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Room temperature and sensitive determination of haloanisoles in wine using vacuum-assisted headspace solid-phase microextraction
Journal of Chromatography A, 1602 (2019) 142–149
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Room temperature and sensitive determination of haloanisoles in wine using vacuum-assisted headspace solid-phase microextraction Maria Vakinti, Sofia-Maria Mela, Elena Fernández, Elefteria Psillakis ∗ Laboratory of Aquatic Chemistry, School of Environmental Engineering, Polytechnioupolis, Technical University of Crete, GR-73100, Chania, Crete, Greece
a r t i c l e
i n f o
Article history: Received 3 January 2019 Received in revised form 20 March 2019 Accepted 21 March 2019 Available online 23 March 2019 Keywords: Wine analysis Haloanisoles Vac-HSSPME 2,4,6-Trichloroanisole Headspace sampling
a b s t r a c t Headspace solid-phase microextraction (HSSPME) is a widespread technique used to extract trace amounts of haloanisoles from wine samples. A major challenge to overcome is the high ethanol content in wines that affects the solubilities of haloanisoles and reduces their headspace abundance. To overcome this obstacle and meet sensitivity requirements, reported HSSPME procedures typically suggest heating the wine samples and/or sampling for extended times. The present work proposes the use of vacuum-assisted HSSPME (Vac-HSSPME) to accelerate the extraction kinetics whilst sampling at room temperature. Although ethanol affected the physico-chemical properties of the target analytes, these changes were not sufficient to prevent the positive effect of vacuum on HSSPME sampling. To demonstrate the benefits of adopting the vacuum approach, Vac-HSSPME and regular HSSPME methods were independently optimized and the results were compared at all times. The effect of ethanol under each pressure condition was also discussed. Under the optimum conditions found, Vac-HSSPME sampling for 30 min at room temperature at 25 ◦ C yielded lower detection limits (0.13 to 0.19 ng L−1 ) than those obtained with regular HSSPME sampling for 30 min at 55 ◦ C (0.26 to 0.76 ng L−1 ). The proposed VacHSSPME method was successfully applied to quantify haloanisoles in bottled red wines and a discussion on the effect of wine volatiles was included. The standard addition method was used to minimize matrix effects. The increase in total pressure due to the presence of ethanol and other volatile wine components did not reduce the positive effect of vacuum on HSSPME. Nonetheless, in accordance to past HSSPME methods, the limits of detection and quantification were affected due to the noise level increase and analyte interaction with matrix. The proposed Vac-HSSPME procedure was applied to twelve bottled red wines and one sample was found positive on 2,4,6-trichloronanisole. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Haloanisoles are well-known for creating a musty/moldy offaroma in wines that is rejected by consumers, causing important economic losses for the wine industries [1]. The problem is known as “cork taint” and has been traditionally related to the use of cork stoppers; though different studies have proven other sources of contamination (e.g., containers, oak barrels or wooden structures in cellars) [2,3]. Under certain temperature and humidity conditions, haloanisoles are produced by the microbiological methylation of halophenols, which are frequently used as fungicides, herbicides, wood preservatives and cork bleaching agents [2]. The main compounds responsible for the musty odor in wines are 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA),
∗ Corresponding author. E-mail address: [email protected] (E. Psillakis). https://doi.org/10.1016/j.chroma.2019.03.047 0021-9673/© 2019 Elsevier B.V. All rights reserved.
pentachloroanisole (PCA) and 2,4,6-tribromoanisole (TBA), being TCA detected in almost 80% of the positive samples [1,4]. There are no toxicological studies indicating a risk to the consumers; however, humans have very low sensory thresholds and haloanisoles can be perceived in alcoholic solutions at very low levels (i.e., ng L−1 ). These sensory thresholds depend on individuals as well as wine composition and vary between 0.03–50 ng L−1 for TCA, 5–35 ng L−1 for TeCA, 10 g L−1 for PCA and 2–7.9 ng L−1 for TBA [1]. The determination of trace amounts of haloanisoles is therefore an important issue for enological industry in order to guarantee the quality of wine, keep the brand recognition and avoid financial losses. In the past, different publications reported the determination of haloanisoles in wine samples and successfully addressed the issue of reaching very low detection limits in such a complex matrix [1]. The majority of reported methodologies include a sample preparation step for the isolation and preconcentration of target analytes prior to different separation/detection analyti-
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cal techniques, mainly gas chromatography (GC) coupled to mass spectrometry or electron capture detection (ECD) [1,3,5]. Both liquid-phase and solid-phase (micro)extraction techniques have been extensively studied, being headspace solid-phase microextraction (HSSPME) the most widespread method used [1,3]. In HSSPME, a fused-silica fiber coated with a polymeric film is exposed in the headspace above the sample leading to a three-phase system in which volatile or semi-volatile analytes partition between the sample, headspace and polymeric film [6]. The time needed to reach the equilibrium can vary from minutes to hours, mainly depending on the physico-chemical properties of the target analytes [7]. There are different means to reduce equilibration times, and these include agitating the sample, maximizing the sample/headspace interface, heating the sample and/or cooling the fiber as seen in the cold-fiber SPME approach [6,8]. More recently, headspace sampling under reduced-pressure conditions was suggested as an alternative approach to accelerate HSSPME kinetics [9,10]. Lowering the sampling pressure during the pre-equilibrium stage of HSSPME was found to improve the evaporation rates of compounds with a low Henry´s Law constant (KH ), as for these analytes the rate-limiting step was mass transfer in the thin gas-film adjacent to gas/sample interface. Vacuum-assisted HSSPME (Vac-HSSPME) evolved from this approach [11] and in all reported Vac-HSSPME methods, the presence of an air-evacuated headspace accelerated extraction kinetics and led to high extraction efficiencies at short sampling times and mild temperatures [10,12]. The determination of haloanisoles in wine samples with HSSPME typically requires long extraction times and/or elevated sampling temperatures [1,3]. Adopting the Vac-HSSPME approach for this type of analyses could be particularly beneficial as the low sampling pressure has the potential to accelerate the extraction kinetics and eliminate the need for heating the sample. However, similarly to regular HSSPME, wine samples represent a challenging type of matrix to analyze as the high ethanol content and other volatile wine components are expected to induce changes in analyte’s solubility and as such, partitioning with the headspace [13,14]. Past HSSPME reports also concluded that ethanol and other wine volatiles compete with haloanisoles for binding sites in porous SPME fibers and this effect can be intensified under vacuum conditions [14–16]. More importantly, the wine volatiles (including ethanol) will increase the total pressure in the sampler and this pressure accumulation has the potential to reduce the positive effect of vacuum on HSSPME. Examining the behavior and performance of Vac-HSSPME with wine samples is therefore of great interest in terms of exploring new insights of the method next to reporting a new powerful SPME-based analytical tool. It is noted that the performance of vacuum-assisted sorbent extraction (VASE) was recently reported for extracting volatile phenols from beer samples under low pressure conditions [17]. In this report, optimization of the method was performed using spiked water solutions and quantitative analysis proceeded using smoked beers and light Pilsner type beer samples. Although the use of VASE resulted in a sensitive analytical tool, the performance of the method was not compared to that under regular pressure conditions, and details on the influence of the matrix components on the pressure conditions were not included. In this article, we expand the applicability of Vac-HSSPME and report a fast, room temperature and sensitive method used for the determination of haloanisoles in wine samples. To demonstrate the benefits of adopting the vacuum approach, a comparative study between Vac-HSSPME and regular HSSPME was carried out. The effect of ethanol content under each pressure condition was discussed in details. The two methods were independently optimized and their performance was assessed. Finally, the effect of matrix upon extraction was evaluated and the proposed Vac-HSSPME pro-
