Stable Isotope Dilution Analysis Research Articles

Furfuryl alcohol is a precursor for furfurylthiol in coffee

This study investigated the role of furfuryl alcohol (FFA) in the formation of furfurylthiol (FFT), the most important odorant in roasted coffee, using in-bean and spiking experiments. Green beans were spiked with FFA, and after roasting FFT was quantified by stable isotope dilution analysis. The FFT level in the roasted beans increased dose-dependently with addition of FFA. Additionally, beans were spiked with isotopically labelled d2-FFA which generated isotopically labelled d2-FFT after roasting. However, no labelled furfural was observed. The results unambiguously show that FFA serves as a precursor of FFT in coffee. On the other hand, the data indicate that furfural stems not from oxidation of FFA and plays no major role as precursor for FFT formation during coffee roasting. The suggested formation pathway leads from FFA to the furfuryl cation, then protein-bound S-furfuryl-l-cysteine and by subsequent elimination to FFT.

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3‘-yl)-2‘-desoxyguanosine (E3′N2dG) and N6-(isoestragole-3‘-yl)-desoxyadenosine (E3′N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3′N6dA at 10 μM, E3′N2dG at 1μM estragole). E3′N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 μM after 24 h and < 0.5 μM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 μM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 μM estragole led to a 3.2 fold and 300 μM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a ‘practical threshold’ dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.

High fractional excretion of glycation adducts is associated with subsequent early decline in renal function in type 1 diabetes

Increased protein glycation, oxidation and nitration is linked to the development of diabetic nephropathy. We reported levels of serum protein glycation, oxidation and nitration and related hydrolysis products, glycation, oxidation and nitration free adducts in patients with type 1 diabetes (T1DM) during onset of microalbuminuria (MA) from the First Joslin Kidney Study, a prospective case–control study of patients with T1DM with and without early decline in GFR. Herein we report urinary excretion of the latter analytes and related fractional excretion values, exploring the link to MA and early decline in GFR. We recruited patients with T1DM and normoalbuminuria (NA) (n = 30) or new onset MA with and without early GFR decline (n = 22 and 33, respectively) for this study. We determined urinary protein glycation, oxidation and nitration free adducts by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC–MS/MS) and deduced fractional excretion using reported plasma levels and urinary and plasma creatinine estimates. We found urinary excretion of pentosidine was increased ca. twofold in patients with MA, compared to normoalbuminuria (0.0442 vs 0.0103 nmol/mg creatinine, P < 0.0001), and increased ca. threefold in patients with early decline in GFR, compared to patients with stable GFR (0.0561 vs 0.0176 nmol/mg creatinine, P < 0.01). Urinary excretion of all other analytes was unchanged between the study groups. Remarkably, fractional excretions of 6 lysine and arginine-derived glycation free adducts were higher in patients with early decline in GFR, compared to those with stable GFR. Impaired tubular reuptake of glycation free adducts by lysine and arginine transporter proteins in patients with early GFR decline is likely involved. We conclude that higher fractional excretions of glycation adducts are potential biomarkers for early GFR decline in T1DM and MA. Measurement of these analytes could provide the basis for identifying patients at risk of early decline in renal function to target and intensify renoprotective treatment.

Dry-Hopping to Modify the Aroma of Alcohol-Free Beer on a Molecular Level-Loss and Transfer of Odor-Active Compounds.

