Gas Chromatography Combustion Isotope Ratio Mass Spectrometry Research Articles

Two-Dimensional Liquid Chromatography Purified GC/C/IRMS Doping Control Method: Analysis of Endogenous and Exogenous Sources in Urine Samples from Asian Subjects Administered a Low Dose of AICAR

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation. MethodIn this study, an automated 2D-HPLC separation technique was used to separate and purify AICAR and endogenous reference substances in urine samples. Then, AICAR was derivatized with 3-TMS as the main derivative product, while the endogenous reference compounds remained in their original form. Subsequently, the developed GC/C/IRMS method was utilized for the detection of the target and reference substances. Followed, we evaluated the applicability of this method using urine samples from two Asian males administered a low dose of AICAR (3 grams). ResultsThe advantages of this study include: 1) reduced sample pretreatment time: the established 2D-HPLC separation method can separate the target and endogenous reference substances in one step; 2) low interference: the isotope chromatograms have low background interference, and the separation of endogenous reference substances is purer; 3) more accurate result calculations: this method only requires derivatization and result correction for AICAR, with the endogenous reference substances measured in their original form, reducing biases from corrections of multiple substances. The detection method performed well, with a concentriton limit of 2500 ng/mL, meeting the needs of routine detection concentrations. The CIR results from volunteer samples indicated that samples collected within 16 hours post-administration exceeded the threshold set in the literature. ConclusionThis study successfully established a 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing the internal and external sources of AICAR. After administering a low dose of AICAR to the Asian population, exogenous drug characteristics were manifested within 16 hours. This observation, when compared to the 40-hour detection window cited in the literature, suggests that the length of the detection window is positively correlated with the dosage of the test drug.

Evaluation of analytical columns suitable for high-temperature liquid-chromatography-isotope-ratio-mass-spectrometry analysis of anabolic steroids

Isotope ratio mass spectrometry (IRMS) can be used to determine the carbon isotope ratio of anabolic steroids. For example, in sports doping and food safety control, it enables determining an endogenous or synthetic origin of anabolic steroids. Generally, the steroids of interest are purified by liquid chromatography (LC) and analysed by gas chromatography combustion IRMS. LC-IRMS is not used since only mobile phases without carbon atoms can be used. For analysing mid-to apolar compounds, heated water can be used as an eluent as it has a similar polarity to a weak polar organic solvent. The silica-based columns are not robust enough at elevated temperatures in aqueous conditions. However, modified silica particles, metal oxides coated with polymers, and porous graphitic carbon are promising column materials for high-temperature LC (HT-LC) applications. Here, the stability of the stationary phase is crucial, and their chromatographic performance needs to be evaluated under the conditions mentioned above for anabolic steroid separations. Six columns using temperatures up to 200 °C were assessed, and only two were found to be appropriate. The ZirChrom-PBD column can be used for HT-LC-IRMS research purposes but is not recommended for routine laboratory practice applications due to the substantial loss of retention and resolution over time at elevated temperatures. Sachtopore-RP columns are the only suitable option for routine HT-LC-IRMS applications, even though they suffer from peak broadening over time when operating at elevated temperatures

Two-dimensional high performance liquid chromatography purification of underivatized urinary prednisone and prednisolone for compound-specific stable carbon isotope analysis.

The gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS) confirmation procedure for prednisone (PS) and prednisolone (PSL) is still a great challenge for the doping control laboratory due to the many structurally similar steroids present in urinary matrices. This study aims to establish an innovative online two-dimensional high performance liquid chromatography (2D-HPLC) purification method for measuring the carbon isotope ratios (CIRs) and achieving the identification of the synthetic forms of these two endogenous anabolic androgenic steroids (EAASs). Initially, the one-dimensional chromatographic column was used to separate and purify endogenous reference compounds (ERCs), and the co-elution fluids containing PS and PSL were switched to a two-dimensional chromatographic column for further purification through an online transfer system. Then the purified compounds were analyzed using GC/C/IRMS after sample pretreatments. The results showed that the minimum detection concentration of PS and PSL reached 30 ng mL-1, and no isotope fractionation occurred during the entire collection and preparation process. This method has been validated with the WADA technical document and showed good sensitivity and selectivity, demonstrating its practical applicability for urine samples in doping control laboratories.

