Evaluation of an artificial intelligence-facilitated sperm detection tool in azoospermic samples for use in ICSI.

RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, n = 4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, n = 4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30  ×  10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.

Rapid headspace solid-phase microextraction sheets with direct analysis in real time mass spectrometry (SPMESH-DART-MS) of derivatized volatile phenols in grape juices and wines.

Volatile phenols possess "smoky, spicy" aromas and are routinely measured in grapes, wines and other foodstuffs for quality control. Routine analyses of volatile phenols rely on gas chromatography - mass spectrometry (GC-MS), but slow throughput of GC-MS can cause challenges during times of surge demand, i.e. following 'smoke taint' events involving forest fires near vineyards. Parallel extraction of headspace volatiles onto sorbent sheets (HS-SPMESH) followed by direct analysis in real time mass spectrometry (DART-MS) is a rapid alternative to conventional GC-MS approaches. However, HS-SPMESH extraction is poorly suited for lower volatility odorants, including volatile phenols. This work reports development and validation of an HS-SPMESH-DART-MS approach for five volatile phenols (4-ethylphenol, 4-ethylguiacol, guaiacol, 4-methylguaiacol, and cresols). Prior to HS-SPMESH extraction, volatile phenols were acetylated to facilitate their extraction. A unique feature of this work was the use of d6-Ac2O as a derivatizing agent to overcome issues with isobaric interferences inherent to chromatography-free MS techniques. The use of alkaline conditions during derivatization resulted in cumulative measurement of both free and bound forms of volatile phenols. The validated HS-SPMESH-DART-MS method achieved a throughput of 24 samples in ∼60 min (including derivatization and extraction time) with low limits of detection (<1 µg L-1) and good repeatability (3-6% RSD) in grape and wine matrices. Validation experiments with smoke-tainted grape samples indicated good correlation between total (free + bound) volatile phenols measured by HS-SPMESH-DART-MS and a gold standard GC-MS method.

Microfluidics facilitating the use of small extracellular vesicles in innovative approaches to male infertility.

Sperm are transcriptionally and translationally quiescent and, therefore, rely on the seminal plasma microenvironment for function, survival and fertilization of the oocyte in the oviduct. The male reproductive system influences sperm function via the binding and fusion of secreted epididymal (epididymosomes) and prostatic (prostasomes) small extracellular vesicles (S-EVs) that facilitate the transfer of proteins, lipids and nucleic acids to sperm. Seminal plasma S-EVs have important roles in sperm maturation, immune and oxidative stress protection, capacitation, fertilization and endometrial implantation and receptivity. Supplementing asthenozoospermic samples with normospermic-derived S-EVs can improve sperm motility and S-EV microRNAs can be used to predict non-obstructive azoospermia. Thus, S-EV influence on sperm physiology might have both therapeutic and diagnostic potential; however, the isolation of pure populations of S-EVs from bodily fluids with current conventional methods presents a substantial hurdle. Many conventional techniques lack accuracy, effectiveness, and practicality; yet microfluidic technology has the potential to simplify and improve S-EV isolation and detection.

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.

Prednisolone and enoxaparin (clexane) therapy ('the Bondi protocol') for repeated IVF failure.

PROBLEM: What is the impact of an empirical immune therapy protocol of prednisolone and enoxaparin (clexane) (the 'Bondi protocol') on women with repeated in vitro fertilization (IVF) failure? METHOD OF STUDY: This was a retrospective review of live birth outcomes conducted on all transfer cycles performed by a single clinician (GS) at IVFAustralia between February 2016 and April 2020. This study consisted of 1786 transfer cycles, including 460 cycles treated with the Bondi protocol and 1326 without. Women with repeated IVF failure were given the Bondi protocol based on blood NK cell activity. Primary outcome was live birth and statistical analysis was performed with GraphPad Prism software with significance for P-values < .05. RESULTS: Overall 'Bondi' and 'normal' protocol cycles had similar rates of IVF/ICSI, fresh/frozen transfers and use of preimplantation genetic testing (PGT). Women given the Bondi protocol were older, had more previous cycles and had higher blood NK cell activity. There was no significant difference in live birth rates (Bondi 26%, normal 28%). Bondi protocol live birth rates per transfer cycle were as high as 40% in patients under 38 years old. The Bondi protocol was more effective as NK activity increased from 'normal' to 'borderline' to 'high'. For high NK cell activity levels, live birth rates were over four times higher for women on the Bondi protocol (28%) than those on normal protocols (6%, P = .0007). CONCLUSION: This study describes a simple and relatively safe immune therapy protocol that may improve IVF success rates in women with evidence of immune dysfunction.

