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Plant tissue water is the source of oxygen and hydrogen in organic biomatter. Recently, we demonstrated that the stable hydrogen isotope value (δ(2)H) of plant methoxyl groups is a very reliable and easily available archive for the δ(2)H value of this tissue water. Here we show in a model experiment that the δ(2)H values of methoxyl groups remain unchanged after water loss during storage of fruits and vegetables under controlled conditions, while δ(2)H and δ(18)O values of tissue water increase. This enhancement is plant-dependent, and the correlation differs from the meteoric water line. The δ(18)O value is better correlated to the weight decrease of the samples. Therefore, we postulate that the δ(2)H value of methoxyl groups and the δ(18)O value of tissue water are suitable parameters for checking postharvest alterations of tissue water, either addition or loss.

Mit Isotopenverhaltnis-Massenspektrometrie (IRMS), gekoppelt mit Kapillar-GC, gelingt es, zwischen synthetischem, halbsynthetischem und naturlichem Vanillin aus Vanille-Varietaten zu unterscheiden. Derartige Untersuchungsmethoden kann die amtlichen Lebensmitteluberwachung allerdings aus Kostengrunden bisher nur in begrenztem Umfang nutzen.

Vanilla still remains one of the most important and widely used flavours in the food industry and is also extensively employed by fragrance and pharmaceutical manufacturing companies. Natural vanilla flavour, extracted from the pods of the tropic orchid vanilla, is considerably more expensive than synthetic vanillin. The disparity of prices between natural vanillin and that derived from other sources has given rise to many cases of fraudulent adulteration, and for more than 30 years, strenuous efforts have been made to authenticate sources of vanillin. Stable isotope analysis is one of the most powerful analytical tools to distinguish between natural vanillin and that originating from other sources. Recently, a rapid and precise method for analysis of both δ13C and δ2H values of plant methoxyl groups has been published. Here, we report an application of the method for the control of authenticity of vanillin. Carbon and hydrogen stable isotope values of the vanillin molecule and vanillin methoxyl groups of vanillin samples of different origins including authentic and synthetic samples were measured. The results clearly show that use of this approach provides a rapid and reliable authenticity assessment of vanillin. The technique used for these studies is robust and rapid, involves minimum sample preparation and requires only a small amount of vanillin sample, usually 1 mg for stable carbon and 4 mg for stable hydrogen analysis.

Stable isotope ratios of individual plant components have become a valuable tool for the determination of the geographical origin and authenticity of foodstuff. A recently published method with considerable potential in this context is the measurement of the deuterium/hydrogen (D/H) isotope ratios of plant matter methoxyl groups. The method entailed cleavage of methyl ethers or esters with hydriodic acid (HI) to form gaseous methyl iodide (CH3I) and then measurement of the d 2 H value of this gas. Here, as a follow up to a previous study, we describe a method for the rapid and precise d 13 C analysis of plant matter methoxyl groups using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Conditions for sample preparation were investigated for isotope discrimination effects, the GC conditions optimized, the reproducibility of the measurement of standards undertaken, and the precision of the method defined. The reproducibility of the d 13 C value determined for a CH3I standard on 20 consecutive measurements was found to be 0.17%. The method was also tested on four methoxyl-rich plant components: vanillin, lignin, wood and pectin. The analytical precision obtained, expressed as the average standard deviation, for these compounds was found to be better than 0.13%. The described procedure which is simple and rapid, allowing preparation and analysis of a sample within 1h, produces accurate and reproducible isotopic measurements. We suggest that this validated d 13 C method when employed together with the recently published d 2 H method for two-dimensional stable isotope studies of organic matter

Since the ban on growth-promoting antibiotics throughout the EU essential oils are used increasingly as feed additives. In order to ensure a reliable authenticity assessment of those oils appropriate methods were developed. 2H/1H−, 13C/12C− and 18O/16O isotope ratio mass spectrometric analysis of carvacrol from origanum and savory oil, thymol from thyme oil and trans-anethole from fennel oil in combination with IRMS-analysis of minor components and differential analysis is reported. This is the only reliable method to assess the authenticity of these spice oils, provided that an adulteration with major components should be detected. The major component IRMS-measurements is a powerful tool in the authenticity assessment of savory- and thyme oil. In case of origanum and fennel oils a comprehensive analysis including major and minor component-, differential-IRMS as well as enantioselective analysis is essential.

