Stable Isotope Dilution Analysis Research Articles

Characterization of important odorants from Persian silk tree flowers, Albizia julibrissin

AbstractThe Persian silk tree (Albizia julibrissin) has delicate flowers with a pleasant aroma profile, displaying notes of hyacinth and rose with subtle nuances of citrus, coconut and spice. In this study, a total of 29 odorants were identified in the flowers from a single A. julibrissin selection by the application of a cold‐solvent extraction (CSE), followed by solvent‐assisted flavour evaporation (SAFE) and aroma extract dilution analysis (AEDA). Subsequently, nine odorants with flavour dilution (FD) factors ≥16 were quantitated by stable isotope dilution analysis (SIDA), and odour activity values (OAVs) were calculated. Odorants with OAV ≥1 included 2‐phenylacetaldehyde (OAV 3638), linalool (OAV 797), eugenol (OAV 231), geraniol (OAV 161), 1H‐indole (OAV 93), β‐ionone (OAV 31), 2‐phenylethanol (OAV 11) and γ‐octalactone (OAV 1). An aroma simulation model was prepared based on the quantitation data, and it closely matched the aroma of the flower isolate. Chiral chromatography was performed, and (S)‐(+)‐linalool was the only enantiomer detected. This study advances our understanding of A. julibrissin flower aroma chemistry and lays the groundwork for future investigations for the development of delicate floral aromas for flavour and fragrance applications.

Characterization of Odorants in a Commercial Culinary Sage (Salvia officinalis L.) and Several Cultivars.

Culinary sage, Salvia officinalis L., is a popular spice plant commonly used throughout the world. In this study, 35 odorants were identified in dried sage via solvent-assisted flavor evaporation (SAFE) and aroma extract dilution analysis (AEDA), including 9 that were identified in sage for the first time. Fifteen odorants were quantitated by stable isotope dilution analysis (SIDA), and their odor activity values (OAVs) were determined. Odorants with high OAVs included (2E,6Z)-nona-2,6-dienal, 1,8-cineole, and β-myrcene. A formulated aroma simulation model closely matched the aroma profile of an aqueous infusion of dried sage. Enantiomeric proportions of selected odorants were determined by chiral gas chromatography. Furthermore, 6 different sage cultivars were grown in the greenhouse, dried under the same conditions, and analyzed. Sensory analysis determined that all cultivars were dominated by an herbaceous sensory attribute and had varying intensities of eucalyptus, mint, clove, pine, green, earthy, floral, and citrus notes. Cultivars with varying intensities of herbaceous, eucalyptus, pine, and green sensory notes correlated with the OAVs of α-thujone/β-thujone, 1,8-cineole, α-pinene, and (2E,6Z)-nona-2,6-dienal, respectively. This study identified the odorants driving the sensory profiles of different sage cultivars and serves as a foundation for future studies on the aroma chemistry of culinary sage.

Distribution and transition of aroma-active compounds in dark chocolate model systems under conching conditions

Understanding mechanisms inside the conche is inevitable for targeted conching. Therefore, distribution and transition of aroma-active volatiles (acetic acid, benzaldehyde, (R,S)-(±)-linalool, 2,3,5,6-tetramethylpyrazine, 2-phenylethanol, and 2-phenylethyl acetate) between the different components of dark chocolate (cocoa butter, cocoa particles, sugar particles) were studied. Different model systems were designed and aroma-analytically analyzed by stable isotope dilution analysis. Diffusion mechanisms of selected aroma-active compounds within the chocolate mass depended on their physico-chemical properties and the mass composition, such as fat content and crystallization state of the sugar particles. The compound accumulation in the fat phase increased with decreasing compound polarity and increasing fat content. In the presence of cocoa particles, a 1.5-fold fat content resulted in a 1.6-fold higher proportional acetic acid concentration in the fat phase. Further, total acetic acid concentrations raised in all model systems containing crystalline sugar or cocoa particles (by 13.8–56.9 %), indicating the formation of free acetic acid.