Swellable Sorbent Coatings for Parallel Extraction, Storage, and Analysis of Plant Metabolites.

Quantitative and qualitative measurements of trace-level analytes in plants or foodstuffs, e.g., secondary metabolites like carotenoids, are often performed at centralized core facilities or off-site laboratories. However, preparation, storage, and/or transport of both intact samples and sample extracts may be cumbersome and complicated, especially for air-sensitive analytes. We describe the development of inexpensive swellable microextraction (SweME) devices for extraction and storage of nonpolar analytes. SweME devices consist of a thin layer of poly(dimethylsiloxane) (PDMS) grafted onto a stainless steel support. Pretreating the SweME device with small volumes of the organic solvent causes the PDMS to swell. The swollen SweME device can then be immersed directly into complex matrices for absorptive extraction of low-molecular-weight, nonpolar analytes. Following storage, analytes can be solvent-desorbed prior to characterization. Proof-of-principle work with carotenoids from tomatoes and carrots demonstrates that SweME is appropriate for semiquantitative analyses and increases the stability of air-sensitive analytes during storage at ambient temperatures as compared to the solvent extracts. Carotenoid profiles (fractional carotenoid contributions) from tomato and carrot samples were well correlated between SweME and liquid-liquid extraction (R2 = 0.97 and 0.94). Lycopene, the most abundant carotenoid in tomatoes, saw a less than 20% decrease in extracted mass during 1 month of ambient SweME storage. Extractions and desorptions can be run in parallel using multiwell plates. In summary, swelled sorbent extraction with SweME devices is a convenient and inexpensive approach for isolation and storage of analytes in complex matrices and may be particularly well suited for evaluating large numbers of plant samples through external laboratories.

Stable QTL for malate levels in ripe fruit and their transferability across Vitis species.

Malate is a major contributor to the sourness of grape berries (Vitis spp.) and their products, such as wine. Excessive malate at maturity, commonly observed in wild Vitis grapes, is detrimental to grape and wine quality and complicates the introgression of valuable disease resistance and cold hardy genes through breeding. This study investigated an interspecific Vitis family that exhibited strong and stable variation in malate at ripeness for five years and tested the separate contribution of accumulation, degradation, and dilution to malate concentration in ripe fruit in the last year of study. Genotyping was performed using transferable rhAmpSeq haplotype markers, based on the Vitis collinear core genome. Three significant QTL for ripe fruit malate on chromosomes 1, 7, and 17, accounted for over two-fold and 6.9 g/L differences, and explained 40.6% of the phenotypic variation. QTL on chromosomes 7 and 17 were stable in all and in three out of five years, respectively. Variation in pre-veraison malate was the major contributor to variation in ripe fruit malate (39%), and based on two and five years of data, respectively, their associated QTL overlapped on chromosome 7, indicating a common genetic basis. However, use of transferable markers on a closely related Vitis family did not yield a common QTL across families. This suggests that diverse physiological mechanisms regulate the levels of this key metabolite in the Vitis genus, a conclusion supported by a review of over a dozen publications from the past decade, showing malate-associated genetic loci on all 19 chromosomes.

Quantification of protein by acid hydrolysis reveals higher than expected concentrations in red wines: Implications for wine tannin concentration and colloidal stability.

