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Two proanthocyanidin (PA) fractions, one (Sdp3) with the mean degree of polymerization (mDP) of 3 and the other (Sdp9) with mDP of 9, were obtained from a Vitis vinifera cv. Shiraz grape seed extract. The PA fractions were directly analyzed by electrospray ionization mass spectrometry (ES-MS) and negative ion mass spectra were recorded. The mass spectrum of Sdp3 exhibited only singly charged ions corresponding to the molecular mass of PA with a degree of polymerization (DP) up to 9 (nonamers). In contrast, Sdp9 yielded rather complex mass spectra featuring ions with single [M - H](-), double [M - 2H](2-) and triple [M - 3H](3-) charge representing the molecular masses of PAs up to a DP of 28. In addition, the degree of galloylation per procyanidin (DG) was observed to be up to 5 (pentagallates) in Sdp3 and 8 (octagallates) in the Sdp9. This is the first evidence obtained by mass spectrometry for the distribution of grape seed PAs with such a high degree of polymerization and a broad diversity of galloylation. ES-MS data together with the complementary information provided by acid hydrolysis provides a detailed picture of the composition of grape seed PAs.

The volatile phenols guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol, o-, m-, and p-cresol, as well as their glycoconjugates, have previously been shown to be present in elevated concentrations in smoke-tainted wine. Sensory descriptive analysis experiments, with addition of free volatile phenols in combination with their glycosidically bound forms, were used to mimic smoke taint in red wines. The addition of volatile phenols together with glycoconjugates gave the strongest off-flavor. The hydrolysis of glycosidically bound flavor compounds in-mouth was further investigated by in vitro and in vivo experiments. The results indicate that enzymes present in human saliva are able to release the volatile aglycones from their glycoconjugates even under low pH and elevated ethanol conditions, confirming that in-mouth breakdown of monosaccharide and disaccharide glycosides is an important mechanism for smoke flavor from smoke affected wines, and that this mechanism may play an important general role in the flavor and aftertaste of wine.

Continuing our studies on the formation of tryptophan-derived glycoconjugates, we observed that the reactivity of tryptophan was significantly higher towards D-ribose in comparison to D-glucose. Detailed analysis of the reaction products obtained from tryptophan and the pentose sugars D-ribose, D-xylose, and L-arabinose by means of HPLC-mass spectrometry revealed the formation of several C-glycosyl derivatives, N-glycosides, glyco-tetrahydro-β-carbolines, and of carbohydrate-derived β-carbolines. Following the isolation of these compounds from model reactions, the structure elucidation was accomplished by NMR spectroscopy. Finally, we proved the presence of these novel tryptophan derivatives in various food samples by HPLC-MS analysis.

Numerous carbohydrate-derived beta-carbolines have been identified for the first time in model reactions of tryptophan with glucose and ribose, as well as in food samples. Extending these structural studies, we performed detailed investigations to elucidate the corresponding intermediates and formation pathway of these alkaloids. Degradation experiments with purified tryptophan glycoconjugates established that only glyco-tetrahydro-beta-carboline-3-carboxylic acids, and not the N-glycosides nor the C-glycoconjugates represented the direct precursors of carbohydrate-derived beta-carbolines. In addition, the significance of the oxidative decarboxylation reaction as the initial step for formation of 1-substituted beta-carbolines was proven. Finally, the stereochemistry of the carbohydrate-derived side chain was studied by means of CD spectroscopy and HPLC-CD experiments. These detailed stereochemical analyses yielded experimental evidence for the racemization steps required for formation of the carbohydrate-derived harman alkaloids and confirmed the proposed reaction pathway.

An efficient evaluation procedure for the chemical screening and on-line structural elucidation of dimeric naphthylisoquinoline alkaloids has been developed. The method is based on the lead tetraacetate oxidation of the central binaphthalene core of the alkaloids. UV spectra of the extracts after addition of the oxidant show, in the presence of naphthylisoquinoline dimers, the appearance of a characteristic long-wavelength absorption indicative of dinaphthoquinones. The efficiency and relevance of the method has been demonstrated in the discovery of a constitutionally and configurationally new dimeric naphthylisoquinoline alkaloid, named ancistrogriffithine A (4), from the previously uninvestigated Asian liana Ancistrocladus griffithii. After verification of this screening result by LC−ESI-MS/MS, the constitution and the relative configuration of the compound were elucidated on line, by LC−NMR and LC−CD on the extract. Using an LC−NMR-WET-ROESY experiment, it was possible for the first time to determine the relative axial configuration of a natural biaryl compound on line, by observing long-range ROE interactions. Finally, an oxidative degradation right on the extract delivered the absolute configuration of 4, without isolation of the alkaloid. Ancistrogriffithine A is the as yet only dimeric naphthylisoquinoline from an Asian Ancistrocladaceae plant.

