Profiling of Phenolic Metabolites in Feces from Menopausal Women after Long-Term Isoflavone Supplementation.

Phenolic compounds were screened by UPLC-ESI-MS/MS in the feces of 15 menopausal women before and after long-term isoflavone treatment. In total, 44 compounds were detected. Large intertreatment, interindividual, and intersample variations were observed in terms of the number of compounds and their concentration. Four compounds, the aglycones daidzein and genistein and the daidzein derivatives dihydrodaidzein and O-desmethylangolensin, were associated with isoflavone metabolism; these were identified only after the isoflavone treatment. In addition, 4-ethylcatechol, 3-hydroxyphenylacetic acid, and 3-phenylpropionic acid differed significantly in pre- and postintervention samples, whereas the concentration of 4-hydroxy-5-phenylvaleric acid showed a trend toward increasing over the treatment. The phenolic profiles of equol-producing and -non-producing groups were similar, with the exceptions of 3-hydroxyphenylacetic acid and 3-phenylpropionic acid, which showed higher concentrations in equol-non-producing women. These findings may help to trace isoflavone-derived metabolites in feces during isoflavone interventions and to design new studies to address their biological effects.

Phenolic Assesment of Uncaria tomentosa L. (Cat's Claw): Leaves, Stem, Bark and Wood Extracts.

The phenolic composition of extracts from Uncaria tomentosa L. from different regions of Costa Rica was studied using advanced analytical techniques such as UPLC/TQ-ESI-MS and (13)C-NMR. Samples from leaves, stems, bark and wood (n = 22) were subjected to extraction to obtain phenolic and alkaloid extracts, separately. Comparatively, higher values of total phenolic content were observed for leaves, stems and bark (225-494 gallic acid equivalents/g) than for wood extracts (40-167 gallic acid equivalents/g). A total of 32 non-flavonoid and flavonoid compounds were identified in the phenolic extracts: hydroxybenzoic acids (benzoic, salicylic, 4-hydroxybenzoic, prochatechuic, gallic, syringic and vanillic acids), hydroxycinnamic acids (p-coumaric, caffeic, ferulic and isoferulic acids), flavan-3-ols monomers [(+)-catechin and (-)-epicatechin)], procyanidin dimers (B1, B2, B3, B4, B5, B7 and two other of unknown structure) and trimers (C1, T2 and one of unknown structure), flavalignans (four unknown structures pertaining to the cinchonain family) and propelargonidin dimers (four unknown structures, reported for the first time in U. tomentosa). Additionally, alkaloid extracts obtained from the plant residue after phenolic extraction exhibited a content of tetracyclic and pentacyclic alkaloids ranging between 95 and 275 mg/100 g of dry material for bark extracts, and between 30 and 704 mg/100 g for leaves extracts. In addition, a minor alkaloid was isolated and characterized, namely 18,19-dehydrocorynoxinoic acid. Our results confirmed the feasibility of U. tomentosa as a suitable raw material for obtaining phenolic- and alkaloid-rich extracts of potential interest.

Comparative in vitro fermentations of cranberry and grape seed polyphenols with colonic microbiota.

In this study, we have assessed the phenolic metabolism of a cranberry extract by microbiota obtained from the ascending colon and descending colon compartments of a dynamic gastrointestinal simulator (SHIME). For comparison, parallel fermentations with a grape seed extract were carried out. Extracts were used directly without previous intestinal digestion. Among the 60 phenolic compounds targeted, our results confirmed the formation of phenylacetic, phenylpropionic and benzoic acids as well as phenols such as catechol and its derivatives from the action of colonic microbiota on cranberry polyphenols. Benzoic acid (38.4µg/ml), 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid (26.2µg/ml) and phenylacetic acid (19.5µg/ml) reached the highest concentrations. Under the same conditions, microbial degradation of grape seed polyphenols took place to a lesser extent compared to cranberry polyphenols, which was consistent with the more pronounced antimicrobial effect observed for the grape seed polyphenols, particularly against Bacteroides, Prevotella and Blautia coccoides-Eubacterium rectale.

Faecal metabolomic fingerprint after moderate consumption of red wine by healthy subjects.

