Enantioselective Multidimensional Gas Chromatography-mass Spectrometry Research Articles

Relationship between the terpene enantiomeric distribution and the growth cycle of lemon fruit and comparison of two extraction methods

Lemon essential oil is one of the most widely used natural flavours in the food and beverage industries. Knowledge of the enantiomeric ratio in flavours has gained relevance because they may indicate differences in their organoleptic characteristics or their authenticity. The study was carried out during the growth of the lemon fruit to evaluate the possible variations of the enantiomeric ratio of four characteristic terpenes: α-pinene, β-pinene, linalool and limonene. Two extraction methods were applied to extract the terpenes: simultaneous distillation–extraction at reduced pressure (V-SDE) and solid-phase microextraction; the results were equivalent whatever the method applied. In both cases, the target compounds were obtained from the lemon peel. Enantiomeric profiles were obtained by enantioselective multi-dimensional gas chromatography–mass spectrometry (enantio-MDGC-MS). Consistent enantiomeric ratios of α-pinene, β-pinene and limonene were observed; however, the enantiomeric ratio of linalool varied significantly according to the growth stage of the lemons in a spontaneous and natural way.

Enantiomeric distribution of odour-active epoxyaldehydes in yuzu (Citrus junos Sieb. ex Tanaka)

The odour-active epoxyaldehydes cis-4,5-epoxy-(E)-2-decenal (1), trans-4,5-epoxy-(E)-2-decenal (2), and trans-4,5-epoxy-(E,Z)-2,7-decadienal (3) are present in yuzu (Citrus junos Sieb. ex Tanaka) volatile oil. These epoxyaldehydes have two chirality centres at the C4 and C5 positions in the epoxide moiety. To date, little is known about the enantiomeric distributions of the expoxyaldehydes in natural products. In this study, we examined the enantiomeric distributions of the epoxyaldehydes in yuzu volatile oil using multidimensional gas chromatography. The standard substrates used for 1 and 3 were synthesised enantioselectively using the Katsuki-Sharpless epoxidation. Standard substrates used for 2 were made via the asymmetric organocatalytic epoxidation with a proline-derivative catalyst. The 4S,5R- and 4R,5S-enantiomers of 1 were obtained in moderate enantiomeric excess (44–66 % ee), while 4S,5S- and 4R,5R-enantiomers of 2 and 3 were obtained in high enantiomeric excess (2, 90–92 % ee; 3, 96 % ee). Enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS) revealed that the chiral centres at C4 and C5 in the cis-form (1) have the (4R,5S) absolute configuration, while trans-forms (2 and 3) have the (4S,5S) configuration. The finding of the absolute configuration at C5 (S) suggests that the optically active epoxyaldehydes may be formed along with retention of the S configuration at C13 of the lipid hydroperoxides 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) or 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) during enzymatic lipid oxidation. Finally, we propose a pathway for the formation of the optically active epoxyaldehydes in yuzu.

Occurrence of 2,2,4-trimethyl–1,3-pentanediol monoisobutyrate (Texanol®) in foods packed in polystyrene and polypropylene cups

After simultaneous distillation–extraction (SDE) of foods packed in polystyrene (n = 77) and polypropylene cups (n = 42) from 61 different suppliers, coupled capillary gas chromatography–mass spectrometric (HRGC–MS) analyses indicated the presence of diastereomers of 2,2,4-trimethyl–1,3-pentanediol monoisobutyrate (TMPD-MIB; Texanol®), a known coalescent of paints and printing inks. The contaminant was found in 55 and 50% of the polystyrene and polypropylene packed samples, respectively. Amounts ranged 1.2–64.5 µg kg−1 in polystyrene cups (average 25.1 µg kg−1) and 0.9–45.7 µg kg−1 in polypropylene cups (average 10.8 µg kg−1). The origin of Texanol® in the printed plastic cups was demonstrated by separate HRGC–MS analysis, showing amounts in the higher µg kg−1 range. In addition, the presence of two pairs of enantiomers, both found to be racemic by enantioselective multi-dimensional gas chromatography–mass spectrometry (enantio-MDGC–MS), excluded it being of natural origin. The detection limit of overall procedure (DLOP) and the reliable quantification limit (RQL) were 0.2 and 0.9 µg kg−1, respectively. As the diester, 2,2,4-trimethyl–1,3-pentanediol diisobutyrate (TXIB), is on the EU list of regulated substances (restricted to single-use gloves only) with a migration limit of 5 mg kg−1 in food and is metabolised rapidly by hydrolysis, the observed migration of the monoester Texanol® at the µg kg−1 level poses no risk of adverse effects.

