2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype
Introduction
2-Methoxy-3-isobutylpyrazine (MIBP) (1), 2-methoxy-3-isopropylpyrazine (MIPP), and 2-methoxy-3-sec-butylpyrazine (MPs-B) were reported in Freon extracts of Vitis vinifera L. Sauvignon blanc fruit (Augustyn et al., 1982) and confirmed using gas chromatography–mass spectrometry (GC–MS) following distillation and extraction of wines (Harris et al., 1987). Of these three methoxypyrazines, MIBP (1) (Fig. 1), which has a bell pepper aroma, is considered the most important because of its very low aroma threshold (2 ng L−1 in water) (Buttery et al., 1969) and relatively high concentration in grapes and wines (e.g., Lacey et al., 1991). Augustyn et al. (1982) proposed that MIBP (1) was key to the characteristic ‘asparagus-like, green, grassy, bell pepper-like’ aroma of Sauvignon blanc wines, and they cited Bayonove et al. (1975) as having made a similar suggestion – that MIBP (1) was responsible for the characteristic ‘green note’ in Cabernet Sauvignon grapes and wines. Since that early work, methods for quantifying volatiles have improved considerably, and the importance of understanding factors that impact MIBP levels in both Sauvignon blanc and in Cabernet-type wines has increased. In Cabernet Sauvignon, a high MIBP (1) concentration in grapes may have a negative impact on wine aroma quality (Allen and Lacey, 1999).
Headspace solid phase microextraction (HS-SPME) combined with GC–MS is widely used for analysis of volatiles in food and beverage samples because it is rapid and easily automated (Ebeler, 2001, Pawliszyn, 1997). Chapman et al. (2004) developed a HS-SPME-GC–MS method for analysis of MIBP (1) in wines with an accuracy of >95%, relative standard deviation (RSD) of <12%, and a limit of quantitation (LOQ) of 5 ng L−1; however, the method was not validated in a grape or juice matrix. Similar approaches to quantifying MIBP (1) in grape berries have been described but they required long extraction times (Belancic and Agosin, 2007, Sala et al., 2000) or lacked sufficient sensitivity (Hartmann et al., 2002) for our application. Recently, Ryona et al., 2008, Ryona et al., 2009 measured MIBP (1) in pulverized Cabernet franc grapes using HS-SPME combined with two-dimensional comprehensive gas chromatography coupled to a time-of-flight mass spectrometer (GC × GC-TOF MS). The GC × GC analysis improved separation from matrix interferences (Ryan et al., 2005, Ryona et al., 2008, Ryona et al., 2009), however, absolute recoveries for the method were not reported. Use of MS for detection allows stable isotope labeled internal standards to be used which can significantly improve the accuracy and precision of the MIBP (1) analysis compared to use of a chemically similar, but not identical internal standard (Allen et al., 1994). However, for previous MIBP (1) analyses of grape berries, the internal standard was added after the berries had been homogenized and diluted (Belancic and Agosin, 2007, Ryona et al., 2008, Ryona et al., 2009), so analyte recovery losses during these steps could not be accounted for.
Here, we describe a HS-SPME-GC–MS method for MIBP (1) analysis in grapes; we evaluated the effects of different grape sample preparation conditions and the effects of grape composition (soluble solids and pH) on the accuracy and precision of the method. The method was then used to survey 29 cultivars for the presence of MIBP (1) in fruit and to evaluate whether it is translocated from leaves to fruit using the reciprocal grafting technique of Gholami et al. (1995) who demonstrated that monoterpenes are not translocated from leaves to berries.
Section snippets
Grape sample preparation
During initial method validation, sample preparation methods were compared using either frozen or thawed whole berries and skins only from frozen or thawed berries. The peak area of the MIBP (1) quantification ion (m/z = 124) was used to compare the different sample preparation methods for their ability to release MIBP (1) from the grape berries and skins (data not shown). Measurable differences in its amount were not observed in the headspace of the supernatants obtained with the different
Discussion
In this study, a method for extracting and measuring MIBP (1) concentration in whole berries throughout maturation was developed that includes use of standard additions to eliminate unacceptable noise in the MIBP (1) response curves in unripe fruit. The method was used to evaluate 29 cultivars for the presence of MIBP (1) in unripe and ripe fruit, and to show that the fruit genotype, and not the shoot genotype, determines the presence or absence of MIBP (1) in ripe fruit.
Concluding remarks
We developed a method for analysis of MIBP (1) in whole grape berries that is accurate (spiked recoveries of >91%), reproducible (RSD <10%), and sensitive (LOQ = 2 ng L−1). However, the grape matrix, particularly for pre-véraison and véraison samples, can influence the accuracy and precision of the analysis, therefore, the method of standard additions is recommended for quantification. Reciprocal grafting experiments demonstrated that MIBP (1) is produced in the berries of Cabernet Sauvignon and is
General experimental procedures
MIBP (99% pure; Fig. 1) for preparation of standards was purchased from Sigma Chemical Co. (St. Louis, MO). The internal standard 2-(2D3)-methoxy-3-isobutylpyrazine (dMIBP1a; Fig. 1) was obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada, 98% atom% D). SPME fibers (23 gauge, 2 cm divinylbenzene/Carboxen™/polydimethylsiloxane (DVB/CARB/PDMS)) were purchased from Supelco, Bellafonte, PA. An Agilent 6890 GC with a 5973MSD (Agilent, Santa Clara, CA) and Gerstel MPS2 autosampler (Gerstel Inc.,
Acknowledgements
This work was supported with funds from the American Vineyard Foundation and the Viticulture Consortium.
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