Faecal metabolome contains information on the metabolites found in the intestine, from which knowledge about the metabolic function of the gut microbiota can be obtained. Changes in the metabolomic profile of faeces reflect, among others, changes in the composition and activity of the intestinal microorganisms. In an effort to improve our understanding of the biological effects that phenolic compounds (including red wine polyphenols) exert at the gut level, in this foodomic study we have undertaken a metabolome characterization of human faeces after moderate consumption of red wine by healthy subjects for 4 weeks. Namely, a nontargeted metabolomic approach based on the use of UHPLC-TOF MS was developed to achieve the maximum metabolite information on 82 human faecal samples. After data processing and statistical analysis, 37 metabolites were related to wine intake, from which 20 could be tentatively or completely identified, including the following: (A) wine compounds, (B) microbial-derived metabolites of wine polyphenols, and (C) endogenous metabolites and/or others derived from other nutrient pathways. After wine consumption, faecal metabolome was fortified in flavan-3-ols metabolites. Also, of relevance was the down regulation of xanthine and bilirubin-derived metabolites such as urobilinogen and stercobilin after moderate wine consumption. As far as we know, this is the first study of the faecal metabolome after wine intake.

Moderate consumption of red wine can modulate human intestinal inflammatory response.

In this study, 24 immune markers were analyzed in feces from healthy volunteers (n = 34) before and after consumption of a red wine (12% ethanol, 1758 mg/L total polyphenols) for 4 weeks. Analysis of the data permitted the differentiation of a six-volunteer subgroup showing unusually high basal values of cytokines. For this subgroup, consumption of wine significantly (P < 0.05) reduced the content of 16 markers to usual values, especially noticeable for those cytokines that promote initial inflammation (TNF-α, IL-6, and IFN-γ). On the contrary, no significant differences in the concentration of any immune marker were observed after wine consumption for the rest of the volunteers. Additionally, significant and negative correlations among cytokines IFN-γ, IL-8, and IL-6 and the total fecal content of phenolic metabolites were found for the high-cytokines-values subgroup, before wine intake. This study shows, for the first time, that moderate consumption of red wine could modulate inflammatory intestinal response in vivo.

Understanding the role of saliva in aroma release from wine by using static and dynamic headspace conditions.

The aim of this work was to determine the role of saliva in wine aroma release by using static and dynamic headspace conditions. In the latter conditions, two different sampling points (t = 0 and t = 10 min) corresponding with oral (25.5 °C) and postoral phases (36 °C) were monitored. Both methodologies were applied to reconstituted dearomatized white and red wines with different nonvolatile wine matrix compositions and a synthetic wine (without matrix effect). All of the wines had the same ethanol concentration and were spiked with a mixture of 45 aroma compounds covering a wide range of physicochemical characteristics at typical wine concentrations. Two types of saliva (human and artificial) or control samples (water) were added to the wines. The adequacy of the two headspace methodologies for the purposes of the study (repeatability, linear ranges, determination coefficients, etc.) was previously determined. After application of different chemometric analysis (ANOVA, LSD, PCA), results showed a significant effect of saliva on aroma release dependent on saliva type (differences between artificial and human) and on wine matrix using static headspace conditions. Red wines were more affected than white and synthetic wines by saliva, specifically human saliva, which provoked a reduction in aroma release for most of the assayed aroma compounds independent of their chemical structure. The application of dynamic headspace conditions using a saliva bioreactor at the two different sampling points (t = 0 and t = 10 min) showed a lesser but significant effect of saliva than matrix composition and a high influence of temperature (oral and postoral phases) on aroma release.

Impact of the nonvolatile wine matrix composition on the in vivo aroma release from wines.