Flavour extracts of different blackberry cultivars were investigated in a view of authenticity assessment, using multielement-/multicomponent MDGC–IRMS and enantioselective MDGC analysis. In conclusion enantioselective analysis of heptan-2-ol and trans-linalool oxide (fur.) in conjunction with multielement MDGC-C/P-IRMS analysis were proved to be useful tools in the authenticity assessment of blackberry flavours.

By reductive glyceride cleavage food fats of different origin, converted to free glycerol and to fatty alcohols of the corresponding fatty acids, were investigated. Multielement IRMS-analysis (δ13C, δ18O) of the decomposition products was performed in order to develop GC-IRMS methods to the authenticity assessment of food fats. For this reason glycerol from olive oils of definite origin, as well as glycerol of commercially available edible fats and oils, was analysed. Furthermore, the determination of 18O/16O and 13C/12C isotope ratios of commercial glycerols from different origins was achieved. In addition, δ13CV‐PDB values of fatty alcohols corresponding to genuine fatty acid moieties were measured and their usability in view of fat authenticity assessment is discussed.

The stereoisomers of linalool and lilac aldehyde/alcohol were determined in the flower scent of 15 plant species using enantioselective multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS). Both linalool and all 8 stereoisomers of lilac alcohol and lilac aldehyde were detected, and there was a species-specific pattern. Single stereoisomers were collected by micropreparative-enantio-MDGC and were electrophysiologically tested on antennae of the noctuid moth Hadena bicruris, a species known to rely on lilac aldehyde for finding its host plant. The moth responded to all 8 stereoisomers, though only four stereoisomers were found in the scent of its host plant. The moth was less sensitive to some isomers than to others.

The determination of glycerol and 2,3-butanediol by photometric or enzymatic methods is well established. This paper reports on the direct assessment of glycerol and stereoselective analysis of 2,3-butanediol isomers in wine using capillary GC without any derivatisation. A “model wine” and commercially available wines, as well as wines of definite origin were investigated. The contents of glycerol and 2,3-butanediol and the ratio of (R,R)- and meso-2,3-butanediol were determined. Capillary GC has proved to be a reliable alternative in glycerol assessment from wine, thus a GC-IRMS method for authenticity assessment of glycerol was developed.

The biosynthesis of the monoterpene (S)-linalool and the sesquiterpene trans-(S)-nerolidol in fruits of Fragaria x ananassa Duch. cv. Eros and Florence and of the monoterpene (-)-alpha-pinene in Fragaria vesca was investigated by in vivo feeding experiments with [5,5-2H2]mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-d-xylulose (d2-DOX). The feeding experiments indicate that (S)-linalool and trans-(S)-nerolidol in Fragaria x ananassa Duch. and (-)-alpha-pinene in F. vesca are exclusively synthesized via the cytosolic mevalonic acid pathway without any contribution from the plastidial 1-deoxy-D-xylulose/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) route. Inhibition experiments revealed that even the presence of mevastatin, an export of plastid-derived isopentyl diphosphate/dimethylallyl diphosphate, cannot be induced. However, the enantioselective analysis shows that in Fragaria x ananassa Duch. cv. Eros and Florence both linalool enantiomers are present and that only (S)-linalool is labeled after administration of d2-MVL. Therefore, the origin of (R)-linalool in these fruits remains unknown. Contrarily, in Fragaria x ananassa Duch. foliage (R)-linalool is the dominant enantiomer. Feeding experiments revealed an incorporation of d2-MVL and d2-DOX at equal rates exclusively into (S)-linalool. Only in F. vesca foliage, where (R)-linalool is present at high enantiomeric purity (ee90%), is a de novo biosynthesis of the (R)-enantiomer via the DOXP/MEP pathway detectable. These results demonstrate a complex intraplant variation of (R)- and (S)-linalool biosynthesis via the cytosolic and plastidial route.

The terpene biosynthesis in leaves of Vitis vinifera L. cv. Morio Muskat was studied using methyl jasmonate to induce defensive responses in vivo. The experiments demonstrated the strong activation of the de novo biosynthesis of terpenoids via the octadecanoid-signaling cascade and release of the compounds to the gas phase. Feeding experiments with [5,5-2H2]-1-deoxy-d-xylulose and [5,5-2H2]mevalonic acid lactone allowed the investigation of the dynamic allocation of resources via the mevalonic acid and 1-deoxy-d-xylulose/2-C-methyl-d-erythritol 4-phosphate (DOXP/MEP) pathway under induced conditions and after treatment with the specific inhibitors mevastatin and fosmidomycin. The experiments reveal that monoterpenes are almost exclusively synthesized via the DOXP/MEP pathway, whereas sesquiterpenes are generated via both pathways at approximately equal rates. The biosynthesis of the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene was not affected by mevastatine or fosmidomycin. Keywords: Vitaceae; 1-deoxy-d...