Identification of ethyl vanillin in strawberry (Fragaria × ananassa) using a targeted metabolomics strategy: From artificial to natural

Improving flavor can be an important goal of strawberry through breeding that is enhanced through the accurate identification and quantification of flavor compounds. Herein, a targeted metabolomics strategy was developed using liquid–liquid extraction, an in-house standard database, and GC–MS/MS analysis. The database consisted of key food odorants (KFOs), artificial flavor compounds (AFCs) and volatiles. A total of 131 flavor compounds were accurately identified in Medallion® ‘FL 16.30-128′ strawberry. Importantly, ethyl vanillin was identified for the first time in natural food. Multiple techniques, including GC–MS, GC–MS/MS and UPLC-MS/MS were applied to ensure the identification. The ethyl vanillin in the Medallion® samples were determined in a range of concentrations from 0.070 ± 0.0006 µg/kg to 0.1372 ± 0.0014 µg/kg by using stable isotope dilution analysis. The identification of ethyl vanillin in strawberry implys the future commercial use a natural flavor compound and the potential to identify genes and proteins associated with its biosynthesis.

Characterizing and Decoding the Effects of Different Fermentation Levels on Key Aroma Substances of Congou Black Tea by Sensomics.

Fermentation is the key technology for black tea aroma formation. The key aroma substances of black tea at different fermentation stages (unfermented (WDY), underfermented (F1H), fully fermented (F4H), and overfermented (F8H)) were characterized by the methodology of Sensomics. Aroma extract dilution analysis was performed on volatile fractions extracted by using solvent-assisted flavor evaporation and solid-phase microextraction, yielding 93 odor-active areas. Internal standard method plus stable isotope dilution analysis was used for quantitative analysis. The omission experiment identified 23 aroma substances. Further reduction and addition experiments revealed phenylacetaldehyde, (E,E)-2,4-heptadienal, geraniol, linalool, β-damascenone, 2-methylbutyraldehyde, dimethyl sulfide, and isovaleraldehyde with odor activity values (OAV) > 100 as the characteristic aroma components of F4H and also as the main contributors to aroma differences between different fermentation degrees. The green odor of (E,E)-2,4-heptadienal was highlighted in WDY and F1H relative to that in F4H due to the lower contribution of phenylacetaldehyde and β-damascenone in the former two samples. Additionally, excessive OAV increase of fatty aldehydes in F8H masked its similar floral and fruity aroma.

Stable Isotope Dilution Analysis (SIDA) to Determine Metabolites of Furan and 2-Methylfuran in Human Urine Samples: A Pilot Study.

Furan and 2-methylfuran (2-MF) are food contaminants that are classified as potentially carcinogenic to humans. The main source of exposure for adults via food is coffee consumption. Furan and 2-MF are volatile, which complicates exposure assessment because their content measured in food prior to consumption does not afford a reliable dosimetry. Therefore, other ways of exposure assessment need to be developed, preferably by monitoring exposure biomarkers, e.g., selected metabolites excreted in urine. In this study, cis-2-buten-1,4-dial (BDA)-derived urinary furan metabolites Lys-BDA (l-2-amino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)hexanoic acid), AcLys-BDA (l-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)hexanoic acid) and GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinyl-glycine cyclic sulfide), as well as acetyl acrolein (AcA, 2-oxo-pent-2-enal)-derived metabolites Lys-AcA (l-2-(acetylamino)-6-(2,5-dihydro-5-methyl-2-oxo-1H-pyrrol-1-yl)-hexanoic acid) and AcLys-AcA (l-2-amino-6-(2,5-dihydro-5-methyl-2-oxo-1H-pyrrol-1-yl)-hexanoic acid) and their stable isotopically labeled analogs, were synthesized and characterized through NMR and MS, and a stable isotope dilution analysis (SIDA) with UPLC-ESI-MS/MS was established. As a proof of concept, urinary samples of a four-day human intervention study were used. In the frame of this study, ten subjects ingested 500 mL of coffee containing 0.648 µmol furan and 1.059 µmol 2-MF. Among the furan metabolites, AcLys-BDA was the most abundant, followed by Lys-BDA and GSH-BDA. Exposure to 2-MF via the coffee brew led to the formation of Lys-AcA and AcLys-AcA. Within 24 h, 89.1% of the ingested amount of furan and 15.4% of the ingested amount of 2-MF were detected in the urine in the form of the investigated metabolites. Therefore, GSH-BDA, Lys-BDA, AcLys-BDA, Lys-AcA and AcLys-AcA may be suitable as short-term-exposure biomarkers of furan and 2-MF exposure.