The impact of the nonvolatile wine matrix composition on the retronasal aroma release of four volatile compounds added to different types of wines has been evaluated. For this purpose, a tailor-made retronasal aroma trapping device (RATD) was used to entrap the exhaled breath of six panelists previously trained in a specific consumption procedure. Five wines of different composition (white wine, sparkling white wine, young red wine, aged red wine, and a sweet wine) were evaluated. Prior to the evaluation, with the exception of the sweet wine, the wines were adjusted to the same ethanol content and aromatized with a mixture of four target volatile compounds. Aroma release data were submitted to multivariate statistical analysis in order to relate wine chemical composition and aroma release during wine drinking. Results showed interindividual differences and a clustering of panelists among lower and higher aroma releasers, which was in agreement to the differences in their breathing capacity. A significant influence of the matrix composition in the low aroma releasers group during wine consumption was observed. The consumption of red wines provoked a significantly higher aroma release than the consumption of white and sweet wines. From the chemical composition determined in the wine samples (pH, total acidity, total polyphenols, neutral polysaccharides, residual sugar, and nitrogenous compounds), the amount of total polyphenols was better correlated with the observed effect.

Lactobacillus plantarum IFPL935 favors the initial metabolism of red wine polyphenols when added to a colonic microbiota.

This work aimed to unravel the role of Lactobacillus plantarum IFPL935 strain in the colonic metabolism of a polyphenolic red wine extract, when added to a complex human colonic microbiota from the dynamic simulator of the human intestinal microbial ecosystem (SHIME). The concentration of microbial-derived phenolic metabolites and microbial community changes along with fermentative and proteolytic activities were monitored. The results showed that L. plantarum IFPL935 significantly increased the concentration of the initial microbial ring-fission catabolite of catechins and procyanidins, diphenylpropanol, and, similarly, 4-hydroxy-5-(3'-hydroxyphenyl)valeric acid production. Overall, the addition of L. plantarum IFPL935 did not have an impact on the total concentration of phenolic metabolites, except for batches inoculated with colonic microbiota from the effluent compartment (EC), where the figures were significantly higher when L. plantarum IFPL935 was added (24 h). In summary, the data highlighted that L. plantarum IFPL935 may have an impact on the bioavailability of these dietary polyphenols. Some of the microbial-derived metabolites may play a key role in the protective effects that have been linked to a polyphenol-rich diet.

Profiling of microbial-derived phenolic metabolites in human feces after moderate red wine intake.

A controlled and randomized trial study involving 41 healthy volunteers (33 intervention and 8 control subjects) was performed in order to establish changes in the microbial-derived phenolic metabolite profile of feces after moderate consumption of red wine (250 mL/day, 4 weeks). Out of the 35 phenolic metabolites identified, 10 compounds (mainly benzoic and 4-hydroxyvaleric acids) showed statistically significant increases (P < 0.05) after the wine intake. Also, the total phenolic metabolites content was significantly (P < 0.05) higher in the samples after the wine intake (625 ± 380 µg/g feces) in comparison to the samples before (358 ± 270 µg/g feces), and a tentative distribution of the volunteers into three groups could be established: <500, 500-1000, and >1000 µg/g feces. These results suggest that a different gut microbial capacity to metabolize wine polyphenols exists among the human population, as observed for polyphenols from other sources.

Comparative study of microbial-derived phenolic metabolites in human feces after intake of gin, red wine, and dealcoholized red wine.

The analysis of microbial phenolic metabolites in fecal samples from in vivo studies is crucial to understanding the potential modulatory effects derived from polyphenol consumption and its overall health effects, particularly at the gut level. In this study, the composition of microbial phenolic metabolites in human feces collected after regular consumption of either red wine, dealcoholized red wine, or gin was analyzed by UPLC-ESI-MS/MS. Red wine interventions produce a change in the content of eight phenolic acids, which are probably derived from the catabolism of flavan-3-ols and anthocyanins, the main flavonoids in red wine. Moreover, alcohol seemed not to influence the formation of phenolic metabolites by the gut microbiota. A principal component analysis revealed large interindividual differences in the formation of microbial metabolites after each red wine polyphenol intervention, but not after the gin intervention, indicating differences in the gut microbial composition among subjects.

Assessment of the impact of the addition of antimicrobial plant extracts to wine: volatile and phenolic composition.

