Analysis of Rotundone in Japanese Syrah Grapes and Wines Using SBSE with Heart-Cutting Two-Dimensional GC/MS

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1 AJEV Papers in Press. Published online April 24, Research Note Analysis of Rotundone in Japanese Syrah Grapes and Wines Using SBSE with Heart-Cutting Two-Dimensional GC/MS Hideki Takase, 1,2 Kanako Sasaki, 1 Hideyuki Shinmori, 3 Akira Shinohara, 3 Chihiro Mochizuki, 3 Hironori Kobayashi, 4 Hiroshi Saito, 4 Hironori Matsuo, 4 Shunji Suzuki, 2 * and Ryoji Takata 1 1 Laboratory, New Product & Process Developments, Mercian Corporation, Jonan, Fujisawa, Kanagawa , Japan; 2 Institute of Enology and Viticulture, University of Yamanashi, Kitashin, Kofu, Yamanashi , Japan; 3 Interdisciplinary Graduate School of Medical and Engineering, University of Yamanashi, Takeda, Kofu, Yamanashi , Japan; and 4 Château Mercian, Shimoiwasaki, Katsunuma, Koshu, Yamanashi , Japan. *Corresponding author (suzukis@yamanashi.ac.jp; tel: ; fax: ) Manuscript submitted Oct 2014, Jan 2015, accepted Feb 2015 Copyright 2015 by the American Society for Enology and Viticulture. All rights reserved. Abstract: Rotundone, an oxygenated sesquiterpene, is responsible for the peppery aroma in wines. We have developed an effective and simple method to determine trace levels of rotundone in grapes and wines. The method uses stir bar sorptive extraction with heart-cutting two- dimensional gas chromatography/mass spectrometry but without a pre-extraction process, such as solid-phase extraction. The limits of detection and quantitation for rotundone were 2.1 and 6.5 ng/kg in grape, and 2.4 and 7.2 ng/l in wine. The recoveries of rotundone from Cabernet Sauvignon grape and wine were 106% (spiked with 120 ng/kg for grape) and 96% (spiked with 120 ng/l for wine), respectively. We demonstrated that rotundone concentrations in Syrah grapes and wines from Ueda were higher (up to 2342 ng/kg and 232 ng/l, respectively) than the maximal rotundone concentrations in Australian Syrah grapes and wines. Those findings indicate that some environmental factors in Japan may have a positive influence on rotundone accumulation in Syrah grapes, and underscore the importance of studying regional characteristics of and rotundone accumulation in Syrah grapes. Key words: aroma, heart-cutting two-dimensional GC/MS, rotundone, SBSE, Syrah, Vitis vinifera 1

2 Introduction Rotundone is an oxygenated sesquiterpene that is responsible for the peppery aroma in grapes, wines, herbs, and spices (Fig. 1) (Wood et al. 2008). Owing to its low detection threshold (16 ng/l in red wine and 8 ng/l in water), rotundone produces a strong aroma in wines (Wood et al. 2008). Rotundone is found in several grape varieties, particularly Syrah, Mourvèdre, and Durif from Australia (Herderich et al. 2012, Wood et al. 2008), and Schioppettino, Vespolina, and Grüner Veltliner from Europe (Caputi et al. 2011, Mattivi et al. 2011). Previous reports had suggested that higher levels of rotundone were accumulated in grapes grown in cooler vintages and regions (Caputi et al. 2011, Herderich et al. 2012). This might indicate that regional atmospheric characteristics can give a distinctive peppery aroma to wines via rotundone accumulation in grapes. On the other hand, the winemaking process for peppery aroma wines should be improved as the rotundone concentration in wines after such winemaking processes as fermentation and filtration is relatively low (Caputi et al. 2011). The determination of rotundone has been carried out using solid-phase microextraction (SPME) with gas chromatography/mass spectrometry or gas chromatography/tandem mass spectrometry (GC/MS or GC/MS/MS) systems (Caputi et al. 2011, Geffroy et al. 2014, Mattivi et al. 2011, Siebert et al. 2008). However, the pre-extraction process accomplished with a solid- phase extraction (SPE) cartridge prior to SPME requires a large amount of sample, for example approximately 100 ml of wine, to analyze trace levels of rotundone in grapes and wines. Furthermore, a turbid sample such as grape berry extract frequently clogs the SPE cartridge, resulting in waste of time. The stir bar sorptive extraction (SBSE) method was developed for the extraction and enrichment of organic compounds from aqueous samples (Baltussen et al. 1999), and it has been applied to the trace analysis of various aroma compounds in grapes and wines (Fang and 2