There are mainly two options for the dealcoholization of beer: evaporation of ethanol by heat treatment, whereby desired aroma-active compounds are also removed, and stopped fermentation that leads to beers still containing high amounts of unfermented sugar in parallel with lower amounts of aroma-active fermentation products. Thus, dry-hopping could be an opportunity to compensate for these aroma deficiencies. Therefore, following the sensomics approach, odorants were characterized in dry-hopped (Hallertauer Mandarina Bavaria, Hallertauer Cascade, or Hallertauer Mittelfrüh) top- and bottom-fermented alcohol-free beers either after thermal dealcoholization or stopped fermentation. Twenty-three odorants were quantitated via stable isotope dilution analysis, and odor activity values (OAVs; ratio of concentration to odor threshold) were calculated. Thermally dealcoholized samples showed high losses (up to 100%) of key odorants like 3-methyl-1-butanol or 3-methylbutyl acetate. During stopped fermentation, aroma compounds like ethyl butanoate or 2-phenylethanol were formed in relevant concentrations, leading to OAVs ≥ 1, but the amounts were significantly lower compared to beers with normal alcohol contents. For hop-derived odorants (linalool, geraniol, myrcene, and esters), transfer rates between 20 and 90% were found, leading to OAVs ≥ 1 in beer. Furthermore, hop addition apparently induced the formation of ethyl esters of hop-derived monocarboxylic acids.

Occurrence of allergen proteins in wines from Chilean market

ABSTRACT Casein and ovalbumin are proteins commonly used as wine fining agents that may trigger allergic reactions in susceptible individuals. Therefore, their occurrence in wines could become a health risk, moreover when their presence is not reported. The objective of this work was to determine the occurrence of casein and ovalbumin in Chilean wines. Proteins were extracted by ultrafiltration and digested applying an optimised ultrasound-assisted method. Peptides were quantified by validated tandem mass spectrometry method using stable isotope dilution analysis. Optimal digestion was achieved in 3 minutes at a 1:10 enzyme protein ratio. The method was validated following ICH guidelines showing determination coefficients R2 ≥ 0.99, repeatability, and intermediate precision with RSD values <1.95% and recoveries from 89.8% to 115.1% (RSD < 5.84%). The method was applied to analyse 60 wine samples. Fifty-six samples showed quantifiable levels, from which 28% presented a total casein and ovalbumin concentration equal or higher to the European limit (0.25 mg L−1).

Myco-DES: Enabling Remote Extraction of Mycotoxins for Robust and Reliable Quantification by Stable Isotope Dilution LC-MS/MS.

Mycotoxins remain a global threat to human and animal health, especially in countries lacking effective measures to detect and control contaminated commodities. As the quantification of mycotoxins usually relies on complex and expensive techniques, the availability of suitable instrumentation is often a bottleneck in reliable mycotoxin detection. As part of our research toward strategies offering widespread access to mycotoxin analysis while cutting down on costs, we present a new extraction and quantification protocol combining materials originally designed for dried blood spot analysis with stable isotope dilution analysis. Its key benefits are that extraction of mycotoxins can be carried out at remote sites and by minimally trained personnel, while quantification will take place in specialized central laboratories simply connected by regular, paper-based mail. As a proof of concept, aflatoxins, ochratoxin A, and deoxynivalenol were extracted from cereal-based foodstuffs, fixed on paper cards for transport, and successfully quantified after re-extraction by stable isotope dilution LC-MS/MS analysis. Several materials (cellulose/polyethylene terephthalate/glass fiber, nontreated/chemically treated) as well as possible transport and storage conditions (temperature, humidity) were evaluated. The final myco-DES (dried extract spots) protocol allows quantification of mycotoxin levels currently recognized as safe (aflatoxin B1: 2 μg/kg, ochratoxin A: 3 μg/kg, deoxynivalenol: 500 μg/kg) after a storage of up to 4 weeks under tropical climate conditions (40 °C, 75% relative humidity).

