Inter-laboratory comparison of elemental analysis and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Part I: delta13C measurements of selected compounds for the development of an isotopic Grob-test.

This study was directed towards investigating suitable compounds to be used as stable isotope reference materials for gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) calibration. Several compounds were selected from those used in the 'Grob-test' mixture. Oxygen- and nitrogen-containing substances were added to these compounds to allow the mixture to be used as a possible multi-isotopic calibration tool for 2H/1H, 13C/12C, 15N/14N and 18O/16O ratio determinations. In this paper we present the results of delta13C measurements performed by the consortium of the five laboratories taking part in this inter-calibration exercise. All the compounds were individually assessed for homogeneity, short-term stability and long-term stability by means of EA-IRMS, as required by the bureau communitaire de reference (BCR) Guide for Production of Certified Reference Materials. The results were compared then with the GC-C-IRMS measurements using both polar and non-polar columns, and the final mixture of selected compounds underwent a further certification exercise assessing limits of accuracy and reproducibility under specified GC-C-IRMS conditions.

Authenticity assessment: a permanent challenge in food flavor and essential oil analysis.

Both phenomena, enantioselectivity as well as isotope discrimination during biosynthesis, may serve as "endogenous" parameters, provided that suitable methods and comprehensive data from authentic sources are available. This review reports on enantioselective capillary gas chromatography and online methods of isotope-ratio mass spectrometry in the authentication of food flavor and essential oil compounds, referring to literature references published in the last decade.

Stereoselective analysis of 2-hydroxysebacic acid in urine of patients with Zellweger syndrome and of premature infants fed with medium-chain triglycerides.

The chiral metabolite 2-hydroxysebacic acid (2-HS) is considered to be an important diagnostic marker for peroxisomal disorders. The pathway of formation of 2-HS, excreted in increased amounts in patients with peroxisomal diseases, is not absolutely clear. Moreover, there is no information about the enantiomeric distribution of 2-HS in human urine. Here, we describe the stereodifferentiation of 2-HS in urine samples of nine patients with Zellweger syndrome (ZS), and for the first time in urine samples of premature infants fed a medium-chain triglyceride (MCT)-containing diet. Using enantioselective multidimensional gas chromatography-mass spectrometry, an increased excretion of 2R-HS was observed in all investigated ZS patients. 2-HS was also present in urine samples of premature infants fed MCT. Analogously to the ZS patients, a dominant 2R-HS excretion in the urine samples of the premature infants was identified. The formation of 2-HS is expected to result from the same or similar pathways as described for ZS patients. Additionally, we determined the absolute configuration of urinary 3-hydroxysebacic acid (3-HS) in the cases investigated. The enantioselective analysis provides further information for the diagnosis and treatment of patients with impaired peroxisomal fatty acid oxidation. Further insight into the metabolic origin and the biochemical pathway leading to these urinary metabolites is provided.

Different stereoselectivity in the reduction of pulegone by Mentha species.

Aqueous solutions of [2H]-labeled pulegone enantiomers were fed to Mentha spicata ssp. spicata L. and Mentha spicata ssp. crispata L. shoot tip and first leaf pair. After solid phase microextraction the essential oil was analysed with enantioselective multidimensional gas chromatography/mass spectrometry. Both Mentha spicata species were able to convert labelled (1R)- and (1S)-pulegone at the same rate into the corresponding menthone and isomenthone, indicating an unspecific process. The reduction of both pulegone enantiomers preferably led to the cis-stereoisomers. The observed stereoselectivity is completely different from those of pulegone reduction by Mentha x piperita L.

Menthone and isomenthone biosynthesis in Pelargonium tomentosum Jacq.

Pelargonium tomentosum was fed with aqueous solutions of different deuterium labelled monoterpenoid precursors. After headspace extraction with solid phase microextraction (SPME) the essential oil was analyzed with enantioselective multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS). Labelled piperitone with different enantiomeric distributions led to labelled (-)-isomenthone and labelled (+)-menthone in the same ratio as genuine (-)-isomenthone and (+)-menthone. Furthermore, labelled pulegone, piperitenone, piperitole and citronellyl glycopyranoside were investigated.

[Enantiomeric monoterpenes in ether oil from Achillea millefolium s. I.--a taxonomically useful marker?]. / Enantiomere Monoterpene im ätherischen Ol von Achillea millefolium s. l.--eine zusätzliche taxonomische Bestimmungshilfe?

