Ratio Mass Spectrometry Analysis Research Articles

Organo-mineral associations and size-fractionated colloidal organic carbon dynamics in a redox-controlled wetland

The formation of organo-mineral associations serves as a crucial mechanism for stabilizing soil organic matter, particularly for determining the fate of soil organic carbon (OC) in redox dynamic wetlands characterized by high C content. Despite its importance, few studies have assessed the retention, transformation, and transport of colloids (1–1000 nm) and colloidal OC (COC) in those environments. This leaves a crucial knowledge gap concerning the quantities of colloids and COC and their molecular compositions, especially considering the significant role of metabolically active depressional wetlands that may play as biogeochemical hotspots for C cycling. To address this gap, we conducted a study in a Delmarva Bay depressional wetland located in Blackbird State Forest, Delaware, USA. We established a transect encompassing upland (U), transition (T), and wetland (W) zones based on seasonal hydrologic conditions. We installed piezometers at 50 cm, 100 cm, and 200 cm depths within each zone and collected pore-water samples from 11/2017 to 05/2019. We then fractionated these samples into dissolved (<2.3 nm), natural nanoparticle (NNP, 2.3–100 nm), fine colloid (100–450 nm), and particulate (450–1000 nm) fractions via sequential centrifugation and ultrafiltration, and quantified their concentration and molecular composition. We observed variations in the concentration and molecular composition of soil COC both vertically at different soil depths and horizontally along a redox gradient from the U-T-W transect. The sum of the NNP and fine colloid fractions accounted for 47±20% of the operationally defined “dissolved” (<450 nm) fraction. Isotope ratio mass spectrometry and X-ray photoelectron spectroscopy analyses further revealed that the NNP fraction is more enriched in heavier δ13C isotope and oxidized carbonyl/carboxyl C functional groups (C=O, p<0.05) with significantly higher surficial atomic percentages of C (p<0.01), N (p<0.01), and Mg:Al ratios (p<0.05), and lower atomic percentages of Al (p<0.01) and Si (p<0.01) compared to the larger particles. The formation of cation bridges and/or hydrogen bonds between carboxylic OC and phyllosilicate minerals likely dominated the mineral-OC association in the NNP fraction. The higher surface C enrichment in the particulate fraction compared to the NNP fraction suggests a patchy distribution of more plant-derived OC through organo-organic interactions in the larger fractions. Along the transect, Zone U exhibited significant enrichment of heavier δ13C isotope and lower SUVA values than the Zones T and W indicating inefficient decomposition and dissociation of SOM from minerals due to the reductive dissolution of Fe and Al oxides under anoxic conditions. Furthermore, an increase in low molecular weight microbial metabolites with reduced aromatic OC content was observed at deeper depths in Zones U and T. Our study provides new insights into the concentration and molecular composition of size-fractionated COC, highlighting the need to separately consider the NNP and fine colloid fractions from the operationally defined “dissolved” phase. This is crucial for assessing the biogeochemical cycling of OC in redox-active wetlands, a pressing need amidst the growing concerns about the impacts of climate change.

Identification of endogenous and exogenous semicarbazide (SEM) in crustacea aquatic products using compound-specific nitrogen stable isotope ratio analysis (NSIRA)

Endogenous SEM (SEMend) naturally occurs in crustacea aquatic products, it confuses the legal verdict of exogenous SEM (SEMex) degraded by antibiotic nitrofurazone abuse. In the present study, compound-specific gas phase chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis coupled with a two-step pre-column derivatization was developed for determining the nitrogen stable ratio (δ15N) of SEM in shrimp and crab. The optimal derivatization and solid phase extraction (SPE) condition of SEM were screened by Plackett-Burman design and central composite design (CCD) experiment. The nitrogen isotope ratio analysis (NSIRA) of SEM in two types of crustaceans proved that its δ15N values significantly decreased from 7.0‰ to 2.0‰ once nitrofurazone being used. Analytical results found that the δ15N value of SEMex in vivo was consistent its nitrofurazone precursor but the value of SEMend was dependent on N-containing components (e.g., tissue protein), ANOVA showed significant difference between two types of SEM (p < 0.05). Based on the δ15N variations between SEM itself and its nitrofurazone precursor, tissue protein of crustaceans, the percentages of SEMex and SEMend can be calculated for its source apportionment in vivo. The limit of detection of SEM is down to 0.21 μg kg−1 and 0.54 μg kg−1, and the identification lower limit of SEMex are accurate at 6.27% and 0% for two types of crustaceans. The strategy first realized the independent determination of SEMex and SEMend in crustacea aquatic products, which explained that high-level SEM in crustaceans can’t as the legal verdict of nitrofurazone abuse in aquaculture.

