IRMS detection of testosterone manipulated with C labeled standards in human urine by removing the labeled C

Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ¹³C value). However, ¹³C labeled standards can be used to control the δ¹³C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the ¹³C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ¹³C values between Andro and ANAD (Δδ¹³C_Andro–ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different ¹³C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ¹³C_Andro–ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ¹³C_Andro–ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-¹³C labeled standards.     

Baseline isotopic data of polyhalogenated compounds

The δ²H- and δ¹³C-values of polyhalogenated compounds were determined by EA-IRMS. Most of the compounds were related to the chloropesticides DDT and its metabolites, hexachlorocyclohexanes, and toxaphene, as well as several polybrominated compounds such as bromophenols and -anisoles. δ²H-values ranged between −235‰ and +75‰ whereas δ¹³C-values were found in the range −22‰ to −38‰. No correlation between δ²H- and δ¹³C-values could be identified. Comparative analysis clarified that bromophenols and the corresponding bromoanisoles may vary in their isotopic distribution. ²H NMR was used to quantify abundances of ²H isotopomers. Quantification of isotopomers of 2,4-dibromophenol and 2,4-dibromoanisole proved that both compounds from different suppliers do not originate from the same source. Differences in the δ²H-values of two toxaphene products were further investigated by the synthesis of products of different degree of chlorination from camphene. It was shown that the δ¹³C-values remained mostly unaltered as was expected since no carbon is lost in this procedure. However, the reaction products became enriched in ²H with increasing degree of chlorination. Different δ²H-values of the starting material will also impact the δ²H-values of the chlorination products.     

Conditions to obtain precise and true measurements of the intramolecular C distribution in organic molecules by isotopic C nuclear magnetic resonance spectrometry

Intramolecular ¹³C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic ¹³C NMR spectrometry provides a general tool for measuring the position-specific ¹³C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal ¹³C distribution, and (ii) an approach to determining the “absolute” position-specific ¹³C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the ¹³C frequency range of the studied molecule, i.e. the chemical shift range. The “absolute value” and, therefore, the trueness of the ¹³C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH₃ by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py–irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py–irm-MS (thus, the “true” value) while the other showed a bias, which was dependent to the range covered by the resonance frequencies of the molecule. Therefore, the former can be used directly for studying isotope affiliations, while the latter can only be used directly for comparative data, for example in authenticity studies, but can also be used to obtain the true values by applying appropriate correction factors. The present study assesses several key protocol steps required to enable the determination of position-specific ¹³C content by isotopic ¹³C NMR, irrespective of the NMR spectrometer: parameters to be adjusted, performance test using [1,2-¹³C₂]acetic acid, generation of correction factors.     

Evidence of C non-covalent isotope effects obtained by quantitative C nuclear magnetic resonance spectroscopy at natural abundance during normal phase liquid chromatography

Quantitative isotopic ¹³C NMR at natural abundance has been used to determine the site-by-site ¹³C/¹²C ratios in vanillin and a number of related compounds eluted from silica gel chromatography columns under similar conditions. Head-to-tail isotope fractionation is observed in all compounds at the majority of carbon positions. Furthermore, the site-specific isotope deviations show signatures characteristic of the position and functionality of the substituents present. The observed effects are more complex than would be obtained by simply summing the individual effects. Such detail is hidden when only the global ¹³C content is measured by mass spectrometry. In particular, carbon positions within the aromatic ring are found to show site-specific isotope fractionation between the solute and the stationary phase. These interactions, defined as non-covalent isotope effects, can be normal or inverse and vary with the substitution pattern present.     

Oxidation of cyclopentane-1,2-dione: a study with O labeled reagents

The asymmetric oxidation of 3-alkyl-cyclopentane-1,2-diones with the Ti(OⁱPr)₄/tartaric ester/t-BuOOH complex, which gives, in a cascade process, highly enantiomerically enriched γ-lactone acids, was studied by ¹⁸O isotopic labeling in the substrate and in the oxidant. The path of the labeled atoms was followed by ¹³C NMR spectroscopy. It was found that the oxidative ring cleavage of 1,2-dione proceeds via a Baeyer-Villiger-type oxidation mechanism.     

