Chemical characterization and genetic relationships among Ocimum basilicum L. cultivars

Twenty-seven Ocimum basilicum cultivars were subjected to a chemical characterization of essential oil components by gas chromatography/mass spectrometry (GC/MS) and a genetic characterization using the amplified fragment-length polymorphism (AFLP) technique. Since the same 27 accessions had previously been classified into six morphotypes, these analyses allowed us to make detailed comparisons of chemistry, genetics, and morphology. The chemical composition and morphology of the studied cultivars appeared to have a strong genetic component. The AFLP analysis revealed a distinction between the green and purple morphotypes. The green morphotypes predominantly utilized the terpene biosynthetic pathway, while most purple morphotypes primarily utilized the phenylpropene biosynthetic pathway. The GC/MS analysis led to identification of 87 volatiles. Among the 27 cultivars, five chemotypes were identified. A detailed characterization of the essential oil constituents indicated the existence of both specific combinations of compounds and 'private' compounds with the potential to be used in many aspects of human life. The established relationship between a genetic profile, chemical composition, and morphology represents an important step in future breeding programs and in the cultivation of this species.     

Electrophysiological and behavioral identification of a volatile blend involved in host location of female strawberry sap beetle,Stelidota geminata

The strawberry sap beetle (SSB), Stelidota geminata (Say) (Coleoptera: Nitidulidae), is a serious direct pest of strawberries in the northeastern USA. To date, however, no food or pheromone‐based attractants for SSB have been identified. A combination of solid‐phase microextraction, adsorbent sampling, gas chromatography coupled with electroantennographic detection (GC–EAD), and gas chromatography–mass spectrometry was used to identify volatile compounds from strawberry fruit acting as behavioral attractants for female SSB. Consistent EAD activity was obtained for 16 ester compounds. In Y‐tube olfactometer bioassays, the blend of these 16 compounds mixed at a ratio observed from strawberry headspace was significantly more attractive to adult female SSB than a control. Female SSB showed no difference in response levels between the 16‐component blend and a strawberry volatile adsorbent extract. Our data indicate that the combination of the ethyl acetate with some or all of the remaining 15 compounds is necessary for this ester blend to be attractive to female SSB. Previously identified host volatiles found for other species of sap beetles included mostly alcohols associated with over‐ripe fruit rather than the esters identified from ripe strawberries for SSB. A highly attractive synthetic food odor will be useful for developing new management options for SSB.     

Analysis of Tobacco Headspace Volatiles Using Tenax GC or Active Carbon†

Two simple methods, the Tenax GC method and the active carbon method, have been developed to analyze the headspace volatiles of tobacco. The headspace volatiles swept by helium from cut tobacco were collected by either Tenax GC or active carbon and were analyzed by gas chromatography after desorption by heating the Tenax GC or by extracting the active carbon with dichloromethane. The gas chromatograms of the headspace volatiles of flue-cured tobacco obtained by these two methods differed. Both methods showed good reproducibility and were applied to the analysis of the headspace volatiles of American and Japanese flue-cured tobacco samples. By principal component analysis, three or four principal components were. extracted from the 15 selected peaks of the headspace volatiles of these samples that had been obtained from both the Tenax GC and the active carbon methods.     

Evaluation of enantioselective gas chromatography for the determination of minute deviations from racemic composition of alpha-amino acids with emphasis on tyrosine: accuracy and precision of the method

Whereas the determination of high enantiomeric fractions (EF) of chiral compounds is very well established, the accurate determination of small deviations from racemic compositions has not yet received much attention despite its relevance to studies dealing with the origin of homochirality, where only small initial enantiomeric bias is expected. Racemic samples of representative alpha-amino acids were derivatized as N-(O,S)-trifluoroacetyl/ethylesters and analyzed by enantioselective gas chromatography (GC) on fused silica capillaries coated with the chiral stationary phases (CSPs) Chirasil-D-Val, Chirasil-L-Val, and Lipodex E with GC/FID and GC/MS detection. The validation (accuracy and precision) of the determination of the enantiomeric fraction EF of the D-enantiomer in racemic or near-racemic compositions for 10 DL-alpha-amino acids obtained from commercial sources has been carried out. Emphasis is given to DL-tyrosine, the enantiomers of which have recently been claimed to show different crystallization properties. Values of EF obtained from GC measurements using CSPs were compared with those from CE using chiral mobile phase additives. While the precision of the GC method is generally better than 0.08% for all DL-alpha-amino acids studied, accuracy (trueness) of determination of amino acids with polar side chains is poorer than expected from the precision as a result of systematic errors. The accuracy determined relied on measurements on two oppositely configurated CSPs.     

Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization

Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.     

The determination of (-)-(S)- and (+)-(R)-ifosfamide in plasma using enantioselective gas chromatography: a validated assay for pharmacokinetic and clinical studies

An enantioselective gas chromatographic method has been developed and validated for the determination of the plasma concentration of the enantiomers of the anticancer drug ifosfamide (IFF). In this approach, the IFF enantiomers are separated from the plasma matrix by solid phase extraction, chromatographically resolved by gas chromatography on a chiral stationary phase, and detected by mass selective detection using selective ion monitoring. The assay has been validated for routine clinical and pharmacokinetic use and has a limit of detection in plasma of 250 ng/ml of each isomer.     

Chirality of organophosphorus pesticides: Analysis and toxicity

Although the importance of chirality in organophosphorus compounds (OPs) is well recognized in relation to their biological effects, as with most chiral pesticides, OPs are generally marketed, used and released to the environment as racemates (i.e., equimolar mixtures of enantiomers). In addition, research on enantioselective environmental fate and effects of chiral OPs is still limited, particularly in the evaluation of enantioselectivity in their environmental degradation. A large number of OPs are chiral compounds, and yet enantioselectivity in their environmental fate and effects is rarely addressed. This paper highlights the current state of knowledge on the environmental occurrence and behavior of chiral OP pesticides. Developments in enantioselective analytical techniques, specifically gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), as applied in the evaluation of enantiomer-specific fate and effects of chiral OPs, are also discussed.     

Enhancement of flavour biosynthesis from strawberry (Fragaria x ananassa) callus cultures by Methylobacterium species

Two important character-impact compounds of strawberry flavour, the furanones 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF) and 2,5-dimethyl-4-methoxy-2H-furan-3-one (mesifuran) were synthesized by strawberry tissue cultures derived from a cultivated species (Fragaria × ananassa, cv. Elsanta) after these were treated with Methylobacterium extorquens. These flavour compounds were analysed by HPLC-UV and their levels were compared in the treated and control tissues. In Methylobacterium extorquens treated callus cultures DMHF and mesifuran levels were 5.9 and 11.4 μg/g of fresh weight of callus respectively, compared to zero in the untreated ones. When Methylobacterium extorquens was fed with 1,2-propanediol, 2-hydroxy-propanal (lactaldehyde) was formed. This bacterial oxidation of 1,2-propanediol to lactaldehyde linked with the presence of 1,2-propanediol in strawberry suggests that the increased levels of the two furanones in the treated strawberry cultures is the result of Methylobacterium extorquens oxidative activity on 1,2-propanediol and the bioconversion by the plant cells of this oxidation product, lactaldehyde to DMHF and mesifuran. This revised version was published online in June 2006 with corrections to the Cover Date.     

Flower Volatiles,Crop Varieties and Bee Responses

Pollination contributes to an estimated one third of global food production, through both the improvement of the yield and the quality of crops. Volatile compounds emitted by crop flowers mediate plant-pollinator interactions, but differences between crop varieties are still little explored. We investigated whether the visitation of crop flowers is determined by variety-specific flower volatiles using strawberry varieties (Fragaria x ananassa Duchesne) and how this affects the pollination services of the wild bee Osmia bicornis L. Flower volatile compounds of three strawberry varieties were measured via headspace collection. Gas chromatography showed that the three strawberry varieties produced the same volatile compounds but with quantitative differences of the total amount of volatiles and between distinct compounds. Electroantennographic recordings showed that inexperienced females of Osmia bicornis had higher antennal responses to all volatile compounds than to controls of air and paraffin oil, however responses differed between compounds. The variety Sonata was found to emit a total higher level of volatiles and also higher levels of most of the compounds that evoked antennal responses compared with the other varieties Honeoye and Darselect. Sonata also received more flower visits from Osmia bicornis females under field conditions, compared with Honeoye. Our results suggest that differences in the emission of flower volatile compounds among strawberry varieties mediate their attractiveness to females of Osmia bicornis. Since quality and quantity of marketable fruits depend on optimal pollination, a better understanding of the role of flower volatiles in crop production is required and should be considered more closely in crop-variety breeding.     

Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Direct stereoselective assay of fluoxetine and norfluoxetine enantiomers in human plasma or serum by two-dimensional gas-liquid chromatography with nitrogen-phosphorus selective detection

A method was developed and validated for the direct enantioselective assay of fluoxetine and norfluoxetine in human plasma or serum by two-dimensional capillary gas-liquid chromatography (GC). A Rtx-1 fused-silica capillary (15 mx0.25 mm I.D., 1.0 micrometer film thickness) and a hydrodex-beta-6-TBDM fused-silica capillary (25 mx0.25 mm I.D., 0.25 micrometer film thickness) were used. A three-step liquid-liquid extraction was used for sample preparation with fluvoxamine and nisoxetine as internal standards. The method provided linear calibration between about 5 and 250 ng/ml for (R)- and (S)-fluoxetine as well as 15 and 250 ng/ml for (R)- and (S)-norfluoxetine. The limits of detection were about 1.5 and 6 ng/ml, respectively. Intra-day precision (coefficient of variation) was estimated as being between 5.4 and 12.7% at plasma levels of 25, 100 and 200 ng/ml for the four enantiomers. Inter-day precision was between 5.3 and 9.1% at 100 ng/ml. The enantioselective separation of some racemic psychopharmaceuticals was tested with various cyclodextrin GC-capillaries. Advantages and disadvantages of direct enantioselective GC are discussed for the assay of racemic psychopharmaceuticals. Samples from a patient who was treated with racemic fluoxetine were measured. In agreement with literature, plasma levels of the (R)-enantiomers of fluoxetine and norfluoxetine were considerably decreased in comparison to the (S)-enantiomers.     

Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

The hyphenation of enantioselective capillary gas chromatography and mass spectrometry is not always sufficient to distinguish between structural isomers, thus requiring peak identification by NMR spectroscopy. Here the first online coupling of enantioselective capillary gas chromatography with proton nuclear resonance spectroscopy is described for the unfunctionalized chiral alkane 2,4‐dimethylhexane resolved on octakis(6‐O‐methyl‐2,3‐di‐O‐pentyl)‐γ‐cyclodextrin at 60°C. NMR allows constitutional and configurational isomers (diastereomers and enantiomers) to be distinguished. Enantiomers display identical spectra at different retention times, which enable an indirect identification of these unfunctionalized alkanes. The presented method is still at an early development stage, and will require instrumental optimization in the future. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Thuja occidentalis: identification of volatiles and electroantennographic response by the invasive cedar bark beetle,Phloeosinus aubei