Identification of Important Aroma Components and Sensory Profiles of Minimally Processed (Unroasted) and Conventionally Roasted Dark Chocolates.

Roasting is an important unit operation for the development of characteristic chocolate aroma during manufacturing. However, there is an increase in interest in minimally processed chocolate products due to their potential positive health benefits. The odor-important compounds and sensory characteristics of minimally processed (unroasted) and conventionally roasted dark chocolates were determined by gas chromatography-olfactometry, aroma extract dilution analysis (AEDA), and stable isotope dilution analysis (SIDA). Except for acetic acid, all odorants had higher odor-activity values (OAVs) in roasted chocolate. Acetic acid, developed during fermentation and drying, had the highest OAV in both chocolates but was better preserved in unroasted chocolate. Compounds making a greater aroma impact on roasted chocolate compared with unroasted chocolate included dimethyl trisulfide, 2-ethyl-3,5-dimethylpyrazine, and 3-methylbutanal. Nine significant sensory attributes in unroasted and roasted chocolates were identified. Vinegar (aroma) and roasted (aroma and aroma by mouth), sweet (taste), and hardness (texture) attributes differed between unroasted and roasted chocolates. The results of this study enforce the embracement of low thermal processes to showcase the inherent flavor potential of cacao beans but also to support the concept of chocolate "terroir" by potentially preserving important aroma compounds developed during fermentation.

Toward High-Throughput Analysis of Aroma Compounds Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry: Screening of Key Food Odorants in Various Foods.

Recent studies show the immense capacities of the unified quantitation of aroma and taste compounds using liquid chromatography-mass spectrometry (LC-MS). The goal of this study was to highlight the broad application of this unified method. Thus, a stable isotope dilution analysis quantification method of the most important key food odorants in various food categories by LC-MS was developed. Using the well-known derivatization agent 3-nitrophenylhydrazine for carbonyl derivatization and a newly developed approach for alcohol and thiol derivatization, a method for the quantitation of 20 key food odorants was established. Intraday precision was determined to be ≤26%, and interday precision was between 24 and 31%. Limits of quantitation were determined between 0.014 and 283 μg/kg. The work shows that a wide array of aroma compounds can be analyzed accurately by LC-MS.

Quantification of Nτ -Methylhistidine and Nπ-Methylhistidine in Chicken Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

The concentration of Nτ-methylhistidine in plasma provides an index of skeletal muscle protein breakdown. This study aimed to establish a quantitative method for measuring the concentrations of Nτ-methylhistidine and its isomer Nπ-methylhistidine in chicken plasma, using liquid chromatography-tandem mass spectrometry with stable isotope dilution analysis. The acceptable linear ranges of detection were 1.56-50.00 μmol/L for Nτ-methylhistidine and 0.78-25.00 μmol/L for Nπ-methylhistidine. The proposed method detected changes in the plasma levels of Nτ-methylhistidine and Nπ-methylhistidine in response to fasting and re-feeding. These results suggest that the method developed in this study can be used for the simultaneous measurement of Nτ-methylhistidine and Nπ-methylhistidine in chicken plasma.

Formation of furanoic compounds in model systems with saccharides, amino acids, and fatty acids, analyzed with headspace-solid phase micro-extraction-gas chromatography–tandem mass spectrometry

Furan and furan derivatives are processing contaminants found in thermally processed food, such as coffee or canned products. For the determination of furan and ten derivatives, a sensitive headspace-solid phase micro-extraction-gas chromatography–tandem mass spectrometry method with stable isotope dilution analysis was developed. Different precursors such as saccharides, saccharide/amino acid mixtures, polyunsaturated fatty acids (PUFA), and oils were heat processed to investigate the formation of furanoic compounds via Maillard reaction and lipid oxidation. Therefore, a low moister (silica gel with 10% water content) and an aqueous (phosphate buffer, pH 4.5, 7, and 9) model system were chosen to study the effect of water content and pH value on the formation process. The formation of different furan derivatives could be attributed to distinct precursors; furfuryl alcohol was highly formed from 1,4-linked disaccharides, whereas PUFA formed the highest amounts of 2-ethylfuran and 2-pentylfuran.