3 Qian 2006, Franc et al. 2009). SBSE method has some advantages compared with SPE method. It is necessary to condition the SPE cartridge with several solvents just before use, while the conditioning of stir bar is easy at any time. The samples are punctually analyzed using SBSE method, because clogging with the turbid sample is never occurred. The objective of this study was to develop an effective and simple method to determine trace levels of rotundone in grapes and wines. The method uses SBSE with heart-cutting two- dimensional GC/MS without pre-extraction by SPE. In addition, we demonstrate that Syrah grapes cultivated in Japan accumulate a relatively large amount of rotundone. Chemicals. Materials and Methods Rotundone was synthesized according to a previous report (Mattivi et al. 2011). 1 H nuclear magnetic resonance (NMR, 400 MHz) and 13 C NMR (100MHz) spectra were acquired with a Bruker AVANCE 400 spectrometer, and measurements were conducted in deuterated chloroform (CDCl 3 ) at ambient temperature. Chemical shifts were recorded in parts per million (ppm). The mass spectrum and the NMR spectra of synthesized rotundone were identical with those reported in the literature. d 5 -Rotundone was synthesized according to a previous report (Siebert et al. 2008) and identified by mass measurement. All chemicals required for the synthesis, CDCl 3 for NMR, HPLC-grade ethanol, HPLC- grade methanol, dichloromethane, acetone, n-hexane, n-pentane, ethyl acetate, and tartaric acid were purchased from Sigma-Aldrich Japan (Osaka, Japan). Milli-Q water was obtained from a Milli-Q purification system (Merck Millipore, Tokyo, Japan). 3

4 Grapes and wines. Grapevines were grown in several regions in Yamanashi and Nagano Prefectures in Japan. Vitis vinifera cvs. Koshu grapes cultivated in Nirasaki (lat. 35º41'6"N; long. 138º22'9"E; 540 m a.s.l.), hybrid grape variety Muscat Bailey A [V. labrusca (Bailey) V. vinifera (Muscat Hamburg)] grapes cultivated in Fuefuki (lat. 35º38'51"N; long. 138º41'54"E; 350 m a.s.l.), Cabernet Sauvignon and Syrah grapes cultivated in Koshu (lat. 35º39'0"N; long. 138º44'42"E; 600 m a.s.l.), and Merlot, Sauvignon Blanc, and Syrah grapes cultivated in Ueda (lat. 36º20'35"N; long. 138º18'5"E; 640 m a.s.l.) were harvested at maturity in the 2012 growing season. After destemming, the berries were immediately stored at -30ºC. Wines made from Syrah grapes were purchased from local stores in Tokyo, Kanagawa Prefecture, and Yamanashi Prefecture in Japan, and stored at 20ºC. Sample preparation. For the extraction of rotundone from grape berries, approximately 200 g of frozen grape berries was homogenized in a mill (IFM-620DG, Iwatani Co., Osaka, Japan) for 30 seconds under minimal seed breakage conditions, as described in a previous report (Siebert et al. 2008). After adding 100 μl of d 5 -rotundone (100 μg/l) as the internal standard, 10 g of the homogenized mixture was extracted with 20 ml of n-pentane/ethyl acetate (9:1) by shaking for 1 h with a shaker at room temperature. The extract was centrifuged at 3,000 rpm for 10 min. The organic solvent was carefully removed by nitrogen gas purging. The residue was dissolved in 500 μl of ethanol, and 4.5 ml of aqueous tartrate buffer (ph 3.2) was added thereafter. For the analysis of wine sample, 5 ml of wine was used as is after adding 100 μl of d 5 -rotundone (100 μg/l) as the internal standard. The prepared grape samples and wine samples were subjected to SBSE and two-dimensional GC/MS analyses. 4

