Optimized Solid-Phase Mesh-Enhanced Sorption from Headspace (SPMESH) for Rapid Sub-ng/kg Measurements of 3-Isobutyl-2-methoxypyrazine (IBMP) in Grapes.

Parallel extraction of headspace volatiles from multiwell plates using sorbent sheets (HS-SPMESH) followed by direct analysis in real-time high-resolution mass spectrometry (DART-HRMS) can be used as a rapid alternative to solid-phase micro-extraction (SPME) gas-chromatography mass-spectrometry (GC-MS) for trace level volatile analyses. However, an earlier validation study of SPMESH-DART-MS using 3-isobutyl-2-methoxypyrazine (IBMP) in grape juice showed poor correlation between SPMESH-DART-MS and a gold standard SPME-GC-MS around the compound's odor detection threshold (<10 ng/kg) in grape juice, and lacked sufficient sensitivity to detect IBMP at this concentration in grape homogenate. In this work, we report on the development and validation of an improved SPMESH extraction approach that lowers the limit of detection (LOD < 0.5 ng/kg), and regulates crosstalk between wells (<0.5%) over a calibration range of 0.5−100 ng/kg. The optimized SPMESH-DART-MS method was validated using Cabernet Sauvignon and Merlot grape samples harvested from commercial vineyards in the central valley of California (n = 302) and achieved good correlation and agreement with SPME-GC-MS (R2 = 0.84) over the native range of IBMP (<0.5−20 ng/kg). Coupling of SPMESH to a lower resolution triple quadrupole (QqQ)-MS via a new JumpShot-HTS DART source also achieved low ng/kg detection limits, and throughput was improved through positioning stage optimizations which reduced time spent on intra-well SPMESH areas.

Correlating Sensory Assessment of Smoke-Tainted Wines with Inter-Laboratory Study Consensus Values for Volatile Phenols.

Vineyard exposure to wildfire smoke can taint grapes and wine. To understand the impact of this taint, it is imperative that the analytical methods used are accurate and precise. This study compared the variance across nine commercial and research laboratories following quantitative analysis of the same set of smoke-tainted wines. In parallel, correlations between the interlaboratory consensus values for smoke-taint markers and sensory analyses of the same smoke-tainted wines were evaluated. For free guaiacol, the mean accuracy was 94 ± 11% in model wine, while the free cresols and 4-methylguaiacol showed a negative bias and/or decreased precision relative to guaiacol. Similar trends were observed in smoke-tainted wines, with the cresols and glycosidically bound markers demonstrating high variance. Collectively, the interlaboratory results show that data from a single laboratory can be used quantitatively to understand smoke-taint. Results from different laboratories, however, should not be directly compared due to the high variance between study participants. Correlations between consensus compositional data and sensory evaluations suggest the risk of perceivable smoke-taint can be predicted from free cresol concentrations, overcoming limitations associated with the occurrence of some volatile phenols, guaiacol in particular, as natural constituents of some grape cultivars and of the oak used for barrel maturation.

Quantification of Polyfunctional Thiols in Wine by HS-SPME-GC-MS Following Extractive Alkylation.

Analyses of key odorous polyfunctional volatile thiols in wines (3-mercaptohexanol (3-MH), 3-mercaptohexylacetate (3-MHA), and 4-mercapto-4-methyl-2-pentanone (4-MMP)) are challenging due to their high reactivity and ultra-trace concentrations, especially when using conventional gas-chromatography electron impact mass spectrometry (GC-EI-MS). We describe a method in which thiols are converted to pentafluorobenzyl (PFB) derivatives by extractive alkylation and the organic layer is evaporated prior to headspace solid phase microextraction (HS-SPME) and GC-EI-MS analysis. Optimal parameters were determined by response surface area modeling. The addition of NaCl solution to the dried SPME vials prior to extraction resulted in up to less than fivefold improvement in detection limits. Using 40 mL wine samples, limits of detection for 4-MMP, 3-MH, and 3-MHA were 0.9 ng/L, 1 ng/L, and 17 ng/L, respectively. Good recovery (90%-109%) and precision (5%-11% RSD) were achieved in wine matrices. The new method was used to survey polyfunctional thiol concentrations in 61 commercial California and New York State wines produced from V. vinifera (Riesling, Gewürztraminer, Cabernet Sauvignon, Sauvignon blanc and non-varietal rosé wines), V. labruscana (Niagara), and Vitis spp. (Cayuga White). Mean 4-MMP concentrations in New York Niagara (17 ng/L) were not significantly different from concentrations in Sauvignon blanc, but were significantly higher than 4-MMP in other varietal wines.