Ultraviolet spectra determination and computational analysis of 44 E/Z steroid isomers in dried blood spot.

The dried blood spot (DBS) is a novel alternative matrix used in 2022 Beijing Winter Olympics and Paralympics. It is capable of distinguishing anabolic androgenic steroid (AAS) esters without the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) confirmation. In this study, a method for detection of 22 anabolic steroid esters in DBS based on ultra-high liquid chromatography-mass spectrometry (UPLC-MS) with parallel reaction monitoring (PRM) was developed and validated. Methoxylamine was used as the derivatization reagent to improve the sensibility. Specificity, limit of detection (LOD), linearity, stability, robustness, and carryover were evaluated. Steroid esters are nine testosterone esters, six nandrolone esters, five boldenone esters, methenolone enanthate, and trenbolone acetate. UV spectra were determined by HPLC. And density functional theory (DFT) calculation methods could provide theoretical UV spectra data. Three basis set of B3LYP/6-31G(d), B3LYP/6-31+G(d, p), and WB97XD/6-31+G(d, p) were used for the geometry optimizations and TD-DFT calculation. The average deviation (%RD) of B3LYP/6-31+G(d) for all 44 ester oximes are less than 3.0%. This study for the first time provides a method to tentatively identify the 44 E/Z configurations of steroid oxime products.

δ13C-Ethanol as a Potential Exclusionary Criterium for the Authentication of Scotch Whiskies in Taiwan: Normal vs. 3-Parameter Lognormal Distributions of δ13C-Ethanol Found in Single Malt and Blended Scotch Whiskies

With the difference in the photosynthesis process between C3- and C4-plants, the 13C/12C stable isotope ratio of ethanol, i.e., δ13C-ethanol, can potentially be a basis for the discrimination of Scotch whiskies derived from different raw materials. This study analyzed 51 authentic single malt Scotch whiskies and 34 authentic blended Scotch whiskies by gas chromatography–combustion–isotope ratio mass spectrometry (GC-C-IRMS) and examined the resulting data by a series of fitting distribution processes. The evaluation result demonstrated that δ13C-ethanol distribution of single malt Scotch whiskies fitted both normal and 3-parameter lognormal distribution. For blended Scotch whiskies, however, the data distribution of δ13C-ethanol conformed with a 3-parameter lognormal distribution rather than a normal one. Moreover, 99.7% of the confidence intervals (CI) of δ13C-ethanol for single malt Scotch whiskies would define between −23.21‰ to −30.07‰ for 3-parameter lognormal distribution, while from −11.19‰ to −28.93‰ for blended Scotch whiskies on the basis of the statistical properties. The simulative adulterated Scotch whiskies using more than 30% C4-derived edible distilled spirits can be effectively discriminated by means of CI of δ13C-ethanol. Since the addition of rectified spirits produced from the C4 plant has been found in some cases of seized Scotch whiskies in Taiwan, establishing a CI of δ13C-ethanol would be valuable for the purpose of Scotch whisky authentication.