The first report on the use of deuterium-labelled water in analysing the splash phenomenon

Splash is a widely studied phenomenon, partly forming the phenomenon of erosion. Until now, tracing the origin of splash water was a highly error-prone, or impossible, task. We propose a method for tracking splash water by means of stable isotope ratio mass spectrometry (IRMS) analysis. We tested the method on a water-water model, checking the effect of drop height, water layer thickness and the effect of water evaporation. It turns out that the drop height and the thickness of the water layer have practically no effect on the origin of the water in the splash, and all the water comes from the vessel, not from the drops. The only exception is when a thin layer of water is impacted with a drop, in which case 23% of the water also comes from the drop. The proposed method could be used to study more complex systems such as water-soil.

Extraction possibilities of lipid fraction and authentication assessment of chaga (Inonotus obliquus)

Chaga (Inonotus obliquus) (Fr.) Pilát is a black perennial fungus that grows on adult birch trunks and has been traditionally used as a health promoting remedy in different countries. The lipophilic compounds of chaga were isolated applying conventional (Soxhlet), intensive (ultrasound assisted, accelerated solvent extraction) and environmentally friendly (supercritical fluid) extraction methods utilizing both polar and non-polar solvents. Soxhlet extraction showed the highest inotodiol yield (231 mg/100 g chaga) using cyclohexane. For authentication of chaga samples, isotope ratio mass spectrometry (IRMS) and multi-elemental analysis was performed. The extraction yield and profile of lipids, sterols and triterpenoids depend on the chaga origin. The amount of lipids in chaga samples varied in the range from 0.5 to 1.0%. The antimicrobial activity of chaga extracts against pathogenic and opportunistic pathogenic microorganisms was demonstrated. The results showed that extraction using supercritical CO2 delivered yields comparable to those obtained by conventional and intensive extraction techniques. Amount of target compounds (sterols and triterpenoids) obtained using supercritical CO2 extraction could represent the future for sustainable industrial-scale production of biologically active ingredients with antimicrobial activity. Authentication using IRMS allowed to differentiate between natural (fruiting bodies) and cultivated (mycelium) chaga.

Comparison of isotope ratio mass spectrometry and cavity ring-down spectroscopy procedures and precision of the doubly labeled water method in different physiological specimens.

This study determines if saliva collection procedures for the doubly labeled water (DLW) method, used for measuring total energy expenditure (TEE), are comparable to urine and plasma collection. Both the cavity ring-down spectroscopy (CRDS) and isotope ratio mass spectrometry (IRMS) analyses techniques are compared. Saliva specimens were collected from participants for the DLW method. The specimens were collected under different conditions: after consumption of tap water, after chewing gum, and during exposure to conditions of high and low relative humidity. The isotopes in saliva were compared with simultaneous plasma and urine collection. TEE calculated from saliva and analyzed using CRDS was compared to that of plasma analyzed using IRMS. The within-individual variances were not significantly different between the saliva specimens (0.4‰) and plasma (0.3‰). After the oral dose of DLW, the saliva specimens displayed a shorter equilibration time to urine. When participants consumed 500 mL of tap water, the enrichment of saliva specimens reached a new plateau value faster than urine. Saliva collection exposed to high ambient humidity conditions was slightly less enriched as compared to low-humidity conditions while urine enrichment was unaffected. In contrast, whereas the within-individual effects of gum chewing during saliva collection on 18 O were unaffected, the abundance of 2 H in saliva was slightly lower after chewing the gum. The within-individual difference between TEE calculated from saliva and that calculated from plasma analyzed using IRMS did not differ from zero, and the standard deviation was not different from that predicted by a propagation of error analysis based on analytical error alone. Our findings support using saliva specimens for the DLW method. The analysis of plasma and urine, however, requires reducing the memory effect resulting from contaminants. Also, it should be performed in a manner that minimizes exposure to air where specimens may be exposed to evaporation or contamination from water vapor during sampling.