The metabolism of deuterium labeled geraniol in grape mesocarp of Vitis vinifera L. cv. Scheurebe was studied by in vivo-feeding experiments. Stereoselective reduction to (S)-citronellol, E/Z-isomerization to nerol, oxidation to neral/geranial and glycosylation of the corresponding monoterpene alcohols could be demonstrated. Time course studies including the determination of conversion rates revealed that the activity of these secondary transformations of monoterpenes is dependent on the ripening stage and can be distinguished from the development of the primary monoterpene synthase activities by the sharp increase at the end of the ripening period. The stereoselective biosynthesis of the potent odorant cis-(2S,4R)-rose oxide from labeled geraniol in grape berry mesocarp is demonstrated as well. Since (S)-citronellol is the precursor of cis-(2S,4R)-rose oxide it can be concluded that especially the last part of the ripening period is important for the generation of this potent odorant. This finding confirms the conclusion that a higher concentration of flavor compounds could be established in the berries by leaving the fruit on the vine for extended periods.

The biosynthesis of the monoterpenes terpinolene and myrcene and the sesquiterpene beta-caryophyllene in roots and leaves of two carrot varieties (Daucus carota L. cultivars Bolero and Kazan) were investigated by in vivo feeding experiments with [5,5-2H2]-mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-D-xylulose (d2-DOX). The volatiles of the tissues were extracted by stir bar sorptive extraction and analyzed using thermal desorption-multidimensional gas chromatography-mass spectrometry. The experiments demonstrate independent de novo-biosynthesis of terpenoids in carrot roots and in carrot leaves. In both plant tissues monoterpenes are biosynthesized exclusively via the 1-deoxy-D-xylulose/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP) pathway, whereas sesquiterpenes are generated by the classical mevalonic acid pathway as well as by the DOXP/MEP route. A more detailed investigation of carrot root tissues revealed that the biosynthesis of terpenes is mainly localized in the phloem. Nevertheless, in xylem a de novo-biosynthesis of terpenes was detectable as well, even in the absence of oil ducts in this tissue.

δ13CV-PDB, δ2HV-SMOW and δ18OV-SMOW multielement isotope ratio mass spectrometry analysis of linalool and linalyl acetate, the main components of lavender oils, is reported. Self-prepared and commercially available lavender oils, as well as samples of linalool and linalyl acetate labelled as synthetic and natural products respectively, were investigated. Multielement/multicomponent isotope ratio mass spectrometry analysis and—as far as possible—in conjunction with enantioselective analysis is judged to be the most comprehensive basis of authenticity assessment in flavour and essential oil analysis.

The combination of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) and gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-P-IRMS) is applied to the authenticity assessment of cinnamaldehyde from various sources. For that reason, cinnamon oils were self-prepared by steam distillation from three different varieties of cinnamon bark on the market, C. ceylanicum (ceylon), C. cassia (cassia) and C. burmanii (cassia vera). Furthermore, the so-called wood cinnamon was investigated, which is produced from the outer bark of older branches of cinnamon of minor quality. Self-prepared oils were analysed from commercial cinnamon powder. In addition several commercial samples of cinnamon oil and cinnamaldehyde, some of them declared to be natural, were investigated. δ 2ΗV-SMOW and δ 13CV-PDB values of cinnamaldehyde were determined and characteristic authenticity ranges were deduced, allowing the differentiation between synthetic and natural samples. By correlation of both the δ 2ΗV-SMOW and δ 13CV-PDB values, characteristic authenticity ranges were defined for ceylon, cassia and wood cinnamon. The δ 2ΗV-SMOW and δ 13CV-PDB values of cassia vera samples are in the range of cassia. By comparing the δ 2ΗV-SMOW values of different self-prepared samples (ground bark, distillate) of cinnamon determined by TC/EA-IRMS with the corresponding GC-IRMS values, online GC-IRMS methods are proved to be essential in the authentication of complex natural products.

