Application of headspace solid-phase microextraction (HS-SPME) coupled with high-resolution gas chromatographic (HRGC) analysis was studied for determining lactones in wines. Six different SPME fibers were tested, and the influence of different factors such as temperature and time of desorption, ionic strength, time of extraction, content of sugar, ethanol, tannins and anthocyanins, and pH and influence of SO2 were studied. The proposed HS-SPME-GC method is an appropriate technique for the quantitative analysis of
Among volatile components of wine, lactones and particularly the
Lactone smell is usually described as “fruity” or “coconut-like, fruity” (
Lactones are among the most important compounds contributing to the sensory characteristics of wines aged in oak wood. Whiskey lactones and some volatile phenols coming directly from wood have been recognized as important odor active compounds in Madeira wines [
They are already present in natural oak and their content increases due to ageing. From an organoleptic point of view, they are the most important lactones extractable from oak casks. Furthermore, they have been reported as potential aging markers in Madeira wines [
Oak species, geographical origin, silvicultural treatment of tree, and processing of wood have influence on volatile composition of barrel wood. These volatile compounds are susceptible to migrate from oak wood to wine. Although the volatile composition of wine undergoes an evolution during bottle aging, this is carried out in such a way that the most important characteristics spread into wine from wood and remain until the end of bottle aging. These conclusions emphasize the importance of species and geographical origin of oak wood in the volatile composition of wines during aging [
Other less studied lactones such as pantolactone and the 4-carbethoxy-
Chemical structure defines their sensorial and chemical properties [
Different methods have been proposed for extraction of wine. Analytical methods for gas chromatography determination of lactones need a previous concentration step due to the low concentrations existing in wine [
A great number of wine aroma compounds have been characterised as lactones, many of them extracted by SPME. Published works account for particular compounds like diacetyl [
The aim of this work was to apply the GC-MS technique combined with automatic headspace (HS) SPME to develop a new method to determine a set of lactones in wine (
The following lactones were studied (CAS number in brackets):
Individual stock standard solution in ethanol of
All parameters have been optimized using a synthetic wine solution containing concentrations of lactones. Table
Concentrations of synthetic solutions containing lactones.
Compound | Concentrations ( |
||
---|---|---|---|
Low level | High level | Optimization | |
|
14400 | 36200 | 20300 |
|
146 | 365 | 98.9 |
|
494 | ||
Whiskey-lactone I | 48.3 | 144 | 100 |
|
3.96 | 9.90 | 9.9 |
Whiskey-lactone II | 48.3 | 144 | 100 |
|
19.8 | 59.5 | 100 |
|
3.83 | 9.56 | 9.9 |
|
124 | 310 | 96.8 |
|
1.92 | 4.80 | 9.9 |
3,4-Dimethylphenol | 491 |
Either individual stock standard solutions or real wine samples were prepared in 2 mL vials adding 0.77 mL of sample and 0.03 mL of internal standard solution. The vials were tightly capped with PTFE-lined cap and shaken for 10 min at 200 min−1.
Regularly verified pipettes and class A volumetric flasks were used in solution preparation. A precision balance (Sartorius BP 210-S), a pH meter (WTW, pH 197-S), Milli-Q gradient A10 (Millipore), and a mechanical shaker (Selecta, Rotabit) were used in the study.
Six fibers coated with different stationary phases and various film thicknesses were purchased from Supelco (Bellefonte): polydimethylsiloxane 100
The analyses were carried out on a 3800 GC gas chromatograph equipped with an 8200 Standalone autosampler, a 1079 split/splitless injector, and a mass spectrometry detector Saturn 2000 (Varian, Walnut Creek, CA, USA). Injections were performed in splitless mode, using a 0.75 mm I.D. liner which improved GC resolution. Ionization mode used was electronic impact.
Separations were performed using a DB-WAXETR capillary column (60 m, 0.25 mm I.D., 0.5
The GC-MS transfer line temperature was 240°C. The MS operated in electron impact mode at 70 eV and collected data at a rate of 1.0 scans/s over a mass range of m/z 25–350. The ion source temperature was 200°C, the detector voltage was set to 1500 V, and the detector temperature was 300°C.
Chromatographic conditions have been set using both synthetic solutions and wine samples to ensure a good chromatographic resolution and no coelution of compounds.
Extraction time and reproducibility, extraction temperature, desorption time and temperature, and ionic strength were optimized as a need of establishing basic instrumental parameters and simultaneously for selecting the appropriate SPME fiber.
The following steps are designed to reveal and correct possible matrix effect due to specific parameters such as phenolics, sugar, pH, sulphur dioxide, and ethanol.