Construction of IsoVoc Database for the Authentication of Natural Flavours.

Flavour is an important quality trait of food and beverages. As the demand for natural aromas increases and the cost of raw materials go up, so does the potential for economically motivated adulteration. In this study, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis of volatile fruit compounds, sampled using headspace-solid phase microextraction (HS-SPME), is used as a tool to differentiate between synthetic and naturally produced volatile aroma compounds (VOCs). The result is an extensive stable isotope database (IsoVoc—Isotope Volatile organic compounds) consisting of 39 authentic flavour compounds with well-defined origin: apple (148), strawberry (33), raspberry (12), pear (9), blueberry (7), and sour cherry (4) samples. Synthetically derived VOCs (48) were also characterised. Comparing isotope ratios of volatile compounds between distillates and fresh apples and strawberries proved the suitability of using fresh samples to create a database covering the natural variability in δ13C values and range of VOCs. In total, 25 aroma compounds were identified and used to test 33 flavoured commercial products to evaluate the usefulness of the IsoVoc database for fruit flavour authenticity studies. The results revealed the possible falsification for several fruit aroma compounds.

Organic matter removal for continuous flow isotope ratio mass spectrometry analysis of carbon and oxygen isotope compositions of calcite or dolomite in organic‐rich samples

Abstractδ13C and δ18O values of carbonate in organic‐rich samples such as soils, biofilms, and lake sediments have been scarcely used so far in paleo‐environmental, biomineralization, or diagenetic studies because organic matter in high proportions is suspected to alter carbonate isotope analysis. Yet, with the improvement of analytical capabilities and in particular the use of CF‐IRMS, this may not be an issue anymore. To evaluate this, δ13C and δ18O values of calcite or dolomite mixed in various proportions with yeast (used here as a model for immature organic matter) were measured. The results indicate that measurements of calcite or dolomite δ13C and δ18O by CF‐IRMS require organic matter removal only when its weight proportion exceeds that of carbonates. Yeast generated an unidentified molecule during phosphoric acid digestion at 25°C (none at 80°C), which shifted the carbonate δ18O values for yeast proportions higher than 50%. It also generated CO2, but with a noticeable shift in δ13C and δ18O values only at 80°C (none at 25°C) and for yeast proportions higher than 75%. Three methods of organic matter removal were tested: the well‐established NaOCl and H2O2 wet treatments as well as low temperature plasma ashing (LTA) so far seldom examined. LTA removed the isotope shifts, although imperfectly for yeast proportions of 95%, without carbonate dissolution. NaOCl efficiently removed the isotope shifts in all cases but dissolved part of the calcite and dolomite. H2O2 treatment caused severe dissolution of calcite and dolomite while worsening the δ13C and δ18O shifts and should thus be avoided.

A survey of novel MDA and MDMA precursors by isotope ratio mass spectrometry

Presented is a survey of the hydrogen, carbon and oxygen isotope profiles of traditional and novel precursors and pre-precursors used for the synthesis of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethylamphetamine (MDMA). The purpose of this survey was to determine what variations exist in the stable isotope profiles of compounds from which these drugs can be produced.The control of the traditional precursors safrole, isosafrole, piperonal and 3,4-methylenedioxyphenyl-2-propanone (MDP2P) has seen clandestine laboratories utilising some novel alternative chemicals. Helional has emerged primarily as a precursor to MDA and although recently legislated in Queensland, Australia, remains largely uncontrolled in most jurisdictions. Results for the uncontrolled pre-precursors catechol, vanillin and vanillic acid are also presented in this survey.In total 45 precursor samples were collected, with a focus on helional that is readily available online through fragrance supply companies. The identity and purity of all samples was confirmed using a range of techniques. Stable isotope profiles were obtained by isotope ratio mass spectrometry (IRMS) analysis. These results showed a greater variation in stable isotope profiles than previously reported in MDA/MDMA precursors. These results suggest that stable isotope profiling may be used to compare and differentiate precursors used in the production of MDA/MDMA. IRMS may also allow for the grouping or discrimination of MDA and MDMA, especially when synthesised from these novel precursors or pre-precursors.