Syringa vulgaris L. inflorescences were fed with aqueous solutions of regioselectively deuterated compounds assumed to be precursors of lilac aldehyde and lilac alcohol, respectively. Volatiles were extracted by stir bar sorptive extraction (SBSE) and analyzed using enantioselective multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS); deuterium-labeled lilac aldehydes and lilac alcohols were separated from unlabeled stereoisomers on a fused silica capillary column, coated with heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin (DIME-beta-CD) (30%) in SE 52 (70%), as the chiral stationary phase. Feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone 22 and [5,5-(2)H(2)]deoxy-d-xylose 23 indicate that the novel mevalonate independent 1-deoxy-d-xylose 5-phosphate/2C-methyl-d-erythritol 4-phosphate pathway is the dominant metabolic route for biosynthesis in lilac flowers. Additionally, bioconversion of deuterium-labeled d(5)-(R/S)-linalool 3, d(6)-(R)-linalool 21, d(5)-(R/S)-8-hydroxylinalool 6, d(5)-(R/S)-8-oxolinalool 7, d(5)-lilac aldehydes 8-11 and d(5)-lilac alcohols 12-15 into lilac during in vivo feeding experiments was investigated and the metabolic pathway is discussed. Incubation of petals with an aqueous solution of deuterated d(5)-(R/S)-linalool 3 indicates an autonomic terpene biosynthesis of lilac flavor compounds in the flower petals of lilac.

Gas chromatography-pyrolysis-isotope mass spectrometry (GC-P-IRMS) is applied to the authenticity assessment of lavender oils. For this reason, self-prepared lavender oils, samples of linalool and linalyl acetate labeled as synthetic and natural, and commercially available lavender oils have been investigated. 2H/1H isotope ratios of linalool and linalyl acetate have been determined, and characteristic ranges of authenticity were deduced. Samples of natural linalool and linalyl acetate are clearly differentiated from those of synthetic origin by their δ2HV-SMOW values. Besides enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS), online GC-P-IRMS has proven to be an additional and powerful tool in the detection of lavender oils adulteration.

The sesquiterpene germacrene D and two deuterium-labeled isotopomers were analyzed by enantio-multidimensional gas chromatography with tandem mass spectrometry in order to investigate mass spectral fragmentation mechanisms. The deuterated analogues were derived from in vivo feeding experiments with 1-[5,5-D2]deoxy-D-xylulose-5-phosphate using intact plant material of Solidago canadensis and were formed by biosynthesis via the methylerythritol phosphate pathway.

2-Methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-but-2-enol is a synthetic fragrance with a strong sandalwood-like odor. It was found that the (R)-enantiomer is exclusively responsible for the sandalwood-like odor, whilst the (S)-enantiomer has only a faint, non-specific odor. Consequently, enantioselective analysis is a powerful tool in sensory quality control of 2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-but-2-enol. The industrial synthesis starts from u-campholene aldehyde via 2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-but-2-enal. This is why the enantiomeric purity of 2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-but-2-enol depends on the purity of u-campholene aldehyde as starting material. Hence, an enantioselective analysis of u-campholene aldehyde, 2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-but-2-enal, and 2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)-but-2-enol was achieved by enantioselective gas chromatography using tert-butyldimethylsilylated-cyclodextrin derivatives as chiral GC phases.

The recently introduced solventless extraction technique, the Stir Bar Sorptive Extraction (SBSE), was applied for the enantioselective analysis of chiral flavour compounds in strawberries and their processed products using enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS). Using this rapid method the usual preparation steps for complex matrices such as liquid/liquid extraction, solid phase extraction or distillation techniques can be omitted. The analysed chiral compounds showed fruit-specific characteristic enantiomeric ratios, irrespective of food processing techniques employed. Hence, it is possible to differentiate the flavour of authentic and aromatised strawberry products.

Stir bar sorptive extraction (SBSE), a recently introduced solventless extraction technique, was applied for the enantioselective analysis of chiral monoterpenes in tea tree oil, eucalyptus oil and thyme oil using enantioselective multidimensional gas chromatography–mass spectrometry (enantio-MDGC–MS). In addition, headspace sorptive extraction (HSSE) and in vivo headspace sorptive extraction (in vivo HSSE) of the chiral monoterpenes in three aromatic and medicinal plants, thyme [Thymus vulgaris (L.)], eucalyptus [Eucalyptus globulus (L.)] and tea tree [Melaleuca alternifolia (C.)], was investigated and the results of the different sampling techniques were compared and discussed. This method was utilized in order to establish characteristic authenticity profiles of the essential oils. Moreover, the enantiomeric ratios of the major and minor chiral components emitted in living plants were determined. Copyright © 2001 John Wiley & Sons, Ltd.