The Achillea millefolium complex is a group of taxonomically hardly separable species. Yarrow has the tendency to hybridize and to vary in phenotype. An obvious characterization of the species or hybrids is not just important for the taxonomical distinction but also for a reliable assessment of herbal drug quality. Most of the Achillea plants are still gathered from natural populations. According to the variation in phenotype, mixtures with Achillea species, which contain allergy setting compounds, often cannot be determined. Morphometric investigations exclusively do not replace a further chemical characterization. Therefore we tried to assess the composition of the chiral monoterpenes alpha-pinene, beta-pinene and sabinene. They were selected because of their frequency in the essential oil of Achillea species. By method of M.C.S.S. (Moving Capillary Stream Switching) the differentiation of stereoisomeres succeeded directly from the essential oil, which was distilled or extracted by headspace trapping at room temperature. The enantiomeric distribution neither depends on the method of extraction, nor on the habitat or the developmental stage of Yarrow. Since the compositions of enantiomeres from several Achillea species and their hybrides are of different pattern, it seemed to represent an additional marker. Though investigations of all species within the Achillea millefolium group and possibly of further chiral compounds are necessary to ensure these results.

Stereodifferentiation of 3-hydroxyisobutyric- and 3-aminoisobutyric acid in human urine by enantioselective multidimensional capillary gas chromatography-mass spectrometry.

The chiral metabolites 3-hydroxyisobutyric acid (HIBA) and 3-aminoisobutyric acid (AIBA) are intermediates in the pathways of L-valine and thymine and play an important role in the diagnosis of the very rare inherited metabolic diseases 3-hydroxyisobutyric aciduria (McKusick 236975) and methylmalonic semialdehyde dehydrogenase deficiency (McKusick 603178-MSDD). Until now only a few approaches have been made in enantioselective analysis of HIBA and AIBA and for that reason very little information is available on enantiomeric ratios of these metabolites in man. This paper reports on the simultaneous stereodifferentiation of HIBA and AIBA in human urine as corresponding N(O)-methoxycarbonyl methyl esters by derivatization with methyl chloroformate (MCF) using enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC/MS) with heptakis-(2, 3-di-O-methyl-6-O-tert.-butyl-dimethylsilyl)-beta-cyclodextrin as the chiral stationary phase. During this investigation urine samples from different patients and healthy controls were analyzed in order to reveal characteristic enantiomeric patterns of these metabolites. A trend of dominating R-HIBA excretion in the control urine samples investigated was observed. An excretion of more than 80% S-HIBA was found in the urines of two patients with ketonemic vomiting. There are some clues indicating a possible renal reabsorbtion of S-HIBA similar to those of S-AIBA. Furthermore, there was a significant finding with regard to the enantiomeric distribution of AIBA in a patient with MSDD - a markedly increased excretion of the S-enantiomer in contrast to the other samples. Using the enantiomeric ratios of AIBA, a previously investigated case of benign methylmalonic aciduria (bMMA) could be excluded from the diagnosis of MSDD.

Biosynthesis of menthofuran in Mentha x piperita: stereoselective and mechanistic studies.

Mentha x piperita shoot tips and first leaf pair were fed with aqueous solutions of [(2)H(2)]- and [(2)H(2)]/[(18)O]-labeled pulegone. The essential oil was analyzed by solid phase microextraction and enantioselective multidimensional gas chromatography/mass spectrometry. After feeding experiments with labeled pulegone racemate, both labeled (S)-menthofuran and (R)-menthofuran were detectable simultaneously together with genuine (R)-menthofuran. It could be shown that both labeled pulegone enantiomers are converted by Mentha x piperita to the corresponding labeled menthofuran enantiomers, favoring the labeled analogue of the nongenuine (S)-pulegone. The oxygen in menthofuran is introduced by enzymatic oxidation of pulegone, as concluded from feeding experiments with mixed labeled [(2)H(2)]/[(18)O]pulegone.

Biogenetic studies in Mentha x piperita. 1. deuterium-labeled monoterpene ketones: synthesis and stereoselective analysis.

Pulegone, menthone, and isomenthone isotopomers are synthesized as regioselectively deuterated d(5)- and d(8)-stereoisomers. Deuterium-labeled menthone and isomenthone enantiomers are analyzed using enantioselective multidimensional gas chromatography/mass spectrometry. The deuterated stereoisomers of menthone and isomenthone are separated from the unlabeled menthone and isomenthone on a glass capillary column, coated with 50% octakis(2, 3-di-O-butyryl-6-O-tert- butyldimethylsilyl)-gamma-cyclodextrin in OV 1701vi as the chiral stationary phase. These deuterium-labeled monoterpene ketones are proved to be highly valuable substrates in biosynthetic studies of terpenoid compounds.

Biogenetic studies in Mentha x piperita. 2. Stereoselectivity in the bioconversion of pulegone into menthone and isomenthone.