Organic contamination in online laser-based plant stem and leaf water isotope measurements for pre-extracted samples

ABSTRACT Water stable isotopes have been widely used as natural tracers to investigate soil–plant–atmosphere interactions. Recent developments in induction module cavity ring-down spectroscopy (IM-CRDS) have made it possible to rapidly complete isotope analyses, and to combust co-extracted organic compounds at the same time. However, the agreement between IM-CRDS and isotope ratio mass spectrometry (IRMS) analyses has generally been poor and was primarily attributable to spectral interference of IM-CRDS. Here we evaluated the impacts of organic contamination on the isotope ratios using IM-CRDS with two different methods. No spectral interference was observed for solid samples measured directly by IM-CRDS, whereas clear organic contamination occurred in isotope analyses for pre-extracted plant stem and leaf samples. Our results demonstrate that IM-CRDS can fully combust co-extracted organic compounds by in-line oxidation in the direct measurement of solid samples, although this may not guarantee that the IM-CRDS can obtain better isotopic data than IRMS. It may be risky to evaluate the performance of IM-CRDS by measuring pre-extracted water samples because cryogenic vacuum distillation is likely to introduce extra organic compounds, which may not be fully removed during subsequent IM-CRDS measurement. In addition, spectral variables are useful for post-processing corrections.

Chemical attribution of the homemade explosive ETN - Part II: Isotope ratio mass spectrometry analysis of ETN and its precursors

In this follow-up study the collaboration between two research groups from the USA and the Netherlands was continued to expand the framework of chemical attribution for the homemade explosive erythritol tetranitrate (ETN). Isotope ratio mass spectrometry (IRMS) analysis was performed to predict possible links between ETN samples and its precursors. Carbon, nitrogen, hydrogen and oxygen isotope ratios were determined for a wide variety of precursor sources and for ETN samples that were prepared with selected precursors. The stability of isotope ratios of ETN has been demonstrated for melt-cast samples and two-year old samples, which enables sample comparison of ETN in forensic casework independent of age and appearance. Erythritol and nitric acid (or nitrate salt) are the exclusive donor of carbon and nitrogen atoms in ETN, respectively, and robust linear relationships between precursor and the end-product were observed for these isotopes. This allowed for defining isotopic enrichment ranges for carbon and nitrogen that support the hypothesis that a given erythritol or nitrate precursor was used to synthesize a specific ETN batch. The hydrogen and oxygen atoms in ETN do not originate from one exclusive donor material, making linkage prediction more difficult. However, the large negative enrichments observed for both isotopes do provide powerful information to exclude suspected precursor materials as donor of ETN. Additionally, combing the isotopic data of all elements results in a higher discrimination power for ETN samples and its precursor materials. Combining the findings of our previously reported LC–MS analysis of ETN with this IRMS study is expected to increase the robustness of the forensic comparison even further. The partially nitrated impurities can provide insight on the synthesis conditions while the isotope data contain information on the raw materials used for the production of ETN.

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine.

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid-liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.

Refining the termination age of the Cryogenian Sturtian glaciation in South China

Zhou et al. (2004) reported a zircon U-Pb isotope dilution – thermal ionization mass spectrometry (ID-TIMS) age of 662.9 ± 4.3 Ma from the basal Datangpo Formation of the Cryogenian System in South China. It was the first zircon U-Pb age to constrain both the termination of Sturtian glaciation and the onset of Marinoan glaciation, despite its much larger uncertainty expected for modern ID-TIMS analysis. In this study, we carried out U-Pb chemical abrasion – isotope dilution – isotope ratio mass spectrometry (CA-ID-IRMS) analysis on the same sample, yielding a precise 206Pb/238U date of 659.96 ± 0.46 Ma. Bayesian eruption age estimate indicates that the ash layer was most likely deposited at 659.23 ± 1.24 Ma. The present study suggests that the Sturtian glaciation terminated before ∼660 Ma. Together with previously reported radiometric dates, the precise U-Pb age presented herein helps refine the current Cryogenian chronology.

Improved isotopic model based on 15 N tracing and Rayleigh-type isotope fractionation for simulating differential sources of N2 O emissions in a clay grassland soil.