Young plants of Agathosma crenulata (L.) Pillans were stem fed with aqueous solutions of 2H2 and 18O/2H2-labelled monoterpene ketone precursors. The essential oil was extracted by solid phase microextraction and subsequently analysed with enantioselective multidimensional gas chromatography–mass spectrometry. Both labelled pulegone precursors were converted into corresponding labelled menthone, isomenthone and menthofuran with different enantioselectivity. Feeding experiments with 18O/2H-labelled pulegone proved an enzymatic conversion of pulegone to menthofuran and the loss of the pulegone oxygen. Stereoselective analysis of all four isopulegone stereomers using octakis(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin or heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin allowed the detection of enantiopure (1S)-cis- and (1S)-trans-isopulegone (>99%) in A. crenulata and buchu samples. Labelled isopulegone is discussed as a suitable pulegone precursor. Feeding experiments regarding the biosynthesis of 3-oxo-p-menthan-8-thiol and 3-oxo-p-menthan-8-thiol acetate are also reported. Copyright © 2001 John Wiley & Sons, Ltd.

Several cyclodextrin derivatives dissolved in different polysiloxanes have been investigated with regard to their suitability for achieving simultaneous enantioseparation of kavain, dihydrokavain, methysticin, and dihydromethysticin by high resolution gas chromatography. Enantioseparation of these four chiral kavapyrones is achieved using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin dissolved in a mixture of SE-30/SE-52 (1:1) as the chiral stationary phase.

Separation factors and thermodynamic data for the separation of various chiral analytes on different di-O-tert-butyldimethyl-silylated cyclodextrin derivatives are collected and described. Modifying the substitution pattern of the tert-butyldimethylsilyl group in position 2 and 3 or changing from β- to γ-cyclodextrin significantly affects the separation properties of the cyclodextrin derivatives.

3-Mercapto-2-methylpropanol, an odorous compound recently identified in red wines, was extracted from a red wine variety, using a low-temperature vacuum distillation coupled with a specific reversible thiol capture. The analysis of the diacetylated wine extract by enantioselective multidimensional gas chromatography with mass spectrometry detection revealed the presence of only one enantiomer, (R)-3-mercapto-2-methylpropanol. Although both of the optical isomers are characterized by the same broth and sweat odour, they have very different odour thresholds. The amounts of (R)-3-mercapto-2-methylpropanol in young Cabernet-Sauvignon and Merlot wines can exceed its perception threshold, thus suggesting its contribution to the aroma of these wines.

Enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS) is a valuable tool for the differentiation of enantiomers from complex matrices when present in trace amounts. The separation of chiral compounds provides further information on the diagnosis of diseases, and on normal and abnormal biochemical pathways. The formation of the normal urinary metabolite 3-hydroxy-2-methylbutanoic acid (HMBA), excreted in abnormally high amounts in beta-ketothiolase deficiency, is not absolutely clarified. Metabolic pathways involving this metabolite are isoleucine catabolism, as well as presumably beta-oxidation of fatty acids and ketogenesis. The latter two pathways are distinguishable in their enantioselectivity. Enantioselective analysis gives further information on interfering metabolic pathways and the selectivity of the enzyme(s) forming HMBA. Different ratios of the stereoisomers of HMBA in control urine samples and patients with beta-ketothiolase deficiency were detected. Analogous to HMBA urinary 3-hydroxybutanoic acid (HBA) was investigated in several diseases. The formation of HBA and HMBA is expected to result from the same or similar metabolic pathways. Differences in the enantiomeric ratio of HMBA may originate from the enantioselectivity of different enzyme systems.

The synthesis of octakis(2,3-di-O-propionyl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin and its use as chiral stationary phase in capillary gas chromatography is described. Separation factors and thermodynamic parameters of several chiral compounds on two columns with different cyclodextrin content and different polysiloxanes as diluting phase are investigated. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 482–492, 2000