A simple rapid method to precisely determine 13C/12C ratios of plant methoxyl groups

Stable isotope ratios of individual plant components have become a valuable tool for the determination of the geographical origin and authenticity of foodstuff. A recently published method with considerable potential in this context is the measurement of the deuterium/hydrogen (D/H) isotope ratios of plant matter methoxyl groups. The method entailed cleavage of methyl ethers or esters with hydriodic acid (HI) to form gaseous methyl iodide (CH₃I) and then measurement of the δ²H value of this gas. Here, as a follow up to a previous study, we describe a method for the rapid and precise δ¹³C analysis of plant matter methoxyl groups using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Conditions for sample preparation were investigated for isotope discrimination effects, the GC conditions optimized, the reproducibility of the measurement of standards undertaken, and the precision of the method defined. The reproducibility of the δ¹³C value determined for a CH₃I standard on 20 consecutive measurements was found to be 0.17‰. The method was also tested on four methoxyl‐rich plant components: vanillin, lignin, wood and pectin. The analytical precision obtained, expressed as the average standard deviation, for these compounds was found to be better than 0.13‰. The described procedure which is simple and rapid, allowing preparation and analysis of a sample within 1 h, produces accurate and reproducible isotopic measurements. We suggest that this validated δ¹³C method when employed together with the recently published δ²H method for two‐dimensional stable isotope studies of organic matter containing methoxyl groups will be of considerable value, e.g. for proving the authenticity of foodstuff. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative analysis of deuterium using the isotopic effect on quaternary C NMR chemical shifts

Quantitative analysis of specifically deuterated compounds can be achieved by a number of conventional methods, such as mass spectroscopy, or by quantifying the residual ¹H NMR signals compared to signals from internal standards. However, site specific quantification using these methods becomes challenging when dealing with non-specifically or randomly deuterated compounds that are produced by metal catalyzed hydrothermal reactions in D₂O, one of the most convenient deuteration methods. In this study, deuterium-induced NMR isotope shifts of quaternary ¹³C resonances neighboring deuterated sites have been utilized to quantify the degree of isotope labeling of molecular sites in non-specifically deuterated molecules. By probing ¹³C NMR signals while decoupling both proton and deuterium nuclei, it is possible to resolve ¹³C resonances of the different isotopologues based on the isotopic shifts and the degree of deuteration of the carbon atoms. We demonstrate that in different isotopologues, the same quaternary carbon, neighboring partially deuterated carbon atoms, are affected to an equal extent by relaxation. Decoupling both nuclei (¹H, ²H) resolves closely separated quaternary ¹³C signals of the different isotopologues, and allows their accurate integration and quantification under short relaxation delays (D1 = 1 s) and hence fast accumulative spectral acquisition. We have performed a number of approaches to quantify the deuterium content at different specific sites to demonstrate a convenient and generic analysis method for use in randomly deuterated molecules, or in cases of specifically deuterated molecules where back-exchange processes may take place during work up.     

The natural abundance of C with different agricultural management by NIRS with fibre optic probe technology

In the present study the natural abundance of ¹³C is quantified in agricultural soils in Mexico which have been submitted to different agronomic practices, zero and conventional tillage, retention of crop residues (with and without) and rotation of crops (wheat and maize) for 17 years, which have influenced the physical, chemical and biological characteristics of the soil. The natural abundance of C13 is quantified by near infrared spectra (NIRS) with a remote reflectance fibre optic probe, applying the probe directly to the soil samples.Discriminate partial least squares analysis of the near infrared spectra allowed to classify soils with and without residues, regardless of the type of tillage or rotation systems used with a prediction rate of 90% in the internal validation and 94% in the external validation. The NIRS calibration model using a modified partial least squares regression allowed to determine the δ¹³C in soils with or without residues, with multiple correlation coefficients 0.81 and standard error prediction 0.5‰ in soils with residues and 0.92 and 0.2‰ in soils without residues. The ratio performance deviation for the quantification of δ¹³C in soil was 2.5 in soil with residues and 3.8 without residues. This indicated that the model was adequate to determine the δ¹³C of unknown soils in the −16.2‰ to −20.4‰ range. The development of the NIR calibration permits analytic determinations of the values of δ¹³C in unknown agricultural soils in less time, employing a non-destructive method, by the application of the fibre optic probe of remote reflectance to the soil sample.     