The incidence of esophageal adenocarcinoma is increasing rapidly. In rats, surgically induced duodenoesophageal reflux is carcinogenic. One proposed mechanism of carcinogenesis is based on the reaction of physiological bile acids with nitrite to produce carcinogenic N:-nitroso amides. To test this hypothesis, duodenal juice was analyzed for endogenously formed N:-nitroso bile acids and its genotoxicity was determined. Esophagojejunostomy was performed on 15 Sprague-Dawley rats to produce duodeno-esophageal reflux. At the time of surgery and 2 and 6 weeks later, duodenal contents were aspirated and analyzed immediately. High performance liquid chromatography coupled to tandem mass spectrometry was used to detect bile acids and their nitroso derivates. Genotoxicity was assessed using a micronucleus test. The characteristic pattern of bile acid derivatives, with taurocholic acid (TCA) and glycocholic acid (GCA) as the predominant conjugates, was detected in all samples. However, even selective reaction monitoring experiments failed to demonstrate the presence of any N:-nitroso-TCA or N:-nitroso-GCA. In addition, other nitroso derivatives could not be detected in any of the samples by neutral loss experiments monitoring the loss of nitric oxide (detection limit 0.1% of the concentration of TCA). All samples were cytotoxic, but neither the preoperative nor the postoperative samples were genotoxic. Duodenal juice was cytotoxic but not genotoxic. Tumorigenesis of esophageal adenocarcinoma in the rodent model could not be linked to a specific carcinogen, especially not to nitroso bile acids. Chronic inflammation is likely to be the mechanism of carcinogenesis by duodenogastric reflux.

EPR and ESI-MS/MS evidence is presented that in the absence of an olefinic substrate the reaction between the MnIII(salen) complexes A1 (X = Cl) and A2 (X = PF6) and PhIO or NaOCl as oxygen sources...

[reaction: see text] The catalytic oxidation of the allylic alcohols 1d-n with iodosobenzene diacetate, mediated by the [Cr(III)(salen)]X complex, affords the respective enones in excellent chemoselectivity for Cl(-) as counterion [complex A(Cl)], while for the counterions TfO(-) [complex A(TfO)] and PF(6)(-) [complex A(PF(6)())] nearly equal amounts of enone and epoxide are observed. This counterion-dependent oxidation of allylic alcohols by Cr(III)(salen) complexes is rationalized in terms of Lewis acid catalysis by the complex A(Cl) and redox catalysis for A(TfO) and A(PF(6)()).

Using sterically hindered 2,9-diarylphenanthrolines (I) as well as unsubstituted or 4,7-disubstituted phenanthrolines (II) the first preparation of open chain heteroleptic bis(phenanthroline) complexes [M(I)(II)]n+ with M = Ag+ or Zn2+ has been realised. Their structure was confirmed by electrospray MS, NMR and in one case X-ray data. With two sets of phenanthroline ligands, three stereoisomers of [M(I)(II)]+ were detected in the silver(I) and for comparative reasons in the corresponding copper(I) complexes. On the basis of NMR experiments these were assigned as in-in, in-out and out-out isomers.