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cedure was used for the determination of haloanisoles in bottled red wine samples. 2. Material and methods 2.1. Chemicals, material and samples TCA, TeCA, PCA and TBA were all supplied by Neochema GmbH (Bodenheim, Germany) and were greater than 98% in purity. Stock solutions were prepared in methanol and stored in the dark at 4 ◦ C. Pesticide residue grade methanol and L(+)-tartaric acid (99.5% purity) were purchased from Sigma-Aldrich (Steinheim, Germany) and highest purity ethanol was obtained from Honeywell (Seelze, Germany). Sodium hydroxide was supplied by Merck (Darmstabt, Germany). Milli-Q water was prepared on a water purification system (Barnstead EASYpure II) supplied by Thermo Scientific (Dubuque, USA). A bench pH/mV meter from Hanna Instruments (Woonsocket, USA) was used to measure the pH. The total pressure in the headspace of the liquid samples was measured using a home-made vacuum meter assembled at the University of Waterloo, Canada. A synthetic wine solution was used to simulate the effect of wine matrix on extraction. The solution was prepared by dissolving l(+)-tartaric acid (5 g L−1 ) in a hydroalcoholic solution (13% v/v ethanol) [15,18]. The pH of the solution was adjusted to 3.5 using an aqueous solution of NaOH. Calibration curves were constructed in synthetic wine and red wine samples. Commercial red wine packaged in tetra pack cartridges was purchased from a local supermarket and was found to be free of the target haloanisoles. This wine was used for initial investigations on the effect of matrix. Twelve red wines, produced between 2014 and 2016, were analyzed using the proposed Vac-HSSPME analytical method. Six of these bottled wines were supplied by a winery located in the prefecture of Chania, Crete, Greece and the rest from another winery in Herakleion, Crete, Greece. The pH of all real wine samples was found to range from 3.2−3.8. The ethanol content in the wines obtained from the winery in Chania and Heraklion was 14.5 and 13.5% v/v respectively. 2.2. Vac-HSSPME and regular HSSPME procedures ®
A crimp-top Mininert valve (Sigma-Aldrich) was modified according to a published procedure [19] and a cylindrical Thermogreen® LB-1 septum (Supelco) with half-hole (6 mm diameter × 9 mm length) and O-ring (10 mm internal diameter) were fitted. Upon modification, the Mininert valve could provide gastight seal to 20 mL crimp-top glass SPME vials (Supelco). For Vac-HSSPME, the air inside the sampling device containing a magnetic polytetrafluoroethylene (PTFE) stir bar (10 mm × 6 mm) was evacuated for 1 min prior introducing the liquid sample, using a VP 2 Autovac pumping unit (7 mbar = 0.007 atm ultimate vacuum without gas ballast) manufactured by Vacuubrand GmbH & Co. KZ (Wertheim, Germany). A 10 mL gastight syringe (SGE, Australia) was used to introduce the 5 mL samples in the sample container. The device and sample were then mounted on top of a magnetic stir plate (Heidolph MR-Standard, Germany) inside a heating water bath equipped with a stainless steel temperature sensor. The sample was allowed to equilibrate with the headspace for 10 min [20] at the temperature set for extraction and under a 1000 rpm agitation speed. Then, the fiber of an SPME fiber/holder assembly (Supelco, Bellefonte, PA) was exposed to headspace and sampling was performed under 1000 rpm agitation speed and at a preset sampling temperature. Based on previous reports, the 65-m polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fiber (Supelco, Bellefonte, PA) was used for extraction offering
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Table 1 Main physicochemical properties of the four haloanisoles investigated here. The estimated values were taken from [22]. Analyte
Molecular Weight
Vapor pressure 25 ◦ C (mm Hg) a
KH (atm m3 mol−1 ) b
Log Kow
TCA TeCA PCA TBA
211.47 245.92 280.36 344.82
1.93 10−2 3.19 10−4 3.44 10−4 4.72 10−4
4.55 10−4 4.72 10−4 3.59 10−4 4.28 10−6
4.11 4.75 5.45 4.48
a b
1 mm Hg = 133.322 Pa. 1 atm = 1.01 105 Pa.
high sensitivity and precision [5,16]. Upon completion of the sampling procedure, the fiber was retracted and transferred for thermal desorption and analysis to a GC-ECD. During the initial stage of optimization, the GC desorption time was optimized to prevent carry over effects. The desorption times tested were 5, 10 and 15 min without changing the 5-min purge time in the GC injector. Based on the results a 10 min long GC desorption time was set as optimum when sampling spiked simulated wine. This period was extended to 15 min after optimization when sampling complex wine samples, which is in accordance with past investigations [20]. The sample container was emptied and cleaned after pressure equilibration. The Thermogreen septum was replaced daily to avoid pressure loss due to septum damage. Prior to starting an analytical sequence, the SPME fiber was conditioned for 10 min in the GC injector. Blanks were run periodically to ensure the absence of carry over between runs. All extractions were run in triplicate. For regular HSSPME the air-evacuation step was omitted. Heating synthetic wine and bottled wine samples at elevated temperatures resulted in pressure build-up inside the vial-modified Mininert valve extraction apparatus that could displace the Thermogreen septum. For this reason 20 mL screw top vials, metallic caps with a hole and polytetrafluoroethylene (PTFE)/silicone septa (Restek, Bellefonte, USA) were used instead. All experiments were run in triplicate. The optimum conditions found for Vac-HSSPME were: 5 mL sample; PDMS/DVB fiber; 10 min incubation time; 30 min HSSPME sampling; 25 ◦ C sampling temperature; 1000 rpm agitation speed; the GC desorption time was 10 min for synthetic wine samples and 15 min for wine samples. The optimum conditions found for regular HSSPME were: 5 mL sample; PDMS/DVB fiber; 10 min incubation time; 30 min HSSPME sampling; 55 ◦ C sampling temperature; 1000 rpm agitation speed; the GC desorption time was 10 min for synthetic wine samples and 15 min for wine samples. 2.3. GC-ECD analysis A Shimadzu model GC-17 A, GC-ECD was used. The separation TM was achieved using a J & W DB-5MS UI column (30 m × 0.250 mm I.D., 0.25 m of film thickness) from Agilent Technologies (Sanda Clara, USA). A flow rate of 1 mL·min−1 of helium (carrier gas) with a column head pressure of 100 kPa was required for the separation of the analytes. The analyses were performed under the splitless injection mode at 270 ◦ C with the split closed for 5 min. The GC oven temperature program was the following: initially at 90 ◦ C for 5 min, then increased at a rate of 20 ◦ C·min−1 up to 280 ◦ C where it was held for 5 min. The total analysis time was 19.5 min. The temperature of the ECD was kept at 280 ◦ C. 3. Results and discussion 3.1. The effect of ethanol on Vac- and regular HSSPME The estimated values of the main physicochemical properties of the four studied haloanisoles are presented in Table 1 [22]. All tar-
Table 2 Total pressure in the headspace above water and ethanol:water samples under vacuum and regular pressure conditions. Pressure values given in mbar (1 mbar = 100 Pa). A 7 mbar ultimate vacuum pump pressure limit was considered for Vac-HSSPME.
Temperature
25 ◦ C 35 ◦ C 45 ◦ C 55 ◦ C
Vac-HSSPME
Regular HSSPME
Water
Ethanol:Water
Water
Ethanol:Water
(mbar)
(mbar)
(mbar)
(mbar)
39 63 103 164
46 77 126 201
1032 1056 1096 1157
1039 1070 1119 1194
get analytes were amenable to Vac-HSSPME sampling from water samples since their KH values satisfied the KH criterion established in the past and used for predicting the positive effect of vacuum on HSSPME [10,12]. In particular, TBA was a low KH compound with gas-phase resistance controlling more than 95% of the evaporation rate from the liquid sample to the headspace; whereas the KH values of TCA, TeCA and PCA were below the threshold value for intermediate KH compounds, where gas-phase resistance starts to control more than 50% of the evaporation rate [10,12]. Previous HSSPME optimization studies using aqueous ethanol model solutions and alcoholic beverages indicated that the high ethanol content reduced the extraction efficiency of HSSPME [13,14]. It was suggested that at these concentrations, ethanol acted as a co-solvent and had a direct impact on the solubility of other wine components including haloanisoles [14]. As a result, the headspace abundance of these compounds was affected and changes associated with the compound-specific partition coefficient were recorded [14]. The reduced HSSPME efficiency was also related to ethanol molecules and other components of the volatile fraction of wines directly competing with haloanisoles for binding sites in porous SPME fibers [13,14]. The determination of haloanisoles in wine samples with HSSPME is therefore a challenging task, as trace amounts of haloanisoles have to be extracted from a complex matrix. For Vac-HSSPME additional concerns existed over the ethanol content, since the presence of ethanol would increase the total pressure in the sample vial. As an example, Table 2 shows the contribution of ethanol in the total pressure by giving the calculated total pressure values at different temperatures when sampling water and ethanol:water samples with Vac-HSSPME and regular HSSPME. In these calculations, the sum of the partial pressures of haloanisoles being ∼0.03 mbar (1 mbar = 100 Pa) was neglected and the 7 mbar ultimate vacuum pump pressure limit was considered for Vac-HSSPME. As seen, the presence of ethanol increases the total pressure in the headspace and the effect is more pronounced when heating the sample due to the related increase in the partial pressures of water and ethanol. However, this pressure accumulation seems not to be sufficient to cancel the low pressure conditions necessary for successful Vac-HSSPME sampling. In order to investigate the effect of the hydroalcoholic matrix on Vac- and regular HSSPME, analytes were extracted from synthetic wine solution (13% v:v, ethanol:water) and pure water at
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Fig. 1. Effect of matrix composition: (i) synthetic wine and (ii) pure water without any pH adjustment on the extraction of the four haloanisoles investigated under reduced-pressure conditions (Vac-HSSPME) and atmospheric pressure conditions (HSSPME). Other experimental conditions: 5 mL sample solutions spiked at 15 ng L−1 with haloanisoles; 10 min incubation time; 30 min sampling time; 25 ◦ C sampling temperature; 1000 rpm agitation speed.