RationaleIsotopic signatures of N2O can help distinguish between two sources (fertiliser N or endogenous soil N) of N2O emissions. The contribution of each source to N2O emissions after N‐application is difficult to determine. Here, isotopologue signatures of emitted N2O are used in an improved isotopic model based on Rayleigh‐type equations.MethodsThe effects of a partial (33% of surface area, treatment 1c) or total (100% of surface area, treatment 3c) dispersal of N and C on gaseous emissions from denitrification were measured in a laboratory incubation system (DENIS) allowing simultaneous measurements of NO, N2O, N2 and CO2 over a 12‐day incubation period. To determine the source of N2O emissions those results were combined with both the isotope ratio mass spectrometry analysis of the isotopocules of emitted N2O and those from the 15N‐tracing technique.ResultsThe spatial dispersal of N and C significantly affected the quantity, but not the timing, of gas fluxes. Cumulative emissions are larger for treatment 3c than treatment 1c. The 15N‐enrichment analysis shows that initially ~70% of the emitted N2O derived from the applied amendment followed by a constant decrease. The decrease in contribution of the fertiliser N‐pool after an initial increase is sooner and larger for treatment 1c. The Rayleigh‐type model applied to N2O isotopocules data (δ15Nbulk‐N2O values) shows poor agreement with the measurements for the original one‐pool model for treatment 1c; the two‐pool models gives better results when using a third‐order polynomial equation. In contrast, in treatment 3c little difference is observed between the two modelling approaches.ConclusionsThe importance of N2O emissions from different N‐pools in soil for the interpretation of N2O isotopocules data was demonstrated using a Rayleigh‐type model. Earlier statements concerning exponential increase in native soil nitrate pool activity highlighted in previous studies should be replaced with a polynomial increase with dependency on both N‐pool sizes.

Photobacterium sp. NNA4, an efficient hydroxylamine-transforming heterotrophic nitrifier/aerobic denitrifier

An efficient heterotrophic nitrifying/aerobic denitrifying strain, Photobacterium sp. NNA4 was isolated from a recirculating aquaculture system (RAS). NNA4 was capable of utilizing ammonia, nitrate or nitrite as sole N-source with maximal removal rates of 12.5mg/L/h for NH4+N, 16.4mg/L/h for NO3--N, and 4.5mg/L/h for NO2--N, respectively. Optimal nitrification conditions were: sodium succinate as C-source, 30-37°C, NaCl 1-4%, pH 7.0-8.0, dissolved oxygen 5.89mg/L, C/N>10. Gas chromatography/mass spectrometry and gas chromatography/isotope ratio mass spectrometry analyses showed that N2 and N2O were aerobic denitrification products of nitrite and nitrate. NNA4 could tolerate high concentration of hydroxylamine and displayed efficient hydroxylamine-transforming capability. Hydroxylamine oxidoreductase activity using potassium ferricyanide as electron acceptor was 0.042U. Results revealed that strain NNA4 could oxidize NH2OH directly to N2O at aerobic conditions. In view of its high removal ability of inorganic nitrogen pollutants and broad salinity tolerance range, NNA4 has great potential in denitrification treatment of types of wastewater with either low salinity (e.g., municipal facilities) or high salinity (e.g., aquaculture, seafood processing).

Early pottery use across the Baltic – A comparative lipid residue study on Ertebølle and Narva ceramics from coastal hunter-gatherer sites in southern Scandinavia, northern Germany and Estonia

The Late Mesolithic Ertebølle and Narva cultures (6th – 5th/4th millennium BC) in the southwest and eastern Baltic, respectively, shared similar vessel types, namely pointed-based pots and oval bowls. As a consequence, this phenomenon raised questions about inter-cultural connections across the Baltic and possible influence for the production of pottery from the Narva to the Ertebølle hunter-gatherers. Whereas the two pottery traditions were shown to be different with regards to raw materials and manufacture, in this study we further attempt a comparison on the basis of function using a lipid residue analysis approach. The aim is to examine whether typological analogies were based on common functional requirements. This paper presents new evidence for the use of Ertebølle ceramics in the southwest Baltic from the analysis of pottery samples from a number of coastal sites in southern Sweden (Scania) and eastern Denmark (Lolland). Gas chromatography-mass spectrometry (GC–MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-c-IRMS) analysis were performed on the absorbed lipid residues to determine their structural characteristics and the stable carbon isotopic composition of selected fatty acids. Results are discussed and compared with analogous published data of Narva ceramics from Estonia. Data from other coastal sites in Denmark and northern Germany are also included for wider comparison. Based on our findings, we conclude that despite little variability in the isotope values of residues, Ertebølle and Narva pots did not serve the same functional demands, and different motives led to their production. Whilst the Narva ceramics appear to have had a specialized role in processing aquatic products, the Ertebølle were more multi-purpose vessels, used also for terrestrial animal and plant resources.

