Changes in 13C/12C of oil palm leaves to understand carbon use during their passage from heterotrophy to autotrophy

The carbon isotope composition of leaf bulk organic matter was determined on the tropical tree Elaeis guineensis Jacq. (oil palm) in North Sumatra (Indonesia) to get a better understanding of the changes in carbon metabolism during the passage from heterotrophy to autotrophy of the leaves. Leaf soluble sugar (sucrose, glucose and fructose) contents, stomatal conductance and dark respiration, as well as leaf chlorophyll and nitrogen contents, were also investigated. Different growing stages were sampled from leaf rank ?6 to rank 57. The mean values for the δ¹³C of bulk organic matter were ?29.01 ± 0.9‰ for the leaflets during the autotrophic stage, ?27.87 ± 1.08‰ for the petioles and ?28.17 ± 1.09‰ for the rachises, which are in the range of expected values for a C₃ plant. The differences in δ¹³C among leaf ranks clearly revealed the changes in the origin of the carbon source used for leaf growth. Leaves were ¹³C‐enriched at ranks below zero (around ?27‰). During this period, the ‘spear’ leaves were completely heterotrophic and reserves from storage organs were mobilised for the growth of these young emerging leaves. ¹³C‐depletion was then observed when the leaf was expanding at rank 1, and there was a continuous decrease during the progressive passage from heterotrophy until reaching full autotrophy. Thereafter, the δ¹³C remained more or less constant at around ?29.5‰. Changes in sugar content and in δ¹³C related to leaf ranks showed an interesting similarity of the passage from heterotrophy to autotrophy of oil palm leaves to the budburst of some temperate trees or seed germination reported in the literature. Copyright © 2009 John Wiley & Sons, Ltd.     

A method for 13C‐labeling of metabolic carbohydrates within French bean leaves (Phaseolus vulgaris L.) for decomposition studies in soils

The molecular composition of plant residues is suspected to largely govern the fate of their constitutive carbon (C) in soils. Labile compounds, such as metabolic carbohydrates, are affected differently from recalcitrant and structural compounds by soil‐C stabilisation mechanisms. Producing ¹³C‐enriched plant residues with specifically labeled fractions would help us to investigate the fate in soils of the constitutive C of these compounds. The objective of the present research was to test ¹³C pulse chase labeling as a method for specifically enriching the metabolic carbohydrate components of plant residues, i.e. soluble sugars and starch. Bean plants were exposed to a ¹³CO₂‐enriched atmosphere for 0.5, 1, 2, 3 and 21 h. The major soluble sugars were then determined on water‐soluble extracts, and starch on HCl‐hydrolysable extracts. The results show a quick differential labeling between water‐soluble and water‐insoluble compounds. For both groups, ¹³C‐labeling increased linearly with time. The difference in δ¹³C signature between water‐soluble and insoluble fractions was 7‰ after 0.5 h and 70‰ after 21 h. However, this clear isotopic contrast masked a substantial labeling variability within each fraction. By contrast, metabolic carbohydrates on the one hand (i.e. soluble sugars + starch) and other fractions (essentially cell wall components) on the other hand displayed quite homogeneous signatures within fractions, and a significant difference in labeling between fractions: δ¹³C = 414 ± 3.7‰ and 56 ± 5.5‰, respectively. Thus, the technique generates labeled plant residues displaying contrasting ¹³C‐isotopic signatures between metabolic carbohydrates and other compounds, with homogenous signatures within each group. Metabolic carbohydrates being labile compounds, our findings suggest that the technique is particularly appropriate for investigating the effect of compound lability on the long‐term storage of their constitutive C in soils. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation and Total Synthesis of Kirkamide,an Aminocyclitol from an Obligate Leaf Nodule Symbiont