Protein is reportedly negligible in most red wines, due to its loss following co-precipitation with phenolic substances. A method for protein quantification in red wine was developed which overcame analytical interference from phenolic substances, based on ethanol precipitation, followed by acid-hydrolysis and amino acid quantification. Protein concentration was surveyed in a range of red wines produced from V. vinifera and interspecific (Vitis spp) hybrids, revealing higher than expected concentrations, ranging from 23 mg/L ± 2.57 to 380 mg/L ± 16. The results showed that tannin extracted from grapes remains soluble in wine in the presence of protein even at high protein (>100 mg/L) and tannin (>500 mg/L) concentrations. As a further consequence of this, the particle size and concentration of colloids within high- and low-protein wines were similar, independent of protein or tannin concentration. Higher wine tannin concentration was also correlated with increased heat stability of wine protein.

Berry Anthocyanin, Acid, and Volatile Trait Analyses in a Grapevine-Interspecific F2 Population Using an Integrated GBS and rhAmpSeq Genetic Map.

Increased map density and transferability of markers are essential for the genetic analysis of fruit quality and stress tolerance in interspecific grapevine populations. We used 1449 GBS and 2000 rhAmpSeq markers to develop a dense map for an interspecific F2 population (VRS-F2) that was derived by selfing a single F1 from a Vitis riparia x 'Seyval blanc' cross. The resultant map contained 2519 markers spanning 1131.3 cM and was highly collinear with the Vitis vinifera 'PN40024' genome. Quantitative trait loci (QTL) for berry skin color and flower type were used to validate the map. Four rhAmpSeq transferable markers were identified that can be used in pairs (one pistillate and one hermaphroditic) to predict pistillate and hermaphrodite flower type with ≥99.7% accuracy. Total and individual anthocyanin diglucoside QTL mapped to chromosome 9 near a 5-O-GLUCOSYLTRANSFERASE candidate gene. Malic acid QTL were observed on chromosome 1 and 6 with two MALATE DEHYRDROGENASE CYTOPLASMIC 1 and ALUMINUM-ACTIVATED MALATE TRANSPORTER 2-LIKE (ALMT) candidate genes, respectively. Modeling malic acid identified a potential QTL on chromosome 8 with peak position in proximity of another ALMT. A first-ever reported QTL for the grassy smelling volatile (E)-2-hexenal was found on chromosome 2 with a PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE candidate gene near peak markers.

The Genetic Basis of Anthocyanin Acylation in North American Grapes (Vitis spp.).

Hydroxycinnamylated anthocyanins (or simply 'acylated anthocyanins') increase color stability in grape products, such as wine. Several genes that are relevant for anthocyanin acylation in grapes have been previously described; however, control of the degree of acylation in grapes is complicated by the lack of genetic markers quantitatively associated with this trait. To characterize the genetic basis of anthocyanin acylation in grapevine, we analyzed the acylation ratio in two closely related biparental families, Vitis rupestris B38 × 'Horizon' and 'Horizon' × Illinois 547-1, for 2 and 3 years, respectively. The acylation ratio followed a bimodal and skewed distribution in both families, with repeatability estimates larger than 0.84. Quantitative trait locus (QTL) mapping with amplicon-based markers (rhAmpSeq) identified a strong QTL from 'Horizon' on chromosome 3, near 15.85 Mb in both families and across years, explaining up to 85.2% of the phenotypic variance. Multiple candidate genes were identified in the 14.85-17.95 Mb interval, in particular, three copies of a gene encoding an acetyl-CoA-benzylalcohol acetyltransferase-like protein within the two most strongly associated markers. Additional population-specific QTLs were found in chromosomes 9, 10, 15, and 16; however, no candidate genes were described. The rhAmpSeq markers reported here, which were previously shown to be highly transferable among the Vitis genus, could be immediately implemented in current grapevine breeding efforts to control the degree of anthocyanin acylation and improve the quality of grapes and their products.

Rapid Analysis of Volatile Phenols from Grape Juice by Immersive Sorbent Sheet Extraction Prior to Direct Analysis in Real-Time Mass Spectrometry (DART-MS).