Methylcitric acid (2-hydroxybutane-1,2,3-tricarboxylic acid–MCA) is a structural analogue of citric acid, but due to an additional methyl group it is a chiral molecule with two stereogenic centers and thus four stereoisomers are conceivable. MCA occurs naturally as prominent metabolite in body fluids of patients with inherited metabolic diseases such as propionic acidemia, methylmalonic aciduria, or holocarboxylase synthetase deficiency. Therefore methylcitric acid is considered to be an important diagnostic marker for these diseases. MCA is most likely produced from accumulated propionyl-CoA in these diseases by the enzyme si-citrate synthase from the citric acid cycle; however, there are other enzymes known which could catalyze the same reaction with different stereoselectivity, such as re-citrate synthase or the more specific enzyme methylcitrate synthase, found in microorganisms. Almost all methods dealing with MCA in the literature are non-enantioselective. For that reason there is no information about occurrence of MCA enantiomers in healthy people, patients with propionic acidemia, methylmalonic aciduria, or holocarboxylase synthetase deficiency and about value of enantiomeric distribution for diagnosis and long-term treatment. The enantioselective analysis of MCA as corresponding trimethyl ester was achieved by enantioselective multidimensional gas chromatography coupled with mass spectrometry using heptakis-(2,3-di-O-methyl-6-O-tert-butyl-dimethylsilyl)-β-cyclodextrin as chiral stationary phase. The described method allows a reliable screening of MCA in complex matrices like urine without time consuming sample preparation and with mass selective detection. During this investigation urine samples from various patients and healthy controls were analyzed. As concluded, MCA is a good diagnostic marker and can be easily measured by the method presented. Only the two stereoisomers (2S,3R) and (2S,3S) were detectable in patients and healthy controls. The varying ratios of these stereoisomers cannot presently be correlated with the health status of patients, although there are some indications that this might be possible. However, the quantitative levels of MCA, determined as the ratio of MCA absolute peak area divided by 1,000 to the creatinine contents of urine samples in this investigation, showed a dependence on the state of health and MCA would thus also be a possible marker for long-term treatment. Such a substance is of major interest nowadays since there are different studies searching for such a long-term marker in propionic acidemia or methylmalonic aciduria.

Enantioselective gas chromatography and/or 13C/12C isotope ratio analysis are suitable tools for the authenticity assessment of the fusel alcohols, 2- and 3-methylbutanol (1 and 2, respectively). The chiral compound, 1 is biosynthesised almost completely as the (S)-enantiomer, regardless of which carbohydrate source is used for fermentation. The type of CO2 fixation and some plant-specific influences were of prime importance to the 13C/12C isotope ratios of the starting materials in alcoholic fermentation, and the δ13C values of 1 and 2 differed significantly. In general, the δ13C values of 2 were about 4–5o lower than those of 1 produced via the same fermentation process. 13C/12C isotope ratio analysis results can be used to determine between fusel alcohols produced from different sources, and provides a new and valuable method of authenticity assessment.

The δ13CPDB values of characteristic flavour compounds of black Ceylon, Darjeeling and Assam teas were determined using gas chromatography-isotope ratio mass spectrometry. The influence of different harvest times, years and various cultivation regions on the δ13CPDB and the δ13C cis linalool oxide (fur.) values were investigated. By comparison with suitable internal isotopic standards, adulterations with methyl salicylate were detectable.

Maple syrup urine disease is an autosomal recessive inherited disorder of branched-chain amino acid metabolism due to deficiency of the branched-chain alpha-keto acid dehydrogenase complex. The disease was originally named after the characteristic sweet aroma, reminiscent of maple syrup, present in the body fluids of affected patients. Until now, the substance responsible for the odour has not been positively identified. Using enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS), we could demonstrate that 4,5-dimethyl-3-hydroxy-2[5H]-furanone (sotolone), a well-known flavour impact compound present in fenugreek and lovage, was present in urine from seven patients with maple syrup urine disease. Urine samples from healthy control persons lacked sotolone. We have shown that sotolone is responsible for the characteristic odour of maple syrup urine disease and, since maple syrup also contains sotolone, the naming of this disease appears to be correct.

The even numbered γ(δ)-thionolactones (C6–C12) were investigated, using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)- and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as chiral stationary phases in capillary gas chromatography. The odor characteristics of γ(δ)-thionolactone enantiomers were investigated by enantioselective gas chromatography/olfactometry.