The new C₇N aminocyclitol kirkamide ( 1 ) was isolated from leaf nodules of the plant Psychotria kirkii by using a genome‐driven ¹H NMR‐guided fractionation approach. The structure and absolute configuration were elucidated by HRMS, NMR, and single‐crystal X‐ray crystallography. An enantioselective total synthesis was developed, which delivered kirkamide ( 1 ) on a gram scale in 11 steps and features a Ferrier carbocyclization and a Pd‐mediated hydroxymethylation. We propose that kirkamide is synthesized by Candidatus Burkholderia kirkii, the obligate leaf symbiont of Psychotria kirkii. Kirkamide ( 1 ) was shown to be toxic to aquatic arthropods and insects, thus suggesting that bacterial secondary metabolites play a protective role in the Psychotria/Burkholderia leaf nodule symbiosis.     

Isolation and Total Synthesis of Kirkamide,an Aminocyclitol from an Obligate Leaf Nodule Symbiont

The new C₇N aminocyclitol kirkamide ( 1 ) was isolated from leaf nodules of the plant Psychotria kirkii by using a genome‐driven ¹H NMR‐guided fractionation approach. The structure and absolute configuration were elucidated by HRMS, NMR, and single‐crystal X‐ray crystallography. An enantioselective total synthesis was developed, which delivered kirkamide ( 1 ) on a gram scale in 11 steps and features a Ferrier carbocyclization and a Pd‐mediated hydroxymethylation. We propose that kirkamide is synthesized by Candidatus Burkholderia kirkii, the obligate leaf symbiont of Psychotria kirkii. Kirkamide ( 1 ) was shown to be toxic to aquatic arthropods and insects, thus suggesting that bacterial secondary metabolites play a protective role in the Psychotria/Burkholderia leaf nodule symbiosis.     

Development of an advanced on‐line position‐specific stable carbon isotope system and application to methyl tert‐butyl ether

We present an advanced system for on‐line position‐specific carbon isotope analysis. The main limitation of on‐line intramolecular isotope ratio measurements has been that optimal pyrolytic fragments are obtained mostly at temperatures where the analyte has not completely reacted. As a result of undetermined isotopic fractionation, the isotopic signatures of the pyrolysis products are not strictly equal to these of the equivalent moieties in the parent molecule. We designed a pyrolytic unit in which both temperature and reaction time are variable parameters, enabling determination of the enrichment factor of the pyrolysis at optimal temperature by construction of a Rayleigh plot. In the case of methyl tert‐butyl ether (MTBE) presented here, a ‘pre‐pyrolysis’ fractionation of MTBE leading to a depletion of 0.9‰ was discovered and the enrichment factor of the optimal pyrolysis reaction was determined at −1.7‰. Absolute δ¹³C values of two functional groups of MTBE – the methoxy group and the 2‐methylpropane group – could be determined with 95% confidence intervals of 0.4‰ and 0.5‰, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Atmospheric pressure laser desorption/ionization of plant metabolites and plant tissue using colloidal graphite

Colloidal graphite is a promising matrix for atmospheric pressure laser desorption/ionization mass spectrometry. Intact [M+H]⁺ and [M–H]^? ions are readily produced from a wide range of small molecule plant metabolites, particularly anthocyanins, fatty acids, lipids, glycerides, and ceramides. Compared with a more traditional organic acid matrix, colloidal graphite provides more efficient ionization for small hydrophobic molecules and has a much cleaner background spectrum, especially in negative ion mode. Some important metabolites, e.g., fatty acids and glycosylated flavonoids, can be observed from Arabidopsis thaliana leaf and flower petal tissues in situ. Copyright © 2010 John Wiley & Sons, Ltd.     