The effect of ripening rates on the composition of Cabernet Sauvignon and Riesling wines: Further insights into the sugar/flavor nexus.

Grape ripening accelerates under warmer and drier conditions, resulting in the accumulation of sugars ('technological' maturity) being decoupled from phenolic and aromatic composition. This study investigated the effect of different rates of ripening on the composition of Cabernet Sauvignon and Riesling wines. Manipulating crop load and irrigation led to distinct rates of berry ripening. In the resulting wines, reduced crop load affected the aroma composition, altering the profile and abundance of grape-derived compounds and fermentative esters. Phenolic composition was impacted by the irrigation regime, with color and tannin increased by late season irrigation. In Cabernet Sauvignon, the combination of treatments led to the largest ripening delay (3 weeks), resulting in less green and more fruity compounds, and improved phenolic composition. By mapping grape and wine metabolites and exploring their relationship, the outcomes of this study demonstrate the importance of ripening rates in determining wine quality.

Crop Load and Plant Water Status Influence the Ripening Rate and Aroma Development in Berries of Grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon.

Wine made from grapes subjected to accelerated ripening, an increasingly frequent phenomenon occurring in many wine regions due to peaks of heat and water stress, displays higher alcohol levels and lacks balance with color and flavor compounds. Herein, the rate of sugar accumulation of grapes was manipulated by varying the crop load and irrigation regime and the development of secondary metabolites was monitored by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). A 3-week delay in ripening correlated to an increase in the concentration of some monoterpenes and norisoprenoids and a greater decrease of green aroma compounds. Delayed ripening had a positive impact on the phenolic composition of grapes, displaying higher contents of total anthocyanins, total phenolics, quercetin glycosides, and polymeric pigments. A map of the chemical composition of grapes close to harvest allowed discrimination of compounds mainly responsive to delayed ripening from those driven by crop load or irrigation.

Polymeric Sorbent Sheets Coupled to Direct Analysis in Real Time Mass Spectrometry for Trace-Level Volatile Analysis-A Multi-Vineyard Evaluation Study.

Etched polymeric sorbent sheets (solid-phase mesh-enhanced sorption from headspace (SPMESH) sheets) were recently described as an alternative to solid-phase microextraction (SPME) for rapid, parallel, multi-sample extraction and pre-concentration of headspace volatiles. In this report, a workflow was evaluated based on SPMESH sheet extraction followed by direct analysis in real time-mass spectrometry (DART-MS) using grape samples harvested from multiple commercial vineyards at different maturities. SPMESH sheet-DART-MS(-MS) was performed on two grape-derived odorants related to wine quality: 3-isobutyl-2-methoxypyrazine (IBMP) in Cabernet Sauvignon and Merlot grape homogenate (n = 86 samples) and linalool in Muscat-type grape juice samples (n = 18 samples). As part of the optimization process, an MS-MS method was developed for IBMP and an equilibration procedure prior to extraction was established for homogenate samples. Following optimization, we achieved good correlation between SPMESH sheet-DART-MS and SPME-GC-MS for both IBMP (range by GC-MS = < 2 ng/L to 28 ng/L, R2 = 0.70) and linalool (range by GC-MS = 135 to 415 µg/L, R2 = 0.66). The results indicate SPMESH sheet-DART-MS is suitable for rapid measurements of trace level volatiles in grapes.