25 ◦ C. During Vac-HSSPME, the total pressure in the headspace of the synthetic wine solutions was measured before sample introduction and after the sample equilibration step and a ∼50 mbar increase was recorded. The results, given in Fig. 1, confirmed the expected negative effect of ethanol on the extraction of all target analytes under each sampling pressure. To facilitate the interpretation of the results the calculated synthetic wine/water peak area ratios for each analyte under each pressure condition are also given in Table ESM-1 in the electronic supplementary material. The low ratio values obtained under each pressure condition (ranged between 0.4−0.5) confirmed that the presence of ethanol affected the water solubility of target analytes and reduced their abundance in the headspace. Moreover, the ratio values for each analyte under vacuum and regular pressure conditions were close, suggesting that at 25 ◦ C the negative effect of ethanol was relatively independent of the total pressure in the headspace. Although the reduction in extraction efficiencies when moving from aqueous to synthetic wine solutions revealed the challenges associated when sampling wine samples, Fig. 1 also showed that for each solution Vac-HSSPME accelerated extraction rates and improved the extraction efficiencies compared to regular HS-SPME. To this end, the relative enhancements in extraction efficiencies when lowering the sampling pressure, shown in Table ESM-2, were similar in both matrices for all target analytes, possibly with the exception of TBA. It therefore appears, that despite the changes in the properties of the target analytes (i.e. solubility and liquid-gas phase partitioning), the gas-phase resistance continued to control the evaporation rate of each target analyte and that under the present experimental conditions, the increment in total pressure was not sufficient to suppress the positive effect of vacuum on HSSPME sampling. 3.2. The effects of sample volume and agitation on Vac- and regular HSSPME Sample volumes ranging from 5 to 9 mL were then extracted under each sampling pressure. The results, given in Fig. ESM-1 of the electronic supplementary material, showed that under each pressure condition no significant changes on SPME sorption kinetics were taking place when increasing the sample volume [10]. Based on these studies, the lowest sample volume tested (5 mL) was chosen as optimum so as to minimize the influence of the matrix [17]. Next, the effect of agitation rate was tested for 0, 500 and 1000 rpm under each sampling pressure and the results are given in Fig. ESM-2 of the electronic supplementary material. As expected
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Fig. 2. Effect of temperature on the extraction of the four haloanisoles investigated under reduced-pressure conditions (Vac-HSSPME) and atmospheric pressure conditions (HSSPME). Other experimental conditions: 5 mL synthetic wine solution spiked at 25 ng L−1 with haloanisoles; 10 min incubation time; 20 min sampling time; 1000 rpm agitation speed.
sample agitation, facilitated analyte transport from the bulk solution to the SPME fiber [10] and the 1000 rpm agitation speed was chosen as optimum. 3.3. The effects of temperature and extraction time on Vac- and regular HSSPME Sample heating is commonly used in HSSPME methodologies as a viable approach to enhance the extraction kinetics [7]. For halonisole determination, published results on the influence of temperature on HSSPME vary [1,16,20] with the majority of reports suggesting heating the sample during extraction [1]. Here, the effect of temperature on Vac- and regular HSSPME was evaluated between 25 and 55 ◦ C and for a 20 min sampling time. The results shown in Fig. 2, reveal that for regular HSSPME, increasing the sampling temperature improved the extraction performance. For Vac-HSSPME, signals increased with temperature between 25 and 35 ◦ C, whereas further heating led to decreased extraction efficiencies. Past reports suggested that the SPME fiber coating will uptake gas molecules much faster under low sampling pressures relative to regular HSSPME, since the portion of molecules in an air-evacuated headspace colliding with the fiber will be much larger than that in the presence of air [19,23]. Here, heating the sample during VacHSSPME increased the amount of water and ethanol molecules in the headspace. It was therefore assumed that at higher temperatures, the collisions of these molecules with the adsorbent coating surface were enhanced compared to regular HSSPME sampling, leaving fewer sites available for analytes to adsorb. Based on Fig. 2, the optimum temperatures for Vac- and regular HSSPME were 35 ◦ C and 55 ◦ C, respectively. Despite the superior performance of Vac-HSSPME at 35 ◦ C, the 25 ◦ C sampling temperature was not excluded as an option. Sampling at 25 ◦ C (i.e., room temperature) is particularly advantageous to maintain sample composition and minimize the evaporation of potential volatile components acting as matrix interferences during real wine samples analysis. At the same time, extraction efficiencies with Vac-HSSPME at 25 ◦ C were similar to those recorded with regular HSSPME at 55 ◦ C. For these reasons, 25 and 35 ◦ C were chosen for subsequent Vac-HSSPME investigations and 55 ◦ C for regular HSSPME. Fig. 3 shows the extraction time profiles obtained under reduced (at 25 and 35 ◦ C) and atmospheric pressure conditions (at 55 ◦ C). With regular HSSPME at 55 ◦ C, the extraction time profiles of TCA and TeCA reached equilibrium for sampling times larger than 45 and 60 min, respectively. With Vac-HSSPME, TCA was the only ana-
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regular HSSPME at 55 ◦ C was lower than those extracted under pre-equilibrium conditions with Vac-HSSPME at 25 or 35 ◦ C. It is noted that the possibility of an enhanced competition for SPME fiber binding sites taking place at 55 ◦ C between the target analytes and the increased amount of ethanol molecules cannot be excluded. For PCA and TBA, longer equilibration times were expected since they possess the lowest KH values studied here, and PCA is the most hydrophobic compound (see Table 1). In accordance, Fig. 3 shows that equilibrium was not reached after 90 min of Vac- or regular HSSPME sampling, and that the positive effect of sampling at reduced pressure conditions remained important, especially for PCA, even at extended extraction times. Based on the current findings, a 30 min was selected for both Vac- and regular HSSPME, as a compromise between sensitivity and extraction time, and after considering the GC run time. At this sampling point, the relative improvement expressed as Vac-HSSPME/HSSPME peak area ratios, ranged from 1.2 to 1.6 for Vac-HSSPME at 25 ◦ C over HSSPME at 55 ◦ C and 1.7–2.4 for Vac-HSSPME at 35 ◦ C over HSSPME at 55 ◦ C (Table ESM-3 in the electronic supplementary material). In order to proceed with the evaluation of the analytical performances of the methods a selection on the sampling temperature for Vac-HSSPME had to be made at this point. Wine samples free of target analytes were spiked and submitted to Vac-HSSPME at 25 and 35 ◦ C in triplicate. Representative chromatograms obtained after Vac-HSSPME sampling of wine samples at 25 and 35 ◦ C are given in Fig. ESM-3 in the electronic supplementary material. The results showed that the improvement in extraction efficiencies after applying mild heating was somewhat cancelled when moving from simulated wine to complex real wine matrix. This observation suggested that the volatile components of wine (other than ethanol) were interfering with Vac-HSSPME sampling even when mild heating was applied. It was therefore decided to proceed with 25 ◦ C as the optimum sampling temperature for Vac-HSSPME. It is noted that the effect of matrix on both Vac- and regular HSSPME will be discussed in details in a following section. 3.4. Analytical performance of the optimized Vac-HSSPME and regular HSSPME procedures using simulated wine
Fig. 3. Effect of time on the extraction of the four haloanisoles investigated under (i) reduced-pressure conditions (Vac-HSSPME) at 25 ◦ C (ii) reduced-pressure conditions (Vac-HSSPME) at 35 ◦ C and (iii) atmospheric pressure conditions (HSSPME) at 55 ◦ C. Other experimental conditions: 5 mL synthetic wine solution spiked at 15 ng L−1 with haloanisoles; 10 min incubation time; 1000 rpm agitation speed.
