To solve problems of sensitivity, repeatability and multi-step extraction related to 3-isobutyl-2-methoxypyrazine (IBMP) determination in wines, a simple method based on the novel combination of solid-phase microextraction and stable isotope dilution assay is presented. Among the parameters that affect this type of extraction, five of them have been optimised since the other parameters have common values or do not require optimisation (e.g. addition of sodium chloride at saturated concentration) and so were fixed. Vial volume, sample volume/vial volume ratio, pH, adsorption time and temperature have been optimised by means of two experimental designs. After extraction, quantification was performed by stable isotope dilution with gas chromatography-tandem mass spectrometry ([]-IBMP as internal standard). The final procedure allowed quantification far below IBMP’s sensory threshold (1 ng l−1 versus 15 ng l−1) with a 4% standard deviation. This method has been applied to experimental Fer servadou wines. Comparison of IBMP contents confirmed the efficiency of some viticultural and enological techniques on the herbaceous flavour decrease, such as prior fermentation maceration at high temperature (70 °C) and the use of a reflective carpet on viticultural soil

Nepveu, Françoise

Peres-Lucchese, Yolande

Prouteau, Cécile

Renard, Romain

Schneider, Rémi

Vaca-Garcia, Carlos

Open Archive Toulouse Archive Ouverte

OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible Any correspondence concerning this service should be sent  to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr This is an author’s version published in: http://oatao.univ-toulouse.fr/23246To cite this version:Prouteau, Cécile  and Schneider, Rémi and Peres-Lucchese, Yolande  and Nepveu, Françoise and Renard, Romain and Vaca-Garcia, Carlos  Improving headspace-solid-phase microextraction of 3-isobutyl-2-methoxypyrazine by experimental design with regard to stable isotope dilution gas chromatography–mass spectrometric analysis of wine. (2004) Analytica Chimica Acta, 513 (1).223-227. ISSN 0003-2670Official URL: https://doi.org/10.1016/j.aca.2003.11.083 Improving headspace-solid-phase microextraction of3-isobutyl-2-methoxypyrazine by experimental design withregard to stable isotope dilution gas chromatography–massspectrometric analysis of wineCécile Prouteau a,∗, Rémi Schneider b, Yolande Lucchese a, Françoise Nepveu c,Romain Renard d, Carlos Vaca-Garcia ea ENSIACET Laboratoire de Controˆle, 118 rte de Narbonne 31077 Toulouse Cedex 4, Franceb ITV France, détaché à l ‘INRA-UMR-SPO, 2 place Viala, 34060 Montpellier, Francec Laboratoire Pharmacochimie des Substances Naturelles et Pharmacophores Redox (UMR IRD-UNC-UPS U152),Faculté des Sciences Pharmaceutiques, Université Paul Sabatier, 35, chemin des Maraıˆchers, F-31062 Toulouse Cedex 4, Franced Station régionale ITV Midi-Pyrénées V’innopoˆle Brames-Aı¨gues BP22 81310 Lisle/Tarn, Francee ENSIACET Laboratoire de Chimie Agro-Industrielle, 118 rte de Narbonne 31077 Toulouse Cedex 4, FranceAbstractTo solve problems of sensitivity, repeatability and multi-step extraction related to 3-isobutyl-2-methoxypyrazine (IBMP) determination in wines, a simple method based on the novel combination of solid-phase microextraction and stable isotope dilution assay is presented. Among the parameters that affect this type of extraction, ﬁve of them have been optimised since the other parameters have common values or do not require optimisation (e.g. addition of sodium chloride at saturated concentration) and so were ﬁxed. Vial volume, sample volume/vial volume ratio, pH, adsorption time and temperature have been optimised by means of two experimental designs. After extraction, quantiﬁcation was performed by stable isotope dilution with gas chromatography-tandem mass spectrometry ([2H2]-IBMP as internal standard). The ﬁnal procedure allowed quantiﬁcation far below IBMP’s sensory threshold (1 ng l−1 versus 15 ng l−1) with a 4% standard deviation. This method has been applied to experimental Fer servadou wines. Comparison of IBMP contents conﬁrmed the efﬁciency of some viticultural and enological techniques on the herbaceous ﬂavour decrease, such as prior fermentation maceration at high temperature (70 ◦C) and the use of a reﬂective carpet on viticultural soil.Keywords: Solid-phase microextraction; Stable isotope dilution; Experimental design; Wine; 3-Isobutyl-2-methoxypyrazine1. IntroductionThe Fer servadou grape variety (Vitis vinifera cv. Fer ser-vadou) comes from south-west France. This variety belongsto the same family as Cabernet Sauvignon. Among its va-rietal key aroma compounds, 3-alkyl-2-methoxypyrazineshave been identiﬁed, in particular 3-isobutyl-2-methoxypy-razine (IBMP), known for its contribution to the green bellpepper odour. This compound is present at very low con-∗ Corresponding author. Tel.: +33-5-34-61-52-83;fax: +33-5-34-61-52-53.E-mail address: cecile.prouteau@ensiacet.fr (C. Prouteau).centrations (10–50 ng l−1 in wines and up to 80 ng l−1 ingrapes) but