Reading patterns of proteome damage by glycation, oxidation and nitration: quantitation by stable isotopic dilution analysis LC-MS/MS.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides a high sensitivity, high specificity multiplexed method for concurrent detection of adducts formed by protein glycation, oxidation and nitration, also called AGEomics. Combined with stable isotopic dilution analysis, it provides for robust quantitation of protein glycation, oxidation and nitration adduct analytes. It is the reference method for such measurements. LC-MS/MS has been used to measure glycated, oxidized and nitrated amino acids - also called glycation, oxidation and nitration free adducts, with a concurrent quantitation of the amino acid metabolome in physiological fluids. Similar adduct residues in proteins may be quantitated with prior exhaustive enzymatic hydrolysis. It has also been applied to quantitation of other post-translation modifications, such as citrullination and formation of Nε-(γ-glutamyl)lysine crosslink by transglutaminases. Application to cellular and extracellular proteins gives estimates of the steady-state levels of protein modification by glycation, oxidation and nitration, and measurement of the accumulation of glycation, oxidation and nitration adducts in cell culture medium and urinary excretion gives an indication of flux of adduct formation. Measurement of glycation, oxidation and nitration free adducts in plasma and urine provides for estimates of renal clearance of free adducts. Diagnostic potential in clinical studies has been enhanced by the combination of estimates of multiple adducts in optimized diagnostic algorithms by machine learning. Recent applications have been in early-stage detection of metabolic, vascular and renal disease, and arthritis, metabolic control and risk of developing vascular complication in diabetes, and a blood test for autism.

Determination of di-n-butyl adipate (DnBA) metabolites as possible biomarkers of exposure in human urine by online-SPE-LC-MS/MS

Di-n-butyl adipate (DnBA) is an alternative to the anti-androgenic and strictly regulated di-n-butyl phthalate (DnBP) used as a cosmetic ingredient, plasticizer, and in various articles of everyday life. Hence, exposures of the general population have to be expected. Currently, biomarkers of DnBA exposure and methods for their determination are not available. Here, we describe a sensitive, rugged and precise analytical method for the determination of the DnBA monoester metabolite mono-n-butyl adipate (MnBA), as well as its potential downstream metabolites 3-hydroxy-mono-n-butyl adipate (3OH-MnBA) and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA) in human urine. Glucuronic acid conjugates present in urine were deconjugated using a pure β-glucuronidase. The metabolites were then analyzed by liquid chromatography on a C18 column with superficially porous particles coupled to electrospray ionization-triple quadrupole-tandem mass spectrometry, applying online turbulent flow chromatography for analyte enrichment and matrix depletion (online-SPE-LC-MS/MS). The metabolites were quantified using stable isotope dilution analysis with limits of quantification of 0.05 µg/L (MnBA), 0.1 µg/L (3OH-MnBA), and 0.5 µg/L (3cx-MnPrA). Method imprecision in urinary matrix was below 7% (coefficient of variation) for all analytes. Mean relative recoveries were between 93% and 107%. The suitability of the DnBA metabolites as biomarkers of exposure was demonstrated after dermal application of a commercially available sunscreen containing DnBA. Maximum concentrations were reached 6.5 h after dose (219 µg/L 3cx-MnPrA, 91 µg/L MnBA, and 3.9 µg/L 3OH-MnBA). Elimination kinetics were similar for all three metabolites. We were able to quantify 3cx-MnPrA and MnBA until 4 d after sunscreen application. In a sample set of 35 urine samples from the general German population, 3cx-MnPrA was quantified in 94% (median 2.54 µg/L, maximum 78.3 µg/L) and MnBA in 3% (median < LOQ, maximum 0.18 µg/L) of the samples. The method will be applied in future human metabolism and human biomonitoring population studies.

Targeted metabolomics of pellicle and saliva in children with different caries activity