Figure
TIC chromatogram obtained with a PA fiber for a synthetic wine spiked with the different analytes.
Table
Retention time, molecular weight and enthalpy of vaporization [
Compound | Retention time (min) | Molecular weight (g/mol) |
|
---|---|---|---|
|
15.97 | 86.04 |
|
|
17.09 | 114.14 |
|
|
18.60 | 128.17 |
|
Whiskey-lactone I |
19.73 | 156.22 | 48.36 |
|
20.31 | 142.20 |
|
Whiskey-lactone II |
20.74 | 156.22 | 48.36 |
|
21.67 | 156.22 |
|
|
23.14 | 170.25 |
|
3,4-Dimethylphenol (IS) | 23.86 | 122.16 |
|
|
24.01 | 170.25 |
|
|
24.73 | 184.28 |
|
Because of the kinetic nature of the extraction process, it is heavily influenced by fiber type and extraction time. Optimization of both parameters is the first step when building a microextraction method. Since the final aim of this work is to determine the analytes in sweet wines, which can have a high content in sugars (up to 200 g/L) and other many compounds, direct immersion mode leads to a rapid degradation of the fiber surface. To avoid this effect, all the studies were performed in headspace mode.
In order to establish optimal extraction parameters, the six fibers named above were studied. Experiences were made varying extraction time from 15 min to 90 min using a spiked synthetic wine.
Figure
Extraction profiles of the different analytes versus extraction time for the different fibers.
CAR/PDMS fiber was immediately discarded because it offered very wide and low peaks shapes resulting in a poor peak resolution with lactone peaks overlapping between them.
As can be seen,
Almost all fibers show a fast initial increase in extraction during the first 15 min, and then it slows down until 45 min. After these time differences among compounds appear, some compounds like
The extraction performance increases all over homologous series of n-
PDMS fiber presents the lower extraction ability for
A reproducibility study was made injecting five times a wine synthetic solution spiked with lactones (Table
RSD (%) (
Compound | PA | DVB/CAR/PDMS | PDMS/DVB | CW/DVB |
---|---|---|---|---|
|
8.14 | 36.43 | 14.41 | 8.47 |
|
8.24 | 11.76 | 6.56 | 8.94 |
Whiskey-lactone I | 1.26 | 31.44 | 1.47 | 4.33 |
|
1.72 | 26.08 | 2.45 | 2.56 |
Whiskey-lactone II | 2.19 | 27.04 | 3.39 | 4.27 |
|
1.40 | 46.96 | 1.80 | 0.56 |
|
2.45 | 58.36 | 4.40 | 3.24 |
|
2.91 | 16.77 | 5.23 | 3.71 |
|
2.78 | 62.05 | 3.96 | 3.37 |
As can be observed, DVB/CAR/PDMS shows the worst RSD values for most compounds; so it was discarded for the rest of the studies.
Focusing on the rest of the three fibers, all of them present good overall reproducibility. Nevertheless, the value of 14.41 for
Finally, PA and CW/DVB present a similar behaviour in terms of extraction and reproducibility; so any of them would be adequate. As CW/DVB has been discontinued by the manufacturer, PA fiber was selected as the best fiber for extracting lactones in wine samples. So, the rest of this study was done using PA fiber.
Extraction temperature plays an important role in extraction but in two opposite ways. Increasing temperature produces desorption of molecules on the fiber decreasing sensibility. Simultaneously increasing temperature modifies liquid-gas equilibrium enriching gas phase with analytes [
An extraction temperature, study temperature, was done using a synthetic wine spiked with lactones. Temperature was set to 60°C, 42°C, and 25°C using 45 min extraction time. Results of normalized peak area (peak area/concentration) versus temperatures are shown in Figure
Temperature influence on the extraction of the different analytes on a PA fiber.
Figure
Desorption time and temperature were also tested within the range recommended by manufacturer. Injections were made at 250°C and 300°C desorption temperatures and 2 min, 5 min, and 10 min desorption time using the rest of selected parameters. Results are shown in Table
Normalized peak areas at different temperatures and desorption time.