IBMP has a high odorant potency since its sen-sory threshold was reported to be 10 ng l−1 in red wines [1].To diminish the IBMP concentration, wine makers eval-uated different techniques based on thermolability andphotosensitivity of IBMP [2], such as prior fermentationmaceration at high temperature or the use of a reﬂectivecarpet on viticultural soil. A reliable analysis for IBMPis then needed for evaluating effects of those technicaldevelopments and also environmental factors.Detection of volatiles at low concentrations (ng l−1) re-quires an extraction-concentration step coupled to a verysensitive analytical technique. Several extraction-concentra- doi:10.1016/j.aca.2003.11.083tion methods have been used: extraction by cation-exchangeresin, liquid–liquid extraction [3] and a method based onheadspace-solid-phase microextraction (HS-SPME) afterconcentration by evaporation [4]. All these extractionsare multi-step, time-consuming and laborious. The analy-sis can be performed either by gas chromatography (GC)with nitrogen–phosphorus detection (NPD) (non-labelledinternal standard) or by GC-mass spectrometry (MS) usingdeuterium labelled [2H2]-IBMP. Recently, an attempt tosimplify the extraction step to only one, SPME, followedby GC-NPD for analysis encountered sensitivity and re-peatability problems (coefﬁcient of variation = 8–18%)[5]. To solve these problems and also to permit automa-tion, we chose to combine SPME with stable isotopedilution GC. This type of calibration, using deuterium la-belled [2H2]-IBMP as internal standard, is performed byGC-MS/MS.This combination seems to be appropriate since the twomolecules (target and standard) react in the most similarbehaviour with the extraction ﬁbre. After optimisation of theSPME conditions, the described method has been applied towine samples.2. Experimental2.1. Reagents3-Isobutyl-2-methoxypyrazine [24683-00-9] was suppliedby Aldrich. The purity of the standard was >99%. The la-belled [2H2]-IBMP was synthesized as described in [6].Stock solutions of 1 g l−1 were prepared in RP Normapurabsolute ethanol (Prolabo), and stored in the dark at +4 ◦C.To adjust the pH, potassium hydroxide (85% ACS reagent,Aldrich) at 200 g l−1 was used.A wine model solution contained in 1 l of demineralisedwater 6 g of glycerol (Aldrich), 2.5 g of l-(+)-tartaric acid(Aldrich), 3 g of l-(+)-lactic acid (Fluka), 1 g of potassiumphosphate (Aldrich), 11% absolute ethanol (Prolabo), pHadjusted to 3.5.2.2. SPME procedureAn SPME manual device and 65mm polydimethylsil-oxane-divinylbenzene (PDMS-DVB) ﬁbres were purchasedfrom Supelco. To prevent contamination, each ﬁbre was con-ditioned and cleaned before and after use by inserting it intothe GC injector at the recommended temperature. A visualdaily control is assumed (colour, coating state . . . ).For optimisation, parameters were ﬁxed as mentioned inthe experimental design section and as described in Table 1.The ﬁnal procedure for calibration was as follows: 25mlof a wine solution was adjusted to the optimum pH (6.6)with potassium hydroxide solution (200 g l−1) and spikedwith 10.5 ng l−1 [2H2]-IBMP. Ten millilitres of this solutionwas transferred into a 20ml vial containing 3 g of sodiumTable 1SPME optimised parameters values for experimental designaParameter Previous methodfor musts [7]Geometry AdsorptionVs/Vv (ml) 0.5 Variable 0.5Vv (ml) 20 Variable 20pH 8.5 8.5 VariableAdsorption temperature(◦C)40 40 VariableAdsorption time (min) 240 240 Variablea Saturated NaCl, magnetic stirring, desorption at 240 ◦C, 5min des-orption time.chloride and a magnetic stirrer. The vial was immediatelycapped with a PTFE-faced silicone septum/aluminium crimpcap. The extraction was performed in the headspace over165min at 27 ◦C, with constant stirring.2.3. Experimental designAmong all the parameters affecting the extraction quality,most have identical values in the published SPME methods.This is the case with salt concentration, which is commonlysodium chloride at saturation, and with magnetic stirring. Inthe same way, a compromise between desorption time andtemperature should be achieved in order to avoid carry-over.Therefore, only ﬁve parameters have to be optimised: vialvolume (Vv), ratio between the sample volume and vialvolume (Vs/Vv), sample pH, adsorption temperature andadsorption time. Instead of studying the ﬁve parameterstogether, this was simpliﬁed by dividing them into twogroups, by grouping the interdependent parameters.Vv and Vs/Vv were evaluated together using a two-factorfull factorial experimental design. They deﬁne the geome-try of the headspace but do not facilitate molecules to passfrom aqueous phase to the headspace. Vial volume choicewas restricted to 20 and 50ml. These volumes are differentenough and contain a reasonable sample quantity. Vs/Vv ad-viced by the constructor is ca. 75%. This value and a smallerone were selected. Table 2 shows the experimental domain.The matrix of the experiments is presented in Table 3.Table 2Experimental domain for “geometry” experimental designFactor Parameter Level −1 