Evaluating the formation of tryptophan glycoconjugates other than well-established Amadori rearrangement products, HPLC-tandem MS (MS/MS) analysis of human urine collected from several healthy individuals proved the presence of one distinct tryptophan C-glycosyl compound [Horiuchi, Yonekawa, Iwahara, Kanno, Kurihara and Fujise (1994) J. Biochem. (Tokyo) 115, 362-366]. After isolation, unambiguous identification of this novel tryptophan metabolite as 2-(α-mannopyranosyl)-L-tryptophan was achieved by tandem MS combined with NMR spectroscopy including homonuclear COSY, heteronuclear multiple-bond connectivity and 1H-detected heteronuclear multiple-quantum coherence experiments. Remarkably, a thorough evaluation of vicinal proton-proton coupling constants in different solvents and nuclear Overhauser effect experiments demonstrate the predominant axial orientation of the hydroxymethyl group of the hexopyranosyl residue. Likewise this spatial arrangement indicates that the respective α-anomeric C-mannosylhexopyranose is preferentially adopting a 1C4 conformation in acidic methanol. Whereas only one distinct tryptophan mannoconjugate could be observed in human urine, HPLC-MS/MS analysis of food samples for the first time led to the identification of numerous N1-(β-D-hexopyranosyl)-L-tryptophan, 2-(β-D-hexopyranosyl)-L-tryptophan and 1-(1,2,3,4,5-p e n t a h y d r o x y p e n t - 1 - yl) - 1, 2, 3, 4 - t e t r a h y d r o - β - c a r b o l i n e - 3 -carboxylic acid derivatives derived from the condensation of tryptophan with aldohexoses. Taking into consideration the significant differences between profiles and configurations of tryptophan glycoconjugates originating from dietary sources and human urine, C-2 mannosylation of tryptophan residues [de Beer, Vliegenthart, Loeffler and Hofsteenge (1995) Biochemistry 34, 11785-11789] represents a novel enzymic pathway in tryptophan metabolism in humans.

Two new indolocarbazole alkaloids, 3-hydroxy-3'-demethoxy-3'-hydroxystaurosporine (5) and 11-hydroxy-4'-N-demethylstaurosporine (6), were isolated from the marine ascidian Eudistoma toealensis and its predator, the marine flatworm Pseudoceros sp. In addition, five known derivatives were isolated in their protonated states, which caused the pyran-ring system to adopt a boat conformation. The structures were determined by 1D and 2D homonuclear and (1)H-detected heteronuclear NMR spectroscopy and from comparisons with published data. The heteronuclear correlations were necessary to establish reliable data for the structure elucidation.

Ochratoxin A (OTA) is a nephrotoxic and nephrocacinogenic mycotoxin commonly produced by several ubiquitousAspergillus andPenicillium species. OTA is found predominantly in cereals and derived products, and therefore OTA may be transferred into beer from contaminated grain. Thirty-five samples of German beers were analyzed: OTA was extracted with toluene and purified by solid phase extraction using silica cartridges. OTA was determined by high-performance liquid chromatography with fluorescence detection. Since of fluorescence detection is not specific in this instance, the beer samples were also analyzed by high-performance liquid chromatography electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). OTA levels in the range of 0.1–0.2 μg L−1 were found in 9 beer samples, 21 samples contained trace amounts of OTA (

Synthesis of six new flavor sulfates (benzyl sulfate, 2-phenylethyl sulfate, 2,5-dimethyl-4-hydroxy-3(2H)-furanone sulfate, α-ionol sulfate, vomifoliol sulfate, linalyl sulfate) was performed in order to screen for these compounds in plants. Structural elucidation was performed by NMR spectroscopy and a screening method developed by using high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS). The results obtained with various plant tissues indicate that sulfation of flavor compounds is a common pathway in plant metabolism.

A column liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI-MS–MS) method is described for the analysis of ochratoxin A (OTA) in human food. In combination with selected reaction monitoring (SRM) detection limits of 20 pg (50 fmol) OTA on column were established by the use of model solutions. The present LC–ESI-MS–MS method allows the sensitive and selective monitoring of OTA in complex matrices.

Ten heterocyclic aromatic amines (HAA) (1)–(10) were analyzed in commercially available meat products and fish. After sample preparation by Extrelut treatment and subsequent solid phase extraction applying propylsulphonic and C18 silica cartridges, HPLC-ESI-MS-MS using selected reaction monitoring (SRM) and d3-PhIP and d3-MeIQx as internal and external standards, respectively, revealed the widely distributed presence of PhIP (8) and MeIQx (4), ranging from 0.1 to 5.3 ng g−1 and 0.1 to 5.2 ng g−1, respectively. Lower amounts were found for 4,8-DiMeIQx (5) and 7,8-DiMeIQx (6), ranging from 0.2 to 2.0 ng g−1 and 0.1 to 0.2 ng g−1, respectively. The other HAA under study, i.e. IQ, MeIQ, 4,7,8-TriMeIQx, Glu-P-1, and Glu-P-2 were not determinable under the experimental conditions used (determination limit 0.1 ng g−1).