lyte that reached equilibrium after 45 min of sampling at 25 ◦ C and 30 min extraction at 35 ◦ C. Heating the sample generally accelerates extraction kinetics so that analytes can reach equilibrium faster. Although regular HSSPME at 55 ◦ C proved advantageous to this end, the final amount of TCA extracted at equilibrium was lower compared to that with Vac-HSSPME. This is in agreement with previous reports, where elevated sample temperatures were found to reduce analyte recovery by shifting both the sample-headspace and the fiber-headspace equilibrium to favor the gas phase [7]. It was therefore assumed that at 55 ◦ C the distribution constant between the headspace and the fiber coating decreased, reducing the amount of TCA extracted at equilibrium. The results presented in Fig. 3 suggest that heating the sample also reduced the extraction efficiency of TeCA, as the equilibrium amount extracted with
The analytical performances of the optimized Vac- and regular HSSPME procedures were evaluated using simulated wine as a solvent. The main analytical parameters are summarized in Table 3 and the graphical representations of the calibration curves for each analyte are given in Fig. ESM-4 of the electronic supplementary material. The calibration curves showed good linearity in a wide concentration range, with coefficients of determination varying between 0.994 and 0.998 (n = 7), and between 0.997 and 0.999 (n = 6) for Vac- and regular HSSPME, respectively. The intraday precision was evaluated by six replicate analysis of synthetic wine standards spiked at two different concentration levels for each extraction procedure. The obtained relative standard deviations (RSDs) ranged from 4.0–10.9% with Vac-HSSPME and from 4.8–10.6% with regular HSSPME. Limits of detection (LODs) were estimated according to three times signal-to-noise-ratio (S/N) criterion. LODs ranged from 0.13 to 0.19 ng L−1 for Vac-HSSPME and from 0.26 to 0.76 ng L−1 for regular HSSPME. Based on the present results, sensitivity decreased from Vac-HSSPME to regular HSSPME (Fig. ESM-4). Nonetheless, with both methods the LODs were low enough to reach the sensory threshold levels at which haloanisoles can be perceived. In general, previously published regular HSSPME methods suggested heating the sample when choosing short extraction times or sampling the headspace for extended times when applying room or mild temperatures [1]. Here, the LODs obtained with Vac-HSSPME were better than or similar to published regular HSSPME methods using porous SPME fibers and a similar analytical instrumentation [1,24,25]. The
9.5 7.4 5.6 6.8 0.44 0.37 0.26 0.66
c
a
b
Coefficient of determination, number of calibration points (n) in parenthesis. Limit of detection evaluated as S/N = 3. Intra-day precision expressed as relative standard deviation (n = 6) and given for two concentration levels.
5.8 4.0 4.1 4.2 10.9 4.6 8.8 8.0 0.16 0.18 0.19 0.13 0.998 0.998 0.998 0.994 9.49 11.20 12.56 11.85 TCA TeCA PCA TBA
0.25-25 0.25-25 0.25-25 0.25-25
Low level (0.5 ng L−1 )
Linear range (ng L−1 )
Retention time (min)
Vac-HSSPME
r2 (n = 7) a
LODb (ng L−1 )
Precision (RSD, %)
c
High level (10 ng L−1 )
0.75-25 0.75-25 0.75-25 0.75-25
0.998 0.999 0.999 0.997
r2 (n = 6) a Linear range (ng L−1 )
Regular HSSPME
3.5. Analysis of wine samples using Vac-HSSPME
Analyte
Table 3 Analytical performance of the optimized Vac-HSSPME and regular HSSPME procedures.
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proposed Vac-HSSPME procedure therefore possessed the great advantage of sampling for short sampling times and at room temperature, eliminating thus the need for heating the sample.
10.6 4.8 6.7 4.8
Low level (1 ng L−1 )
LODb (ng L−1 )
Precision (RSD, %)c
High level (15 ng L−1 )
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Past investigations concluded that a broad spectrum of compounds occurring in wine can interact with the extraction of haloanisoles. To this end, the liquid-gas phase partitioning of volatile wine components can be influenced by other wine constituents, such as polysaccharides, proteins and polyphenols. In particular, wine polyphenols have attracted much attention, because of their ability to interact with proteins as well as the volatile substances present in wine [5]. For this reason, wine matrix classification is typically based on polyphenol content with white wine assumed to be a matrix of low interaction and red wine a matrix with high matrix effect [15,26]. For TCA determination it has been proposed that the approach of low/high polyphenol contents might be too simple, and instead it was suggested that the nature rather than the concentration of polyphenolic compounds most likely affects TCA recoveries [26]. At the same time, the different ethanol concentrations found in wines will affect HSSPME extraction efficiencies of haloanisoles, given that several past investigations conclude that peak areas decrease with increased ethanol content in wine samples [16,20,24]. The determination of haloanisoles in wine by HSSPME is therefore a matrix effect-dependent analytical procedure and the use of a suitable internal standard or the method of standard additions can minimize or avoid this effect [5,15,16,26]. To investigate matrix effects on the proposed Vac-HSSPME procedure commercial red wine samples were tested. Initial blanks using the proposed Vac-HSSPME method showed no detectable amounts of target analytes but the noise level was increased due to matrix complexity [26]. The total pressure in the sampler was measured before sample introduction and after the sample equilibration step and a ∼80 mbar increase was recorded. Compared to the total pressure measured in the headspace of the synthetic wine solution, the additional ∼30 mbar increase accounted for the presence of other volatile wine components. The same wine was then spiked at 10 ng L−1 and submitted to Vac-HSSPME analysis at 25 ◦ C. As expected, a strong matrix effect was observed and measured analyte peak areas were affected due to the interaction with the matrix. Extractions were repeated this time using regular HSSPME at 55 ◦ C and the resulting Vac-HSSPME/regular HSSPME peak area ratios ranged from 1.2 to 1.5 for target analytes, similar to those obtained when using simulated wine (Table ESM-3). The latter demonstrated that the additional increment in total pressure when sampling real wine samples was not affecting the relative performance of Vac-HSSPME over regular HSSPME. Subsequent investigations consisted of using the standard addition method to test the analytical performance of the method in the same wine sample. The resulting calibration curves showed good linearity between 1.5 and 25 ng L−1 , and the coefficients of determination were 0.997, 0.997, 0.991 and 0.991 for TCA, TeCA, PCA and TBA respectively (n = 5). In accordance with previous reports, calibration curves reflected a decrease sensitivity (slope) from simulated water to real wine (Fig. ESM-4) and was related to the complexity of the matrix. LODs were estimated according to three times signalto-noise-ratio (S/N) criterion and were 0.43, 0.45, 0.31 and 0.64 ng L-1 for TCA, TeCA, PCA and TBA respectively. As expected, the proposed method showed a good linearity in real wine samples, but the limits of detection and quantification were affected due to the noise level increase and analyte interaction with matrix. Twelve bottled red wines were then analyzed using the proposed Vac-HSSPME. In almost all of the samples, there were no detectable amounts of haloanisoles, but in one red wine sample
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References
Fig. 4. Overlaid Vac-HSSPME-GC-ECD chromatograms of the red wine found positive in TCA: (i) spiked at 10 ng L−1 with haloanisoles and (ii) blank showing trace amounts TCA (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
TCA content was detected. Fig. 4 shows the chromatograms of the TCA positive red wine sample before and after adding 10 ng L−1 of haloanisoles. The standard addition method was then chosen and samples were spiked and multiply extracted. Based on these studies, the TCA concentration in the positive sample was 3.2 ± 0.2 ng L−1 (n = 3). This value was within the perception level for TCA (ranging between 0.03–10.0 ng L−1 ) and below the 10 ng L−1 concentration threshold where a defect in wine is produced [16]. 4. Conclusions The determination of trace amounts of haloanisoles in complex wine samples has represented a challenge for Vac- and regular HSSPME. The high ethanol content affected the physico-chemical properties of the target analytes and their partitioning with the headspace. For Vac-HSSPME additional concerns existed over the presence of ethanol and other volatile wine compounds as they would increase the total pressure in the headspace. The present work demonstrated that these changes were not sufficient to suppress the positive effect of vacuum in HSSPME. The optimized Vac-HSSPME procedure proved to be a fast, room temperature and sensitive method that could be used for the determination of haloanisoles in wine samples. The need for heating the sample at elevated temperatures as seen in the optimized HSSPME procedure was eliminated. Sampling at room temperature is particularly advantageous to maintain sample composition and minimize the evaporation of potential volatile components acting as matrix interferences during real wine sample analysis. Acknowledgments The authors are grateful to the wineries of Diamantakis (Herakleion, Crete, Greece) and Manousakis (Chania, Crete, Greece) for supplying the wine samples. The authors also wish to thank Professor Janusz Pawliszyn (University of Waterloo, Canada) for supplying the vacuum meter. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 03.047.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Room temperature and sensitive determination of haloanisoles in wine using vacuum-assisted headspace solid-phase microextraction Maria Vakinti, Sofia-Maria Mela, Elena Fernández, Elefteria Psillakis ∗ Laboratory of Aquatic Chemistry, School of Environmental Engineering, Polytechnioupolis, Technical University of Crete, GR-73100, Chania, Crete, Greece