Level +1X1 Vial volume (ml) 20 50X2 Sample volume/vial volume 0.5 0.75Table 3Two-factors full factorial experimental designExperiment X1 X21 −1 −12 +1 −13 −1 +14 +1 +1Table 4Three-factors Doelhert experimental design (13 assays plus 2 centre rep-etitions)Experiment X1 X2 X31 0 0 02 1 0 03 0.5 0.866 04 −0.5 0.866 05 −1 0 06 −0.5 −0.866 07 0.5 −0.866 08 0.5 0.289 0.8169 −0.5 0.289 0.81610 0 −0.577 0.81611 0.5 −0.289 −0.81612 −0.5 −0.289 −0.81613 0 0.577 −0.81614 0 0 015 0 0 0After the “geometry” experimental design, a second ex-perimental design was planned to evaluate the last three pa-rameters. A three-factor Doelhert design was used and 2centre points were added to ensure enough degrees of free-dom for experimental error evaluation (Table 4). After attri-bution of parameters to factors according to the number oflevels, the experimental domain was determined and listedin Table 5.Each design was tested with the same ﬁbre on a winemodel solution spiked with IBMP at 1mg l−1. Quality eval-uation was performed through a GC-NPD analysis, by mea-suring of IBMP peak area. The higher the peak area, the bet-ter is the extraction. Data analysis was performed by meansof the statistical software Mathcad® (Mathsoft, Cambridge,UK).2.4. GC analysis2.4.1. HS-SPME-GC-NPD analysis for HS-SPMEoptimisationSince, under same experimental conditions, [2H2]-IBMPbehaved similarly to IBMP, this study was restricted toIBMP only. Chromatographic analysis was performedwith a Trace 2000 (Thermoﬁnnigan) equipped with anitrogen–phosphorus detector (NPD) system. The NPDtemperature was 300 ◦C. The make-up gas was nitrogenﬂowing at 15mlmin−1. The source current was adjusted to13 pA and the polarisation tension was ﬁxed at 3.5V.Table 5Experimental domain for Dolhert experimental designFactor Parameter Level −1 Level 0 Level +1X1 Adsorption time (min) 60 180 300X2 Adsorptiontemperature (◦C)30 55 80X3 pH 6 7.5 9Detector gas ﬂows were: 1.7mlmin−1 H2, 15mlmin−1N2 and 60mlmin−1 air. The carrier gas was hydrogen ﬂow-ing at 1mlmin−1. The injection was done in the splitlessmode for 3min at 240 ◦C.The oven temperature was programmed as follows: 50 ◦C(3min), then a ﬁrst ramp (40 ◦Cmin−1) to 90 ◦C, then asecond ramp (4 ◦Cmin−1) to 140 ◦C. The ﬁnal temperaturewas raised to 230 ◦C at 20 ◦Cmin−1 and was maintained for10min.A BPX5 (30m × 0.32mm × 0.25mm) capillary columnwas used. Data handling was performed by Chromquest soft-ware (Thermoﬁnnigan, Courtaboeuf, France).2.4.2. HS-SPME-GC-MS/MS for analysisAnalysis was carried out using a Varian Saturn 2000 gaschromatograph-ion trap mass spectrometer (GC-ITMS). ADBWAX-ETR (30m×0.25mm×0.5mm) capillary columnwas used. Desorption was realised in the splitless mode over5min at 240 ◦C, with CO2 cryogenic focussing at the top ofthe column.The oven temperature program is the same as alreadydescribed.The carrier gas was helium N60 ﬂowing at 1.0mlmin−1.The temperatures of the transfer line and ion trap were 170and 150 ◦C, respectively. Detection was performed by elec-tron impact (EI) MS/MS in the multiple reactions monitor-ing mode, with the following parameters: scan channel 1,parent ion m/z 124 with an isolation window of 1 amu innon-resonant mode, excitation storage 40.7 m/z, excitationamplitude of 38 eV; scan channel 2, parent ion m/z 126 withan isolation window of 1 amu in non-resonant mode, exci-tation storage 41.4 m/z, excitation amplitude 40 eV. Naturaland deuterated IBMP were quantiﬁed using the ions m/z 95and 97, respectively. The ions m/z 81, 109, 124 and m/z 81,109, 126 were used as qualiﬁers.2.5. Wine samplesFer servadou experimental wines were made at the ex-perimental cellar of ITV at Gaillac. They are derived fromdifferent plots and from different harvest dates. All sampleswere stored in dark bottles at +4 ◦C until analysed.3. Results and discussion3.1. Experimental design3.1.1. Geometric factorsTwo vial volumes were tested: 50 and 20ml. Ratios Vs/Vvof 75 and 50% were also tested. The results are shown inFig. 1. No modelling was sought. The highest response wastracked and tested to show if it was signiﬁcantly differentfrom the others.By means of the Student’s t-test and standard deviation(n = 5 on assay 1), the equivalence between answers Y1,Fig. 1. “Geometry” experimental design results (Yi response = IBMPpeak area).the highest response, and Y2 and Y4, was established. Thepoor extraction of assay 3 can be explained by the inad-equate conﬁguration of extraction: a long and narrow vialcompared to a large volume of sample. Magnetic stirring wasnot sufﬁcient to homogenise the sample and the headspacewas too conﬁned. Among assays 1, 2 and 4, few samplequantity consumer conditions were retained. So extractionwill proceed in a 20ml vial sample ﬁlled to 50%.3.1.2. Adsorption factorsRising temperatures deﬁned the order of experiments.Data underlined that the temperature domain was too high;in fact the IBMP peak area decreased strongly when thetemperature exceeded 48 ◦C. This can be explained by thethermolability of the molecule and by preferential moleculetransfer from