Occurrence of glycidyl esters in infant formula products on the Canadian market between 2015 and 2019

Glycidyl fatty acid esters (GEs) are processing contaminants formed during refining steps of vegetable oils. ‘In vivo’ hydrolysis of GEs releases potentially carcinogenic and genotoxic glycidol (2,3-epoxy-1-propanol). Occurrence of GEs in vegetable oils used for infant formula manufacturing may pose a potential health concern for formula-fed infants. Refined oils are commonly used as the main fat ingredient in formula manufacturing. For this study, different infant formula products (powders, concentrates and ready-to-feed formula products) were purchased and analysed in 2015 (35 samples) and 2019 (33 samples). Seven individual GEs were analysed by LC-MS/MS via direct approach by stable isotope dilution analysis, and total bound glycidol concentrations were calculated. Concentrations of bound glycidol in reconstituted formula reached maxima of 40.3 ng/g in the 2015 samples and 31.5 ng/g in the samples collected in 2019, with respective means of 8.7 ng/g and 6.7 ng/g. The analysed bound glycidol concentrations are comparable with concentration ranges from other studies, but are higher than observed in studies from the European market. Temporal trend data show a reduction of bound glycidol concentrations in 2019. GE concentrations were compared across different manufacturers.

Aroma-active volatiles and rheological characteristics of the plastic mass during conching of dark chocolate

Chocolate conching is a highly complex, thermomechanical process that transforms the aroma and flow properties of a dry starting material. Different conched plastic masses of dark chocolate were characterized. Rheological characterization of plastic masses was performed for the first time using a closed cavity rheometer (CCR11CCR: Closed cavity rheometer.). In free cocoa butter derived from the plastic masses, acetic acid, benzaldehyde, (R,S)-(±)-linalool, 2,3,5,6-tetramethylpyrazine, and 2-phenylethanol were quantified by stable isotope dilution analysis (SIDA22SIDA: stable isotope dilution analysis.) and gas chromatography-mass spectrometry. During the conching process, the amount of free cocoa butter increased possibly due to de-agglomeration. The complex viscosity of the plastic mass decreased as a function of conching time. Regarding aroma refinement, the concentrations of all five aroma-active volatiles decreased with increasing conching duration, albeit to varying degrees. The level of acetic acid showed the most pronounced decrease of about 60%, whereas linalool exhibited the lowest decrease in concentration, up to 26%. Overall, a lower polarity or boiling point of the aroma-active volatiles was linked to a stronger decrease in concentration during conching. These data illustrate the influence of conching on texture and the respective aroma changes, which deepens understanding of the conching effect on the sensory quality of dark chocolate.

Metabolic folate profiling as a function of time during cultivation suggests potential C2-metabolism in Saccharomyces cerevisiae

Yeasts are reported to be rich in folates, a group of vitamers known to be involved in several biosynthetic reactions such as methylation reactions, oxidation and reduction processes, and nucleotide synthesis. Not being able to synthesize folates, humans rely on external folate supply. Here, we show the application of LC/MS-MS methods using SIDA (stable isotope dilution analysis) assays for the quantitative analysis of different folate mono- and polyglutamates during growth of Saccharomyces cerevisiae. Molecular networking (MN) was applied for detailed analysis of further folate metabolites. Highest folate contents of 13,120 μg/100 g were observed after 20 h of cultivation. The main vitamers 5-CH3-H4folate and H4folate decreased during cultivation, while 5-CHO-H4folate increased during cultivation. The hexa- and heptaglutamate of 5-CH3-H4folate accounted for >96% of the total 5-CH3-H4folate content. A shift of the major polyglutamate from hexa- to heptaglutamate was observed after 29 h. MN unraveled two groups of novel folates which could be assigned to a potentially existing C2-metabolism in yeast. In detail, 5,10-ethenyl-tetrahydrofolate and a further CO-substituted 5-CH3-H4folate were identified as hexa- and heptaglutamates. The latter was neither identified as 5-acetyl-tetrahydrofolate nor as EthylFox, the oxidation product of 5-ethyl-tetrahydrofolate. The structure needs to be elucidated in future studies.