Methyl-branched volatiles in apples and processed apples are investigated. The ratio of 3-methyl-branched and 2-methyl-branched flavor compounds was determined and the enantiomeric distribution of the latter investigated. The enantiomeric ratios of 2-methyl-branched volatiles are analyzed, using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as an efficient enantioselective stationary phase in capillary GC. 2-Methylbutanoic acid and the corresponding esters (methyl−n-hexyl ester), 2-methylbutanol, and 2-methylbutyl acetate were detected as genuine flavor compounds favoring (S)-configuration and high enantiomeric purity (>99%). 3-Methylbutanoic acid, 3-methylbutanol, and 3-methylbutyl acetate are proved to be not genuine compounds of the apple aroma but fermentation products. Keywords: Apple flavor; methyl-branched volatiles; enantioselective capillary gas chromatography; enantioselective multidimensional gas chromatography (enantio-MDGC)

Enantioselective multidimensional gas chromatography-mass spectrometry is a valuable tool for the enantioselective analysis of compounds from complex matrices. Samples are separated initially on a precolumn and selected substances then transferred on-line to a main-column coated with suitable chiral stationary phase for enantioselective analysis with subsequent mass selective detection. In this paper the method is used as an adjunct to urinary organic acid analysis to provide information in patients with suspected inborn errors of metabolism. Lactic acid, alpha-hydroxyisocaproic acid, 3-phenyllactic acid and 3-(4-hydroxyphenyl)-lactic acid are separated in a single run. In addition, the enantioselective analysis of pyroglutamic acid is presented. D-Enantiomers of amino acids and alpha-hydroxycarboxylic acids derived from amino acids, point to a bacterial origin whereas the L-enantiomer is predominantly of endogenous origin. Therefore the enantiomeric ratio of chiral metabolites is an important parameter for the understanding of metabolic processes.

By feeding experiments with labeled [1,1- 2 H 2 , 18 O]citronellol, it could be shown that Pelargonium graveolens is able to convert the fed precursor into cis-/trans-rose oxide. The labeling pattern and diastereomeric ratio of the resulting cis-/trans-rose oxides are in agreement with a mechanism which includes the enzymatic oxidation of citronellol in the allylic position and subsequent cyclization of the resulting diol to cis-/trans-rose oxide with retention of the oxygen of [1,1- 2 H 2 , 18 O]citronellol.

The direct enantioseparation of 2-methylbutanal, 2-methylpentanal, 2-ethylhexanal, 3-methylpentanal and 4-methylhexanal was achieved using enantioselective gas chromatography and the chiral stationary phase heptakis-[2,3-di-O-acetyl-6-O-tert.butyldimethylsilyl]-β-cyclodextrin (DIAC-TBDMS-β-CD). Odour characteristics and threshold values of detection for compounds were evaluated by enantioselective gas chromatography/olfactometry. Enantiopure reference compounds with defined absolute configurations were synthesized starting from the corresponding alcohols or acids which were commercially available or prepared in the laboratory.

Multidimensional gas chromatography coupled on-line with isotope ratio mass spectrometry (MDGC-IRMS) is presented as a powerful method for origin-specific analysis. Scope and limitations of this sophisticated coupling technique are discussed with regard to accuracy and precision.

We know that many familiar objects such as gloves, shoes and screws are non identical with their mirror images. The same applies to certain chemical compounds. These compounds show a degree of asymmetry in their structure which means that when looked at in a mirror, their images are not superimposable--they are chiral compounds and have two forms (enantiomers). Until recently biochemists have only been concerned with perhaps one form, other forms having been ignored primarily due to analytical difficulties. We now know of inborn errors of metabolism where one form of a pathological metabolite predominates, thus requiring precise enantiomeric analysis. This article provides a background to the field of enantiomers and their analysis as demonstrated by examples of inborn errors of metabolism and hints at the possible discovery of as yet unidentified metabolic pathways.Recent advances in the field of enantiomeric metabolite analysis, in particular the use of enantioselective gas chromatography-mass spectrometry, have an increasingly important role to play, not only in the diagnosis of inborn errors of metabolism, but also to increase our understanding of normal physiological processes.

Mechanistic aspects of the biogenesis of the chiral monoterpenoid rose oxide in Pelargonium graveolens L'Heritier are investigated using deuterium-labelled precursors. After administration of the precursors using the cut-stem method, the dynamic headspace extracts of the plants are analysed using multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS). It is unequivocally shown that this plant is able to convert citronellol and menthocitronellol into cis-/trans-rose oxide. Menthocitronellol is converted into rose oxide with a clearly detectable enantiodiscrimination. These facts may be explained with the presence of an oxidase, which is able to oxidize citronellol and menthocitronellol in allylic position. A photooxygenation mechanism including singlet oxygen as the oxidizing agent is rather unlikely. Chirality 10:229–237, 1998. © 1998 Wiley-Liss, Inc.

