Simultaneous separation, clean-up and analysis of musty odorous compounds in wines by on-line coupling of a pervaporation unit to gas chromatography–tandem mass spectrometry

Analytica Chimica Acta 516 (2004) 165–170

Simultaneous separation, clean-up and analysis of musty odorous compounds in wines by on-line coupling of a pervaporation unit to gas chromatography–tandem mass spectrometry J.L. Gómez-Ariza∗ , T. Garc´ıa-Barrera, F. Lorenzo Departamento de Qu´ımica y Ciencia de los Materiales, Facultad de Ciencias Experimentales, Universidad de Huelva, Campus de El Carmen, 21007 Huelva, Spain Received 17 December 2003; received in revised form 16 March 2004; accepted 17 March 2004 Available online 25 May 2004

Abstract A procedure for fast, reliable and precise analysis of 2,4,6-trichloroanisole (TCA) and other anisoles [2,6-dichloroanisole (DCA) and 2,4,6-tribomoanisole (TBA)] in cork-tainted wines has been performed. The method is based on compounds separation by pervaporation (PV) and later on-line clean-up, using a Carbofrit packed liner (CPL) fitted to the gas chromatograph–mass spectrometer injector (PV–CPL–GC–MS). Detection limit was about 54 pg for TCA (5 ng l−1 when aliquots of 10 ml were used in the analysis). These results were comparable with those obtained from the coupling pervaporation solid-phase cryogenic trap–thermal desorption GC–MS (PV–CT–TD–GC–MS) and solid-phase microextraction gas chromatography. Precision of PV–CPL–GC–MS, expressed as relative standard deviation, was 5.2, 2.0 and 6.2% for DCA, TCA and TBA, respectively. Linear range of calibration curves ranged from quantification limit to 2 ng for all the anisoles. In addition, the method based on the PV–CPL–GC–MS coupling was more rapid and reliable than that using PV–CT–TD–GC–MS, due to the one-step direct on-line introduction of analytes from the PV unit to the GC–MS system facilitated by CPL, which increases the analysis precision and sample throughput. © 2004 Elsevier B.V. All rights reserved. Keywords: Anisoles; Wine; Pervaporation; GC–MS; Taint; Off-flavour; Musty compounds; Carbofrit

1. Introduction An important problem for wine and cork industries is the appearance of a mouldy/musty off flavour called “cork taint” that affect between 0.1 and 10% of European bottled wines [1], which represent a great economical concern. Recent studies have concluded that the main compound responsible for this odour is 2,4,6-trichloroanisole (TCA) [2], although other compounds such as 2,6-dichloroanisole (DCA), 2,4,6-tribromoanisole (TBA), geosmin, guaiacol, 2-methylisoborneol, 1-octen-3-one, 1-octen-3-ol and chlorophenolic compounds may also contribute [3,4]. The human sensory threshold for this type of compounds in wine is in the range of 1.4–10.0 ng l−1 [5], concentrations that are beyond the sensitivity of most analytical systems if

∗

Corresponding author. Tel.: +34-959-019968; fax: +34-959-019942. E-mail address: [email protected] (J.L. G´omez-Ariza).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.03.083

a preconcentration step is not introduced. Therefore, several preconcentration methods have been proposed for this purpose such as liquid–liquid extraction with recoveries in the range 43–72% [6], supercritical fluid extraction (SFE) for cork stoppers [7], and solid-phase microextraction (SPME) for TCA isolation from wines [3], with a significative increases in recovery and improvements in accuracy, sensitivity and sample throughput. Pervaporation (PV) constitutes a reliable alternative for volatile compounds analysis from liquid and solid samples, which compares favourably with headspace for analyte extraction and introduction in some analytical instrumental devices, especially gas chromatography [8]. The pervaporation unit consists of two chambers separated by a hydrophobic membrane. The analytes pass through the membrane and they are driven to the upper chamber where an inert gas flow drives them to the GC–MS. Pervaporation has been applied to the analysis of organometallic contaminants [9,10], volatile organic compounds (VOCs) [11], and