5 For the analyses of general fruit components, juice was prepared by homogenizing grape berries in the mill followed by pressing to 60% of the total berry weight. Total soluble solids in juice were measured with a refractometer (Pocket PAL-1, ATAGO Co., Tokyo, Japan) and expressed as Brix. Titratable acidity (TA) was determined by adding 10 ml of distilled water to 10 ml of juice and subjecting the solution to neutralization titration using 0.1 N NaOH. TA was expressed as g tartaric acid/l. Stir bar sorptive extraction (SBSE). A stir bar coated with 24 μl of polydimethylsiloxane (PDMS, Twister, GERSTEL GmbH, Mülheim an der Ruhr, Germany) was conditioned for 60 min at 300ºC in a flow of nitrogen. The grape or wine samples were transferred to 10 ml headspace vials, and the vials were sealed with a screw cap after the addition of a stir bar. SBSE was performed at room temperature for 60 min by stirring at 1,500 rpm. The stir bar was washed with Milli-Q water, dried with lint-free paper, and introduced to a glass thermal desorption liner. The glass liner was placed in a thermal desorption unit. The stir bar was reconditioned by soaking in methanol/dichloromethane (1:1) for 24 h, dried at room temperature, and stored carefully until next use. Heart-cutting two-dimensional GC/MS analysis. GC/MS analysis was performed with a gas chromatograph (7890A GC system, Agilent Technologies, CA, USA) connected to a flame ionization detector (FID) for the first dimension detection, and an Agilent CFT Deans switch and a mass spectrometric detector (5975C inert XL MSD with Triple-Axis Detector, Agilent Technologies) for the second dimension detection. This system was equipped with a thermal desorption unit (TDU, GERSTEL GmbH), a programmed temperature vaporizing injector (CIS4, GERSTEL GmbH), and an Agilent Low Thermal Mass II Module (LTM II, Agilent Technologies). 5

6 For thermal desorption, the TDU was programmed from 40ºC (held for 0.5 min) to 300ºC (held for 3 min) at 200ºC/min with 50 ml/min desorption flow. The transfer capillary temperature was fixed at 300ºC. Desorbed compounds were trapped at 10ºC on a Tenax TA packed liner in the CIS4. After desorption, the CIS4 was programmed from 10ºC to 250ºC (held for 1 min) at 720ºC/min to inject trapped compounds onto the column in the splitless mode with the splitless time of 2 min. Separations were performed on a BP20 column (50 m 0.22 mm 0.25 μm; Trajan Scientific and Medical, Melbourne, Australia) as the first dimension column and a DB-1 column (10 m 0.18 mm 0.40 μm; Agilent Technologies) as the second dimension column with helium carrier gas at the constant flow rate of 1 ml/min. The GC oven temperature for the BP20 column was programmed from 60ºC (held for 3 min) to 240ºC (held for 17 min) at 5ºC/min, and FID was kept at 250ºC. The LTM II oven temperature for the DB-1 column was programmed from 35ºC (held for 40.8 min) to 250ºC (held for 5 min) at 20ºC/min. The injected compounds were separated by the BP20 column and the column was heart-cut from 37 to 40 min with the Deans switch. The heart-cut region was transferred to the DB-1 column connected to MS. MS measurements were performed in the scan mode and the selected ion monitoring (SIM) mode using electron ionization at 70 ev. The MS transfer line was held at 250ºC. Scan range was set from m/z 40 to 300. For SIM, the ions monitored at the dwell time of 40 ms were m/z 147, 161, 203, 208, 218, and 223. The target ions were m/z 218 for rotundone and m/z 223 for d 5 - rotundone, and other ions were used as qualifiers as described previously (Siebert et al. 2008). 6