Quick analysis of composition of semi-aromatic copolyamide via 13C NMR study

Standard high resolution ¹³C NMR spectra of PA10T, PA6T, PA106, and PA66 were obtained by a nonacidic solvent mixture of HFIP and CDCl₃. Several chemical shifts were found extremely sensitive to the polyamide type. According to the standard spectra, semi-aromatic copolyamides comprising PA10T, PA6T, PA106, and PA66 units could be distinguished. The ratio of each polyamide component in the copolyamide was determined through the integration of the methylene carbon peak associated with the amine group. ¹³C NMR analysis results were consistent with the theoretical values and copolyamide hydrolysis test results, making ¹³C NMR analysis quite reliable on the quick composition analysis of semi-aromatic copolyamides. Based on this technique, several commercial semi-aromatic copolyamides were further examined and their compositions were easily determined.     

Quality assessment of Radix Codonopsis by quantitative nuclear magnetic resonance

Radix Codonopsis (Dangshen) is a famous traditional Chinese medicine and has long been used for replenishing energy deficiency, strengthening the immune system, lowering blood pressure and improving appetite in China, Japan and Korea. A highly specific quantification method using ¹H NMR has been developed for the simultaneous determination of novel quaternary ammonium alkaloids codotubulosine A and B, adenosine and 5-(hydroxymethyl)furfural in Radix Codonopsis materials Codonopsis pilosula, C. pilosula var. modesta, C. tangshen, C. tubulosa, C. subglobosa, C. clematidea, C. lanceolota and Campanumoea javanica collected from different regions of China and Taiwan. A solid-phase extraction with C-18 cartridge followed by elution with water can easily remove sugars the major components that may affect the determination of target constituents. In the ¹H NMR spectrum, the signals of N-CH₃ of codotubulosine A (δ 2.75) and B (δ 2.83), H-8 of adenosine (δ 8.15), and CHO signal of 5-(hydroxymethyl)furfural (δ 9.49) are well separated from other signals in [²H₄]methanol. The quantity of the compounds was calculated by the relative ratio of the integral values of the target peaks of each compound to the known amount of internal standard pyrazine. The described NMR method is found to be relatively simple, specific, precise and accurate for the quality control of Radix Codonopsis herbs and no reference compounds are required for calibration curves, in comparison to conventional HPLC methods, for instance.     

Solid-state Na and C NMR characterization of Na3C60

Two separate samples of Na₃C₆₀ were prepared by direct reaction of C₆₀ with sodium metal vapor, and subjected to different annealing times of 10 days and 16 days. Solid-state ¹³C and ²³Na NMR, along with elemental analysis, powder X-ray diffraction (XRD) and Raman spectroscopy, were used to characterize both samples. The Raman spectra of both materials have a single peak at 1447 cm⁻¹ which correspond to the A_g peak of C₆₀³⁻, consistent with the stoichiometry of Na_xC₆₀ with x=3. The powder XRD patterns are also virtually identical for both samples. However, solid-state ²³Na and ¹³C NMR spectra of the two samples are significantly different, suggesting a relationship between annealing times and the final structure of the alkali fulleride. Variable-temperature ²³Na magic-angle spinning (MAS) NMR experiments reveal the existence of two or three distinct sodium species and reversible temperature-dependent diffusion of sodium ions between octahedral and tetrahedral interstitial sites. ¹³C MAS NMR experiments are used to identify resonances corresponding to free C₆₀ and fulleride species, implying that the samples are segregated-phase materials composed of C₆₀ and non-stoichiometric Na₃C₆₀. Variable-temperature ¹³C MAS NMR experiments reveal temperature-dependent motion of the fullerides.     