The δ13CPDB values of characteristic flavor components of mandarin essential oils have been measured by gas chromatography/isotope ratio mass spectrometry. For genuine mandarin oils a characteristic authenticity profile was established and used for the authenticity control of commercially available mandarin oils. In addition to cold-pressed mandarin essential oils, distilled mandarin and sweet orange oils were investigated to assess blends of cold-pressed mandarin oils with these products. Keywords: GC/IRMS; δ13CPDB values; authenticity profile; authenticity control; mandarin essential oil; Citrus reticulata Blanco

Using enantioselective multidimensional gas chromatography with heptakis[2,3-di-O-acetyl-6-O-(tert-butyldimethylsilyl)]-β-cyclodextrin as the chiral stationary phase, the simultaneous analysis of the 3-butylphthalide enantiomers and all eight 3-butylhexahydrophthalide stereoisomers was achieved. Enantiomeric distributions of compounds in celery, celeriac, celery seed, and fennel extracts were elucidated. Keywords: Enantioselective multidimensional gas chromatography (enantio-MDGC); 3-butyl(hexahydro)phthalide stereoisomers; celery; celeriac; fennel

cis- and trans-Rose oxide ketones are prepared enantioselectively using (R)-3-methylpentanedioic acid monomethyl ester as a chiral building block. All four stereoisomers of rose oxide ketone can be analysed simultaneously by enantioselective GC using heptakis-(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin in OV-1701-vi as the chiral stationary phase. The enantiomeric distribution of cis- and trans-rose oxide ketone in geranium oils of different origin was investigated and found to correlate with the enantiomeric ratios of cis- and trans-rose oxide. A pathway for the biogenesis of rose oxide ketone in Pelargonium species is proposed, including rose oxide as the precursor of rose oxide ketone. © 1997 John Wiley & Sons, Ltd.

The analysis of the δ13CPDB-values and the enantiomeric distribution in combination with the composition of the essential oil of dill (Anethum graveolens L.) at various developing stages of different plant parts allows us to draw valuable conclusions about the biosynthesis of the monoterpenes in the entire plant. The composition of the essential oil is different for the various plant parts and changes significantly during the ripening of the dill umbels. While α-phellandrene, β-phellandrene, dill ether and carvone occur enantiomerically pure in all investigated plant parts and during all stages of maturity, both enantiomers of limonene are detected. The enantiomeric composition of limonene is different for the various plant parts and changes during the development of the umbels. The δ13CPDB-values of the different monoterpenes also change with development. (R)-Limonene and carvone show absolutely the same behaviour in their δ13CPDB-values during the whole development of the umbels up to the seeds. In addition the δ13CPDB-values themselves are absolutely the same. Also α-phellandrene and dill ether show the same behaviour in their δ13CPDB-values, but the δ13CPDB-values themselves differ around 1.5–2‰. Based on the results of these investigations, a biochemical pathway of the monoterpenes in dill is postulated, according to which a very close biogenetic relationship is established for limonene and carvone and also for α-phellandrene and dill ether. © 1997 John Wiley & Sons, Ltd.

Using enantioselective gas chromatography and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (DIAC-TBDMS-β-CD) as the chiral stationary phase, the direct enantioseparation of 4-tert-butyl-α-methyldihydrocinnamaldehyde was achieved. The threshold values and odour characteristics of the enantiomers were investigated by enantioselective gas chromatography/olfactometry. In order to elucidate stereochemical features, the carbonyl-function was oxidized to the corresponding acid and diastereomeric amides were generated with (S)-2-amino-2-phenyl-ethanol [L(+)-α-phenylglycinol] as the enantiopure reagent. After separation and isolation by high-performance liquid chromatography, absolute configurations were deduced from X-ray structure elucidation of a pure stereoisomer. Amide cleavage, reduction and selective oxidation yielded the enantiomers of 4-tert-butyl-α-methyldihydrocinnamaldehyde.

The δ13CPDB and δ15NAIR values of methyl N-methylanthranilate from mandarin essential oils have been measured by capillary gas chromatography, on-line coupled with isotope ratio mass spectrometry (GC/IRMS). Cold-pressed and distilled mandarin peel oils of different origins and petitgrain oils mandarinier as well as reference substances of methyl N-methylanthranilate and methyl anthranilate were investigated. The combination of the δ13C and δ15N values is useful in the authenticity control of mandarin oils. Keywords: GC/IRMS; δ13CPDB and δ15NAIR values; methyl N-methylanthranilate; mandarin essential oil; Citrus reticulata Blanco