Poly(dimethylsiloxane)-based thin-film sorbent sheets (SPMESH) have previously been used for parallel headspace (HS) extraction prior to direct analysis in real-time mass spectrometry (DART-MS) for rapid quantitation of odorants in complex matrices. However, HS-SPMESH extraction is poorly suited for less volatile odorants, e.g., volatile phenols. This report describes modifications to the previous SPMESH extraction device, which make it amenable to parallel extraction of low-volatility analytes from multiwell plates under direct immersion (DI) conditions. Optimization and validation of the DI-SPMESH-DART-MS approach were performed on four volatile phenols (4-ethylphenol, 4-ethylguaiacol, 4-methylguaiacol, and guaiacol) of relevance to the quality of grape juices. Negative-ion mode DART-MS spectra showed a series of oxygenated adducts [M + nO - H]- for all analytes, but isobaric interferences could be limited for three of the four analytes by selecting an appropriate MS/MS transition. Signal suppression from nonvolatiles (sugars, acids) could be overcome by a rinse step. DI-SPMESH-DART-MS analysis of 24 samples could be performed in ∼45 min (30 min extraction, 16 min DART analysis) with 0.5-3 µg/L detection limits in aqueous and model juice solutions. In real grape juices (n = 5 cultivars), good accuracy (72-137%) could be achieved for two of the four volatile phenols initially investigated, 4-ethylphenol and 4-ethylguaiacol. However, poor accuracy was observed for guaiacol in some cultivars, and 4-methylguaiacol could not be quantitated due to interferences with other volatile phenols. Despite these limitations, DI-SPMESH-DART-MS/MS may be useful for prescreening a large number of samples prior to more selective conventional analyses.

Brine-Releasable Hydrogen Sulfide in Wine: Mechanism of Release from Copper Complexes and Effects of Glutathione.

Copper-sulfhydryl complexes in wine can be disrupted by addition of brine to release free hydrogen sulfide (H2S), and the resulting "brine-releasable H2S" is reported to correlate with formation of H2S during bottle storage. However, both the mechanism of the brine-release assay and factors affecting the stability of copper sulfhydryls under brine-release conditions are not well understood. By varying brine composition and concentration, it is shown that release of copper-complexed H2S requires the presence of a halide (Cl- and Br-) and is not due to a general "salting-out" effect. Release of copper-complexed H2S by the brine dilution assay is highly temperature-dependent. When H2S and Cu(II) are added to a model wine, brine-releasable H2S decreases markedly (∼10-fold) after a 20 min incubation period prior to performing the brine-release assay. In commercial wines, the fraction of added H2S recovered through the brine-release assay was correlated with the initial glutathione (GSH) concentration (r2 = 0.58) but not with initial Cu. Negligible additional release of H2S from organopolysulfanes was observed following addition of a disulfide-reducing agent (tris(2-carboxyethyl)phosphine). As previous studies have reported a correlation between H2S formed under brine-release conditions and normal storage, these results suggest that the susceptibility of a wine to form latent copper-sulfhydryl precursors of H2S following copper addition is dependent on the concentration of sulfhydryls like GSH.

Natural killer cells and reproductive success.

Natural killer (NK) cell assessment has been attempted since the 1990s and, apart from antibody testing, is probably the commonest immune test available to clinicians. It is clear that simple enumeration of uterine NK cells by immunohistochemistry is inadequate, although better methodology such as flow cytometry may prove to be more beneficial in the future. Blood testing is an appealing noninvasive test that may be a marker for immune dysfunction, rather than a guide to uterine numbers per se. It is currently performed in women with repeated reproductive failure and should be done using tests of activation. Patients value investigation and clinicians should prefer it to blind empirical immune therapy in repeated reproductive failure cases. But, in addition to blood NK testing, a fundamental new NK genetic test (the KIR/HLA-C interaction) is likely to provide an even more effective diagnostic tool. Insights from KIR/HLA-C analysis imply that new immune therapy trials will need to take KIR/HLA-C results into account.