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pesticides [12], as well as in study of volatile compounds in food, such as trimethylamine in fish [13], and the selective determination of pectinesterase activity in fruits [14]. Pervaporation is a versatile technique that can be coupled with a solid-phase cryogenic trapping–thermal desorption unit (PV–CT–TD) for very low level analytes determination in complex matrices [10]. Recently, it has been proposed for the analysis of DCA, TCA and TBA in wines at levels below 40 or 5 ng l−1 , when the CT–TD post-pervaporation step is used [15]. Carbon-base packed liners have been used for simultaneous improvement of detection limits and sample clean-up. The commercial product used in the liner (Carbofrit) consisted in a carbon material of open structure and low retention power. It has been reported for the introduction of large volumes of water containing samples into a GC–MS for the analysis of methyl esters of the fatty acids (C6 –C28 ) [16], therapeutic agents extracted from rat plasma [17] and analysis of pesticides in fruits and vegetables [18]. Anisoles exhibit a lightly polar character that induce their fixation in the Carbofrit and are thermally desorbed when a temperature ramp is applied to the injector. In this work, a Carbofrit packed liner was used in order to improve the detection limits and clean-up wine samples prior to their introduction into a GC–MS system. The results were suitably compared with those from a PV–CT–TD system. Fig. 1. Pervaporation laboratory-made device.

2. Experimental 2.1. Standard solutions and reagents Milli-Q water (Millipore, Waford, UK) was used for intermediate standard solutions. Ethanol absolute (ROMIL-SpS) was obtained from Teknokroma (Barcelona, Spain). 2,6-Dichloroanisole (97%), 2,4,6-trichloroanisole (99%), 2,4,6-tribromoanisole (99%) and lindane (97%), used as internal standard [19], were purchased from Aldrich (Steinheim, Germany). Stock solutions were prepared at concentrations of 514, 512, 514, 720 ␮g l−1 in ethanol for 2,6-dichloroanisole, 2,4,6-trichloroanisole, 2,4,6-tribromoanisole and lindane, respectively. Intermediate solutions were prepared by dissolving appropriate volumes of stock solutions in 12% (v/v) ethanol–water. All standard solutions were stored in the dark at 4 ◦ C until analysis. 2.2. Instrumentation The instrumental coupling for anisoles analysis consists of a high pressure injection valve (Rheodyne, USA), a laboratory-made pervaporation unit, and a GC–MS system (Varian Ibérica, Barcelona, Spain). The pervaporation module consisted of a lower compartment, where the sample was placed, an upper compartment in which the carrier gas collected the volatile analytes, and a hydrophobic membrane (PTFE membrane, 1.5 mm thick and

40 mm diameter, Trace Biotech, Braunschweig, Germany) that separates both compartments placed on a support. The volume of the chambers could be selected by putting spacers between the membrane support and the corresponding compartment. The two chambers were aligned with the membrane support using two metallic bars. The whole module was placed between two aluminium supports and four long screws closed the system tightly. A Minipuls-3 peristaltic pump (Gilson, France) was used for liquid sample introduction. The scheme of a pervaporation device is shown in Fig. 1. Volatile compounds were analysed using a Varian Model 3800 gas chromatograph paired with a Saturn 2000 ion-trap mass spectrometry detector (Varian, Sunnyvale, CA, USA). The gas chromatograph was fitted with a fused-silica capillary column with a VF-5 ms stationary phase and dimensions: 30 m × 0.25 mm i.d., 0.25 ␮m film thickness (Factor Four CPSIL-8, Varian Ibérica). A split liner (3.4 mm i.d.) packed with 0.5 cm of Carbofrit (Restek, Bellefonte, PA, USA) was used into the injector port. 2.3. Procedures 2.3.1. Chromatographic analysis The carrier gas was helium at a flow rate of 1 ml min−1 . Pervaporation outlet was directly coupled to a split–splitless injector with a Carbofrit packed liner: initial temperature 50 ◦ C ramped to 310 ◦ C at 200 ◦ C min−1 . Split valve was

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Table 1 MS–MS parameters for anisoles analysis Anisole compound

Precursor ion

Product ion

2,6-Dichloroanisole 2,4,6-Trichloroanisole 2,4,6-Tribromoanisole Lindane (internal standard)