Timing of cluster light environment manipulation during grape development affects C13 norisoprenoid and carotenoid concentrations in Riesling.

Sunlight exposure of winegrape clusters is frequently reported to increase C(13)-norisoprenoids in resulting wines, but the timing and mechanism of this influence is not well understood. Fruit zone leaf removal was applied to Vitis vinifera cv. Riesling at three timings: 2, 33 and 68 days past berry set (PBS), and compared to an untreated control. Free and total 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), vitispirane and beta-damascenone were measured in juice and wines, and carotenoid profiles were determined in grapes at midseason and maturity. Significantly higher total TDN was observed in grapes from the 33-day PBS treatment compared to the control and other treatments (195 microg/L vs 54-87 microg/L). Total vitispirane in juice was also significantly increased in the 33-day PBS treatment, while total beta-damascenone was reduced in the 68-day PBS treatment compared to the control. Existing HPLC protocols were modified to allow for quantification of zeaxanthin in V. vinifera berries, and zeaxanthin was determined to be significantly higher in the 33-day PBS treatment than the control or other treatments (p < 0.05). Total TDN in juice correlated with free TDN in wine, with 11.0% +/- 2.5% of total juice TDN converted to free TDN in wine. In contrast, total vitispirane increased significantly during fermentation, and was not correlated with vitispirane in juice. In summary, leaf removal at 33 days PBS significantly increased zeaxanthin in Riesling grapes midseason, total TDN and vitispirane in the juice of mature Riesling grapes, and free and total TDN in finished wine, while earlier or later leaf removal had no effect.

Rapid measurement of 3-alkyl-2-methoxypyrazine content of winegrapes to predict levels in resultant wines.

We describe an optimized protocol for analysis of the herbaceous smelling 3-alkyl-2-methoxypyrazines (MPs) in whole berries that predicts MPs in resultant red wines. Berries are homogenized by bead-milling with a deuterated standard prior to headspace solid phase microextraction (HS-SPME) and quantification by two-dimensional gas chromatography time-of-flight-mass-spectrometry (GCxGC-TOF-MS). In the case of 3-isopropyl-2-methoxypyrazine (IPMP), GCxGC-TOF-MS successfully resolved interferences that coeluted with the analyte in the first dimension. HS-SPME parameters (pH, queue time, incubation time, extraction time, extraction temperature) were optimized by a statistical experimental design. Good method accuracy was observed (consistent ratio of unlabeled analyte to labeled standard) at 10 min extraction times when 80 degrees C extraction temperatures were employed, although increasing sensitivity was observed for longer extraction times (up to 140 min). Standard addition of 3-isobutyl-2-methoxypyrazine (IBMP) and IPMP into preveraison and harvest ripe berry matrices showed good linearity (r(2) >0.99 in all cases), with limits of detection ranging from 0.6 to 1.8 pg/g. The protocol was validated by comparing IBMP in 16 lots of Cabernet Franc berries (range = undetectable to 18.4 pg/g) to the resulting wines (range = undetectable to 14.5 pg/g). Berry and wine MP content were strongly correlated, (r(2) = 0.97, p < 0.0001). Following correction for CO(2) loss, the observed concentration of IBMP in wines was 67 +/- 13% of the IBMP concentration observed in berries.

Effects of cluster light exposure on 3-isobutyl-2-methoxypyrazine accumulation and degradation patterns in red wine grapes ( Vitis vinifera L. Cv. Cabernet Franc).