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.

The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor.

Modern tomatoes have narrow genetic diversity limiting their improvement potential. We present a tomato pan-genome constructed using genome sequences of 725 phylogenetically and geographically representative accessions, revealing 4,873 genes absent from the reference genome. Presence/absence variation analyses reveal substantial gene loss and intense negative selection of genes and promoters during tomato domestication and improvement. Lost or negatively selected genes are enriched for important traits, especially disease resistance. We identify a rare allele in the TomLoxC promoter selected against during domestication. Quantitative trait locus mapping and analysis of transgenic plants reveal a role for TomLoxC in apocarotenoid production, which contributes to desirable tomato flavor. In orange-stage fruit, accessions harboring both the rare and common TomLoxC alleles (heterozygotes) have higher TomLoxC expression than those homozygous for either and are resurgent in modern tomatoes. The tomato pan-genome adds depth and completeness to the reference genome, and is useful for future biological discovery and breeding.

Spatially Resolved Headspace Extractions of Trace-Level Volatiles from Planar Surfaces for High-Throughput Quantitation and Mass Spectral Imaging.

The use of headspace thin-film microextraction devices (SPMESH) for parallel extraction of trace-level volatiles prior to direct analysis in real-time mass spectrometry (DART-MS) has been reported previously, in which volatiles were extracted from samples in multi-well plates. In this report, we demonstrate that headspace extraction of volatiles by SPMESH sheets can be performed directly from planar surfaces. When coupled with DART-MS, this approach yields volatile mass spectral images with at least 4 mm resolution. When samples were spotted onto general-purpose silica gel thin-layer chromatography (TLC) plates, the SPMESH extraction could reach equilibrium within 2-4 min and 48 samples could be extracted and analyzed in 14 min. Because volatilization of analytes from TLC plates was very rapid, SPMESH extraction was delayed by the addition of 5% polyethylene glycol. Good linearity was achieved in the microgram per liter to milligram per liter range for four odorants (3-isobutyl-2-methoxypyrazine, linalool, methyl anthranilate, and o-aminoacetophenone) in several matrices (water, 10% ethanol, juice, and grape macerate) using 5 µL sample sizes. Detection limits as low as 50 pg/spot (10 µg/L in grape macerate) could be achieved. In contrast to many reports on headspace solid-phase microextraction, negligible matrix effects were observed for ethanol and grape macerates compared to water. SPMESH can preserve volatile images from planar surfaces, and SPMESH-DART-MS from TLC plates is well-suited for rapid trace volatile analysis, especially with small sample sizes.

Loss and formation of malodorous volatile sulfhydryl compounds during wine storage.

Volatile sulfur compounds (VSCs), particularly low molecular weight sulfhydryls like hydrogen sulfide (H2S) and methanethiol (MeSH), are often observed in wines with sulfurous off-aromas. Recent work has shown both H2S and MeSH can increase up to a few µM (> 40 µg/L) during anoxic storage, but the identity of the latent sources of these sulfhydryls is still disputed. This review critically evaluates the latent precursors and pathways likely to be responsible for the loss and formation of these sulfhydryls during wine storage based on the existing enology literature as well as studies from food chemistry, geochemistry, biochemistry, and synthetic chemistry. We propose that three precursor classes have sufficient concentration and metastability to serve as latent sulfhydryl precursors in wine: 1) transition metal-sulfhydryl complexes, particularly those formed following Cu(II) addition, which are released under anoxic conditions through an unknown mechanism; 2) asymmetric disulfides, polysulfanes, and (di)organopolysulfanes formed through transition-metal mediated oxidation (e.g., Cu(II)) of sulfhydryls or pesticide degradation, and released through sulfitolysis, metal-catalyzed thiol-disulfide exchange or related reactions; 3) S-alkylthioacetates, primarily formed during fermentation, and releasable hydrolytically. Some evidence also exists for S-amino acids serving as precursors. Based on these findings, we propose a "decision tree" approach to choosing appropriate strategies for managing wines with sulfurous off-aromas.