The atropodiastereomeric dimeric naphthylisoquinoline alkaloids, michellamines A ( 1a ), B ( 1b ) and C ( 1c ), together with their monomers, korupensamines A ( 2a ) and B ( 2b ), were investigated using electrospray ionization tandem mass spectrometry coupled to liquid chromatography (LC–ESI-MS–MS). From the spectra obtained, characteristic product ions were chosen to monitor the chromatographic separation achieved on an RP-18 column. Under acidic conditions required for chromatographic analysis, the monomeric alkaloids 2a and 2b yielded protonated molecules [M+H] + , while the dimers, the michellamines, exhibited doubly protonated [M+2H] 2+ molecules. In addition, the coeluting alkaloids 1b and 2b were identified unambiguously by means of tandem mass spectrometry. Thus, together with the retention times of the alkaloids, the product ion spectra allowed us the identification of michellamines in the presence of their presumed biogenetic monomeric precursors. Application of the HPLC–MS–MS method successfully proved the enzymatic formation of michellamine C ( 1c ) by in vitro dimerization of korupensamine B ( 2b ).

Based on electrospray ionization – tandem mass spectrometry coupled to liquid chromatography (HPLC-ESI-MS/MS) a method for separation and selective detection of 1,2,3,4-tetrahydro-β-carboline derivatives (THC) was developed. Retro-Diels Alder (RDA) fragmentation of the tetrahydropyrido moiety resulted in the characteristic neutral loss of 73 amu for tryptophan-derived THC-3-carboxylates. Accordingly, Pictet-Spengler condensation products of tryptamin exhibited product ions formed by loss of 29 amu. However, THC-1-carboxylates as obtained by reaction of tryptamin with α-oxo acids also yielded product ions [M+H-73]+, apparently originating from the combination of RDA-cleavage plus subsequent decarboxylation. As result, one had to consider the possibility of false-positive identification of THC-3-carboxylates in presence of isomeric THC-1-carboxylates. In order to overcome these analytical pitfalls, the unequivocal identification of trace amounts of THC-3-carboxylates by HPLC-MS/MS required the chromatographic separation of isomeric THC prior to selected reaction monitoring (SRM). Utilizing SRM, limits of detection for various THC were established in the 10 ng mL–1 range. Subsequent analysis of food samples like seasoning sauce and yeast extract by HPLC-ESI-MS/MS revealed the presence of tryptamin-derived 1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid, 1-methyl-tetrahydro-β-carboline-1-carboxylic acid, 1-carboxyethyl-tetrahydro-β-carboline and 1-carboxyethyl-tetrahydro-β-carboline-1-carboxylic acid beside established THC-3-carboxylates and -1,3-dicarboxylates.

Chromatographic separation of phosphorylated carbohydrates was achieved on a β-cyclodextrin (CD) bonded HPLC column. Applying acetonitrile–aqueous ammonium acetate gradient elution, the separation of sugar phosphates relied on the combination of anion-exchange and hydrophilic interaction properties of CD bonded phases in the presence of ammonium ions. Furthermore, this solvent system allowed on-line coupling to electrospray ionization mass spectrometry (ESI-MS) and direct structural characterisation of carbohydrate phosphates by tandem mass spectrometry (MS–MS). In addition to the analysis of various pentose and hexose phosphates, the HPLC–ESI-MS–MS method was successfully applied to demonstrate the enzymatic formation of D-1-deoxyxylulose 5-phosphate from pyruvate and glyceraldehyde phosphate catalysed by yeast transketolase.

2,5-Dimethyl-4-hydroxy-3[2H]furanone (Furaneol, DMHF) [3658-77-3], an important flavour constituent of strawberry fruit, was administered to four male and two female volunteers using fresh strawberries as a natural DMHF source. The amount excreted was determined by measuring urinary levels of DMHF and DMHF glucuronide. DMHF glucuronide was synthesized and the structure elucidated by mens of 1H, 13C and two dimensional nuclear magnetic resonance, as well as mass spectral data. Identification and quantification of DMHF glucuronide in human urine were achieved after solid phase extraction on XAD-2 using reverse-phase reverse-phase HPLC with either on-line UV/VIS or electrospray tandem mass spectrometry detection. Male and female volunteers excreted 59-69% and 81-94%, respectively, of the DMHF dose (total of free and glycosidically bound DMHF in strawberries) as DMHF glucuronide in urine within 24 hr. The amount of DMHF excretion was independent of the dose size and the ratio of free to glycosidically bound forms of DMHF in strawberry fruit. DMHF, DMHF glucoside and its 6'-O-malonyl derivative, naturally occurring in strawberries, were not detected in human urine.