Characterization of the key odorants of ripe Capparis spinosa L. fruit and changes in concentrations after storage by means of molecular sensory science

To characterize the key odorants in ripe Capparis spinosa L. fruit (RCF) and to investigate changes in the odorants after storage, volatiles were captured by both solid-phase microextraction and solvent extraction combined with solvent-assisted flavor evaporation; a total of 22 aroma-active regions were detected in the isolates by gas chromatography–olfactometry. Of 19 odor-active compounds identified positively, 13 odorants were found in RCF for the first time; their flavor dilution factors ranging from 1 to 512 were measured by an aroma extract dilution assay. Eleven odorants were determined as the key odorants by stable isotope dilution analysis, odor activity value (OAV) calculation, and recombination experiments. To further investigate the changes in odorants after storage, their concentrations and OAVs were determined. The results showed that the number of key odorants did not change before and after storage, but the concentrations of octanoic acid, sotolon, and 3-methylbutanoic acid increased markedly.

New Mass Spectrometric Approach to Quantify the Major Isoallergens of the Apple Allergen Mal d 1.

Patients who suffer from birch pollinosis often develop adverse reactions to the consumption of fresh apples due to the structural similarity of the allergens Bet v 1 and Mal d 1 from birch and apples, respectively. A different allergenic potential for Mal d 1 isoallergens is postulated, but approaches to quantify the Mal d 1 isoallergen-specific are missing. Therefore, a bottom-up proteomics approach was developed to quantify Mal d 1 by stable isotope dilution and microHPLC-QTOF analyses. Marker peptides for individual isoallergens (Mal d 1.01-Mal d 1.03 and Mal d 1.06), combinations thereof (Mal d 1.01 + 1.02, Mal d 1.02 + 1.06, and Mal d 1.04 + 1.05), and two global marker peptides, comprising Mal d 1.01 + 1.02 + 1.04 + 1.05 and Mal d 1.03 + 1.06 + 1.07 + 1.08 + 1.09, were identified. By the use of an extraction standard (r-Mal d 1_mut), an optimized protocol for extraction and tryptic digestion of apple proteins was developed, and the variety-specific extraction efficiency was monitored for the flesh and peel of apples. The Mal d 1 contents in flesh and peel of five commercial apple breeds and four apple varieties from orchard meadows were quantified isoallergen-specific.

The Sensory-Directed Elucidation of the Key Tastants and Odorants in Sourdough Bread Crumb.

Sourdough bread is highly enjoyed for its exceptional flavor. In contrast to bread crust, which has been investigated intensively, the knowledge on bread crumb is rather fragmentary. In this study, the taste-active compounds of sourdough bread crumb were identified and quantified. By means of recombination experiments and omission tests, the authentic flavor signature of sourdough rye bread crumb was decoded and recreated with ten key tastants and eleven key odorants. Based on the final taste and aroma recombinants, a fast and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method using stable isotope dilution analysis (SIDA) was developed and validated. Due to prior derivatization using 3-nitrophenylhydrazine (3-NPH), key tastants and odorants in bread crumb could be quantified simultaneously in a single UHPLC run. The identified key flavor compounds in combination with the developed UHPLC-MS/MS method could offer the scientific basis for a knowledge-based optimization of the taste and odor of sourdough bread.

Studies about the Dietary Impact on "Free" Glycation Compounds in Human Saliva.

Glycation reactions play a key role in post-translational modifications of amino acids in food proteins. Questions have arisen about a possible pathophysiological role of dietary glycation compounds. Several studies assessed the metabolic fate of dietary glycation compounds into blood and urine, but studies about saliva are rare. We investigated here the dietary impact on salivary concentrations of the individual Maillard reaction products (MRPs) N-ε-fructosyllysine, N-ε-carboxymethyllysine (CML), N-ε-carboxyethyllysine (CEL), pyrraline (Pyr), and methylglyoxal-derived hydroimidazolone 1 (MG-H1). Quantitation was performed using stable isotope dilution analysis (LC-MS/MS). We describe here, that a low MRP diet causes a significant lowering of salivary levels of Pyr from 1.9 ± 0.4 ng/mL to below the LOD and MG-H1 from 2.5 ± 1.5 ng/mL to 0.7 ± 1.8 ng/mL. An impact on the salivary protein fraction was not observed. Furthermore, salivary Pyr and MG-H1 levels are modified in a time-dependent manner after a dietary intervention containing 1.2 mg Pyr and 4.7 mg MG-H1. An increase in mean salivary concentrations to 1.4 ng/mL Pyr and 4.2 ng/mL MG-H1 was observed within 30–210 min. In conclusion, saliva may be a useful tool for monitoring glycation compound levels by using Pyr and MG-H1 as biomarkers for intake of heated food.