BACKGROUND: Antimicrobial plant phenolic-rich extracts have been proposed as alternative to sulfites in the control of the malolactic fermentation (MLF) during winemaking. This addition may affect wine organoleptic properties. In this paper, we have investigated the changes in wine volatile and phenolic composition, after MLF, of a red wine treated with antimicrobial extracts from eucalyptus leaves and almond skins. RESULTS: Although addition of both extracts led to statistically significant changes (P < 0.05) in the concentration of several esters, alcohols, C13 nor-isoprenoids and volatile phenols, only few of these volatile compounds showed values of odour activity > 1 aroma unit; that is to say, whose concentrations were higher than their corresponding odour thresholds. With regard to phenolic compounds, the addition of both extracts did not significantly modify the content of anthocyanins, which predicts minor changes in wine colour. However, the content of non-anthocyanin phenolics was significantly higher in the wines treated with antimicrobial extracts, especially for flavonols, being the dose-over-taste factor for these wines significantly higher. Finally, principal component analysis showed that wines were mainly differentiated on the basis of whether MLF was conducted or not, and its method of performance (inoculated/spontaneous). CONCLUSION: Addition of antimicrobial extracts leads to some compositional changes in the wine, whose relevance needs to be addressed in future experiments, including wine sensorial analysis.

Immunoaffinity, capillary electrophoresis, and statistics for studying intact alpha 1-acid glycoprotein isoforms as an atherothrombosis biomarker.

Variations in the amino acid sequence, glycosylation, and/or other posttranslational modifications in glycoproteins give rise to different molecules of the glycoprotein called forms. Qualitative and/or quantitative alterations in these forms are related to pathophysiological situations in the individuals. In this study, a methodology to analyze these differences in forms of the alpha 1-acid glycoprotein (AGP) between healthy individuals and patients with two different vascular diseases is detailed. The whole methodology includes a sample preparation method based on immunochromatography, a capillary electrophoresis method for separation of AGP peaks (isoforms), and statistical methods (Linear Discriminant Analysis) for sample classification. As a result, it is shown that the methodology proposed allows studying the role of AGP isoforms as potential vascular disease biomarkers.

In vitro fermentation of grape seed flavan-3-ol fractions by human faecal microbiota: changes in microbial groups and phenolic metabolites.

With the aim of investigating the potential of flavan-3-ols to influence the growth of intestinal bacterial groups, we have carried out the in vitro fermentation, with human faecal microbiota, of two purified fractions from grape seed extract (GSE): GSE-M (70% monomers and 28% procyanidins) and GSE-O (21% monomers and 78% procyanidins). Samples were collected at 0, 5, 10, 24, 30 and 48 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and for analysis of phenolic metabolites. Both GSE-M and GSE-O fractions promoted growth of Lactobacillus/Enterococcus and decrease in the Clostridium histolyticum group during fermentation, although the effects were only statistically significant with GSE-M for Lactobacillus/Enterococcus (at 5 and 10 h of fermentation) and GSE-O for C. histolyticum (at 10 h of fermentation). Main changes in polyphenol catabolism also occurred during the first 10 h of fermentation; however, no significant correlation coefficients (P > 0.05) were found between changes in microbial populations and precursor flavan-3-ols or microbial metabolites. Together, these data suggest that the flavan-3-ol profile of a particular food source could affect the microbiota composition and its catabolic activity, inducing changes that could in turn affect the bioavailability and potential bioactivity of these compounds.

Toward a predictive model of Alzheimer's disease progression using capillary electrophoresis-mass spectrometry metabolomics.

Alzheimer's disease (AD) is the most prevalent form of dementia with an estimated worldwide prevalence of over 30 million people, and its incidence is expected to increase dramatically with an increasing elderly population. Up until now, cerebrospinal fluid (CSF) has been the preferred sample to investigate central nervous system (CNS) disorders since its composition is directly related to metabolite production in the brain. In this work, a nontargeted metabolomic approach based on capillary electrophoresis-mass spectrometry (CE-MS) is developed to examine metabolic differences in CSF samples from subjects with different cognitive status related to AD progression. To do this, CSF samples from 85 subjects were obtained from patients with (i) subjective cognitive impairment (SCI, i.e. control group), (ii) mild cognitive impairment (MCI) which remained stable after a follow-up period of 2 years, (iii) MCI which progressed to AD within a 2-year time after the initial MCI diagnostic and, (iv) diagnosed AD. A prediction model for AD progression using multivariate statistical analysis based on CE-MS metabolomics of CSF samples was obtained using 73 CSF samples. Using our model, we were able to correctly classify 97-100% of the samples in the diagnostic groups. The prediction power was confirmed in a blind small test set of 12 CSF samples, reaching a 83% of diagnostic accuracy. The obtained predictive values were higher than those reported with classical CSF AD biomarkers (Aß42 and tau) but need to be confirmed in larger samples cohorts. Choline, dimethylarginine, arginine, valine, proline, serine, histidine, creatine, carnitine, and suberylglycine were identified as possible disease progression biomarkers. Our results suggest that CE-MS metabolomics of CSF samples can be a useful tool to predict AD progression.