Pellicle is the initial proteinaceous layer that is formed almost instantaneously on all solid surfaces in the oral cavity. It is of essential relevance for any interactions and metabolism on the tooth surface. Up to now, there is no information on the metabolome of this structure. Accordingly, the present study aims to characterise the metabolomic profile of in-situ pellicle in children with different caries activity for the first time in comparison to saliva. Small molecules such as carbohydrates, amino acids, organic acids, and fatty acids, putatively involved in the formation of caries were quantified using mass spectrometry (MS)-based techniques, such as (stable isotope dilution analysis)-ultra-performance liquid chromatography-tandem MS and gas chromatography/electron ionisation-MS. Pellicle and corresponding saliva samples were collected from caries-active, caries-free and caries-rehabilitated 4- to 6-year-old children. The most abundant analytes in pellicle were acetic acid (1.2–10.5 nmol/cm2), propionic acid (0.1–8.5 nmol/cm2), glycine (0.7–3.5 nmol/cm2), serine (0.08–2.3 nmol/cm2), galactose (galactose + mannose; 0.035–0.078 nmol/cm2), lactose (0.002–0.086 nmol/cm2), glucose (0.018–0.953 nmol/cm2), palmitic acid (0.26–2.03 nmol/cm2), and stearic acid (0.34–1.81 nmol/cm2). Significant differences depending on caries activity were detected neither in saliva nor in the corresponding pellicle samples.

Differentiation between Aroma-Related Bioactives in Native Cold-Pressed Rapeseed Oils with Desired Sensory Attributes and with a Fusty/Musty Off-Flavor Using Multivariate Methods

A systematic quantitation of selected odorants using stable isotope dilution analysis (SIDA) was performed in six native cold-pressed rapeseed oils with desired retention of bioactives and desired sensorial attributes (“positive controls”, PCs), in nine native cold-pressed rapeseed oils eliciting a fusty/musty off-flavor (OFs), and in two rapeseed samples, from which two of the nine fusty/musty off-flavor oils were pressed. These data were used as basis to find marker compounds for the fusty/musty off-flavor by means of multivariate methods. Obtained data clustered pairwise in a heatmap clearly showed that all PCs and OFs were very similar to each other in their own group concerning the selected compounds. Additionally, data were statistically evaluated using principal component analysis (PCA). Based on selected compounds, a high accordance of PCs, in parallel to a clear discrimination to the group of fusty/musty off-flavor oils and to the two rapeseed samples, was achieved. In a biplot, compounds with the highest differences between the two oil groups were identified as 4-methylphenol, 3-methylbutanoic acid, 2-phenylethanol, 2-ethyl-3,6-dimethylpyrazine, and 2-methylbutanoic acid. Thus, these compounds can be seen as marker compounds for the fusty/musty off-flavor oils and the seeds and can, therefore, serve for quality control of the raw material within a quick routine method.

Rapid determination of the free and total hydroxytyrosol and tyrosol content in extra virgin olive oil by stable isotope dilution analysis and paper spray tandem mass spectrometry

A rapid analytical method for the determination of phenylethanoids content in extra virgin olive oil has been developed. The method intends to address the European regulation EU 432/2012 on health claims, which allows to report on the front label of olive oil, the positive health effects due to the consumption of this food. The innovative method is based on paper spray tandem mass spectrometry using deuterated standards. It relies on a two-step analysis, needed to assess the free form of tyrosol and hydroxytyrosol and their ester conjugates after hydrolysis treatment. Different olive oil samples have been analyzed and the classical analytical parameters such as accuracy, LOQ and LOD were calculated from fortified samples. The good values of the latters show the reliability of the new approach, that limits the time of analysis and sample preparation to few minutes.

Nosy confirmation: reconstitution of the characteristic odor of softwood via quantitative analysis and human sensory evaluation.

The significance of wood odors beyond flavoring effects for barbecues and wine aged in barrels has recently been under discussion. Wood has an immense influence on our physical and mental condition through our palate, such as positive health and mood-stimulating effects. As a result of increased public interest, the key odorants from various natural wood species were recently decoded. To gain profound insights into the contribution of single odorants to the overall scent of distinct wood types, this study compares several softwood species (Scots pine, incense cedar, European larch, Norway spruce, and white fir) by odor profile analysis. Nine odor-active constituents, which were previously detected in those woods, were quantified using stable isotope dilution analysis (SIDA). Odor activity values (OAVs) calculated on the basis of odor thresholds (OTs) determined in cellulose revealed hexanal, octanal, (E)-non-2-enal, p-cresol, vanillin, and thymoquinone as dominant odorants for wood odor. Recombination experiments by mixing the odorants in their naturally occurring concentrations in a cellulose matrix confirmed the successful characterization of the key odorants for Scots pine and incense cedar wood.

