15.2)

103.5 (8.0) 96.7 (6.1) 91.9 (8.3) 94.9 (5.0) 78.8 (9.5) 66.1 (4.9)

109.1 (4.9) 109.6 (5.8) 104.2 (8.9) 110.5 (1.8) 87.5 (7.9) 76.2 (7.1)

3. Results and discussion 3.1. Evaluation of the method for the analysis of chloroanisoles and chlorophenols in cork material The assay of both chloroanisoles and chlorophenols from a single cork sample is not an easy task. The chemical differences among the two families of compounds, especially in terms of polarity and the reactivity of the hydroxyl group, had led to the development of methodologies for their separate analysis. Taking advantage of the ability of organic solvents to extract chloroanisoles as well as chlorophenols [23–25] from cork samples, we have developed a methodology with the aim of quantifying all considered analytes present in contaminated cork. Prior to the evaluation of the cork extraction strategy, we checked the efficiency of liquid–liquid extraction for the separation of chloroanisoles from chlorophenols present in a pentane solution. This step is necessary due to the poor chromatographic behaviour of chlorophenols, which is substantially improved when the phenols are transformed into the corresponding acetyl derivates. Although this reaction has been reported to take place in organic media [29], our preliminary studies resulted in better yields when the derivatisation was carried out in aqueous solution. Chlorophenols can be recovered from the organic solvent to an aqueous layer containing 5% of K2 CO3 while the chloroanisoles remain in the organic phase. The recovery results from a standard solution containing 20 ng/mL in the case of the chloroanisoles and 15 ng/mL for the chlorophenols show that the liquid–liquid extraction process is quantitative for all studied analytes (n = 2). The lowest value obtained is for PCP, with a recovery of 81.7% and relative standard deviation of 2.7%.

Once the separation of the chloroanisoles from their direct precursors was evaluated, we tested the overall method, including the extraction of the analytes from cork samples, separation, preconcentration and gas chromatography analysis. Spiked cork with the target analytes at different concentration level was used for these experiments. The spiking procedure was carried out as described in Section 2. In Table 2, we can observe the recovery results for each analyte as well precision expressed as repeatability. These results can be considered excellent taking into account the variability associated with a natural matrix such as cork. Onefactor analysis of variance (ANOVA) was performed to compare the results obtained at different spiking levels. No significant differences at 95% confidence level were observed. From these values a calibration curve was built. As can be seen in Table 3, the fit of the response to the linear calibration was good (r > 0.986) and the limits of detection, calculated based on a signal-to-noise ratio of 3:1, even though slightly higher, agrees well with those Table 3 Parameters of the different calibration curves built (y = a + bx) to quantify the target compounds in cork samples Compound

a (Sa)

b (Sb)

r

LOD (ng/g)

TCA TeCA PCA TCP TeCP PCP

0.05 (0.01) 0.01 (0.02) 0.02 (0.01) 0.04 (0.01) 0.04 (0.01) 0.07 (0.01)

11.2 (0.4) 14.7 (0.6) 14.0 (0.4) 0.56 (0.01) 0.65 (0.02) 0.49 (0.01)

0.987 0.986 0.989 0.992 0.992 0.992

2.2 2.4 4.1 0.5 1.3 1.7

Standards prepared from non-faulty cork spiked at the chosen concentration level.

S. Insa et al. / J. Chromatogr. A 1122 (2006) 215–221

219

Table 4 Recovery results obtained for long-term spiked samples with 20 ng/g of chloranisoles and chlorophenols (n = 3) Compound

TCA TeCA PCA TCP TeCP PCP

Recovery (RSD) (%) 12 h

24 h

103.8 (9.4) 100.4 (9.2) 95.7 (6.9) 95.5 (2.5) 76.3 (2.8) 59.5 (6.4)