176 195 344 183

133 167 329 + 301 148 + 146 + 109

opened at 20:1 ratio from initial time to 0.01 min, then closed during 1 min, and finally opened to 1:50 ratio. The temperature of the GC–MS transfer line was maintained at 280 ◦ C. The temperature of the oven was set at 45 ◦ C for 2 min, subsequently increased to 265 ◦ C with a ramp of 12 ◦ C min−1 , and finally held at 265 ◦ C for 1 min. Full scan electron impact ionisation data were acquired under the following conditions: solvent delay 9 min, 70 eV electron impact energy, emission current 30 ␮A, scan time 1 s per scan, and manifold and trap temperatures 50 and 200 ◦ C, respectively. The automatic gain control was switched on with a target fixed at 20 000 counts. The overall run time consisted of 9 min of delay and one segment, scanning the following range (m/z): 90–400 from 9 to 21.33 min. 2.3.1.1. MS–MS mode. In order to improve sensitivity and selectivity, the option MS–MS was used. When the mass detector operated in MS–MS mode, emission current was fixed at 80 ␮A and scan time at 0.6 s per scan. To program the isolation of precursor ions for every compounds along the chromatographic run, the overall run time was split into five segments scanning the following ranges (m/z): 80–190 in the second segment (9.00–11.30 min); 90–210 in the third segment (11.30–14.00 min); 120–360 in the fourth segment (14.00–15.80) and 80–360 in the fifth segment (15.80–21.33). Precursor ions were isolated using 3 amu isolation window and subjected to collision-induced dissociation (CID) are shown in Table 1. Automated method development toolkit software was used to optimise the CID parameters (low mass cut off and CID voltage) to obtain maximum sensitivity. The excitation storage level was selected at the minimum value that allowed the dissociation of the precursor ion. High CID energies were required due to the stable nature of the selected precursor ions. 2.3.2. Pervaporation coupled to a GC–MS system provided with a packed liner (Approach A) A peristaltic pump was used for liquid sample introduction in the pervaporation device. Nine millilitre of red or white wine were placed into a 10 ml glass vial sealed with a silicone septum and an additional aliquot of 1 ml of wine was injected in the lower chamber of the pervaporation module. A spacer was put below the membrane to separate it from the sample in order to create a headspace. Two Teflon tubes were connected to the sample containing vial through

CID parameters Store level (m/z)

Amplitude (V)

80 90 120 80

69 78 97 72

the septum. One of them was attached to the inlet port of pervaporation device (lower chamber) and the other one was connected to the outlet port to allow the recirculation of the sample. Sample was flushed through the tubes by the peristaltic pump at 5 ml min−1 . The pervaporation module was placed in a water bath at 85 ◦ C and mechanically stirred for 5 min. The high pressure valve was switched and a helium stream (60 ml min−1 ) drove the pervaporated analytes from the upper chamber to the Carbofrit packed liner in the chromatograph injector. 2.3.3. Pervaporation with solid-phase cryogenic trapping–thermal desorption preconcentration (PV–CT–TD) (Approach B) A sorbent type K (58.8% Carbopack B, 35.3% Carboxem 1000 and 5.9% Carboxem 1001) was chosen for analytes preconcentration. A U-shaped stainless steel tube (25 cm length×1 mm i.d.) was packed with the sorbent and placed in an oven–refrigeration system. Carrier gas for pervaporation and sorption was nitrogen at a flow rate of 60 ml min−1 . In the leaching-preconcentration step, temperatures were fixed at 85 ◦ C in the pervaporation chamber and at 0 ◦ C (ice-water bath) in the preconcentration sorption minicolumn. Time needed for the pervaporation process was 5 min. In the desorption step, the helium flow was fixed at 80 ml min−1 and temperature in the minicolumn at 210 ◦ C. The analytes are driven by the helium stream to the chromatograph. A scheme of this approach is shown in Fig. 2.

Fig. 2. Scheme of PV–CT–TD device.

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3. Results and discussion 3.1. Pervaporation variables The main variables controlling the pervaporation process are sweeping gas flow and pervaporation temperature. When the temperature increased, compounds releasing from the matrix is enhanced and sensitivity increased, however, high temperatures do not improve the signal and the precision decreased, probably due to the poorer stabilisation of the bath temperature. Preconcentration time of the analytes in the static gas volume of the headspace constituted by upper pervaporation chamber is also critical. Longer time yielded better results, due to the dynamic character of the acceptor gas that is permanently in contact with the membrane and produces an efficient and continuous diffusion of the analytes from the air gap above the sample to the upper chamber. In order to improve the sensitivity, large volumes of sample (10 ml) were introduced in the pervaporation device using a peristaltic pump. Sample flow rate was optimised in the range 2–5 ml min−1 . Two millilitre per minute is the lowest flow that allows the whole sample volume to pass one time through the lower chamber. When flow rates higher than 5 ml min−1 were used, sample splashes the membrane surface. Therefore, 5 ml min−1 was chosen as optimum flow since, it allows the sample to pass two and a half times by the chamber maintaining the dynamic equilibrium. The use of a high carrier gas flow (He) improves the signal due to the higher amount of analyte introduced in the chromatograph but the optimum value is limited by the dynamic characteristics of the chromatographic injector. All these parameters were studied and optimised in a previous work and were reported elsewhere [15]. First of all, Carbofrit packed liner was conditioned inside the injector port by heating at 300 ◦ C for 2 h. The adsorptive properties of Carbofrit for the analytes allow a progressive introduction of them into the injector before the chromatographic run start. The pervaporated analytes are progressively fixed in this solid phase, which permits the introduction of higher amount of analytes. Optimisation of time necessary for the adsorption of pervaporated analytes in the Carbofrit was performed in the range 5–30 s, and finally fixed at 20 s, shorter and longer times provided lower peak areas. Afterwards, the chromatographic run started. Temperature in the liner was fixed at 50 ◦ C, higher temperatures during the sorption time produce losses of the analytes. Temperature was ramped to 310 ◦ C at 200 ◦ C min−1 . Fast temperature ramps in the injector, increased peak areas, 200 ◦ C was chosen as optimum because no peak tailing was observed. After 1 min, split valve was open at 50:1 ratio. The introduction of Carbofrit improves the inertness of the injector port referred to the standard quartz wool commonly used. With this injection technique very good sensitivity is achieved and the background noise is reduced by absorption of the sample matrix in the liner packing.