Indispensable Amino Acid Digestibility of Moroccan Fava Bean Using the Dual Isotope Method in Healthy Adults

BackgroundAssessment of protein quality is necessary to satisfy the nutritional needs of populations across the world. In addition to indispensable amino acid (IAAs) composition, protein digestibility is a major component of IAA bioavailability, playing a crucial role in human health and affecting the linear growth of children. ObjectivesThis study aimed to evaluate IAA digestibility of fava beans, a legume widely consumed in Morocco using the dual-tracer method. Methods2H-intrinsically labeled Fava beans supplemented with 12 mg/kg BW of 13C spirulina were given to 5 healthy volunteers (3 men and 2 women), aged 25.8 ± 3.3 y, with a mean BMI of 20.0 kg/m2. The meal was spread in small portions and was given hourly throughout 7 h. Blood was sampled at baseline and hourly from 5 to 8 h after meal ingestion. IAA digestibility was evaluated by gas chromatography—combustion—isotope ratio mass spectrometry using the 2H/13C ratio in plasma IAA. Digestible indispensable amino acid ratios (DIAAR) were calculated using the scoring pattern for people older than 3 y. ResultsFava beans had an adequate level of lysine but were limiting in several IAAs, especially methionine. Under our experimental conditions, the average IAA digestibility of fava bean was 61.1% ± 5.2%. Valine had the highest digestibility (68.9% ± 4.3%) and threonine had the lowest (43.7% ± 8.2%). In consequence, the lowest DIAAR was 67% for threonine and only 47% for sulfur amino acids (SAA). ConclusionsThe present study is the first to determine the digestibility of fava bean amino acids in humans. The mean IAA digestibility was moderate, and consequently, we conclude that fava bean provides a limited amount of several IAAs, especially SAA, but adequately for lysine. Preparation and cooking methods of fava beans should be improved to increase digestibility.This study was registered at ClinicalTrials.gov as NCT04866927.

Evaluation of

Context Plant species exhibit different patterns of plant cuticular wax profiles, which can potentially be used as diet composition markers in free-grazing herbivores. Aims Evaluate the suitability of the plant cuticular n-alkanes, long-chain alcohol (LCOH) profiles and carbon stable isotope enrichment (δ13C) of n-alkanes as markers in the estimation of diet composition of grazing animals. Methods Forage samples were collected from 100 representative quadrats of 0.5 m × 0.5 m at 10 m transects and sorted by species and pooled from different quadrats to obtain enough quantities of representative individual species. In total, 10 dominant forage species were identified and analysed for n-alkanes and LCOH by gas chromatography, and the isotopic ratio by using gas chromatography–combustion–isotope ratio mass spectrometry. Principal component analysis (PCA) was used to identify inter-species differences in the concentration patterns of plant wax components. Key results Odd-chain n-alkanes comprised the highest proportion of the total n-alkane concentration, ranging from 79% in Ischaemum afrum to 95% in Haplocarpha hastata. n-Alkanes C31, C29 and C33 were the most abundant with an average of 167, 80 and 61 mg/kg DM, in that order, in all species. Even-chain LCOH comprised the highest proportion of the total LCOH concentration, accounting for 92% in Brachiaria scalaris to 97% in Ischaemum afrum. The dominant even-chain LCOH were C30OH, C32OH, C28OH and C26OH, with an average concentration of 362, 348, 266 and 237 mg/kg DM respectively, across species. The δ13C of n-alkanes showed large variations among forage species, ranging from −19.7‰ in Andropogon amethystinus to −40.6‰ in Trifolium mattirolianum. The result of the PCA showed that 81% of the variance in the pattern of concentrations of n-alkanes was explained by the first two principal components compared with 69.3% and 82.9% in the case of LCOH and δ13C of n-alkanes, respectively. Conclusions Noticeable variations were observed for forage species studied in the patterns of plant wax components. Implications The differences in the patterns of concentrations of n-alkanes, LCOH and δ13C of n-alkanes could be suitable as markers for diet composition estimation of grazing animals.