a r t i c l e
i n f o
Article history: Received 3 January 2019 Received in revised form 20 March 2019 Accepted 21 March 2019 Available online 23 March 2019 Keywords: Wine analysis Haloanisoles Vac-HSSPME 2,4,6-Trichloroanisole Headspace sampling
a b s t r a c t Headspace solid-phase microextraction (HSSPME) is a widespread technique used to extract trace amounts of haloanisoles from wine samples. A major challenge to overcome is the high ethanol content in wines that affects the solubilities of haloanisoles and reduces their headspace abundance. To overcome this obstacle and meet sensitivity requirements, reported HSSPME procedures typically suggest heating the wine samples and/or sampling for extended times. The present work proposes the use of vacuum-assisted HSSPME (Vac-HSSPME) to accelerate the extraction kinetics whilst sampling at room temperature. Although ethanol affected the physico-chemical properties of the target analytes, these changes were not sufficient to prevent the positive effect of vacuum on HSSPME sampling. To demonstrate the benefits of adopting the vacuum approach, Vac-HSSPME and regular HSSPME methods were independently optimized and the results were compared at all times. The effect of ethanol under each pressure condition was also discussed. Under the optimum conditions found, Vac-HSSPME sampling for 30 min at room temperature at 25 ◦ C yielded lower detection limits (0.13 to 0.19 ng L−1 ) than those obtained with regular HSSPME sampling for 30 min at 55 ◦ C (0.26 to 0.76 ng L−1 ). The proposed VacHSSPME method was successfully applied to quantify haloanisoles in bottled red wines and a discussion on the effect of wine volatiles was included. The standard addition method was used to minimize matrix effects. The increase in total pressure due to the presence of ethanol and other volatile wine components did not reduce the positive effect of vacuum on HSSPME. Nonetheless, in accordance to past HSSPME methods, the limits of detection and quantification were affected due to the noise level increase and analyte interaction with matrix. The proposed Vac-HSSPME procedure was applied to twelve bottled red wines and one sample was found positive on 2,4,6-trichloronanisole. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Haloanisoles are well-known for creating a musty/moldy offaroma in wines that is rejected by consumers, causing important economic losses for the wine industries [1]. The problem is known as “cork taint” and has been traditionally related to the use of cork stoppers; though different studies have proven other sources of contamination (e.g., containers, oak barrels or wooden structures in cellars) [2,3]. Under certain temperature and humidity conditions, haloanisoles are produced by the microbiological methylation of halophenols, which are frequently used as fungicides, herbicides, wood preservatives and cork bleaching agents [2]. The main compounds responsible for the musty odor in wines are 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA),
∗ Corresponding author. E-mail address: [email protected] (E. Psillakis). https://doi.org/10.1016/j.chroma.2019.03.047 0021-9673/© 2019 Elsevier B.V. All rights reserved.
pentachloroanisole (PCA) and 2,4,6-tribromoanisole (TBA), being TCA detected in almost 80% of the positive samples [1,4]. There are no toxicological studies indicating a risk to the consumers; however, humans have very low sensory thresholds and haloanisoles can be perceived in alcoholic solutions at very low levels (i.e., ng L−1 ). These sensory thresholds depend on individuals as well as wine composition and vary between 0.03–50 ng L−1 for TCA, 5–35 ng L−1 for TeCA, 10 g L−1 for PCA and 2–7.9 ng L−1 for TBA [1]. The determination of trace amounts of haloanisoles is therefore an important issue for enological industry in order to guarantee the quality of wine, keep the brand recognition and avoid financial losses. In the past, different publications reported the determination of haloanisoles in wine samples and successfully addressed the issue of reaching very low detection limits in such a complex matrix [1]. The majority of reported methodologies include a sample preparation step for the isolation and preconcentration of target analytes prior to different separation/detection analyti-
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cal techniques, mainly gas chromatography (GC) coupled to mass spectrometry or electron capture detection (ECD) [1,3,5]. Both liquid-phase and solid-phase (micro)extraction techniques have been extensively studied, being headspace solid-phase microextraction (HSSPME) the most widespread method used [1,3]. In HSSPME, a fused-silica fiber coated with a polymeric film is exposed in the headspace above the sample leading to a three-phase system in which volatile or semi-volatile analytes partition between the sample, headspace and polymeric film [6]. The time needed to reach the equilibrium can vary from minutes to hours, mainly depending on the physico-chemical properties of the target analytes [7]. There are different means to reduce equilibration times, and these include agitating the sample, maximizing the sample/headspace interface, heating the sample and/or cooling the fiber as seen in the cold-fiber SPME approach [6,8]. More recently, headspace sampling under reduced-pressure conditions was suggested as an alternative approach to accelerate HSSPME kinetics [9,10]. Lowering the sampling pressure during the pre-equilibrium stage of HSSPME was found to improve the evaporation rates of compounds with a low Henry´s Law constant (KH ), as for these analytes the rate-limiting step was mass transfer in the thin gas-film adjacent to gas/sample interface. Vacuum-assisted HSSPME (Vac-HSSPME) evolved from this approach [11] and in all reported Vac-HSSPME methods, the presence of an air-evacuated headspace accelerated extraction kinetics and led to high extraction efficiencies at short sampling times and mild temperatures [10,12]. The determination of haloanisoles in wine samples with HSSPME typically requires long extraction times and/or elevated sampling temperatures [1,3]. Adopting the Vac-HSSPME approach for this type of analyses could be particularly beneficial as the low sampling pressure has the potential to accelerate the extraction kinetics and eliminate the need for heating the sample. However, similarly to regular HSSPME, wine samples represent a challenging type of matrix to analyze as the high ethanol content and other volatile wine components are expected to induce changes in analyte’s solubility and as such, partitioning with the headspace [13,14]. Past HSSPME reports also concluded that ethanol and other wine volatiles compete with haloanisoles for binding sites in porous SPME fibers and this effect can be intensified under vacuum conditions [14–16]. More importantly, the wine volatiles (including ethanol) will increase the total pressure in the sampler and this pressure accumulation has the potential to reduce the positive effect of vacuum on HSSPME. Examining the behavior and performance of Vac-HSSPME with wine samples is therefore of great interest in terms of exploring new insights of the method next to reporting a new powerful SPME-based analytical tool. It is noted that the performance of vacuum-assisted sorbent extraction (VASE) was recently reported for extracting volatile phenols from beer samples under low pressure conditions [17]. In this report, optimization of the method was performed using spiked water solutions and quantitative analysis proceeded using smoked beers and light Pilsner type beer samples. Although the use of VASE resulted in a sensitive analytical tool, the performance of the method was not compared to that under regular pressure conditions, and details on the influence of the matrix components on the pressure conditions were not included. In this article, we expand the applicability of Vac-HSSPME and report a fast, room temperature and sensitive method used for the determination of haloanisoles in wine samples. To demonstrate the benefits of adopting the vacuum approach, a comparative study between Vac-HSSPME and regular HSSPME was carried out. The effect of ethanol content under each pressure condition was discussed in details. The two methods were independently optimized and their performance was assessed. Finally, the effect of matrix upon extraction was evaluated and the proposed Vac-HSSPME pro-