Precise determination of cadmium and lead isotopic compositions in river sediments

A method for the accurate determination of Cd and Pb isotope compositions in sediment samples is presented. Separation of Cd and Pb was designed by using an anionic exchange chromatographic procedure. Measurements of Cd isotopic compositions were carried out by multi-collector inductively coupled plasma mass spectrometer (MC-ICPMS), by using standard-sample bracketing technology for mass bias correction and Pb isotopic ratios were determined by thermal ionization mass spectrometry (TIMS). The factors that affect the accurate and precise Cd isotope compositions analysis, such as instrumental mass fractionation and isobaric interferences, were carefully evaluated and corrected. The Cd isotopic results were reported relative to an internal Cd solution and expressed as the δ^114/110Cd. Five Cd reference solutions and one Pb standard were repeatedly measured in order to assess the accuracy of the measurements. Uncertainties obtained were estimated to be lesser than 0.11‰ (2s) for the δ^114/110Cd value. Analytical uncertainties in 2s for Pb isotopic ratios were better than 0.5‰. The method has been successfully applied to the investigation of Cd and Pb isotope compositions in sediment samples collected from North River in south China.     

Analysis of F and C NMR spectra of tetrafluorophthalic anhydride and its derivatives

¹⁹F and ¹³C NMR spectra of perfluorinated compounds (i.e., tetrafluorophthalic anhydride, its hydroxyl- and amino-derivatives, N-pentafluorophenyltetrafluorophthalimide, and hexafluoroindan-1,3-dione) were analysed. Different signals in NMR spectra were assigned based on the analysis of spin-spin coupling constants. All assignments made were further confirmed by density functional theory (DFT) calculations of ¹³C chemical shifts and J_C,F coupling constants.     

Carbon-13 NMR spectra of coumarin and methoxycoumarins—a reinvestigation of charge density/Chemical Shift Relations

The carbon-13 NMR spectra of coumarin, 6-, 7-, 8-methoxycoumarin, and 5,7-, 7,8-, 5,8- and 6,7-dimethoxycoumarin have been measured and assigned. It is shown that substituent induced chemical shifts S(δ) in the mono- and disubstituted systems correlate well with the HMO atom-atom polarisibilities π_ij of the parent compound: S_i(δ_i) = 80.13 π_ij with a standard deviation of 1.42 ppm and a correlation factor of 0.994. Correlations between δ(¹³C) values and charge densities calculated by various semi-empirical methods are less successful.     

Study and validity of 13C stable carbon isotopic ratio analysis by mass spectrometry and 2H site-specific natural isotopic fractionation by nuclear magnetic resonance isotopic measurements to characterize and control the authenticity of honey

Honey samples were analyzed by stable carbon isotopic ratio analysis by mass spectrometry (SCIRA-MS) and site-specific natural isotopic fractionation measured by nuclear magnetic resonance (SNIF-NMR) to first determine their potentials for characterizing the substance and then to combat adulteration. Honey samples from several geographic and botanical origins were analyzed. The δ¹³C parameter was not significant for characterizing an origin, while the (D/H)_I ratio could be used to differentiate certain single-flower varieties. Application of the official control method of adding a C₄ syrup (AOAC official method 998.12) to our authentic samples revealed anomalies resulting from SCIRA indices that were more negative than −1‰ (permil). A filtration step was added to the experimental procedure and provided results that were compliant with the natural origin of our honey samples. In addition, spiking with a C₄ syrup could be detected starting at 9-10%. The use of SNIF-NMR is limited by the detection of a syrup spike starting only at 20%, which is far from satisfying.     

Crosslinking and ageing of C labelled polyisoprene part 1: Synthesis and polymerisation of 4-C-isoprene

4-¹³C-isoprene was prepared by the Wittig reaction. All reaction steps were optimised using unlabelled compounds. By reaction with triphenyl phosphine, ¹³C labelled methyl iodide afforded labelled methyl-triphenyl phosphine iodide in 84% yield. This reacted with meth acrolein with production of 4-¹³C-isoprene in 64% yield. Labelled polyisoprene was prepared by anionic polymerisation initiated by t-butyl lithium. Based on ¹³CH₃I the overall yield is ca 30%. The polymer was characterized by ¹H and ¹³C NMR spectroscopy. The contribution of each microstructure was [cis 1-4, 72%]; [trans 1-4, 10%]; [3-4, 18%].     