Metabolism and urinary excretion kinetics of di(2-ethylhexyl) adipate (DEHA) in four human volunteers after a single oral dose

Di(2-ethylhexyl) adipate (DEHA) is used as a substitute for the reprotoxic phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP). This study reports the first quantitative data on human in vivo DEHA metabolism and urinary metabolite excretion with the aim of providing tools for DEHA exposure and risk assessments. After DEHA was administered to four healthy volunteers (107−164 μg/kg body weight (bw)), urine samples were continuously and completely collected for 48 h and analyzed for the specific oxidized monoester metabolites mono-2-ethyl-5-hydroxyhexyl adipate (5OH-MEHA), mono-2-ethyl-5-oxohexyl adipate (5oxo-MEHA), and mono-5-carboxy-2-ethylpentyl adipate (5cx-MEPA), as well as for the non-specific hydrolysis product adipic acid (AA) using stable isotope dilution analysis. AA was confirmed as a major (urinary excretion fraction (FUE): 10–40%), yet non-specific DEHA metabolite. 5cx-MEPA was the major specific DEHA metabolite with an FUE of 0.20% (range: 0.17–0.24%). FUEs for 5OH-MEHA and 5oxo-MEHA were 0.07% (0.03–0.10%) and 0.05% (0.01–0.06%), respectively. The three specific metabolites were excreted with two concentration maxima (tmax1 = 1.5–2.3 h, tmax2 = 3.8–6.4 h). Elimination half-lives (t1/2, calculated after the second tmax) for 5cx-MEPA were calculated between 2.1–3.8 h. The majority (98–100%) of metabolites was excreted within 24 h. The FUE of 5cx-MEPA was applied to demonstrate its applicability for calculating daily intakes based on urinary metabolite levels from three pilot populations. Daily intakes were generally far below the tolerable daily intake (TDI) for DEHA (300 μg/kg bw/day). The highest daily intake (114 μg/kg bw/day) was calculated in individuals after consuming food that had been wrapped in DEHA containing cling film.

Urinary Metabolomic Markers of Protein Glycation, Oxidation, and Nitration in Early-Stage Decline in Metabolic, Vascular, and Renal Health.

Glycation, oxidation, nitration, and crosslinking of proteins are implicated in the pathogenic mechanisms of type 2 diabetes, cardiovascular disease, and chronic kidney disease. Related modified amino acids formed by proteolysis are excreted in urine. We quantified urinary levels of these metabolites and branched-chain amino acids (BCAAs) in healthy subjects and assessed changes in early-stage decline in metabolic, vascular, and renal health and explored their diagnostic utility for a noninvasive health screen. We recruited 200 human subjects with early-stage health decline and healthy controls. Urinary amino acid metabolites were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Machine learning was applied to optimise and validate algorithms to discriminate between study groups for potential diagnostic utility. Urinary analyte changes were as follows: impaired metabolic health—increased Nε-carboxymethyl-lysine, glucosepane, glutamic semialdehyde, and pyrraline; impaired vascular health—increased glucosepane; and impaired renal health—increased BCAAs and decreased Nε-(γ-glutamyl)lysine. Algorithms combining subject age, BMI, and BCAAs discriminated between healthy controls and impaired metabolic, vascular, and renal health study groups with accuracy of 84%, 72%, and 90%, respectively. In 2-step analysis, algorithms combining subject age, BMI, and urinary Nε-fructosyl-lysine and valine discriminated between healthy controls and impaired health (any type), accuracy of 78%, and then between types of health impairment with accuracy of 69%-78% (cf. random selection 33%). From likelihood ratios, this provided small, moderate, and conclusive evidence of early-stage cardiovascular, metabolic, and renal disease with diagnostic odds ratios of 6 – 7, 26 – 28, and 34 – 79, respectively. We conclude that measurement of urinary glycated, oxidized, crosslinked, and branched-chain amino acids provides the basis for a noninvasive health screen for early-stage health decline in metabolic, vascular, and renal health.