102.5 (3.2) 99.6 (5.3) 93.7 (5.2) 82.9 (5.7) 69.2 (2.0) 51.3 (1.6)

reported in the literature taking into account the characteristics of this kind of sample [30]. Further experiments were undertaken where spiked cork was allowed to stand for 12 and 24 h before extraction. This longer time allowed the analytes to interact closely with the cork matrix, in a way similar to when they are naturally present in contaminated cork. As can be seen in Table 4, the data for long-term spiked samples compare well with the results obtained in Table 2, although a slight decrease of the recovery is observed. From the one-factor analysis of variance (ANOVA) it can be concluded that long-term spiking does not have a significant effect. Fig. 1a and b illustrates the gas chromatograms obtained from a cork spiked with 20 ng/g of both chloroanisoles and chlorophenols. Peaks of the analytes are clearly separated from other compounds present in the cork matrix. This fact encourages the application of the developed method for a reliable analysis of the major compounds responsible for cork taint. 3.2. Analysis of real cork samples The methodology was applied to the analysis of different types of raw cork material which had been rejected for the manufacture of cork closures because they are susceptible to be contaminated by the analytes under consideration. On the one hand, we chose a cork slab taken from the foot region of the cork oak. It was selected because chlorophenolic-based products have commonly been reported as biocides applied to the base of the trees to control insect pests [7]. On the other hand, we also analysed cork slabs which clearly exhibited the presence of a specific defect called yellow stain, caused by a fungal infection. As shown in Table 5, only trichloro-compounds were detected and consequently quantified in the studied matrices. The unequivocal identification of the analytes was confirmed by

Fig. 1. Examples of chromatograms (GC-ECD) when analysing spiked cork at 20 ng/g concentration: (a) chloroanisoles and (b) chlorophenols.

analysing these samples by gas chromatography coupled to a mass spectrometer detector (see Section 2.7). 3.3. Washing tests for the removal of TCA and TCP from contaminated samples The elimination of compounds responsible for cork taint is of great interest to the cork industry. Well-known cork companies have developed their own methodologies, most of which are patented [31], while other systems are commercially available. In general, factories have focused mainly on removing possible sources of contamination during processing. Various cleaning operations take place at different stages in cork manufacturing, including boiling and washing. According to factory and laboratory studies, certain washing agents have been eliminated from cleaning processes because of their involvement in the formation of compounds responsible for cork taint. Under normal conditions, the cork manufacturing process (above all the boiling stage) reduces the level of TCA [32]. Our contribution here is addressed to the evaluation of simple and inexpensive washing treatments that are already used or can be easily implemented in

Table 5 Concentration of TCA and TCP detected in several cork samples: cork barks which have been in contact with the soil and cork slabs presenting off-flavours related to cork taint and the default designated by yellow stain Detected compound

Concentration (ng/g) (SD) Foot region slab (n = 3)

TCA TCP a

3.3 (0.1) 0.7 (0.4)

This slab was used for the performance of the washing trials.

Slabs with off-flavours and yellow stain At low concentration (n = 3)

At high concentrationa (n = 9)

45.4 (8.2) 4.7 (0.3)

534.9 (66.9) 26.0 (3.4)

220

S. Insa et al. / J. Chromatogr. A 1122 (2006) 215–221

Table 6 Distribution of TCA and TCP after applying a water washing Compound

Initial content in cork (untreated cork)

Final content in cork (washed cork)

Losses due to evaporation (%)

Content in washing water

TCA TCP

534.9 (66.9) 26.0 (3.4)

136.0 (18.9) 10.6 (1.43)

51 9

42.3 (3.4) 10.0 (1.6)

The data are expressed as nanogram (SD).

cork stopper factories because they are based on the employment of standard reagents as washing agents. Cork samples used for these experiments were selected from those slabs which exhibited the higher values of TCA and TCP in Table 5. These samples were analysed several times at intervals over a 1-week period (n = 9) so as to verify that the analyte content in the sample remained constant during the experiments. As was described in Section 2.6, the washing process involves washing cork samples with the appropriate reagent, then airdrying them and, finally, the evaluation of TCP and TCA remaining in the cork. Due to the known volatility of TCA, additional trials were undertaken in order to evaluate possible losses of this compound during the air-drying step. For this purpose, ground cork was allowed to stand in the open air throughout the same period of time as the samples treated. The analysis of this untreated sample gave a 51% loss of TCA and a 9% loss of TCP. Standard deviation was less than 13% (n = 5) showing the good repeatability obtained for this assay. These results will be taken into account when different washing test are compared. We selected Milli-Q water, NaOH solution (0.1 M and 0.01 M) and two commercial products (product 1: peracetic and product 2: sulfamic) used as disinfectant and deodoriser for cork stoppers as washing agents. Washing with water was carried out in two different ways: by shaking in the rotary mixer and by immersing in an ultrasound bath. In Fig. 2 the efficiency of the selected washing treatments is given. We can state that washings performed with NaOH led to a higher elimination of both TCA and TCP. In the first attempt, we decided to apply NaOH at a concentration of 0.1 M since this is the usual concentration when NaOH solutions are used as extractants of phenols from soil samples [33]. The effectiveness of the aqueous alkaline solution is explained by the conversion of the phenolic group into a phenolate. This acid–base reaction converts the analytes into ionic compounds which are more easily recovered from the solid matrix. As can be seen in Fig. 2, this alkaline washing treatment led to a complete removal of TCP from the cork together with a 72% elimination of TCA. The percentages have been calcu-