Oxidation of C-labeled ethyl linoleate monitored and quantitatively analyzed by C NMR

The oxidation of ¹³C-labeled ethyl linoleate (¹³C-EL), a model compound for alkyd resins, was investigated by ¹³C NMR in the presence of Co(II)-2-ethylhexanoate (Co-EH), Mn(acac)₃ (acac = acetylacetonate), and Mn(acac)₃ in combination with 2,2′-bipyridine (bpy), respectively. The use of ¹³C-EL allows us, in an unprecedented way, to reveal the individual evolution of hydroperoxides (ROOH) and peroxy (ROOR) links by ¹³C NMR and to quantify the oxidation intermediates during the oxidation. Mn(acac)₃ appeared to be less effective in decomposing ROOH than Co-EH and the Mn(acac)₃/bpy combination. Quantitative analyses were attempted for a few major ¹³C peaks.     

Biexponential C spin-lattice relaxation behavior of the crystalline region of ethylene copolymers and its origin

In this work, two series of ethylene-dimethylaminoethylmethacrylate (EDAM) and ethylene-methyl acrylate copolymers (EMA) with different comonomer content were studied by high-resolution solid-state ¹³C NMR spectroscopy. Biexponential ¹³C spin-lattice relaxation behaviors of the crystalline region were observed for all copolymer samples either melt-quenched or isothermally crystallized. The relative content of the shorter ¹³C T₁ component to that of the longer ¹³C T₁ component was found to increase with comonomer content. By employing a new pulse sequence which can be considered as a combination of Goldman-Shen's and Torchia's pulse sequences, it was demonstrated that the shorter ¹³C T₁ component is corresponding to the intermediate part of the crystalline region. The thickness of the intermediate part is estimated to be about 0.85 nm.     

Efficient synthesis of H & C labeled benzaldehydes via regio-selective formylation

Benzaldehydes are important building blocks in synthetic organic chemistry that have wide applications for the synthesis of natural products and pharmaceutical drugs. Herein we report a general synthetic methodology for the synthesis of highly functionalized ²H and ¹³C labeled benzaldehydes in transfer of isotopic purity >99% via regio-selective formylation. Regio-selective deprotonation of substituted benzene 1 with LDA/n-BuLi at −78 °C and treatment with DMF-d₇ or EtO-¹³CHO led to the synthesis of 2-deutero-1,3-disubstituted benzaldehydes 2/4 in moderate to good yields. The synthetic methodology described represents a simple yet versatile route to functionalized formyl-deuterated, tritiated ¹³C and ¹⁴C labeled benzaldehydes.     

Stable carbon ((12/13)C) and nitrogen ((14/15)N) isotopes as a tool for identifying the sources of cyanide in wastes and contaminated soils--a method development

The occurrence of iron-cyanide complexes in the environment is of concern, since they are potentially hazardous. In order to determine the source of iron-cyanide complexes in contaminated soils and wastes, we developed a method based on the stable isotope ratios ¹³C/¹²C and ¹⁵N/¹⁴N of the complexed cyanide-ion (CN⁻). The method was tested on three pure chemicals and two industrials wastes: blast-furnace sludge (BFS) and gas-purifier waste (GPW). The iron-cyanide complexes were converted into the solid cupric ferrocyanide, Cu₂[Fe(CN)₆]·7H₂O, followed by combustion and determination of the isotope-ratios by continuous flow isotope ratio mass spectrometry. Cupric ferrocyanide was obtained from the materials by (i) an alkaline extraction with 1 M NaOH and (ii) a distillate digestion. The [Fe(CN)₆]⁴⁻ of the alkaline extraction was precipitated after adding Cu²⁺. The CN⁻ of the distillate digestion was at first complexed with Fe²⁺ under inert conditions and then precipitated after adding Cu²⁺. The δ¹³C-values obtained by the two methods differed slightly up to 1-3‰ for standards and BFS. The difference was larger for alkaline-extracted GPW (4-7‰), since non-cyanide C was co-extracted and co-precipitated. Therefore the distillate digestion technique is recommended when determining the C isotope ratios in samples rich in organic carbon. Since the δ¹³C-values of BFS are in the range of −30 to −24‰ and of −17 to −5‰ for GPW, carbon seems to be a suitable tracer for identifying the source of cyanide in both wastes. However, the δ¹⁵N-values overlapped for BFS and GPW, making nitrogen unsuitable as a tracer.