Development of a rapid multi-mycotoxin LC-MS/MS stable isotope dilution analysis for grain legumes and its application on 66 market samples

Mycotoxin contamination is a well-known problem in grains, nuts, and oilseeds. Similarly, grain legumes (pulses) are affected by fungal infections and, consequently, the formation of mycotoxins as well. However, only little data on mycotoxin occurrence exists for this commodity and only few validated quantification methods for multi-mycotoxin analysis have been reported. In order to effectively assess contamination levels for grain legumes, a high performance liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) was developed, validated for the matrix soybean and tested with further grain legumes. The method implements a QuEChERS-like extraction and a stable isotope dilution assay (SIDA) to facilitate the quantification of nine different mycotoxins (aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin). In a non-representative market sample screening, 66 samples of dry grain legumes from the Dutch and the German market were measured. Incidentally, high levels of ochratoxin A were detected in white beans and green peas with 157 μg/kg and 49.4 μg/kg, respectively. In addition, T-2 toxin was detected in one black lentil sample. A multi-mycotoxin method as a tool for rapid collection of data on mycotoxin levels in grain legumes may proof valuable for evaluating exposure and fate of mycotoxins during the whole value-added (feed and food) chain.

Characterization of Key Aroma Compounds in Pellets of Different Hop Varieties (Humulus lupulus L.) by Means of the Sensomics Approach.

The use of hops in beer brewing is mainly based on its content of bitter acids and aroma compounds. Due to the loss of volatile odorants during wort boiling, the so-called dry hopping is a possibility to intensify the hoppy aroma in the final beer. To clarify the potential of different hop varieties for aroma modulation of beer via dry hopping, key aroma compounds of three different hop varieties were characterized using the sensomics approach. Aroma extract dilution analysis revealed 41 aroma-active compounds, of which 39 were identified via gas chromatography-olfactometry and gas chromatography-mass spectrometry. The highest flavor dilution factor was determined for myrcene with a geranium-like odor. Fourteen substances were quantitated by stable isotope dilution analysis and further two odorants via the internal standard method; all of them revealed odor activity values (OAVs; ratio of concentration to odor threshold) ≥1. Linalool, 3-methylbutanoic acid, myrcene, and dimethyl trisulfide showed the highest OAVs (>1000) in all analyzed hop varieties. For validation of the analytical data, reconstitution models were prepared by adding all quantitated aroma compounds with OAVs ≥ 1 in their naturally occurring concentrations to cellulose as matrix. All three recombinates showed a very high similarity to the aroma profile of the respective hop sample, confirming the correct identification and quantitation of all key aroma compounds.

Fast screening and quantitative mass spectral imaging of thin-layer chromatography plates with flowing atmospheric-pressure afterglow high-resolution mass spectrometry.

Thin-layer chromatography (TLC) was interfaced to high-resolution mass spectrometry (MS) using a flowing atmospheric-pressure afterglow (FAPA) ambient desorption/ionization source. The influence of different TLC stationary phases on the mass spectral signal response and mass spectral image quality in FAPA-MS was carefully investigated. Specifically, a mixture of selected analgesics (acetaminophen), alkaloids (nicotine and caffeine), and steroids (cortisone) was deposited on different stationary phases (silica plates, RP-modified silica plates, CN-modified silica plates, DIOL-modified silica plates, and NH2-modified silica plates), and TLC plates with different thickness (100, 200, 250, 500, 1000, 2000μm) of the stationary phase. After analyte separation, mass spectral imaging was performed of the complete TLC plate via FAPA-MS and the detected ion abundance was compared. It was found that TLC plates with larger particle sizes (10-12μm) and thicker stationary phase layers (e.g., 1000μm and 2000μm) led to higher signals (protonated molecules) compared to smaller particles sizes (6-8μm) and thinner stationary phases (e.g., 100μm and 200μm). Instrumental detection limits in the low ng-range/band were determined for TLC-FAPA-MS of caffeine from RP-modified TLC silica plates. Lastly, a quantitative TLC-FAPA-MS method using stable isotope dilution analysis was developed and applied to the quantification of caffeine in energy drinks. Graphical abstract.