ﬁbre to headspace gas phase. That is the reasonwhy experiments 8 and 9 were carried out at 27 ◦C insteadof 62 ◦C as expected. The results are presented in Table 6.The area prediction calculated model is given bythe following relation: area = 949 503 − 264 033X1 −3 408 018−1 327 344X3−154 891X1X2−333 265X1X3−1 414 976X2X3 − 299 017X12 + 3 006 790X22 −2 759 914X32.Table 6Adjusted matrix of Doelhert experimental design presenting results inrealisation orderExperiment X1 X2 X3 IBMP peak area7 4 33 6.5 63289636 2 33 6.5 60222065 1 55 6.5 9360371 3 55 6.5 76460713 3 69 4.5 02 5 55 6.5 39016014 3 55 6.5 85320711 4 48 4.5 88125112 2 48 4.5 10595353 4 77 6.5 04 2 77 6.5 3372908 4 27 8.5 64559439 2 27 8.5 704940110 3 41 8.5 182864015 3 55 6.5 1035853Fig. 2. Residues analysis. Evaluation of model adjustment. Points are welldistributed on both sides.Coefﬁcient uncertainties: ±126008; ±124190; ±127481;±225742; ±222489; ±310260; ±393057; ±206225;±194143; ±310185.Several indicators were used to evaluate the model ad-justment: R2 (0.9972), adjusted R2 (0.9922) and residuesanalysis (Fig. 2). These values shown that the lack of ad-justment is not too large. The model represents satisfactorilythe IBMP peak area variations as a function of temperature,pH and adsorption time.Next, canonical analysis and iso–response curves allowedthe optimum conditions to be found. Firstly, stationary pointco-ordinates were calculated (−0.38; +0.48; −0.34). Thispoint belongs to the experimental domain, thus an RT-typecanonical analysis will be performed. The results of the anal-ysis permit the conclusion that a pH ca. 6.6, 165min ad-sorption time at 27 ◦C are the optimal conditions. Comparedto previous SPME extractions [4,7], the adsorption time hasbeen considerably reduced without introducing any fastidi-ous and time-consuming sample preparation step.3.2. ValidationFor calibration, linear regression analysis of relative areasversus relative concentrations of [2H2]-IBMP and IBMPwasused. The calibration equation based on 5 points (3 repli-cated) is: areaIBMP/area[2H2]-IBMP = 2.02× [IBMP]+ 0.71(r2 = 0.9884, n = 15).Fig. 3. IBMP amount in Fer servadou wines obtained from differentharvest dates.Table 7IBMP amount in Fer servadou wines obtained from plots where differenttechniques have been usedTechniques [IBMP] (ng l−1)Control sample 14Thin out the leaves under grapes Not detectedPrior-fermentation maceration at 70 ◦C 5Reﬂective carpet 5The limit of detection (LOD) was estimated from the min-imum concentration found in analysed wines. The LOD wascalculated on the basis of a signal/noise ratio of 3. The LODis 1 ng l−1, which is far below the sensory threshold. Moreaccurate precision is not needed. The repeatability (4%) wasestablished from 5 repetitions of an analysis.3.2.1. Analysis of wine samplesSome Fer servadou wines have been analysed by the newmethod. Harvest date inﬂuence (Fig. 3) and viticultural tech-niques (Table 7) impact on the IBMP amount measured.With a late harvest date the IBMP concentration decreaseand can be lower than the sensory threshold. With a lateharvest date the IBMP concentration can be lower than thesensory threshold. Therefore, it is interesting not to harvesttoo early. Furthermore, plots do not have the same “IBMPpotential”, it depends on soil and exposure. A sandy soil (La-grave) will warm up quickly and reﬂect to the grapes ther-mal and luminous energy. This phenomenon favours IBMPdegradation. To reﬂect soil IBMP potential, it would be in-teresting to study Fer servadou plots. It could be used to clas-sify the plots according to their IBMP potential and couldinﬂuence the plots harvest order. Finally, results conﬁrm theefﬁciency of the viticultural (preventive) and enological (cu-rative) techniques used.4. ConclusionsThe method’s sensitivity for detection of IBMP is farbelow the sensory threshold. Moreover the repeatability islower than expected for SPME extraction (CV ca. 12%),this by the use of stable isotope dilution assay. This lattermethod seems to ﬁt very well this kind of extraction. Thesolvent-free method developed will be used in a semi-routineway. It has a lot of applications and will, for example, bevery useful to determine the best harvest date according toaroma maturity.AcknowledgementsThis work has been funded by the “Station régionaleITV Midi-Pyrénées” (Gaillac, France). The authors arealso grateful to UNICOR and Thermoﬁnnigan for theirsupport.References[1] Y. Kotseridis, A. Anocibar Beloqui, A. Bertrand, J.P. Doazan, Am. J.Enol. Vitic. 49 (1998) 44.[2] D. Roujou de Boubée, Doctoral work, Université Victor Segalen Bor-deaux 2, 2000, p. 170.[3] M.J. Lacey, M.S. Allen, R.L.N. Harris, W.V. Brown, Am. J. Enol.Vitic. 42 (1991) 103.[4] C. Sala, M. Mestres, M.P. Marti, O. Busto, J. Guasch, J. Chromatogr.A 953 (2002) 1.[5] P.J. Hartmann, H.M. McNair, B.W. Zoecklein, Am. J. Enol. Vitic. 53(2002) 285.[6] Y. Kotseridis, R. Baumes, G.K. Skrouroumounis, J. Chromatogr. A824 (1998) 71.[7] C. Sala, M. Mestres, M.P. Marti, O. Busto, J. Guasch, J. Chromatogr.A 880 (2000) 93.