Table 2 Performances in anisoles analysis by GC–MS from 12% (v/v) ethanol–water solutions: (a) pervaporation coupled to a Carbofrit packed liner (CPL) (Approach A); (b) pervaporation solid-phase cryogenic trapping–thermal desorption preconcentration (PV–CT–TD) (Approach B); (c) pervaporation without preconcentration Analyte

DL (pg)

R.S.D. (%)

R2

(a) PV–CPL DCA TCA TBA

54.4 54.2 66.2

5.2 2.0 6.2

0.995 0.998 0.996

(b) PV–CT–TD DCA TCA TBA

38.2 42.0 45.7

8.5 6.3 4.7

0.997 0.999 0.998

11.9 6.2 8.5

0.995 0.996 0.998

(c) PV without preconcentration DCA 409 TCA 258 TBA 183

3.2. Performance of anisoles analysis with PV–GC–MS using a CPL 3.2.1. Linearity and limit of quantification The analytical performance of PV–GC–MS coupling with a Carbofrit packed liner (CPL) (Approach A) is shown in Table 2(a). Integrated peak area ratios (peak area analyte/peak area internal standard) were calculated and plotted against the absolute amount of the analytes (pg). The instrumental detection limits (DLs) (calculated as three times the standard deviation of the calibration curve) were 54.4, 54.2 and 66.2 pg for DCA, TCA and TBA, respectively (confidence level 99.5%), corresponding to a lowest concentration detectable in the sample of 5.4 and 6.6 ng l−1 for the compounds formerly cited, if the procedure is applied to 10 ml of sample. Quantification limits (QLs) (calculated as 10 times the standard deviation of the calibration curve) were estimated to be 181.3, 180.7 and 220.7 pg for DCA, TCA and TBA, respectively. Linear calibration curves were obtained from the quantification limit of each standard to 2 ng in all the cases. 3.2.2. Accuracy and precision The relative standard deviation (R.S.D.) of 10 sequential injections of analytes and internal standard solution at 50 ng l−1 in 12% (v/v) ethanol–water mixture were 5.2, 2.0 and 6.2% for DCA, TCA and TBA, respectively. The averaged recoveries in the experiment were 98.8, 99.6 and 94.0% for DCA, TCA and TBA, respectively. Additional recovery studies were carried out in natural tainted wines (red and white), reported to be tainted by sensory trial, after TCA spike. Table 3 shows the results obtained with Approach A and Approach B. Results are quite comparable with standard deviations ranging from 0.3 to 1.8 ng l−1 . The averaged recovery in the spike experiments were 102.8% over a wide concentration range.

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Table 3 Recovery trial for TCA spiked into “tainted“ wines Wine sample

La Mancha (red wine) Rioja (red wine) Rioja (white wine) Valdepeñas (red wine) Condado de Huelva (white wine) a

Approach A

Approach B

Initial measured concentration of TCAa , X ± σ (ng l−1 )

Measured concentration after 20 ng l−1 spikea , X ± σ (ng l−1 )

98.8 ± 1.8 54.2 ± 1.3

120.4 77.7 19.0 38.6 21.5

± ± ± ± ±

2.4 1.7 0.5 0.6 0.3

Mean recovery Initial measured of spike (%) concentration of TCA, X ± σ (ng l−1 ) 108.0 117.5 95.0 88.0 107.5

96.5 ± 6.0 59.4 ± 3.5

Measured concentration after 20 ng l−1 spike, X ± σ (ng l−1 ) 118.3 80.1 17.5 39.8 23.6

± ± ± ± ±

7.3 4.9 0.9 2.7 1.3

Mean recovery of spike (%)

109.0 103.5 87.5 82.0 118.0

Average of three replicates.