AGEomics Biomarkers and Machine Learning-Realizing the Potential of Protein Glycation in Clinical Diagnostics.

Protein damage by glycation, oxidation and nitration is a continuous process in the physiological system caused by reactive metabolites associated with dicarbonyl stress, oxidative stress and nitrative stress, respectively. The term AGEomics is defined as multiplexed quantitation of spontaneous modification of proteins damage and other usually low-level modifications associated with a change of structure and function—for example, citrullination and transglutamination. The method of quantitation is stable isotopic dilution analysis liquid chromatography—tandem mass spectrometry (LC-MS/MS). This provides robust quantitation of normal and damaged or modified amino acids concurrently. AGEomics biomarkers have been used in diagnostic algorithms using machine learning methods. In this review, I describe the utility of AGEomics biomarkers and provide evidence why these are close to the phenotype of a condition or disease compared to other metabolites and metabolomic approaches and how to train and test algorithms for clinical diagnostic and screening applications with high accuracy, sensitivity and specificity using machine learning approaches.

Development of analytical methods to study the effect of malting on levels of free and modified forms of Alternaria mycotoxins in barley

A liquid chromatography tandem mass spectrometry (LC–MS/MS) multi-mycotoxin method was developed for the analysis of the Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), altertoxin I (ATX I), altertoxin II (ATX II), alterperylenol (ALTP), and altenuene (ALT), as well as the modified toxins AOH-3-glucoside (AOH-3-G), AOH-9-glucoside (AOH-9-G), AME-3-glucoside (AME-3-G), AOH-3-sulfate (AOH-3-S), and AME-3-sulfate (AME-3-S) in barley and malt. The toxin tenuazonic acid (TeA) was analyzed separately as it could not be included into the multi-mycotoxin method. Quantitation was conducted by using a combination of stable isotope dilution analysis (SIDA) for AOH, AME, and TeA, and matrix-matched calibration for all other toxins. Limits of detection were between 0.05 µg/kg (AME) and 2.45 µg/kg (ALT), whereas limits of quantitation ranged from 0.16 µg/kg (AME) to 8.75 µg/kg (ALT). Recoveries between 96 and 107% were obtained for the analytes when SIDA was applied, while recoveries between 84 and 112% were found for analytes quantified by matrix-matched calibration. The method was applied for the analysis of 50 barley samples and their respective malts from the harvest years 2016–2020 for their mycotoxin content, showing the overall potential of toxin formation during the malting process. The toxins ALTP and ATX I were mainly found in the malt samples, but not in barley.

Evidence that methoxypyrazine accumulation is elevated in Shiraz rachis grown on Ramsey rootstock, increasing ‘green’ flavour in wine

Background and Aims ‘Green’ sensory characters in Shiraz wines were observed in wines produced from different rootstocks. The compounds responsible were investigated as these attributes are generally seen as negative. Methods and Results GC/MS-olfactometry and stable isotope dilution analysis methods were used to identify and quantify a range of volatile compounds associated with ‘green’ sensory characters in Shiraz wine made from grapes grafted on Ramsey and Dog Ridge rootstocks. These wines were higher in this character compared to wines from Merbein 6262, a low vigour rootstock. GC-olfactometry identified the compound responsible for this intense ‘green’ aroma to be 3-isobutyl-2-methoxypyrazine, which was not detected in Shiraz grape berries but was found at significantly higher concentration in rachis tissue from bunches grown on Ramsey rootstock compared to own-roots. Conclusions Grape rachis inclusion is the most probable cause of the 3-isobutyl-2-methoxypyrazine concentration in the Shiraz wines, and rootstocks can influence the accumulation of methoxypyrazines in the rachis. Significance of the Study Methoxypyrazines in rachis tissues have the potential to impart ‘green’ sensory characters in wines of cultivars not known to produce methoxypyrazines in the berries. Other volatile aroma compounds were found to be affected by rootstocks which may also be useful in targeting rootstocks to give a desired wine style.