Mentha x piperita shoot tips and first leaf pairs were fed with aqueous solutions of different deuterium-labeled pulegone and various enantiomeric distributions. The essential oil was extracted by solid-phase microextraction and analyzed using enantioselective multidimensional gas chromatography/mass spectrometry. The genuine p-menthan-3-ones (-)-menthone and (+)-isomenthone as well as their labeled analogues were analyzed simultaneously. Both enantiomers of labeled pulegone were converted into the corresponding labeled p-menthan-3-ones by Mentha x piperita, indicating an unspecific reduction process. The generation of 4S- and 4R-configured p-menthan-3-ones differed in their stereoselectivities. Labeled (S)-pulegone was reduced by Mentha x piperita more rapidly rather than (R)-pulegone. From a comparison of labeled pulegone enantiomers the bioconversion preferrably led to 4S-configured diastereomers.

Structure elucidation, enantioselective analysis, and biogenesis of nerol oxide in Pelargonium species.

A novel enantioselective synthesis of nerol oxide (3, 6-dihydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran) was used for the determination of the absolute configuration at C-2. The order of elution of the enantiomers on octakis-(2, 3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-gamma-cyclodextrin in OV 1701-vi as the chiral stationary phase in enantioselective GC was determined as (2R) before (2S). Enantioselective multidimensional GC/MS (enantio-MDGC/MS) was used for the determination of the enantiomeric ratios of nerol oxide in different geranium oils. As a result, in all investigated oils nerol oxide occurs as a racemate. The biogenesis of nerol oxide in Pelargonium species was investigated by feeding experiments using deuterium-labeled neryl glucoside as the precursor. The Pelargonium plants were able to convert the fed precursor into racemic nerol oxide, which has to be considered as a "natural racemate".

Conversion of citronellyl diphosphate and citronellyl beta-D-glucoside into rose oxide by Pelargonium graveolens.

Deuterium-labeled citronellyl diphosphate and citronellyl beta-D-glucoside were fed to Pelargonium graveolens. Both precursors were converted into cis-/trans-rose oxide. Citronellyl diphosphate is more effectively converted into rose oxide. The phloem exudate of P. graveolens was analyzed after enzymatic cleavage of the glucosidated and phosphorylated volatiles. It could be shown that glucosidically bound citronellol is translocated in the plant. Phosphorylated citronellol could not be detected.

4,5-dimethyl-3-hydroxy-2[5H]-furanone (sotolone)--the odour of maple syrup urine disease.

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.

Identification of Melissa officinalis Subspecies by DNA Fingerprinting.

The random amplified polymorphic DNA analysis (RAPD) is a method to study genetic variability within and between populations and species on the basis of the amplification of anonymous fragments from genomic DNA templates by means of polymerase chain reaction (PCR). We applied RAPD analysis in order to distinguish medicinal plant subspecies at the level of their genomes. In this study we investigated various samples of two MELISSA subspecies and showed that RAPD analysis is a fast and reliable method to distinguish subspecies on the pharmaceutical market that have been previously classified according to the distribution pattern of compounds present in the lemon balm oil.

Enantioselective multidimensional gas chromatography-mass spectrometry in the analysis of urinary organic acids.

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.

Chiral compounds in metabolism: a look in the molecular mirror.

UNLABELLED: 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. CONCLUSION: 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.

Chiral compounds as indicators of inherited metabolic disease. Simultaneous stereodifferentiation of lactic-, 2-hydroxyglutaric- and glyceric acid by enantioselective cGC.

Urinary organic acid analysis is a vital diagnostic tool in the investigation of patients with suspected inborn errors of metabolism. Recent descriptions of diseases producing abnormal chiral metabolites have provided the need for individual enantiomer analysis. In this paper the isolation and simultaneous stereodifferentiation of lactic, glyceric and 2-hydroxyglutaric acids using capillary gas chromatography on heptakis (2,3-di-O-methyl-6-O-tert.butyldimethylsilyl)-beta-cyclodextrin as the chiral stationary phase is described. The diagnostic potential of this method is demonstrated by the analysis of urine samples from two patients with D-2-hydroxyglutaric aciduria and a dog with D-glyceric aciduria. Due to the lack of specific clinical symptoms in these diseases, enantiomeric analysis is vital for diagnosis enabling prompt therapeutic intervention.

[Stereoisomeric aromatic compounds XIX: Asymmetric reduction of 4(5)-oxocarboxylic acids with baker's yeast]. / Stereoisomere Aromastoffe XIX: Asymmetrische Reduktion von 4(5)-Oxocarbonsäuren mit Bäckerhefe.

Asymmetric reduction of 4(5)-oxocarboxylic acids (esters) by baker's yeast and cyclization in acidic media yields optically active gamma(delta)-lactones. The evaluation of their chirality and optical purity was carried out by HPLC (HRGC) analysis of the corresponding 1,4(1,5)-diols via diastereomeric esters with (R)-Mosher acid (MTPA) and (S)-O-acyllactic acids respectively. By increasing the 4(5) alkyl side chain 4R(5R) configurated gamma(delta)-lactones with high ee-values are generated.