7 Results and Discussion Determination of organic solvents for extraction of rotundone from grape samples. The extraction method of rotundone from grape samples using acetone was reported (Caputi et al. 2011). In this study, first, we compared the extraction methods of rotundone using different organic solvents, such as acetone, n-hexane, n-pentane, and n-pentane/ethyl acetate (9:1). Ten grams of homogenized Syrah grape berries was extracted for 1 h at room temperature by using different organic solvents. The peak area of the quantification ion (m/z 218) for rotundone, which was obtained by the extraction with n-pentane/ethyl acetate (9:1), was the largest among those obtained by the extraction with other organic solvents (data not shown). Consequently, we decided to use n-pentane/ethyl acetate (9:1) as the organic solvent for the efficient extraction of rotundone from grape samples in all experiments. Optimization of SBSE and heart-cutting two-dimensional GC/MS for analysis of rotundone. The principle of SBSE is based on that of SPME. SBSE is able to achieve high sensitivity because of the large amount of the PDMS phase relative to that of SPME. SBSE can be controlled by the partition coefficients of the compounds between the PDMS phase and the aqueous phase (Prieto et al. 2010). As the partition coefficient is correlated with the octanol- water distribution coefficient (K o/w ), theoretical recoveries of the compounds can be predicted from the correlation (David and Sandra 2007). The predicted log K o/w of rotundone is 4.98, as described in a previous report (Mattivi et al. 2011), and the theoretical recovery of rotundone with a stir bar coated with 24 μl of PDMS is 99.8% in a 5 ml sample volume. Next, the conditions for heart-cutting two-dimensional GC/MS were optimized to identify the target ions for rotundone and d 5 -rotundone. The heart-cutting region from 37 to 40 min for 7

8 the quantification of rotundone was determined by the first dimension GC-FID, and the identification of rotundone in the second dimension separation was performed by MS measurement in the scan mode at the retention time of min (Supplemental fig. 1). Taken together, the results suggest that SBSE is well suited to the quantitative analysis of rotundone, and that the calibration of MS in the SIM mode is effective for the quantification of rotundone. Validation of the technique. Calibration curves of rotundone were prepared by using Cabernet Sauvignon grapes and wines from which no rotundone was detected, and those were spiked with rotundone and d 5 -rotundone as the internal standard, respectively. The linearities of the calibration curves were confirmed between 10 and 3000 ng/kg of rotundone in grapes, and between 10 and 1200 ng/l of rotundone in wine. Good linearities (r 2 = in grapes and r 2 = in wine) were obtained from both samples (Table 1). The limit of detection (LOD) and the limit of quantification (LOQ) were defined by the concentration that gave the signal-to-noise ratios (S/N) of 3:1 and 10:1, respectively, and were calculated for Cabernet Sauvignon grapes or wines spiked with 10 ng/kg or 10 ng/l of rotundone, respectively. LOD and LOQ were 2.1 and 6.5 ng/kg in grapes, and 2.4 and 7.2 ng/l in wine, respectively (Table 1). Those levels were similar to the values reported in previous studies (Mattivi et al. 2011, Caputi et al. 2011), suggesting the sensitivity of our method. The recoveries of rotundone from Cabernet Sauvignon grapes spiked with 120 ng/kg of rotundone and wine spiked with 120 ng/l of rotundone were 106 and 96%, and the CV values (n=3) were and 0.017, respectively. 8