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cedure was used for the determination of haloanisoles in bottled red wine samples. 2. Material and methods 2.1. Chemicals, material and samples TCA, TeCA, PCA and TBA were all supplied by Neochema GmbH (Bodenheim, Germany) and were greater than 98% in purity. Stock solutions were prepared in methanol and stored in the dark at 4 ◦ C. Pesticide residue grade methanol and L(+)-tartaric acid (99.5% purity) were purchased from Sigma-Aldrich (Steinheim, Germany) and highest purity ethanol was obtained from Honeywell (Seelze, Germany). Sodium hydroxide was supplied by Merck (Darmstabt, Germany). Milli-Q water was prepared on a water purification system (Barnstead EASYpure II) supplied by Thermo Scientific (Dubuque, USA). A bench pH/mV meter from Hanna Instruments (Woonsocket, USA) was used to measure the pH. The total pressure in the headspace of the liquid samples was measured using a home-made vacuum meter assembled at the University of Waterloo, Canada. A synthetic wine solution was used to simulate the effect of wine matrix on extraction. The solution was prepared by dissolving l(+)-tartaric acid (5 g L−1 ) in a hydroalcoholic solution (13% v/v ethanol) [15,18]. The pH of the solution was adjusted to 3.5 using an aqueous solution of NaOH. Calibration curves were constructed in synthetic wine and red wine samples. Commercial red wine packaged in tetra pack cartridges was purchased from a local supermarket and was found to be free of the target haloanisoles. This wine was used for initial investigations on the effect of matrix. Twelve red wines, produced between 2014 and 2016, were analyzed using the proposed Vac-HSSPME analytical method. Six of these bottled wines were supplied by a winery located in the prefecture of Chania, Crete, Greece and the rest from another winery in Herakleion, Crete, Greece. The pH of all real wine samples was found to range from 3.2−3.8. The ethanol content in the wines obtained from the winery in Chania and Heraklion was 14.5 and 13.5% v/v respectively. 2.2. Vac-HSSPME and regular HSSPME procedures ®
A crimp-top Mininert valve (Sigma-Aldrich) was modified according to a published procedure [19] and a cylindrical Thermogreen® LB-1 septum (Supelco) with half-hole (6 mm diameter × 9 mm length) and O-ring (10 mm internal diameter) were fitted. Upon modification, the Mininert valve could provide gastight seal to 20 mL crimp-top glass SPME vials (Supelco). For Vac-HSSPME, the air inside the sampling device containing a magnetic polytetrafluoroethylene (PTFE) stir bar (10 mm × 6 mm) was evacuated for 1 min prior introducing the liquid sample, using a VP 2 Autovac pumping unit (7 mbar = 0.007 atm ultimate vacuum without gas ballast) manufactured by Vacuubrand GmbH & Co. KZ (Wertheim, Germany). A 10 mL gastight syringe (SGE, Australia) was used to introduce the 5 mL samples in the sample container. The device and sample were then mounted on top of a magnetic stir plate (Heidolph MR-Standard, Germany) inside a heating water bath equipped with a stainless steel temperature sensor. The sample was allowed to equilibrate with the headspace for 10 min [20] at the temperature set for extraction and under a 1000 rpm agitation speed. Then, the fiber of an SPME fiber/holder assembly (Supelco, Bellefonte, PA) was exposed to headspace and sampling was performed under 1000 rpm agitation speed and at a preset sampling temperature. Based on previous reports, the 65-m polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fiber (Supelco, Bellefonte, PA) was used for extraction offering
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Table 1 Main physicochemical properties of the four haloanisoles investigated here. The estimated values were taken from [22]. Analyte
Molecular Weight
Vapor pressure 25 ◦ C (mm Hg) a
KH (atm m3 mol−1 ) b
Log Kow
TCA TeCA PCA TBA
211.47 245.92 280.36 344.82
1.93 10−2 3.19 10−4 3.44 10−4 4.72 10−4
4.55 10−4 4.72 10−4 3.59 10−4 4.28 10−6
4.11 4.75 5.45 4.48
a b
1 mm Hg = 133.322 Pa. 1 atm = 1.01 105 Pa.
high sensitivity and precision [5,16]. Upon completion of the sampling procedure, the fiber was retracted and transferred for thermal desorption and analysis to a GC-ECD. During the initial stage of optimization, the GC desorption time was optimized to prevent carry over effects. The desorption times tested were 5, 10 and 15 min without changing the 5-min purge time in the GC injector. Based on the results a 10 min long GC desorption time was set as optimum when sampling spiked simulated wine. This period was extended to 15 min after optimization when sampling complex wine samples, which is in accordance with past investigations [20]. The sample container was emptied and cleaned after pressure equilibration. The Thermogreen septum was replaced daily to avoid pressure loss due to septum damage. Prior to starting an analytical sequence, the SPME fiber was conditioned for 10 min in the GC injector. Blanks were run periodically to ensure the absence of carry over between runs. All extractions were run in triplicate. For regular HSSPME the air-evacuation step was omitted. Heating synthetic wine and bottled wine samples at elevated temperatures resulted in pressure build-up inside the vial-modified Mininert valve extraction apparatus that could displace the Thermogreen septum. For this reason 20 mL screw top vials, metallic caps with a hole and polytetrafluoroethylene (PTFE)/silicone septa (Restek, Bellefonte, USA) were used instead. All experiments were run in triplicate. The optimum conditions found for Vac-HSSPME were: 5 mL sample; PDMS/DVB fiber; 10 min incubation time; 30 min HSSPME sampling; 25 ◦ C sampling temperature; 1000 rpm agitation speed; the GC desorption time was 10 min for synthetic wine samples and 15 min for wine samples. The optimum conditions found for regular HSSPME were: 5 mL sample; PDMS/DVB fiber; 10 min incubation time; 30 min HSSPME sampling; 55 ◦ C sampling temperature; 1000 rpm agitation speed; the GC desorption time was 10 min for synthetic wine samples and 15 min for wine samples. 2.3. GC-ECD analysis A Shimadzu model GC-17 A, GC-ECD was used. The separation TM was achieved using a J & W DB-5MS UI column (30 m × 0.250 mm I.D., 0.25 m of film thickness) from Agilent Technologies (Sanda Clara, USA). A flow rate of 1 mL·min−1 of helium (carrier gas) with a column head pressure of 100 kPa was required for the separation of the analytes. The analyses were performed under the splitless injection mode at 270 ◦ C with the split closed for 5 min. The GC oven temperature program was the following: initially at 90 ◦ C for 5 min, then increased at a rate of 20 ◦ C·min−1 up to 280 ◦ C where it was held for 5 min. The total analysis time was 19.5 min. The temperature of the ECD was kept at 280 ◦ C. 3. Results and discussion 3.1. The effect of ethanol on Vac- and regular HSSPME The estimated values of the main physicochemical properties of the four studied haloanisoles are presented in Table 1 [22]. All tar-
Table 2 Total pressure in the headspace above water and ethanol:water samples under vacuum and regular pressure conditions. Pressure values given in mbar (1 mbar = 100 Pa). A 7 mbar ultimate vacuum pump pressure limit was considered for Vac-HSSPME.
Temperature
25 ◦ C 35 ◦ C 45 ◦ C 55 ◦ C
Vac-HSSPME
Regular HSSPME
Water
Ethanol:Water
Water
Ethanol:Water
(mbar)
(mbar)
(mbar)
(mbar)
39 63 103 164
46 77 126 201
1032 1056 1096 1157
1039 1070 1119 1194
get analytes were amenable to Vac-HSSPME sampling from water samples since their KH values satisfied the KH criterion established in the past and used for predicting the positive effect of vacuum on HSSPME [10,12]. In particular, TBA was a low KH compound with gas-phase resistance controlling more than 95% of the evaporation rate from the liquid sample to the headspace; whereas the KH values of TCA, TeCA and PCA were below the threshold value for intermediate KH compounds, where gas-phase resistance starts to control more than 50% of the evaporation rate [10,12]. Previous HSSPME optimization studies using aqueous ethanol model solutions and alcoholic beverages indicated that the high ethanol content reduced the extraction efficiency of HSSPME [13,14]. It was suggested that at these concentrations, ethanol acted as a co-solvent and had a direct impact on the solubility of other wine components including haloanisoles [14]. As a result, the headspace abundance of these compounds was affected and changes associated with the compound-specific partition coefficient were recorded [14]. The reduced HSSPME efficiency was also related to ethanol molecules and other components of the volatile fraction of wines directly competing with haloanisoles for binding sites in porous SPME fibers [13,14]. The determination of haloanisoles in wine samples with HSSPME is therefore a challenging task, as trace amounts of haloanisoles have to be extracted from a complex matrix. For Vac-HSSPME additional concerns existed over the ethanol content, since the presence of ethanol would increase the total pressure in the sample vial. As an example, Table 2 shows the contribution of ethanol in the total pressure by giving the calculated total pressure values at different temperatures when sampling water and ethanol:water samples with Vac-HSSPME and regular HSSPME. In these calculations, the sum of the partial pressures of haloanisoles being ∼0.03 mbar (1 mbar = 100 Pa) was neglected and the 7 mbar ultimate vacuum pump pressure limit was considered for Vac-HSSPME. As seen, the presence of ethanol increases the total pressure in the headspace and the effect is more pronounced when heating the sample due to the related increase in the partial pressures of water and ethanol. However, this pressure accumulation seems not to be sufficient to cancel the low pressure conditions necessary for successful Vac-HSSPME sampling. In order to investigate the effect of the hydroalcoholic matrix on Vac- and regular HSSPME, analytes were extracted from synthetic wine solution (13% v:v, ethanol:water) and pure water at
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Fig. 1. Effect of matrix composition: (i) synthetic wine and (ii) pure water without any pH adjustment on the extraction of the four haloanisoles investigated under reduced-pressure conditions (Vac-HSSPME) and atmospheric pressure conditions (HSSPME). Other experimental conditions: 5 mL sample solutions spiked at 15 ng L−1 with haloanisoles; 10 min incubation time; 30 min sampling time; 25 ◦ C sampling temperature; 1000 rpm agitation speed.