Compound-specific carbon isotope analysis of volatile organic compounds in complex soil extracts using purge and trap concentration coupled to heart-cutting two-dimensional gas chromatography–isotope ratio mass spectrometry

Compound-specific carbon isotope analysis (CSIA) is a powerful tool to track the origin and fate of organic subsurface contaminants including petroleum and chlorinated hydrocarbons and is typically applied to water samples. However, soil can form a significant contaminant reservoir. In soil samples, it can be challenging to recover sufficient amounts of volatile organic compounds (VOC) to perform CSIA. Soil samples often contain complex contaminant mixtures and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is highly dependent on good chromatographic separation due to the conversion to a single analyte. To extend the applicability of CSIA to complex volatile organic compound mixtures in soil samples, and to recover sufficient amounts of target compounds for carbon CSIA, we compared two soil extraction solvents, tetraglyme (TGDE) and methanol, and developed a heart-cutting two-dimensional GC-GC-C-IRMS method. We used purge & trap concentration of solvent-water mixtures to increase the amount of analyte delivered to the column and thus lower method detection limits. We optimized purge & trap and chromatographic parameters for twelve target compounds, including one suffering from poor purge efficiency. By using a 30 m thick-film non-polar column in the first and a 15 m polar column in the second dimension, we achieved good chromatographic separation for the target compounds in difficult matrices and high accuracy (trueness and precision) for carbon isotopic analysis. Tetraglyme extraction was shown to offer advantages over methanol for purge & trap concentration, leading to lower target compound method detection limits for CSIA of soil samples. The applicability of the developed method was demonstrated for a case study on soil extracts from a former manufacturing facility. Our approach extends the applicability of CSIA to an important matrix that often controls the long-term fate of contaminants in the subsurface.

A uniform sample preparation procedure for gas chromatography combustion isotope ratio mass spectrometry for all human doping control relevant anabolic steroids using online 2/3-dimensional liquid chromatography fraction collection

Androgenic anabolic steroids are the most misused substances in sports because of their performance-enhancing effects. Often synthetic analogues of endogenously present steroids are administered. To determine their endogenous or exogenous origin, Gas Chromatography Combustion Isotope Ratio Mass Spectrometry (GC-C-IRMS) is used in the field of doping control. Compounds subjected to IRMS analysis must be interference-free, with liquid chromatography fraction collection (HPLC-FC) being the crucial clean-up step. However, this clean-up is challenging, particularly for compounds present at low concentrations in samples with pronounced matrix effects. The compounds of interests for IRMS analyses in doping control are testosterone (T) and its main metabolites (androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol), epitestosterone, 19-norandrosterone (19-NA), boldenone (B) and its main metabolite (BM), formestane (F) and 6αOH-androstenedione (6aOHADION). Currently, the available methods only deal with a selection of the above-mentioned compounds. Some of these compounds (e.g., 19-NA, B, BM, 6aOHADION) are present in very low concentrations, requiring an extensive and dedicated sample clean-up, and this makes it challenging to develop a universal clean-up procedure. Many of these methods require different and multiple offline HPLC-FC setups, which are labour-intensive and time-consuming. That is problematic during, e.g., large sports events, where reporting time is limited (e.g., 72 h). Therefore, in the current work, we developed a uniform online 2D/3D HPLC-FC method, capable of purifying all relevant target compounds in a single run, leading to the fastest clean-up procedure so far (i.e., 31 min for T and its main metabolites; 46 min for 19-NA, F and 6aOHADION; 48 min for B and BM).

Application of Column Chromatography for Accurate Determination of the Carbon Isotopic Compositions of n-alkanes in Diverse Environmental Samples

The carbon isotopic compositions (δ13C) of n-alkanes in various environmental samples have been previously proposed as suitable fingerprints for assessing the origin of organic matter (OM) in diverse environmental systems. However, with respect to using gas chromatography–combustion–isotope ratio mass spectrometry for the carbon isotopic analysis of n-alkanes, analytical uncertainty may often be caused by the co-elution of interfering unsaturated compounds (e.g., aromatic and branched compounds). Hence, we propose a simple but reliable method for purification that uses column chromatography. The performance of two different solid stationary phases (i.e., aluminum oxide and Ag+-impregnated silica) was compared in terms of their capacity to eliminate unsaturated compounds from total hydrocarbons and thus increase the precision of δ13C measurements. Compared to the use of an activated aluminum oxide column, elution from an Ag+-impregnated silica column allows more effective isolation of individual n-alkanes, which results in more precise δ13C measurements for diverse environmental samples. Thus, Ag+-impregnated silica column separation can be effective as a routine experimental technique for increasing the accuracy of the δ13C values for n-alkanes in OM, which includes a large proportion of unsaturated compounds.