lated after subtracting the amount of TCA and TCP lost due to evaporation from the initial content. It is noticeable the high rate of TCA elimination obtained with this treatment, which is probably due to the solubilization of this compound in aqueous solution. In addition, to overcome the possible damage that the NaOH solution may cause to the cork, a more diluted NaOH solution was also tested. Under these conditions, less efficient removals were observed. It is remarkable that a low-cost washing agent such as water is able to reduce the TCA and TCP content to nearly 50%. No improvement was observed when ultrasounds were applied. The worst results were obtained when the washings were carried out with the commercial agents (product 1: peracetic and product 2: sulfamic). The behaviour of these two products regarding the elimination of TCP can be explained taking into account their chemical composition, based on acidic reagents which do not favour the acid–base reaction responsible for the removal of chlorophenols. In the case of TCA, the poor results obtained may be due to the high viscosity of the washing solution that diminished the solubility of 2,4,6-trichloroanisole. The water obtained after the washing treatment was also analysed by applying a headspace solid-phase microextraction gas chromatography method (see Section 2.6) in order to verify the mass balance. Therefore, the sum of the TCA and TCP detected in cork after the treatment plus the amount found in the aqueous solution should be equal to the amount initially present. In Table 6 we show the values obtained. Taking into account the standard deviation values we can say that the data compare rather well, demonstrating the reliability of the figures in Table 6 and the usefulness of the applied approaches. On the whole, our results are in agreement with those indicating a noticeable reduction of TCA level after a boiling step [32], related to the extraction power of pure water. Further experiments will be performed in order to evaluate how the NaOH solutions may affect the performance (flexibility, impermeability, chemical inertness, etc.) of cork stoppers as closures of wine bottles. 4. Conclusion

Fig. 2. Study of the percentage of elimination of TCA and TCP from cork samples applying several washing treatments.

In this work, we have evaluated a method for the determination of chloroanisoles and chlorophenols in cork samples by gas chromatography. The procedure is based on a simultaneous extraction of the target analytes from the cork with an organic solvent followed by a liquid–liquid extraction. The overall methodology has been applied to spiked corks at three concentration levels. Recoveries ranging between 110% and 91% for chloroanisoles and between 111% and 57% for chlorophenols have been obtained. Precision data expressed as relative standard

S. Insa et al. / J. Chromatogr. A 1122 (2006) 215–221

deviations have been on average lower than 10%. Several naturally contaminated samples have been analysed accordingly and have been also employed for the cork washing evaluations. Among the all studied treatments, aqueous sodium hydroxide solutions seem to be the most suitable for the removal of the analytes from contaminated cork samples. Acknowledgments Sara Insa received a grant from the FPU program of the Spanish Ministry of Education, Culture and Sport (Ref. AP20010989). The authors would also like to thank Francisco Oller S.A. for the provision of the cork samples and AECORK (the trade association of the Catalan cork manufacturers) for providing the commercial washing agents. References [1] P. Godden, L. Francis, J. Field, M. Gishen, A. Coulter, P. Valente, P. Høj, E. Robinson, Aust. J. Grape Wine Res. 7 (2001) 64. [2] P. Fuller, Aust. NZ Wine Ind. J. 10 (1995) 58. [3] QUERCUS-Qualitative Experiments to Determine the Components Responsible and Eliminate the Causes of Undesirable Sensory Characteristics in Drinks Stoppered With Cork, European Union and C.E. Li`ege, Contract No. AIR1-CT92-0372, 1996. [4] J.M. Amon, J.M. Vandepeer, R.F. Simpson, Aust. NZ Wine Ind. J. 4 (1989) 62. [5] P. Chatonnet, S. Bonnet, S. Boutou, M.D. Labadie, J. Agric. Food Chem. 52 (2004) 1255. [6] R.F. Simpson, D.L. Capone, M.A. Sefton, J. Agric. Food Chem. 52 (2004) 5425. [7] C. Silva Pereira, J.J. Figueiredo Marques, M.V. San Rom˜ao, Crit. Rev. Microbiol. 26 (2000) 147. [8] S. Rocha, I. Delgadillo, A.J. Ferrer Correia, A.M. Bastos, I. Roseira, A. Guimar˜aes, Aust. NZ Wine Ind. J. 8 (1993) 223. [9] M. Careri, V. Mazzoleni, M. Musci, Molteni R, Chromatographia 53 (2001) 553.