A method for selective detection of 1,2,3,4-tetrahydro-β-carbolinecarboxylic acids (THCCs) was developed based on electrospray ionization−tandem mass spectrometry coupled to liquid chromatography (HPLC−ESI−MS/MS). Low-energy collision-induced dissociation (CID) led to characteristic fragment ions due to neutral loss of 73 amu. Subsequently, constant neutral loss scanning was used for substructure specific screening of THCCs in food samples. Detection limits for HPLC−ESI−MS/MS analysis of THCCs applying neutral loss experiments were established at 100 ng mL-1 (ca. 2.5 pmol on column). Application of this MS/MS method enabled us to detect THCC derivatives derived from Pictet−Spengler condensation of tryptophan with α-oxo acids. Subsequently, diastereomeric 1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid 3a/b, 1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid 4a/b, and 1-(2‘-carboxyethyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid 5a/b were identified in alcoholic beverages, seasoning ...

The enantiomeric distrubution of solerone (5-oxo-4-hexanolide) 1, ethyl 4-hydroxy- 5-oxohexanoate 2, ethyl 5-hydroxy-4-oxohexanoate 3, 4-oxo-5-hexanolide 4, and solerol (5-hydroxy-4-hexanolide) 5 in sherry wines was determined by several HRGC techniques. While gas chromatography-mass spectrometry on chiral cyclodextrin phases (chiral GC-MS) prevented any racemization of α-ketols 2 and 3 caused by keto-enol tautomerization during analysis, multidimensional gas chromatography MDGC (coupled either by “live-T” switching or by “moving column stream switching” MCSS) led to rearranged constitutional-and stereoisomers. The stereochemical results are discussed regarding the biogenesis of sherry constituents 1–5.

A rapid method is described for the identification of fatty acid hydroperoxides by electrospray ionization-tandem mass spectrometry coupled to liquid chromatography without any derivatization required prior to analysis. Localization of fatty acid hydroperoxides in complex mixtures was achieved by monitoring the loss of hydrogen peroxide using constant neutral loss scanning. In the presence of 5 mM NH4OAc in methanol-water, adduct ions [M + NH4]+ were formed almost exclusively, directly revealing the molecular mass of the thermolabile hydroperoxides. In addition, low energy collision-induced dissociation of precursor ions [M + NH4]+ led to characteristic product ions from both the 9- and 13-regioisomers. Thus, electrospray ionization-tandem mass spectrometry provides a straightforward approach for the study of the regioselectivity of lipoxygenase catalysis without any derivatization step required prior to analysis.

By selective modification of serine 221 in the active site, the preceding protease subtilisin carlsberg gained peroxidase activity which was subsequently utilized for the enantioselective reduction of hydroperoxides. Thus, semisynthetic seleno-subtilisin selectively catalyzed the kinetic resolution of four racemic alkyl aryl hydroperoxides p -R 2 -C 6 H 4 CH(OOH)R 1 (R 1 =CH 3 , CH 2 OH, CH(CH 3 ) 2 ; R 2 =H, Cl). The enantiomeric distribution of all products was determined and kinetic parameters were evaluated, indicating seleno-subtilisin as the first semisynthetic enzyme with catalytic efficiency comparable to native enzymes and good enantioselectivity. In order to facilitate further studies, structural motifs responsible for enantioselectivity and efficiency of catalysis were outlined in the discussion, thus providing a rational basis for the selection of suitable substrates.

A biomimetic synthesis of solerone (5-oxo-4-hexanolide, 1) using both enzymatic and acid-catalyzed reactions was performed. Starting from l-glutamic acid 5-ethyl ester (2) enzymatic oxidative deami...