Application of online two-dimensional high-performance liquid chromatography as purification procedure to determine the origin of 19-norandrosterone in urine by gas chromatography-combustion-isotope ratio mass spectrometry.

19-Norandrosterone (19-NA) is the main metabolite of nandrolone and/or its precursors, which can be found naturally in human urine in trace amount. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) confirmation procedure can be used to identify a potential exogenous origin of 19-NA in urine sample. Sample purification for GC-C-IRMS analysis is crucial to the whole confirmation procedure because the concentration of 19-NA in the urine to be tested is very low. Online two-dimensional high-performance liquid chromatography (2D-HPLC) clean-up procedure with high separation capacity is used to isolate and enrich 19-NA as a sample pretreatment process. Linearity, lowest detectable concentration, uncertainty, and selectivity of the method are validated according to the World Anti-doping Agency's (WADA) requirement. Isotope fractionation effect was not observed during the 2D-HPLC purification process. The validated method provides a high efficient and convenient confirmation procedure to determine the origin of 19-NA.

Exploring the metabolic fate of medium-chain triglycerides in healthy individuals using a stable isotope tracer

Medium chain triglyceride (MCT) supplementation is often recommended as treatment for patients with long-chain fatty acid β-oxidation (lcFAO) disorders, since they can be utilized as an energy source without the use of the defective enzyme. However, studies in mice and preterm infants suggest that not all medium-chain fatty acids (MCFA) are oxidized and may undergo elongation to long-chain fatty acids (LCFA). In this single blinded study, we explored the metabolic fates of MCT in healthy individuals using a 13C-labeled MCT tracer. Three healthy males in rest received on two test days a primed continuous infusion of glyceryl tri[1,2,3,4-13C4]-octanoate with either an isocaloric supplementation of 1) exclusively MCT (MCT-only) or 2) a mixture of MCT, proteins and carbohydrates (MCT-mix). Gas chromatography - combustion - isotope ratio mass spectrometry (GC-C-IRMS) was used to determine 13C-enrichment of long-chain fatty acids in plasma and of 13CO2 in exhaled air. When provided as single energy source, an estimated 42% of administered MCT was converted to CO2. In combination with carbohydrates and proteins in the diet, oxidation of MCT was higher (62%). In both diets <1% of 13C-label was incorporated in LCFA in plasma, indicating that administered MCT underwent chain-elongation to LCT. Although the relative MCT oxidation rate was higher when combined with carbohydrates and protein, quantitatively more MCT was oxidized when given an isocaloric meal with solely MCT. As these results were obtained in the resting state opposed to during exercise, it is too early to give a recommendation concerning the use of MCT in lcFAO disorders. The data show that in resting healthy individuals only a very small part of the MCT is traced back as LCFA in plasma, suggesting that MCT treatment does not result in a large LCFA burden, however further research on storage of MCT in tissues is warranted. The study was registered in the Nederlands Trialregister. Protocol ID: Trial NL7417 (NTR7650).

Determination of Arginine δ15N Values in Plant and Animal Proteins by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry.