221

[10] M.F.A. Ferreira Cabral, J. Leal Ferreira, Aust. NZ Grapegrow. Winemak. 449A (2001) 169. [11] M.K. Taylor, T.M. Young, C.E. Butzke, S.E. Ebeler, J. Agric. Food Chem. 48 (2000) 2208. [12] D. Rowe, Aust. NZ Wine Ind. J. 18 (2003) 24. [13] A.P. Pollnitz, K.H. Pardon, D. Liacopoulos, G.K. Skouroumounis, M.A. Sefton, Aust. J. Grape Wine Res. 2 (1996) 184. [14] C. Fischer, U. Fischer, J. Agric. Food Chem. 45 (1997) 1995. [15] R. Juanola, D. Subir`a, V. Salvad´o, J.A. Garcia Regueiro, E. Antic´o, J. Chromatogr. A 953 (2002) 207. [16] R. Juanola, L. Guerrero, D. Subir`a, V. Salvad´o, S. Insa, J.A. Garcia Regueiro, E. Antic´o, Anal. Chim. Acta 513 (2004) 291. [17] N. Campillo, N. Aguinaga, P. Vi˜nas, I. L´opez-Garc´ıa, M. Hern´andezC´ordoba, J. Chromatogr. A 1061 (2004) 85. [18] F. Bianchi, M. Careri, A. Mangia, M. Musci, J. Sep. Sci. 26 (2003) 369. [19] J.L. G´omez-Ariza, T. Garc´ıa-Barrera, F. Lorenzo, A. Gustavo Gonz´alez, Anal. Chim. Acta 540 (2005) 17. [20] O. Ezquerro, M.T. Tena, J. Chromatogr. A 1068 (2005) 201. [21] C. Bayonove, F. Leroy, Simposio Internationale Il sughero in Enologia, Pavia, 1993. [22] S. Insa, V. Salvad´o, E. Antic´o, J. Chromatogr. A 1047 (2004) 15. [23] W.R. Sponholz, H. Muno, Ind. Bevande XXIII (1994) 133. [24] A. Pe˜na-Neira, B. Fern´andez de Sim´on, M.C. Garc´ıa-Vallejo, T. Hern´andez, E. Cadah´ıa, J.A. Suarez, Eur. Food Res. Technol. 211 (2000) 257. [25] P. Chatonnet, D. Labadie, S. Boutou, J. Int. Sci. Vigne Vin 37 (2003) 181. [26] G.J. Soleas, J. Yan, T. Seaver, D.M. Goldberg, J. Agric. Food Chem. 50 (2002) 1032. [27] R. Berthod, O. Flaction, U. Frey, Chimia 58 (2004) 560. [28] S. Insa, E. Besal´u, V. Salvad´o, E. Antic´o, J. Sep. Sci., submitted for publication. [29] M. Ramil Criado, S. Pombo da Torre, I. Rodr´ıguez Pereiro, R. Cela Torrijos, J. Chromatogr. A 1024 (2004) 155. [30] M. Riu, M. Mestres, O. Busto, J. Guasch, J. Chromatogr. A 1107 (2006) 240. [31] M.A. Sefton, R.F. Simpson, Aust. J. Grape Wine Res. 11 (2005) 226. [32] M. Hall, Food Rev. 24 (1997) 21. [33] M.A. Cresp´ın, M. Gallego, M. Valcarcel, Anal. Chem. 71 (1999) 2687.