This survey covers analysis, occurrence, and formation of tryptophanderived compounds in food and edible plants as well as dietary and metabolic aspects of tryptophan derivatives. After summarizing biosynthesis and metabolism of tryptophan, attention is focused on products obtained by oxidative degradation reactions, compounds derived from the Maillard reaction, tetrahydro‐s‐carbolines, and harman derivatives. In addition, formation of heterocyclic aromatic amines and relevance of tryptophan contaminants for the outbreak of the eosinophilia‐myalgia syndrome (EMS) are reviewed.

The development of techniques utilizing atmospheric pressure ionization, namely atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), has pioneered the coupling of liquid chromatography (HPLC) with mass spectrometry in recent years. Both ESI and APCI generate ions from polar and labile biomaterials with remarkable ease and efficiency. In particular, the use of HPLC with tandem mass spectrometry (MS-MS) opens further dimensions in the field of bioorganic analysis. Thus, HPLC-MS-MS provides the tools for direct elucidation of the structure and variety of polar natural compounds in complex matrices. In order to develop efficient and straightforward strategies for the analysis of polar natural products, the potential and the limitations of these hyphenated analytical techniques are discussed using heterocyclic aromatic amines, fumonisins, acylated glycoconjugates and regioisomeric fatty acid hydroperoxides as examples.

Linalool, nerol, geraniol, citronellol and α-terpineol were phosphorylated and the products purified by adsorption chromatography on XAD-2. High performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis using the electrospray ionization technique (ESI) revealed the occurrence of mono-, di-, tri- and tetraphosphate esters of the respective monoterpenes. In extracts obtained by XAD-2 adsorption chromatography followed by anion exchange chromatography from green tea leaves, phosphate and diphosphate esters of linalool and α-terpineol were identified for the first time by ESI LC–MS/MS. The corresponding monoterpene alcohols were analysed by high resolution gas chromatography–mass spectrometry after hydrolysis of the extract using acid phosphatase (EC 3.1.3.2).

A new method has been developed for qualitative and quantitative analysis of fumonisin FB1 and FB2 in corn and corn products. After extractive sample preparation followed by solid phase extraction using a strong anionexchanger, highest selectivity and sensitivity was achieved by high-performance liquid chromatography-electrospray-ionization, tandem mass spectrometry (HPLC-ESI-MS-MS) analysis combined with selected reaction monitoring (SRM). With deuterated fumonisin FB1-d6 as internal standard the routine quantitation limit for FB1 was set at 400 pg (0.8 ng g−1 sample) with a signal-to-noise ratio of 10∶1. Almost all corn grit and corn meal samples under study were found to be contaminated with fumonisin FB1 and FB2 in concentrations ranging from 1 to 200 ng g−1. Lower fumonisin levels were found in cornflakes, popcorn and baby food, while no fumonisins were detected in canned sweet corn.

Sensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL−1 were established by use of HAA model solutions.

The potent flavour compound 2-acetyl-1-pyrroline (1) was identified for the first time in wines spoiled by mousy taint. Identification was verified by comparison of GC and GC-MS data with those of authentic reference material. GC-SNEFF analysis of (1) further confirmed its intense mousy aroma.

3,4-Dimethoxyphenyl β-D-glucopyranoside 1a was identified for the first time in a glycosidic extract obtained from pinuela (Bromelia plumieri Kartens) fruit by adsorption on Amberlite XAD-2 resin and elution with EtOAc and MeOH. After prefractionation of the MeOH eluate using countercurrent chromatography techniques (RLCC and MLCCC) the glycoside was peracetylated and purified by flash chromatography and subsequent HPLC to be characterized as its tetraacetate 1b by NMR and ESI-MS techniques.

The phenolic composition of red wine impacts upon the color and mouthfeel and thus quality of the wine. Both of these characteristics differ depending on the age of a wine, with the purple of young wines changing to brick red and the puckering or aggressive astringency softening in older wines. This study investigated the color parameters, tannin concentrations and tannin composition of a 50 year series of Cabernet Sauvignon wines from a commercial label as well as 30 year series of Cabernet Sauvignon and Shiraz wines from a separate commercial label to assess the impact of wine age on phenolic composition and concentration. The wine color density in wines of 40 to 50 years old was around 5 AU compared with 16 AU of wine less than 12 months old, which correlated well with the concentration of non-bleachable pigments and pigmented polymers. Conversely, the anthocyanin concentrations in 10 year old wines were substantially lower than that of recently bottled wines (around 100 mg/L compared with 627 mg/L, respectively), adding further evidence that non-bleachable pigments including pigmented polymers play a much larger role in long-term wine color than anthocyanins. No age-related trend was observed for tannin concentration, indicating that the widely noted softer astringency of older red wines cannot necessarily be directly related to lower concentrations of soluble wine tannin and is potentially a consequence of changes in tannin structure. Wine tannins from older wines were generally larger than tannins from younger wines and showed structural changes consistent with oxidation.