Membrane permeation continuous‐flow isotope ratio mass spectrometry for on‐line carbon isotope ratio determination

Gaseous membrane permeation (MP) technologies have been combined with continuous‐flow isotope ratio mass spectrometry for on‐line δ¹³C measurements. The experimental setup of membrane permeation‐gas chromatography/combustion/isotope ratio mass spectrometry (MP‐GC/C/IRMS) quantitatively traps gas streams in membrane permeation experiments under steady‐state conditions and performs on‐line gas transfer into a GC/C/IRMS system. A commercial polydimethylsiloxane (PDMS) membrane sheet was used for the experiments. Laboratory tests using CO₂ demonstrate that the whole process does not fractionate the C isotopes of CO₂. Moreover, the δ¹³C values of CO₂ permeated on‐line give the same isotopic results as off‐line static dual‐inlet IRMS δ¹³C measurements. Formaldehyde generated from aqueous formaldehyde solutions has also been used as the feed gas for permeation experiments and on‐line δ¹³C determination. The feed‐formaldehyde δ¹³C value was pre‐determined by sampling the headspace of the thermostated aqueous formaldehyde solution. Comparison of the results obtained by headspace with those from direct aqueous formaldehyde injection confirms that the headspace sampling does not generate isotopic fractionation, but the permeated formaldehyde analyzed on‐line yields a ¹³C enrichment relative to the feed δ¹³C value, the isotopic fractionation being 1.0026 ± 0.0003. The δ¹³C values have been normalized using an adapted two‐point isotopic calibration for δ¹³C values ranging from ?42 to ?10‰. The MP‐GC/C/IRMS system allows the δ¹³C determination of formaldehyde without chemical derivatization or additional analytical imprecision. Copyright © 2009 John Wiley & Sons, Ltd.     

Short‐term dynamics of isotopic composition of leaf‐respired CO2 upon darkening: measurements and implications

Recent advances in understanding the metabolic origin and the temporal dynamics in δ¹³C of dark‐respired CO₂ (δ¹³C_res) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in δ¹³C_res have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in δ¹³C of respiratory substrates; second, post‐photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post‐illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in δ¹³C_res (ranging from 0 to 10‰ diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in δ¹³C_res was negligible in all investigated species. Further, rapid post‐illumination changes in δ¹³C_res (30 min), which increased from 2.6‰ to 5‰ over the course of the day, were examined by positional ¹³C‐labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in δ¹³C_res. However, a Rayleigh fractionation of ¹²C‐discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post‐illumination peak in δ¹³C_res. These results are highly relevant since δ¹³C_res is used in large‐scale carbon cycle studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Multiconstituent identification in root,branch, and leaf extracts of Juglans mandshurica using ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

As a traditional medicinal plant, Juglans mandshurica has been used for the treatment of cancer. Different organs of this plant showed anti‐tumor activity in clinic and laboratory. Comparative identification of constituents in different plant organs is essential for investigation of the relationship between chemical constituents and pharmacological activities. For this aim, the roots, branches, and leaves of J. mandshurica were extracted with 50% v/v methanol and then subjected to ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis conducted under low and high energy. As a result, we have to date identified 111 compounds consisting of 56 tannins, 29 flavonoids, 13 organic acids, 8 naphthalene derivatives, and 5 anthracenes. Five compounds, namely, diquercetin trihydroxy‐truxinoyl‐glucoside, two quercetin kaempferol dihydroxy‐truxinoyl‐glucosides, syringoyl‐tri‐galloyl‐O‐glucose, and dihydroxy‐naphthalene syringoyl‐glucoside, were tentatively identified as new compounds. Of the compounds identified, 76 were found in the root extract, 67 in the branch extract, and 37 in the leaf extract. Only six compounds including four organic acids and two tannins were found in all three extracts. We developed a rapid and sensitive ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry approach to identify multiple constituents of complex extracts without separation and ion selection. The results presented provide useful information on further research of the bioactive compounds of J. mandshurica .     