9 Quantitative analysis of rotundone in grapes cultivated in Japan. Rotundone concentrations of six grape varieties, including Syrah grapes, cultivated in different regions in Japan, were measured using our method. Rotundone concentrations of 2,342 and 1,033 ng/kg were detected in Syrah grapes from Ueda and Koshu, respectively. Rotundone concentrations in Syrah grapes from those two regions were markedly higher than those in the other grape varieties (Table 2). This result supports the previous finding that Syrah is one of the V. vinifera cultivars that have a high concentration of rotundone (Wood et al. 2008). Compared to Koshu, Ueda is a cool region because of its latitudinal position and geographic factors. The climate difference between Ueda and Koshu might influence the accumulation of rotundone in Syrah grapes, as previous reports demonstrated that higher levels of rotundone were accumulated in grapes from cooler regions (Caputi et al. 2011, Herderich et al. 2012). In addition, Japanese Syrah grapes, particularly Syrah grapes from Ueda, had higher rotundone concentration than the maximal concentration (1082 ng/kg) reported in Australian Syrah grapes (Scarlett et al. 2014). Those findings indicate that some environmental factors in Japan may have a positive influence on rotundone accumulation in Syrah grapes. It was recently suggested that rotundone biosynthesis was affected by regional factors, such as soil properties and topography (Scarlett et al. 2014), and soil moisture by irrigation (Geffroy et al. 2014). Further investigations are required to elucidate the environmental factors affecting rotundone biosynthesis in Japan. Comparative analysis of rotundone concentration in Syrah wines. Rotundone concentrations in commercial Syrah wines were quantified by our method (Table 3). In addition to wines made from Syrah grapes cultivated in Ueda, wines from famous Syrah wine producing appellations, such as Côte-Rôtie, Crozes-Hermitage, and Saint-Joseph in France were selected. The concentrations of rotundone in the Syrah wines ranged from 39 to 232 ng/l, and 9

10 were higher than the detection threshold of rotundone, 16 ng/l (Wood et al. 2008). Interestingly, the highest concentration of rotundone (232 ng/l) was detected in Japanese Syrah wine from Ueda (2009 vintage). It was higher than the maximal concentration in Australian Syrah wines (161 ng/l) reported previously (Herderich et al. 2012). This finding supported our hypothesis that some environmental factors in Japan impact rotundone concentration in Syrah grapes. On the other hand, the concentrations of rotundone in Japanese Syrah wines were apparently lower than those in Syrah grapes cultivated in Japan. Not all of rotundone was extracted from grapes during the winemaking process (Caputi et al. 2011). Further enological studies aimed at improving the yield of rotundone from grapes to wine are required for making Syrah wines with a peppery aroma. Conclusion An effective, sensitive, and simple method to determine rotundone concentrations in grapes and wines was developed, which uses SBSE with heart-cutting two-dimensional GC/MS. This method could be punctually applied to the quantitative analysis of rotundone from small amounts of samples, for example 5 ml of wine, without a pre-extraction process. The high concentrations of rotundone in Japanese Syrah grapes and wines indicate that some environmental factors in Japan may have a positive influence on rotundone accumulation in Syrah grapes, and underscore the importance of investigating regional characteristics to sustain the high quality of Syrah grapes. Further studies to understand the mechanism of rotundone biosynthesis in Syrah grapes and control rotundone concentration in Syrah wines may contribute to make a characteristic Syrah wine. 10