Recently, the distribution of the Mediterranean cedar bark beetle, Phloeosinus aubei Perris (Coleoptera: Scolytidae), has expanded to Central Europe. Reported mostly on cypress in the Mediterranean area, potential host plants in the invaded range include other scale‐leafed conifers, such as cultivars of arborvitae, Thuja occidentalis L. To reveal potential kairomonal cues for P. aubei, volatiles of T. occidentalis were collected and analysed by gas chromatography with electroantennographic detection (GC‐EAD). Assignments of chemical structures of antennally active components were carried out by gas chromatography linked to mass spectrometry (GC‐MS) using authentic reference samples. Antennal responses to synthetic samples of the identified compounds were studied by electroantennography (EAG), with antennae of female and male P. aubei. GC‐EAD analysis of head space volatiles of T. occidentalis revealed 22 antennally active compounds, of which 21 were identified. The most abundant components were α‐ and β‐thujone, fenchone, camphor, terpinen‐4‐ol, bornyl acetate and α‐terpinyl acetate, all of which are oxygenated monoterpenes. When EAG activities of synthetic samples were compared, the most intensive responses from female antennae were elicited by a mixture of α‐ and β‐thujone, followed by (–)‐terpinen‐4‐ol, (+)‐camphor, cis‐4‐thujanol, (+)‐sabina ketone, (+)‐terpinen‐4‐ol, isopulegone, (–)‐fenchone, borneol, (3Z)‐hexen‐1‐ol, (–)‐1‐octen‐3‐ol and (+)‐sulcatol. Male antennae responded the most to (–)‐terpinen‐4‐ol and cis‐4‐thujanol followed by the mixture of α‐ and β‐thujone. The next highest responses were elicited by (+)‐camphor, borneol, (+)‐terpinen‐4‐ol, (+)‐sulcatol and (+)‐sabina ketone. Striking differences were found between responses to the enantiomers of fenchone, sulcatol and 1‐octen‐3‐ol, whereas responses to the enantiomers of terpinen‐4‐ol did not differ significantly from each other. Several antennally active volatiles of T. occidentalis have also been reported from cypress and various other members of the Cupressaceae, suggesting that the sensory apparatus of P. aubei may recognize the shared components, which may enable rapid adaptation to new hosts in the invaded areas.     

Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone

Methadone enantiomers and EDDP, the main metabolite of methadone, were separated (R(s) = 2.0 for methadone enantiomers) following liquid-liquid extraction from human serum and urine followed by reverse-phase high-performance liquid chromatography on a derivatized beta-cyclodextrin column and quantified at therapeutic concentrations with ultraviolet detection. Detector response was linear (r(2) > 0.98) to 1,000 and 2,500 ng x mL(-1) for methadone enantiomers and EDDP, respectively. The limit of quantification from a 1-mL biological sample was 2.5 and 5 ng x mL(-1) for methadone enantiomers and EDDP, respectively. Interday variation was <13% and intraday variation was <8% for the analytes of interest. The assay was applied to plasma protein and erythrocyte binding studies and a 96-h pharmacokinetic study in two healthy female volunteers following oral dosing with rac-methadone. The binding of methadone to plasma proteins was enantioselective with the active (-)-(R) enantiomer having the highest free fraction (mean +/- SD: 21.2+/-7.6% vs. 13.3+/-6.2% for (+)-(S)-methadone, n = 8). Binding of methadone to erythrocytes was not apparently enantioselective (38.6+/-1.3% and 38.1+/-1.4% bound for (-)-(R)- and (+)-(S)-methadone, respectively). The pharmacokinetic study revealed enantioselective disposition of methadone in one volunteer but not in the other. EDDP was observed in urine but was only in small or undetectable concentrations in serum. The method is applicable to in vitro and pharmacokinetic studies of rac-methadone disposition in humans.     

Neutral Aroma Constituents in Burley Tobacco

The constituents of the neutral volatiles from air-cured Burley tobacco were studied using distillation, silicic acid column chromatography, preparative gas chromatography and GC–MS. The isolation and identification of 84 compounds are reported of which 27 are newly identified as tobacco constituents and 4 are new natural products.     

Separation and aquatic toxicity of enantiomers of the organophosphorus insecticide trichloronate

Many of the organophosphorus pesticides (OPs) currently used are chiral and therefore consist of mixture of enantiomers. Despite the fact that the biological processes of chiral pesticides are enantioselective, the acute aquatic toxicity of chiral OPs with respect to enantioselectivity has so far received limited research. In this study, the enantiomeric separation and acute aquatic toxicity of trichloronate were investigated. Baseline enantioseparation of trichloronate was successfully achieved using high-performance liquid chromatography on a Chiralcel OJ column, with a mobile phase of n-hexane/n-heptane/ethanol (90/5/5, v/v/v) at the flow rate of 1.0 ml min(-1) and room temperature. The resolved enantiomers were characterized for their optical rotation and by gas chromatography coupled with mass spectrometry. Significant differences were found between the enantiomers in acute aquatic toxicity to Ceriodaphnia dubia and Daphnia magna. The (-)-trichloronate was 8-11 times more toxic to the test organisms than its (+)-form, while the racemate showed intermediate toxicity. These results suggest that assessment of the environmental safety of chiral OPs should take stereospecificity into consideration.     