25 ◦ C. During Vac-HSSPME, the total pressure in the headspace of the synthetic wine solutions was measured before sample introduction and after the sample equilibration step and a ∼50 mbar increase was recorded. The results, given in Fig. 1, confirmed the expected negative effect of ethanol on the extraction of all target analytes under each sampling pressure. To facilitate the interpretation of the results the calculated synthetic wine/water peak area ratios for each analyte under each pressure condition are also given in Table ESM-1 in the electronic supplementary material. The low ratio values obtained under each pressure condition (ranged between 0.4−0.5) confirmed that the presence of ethanol affected the water solubility of target analytes and reduced their abundance in the headspace. Moreover, the ratio values for each analyte under vacuum and regular pressure conditions were close, suggesting that at 25 ◦ C the negative effect of ethanol was relatively independent of the total pressure in the headspace. Although the reduction in extraction efficiencies when moving from aqueous to synthetic wine solutions revealed the challenges associated when sampling wine samples, Fig. 1 also showed that for each solution Vac-HSSPME accelerated extraction rates and improved the extraction efficiencies compared to regular HS-SPME. To this end, the relative enhancements in extraction efficiencies when lowering the sampling pressure, shown in Table ESM-2, were similar in both matrices for all target analytes, possibly with the exception of TBA. It therefore appears, that despite the changes in the properties of the target analytes (i.e. solubility and liquid-gas phase partitioning), the gas-phase resistance continued to control the evaporation rate of each target analyte and that under the present experimental conditions, the increment in total pressure was not sufficient to suppress the positive effect of vacuum on HSSPME sampling. 3.2. The effects of sample volume and agitation on Vac- and regular HSSPME Sample volumes ranging from 5 to 9 mL were then extracted under each sampling pressure. The results, given in Fig. ESM-1 of the electronic supplementary material, showed that under each pressure condition no significant changes on SPME sorption kinetics were taking place when increasing the sample volume [10]. Based on these studies, the lowest sample volume tested (5 mL) was chosen as optimum so as to minimize the influence of the matrix [17]. Next, the effect of agitation rate was tested for 0, 500 and 1000 rpm under each sampling pressure and the results are given in Fig. ESM-2 of the electronic supplementary material. As expected
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Fig. 2. Effect of temperature on the extraction of the four haloanisoles investigated under reduced-pressure conditions (Vac-HSSPME) and atmospheric pressure conditions (HSSPME). Other experimental conditions: 5 mL synthetic wine solution spiked at 25 ng L−1 with haloanisoles; 10 min incubation time; 20 min sampling time; 1000 rpm agitation speed.
sample agitation, facilitated analyte transport from the bulk solution to the SPME fiber [10] and the 1000 rpm agitation speed was chosen as optimum. 3.3. The effects of temperature and extraction time on Vac- and regular HSSPME Sample heating is commonly used in HSSPME methodologies as a viable approach to enhance the extraction kinetics [7]. For halonisole determination, published results on the influence of temperature on HSSPME vary [1,16,20] with the majority of reports suggesting heating the sample during extraction [1]. Here, the effect of temperature on Vac- and regular HSSPME was evaluated between 25 and 55 ◦ C and for a 20 min sampling time. The results shown in Fig. 2, reveal that for regular HSSPME, increasing the sampling temperature improved the extraction performance. For Vac-HSSPME, signals increased with temperature between 25 and 35 ◦ C, whereas further heating led to decreased extraction efficiencies. Past reports suggested that the SPME fiber coating will uptake gas molecules much faster under low sampling pressures relative to regular HSSPME, since the portion of molecules in an air-evacuated headspace colliding with the fiber will be much larger than that in the presence of air [19,23]. Here, heating the sample during VacHSSPME increased the amount of water and ethanol molecules in the headspace. It was therefore assumed that at higher temperatures, the collisions of these molecules with the adsorbent coating surface were enhanced compared to regular HSSPME sampling, leaving fewer sites available for analytes to adsorb. Based on Fig. 2, the optimum temperatures for Vac- and regular HSSPME were 35 ◦ C and 55 ◦ C, respectively. Despite the superior performance of Vac-HSSPME at 35 ◦ C, the 25 ◦ C sampling temperature was not excluded as an option. Sampling at 25 ◦ C (i.e., room temperature) is particularly advantageous to maintain sample composition and minimize the evaporation of potential volatile components acting as matrix interferences during real wine samples analysis. At the same time, extraction efficiencies with Vac-HSSPME at 25 ◦ C were similar to those recorded with regular HSSPME at 55 ◦ C. For these reasons, 25 and 35 ◦ C were chosen for subsequent Vac-HSSPME investigations and 55 ◦ C for regular HSSPME. Fig. 3 shows the extraction time profiles obtained under reduced (at 25 and 35 ◦ C) and atmospheric pressure conditions (at 55 ◦ C). With regular HSSPME at 55 ◦ C, the extraction time profiles of TCA and TeCA reached equilibrium for sampling times larger than 45 and 60 min, respectively. With Vac-HSSPME, TCA was the only ana-
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regular HSSPME at 55 ◦ C was lower than those extracted under pre-equilibrium conditions with Vac-HSSPME at 25 or 35 ◦ C. It is noted that the possibility of an enhanced competition for SPME fiber binding sites taking place at 55 ◦ C between the target analytes and the increased amount of ethanol molecules cannot be excluded. For PCA and TBA, longer equilibration times were expected since they possess the lowest KH values studied here, and PCA is the most hydrophobic compound (see Table 1). In accordance, Fig. 3 shows that equilibrium was not reached after 90 min of Vac- or regular HSSPME sampling, and that the positive effect of sampling at reduced pressure conditions remained important, especially for PCA, even at extended extraction times. Based on the current findings, a 30 min was selected for both Vac- and regular HSSPME, as a compromise between sensitivity and extraction time, and after considering the GC run time. At this sampling point, the relative improvement expressed as Vac-HSSPME/HSSPME peak area ratios, ranged from 1.2 to 1.6 for Vac-HSSPME at 25 ◦ C over HSSPME at 55 ◦ C and 1.7–2.4 for Vac-HSSPME at 35 ◦ C over HSSPME at 55 ◦ C (Table ESM-3 in the electronic supplementary material). In order to proceed with the evaluation of the analytical performances of the methods a selection on the sampling temperature for Vac-HSSPME had to be made at this point. Wine samples free of target analytes were spiked and submitted to Vac-HSSPME at 25 and 35 ◦ C in triplicate. Representative chromatograms obtained after Vac-HSSPME sampling of wine samples at 25 and 35 ◦ C are given in Fig. ESM-3 in the electronic supplementary material. The results showed that the improvement in extraction efficiencies after applying mild heating was somewhat cancelled when moving from simulated wine to complex real wine matrix. This observation suggested that the volatile components of wine (other than ethanol) were interfering with Vac-HSSPME sampling even when mild heating was applied. It was therefore decided to proceed with 25 ◦ C as the optimum sampling temperature for Vac-HSSPME. It is noted that the effect of matrix on both Vac- and regular HSSPME will be discussed in details in a following section. 3.4. Analytical performance of the optimized Vac-HSSPME and regular HSSPME procedures using simulated wine
Fig. 3. Effect of time on the extraction of the four haloanisoles investigated under (i) reduced-pressure conditions (Vac-HSSPME) at 25 ◦ C (ii) reduced-pressure conditions (Vac-HSSPME) at 35 ◦ C and (iii) atmospheric pressure conditions (HSSPME) at 55 ◦ C. Other experimental conditions: 5 mL synthetic wine solution spiked at 15 ng L−1 with haloanisoles; 10 min incubation time; 1000 rpm agitation speed.