Compound | 250°C | 300°C | ||||
---|---|---|---|---|---|---|
2 min | 5 min | 10 min | 2 min | 5 min | 10 min | |
|
2033 | 7644 | 10782 | 3820 | 9353 | 11068 |
|
20681 | 129160 | 155069 | 53359 | 143513 | 155513 |
|
38894 | 185530 | 230138 | 81918 | 209111 | 230259 |
Whiskey-lactone I | 110605 | 364837 | 413042 | 199319 | 387667 | 413463 |
|
111825 | 360337 | 407674 | 197209 | 384203 | 408474 |
Whiskey-lactone II | 90756 | 320900 | 380063 | 183103 | 351145 | 380190 |
|
119260 | 374890 | 417670 | 212936 | 389701 | 418269 |
|
106828 | 327823 | 363834 | 186756 | 339222 | 364786 |
3,4-Dimethylphenol (IS) | 444709 | 999600 | 1090969 | 490860 | 1032004 | 1092175 |
|
16550 | 130347 | 175598 | 49251 | 138751 | 175672 |
|
135335 | 367100 | 412196 | 217489 | 381118 | 411959 |
Values of normalized peak area show increasing values with time for all analytes indicating that short desorption time leads to incomplete desorption.
On the other hand, higher temperature shows higher areas until 10 min desorption time. Taking into account that fiber life is longer at lower desorption temperatures, we selected 250°C as desorption temperature and 10 min as desorption time. Blank injections showed no memory effect in desorption for any analyte.
Ionic strength affects analyte extraction, particularly those of polar character. In order to study this effect, increasing quantities of solid NaCl were added to spiked synthetic wine. Quantities of 0 mL, 80 mL, 160 mL, 200 mL, and 240 mg in 0.77 mL of sample were added to reach 0%, 10.3%, 20.7%, 25.9%, and 31.1% NaCl solutions, respectively. Results are shown in Figure
Influence of ionic strength on the extraction of the different analytes on a PA fiber.
Increasing ionic strength produces an increase in extraction. The best values are those obtained by saturation of NaCl and this condition is selected for further studies.
Wine sample matrix has a wide variety of compounds that can affect extraction process. So it is necessary to study the effect of pH, phenolic compounds, sugar, sulphur dioxide, and ethanol content as influencing extraction process.
Polyphenol content presents wide variations in wines especially from white to red wine. An extraction study was made in order to test its influence in the process.
Synthetic wine spiked solution was prepared with tannins concentrations ranging from 0 g/L to 1 g/L and anthocyanins from 0 g/L to 5 g/L. Obtained results do not show tannins or anthocyanins influence in the extraction.
In the same way, sugar content varies widely from dry to sweet wine reaching even values higher than 200 g/L. Spiking synthetic wine with concentrations up to 200 g/L showed no influence. This is in coincidence with results reported for other compounds [
Sulphur dioxide is a commonly used additive in wine making due to its antiseptic, antioxidant, and antioxidasic properties. Sulphur dioxide added to wine reacts with carbonyl compounds forming the so-called “combined sulphur,” especially with acetaldehyde, changing the expected concentration of free carbonyl compounds. Added sulphur dioxide quantities also change from red to white wine. To study this effect, synthetic wine solutions spiked with lactones and sodium metabisulphite ranging up to 200 mg/L were extracted. All lactones showed no influence in extraction in the range studied.
Behind water, ethanol is the major component in wines. Obviously, ethanol is extracted in fiber and effectively competes with analytes by active positions. This effect has been previously described by several authors [
Normalized peak areas of the different analytes versus alcoholic degree on a PA fiber.
Figure
The rest of lactones including the internal standard
Figure
Relative peak areas versus alcoholic degree.
Method validation was developed in terms of linearity, detection and quantification limits, precision, and matrix effect influence.
Calibration curves were elaborated using eight synthetic wine solutions spiked with lactones, internal standards, and using the parameters selected above. Table
O.O, slope,
Compound | Intercept | Slope |
|
Linear range ( |
---|---|---|---|---|
|
|
|
0.999 | 0.17–60.26* |
|
|
|
0.999 | 11–609 |
Whiskey-lactone I |
|
|
0.999 | 1–401 |
|
|
|
0.999 | 1–21 |
Whiskey-lactone II |
|
|
0.999 | 1–401 |
|
|
|
0.997 | 2–206 |
|
|
|
0.997 | 1–20 |
|
|
|
0.997 | 4–517 |
|
|
|
0.999 | 1–15 |
Detection and quantification limits were calculated as the concentration corresponding to 3 and 10 times signal/noise, respectively. Values are shown in Table
Method repeatability and reproducibility were obtained analyzing 5 replicates of synthetic wine spiked with lactones intermediate concentration of calibration. The 5 replicates were repeated during 3 different days. Results are shown in Table
LOD, repeatability, and reproducibility of method [
Compound | LOD |
LOQ |
Odor threshold |
Repeatability |
Reproducibility |
---|---|---|---|---|---|
|
170.97 | 569.93 | 35000 | 0.63 | 1.93 |
|
10.51 | 35.04 | 359000 | 2.98 | 2.57 |
Whiskey-lactone I | 0.97 | 3.24 | 790 | 3.54 | 4.41 |
|
1.19 | 3.98 | 7 | 4.31 | 3.96 |
Whiskey-lactone II | 0.60 | 2.02 | 67 | 4.13 | 5.25 |
|
2.11 | 7.05 | 30 | 3.24 | 2.73 |
|
0.86 | 2.89 | 88 | 2.50 | 2.78 |
|
4.17 | 13.92 | 386 | 3.19 | 4.56 |
|
0.63 | 2.10 | 60 | 2.88 | 4.45 |
Wine is a complex matrix that includes hundreds of different compounds besides those studied here. So it is necessary to perform a matrix effect study to evidence the existence of extraction interferences [
Three samples of white and red wine were spiked with lactones at two different concentration levels shown in Experimental section. Results are shown in Table
Mean (%) and RSD (%) of recoveries.