Effects of long‐term exposure to elevated CO2 conditions in slow‐growing plants using a 12C‐enriched CO2‐labelling technique

Despite their relevancy, long‐term studies analyzing elevated CO₂ effect in plant production and carbon (C) management on slow‐growing plants are scarce. A special chamber was designed to perform whole‐plant above‐ground gas‐exchange measurements in two slow‐growing plants (Chamaerops humilis and Cycas revoluta) exposed to ambient (ca. 400 µmol mol^?1) and elevated (ca. 800 µmol mol^?1) CO₂ conditions over a long‐term period (20 months). The ambient isotopic ¹³C/¹²C composition (δ¹³C) of plants exposed to elevated CO₂ conditions was modified (from ca. ?12.8‰ to ca. ?19.2‰) in order to study carbon allocation in leaf, shoot and root tissues. Elevated CO₂ increased plant growth by ca. 45% and 60% in Chamaerops and Cycas, respectively. The whole‐plant above‐ground gas‐exchange determinations revealed that, in the case of Chamaerops, elevated CO₂ decreased the photosynthetic activity (determined on leaf area basis) as a consequence of the limited ability to increase C sink strength. On the other hand, the larger C sink strength (reflected by their larger CO₂ stimulatory effect on dry mass) in Cycas plants exposed to elevated CO₂ enabled the enhancement of their photosynthetic capacity. The δ¹³C values determined in the different plant tissues (leaf, shoot and root) suggest that Cycas plants grown under elevated CO₂ had a larger ability to export the excess leaf C, probably to the main root. The results obtained highlighted the different C management strategies of both plants and offered relevant information about the potential response of two slow‐growing plants under global climate change conditions. Copyright © 2008 John Wiley & Sons, Ltd.     

Nicotine,acetanilide and urea multi‐level 2H‐, 13C‐ and 15N‐abundance reference materials for continuous‐flow isotope ratio mass spectrometry

Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the δ values of these reference materials should bracket the isotopic range of samples with unknown δ values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW‐SLAP) and carbonates (NBS 19 and L‐SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA‐IRMS). At present only L‐glutamic acids USGS40 and USGS41 satisfy these requirements for δ¹³C and δ¹⁵N, with the limitation that L‐glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on‐line (i.e. continuous flow) hydrogen reductive gas chromatography‐isotope ratio mass‐spectrometry (GC‐IRMS), (ii) five nicotines for oxidative C, N gas chromatography‐combustion‐isotope ratio mass‐spectrometry (GC‐C‐IRMS, or GC‐IRMS), and (iii) also three acetanilide and three urea reference materials for on‐line oxidative EA‐IRMS for C and N. Isotopic off‐line calibration against international stable isotope measurement standards at Indiana University adhered to the ‘principle of identical treatment’. The new reference materials cover the following isotopic ranges: δ²H_nicotine ?162 to ?45‰, δ¹³C_nicotine ?30.05 to +7.72‰, δ¹⁵N_nicotine ?6.03 to +33.62‰; δ¹⁵N_acetanilide +1.18 to +40.57‰; δ¹³C_urea ?34.13 to +11.71‰, δ¹⁵N_urea +0.26 to +40.61‰ (recommended δ values refer to calibration with NBS 19, L‐SVEC, IAEA‐N‐1, and IAEA‐N‐2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC‐IRMS that are available with different δ¹⁵N values. Comparative δ¹³C and δ¹⁵N on‐line EA‐IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA‐IRMS reference materials. Published in 2009 by John Wiley & Sons, Ltd.     

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates

The natural ¹³C/¹²C isotope composition (δ¹³C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the ¹²C/¹³C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific δ¹³C of individual carbohydrates. Here, we examined two complementary methods for measuring the δ¹³C value of sucrose, glucose and fructose, that is, off‐line high‐performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA‐IRMS) analysis, and gas chromatography‐combustion (GC‐C)‐IRMS. We also used these methods to determine the in vitro ¹²C/¹³C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC～EA‐IRMS, and being sensitive to derivatization conditions, the GC‐C‐IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the ¹²C/¹³C isotope effect is rather small and it is not affected by the use of heavy water (D₂O). Copyright © 2009 John Wiley & Sons, Ltd.     