The study aimed to quantify the effects of grape maturity on wine alcohol, phenolics, flavour compounds and polysaccharides in Vitis vinifera L. cv Cabernet Sauvignon. Grapes were harvested at juice soluble solids from 20 to 26 °Brix which corresponded to a range of wine ethanol concentrations between 12% and 15.5%. Grape anthocyanin and skin tannin concentration increased as ripening progressed, while seed tannin declined. In the corresponding wines, monomeric anthocyanin and wine tannin concentration increased with harvest date, consistent with an enhanced extraction of skin-derived phenolics. In wines, there was an observed increase in yeast-derived metabolites, including volatile esters, dimethyl sulfide, glycerol and mannoproteins with harvest date. Wine volatiles which were significantly influenced by harvest date were isobutyl methoxypyrazine, C 6 alcohols and hexyl acetate, all of which decreased as ripening progressed. The implications of harvest date for wine composition is discussed in terms of both grape composition and yeast metabolism.

Guaiacol and 4-methylguaiacol are well-known as contributors to the flavor of wines made from smoke-affected grapes, but there are other volatile phenols commonly found in smoke from forest fires that are also potentially important. The relationships between the concentration of a range of volatile phenols and their glycoconjugates with the sensory characteristics of wines and model wines were investigated. Modeling of the attribute ratings from a sensory descriptive analysis of smoke-affected wines with their chemical composition indicated the concentrations of guaiacol, o-cresol, m-cresol, and p-cresol were related to smoky attributes. The best-estimate odor thresholds of these compounds were determined in red wine, together with the flavor threshold of guaiacol. Guaiacol β-D-glucoside and m-cresol β-D-glucoside in model wine were found to give rise to a smoky/ashy flavor in-mouth, and the respective free volatiles were released. The study indicated that a combination of volatile phenols and their glycosides produces an undesirable smoke flavor in affected wines. The observation of flavor generation from nonvolatile glycoconjugates in-mouth has potentially important implications.

Methoxyoxime acetates of carbohydrates and partially methylated derivatives were easily separated by high resolution gas chromatography (HRGC) and showed characteristic mass spectra (MS). Thus, the conversion of carbohydrates to the corresponding methoxyoxime acetates offers an attractive derivatization for subsequent HRGC and HRGC/MS analysis of carbohydrates, particularly in glycoconjugates of plant origin.

The novel C13-glucoside β-ionyl-β-D-glucopyranoside 2a was identified in purple passionfruit as a genuine precursor of isomeric megastigma-4,6,8-trienes 1a-d. The identification was verified by comparison of HRGC and HRGC-MS data of the peracetylated natural product with those of an authentic specimen synthesized by a modified Konigs-Knorr procedure.

The presence of glycosides of smoke-derived volatile phenols in smoke-affected grapes and the resulting wines of Chardonnay and Cabernet Sauvignon was investigated with the aid of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). All volatile phenols studied (phenol, p-, m-, and o-cresols, methylguaiacol, syringol, and methylsyringol) could be detected as glycosylated metabolites in smoke-affected grapes in a similar fashion to that previously reported for guaiacol. These phenolic glycosides were found in smoke-affected grapes and wines at significantly elevated levels compared to those in non-smoked control grapes and wines. The extraction of these glycosides from grapes into wine was estimated to be 78% for Chardonnay and 67% for Cabernet Sauvignon. After acid hydrolysis, a large proportion of these phenolic glycosides in grapes (50%) and wine (92%) disappeared but the concentrations of volatile phenols determined by gas chromatography-mass spectrometry (GC-MS) were lower than expected. In the case of wine, the majority of the glycosides of phenol, cresols, guaiacol, and methylguaiacol were decomposed upon acid hydrolysis without releasing their respective aglycones, while syringol and methylsyringol were more effectively released.