In vitro biohydrogenation of 13C-labeled α-linolenic acid in response to ruminal alterations associated with diet-induced milk fat depression in ewes

The basis for marine lipid-induced milk fat depression (MFD) has not been established yet, but recent reports suggest the putative contribution of shifts in the ruminal metabolism of α-linolenic acid (ALA). To test this hypothesis, an isotopic tracer approach was used in batch cultures of rumen microorganisms with inoculum collected from cannulated ewes fed either a total mixed ration without lipid supplementation (control inoculum) or the same diet supplemented with 2% of fish oil, which is known to cause MFD in lactating sheep (FO-MFD inoculum). The [1-13C]ALA was added at a dose of 1% of incubated dry matter and the proportions of 13C-labeled fatty acids (FA) were examined after 24 h of incubation, using complementary gas chromatography and gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS) analyses. Expected differences in FA profiles were confirmed between control and FO-MFD inocula (e.g., large decreases in 18:0 and increases in most 18:1 and 18:2 intermediates, particularly trans isomers, to fish oil supply). The biohydrogenation of 13ALA was extensive and yielded multiple metabolites, with a total of 48 chromatographic peaks showing 13C enrichment, regardless of the inoculum type. However, although ALA was biohydrogenated through common pathways under standard or MFD conditions, large changes in the accumulation of 13C-labeled FA suggest important differences in the relative contribution of each specific route. First, increased accumulation of trans-11-containing FA in FO-MFD incubations was accompanied by a general repression of the trans-13/14 pathway (supported by lower trans-13+14 18:1 or trans-11,trans-13 18:2 proportions), together with a lower production of cis FA (e.g., cis-9, -12, and -15 18:1 and some cis,cis 18:2). Results also downplayed the relevance of the trans-11 to trans-10 shift as an effective marker of diet-induced MFD in sheep, and challenged the involvement of some trans-10 intermediates (e.g., trans-10 18:1 and trans-10,cis-15 18:2) in the low-fat milk syndrome in this species. Conversely, increased abundance of most 18:3 intermediates (including some unidentified isomers) might be representative of ruminal alterations related to fish oil supplementation in ewes. Further research is necessary to examine the potential association between these findings and MFD in lactating animals.

The influence of hydrolysis and derivatization on the determination of amino acid content and isotopic ratios in dual-labeled (13 C, 15 N) white clover.

The cycling of peptide- and protein-bound amino acids (AAs) is important for studying the rate-limiting steps in soil nitrogen (N) turnover. A strong tool is stable C and N isotopes used in combination with compound-specific isotope analysis (CSIA), where a prerequisite for analysis is appropriate methods for peptide and protein hydrolysis and appropriate methods for derivatization of AAs for analysis by gas chromatography (GC). We examined the efficiency of a standard acidic hydrolysis (6 M HCl, 20 h at 110°C) and a fast acidic hydrolysis (6 M HCl, 70 min at 150°C) on the recovery of AAs from a protein standard (bovine serum albumin). The best methods were used on dual-labeled (13 C and 15 N) clover shoot and root juice, divided into four molecular weight (Mw) size fractions. We used NAIP (N-acetyl isopropyl esterification) derivatization for GC/combustion-isotope ratio mass spectrometry (C-IRMS) analysis of AA standards. The NAIP derivatization gave very low limits of detection (LODs) (< 2 pmol) and limits of quantification (LOQs) ranging from 0.55 to 4.89 pmol. Comparing the concentrations of individual AAs in hydrolyzed versus unhydrolyzed clover juice samples of the low Mw size fraction (<1 kDa) showed a significant decline in concentration (p<0.03) for seven AAs after hydrolysis. Despite the decline in AA concentration, we found a linear connection between the obtained atomic fraction (13 C/total carbon and 15 N/total nitrogen) for individual AAs of hydrolyzed versus unhydrolyzed samples. The methodology distinguished differences in atomic fractions across AAs, in individual AAs in Mw size fractions, and between shoot and root samples of experimentally labeled white clover. Specifically, the method separated L-glutamate (Glu) and glutamine (Gln). Thus, for a broader use in plant and soil ecology, we present an optimized methodology for GC/C-IRMS analysis of AAs from organic nitrogen samples enriched with 13 C and 15 N - AA stable isotope probing (SIP).