Streamlined approach for careful and exhaustive aroma characterization of aged distilled liquors

Solvent-assisted flavor evaporation (SAFE) is considered to be the best overall method to produce a “clean” aroma extract to avoid the loss of labile aroma compounds or the formation of thermally generated artifacts during gas chromatographic (GC) analysis. However, SAFE is both time consuming and labor intensive, especially when applied repeatedly for quantitation by stable isotope dilution analysis (SIDA), which requires the addition of isotopes within specific mass ratio ranges relative to target analytes. The streamlined approach described herein allows for accurate quantitation of odor-active components in liquor products with a single SAFE operation. The quantitative results achieved by this method are nearly identical for most odor-active components, except for specific semi-volatile constituents not recovered well by SAFE (e.g., vanillin and syringaldehyde in oak-aged liquors). The streamlined approach provides a simple and convenient way to expedite the careful and exhaustive study of the flavor chemistry of aged liquors.

Characterization of the Key Aroma Compounds in Two Differently Dried Toona sinensis (A. Juss.) Roem. by Means of the Molecular Sensory Science Concept.

A systematic approach for the characterization of the key aroma-active compounds in sun-dried Toona sinensis (SDTS) and vacuum-dried T. sinensis (VDTS) was performed by means of the molecular sensory science concept. A total of 64 aroma-active compounds were identified via gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Aroma extract dilution analysis (AEDA) and static headspace dilution analysis revealed 39 odorants in SDTS and 32 odorants in VDTS with flavor dilution (FD) factors from 8 to 4096, with the highest for vanillin and eugenol in both samples. Stable isotope dilution analysis (SIDA) and an internal standard method were applied to quantitate 42 odorants, revealing 35 compounds in concentrations above their respective odor thresholds in SDTS and 29 compounds in VDTS, respectively. Calculation of odor activity values (OAVs) indicated 2-isopropyl-3-methoxypyrazine, eugenol, and β-ionone with the highest OAVs in both samples. Recombination experiments of the overall aromas of SDTS and VDTS by mixing the odorants with OAVs ≥1 in their naturally occurring concentrations proved the successful identification and quantitation of the respective key odorants.

Characterization of the Key Aroma Compounds in Two Commercial Dark Chocolates with High Cocoa Contents by Means of the Sensomics Approach.

The overall aroma of two orthonasally distinguishable dark chocolates with high cocoa content (90% CC and 99% CC) was decoded using the systematic sensomics approach, including solvent extraction, separation of the volatiles, identification using aroma extract dilution analysis (AEDA) based on gas chromatography-olfactometry (GC-O) combined with gas chromatography-mass spectrometry (GC-MS), quantitation by stable isotope dilution analysis (SIDA), calculation of odor activity values (OAVs), and recombination experiments. Sixty-nine aroma-active compounds were identified and quantitation of 49 compounds revealed 28 odorants in 90% CC and 30 aroma-active compounds in 99% CC with OAVs ≥ 1. Among them, dimethyl trisulfide, acetic acid, 2-methoxyphenol, 3-methylbutanoic acid, phenylacetic acid, vanillin, and linalool showed the highest OAVs. Subsequently, very high similarities of the reconstitution models, containing all odorants with OAVs ≥ 1 in their naturally occurring concentrations in odorless sunflower oil as matrix, proved the correct identification and quantitation of all key odorants in both dark chocolates.