Volatile and sensory characterization of roast coffees - Effects of cherry maturity.

Immature coffee cherries produce roast coffees with lower hedonic scores than those produced from mature cherries, but variation in volatile and sensory characteristics over a range of maturities is not well studied. In this work, cherries from two coffee cultivars (Caturra, Catimor) were sorted into seven maturity stages from fully immature (Stage 1, green) to fully overripe (Stage 7, purple). Volatile profiles of Stage 1 roast coffee had lower concentrations of carbohydrate degradation products and higher concentrations of N-heterocycles and phenols. Differences in volatiles among Stage 2 (partially immature, yellow-green) and subsequent stages were insignificant (p > 0.05) or else minor. Principle component analysis of the volatile data set also distinguished Stage 1 from other stages. Similarly, a trained cupping panel reported significantly lower sensory scores for Stage 1 as compared to Stages 2-7, but few differences among Stages 2-7. Thus, partially mature and overripe cherries may be appropriate for specialty coffee.

Parallel Headspace Extraction onto Etched Sorbent Sheets Prior to Ambient-Ionization Mass Spectrometry for Automated, Trace-Level Volatile Analyses.

Headspace (HS) extraction and preconcentration of volatiles by solid-phase microextraction (SPME) can improve the sensitivity and selectivity of ambient ionization-mass spectrometry approaches like direct analysis in real time (DART), but previous approaches to HS-SPME-DART-MS have been challenging to automate. This report describes the production of inexpensive, reusable solid-phase mesh-enhanced sorption from headspace (SPMESH) sheets by laser-etching mesh patterns into poly(dimethylsiloxane) (PDMS) sheets. Parallel headspace extraction of volatiles from multiple samples can be achieved by positioning the SPMESH sheets over multiwell plates and then attaching to a positioning stage for automated DART-MS quantitation. Using three representative odorants (3-isobutyl-2-methoxypyrazine, linalool, and methyl anthranilate), we achieved µg/L-ng/L detection limits with SPMESH-DART-MS, with the DART-MS step requiring only 17 min for 24 samples. Acceptable repeatability (24% or less day-to-day variation) and excellent recovery from a grape matrix (99-106%) could be achieved. Through use of a Teflon gasket and stainless steel spacers, cross-contamination between the headspaces of adjacent wells could be limited to roughly 1%. Optimum SPMESH extraction and desorption parameters were determined by response surface methodology. In summary, sheet-based SPMESH provides a sensitive, readily automated approach for coupling with DART-MS and achieving high-throughput trace-level volatile analyses.

HS-SPME-GC-MS Analyses of Volatiles in Plant Populations-Quantitating Compound × Individual Matrix Effects.

Headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography⁻mass spectrometry (GC-MS) is widely employed for volatile analyses of plants, including mapping populations used in plant breeding research. Studies often employ a single internal surrogate standard, even when multiple analytes are measured, with the assumption that any relative changes in matrix effects among individuals would be similar for all compounds, i.e., matrix effects do not show Compound × Individual interactions. We tested this assumption using individuals from two plant populations: an interspecific grape (Vitis spp.) mapping population (n = 140) and a tomato (Solanum spp.) recombinant inbred line (RIL) population (n = 148). Individual plants from the two populations were spiked with a cocktail of internal standards (n = 6, 9, respectively) prior to HS-SPME-GC-MS. Variation in the relative responses of internal standards indicated that Compound × Individual interactions exist but were different between the two populations. For the grape population, relative responses among pairs of internal standards varied considerably among individuals, with a maximum of 249% relative standard deviation (RSD) for the pair of [U13C]hexanal and [U13C]hexanol. However, in the tomato population, relative responses of internal standard pairs varied much less, with pairwise RSDs ranging from 8% to 56%. The approach described in this paper could be used to evaluate the suitability of using surrogate standards for HS-SPME-GC-MS studies in other plant populations.