Nitrogen (N) stable isotope techniques are widely used in ecology, archaeology, and forensic science to explore trophic relationships and provenances of organisms and materials, most widely using bulk δ15N values of whole organisms, tissues, or other materials. However, compound-specific isotope values can provide more diagnostic isotope "fingerprints" and specific information about metabolic processes. Existing techniques for nitrogen isotope analysis allow the determination of δ15N values of 14 amino acids (AAs), accounting for ca. 75% of plant protein and collagen N. The majority of remaining N is from arginine, comprising 16 and 14% of collagen and plant protein N, respectively. We therefore aimed to develop a method to detect arginine and determine its δ15N value (δ15NArg) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS), to further contribute to the understanding of the metabolic routing of this important AA. We demonstrate that arginine, as its N-acetyl isopropyl ester, is amenable to GC analysis using a 15 m midpolarity DB-35 column, eluting with baseline resolution from other AAs. The recorded δ15N value by GC-C-IRMS was within the error of that of the underivatized compound determined by elemental analyzer-isotope ratio mass spectrometry (EA-IRMS). The newly developed GC-C-IRMS method was applied to modern plant protein and cattle collagen, enabling their δ15NArg values to be related to AA biosynthesis. Determination of archaeological cattle collagen δ15NArg values confirmed the suitability of this method to provide further insights into past diets and ecosystems. Bulk collagen δ15N value reconstruction including δ15NArg values better reflect the measured bulk values, as the isotopic ratio of 91% of collagen N can now be determined at the compound-specific level.

Carbon isotopic characterization of prednisolone and prednisone pharmaceutical formulations: Implications in antidoping analysis.

Twenty-two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) method. All of them showed typical non-endogenous δ13 C values, except for the Belgian nasal spray, Sofrasolone®, with a less depleted 13 C content (-17.84 ± 0.18‰). Observational studies were performed on two volunteers in therapy with Sofrasolone® to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative δ13 C values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both δ13 C values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre-treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC-C-IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro-11-deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method used routinely for the quali-quantitative detection of glucocorticoids. δ13 C values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius®, a prednisolone oral formulation with a conventional more negative δ13 C value (-29.28 ± 0.25‰). Finally, the potential masking effect that combined therapy with Sofrasolone® and Sintredius® could induce on the IRMS findings was investigated.

Practical considerations for improved reliability and precision during determination of δ15 N values in amino acids using a single combined oxidation-reduction reactor.

There has been increased interest in the measurement of δ15 N values in amino acids (AAs) to gain simultaneous insight into both trophic relationships and the composition of biogeochemical sources used by producers at the base of the food web. A new combustion reactor design in gas chromatography/combustion isotope ratio mass spectrometry (GC/C-irMS) equipment has brought to light variable outcomes in performance, highlighting the need for better information about best practices for new systems. Precision for δ15 N values in amino acids using the single combined oxidation-reduction reactor is improved across a sequence of analyses if the reactor is oxidized for a substantial period (2 h) and subsequently maintained throughout the sequence with 12-17 s seed oxidation before each run during GC/C-irMS. A five-point calibration curve using amino acids with a range of δ15 N values from -2.4‰ to +61.5‰ was used in combination with a 13-15 amino acid mixture to consistently normalize measurements to internationally calibrated reference materials. Combining this oxidation method with normalization techniques using both internal and external standards provided a reliable throughput of ~25 samples per week. It allowed for a reproducible level of precision of <±0.5‰, n = 10 within a derivatized standard mixture across each sequence and an average sample precision of ±0.27‰ n = 3, which is lower than the analytical precision typically associated with δ15 N values for amino acid analysis (<±1‰). A few practical considerations regarding oxidation and conditioning of the combustion reactor allow for increased sequence capacity with the single combined oxidation-reduction reactor. These considerations combined with normalization techniques result in a higher throughput and reduced analytical error during the measurement of δ15 N values in amino acids.

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.