Behavioural response of alate Aphis craccivora Koch (Homoptera: Aphididae) to volatiles from different cowpea cultivars

The cowpea aphid, Aphis craccivora, is a major insect pest of cowpea in Africa. Volatile organic compounds (VOCs) mediate plant–arthropod interactions that could be used in the management of insect pests. In this study, we established the VOC profile involved in the interaction between A. craccivora and four cowpea cultivars, namely Ex‐Luanda, Katumani 80, Machakos 66 and Ken Kunde 1. Behavioural assays were conducted to study host preference and gas chromatography‐mass spectrometry (GC/MS) for chemical analysis of volatiles. In preference assays, alate A. craccivora had no significant preference for any of the four cowpea cultivars tested. However, in the olfactometer assays, the aphids showed a significant preference for odours from cultivar Ex‐Luanda compared to Katumani 80. Machakos 66 and Ken Kunde 1 elicited neutral responses. In pairwise comparisons, alate A. craccivora did not distinguish between odours of respective cowpea cultivars. GC/MS analysis identified 23 compounds in the volatiles of the four cowpea cultivars. Not all compounds were detected in all cowpea cultivars, and the detected compounds amounts varied in each cultivar. Of these, only four compounds (hexanal, (E)‐2‐hexenal, 1‐octen‐3‐ol and p‐xylene) were emitted in significantly different quantities in the four cultivars. A blend of hexanal and (E)‐2‐hexenal added to cowpea cultivar Ex‐Luanda decreased its attractiveness to A. craccivora compared to the control. Our findings showed differential attractiveness of VOCs of cowpea cultivars to A. craccivora, suggesting that VOCs could be used in the management of A. craccivora.     

Enantioselective behaviour of tetraconazole during strawberry wine‐making process

The fate of tetraconazole enantiomers in strawberries during wine‐making process was studied. The residues were determined by ultra‐performance convergence chromatography tandem triple quadrupole mass spectrometry after each process steps. Results indicated that there was significant enantioselective dissipation of tetraconazole enantiomers during the fermentation process. And (?)‐tetraconazole degraded faster than (+)‐tetraconazole. The half‐lives of (?)‐tetraconazole and (+)‐tetraconazole were 3.12, 3.76 days with washing procedure and 3.18, 4.05 days without washing procedure. The processing factors of strawberry wine samples after each step were generally less than 1. In particular, the processing factors of the fermentation process were the lowest. The results could help facilitate more accurate risk assessments of tetraconazole during wine‐making process.     

Odors of Norway spruce (Picea abies L.) seedlings: differences due to age and chemotype

Small conifer seedlings (mini-seedlings) are less damaged by the large pine weevil Hylobius abietis (L.) (Coleoptera: Curculionidae) compared to conventional seedlings. Chemical difference between the seedling types is one possible explanation for this phenomenon. In the present paper, the emissions of volatile organic compounds (VOC) of 7- to 43-week-old Norway spruce [Picea abies (L.) Karst.] seedlings were analyzed. Collection and identification of the volatiles was made by solid phase micro-extraction and gas chromatography mass spectrometry (SPME–GC–MS). The enantiomers of α-pinene and limonene were separated in a two-dimensional GC (2D-GC). Most of the seedlings represented either a limonene- or a bornyl acetate-chemotype. Only minor changes in the volatile composition of the two types of seedlings were found during the first year. Age-related changes, however, were found in the volatiles released by wounded phloem where green leaf volatiles (GLVs) and borneol were the dominated VOC for young seedling. The attractive compound for the pine weevil, α-pinene, was first detected in the phloem emissions of 18- to 22-week-old seedlings. Different storage conditions of the seedlings during the winter/early spring-phase influenced the volatile composition in the phloem. High amount of GLVs was characteristic for the 43-week-old seedlings stored in naturally changing outdoor temperature, but not present in the seedlings winter-stored at a constant temperature of ?4 °C. The possible role of these observed differences in odor emissions between seedlings of different age and physiological status for the feeding preferences of the large pine weevil is discussed.