Comprehensive assessment of the quality of commercial cranberry products. Phenolic characterization and in vitro bioactivity.

Cranberry (Vaccinium macrocarpon) products have been widely recommended in traditional American medicine for the treatment of urinary tract infection (UTI). A total of 19 different commercial cranberry products from American and European markets have been analyzed by different global phenolic methods and by UPLC-DAD-ESI-TQ MS. In addition, in vitro antioxidant capacity and uropathogenic bacterial antiadhesion activity tests have been performed. Results revealed that products found in the market widely differed in their phenolic content and distribution, including products completely devoid of flavan-3-ols to highly purified ones, either in A-type proanthocyanidins (PACs) or in anthocyanins. The product presentation form and polyphenolic profile widely affected the antiadhesion activity, ranging from a negative (nulel) effect to a MIC = 0.5 mg/mL for cranberry powders and a MIC=112 mg/mL for gel capsule samples. Only 4 of 19 products would provide the recommended dose of intake of 36 mg total PACs/day. Of most importance was the fact that this dose would actually provide as low as 0.00 and up to 205 µg/g of procyanidin A2, indicating the lack of product standardization and incongruence between global and individual compound analysis.

Comparative study of the inhibitory effects of wine polyphenols on the growth of enological lactic acid bacteria.

This paper reports a comparative study of the inhibitory potential of 18 phenolic compounds, including hydroxybenzoic acids and their derivatives, hydroxycinnamic acids, phenolic alcohols and other related compounds, stilbenes, flavan-3-ols and flavonols, on different lactic acid bacteria (LAB) strains of the species Oenococcus oeni, Lactobacillus hilgardii and Pediococcus pentosaceus isolated from wine. In general, flavonols and stilbenes showed the greatest inhibitory effects (lowest IC50 values) on the growth of the strains tested (0.160-0.854 for flavonols and 0.307-0.855 g/L for stilbenes). Hydroxycinnamic acids (IC50 > 0.470 g/L) and hydroxybenzoic acids and esters (IC50 >1 g/L) exhibited medium inhibitory effect, and phenolic alcohols (IC50 > 2 g/L) and flavanol-3-ols (negligible effect) showed the lowest effect on the growth of the LAB strains studied. In comparison to the antimicrobial additives used in winemaking, IC50 values of most phenolic compounds were higher than those of potassium metabisulphite for O. oeni strains (e.g., around 4-fold higher for quercetin than for potassium metabisulphite), but lower for L. hilgardii and P. pentosaceus strains (e.g., around 2-fold lower for quercetin). Lysozyme IC50 values were negligible for L. hilgardii and P. pentosaceus, and were higher than those corresponding to most of the phenolic compounds tested for O. oeni strains, indicating that lysozyme was less toxic for LAB than the phenolic compounds in wine. Scanning electron microscopy confirmed damage of the cell membrane integrity as a consequence of the incubation with antimicrobial agents. These results contribute to the understanding of the inhibitory action of wine phenolics on the progress of malolactic fermentation, and also to the development of new alternatives to the use of sulphites in enology.

Targeted analysis of conjugated and microbial-derived phenolic metabolites in human urine after consumption of an almond skin phenolic extract.