Bisphenol A attenuation in natural microcosm: Contribution of ecological components and identification of transformation pathways through stable isotope tracing

Residues of bisphenol A (BPA) are ubiquitously detected in the surface water due to its widespread usage. This study systematically investigated the dissipation and kinetics of BPA under simulated hydrolysis, direct and indirect photolysis, bacterial degradation, microbial degradation and natural attenuation in microcosm. Structural equation modeling (SEM) by using partial least square method in path coefficient analysis suggested that the microbial degradation was the major factor involved in the natural attenuation of BPA. The potential transformation products were identified by using liquid chromatography high-resolution mass spectrometry (LC-HRMS) and stable isotope tracing technique by simultaneous performing gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) and gas chromatography mass spectrometry (GC–MS). A total of fourteen including three novel transformation products of BPA were identified to indicate five possible pathways. An increased yield of labeled (δ13C) CO2 and detection of 13C-labeled phospholipid fatty acids (PLFAs) indicated the mineralization of BPA and possible utilization of BPA or its transformation products by microbes for cellular membrane synthesis, respectively.

Low Temperature Catalytic Combustion Reactors for High Precision Carbon Isotope Measurements in Gas Chromatography Combustion Isotope Ratio Mass Spectrometry

Metal oxide-filled reactors constructed with ceramic tubes or fused silica capillary are widely used for combustion in gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS). However, they tend to be easily cracked or broken and prone to leaks at operating temperatures of ∼950 °C. Here we introduce a modified commercially available catalytic combustion/reduction methanizer to quantitatively convert organics to CO2 for δ13C analysis while retaining chromatographic resolution. These modified "ARC" reactors operate with a transition-metal catalyst that requires a flowing O2 gas to enable complete conversion to CO2 at lower temperature (620 °C) with acceptable reactor life, reduced complexity, and improved robustness. Performance of two versions of the ARC reactors with different combustion volumes was characterized by analysis of steroid and alkane isotopic standard materials. Linearity of steroid isotopic standards ranged from 0.02 to 0.60 ‰/V in the range of 25 to 200 ng of each steroid injected. Precisions and accuracies of measurements for steroids and alkanes had average standard deviations of SD(δ13C) less than ±0.18 ‰ and average accuracy of better than 0.19 ‰ δ13CVPDB. Peak width expansion within both devices were similar to that in traditionally used metal oxide reactors. These data demonstrate for the first time that novel combustion schemes enable operation at lower temperatures as an alternative approach comparable to high temperature techniques to yield high precision δ13C data with GCC-IRMS.

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.

Geographical origin of Chinese wolfberry (goji) determined by carbon isotope analysis of specific volatile compounds

Chinese wolfberry or goji berry (Lycium barbarum) is an important traditional Chinese medicine. Its price and function has a close correlation with its geographical provenance. Illegal mislabeling motivated by commercial gains brings serious food safety problems and damages consumer confidence. In this work, a novel analytical strategy combined with chemometrics statistic tools was developed to determine the geographical origin of wolfberries from different provinces in China. Stable carbon isotopic ratios (δ13C) of wolfberry volatile compounds (i.e. limonene, tetramethylpyrazine, safranal, geranylacetone, and β-ionone) were determined by gas chromatography-combustion-isotope ratio mass spectrometry (GC-IRMS) with headspace-solid phase micro extraction (HS-SPME). Five types of SPME fiber (i.e. DVB/CAR/PDMS, CAR/PDMS, PDMS/DVB + OC, PDMS, and PA), extraction time, temperature and GC-IRMS conditions were comprehensively optimized to obtain the best adsorption of volatile compounds in wolfberry. Method integrity was assessed by comparing volatiles extracted using HS-SPME GC-IRMS with direct injection GC-IRMS and were in good agreement with each other. The geographical variations of volatile compounds using one-way analysis of variance (ANOVA) were explored for individual δ13C values in wolfberry samples from Gansu, Ningxia and Qinghai. Geographical origin of wolfberry was differentiated by linear discrimination analysis (LDA), with an accuracy of 89.16%, 87.77% and 85.87% for these three provinces, respectively. These results showed the combination of SPME and IRMS provides a rapid and valid method to determine the geographical origin of wolfberry.