Improving headspace-solid-phase microextraction of 3-isobutyl-2-methoxypyrazine by experimental design with regard to stable isotope dilution gas chromatography–mass spectrometric analysis of wine

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International audienceTo solve problems of sensitivity, repeatability and multi-step extraction related to 3-isobutyl-2-methoxypyrazine (IBMP) determination in wines, a simple method based on the novel combination of solid-phase microextraction and stable isotope dilution assay is presented. Among the parameters that affect this type of extraction, five of them have been optimised since the other parameters have common values or do not require optimisation (e.g. addition of sodium chloride at saturated concentration) and so were fixed. Vial volume, sample volume/vial volume ratio, pH, adsorption time and temperature have been optimised by means of two experimental designs. After extraction, quantification was performed by stable isotope dilution with gas chromatography-tandem mass spectrometry ([]-IBMP as internal standard). The final procedure allowed quantification far below IBMP’s sensory threshold (1 ng l−1 versus 15 ng l−1) with a 4% standard deviation. This method has been applied to experimental Fer servadou wines. Comparison of IBMP contents confirmed the efficiency of some viticultural and enological techniques on the herbaceous flavour decrease, such as prior fermentation maceration at high temperature (70 °C) and the use of a reflective carpet on viticultural soil

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Improving headspace-solid-phase microextraction of 3-isobutyl-2-methoxypyrazine by experimental design with regard to stable isotope dilution gas chromatography–mass spectrometric analysis of wine

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