Increasing the accuracy of mass isotopomer analysis through calibration curves constructed using biologically synthesized compounds

Mass isotopomer analysis is an important technique to measure the production and flow of metabolites in living cells, tissues, and organisms. This technique depends on accurate quantifications of different mass isotopomers using mass spectrometry. Constructing calibration curves using standard samples is the most universal approach to convert raw mass spectrometry measurements into quantitative distributions of mass isotopomers. Calibration curve approach has been, however, of very limited use in comprehensive analyses of biological systems, mainly suffering from the lack of extensive range of standard samples with accurately known isotopic enrichment. Here, we present a biological method capable of synthesizing specifically labeled amino acids. These amino acids have well‐determined and estimable mass isotopomer distributions and thus can serve as standard samples. In this method, the bacterium strain Methylobacterium salsuginis sp. nov. was cultivated with partially ¹³C‐labeled methanol as the only carbon source to produce ¹³C‐enriched compounds. We show that the mass isotopomer distributions of the various biosynthesized amino acids are well determined and can be reasonably estimated based on proposed binomial approximation if the labeling state of the biomass reached an isotopic steady state. The interference of intramolecular inhomogeneity of ¹³C isotope abundances caused by biological isotope fractionation was eliminated by estimating average ¹³C isotope abundance. Further, the predictions are tested experimentally by mass spectrometry (MS) spectra of the labeled glycine, alanine, and aspartic acid. Most of the error in mass spectrometry measurements was less than 0.74 mol% in the test case, significantly reduced as compared with uncalibrated results, and this error is expected to be less than 0.4 mol% in real experiment as revealed by theoretical analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous analysis of uranium and plutonium using thermal ionization mass spectrometry

Simultaneous isotopic analysis of uranium and plutonium using thermal ionization mass spectrometer coupled to a multi-collector detection assembly with 9 Faraday cups has been reported earlier. Subsequently investigations have been carried out (1) to understand the applicability of correction methodologies available to account for the contribution of²³⁸Pu at²³⁸U and (2) to evaluate the effectiveness of these methodologies on the accuracy of²³⁵U/²³⁸U atom ratio being determined, particularly when samples containing different U/Pu atom ratios. Isotopic fractionation for both U and Pu in the simultaneous isotopic analysis has been compared with the results of the individual analysis of these elements. The different isotopic fractionation factors observed for U were attributed to different conditions of analysis. There was no significant difference in the isotopic fractionation patterns for Pu. The consideration to extend this method to actual samples from our observations on synthetic samples with diferent U/Pu atom ratios containing U and Pu isotopic reference standards is described.     

Stable carbon isotopes as an indicator for soil degradation in an alpine environment (Urseren Valley,Switzerland)

Analyses of soil organic carbon (SOC) content and stable carbon isotope signatures (δ¹³C) of soils were assessed for their suitability to detect early stage soil erosion. We investigated the soils in the alpine Urseren Valley (southern central Switzerland) which are highly impacted by soil erosion. Hill slope transects from uplands (cambisols) to adjacent wetlands (histosols and histic to mollic gleysols) differing in their intensity of visible soil erosion, and reference wetlands without erosion influence were sampled. Carbon isotopic signature and SOC content of soil depth profiles were determined. A close correlation of δ¹³C and carbon content (r > 0.80) is found for upland soils not affected by soil erosion, indicating that depth profiles of δ¹³C of these upland soils mainly reflect decomposition of SOC. Long‐term disturbance of an upland soil is indicated by decreasing correlation of δ¹³C and SOC (r ≤ 0.80) which goes in parallel with increasing (visible) damage at the site. Early stage soil erosion in hill slope transects from uplands to adjacent wetlands is documented as an intermediate δ¹³C value (?27.5‰) for affected wetland soil horizons (0–12 cm) between upland (aerobic metabolism, relatively heavier δ¹³C of ?26.6‰) and wetland isotopic signatures (anaerobic metabolism, relatively lighter δ¹³C of ?28.6‰). Carbon isotopic signature and SOC content are found to be sensitive indicators of short‐ and long‐term soil erosion processes. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of alachlor residues in pepper and pepper leaf using gas chromatography and confirmed via mass spectrometry with matrix protection