The effects of light exposure on 3-isobutyl-2-methoxypyrazine (IBMP) accumulation and degradation in Vitis vinifera L. cv. Cabernet Franc berries were assessed by comparison of shaded and exposed clusters within the same vine throughout a growing season. Twenty-seven vines were shoot-thinned to create regions of high and low cluster-light exposure within each vine. Samples were collected at 10 time points starting from 5 to 130 days postbloom. The experimental design allowed for intravine comparison of IBMP levels between treatments at each time. Vine-to-vine variability of IBMP and the correlation of IBMP to malic acid were also evaluated. Cluster exposure reduced accumulation of IBMP at all preveraison time points by 21-44%, but did not increase postveraison degradation. Significant vine-to-vine variability in IBMP content was observed, with the highest level of IBMP in shaded berries in the most vigorous block of vines. Although IBMP concentration by weight decreased significantly due to dilution just prior to color change (veraison), no significant IBMP degradation per berry occurred until after color change (day 70 postbloom). By contrast, malic acid degradation began prior to color change, and malic acid concentrations were not affected by cluster exposure preveraison, but were affected postveraison. A survey of 13 sites in New York state (Seneca Lake) showed that IBMP concentrations at 2 weeks preveraison were highly correlated (R(2) = 0.936, p < 0.0001) to levels at harvest, whereas classic grape maturity indices at harvest were uncorrelated with IBMP at harvest. In summary, light exposure conditions critically influence IBMP accumulation but not IBMP degradation.

Differential cerebral cortex transcriptomes of baboon neonates consuming moderate and high docosahexaenoic acid formulas.

BACKGROUND: Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels. METHODS AND FINDINGS: Twelve baboons were divided into three formula groups: Control, with no DHA-ARA; "L", LCPUFA, with 0.33%DHA-0.67%ARA; "L3", LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner. CONCLUSIONS: These data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA.

Carbon position-specific isotope analysis of alanine and phenylalanine analogues exhibiting nonideal pyrolytic fragmentation.

Recent advances in gas chromatography combustion-isotope ratio mass spectrometry (GCC-IRMS) has made compound-specific isotope analysis routine, but reports on position-specific isotopic analysis are still scarce. On-line GC-pyrolysis (Py) coupled to GCC-IRMS is reported here for isolation and isotopic characterization of alaninol and phenethylamine, analogues of alanine and phenylalanine, respectively. Ideally, pyrolytic fragments will originate from unique sites within the parent molecule, and isotope ratios for each position within the parent can either be measured directly or calculated from fragment isotope ratios without substantially degrading the analytical precision. Alaninol pyrolysis yielded several fragments, of which CO and CH4 were used for isotope ratio calculations. Isotope labeling experiments showed that CO derived entirely from the C(1) position, while all three positions of alaninol contributed to CH4 (29.0 +/- 0.3% from C(1), 3.6 +/- 0.2% from C(2), and 66.9 +/- 1.1% from C(3)). We demonstrate iterative use of mass balance to calculate isotope ratios from all positions despite the nonideal positional fidelity of CH4. Pyrolysis of phenethylamine generated benzene and toluene fragments. Benzene derived entirely from C(ring), and toluene was proportionately formed from C(3) and C(ring). Relative intramolecular isotope ratios (Deltadelta13C) were calculated directly from delta13C of fragments or indirectly by mass balance. Though the C(3) isotope ratio was calculated from the benzene and toluene fragments, propagation of errors showed that the final precision of the determination was degraded due to the small contribution that C(3) makes to toluene. Samples of each amino acid from four different vendors showed natural variability between sources, especially at the C(1) position of alaninol (range of Deltadelta13C approximately 50 per thousand). The average precision was SD(Deltadelta13C) < 0.20 per thousand for directly measured positions of alaninol and phenethylamine. The precision of indirectly measured positions was poorer (SD(Deltadelta13C) = 0.94 per thousand for alaninol, 6.54 per thousand for phenethylamine) due to propagation of errors. These data demonstrate that GC-Py-GCC-IRMS data can be used to extract high-precision isotope ratios from amino acids despite nonideal positional fidelity in fragments and that natural intramolecular variability in delta13C can be used to distinguish different sources of amino acids.