Multicomponent characterization and differentiation of flash bangers — Part II: Elemental profiling of plastic caps

This study builds on the multicomponent analysis strategy for flash bangers which was previously introduced and where a representative sample set has been collected of a certain type of flash bangers. To expand the forensic strategy, elemental analysis of the plastic caps which are present in these items was performed. Both x-ray fluorescence (XRF) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis was performed to explore the possibilities for differentiation. The inherent inhomogeneity of the plastics resulted in high variations, especially for LA-ICP-MS trace analysis. In addition, due to the lack of suitable reference materials the LA-ICP-MS results can only be used for qualitative comparisons. Although XRF is less sensitive it allows for semi-quantitative analysis and the effect of inhomogeneity is significantly reduced due to the larger sample areas. Therefore, XRF is the method of choice for elemental analysis of intact plastic caps. In this scenario initial differentiation based on visual examination is combined with elemental analysis to obtain the highest degree of discrimination. In post-explosive scenarios, using XRF is not as straightforward due the irregular shapes of the burned plastic cap residues and contamination by explosive residues. For the analysis of these post-explosive caps, LA-ICP-MS proved to be useful for characterization and differentiation. Overall, it was found that blue caps contain a considerable higher amount of elements than the white caps, mainly due to additives related to the coloring process. This limits differentiation for the flash bangers containing white caps. Therefore, isotope ratio mass spectrometry (IRMS) analysis was performed to increase the differentiation potential. Based on carbon and hydrogen isotope ratios additional sets could be distinguished, both for flash bangers containing white and blue caps, that otherwise have similar visual and elemental characteristics. With the elemental and isotopic analysis of the plastic caps, an analysis strategy has been introduced that is not based on the pyrotechnic charge and therefore provides a unique opportunity to perform characterization and differentiation of flash bangers in pre- and post-explosive casework.

Liquid chromatography/isotope ratio mass spectrometry analysis of halogenated benzoates for characterization of the underlying degradation reaction in Thauera chlorobenzoica CB-1T.

Halogenated benzoic acids occur in the environment due to their widespread agricultural and pharmaceutical use. Compound-specific stable isotope analysis (CSIA) has developed over the last decades for investigation of in situ transformation and reaction mechanisms of environmental pollutants amenable by gas chromatography (GC). As polar compounds are unsuitable for GC analysis we developed a method to perform liquid chromatography (LC)/CSIA for halogenated benzoates. LC/isotope ratio mass spectrometry (IRMS) utilizing a LC-Surveyor pump coupled to a MAT 253 isotope ratio mass spectrometer via a LC-Isolink interface was applied. For chromatographic separation a YMC-Triart C18 column and a potassium hydrogen phosphate buffer (150mM, pH7.0, 40°C, 200μL mL-1 ) were used, followed by wet oxidation deploying 1.5molL-1 ortho-phosphoric acid and 200g L-1 sodium peroxodisulfate at 75μL mL-1 . Separation of benzoate and halogenated benzoates could be achieved in less than 40min over a concentration range of 2 orders of magnitude. Under these conditions the dehalogenation reaction of Thauera chlorobenzoica 3CB-1T using 3-chloro-, 3-bromo- and 4-chlorobenzoic acid was investigated resulting in inverse carbon isotope fractionation for meta-substituted benzoic acids and minor normal fractionation for para-substituted benzoic acids. Together with the respective growth rates this led to the assumption that dehalogenation of para-halobenzoic acids follows a different mechanism from that of meta-halobenzoic acids. A new LC/IRMS method for the quantitative determination of halogenated benzoates was developed and used to investigate the in vivo transformation pathways of these compounds, providing some insights into degradation and removal of these widespread compounds by T. chlorobenzoica 3CB-1T .

Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes.

AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), is a naturally occurring substance which is part to the World Anti-Doping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances. Copyright © 2017 John Wiley & Sons, Ltd.