Alachlor residues were determined in pepper and pepper leaf, after 49 days of manufacturer‐recommended single‐ and double‐dose application to the soil and plant. The samples were extracted with acetonitrile, partitioned with n‐hexane, and purified through solid‐phase extraction, and finally detected with a gas chromatography–microelectron capture detector. The linearity of the analytical response across the studied range of concentrations (0.05–4.0 µg/mL) was excellent, obtaining coefficients of determination (r²) of 0.999. Recovery studies were carried out on spiked pepper and pepper leaf samples, at two concentrations levels (0.2 and 1.0 mg/kg), with three replicates performed at each level. Mean recoveries of 73.1–109.0% with relative standard deviations of 1.3–2.3% were obtained. The method was successfully applied to field samples, and alachlor residue was found in pepper (0.02 mg/kg) and pepper leaf (0.03 mg/kg), at levels lower than the maximum residue limits (0.2 mg/kg) set by the Korea Food and Drug Administration. The field‐detected residues were further confirmed with gas chromatography–mass spectrometry with the help of pepper leaf matrix protection. Copyright © 2013 John Wiley & Sons, Ltd.     

On the calibration of continuous,high‐precision δ18O and δ2H measurements using an off‐axis integrated cavity output spectrometer

The ¹⁸O and ²H of water vapor serve as powerful tracers of hydrological processes. The typical method for determining water vapor δ¹⁸O and δ²H involves cryogenic trapping and isotope ratio mass spectrometry. Even with recent technical advances, these methods cannot resolve vapor composition at high temporal resolutions. In recent years, a few groups have developed continuous laser absorption spectroscopy (LAS) approaches for measuring δ¹⁸O and δ²H which achieve accuracy levels similar to those of lab‐based mass spectrometry methods. Unfortunately, most LAS systems need cryogenic cooling and constant calibration to a reference gas, and have substantial power requirements, making them unsuitable for long‐term field deployment at remote field sites. A new method called Off‐Axis Integrated Cavity Output Spectroscopy (OA‐ICOS) has been developed which requires extremely low‐energy consumption and neither reference gas nor cryogenic cooling. In this report, we develop a relatively simple pumping system coupled to a dew point generator to calibrate an ICOS‐based instrument (Los Gatos Research Water Vapor Isotope Analyzer (WVIA) DLT‐100) under various pressures using liquid water with known isotopic signatures. Results show that the WVIA can be successfully calibrated using this customized system for different pressure settings, which ensure that this instrument can be combined with other gas‐sampling systems. The precisions of this instrument and the associated calibration method can reach ～0.08‰ for δ¹⁸O and ～0.4‰ for δ²H. Compared with conventional mass spectrometry and other LAS‐based methods, the OA‐ICOS technique provides a promising alternative tool for continuous water vapor isotopic measurements in field deployments. Copyright © 2009 John Wiley & Sons, Ltd.     

Critical assessment of the applicability of gas chromatography‐combustion‐isotope ratio mass spectrometry to determine amino sugar dynamics in soil