lyte that reached equilibrium after 45 min of sampling at 25 ◦ C and 30 min extraction at 35 ◦ C. Heating the sample generally accelerates extraction kinetics so that analytes can reach equilibrium faster. Although regular HSSPME at 55 ◦ C proved advantageous to this end, the final amount of TCA extracted at equilibrium was lower compared to that with Vac-HSSPME. This is in agreement with previous reports, where elevated sample temperatures were found to reduce analyte recovery by shifting both the sample-headspace and the fiber-headspace equilibrium to favor the gas phase [7]. It was therefore assumed that at 55 ◦ C the distribution constant between the headspace and the fiber coating decreased, reducing the amount of TCA extracted at equilibrium. The results presented in Fig. 3 suggest that heating the sample also reduced the extraction efficiency of TeCA, as the equilibrium amount extracted with
The analytical performances of the optimized Vac- and regular HSSPME procedures were evaluated using simulated wine as a solvent. The main analytical parameters are summarized in Table 3 and the graphical representations of the calibration curves for each analyte are given in Fig. ESM-4 of the electronic supplementary material. The calibration curves showed good linearity in a wide concentration range, with coefficients of determination varying between 0.994 and 0.998 (n = 7), and between 0.997 and 0.999 (n = 6) for Vac- and regular HSSPME, respectively. The intraday precision was evaluated by six replicate analysis of synthetic wine standards spiked at two different concentration levels for each extraction procedure. The obtained relative standard deviations (RSDs) ranged from 4.0–10.9% with Vac-HSSPME and from 4.8–10.6% with regular HSSPME. Limits of detection (LODs) were estimated according to three times signal-to-noise-ratio (S/N) criterion. LODs ranged from 0.13 to 0.19 ng L−1 for Vac-HSSPME and from 0.26 to 0.76 ng L−1 for regular HSSPME. Based on the present results, sensitivity decreased from Vac-HSSPME to regular HSSPME (Fig. ESM-4). Nonetheless, with both methods the LODs were low enough to reach the sensory threshold levels at which haloanisoles can be perceived. In general, previously published regular HSSPME methods suggested heating the sample when choosing short extraction times or sampling the headspace for extended times when applying room or mild temperatures [1]. Here, the LODs obtained with Vac-HSSPME were better than or similar to published regular HSSPME methods using porous SPME fibers and a similar analytical instrumentation [1,24,25]. The
9.5 7.4 5.6 6.8 0.44 0.37 0.26 0.66
c
a
b
Coefficient of determination, number of calibration points (n) in parenthesis. Limit of detection evaluated as S/N = 3. Intra-day precision expressed as relative standard deviation (n = 6) and given for two concentration levels.
5.8 4.0 4.1 4.2 10.9 4.6 8.8 8.0 0.16 0.18 0.19 0.13 0.998 0.998 0.998 0.994 9.49 11.20 12.56 11.85 TCA TeCA PCA TBA
0.25-25 0.25-25 0.25-25 0.25-25
Low level (0.5 ng L−1 )
Linear range (ng L−1 )
Retention time (min)
Vac-HSSPME
r2 (n = 7) a
LODb (ng L−1 )
Precision (RSD, %)
c
High level (10 ng L−1 )
0.75-25 0.75-25 0.75-25 0.75-25
0.998 0.999 0.999 0.997
r2 (n = 6) a Linear range (ng L−1 )
Regular HSSPME
3.5. Analysis of wine samples using Vac-HSSPME
Analyte
Table 3 Analytical performance of the optimized Vac-HSSPME and regular HSSPME procedures.
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proposed Vac-HSSPME procedure therefore possessed the great advantage of sampling for short sampling times and at room temperature, eliminating thus the need for heating the sample.
10.6 4.8 6.7 4.8
Low level (1 ng L−1 )
LODb (ng L−1 )
Precision (RSD, %)c
High level (15 ng L−1 )
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Past investigations concluded that a broad spectrum of compounds occurring in wine can interact with the extraction of haloanisoles. To this end, the liquid-gas phase partitioning of volatile wine components can be influenced by other wine constituents, such as polysaccharides, proteins and polyphenols. In particular, wine polyphenols have attracted much attention, because of their ability to interact with proteins as well as the volatile substances present in wine [5]. For this reason, wine matrix classification is typically based on polyphenol content with white wine assumed to be a matrix of low interaction and red wine a matrix with high matrix effect [15,26]. For TCA determination it has been proposed that the approach of low/high polyphenol contents might be too simple, and instead it was suggested that the nature rather than the concentration of polyphenolic compounds most likely affects TCA recoveries [26]. At the same time, the different ethanol concentrations found in wines will affect HSSPME extraction efficiencies of haloanisoles, given that several past investigations conclude that peak areas decrease with increased ethanol content in wine samples [16,20,24]. The determination of haloanisoles in wine by HSSPME is therefore a matrix effect-dependent analytical procedure and the use of a suitable internal standard or the method of standard additions can minimize or avoid this effect [5,15,16,26]. To investigate matrix effects on the proposed Vac-HSSPME procedure commercial red wine samples were tested. Initial blanks using the proposed Vac-HSSPME method showed no detectable amounts of target analytes but the noise level was increased due to matrix complexity [26]. The total pressure in the sampler was measured before sample introduction and after the sample equilibration step and a ∼80 mbar increase was recorded. Compared to the total pressure measured in the headspace of the synthetic wine solution, the additional ∼30 mbar increase accounted for the presence of other volatile wine components. The same wine was then spiked at 10 ng L−1 and submitted to Vac-HSSPME analysis at 25 ◦ C. As expected, a strong matrix effect was observed and measured analyte peak areas were affected due to the interaction with the matrix. Extractions were repeated this time using regular HSSPME at 55 ◦ C and the resulting Vac-HSSPME/regular HSSPME peak area ratios ranged from 1.2 to 1.5 for target analytes, similar to those obtained when using simulated wine (Table ESM-3). The latter demonstrated that the additional increment in total pressure when sampling real wine samples was not affecting the relative performance of Vac-HSSPME over regular HSSPME. Subsequent investigations consisted of using the standard addition method to test the analytical performance of the method in the same wine sample. The resulting calibration curves showed good linearity between 1.5 and 25 ng L−1 , and the coefficients of determination were 0.997, 0.997, 0.991 and 0.991 for TCA, TeCA, PCA and TBA respectively (n = 5). In accordance with previous reports, calibration curves reflected a decrease sensitivity (slope) from simulated water to real wine (Fig. ESM-4) and was related to the complexity of the matrix. LODs were estimated according to three times signalto-noise-ratio (S/N) criterion and were 0.43, 0.45, 0.31 and 0.64 ng L-1 for TCA, TeCA, PCA and TBA respectively. As expected, the proposed method showed a good linearity in real wine samples, but the limits of detection and quantification were affected due to the noise level increase and analyte interaction with matrix. Twelve bottled red wines were then analyzed using the proposed Vac-HSSPME. In almost all of the samples, there were no detectable amounts of haloanisoles, but in one red wine sample
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References
Fig. 4. Overlaid Vac-HSSPME-GC-ECD chromatograms of the red wine found positive in TCA: (i) spiked at 10 ng L−1 with haloanisoles and (ii) blank showing trace amounts TCA (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
TCA content was detected. Fig. 4 shows the chromatograms of the TCA positive red wine sample before and after adding 10 ng L−1 of haloanisoles. The standard addition method was then chosen and samples were spiked and multiply extracted. Based on these studies, the TCA concentration in the positive sample was 3.2 ± 0.2 ng L−1 (n = 3). This value was within the perception level for TCA (ranging between 0.03–10.0 ng L−1 ) and below the 10 ng L−1 concentration threshold where a defect in wine is produced [16]. 4. Conclusions The determination of trace amounts of haloanisoles in complex wine samples has represented a challenge for Vac- and regular HSSPME. The high ethanol content affected the physico-chemical properties of the target analytes and their partitioning with the headspace. For Vac-HSSPME additional concerns existed over the presence of ethanol and other volatile wine compounds as they would increase the total pressure in the headspace. The present work demonstrated that these changes were not sufficient to suppress the positive effect of vacuum in HSSPME. The optimized Vac-HSSPME procedure proved to be a fast, room temperature and sensitive method that could be used for the determination of haloanisoles in wine samples. The need for heating the sample at elevated temperatures as seen in the optimized HSSPME procedure was eliminated. Sampling at room temperature is particularly advantageous to maintain sample composition and minimize the evaporation of potential volatile components acting as matrix interferences during real wine sample analysis. Acknowledgments The authors are grateful to the wineries of Diamantakis (Herakleion, Crete, Greece) and Manousakis (Chania, Crete, Greece) for supplying the wine samples. The authors also wish to thank Professor Janusz Pawliszyn (University of Waterloo, Canada) for supplying the vacuum meter. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 03.047.
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