11 Literature Cited Baltussen, E., P. Sandra, F. David, and C. Cramers Stir bar sorptive extraction (SBSE), a novel extraction technique for aqueous samples: Theory and principles. J. Microcolumn Sep. 11: Caputi, L., S. Carlin, I. Ghiglieno, M. Stefanini, L. Valenti, U. Vrhovsek, and F. Mattivi Relationship of changes in rotundone content during grape ripening and winemaking to manipulation of the 'peppery' character of wine. J. Agric. Food Chem. 59: David, F., and P. Sandra Stir bar sorptive extraction for trace analysis. J. Chromatogr., A 1152: Fang, Y., and M.C. Qian Quantification of selected aroma-active compounds in Pinot noir wines from different grape maturities. J. Agric. Food Chem. 54: Franc, C., F. David, and G. de Revel Multi-residue off-flavour profiling in wine using stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry. J. Chromatogr., A 1216: Geffroy, O., T. Dufourcq, D. Carcenac, T.E. Siebert, M. Herderich, and E. Serrano Effect of ripeness and viticultural techniques on the rotundone concentration in red wine made from Vitis vinifera L. cv. Duras. Aust. J. Grape Wine Res. 20: Herderich, M.J., T.E. Siebert, M. Parker, D.L. Capone, D.W. Jeffery, P. Osidacz, and I.L. Fransis Spice UP Your Life: Analysis of Key Aroma Compounds in Shiraz. In Flavor Chemistry of Wine and Other Alcoholic Beverages. M.C. Qian and T.H. Shellhammer (eds.), pp American Chemical Society, Washington, DC. Mattivi, F., L. Caputi, S. Carlin, T. Lanza, M. Minozzi, D. Nanni, L. Valenti, and U. Vrhovsek Effective analysis of rotundone at below-threshold levels in red and white wines using solidphase microextraction gas chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 25: Scarlett, N.J., R.G.V. Bramley, and T.E. Siebert Within-vineyard variation in the pepper compound rotundone is spatially structured and related to variation in the land underlying the vineyard. Aust. J. Grape Wine Res. 20: Prieto, A., O. Basauri, R. Rodil, A. Usobiaga, L.A. Fernandez, N. Etxebarria, and O. Zuloaga Stirbar sorptive extraction: A view on method optimisation, novel applications, limitations and potential solutions. J. Chromatogr., A 1217: Siebert, T.E., C. Wood, G.M. Elsey, and A.P. Pollnitz Determination of rotundone, the pepper aroma impact compound, in grapes and wine. J. Agric. Food Chem. 56: Wood, C., et al From wine to pepper: rotundone, an obscure sesquiterpene, is a potent spicy aroma compound. J. Agric. Food Chem. 56:

12 Table 1 Linearity, LOD, and LOQ of rotundone in Cabernet Sauvignon grape and wine. Sample Linear range (ppt a ) Slope Intercept r 2 LOD (ppt a ) LOQ (ppt a ) Grape Wine a ppt (parts per trillion) means the unit of rotundone concentration expressed as ng/kg of grape or ng/l of wine. Table 2 Berry parameters and rotundone concentration in various grapes cultivated in Japan. Nirasaki Fuefuki Koshu Ueda Region Variety Koshu Muscat Bailey A Cabernet Sauvignon Syrah Sauvignon Blanc Merlot Syrah Rotundone (ng/kg) a 60 ± ± 1 21 ± ± ± 2 62 ± ± 149 ºBrix ph TA (g/l) a Data of rotundone concentration are shown as means ± standard deviation of triplicate experiments. Table 3 Rotundone concentrations in Syrah wines from different regions. Country Region/Appellation Vintage Rotundone (ng/l) a France Côte-Rôtie ± 5 Côte-Rôtie ± 11 Côte-Rôtie ± 8 Côte-Rôtie ± 7 Crozes-Hermitage ± 4 Saint-Joseph ± 11 Japan Ueda ± 12 Ueda ± 10 Ueda ± 4 a Data of rotundone concentration are shown as means ± standard deviation of triplicate experiments. 12

13 Figure 1 Chemical structure of ( )-rotundone. Supplemental Figure 1 Two-dimensional chromatogram of rotundone. Six hundred ng/l of rotundone was spiked in Cabernet Sauvignon wine. (A) SIM mode (m/z 218). (B) The mass spectrum of rotundone obtained in the scan mode at the retention time of min. 13