Fermentation of grapes that had been exposed to bushfire smoke can potentially yield unpalatable, smoke-affected wine. Guaiacol and its glucoconjugate were previously found in smoke-affected grapes at an elevated concentration. To find and identify further guaiacol conjugates in smoke-affected grapes, a stable isotope feeding experiment combined with extensive HPLC-MS and MS/MS investigations was carried out. Leaves and berries of a potted grapevine were placed in contact with an aqueous mixture of d(0)- and d(3)-guaiacol for 1-2 days and collected 5 weeks later. Screening for potential guaiacol conjugates in the leaves and berries was facilitated by monitoring the unique mass spectrometric signature of an isotopic doublet separated by 3 Da. Seven different conjugates were detected in leaves and berries and were tentatively identified as mono- and diglycosides of guaiacol. Quantitative analysis demonstrated that the guaiacol conjugates were translocated between leaves and berries to a very limited extent and were also present as low-level natural compounds of untreated leaves and berries. The same guaiacol conjugates were also found at a considerably elevated concentration in leaves and berries obtained from grapevines exposed to bushfire smoke.

HRGC and HRGC-MS identifications of glycosidically bound constituents from starfruit (Averrhoa carambola L.) were achieved in extracts isolated by Amberlite XAD-2 adsorption followed by methanol elution. The majority of aglycones released by almond β-glucosidase hydrolysis were carotenoid degradation products, including megastigma-6,7-diene-3,5,9-triol, an important precursor of the potent aroma compound β-damascenone. Additional aglycones comprised the following ionone derivatives: 4-hydroxy-β-ionol, 3-hydroxy-β-ionol, 4-oxo-β-ionol, 3-hydroxy-β-ionone, 3-oxo-α-ionol, 3-oxo-retro-α-ionol (2 isomers), 3-oxo-4,5-dihydro-α-ionol, 3-oxo-7,8-dihydro-α-ionol (‘Blumenol C’), 3-hydroxy-β-damascone, 3,5-dihydroxy-megastigma-6,7-diene-9-one (‘grasshopper ketone’), 3-hydroxy-5,6-epoxy-β-ionone, 3-hydroxy-5,6-epoxy-β-ionol, 3,4-dihydro-3-hydroxyactinidol, vomifoliol (‘Blumenol A’), 4,5-dihydrovomifoliol, and 7,8-dihydrovomifoliol (‘Blumenol B’). Several of these new starfruit constituents are easily degraded upon heat-treatment at natural pH condition of the fruit pulp, thus rationalizing the formation of a number of C13-aroma compounds, which have recently been reported as starfruit volatiles.

In a polar extract obtained from purple passion fruit (Passiflora edulis Sims) juice, capillary gas chromatography (HRGC), and coupled HRGC techniques, i.e., on-line mass spectrometry (HRGC-MS) and Fourier transform infrared spectroscopy (HRGC-FTIR), revealed for the first time the presence of 3-hydroxy-retro-alpha-ionol. The identification was verified by comparison of HRGC, HRGC-MS, and HRGC-FTIR data of the new natural product with those of an authentic reference compound synthesized from the known C13 keto alcohol, 3-oxo-alpha-ionol. Degradation studies carried out with 3-hydroxy-retro-alpha-ionol showed the easy formation of isomeric edulans under acidic conditions. Possible mechanisms for the formation of the odor active edulans, key flavor components of purple passion fruit juice, are discussed.

Aroma extracts from various plants were screened for acetylenic flavour precursors using on-line HRGC-FTIR spectroscopy. Selected wavelength monitoring (2100–2250 cm−1) revealed for the first time the presence of 3-hydroxy-7,8-didehydro-β-ionone in peelings of lulo syn. naranjilla (Solanum vestissimum D.) fruit. The same compound was also detected as a major aglycone after enzymatic hydrolysis of a glycosidic extract isolated from Vitis vinifera cv. Riesling leaves. The related acetylenic diol, 3-hydroxy-7,8-didehydro-β-ionol, was similarly identified as the aglycone in a glycosidic extract obtained from purple passion fruit (Passiflora edulis Sims) juice. The discovery of these acetylenes is discussed with regard to the formation of β-damascenone in plants.