A single-blind, placebo-controlled, and randomized trial study was carried out with 16 healthy volunteers (7 men and 5 women). The test group ingested an encapsulated almond skin phenolic extract (884 mg of total polyphenols/dose) containing flavan-3-ols, flavonols, and flavanones, whereas the placebo group ingested microcrystalline cellulose. Our aim in this study was to determine changes in the urinary excretion of conjugated and microbial-derived phenolic metabolites before (-2 to 0 h) and after (0-2, 2-6, 6-10, and 10-24 h) intake of the almond polyphenols compared with the placebo group. For the test group, maximum urinary excretion of (epi)catechin and naringenin conjugates derived from phase II metabolism was attained at 2-6 h after consumption of the almond skin extract and excretions differed from the placebo group during this time period (P ≤ 0.0001). However, excretion of conjugated metabolites of isorhamnetin was highest at 10-24 h and did not differ from the placebo group during this time (P > 0.05). Hydroxyphenylvalerolactones reached maximum urinary levels at 6-10 h after consumption of almond polyphenols, and excretion differed from the placebo group during this time period (P = 0.0004). For the test group, excretions of phenolic acids (hydroxyphenylpropionic, hydroxyphenylacetic, hydroxybenzoic, and hydroxycinnamic acids) did not differ from the placebo group at any time period of urine collection (P > 0.05). The findings presented in this work provide evidence concerning the bioavailability of almond skin polyphenols considering the effects of both phase II and microbial metabolism.

Antimicrobial activity of phenolic acids against commensal, probiotic and pathogenic bacteria.

Phenolic acids (benzoic, phenylacetic and phenylpropionic acids) are the most abundant phenolic structures found in fecal water. As an approach towards the exploration of their action in the gut, this paper reports the antimicrobial activity of thirteen phenolic acids towards Escherichia coli, Lactobacillus spp., Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The growth of E. coli ATCC 25922 was inhibited by only four of the phenolic acids tested at a concentration of 1000 microg/mL, whereas pathogenic E. coli O157:H7 (CECT 5947) was susceptible to ten of them. The genetically manipulated E. coli lpxC/tolC strain was highly susceptible to phenolic acids. The growth of lactobacilli (Lactobacillus paraplantarum LCH7, Lactobacillus plantarum LCH17, Lactobacillus fermentum LPH1, L. fermentum CECT 5716, Lactobacillus brevis LCH23, and Lactobacillus coryniformis CECT 5711) and pathogens (S. aureus EP167 and C. albicans MY1055) was also inhibited by phenolic acids, but to varying extents. Only P. aeruginosa PAO1 was not susceptible to any of the phenolic compounds tested. Structure-activity relationships of phenolic acids and some of their diet precursors [(+)-catechin and (-)-epicatechin] were established, based on multivariate analysis of microbial activities. The antimicrobial properties of phenolic acids reported in this paper might be relevant in vivo.

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans.

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism.

API-IT-MS for measuring aroma release from dentifrice products using a device to simulate tooth brushing.

A mechanical tooth brushing device coupled to an atmosphere pressure ionization ion trap mass spectrometer (API-IT-MS) combination has been developed to study the influence of time and dilution on aroma release from a model dentifrice system. API-IT-MS response to nine commonly used dentifrice flavor components was initially studied. Linear regression models were developed based on an exponential dilution method (EDA) to permit quantification of these compounds. Good linear fits were generated for the majority of compounds (R(2) > 0.92). The threshold detection limits were also calculated, and they greatly depended on the type of aroma compound. A brushing device was then coupled to the API-IT-MS and used to monitor the release profile of three aroma components from a model dentifrice system at flavor concentrations ranging from 0.1 to 20 mg g(-1). Large differences in the aroma release patterns were observed for different compounds (limonene, menthone and cinnamic aldehyde) that depended on their physicochemical characteristics (vapor pressure and log P), and on additional factors such as aroma-matrix interactions. In addition, a linear increase in API-IT-MS response with increased flavor concentration up to 1 mg g(-1) flavor was observed, while at higher concentrations, e.g. between 1 and 20 mg g(-1), a plateau in response was noticed. This suggests that at concentrations above 1 mg g(-1) a transition from a purely dissolved state to an emulsified state occurred. This fact influenced the time-dependent characteristics of the release curve (I(max) and t(max)) for the three assayed flavor compounds.