Fig. 3 shows the chromatograms obtained from red wine spiked with 405.5, 404.9, 405.9 and 563.3 pg of DCA, TCA, TBA and lindane, respectively. In Approach A pervaporation with Carbofrit packed liner was used, and in Approach B pervaporation and CT–TD were involved. 3.3. Comparison of pervaporation methods for anisoles analysis Several experimental approaches based on the use of pervaporation have been tested in anisoles analysis from 12% (v/v) ethanol–water solutions. Pervaporation is a very versatile pretreatment system for volatile analytes, such as anisoles, which can be quantitatively separated from solid or liquid matrices using a simple PV extraction. When

low levels of analytes have to be quantified a preconcentration step need to be introduced after the PV extraction to make possible the analysis. In this case a solid-phase cryogenic trapping–thermal desorption (CT–TD) or a Carbofrit packed liner (CPL) fitted to the GC–MS injector are the two choices, when they are on-line connected to the PV outlet. Table 2 shows the analytical features for the three PV extraction methods, when they are applied to the analysis of anisoles from ethanol–water solutions. Detection limits suffer a drastic reduction when some preconcentration procedure is applied; the decrease is more marked for PV–CT–TD although the reduction is similar with PV–CPL which exhibits on the contrary a better precision. Single PV shows more limited sensitivity, especially for DCA, with detection limit about 8- and 11-fold higher

Fig. 3. Chromatograms obtained from red wine spiked with 405.5, 404.9, 405.9 and 563.3 pg of DCA, TCA, TBA and lindane (␥-hexachlorocyclohexane, ␥-HCH), respectively. Approach A: pervaporation with Carbofrit packed liner; Approach B: pervaporation with CT–TD.

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than those for PV–CPL and PV–CT–TD, respectively. For TCA sensitivity using single PV, detection limit is about 5and 6-fold higher respect to the same systems. Therefore, the use of preconcentration is necessary for the analysis of anisole in tainted wine, in order to reach the TCA level usually found in these samples. On the other hand, quantification limits are comparable with those reported in the literature for SPME [5], which has been established about 5 ng l−1 for TCA, however, SPME averaged precision expressed as R.S.D. is about 10%, which is higher than that of both PV–CPL and PV–CT–TD, estimated as 2 and 6.2%, respectively. PV–CT–TD shows detection and quantification limits slightly lower than PV–CPL, but precision of this later is better. This fact can be explained because CPL is a commercial accessory and CT–TD has to be prepared and packed at the laboratory, which undoubtedly affect the method reproducibility. In addition, CT–TD is performed in two steps (trapping and desorption) which are more time-consuming than direct analytes introduction in the GC–MS system performed with CPL. This fact affects the sample throughput which was estimated to be about 2 and 1 samples h−1 using PV–CPL and PV–CT–TD, respectively.

4. Conclusions Pervaporation is a useful alternative to the existing methods for TCA and other anisoles analysis in wine, especially SPME. Generally, on-line preconcentration is necessary to reach the levels of anisoles in wine, and the combination of PV with a Carbofrit packed liner (CPL) fitted to the GC–MS system provide a rapid, sensitive and precise method for the analysis of TCA in this matrix, which compares favourably with PV–CT–TD due to higher precision and sample throughput. Sample throughput has been reported to be 1 sample h−1 using SPME–GC–MS [5] against 2 samples h−1 reported for PV–CPL–GC–MS method. Sensitivity of PV–CPL method is similar to SPME and that from PV–CT–TD is even better, but SPME precision is clearly improved for both pervaporation approaches. Therefore, PV–CPL could be used for routine analysis of TCA in wines to monitor the incidence and causes of cork taint. The procedure could be tested in further studies connected

to other cork-related compounds such as geosmin, guaiacol, 2-methylisoborneol, 1-octen-3-one and 1-octen-3-ol.

Acknowledgements The authors would like to thank “Ministerio de Ciencia y Tecnolog´ıa (MCyT)” for the Grant REN2002-04366-C02-02. F.L. thanks the “Junta de Andaluc´ıa” for a predoctoral grant.

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