Metabolism of linalool and substrate analogs in grape berry mesocarp of Vitis vinifera L. cv. Morio Muscat: demonstration of stereoselective oxygenation and glycosylation

The oxidative metabolism of linalool in Vitis vinifera L. cv. Morio Muscat has been investigated by in vivo feeding experiments using the regioselectively stable isotope-labelled substrates d 5-(3 R/ S)-linalool, d 6-(3 R)-linalool, d 2, 18O-(3 R/ S)-linalool and the substrate analog (3 R)-linalyl methyl ether. The enantiomeric and diastereoisomeric ratios of the metabolites were determined by means of enantioselective multidimensional gas chromatography–mass spectrometry. Stereoselective transformation to diendiol II and the furanoid and pyranoid linalool oxides could be demonstrated. Other metabolites like diendiol I, hotrienol and 8-hydroxy linalool were detectable as well. The studies indicate that the corresponding metabolites were efficiently glycoconjugated. Time course studies including the determination of conversion rates revealed that the activity of these secondary transformations is dependent on the ripening stage. The mass spectrometric analysis of the labelled linalool oxides derived from mixed labelled d 2, 18O-linalool and the stereoselective anaylsis of the metabolite 6-methoxy-2,6-dimethyloct-7-en-2,3-diol, which is derived from the substrate analog (3 R)-linalyl methyl ether, give evidence that the furanoid linalool oxides are generated via two different reaction pathways.

Biogenetic studies in Syringa vulgaris L.: bioconversion of (18)O(2H)-labeled precursors into lilac aldehydes and lilac alcohols.

Syringa vulgaris L. inflorescences, petals, and chloroplasts, isolated from lilac flower petals, were fed with aqueous solutions of (18)O-labeled linalool and [5,5-(2)H(2)]-deoxy-d-xylose (DOX). The chloroplasts of lilac flower petals were isolated after feeding experiments with labeled precursors. Volatiles from the chloroplasts were extracted by stir bar sorptive extraction (SBSE) and analyzed using enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS). Feeding experiments with DOX indicate that the novel mevalonate-independent 1-deoxy-d-xylose 5-phosphate/2C-methyl-d-erythritol 4-phosphate (DOX/MEP) is the decisive pathway of lilac aldehyde and lilac alcohol, respectively. Bioconversion of [(18)O]linalool into lilac aldehyde and lilac alcohol during in vivo feeding experiments was monitored, and the metabolic pathways are discussed.

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.

Authenticity assessment of lavender oils using GC-P-IRMS: 2 H/ 1 H isotope ratios of linalool and linalyl acetate

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.

Enantioselective analysis of monoterpene compounds in essential oils by stir bar sorptive extraction (SBSE)–enantio‐MDGC–MS

AbstractStir 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.

Stir bar sorptive extraction (SBSE)-enantio-MDGC-MS – a rapid method for the enantioselective analysis of chiral flavour compounds in strawberries

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.

Monoterpene biosynthesis inAgathosma crenulata (Buchu)

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.

Enantioselective analysis of ketone bodies in patients with β-ketothiolase deficiency, medium-chain acyl coenzyme A dehydrogenase deficiency and ketonemic vomiting

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 β-ketothiolase deficiency, is not absolutely clarified. Metabolic pathways involving this metabolite are isoleucine catabolism, as well as presumably β-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 β-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.

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)-β-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 markly 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.

Analysis of Methylcitric Acid by Enantioselective Multidimensional Gas Chromatography-Mass Spectrometry

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.

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.

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, α-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 α-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.