Amino sugars in soils have been used as markers of microbial necromass and to determine the relative contribution of bacterial and fungal residues to soil organic matter. However, little is known about the dynamics of amino sugars in soil. This is partly because of a lack of adequate techniques to determine ‘turnover rates’ of amino sugars in soil. We conducted an incubation experiment where ¹³C‐labeled organic substrates of different quality were added to a sandy soil. The objectives were to evaluate the applicability of compound‐specific stable isotope analysis via gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS) for the determination of ¹³C amino sugars and to demonstrate amino sugar dynamics in soil. We found total analytical errors between 0.8 and 2.6‰ for the δ¹³C‐values of the soil amino sugars as a result of the required δ¹³C‐corrections for isotopic alterations due to derivatization, isotopic fractionation and analytical conditions. Furthermore, the δ¹³C‐values of internal standards in samples determined via GC‐C‐IRMS deviated considerably from the δ¹³C‐values of the pure compounds determined via elemental analyzer IRMS (with a variation of 9 to 10‰ between the first and third quartile among all samples). This questions the applicability of GC‐C‐IRMS for soil amino sugar analysis. Liquid chromatography‐combustion‐IRMS (LC‐C‐IRMS) might be a promising alternative since derivatization, one of the main sources of error when using GC‐C‐IRMS, is eliminated from the procedure. The high ¹³C‐enrichment of the substrate allowed for the detection of very high ¹³C‐labels in soil amino sugars after 1 week of incubation, while no significant differences in amino sugar concentrations over time and across treatments were observed. This suggests steady‐state conditions upon substrate addition, i.e. amino sugar formation equalled amino sugar decomposition. Furthermore, higher quality substrates seemed to favor the production of fungal‐derived amino sugars. Copyright © 2009 John Wiley & Sons, Ltd.     

The polyphenolic profiles and antioxidant effects of Agastache rugosa Kuntze (Banga) flower,leaf, stem and root

Agastache rugosa Kuntze (Korean mint) is used as a spice and in folk medicine in East Asia. The present study identified a total of 18 polyphenols from the flower, leaf, stem and roots of this plant using high‐performance liquid chromatography–tandem mass spectrometry. Fourteen of these compounds had not previously been identified in these plant tissues. Each polyphenol was validated in comparison with external calibration curves constructed using structurally related compounds, with determination coefficients >0.9993. The limits of detection and quantification were 0.092–0.650 and 0.307–2.167 mg/L, respectively. Recoveries of 61.92–116.44% were observed at two spiking levels, with 0.91–11% precision, expressed as relative standard deviation (except anthraquinone spiked at 10 mg/L). Hydroxycinnamic acid was the most abundant compound in the root, while the flowers showed the highest total flavonoid level. Antioxidant activities, determined in terms of reducing power, Fe²⁺ chelating activity and the radical scavenging activities using α,α‐diphenyl‐β‐picrylhydrazyl and 2‐2?‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid, increased in a concentration‐dependent manner; the highest activity was identified in the stems, followed by leaves > flowers > roots. These findings indicate that A. rugosa is a good source of bioactive compounds and can be used as a functional food. Copyright © 2015 John Wiley & Sons, Ltd.     

Speciation of essential and toxic elements in edible mushrooms: size‐exclusion chromatography separation with on‐line UV–inductively coupled plasma mass spectrometry detection

Size‐exclusion liquid chromatography was coupled to UV and inductively coupled plasma mass spectrometry (ICP‐MS) for detection to perform elemental speciation studies on different edible mushrooms. Molecular weight (MW) distribution patterns of several elements among different fractions present in various edible mushrooms are presented. The association of the elements with the high and low MW fractions was observed using sequential detection by UV and ICP‐MS. Separation was performed using a Superdex 75 column. Variability of the fractionation patterns with three different extraction media (0.05 mol l^?1 NaOH; 0.05 mol l^?1 HCl; hot water at 60°C) was evaluated for mushroom species. A comparative elemental speciation study was performed in order to determine the differences in the fractionation patterns of silver, arsenic, cadmium, mercury, lead, and tin in Boletus edulis, Agaricus bisporus, and Lentinus edodes. Differences in the fractionation patterns of the elements were found to depend on the mushroom species and the extraction medium. Most of the elements were associated with high mw fractions. It was not possible to assess the trace metal contributions from the mushroom growth media. Copyright © 2004 John Wiley & Sons, Ltd.