Compound | Low level | High Level | ||||||
---|---|---|---|---|---|---|---|---|
Mean | Mean | RSD | RSD | Mean | Mean | RSD | RSD | |
White | Red | White | Red | White | Red | White | Red | |
|
189.1 | 191.9 | 1.45 | 2.56 | 189.4 | 198.6 | 4.23 | 0.14 |
|
88.1 | 86.7 | 3.05 | 5.62 | 88.6 | 88.5 | 1.80 | 0.77 |
Whiskey-lactone I | 144.3 | 145.2 | 9.11 | 1.98 | 144.2 | 143.1 | 1.05 | 1.46 |
|
76.8 | 81.3 | 2.06 | 3.21 | 78.1 | 80.1 | 0.65 | 2.10 |
Whiskey-lactone II | 76.8 | 80.1 | 1.36 | 8.15 | 74.7 | 76.9 | 1.26 | 1.65 |
|
93.2 | 101.6 | 3.73 | 1.79 | 94.2 | 107.0 | 1.26 | 1.05 |
|
136.5 | 142.8 | 0.96 | 3.45 | 137.5 | 147.4 | 2.33 | 0.38 |
|
109.3 | 108.2 | 1.79 | 4.58 | 108.5 | 109.1 | 1.27 | 2.01 |
|
112.0 | 116.5 | 0.99 | 2.10 | 111.0 | 112.6 | 1.31 | 1.17 |
Table
Mean of recoveries and RSD.
Compound | Mean (%) | RSD (%) |
---|---|---|
|
192.3 | 3.01 |
|
88.0 | 2.98 |
Whiskey-lactone I | 144.2 | 4.09 |
|
79.1 | 2.95 |
Whiskey-lactone II | 77.1 | 4.52 |
|
99.0 | 6.23 |
|
141.1 | 3.72 |
|
108.8 | 2.37 |
|
113.0 | 2.27 |
Optimized method was applied to 72 wine samples including white, red, and rosé wines. Table
Concentration mean and SD (
Compound |
White wines1 |
Rosé wines2 |
Red wines3 |
Significative differences | |||
---|---|---|---|---|---|---|---|
Mean | SD | Mean | SD | Mean | SD | ||
|
26287 | 8478 | 25940 | 6553 | 32652 | 6403 | 1–3, 2-3 |
|
200 | 89 | 202 | 72 | 211 | 67 | — |
Whiskey-lactone I | 10.60 | 14.38 | 5.44 | 2.20 | 45.73 | 34.42 | 1–3, 2-3 |
|
5.41 | 2.64 | 5.63 | 2.20 | 6.95 | 3.13 | — |
Whiskey-lactone II | 20.31 | 50.46 | d-nq | — | 138.44 | 111.13 | 1–3, 2-3 |
|
14.77 | 7.39 | 19.98 | 5.64 | 42.09 | 27.45 | 1–3, 2-3 |
|
d-nq | — | 4.16 | 2.91 | 3.85 | 2.65 | 1-2, 1–3 |
|
157 | 72 | 149 | 37 | 228 | 96 | 1–3, 2-3 |
|
nd | — | d-nq | — | d-nq | — | — |
nd: not detected; d-nq: detected not quantified.
As expected,
ANOVA for these samples revealed that
Solid-phase microextraction is a suitable technique for determining concentrations of different lactones in wine matrix. The proposed methodology covers the range of concentrations usually found in wines with an acceptable uncertainty. The use of two internal standards corrects the influence of ethanol content. Matrix effect exists but can be corrected using both standard addition calibration and experimental correction factors, allowing the quantification of all the compounds studied using gas chromatography, mass spectrometry detection, and electronic impact.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work has been funded by the Spanish CICYT (Comisión Interministerial de Ciencia y Tecnología), Project AGL 2003-04911/ALI. The authors acknowledge Bodegas Viñatigo (Tenerife) for sample supply.