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.

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.

Effect of immune responses on breath methane dynamics

Methane (CH4) which can be detected in human breath has long been exclusively associated with anaerobic microbial activity (methanogenesis) in the gastrointestinal tract. However, recent studies challenge this understanding by revealing that CH4 might also be produced endogenously in cells through oxidative–reductive stress reactions. Consequently, variations in breath CH4 levels compared to an individual’s baseline level might indicate enhanced oxidative stress levels, and, therefore, monitoring breath CH4 levels might offer great potential for ‘in vivo’ diagnostics such as disease diagnosis, monitoring the efficacy of treatments, or during the application of personalized medicine. To evaluate the effects from immune responses triggered by infections, inflammations, and induced perturbation by vaccination on CH4 dynamics in breath, two subjects were monitored over a period of almost 2 years. Breath CH4 levels were measured by gas chromatography equipped with a flame-ionization detector. Both subjects exhibited significant deviations (positive and negative, respectively) from their normal CH4 breath levels during periods of potential enhanced immune activity. Deviations from the ‘healthy state’ were indicated by the exceeding of individual CH4 ranges. Moreover, for the first time we could clearly prove CH4 degradation induced through vaccination by measuring stable carbon isotopes of CH4 using gas chromatograph–combustion–isotope ratio mass spectrometry. Hence, breath CH4 concentration and isotopic analyses may be used as a biomarker to evaluate specific immune responses and individual immune states.

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (13C/12C ratio or δ13C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ13C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled 13C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the 13C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and 13C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ13C), validated its utility in diet switch studies.

Volatile organic compounds identification and specific stable isotopic analysis (δ13C) in microplastics by purge and trap gas chromatography coupled to mass spectrometry and combustion isotope ratio mass spectrometry (PT-GC-MS-C-IRMS).

Microplastics (MPs) have become one of the major global environmental issues in recent decades due to their ubiquity in the environment. Understanding MPs source origin and reactivity is urgently needed to better constrain their fate and budget. Despite improvements in analytical methods to characterize MPs, new tools are needed to help understand their sources and reactivity in a complex environment. In this work, we developed and applied an original Purge-&-Trap system coupled to a GC-MS-C-IRMS to explore the δ13C compound-specific stable isotope analysis (CSIA) of volatile organic compounds (VOC) embedded in MPs. The method consists of heating and purging MP samples, with VOCs being cryo-trapped on a Tenax sorbent, followed by GC-MS-C-IRMS analysis. The method was developed using a polystyrene plastic material showing that sample mass and heating temperature increased the sensitivity while not influencing VOC δ13C values. This robust, precise, and accurate methodology allows VOC identification and δ13C CSIA in plastic materials in the low nanogram concentration range. Results show that the monomer styrene displays a different δ13C value (- 22.2 ± 0.2‰), compared to the δ13C value of the bulk polymer sample (- 27.8 ± 0.2‰). This difference could be related to the synthesis procedure and/or diffusion processes. The analysis of complementary plastic materials such as polyethylene terephthalate, and polylactic acid displayed unique VOC δ13C patterns, with toluene showing specific δ13C values for polystyrene (- 25.9 ± 0.1‰), polyethylene terephthalate (- 28.4 ± 0.5‰), and polylactic acid (- 38.7 ± 0.5‰). These results illustrate the potential of VOC δ13C CSIA in MP research to fingerprint plastic materials, and to improve our understanding of their source cycle. Further studies in the laboratory are needed to determine the main mechanisms responsible for MPs VOC stable isotopic fractionation.

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.

Gas Chromatography Combustion Isotope Ratio Mass Spectrometry to Detect Differences in Four Compartments of Simmental Cows Fed on C3 and C4 Diets.

Fatty acids (FAs) metabolism in animals represents an important field of study since they influence the quality and the properties of the meat. The aim of this study is to assess the possibility to discriminate the diets of cows in different animal compartments and to study the fate of dietary FAs in the bovine organism, using carbon isotopic ratios. Five FAs, both essential (linoleic and linolenic) and non-essential (palmitic, stearic, and oleic) in four compartments (feed, rumen, liver, meat) of animals fed two different diets (based on either C3 or C4 plants) were considered. For all compartments, the carbon isotopic ratio (δ13C) of all FAs (with few exceptions) resulted significantly lower in cows fed on C3 than C4 plants, figuring as a powerful tool to discriminate between different diets. Moreover, chemical reactions taking place in each animal compartment result in fraction processes affecting the δ13C values. The δ13CFAs tendentially increase from feed to meat in group C3. On the other hand, the δ13CFAs generally increase from rumen to liver in group C4, while δ13CFAs of rumen and meat are mostly not statistically different. Different trends in the δ13CFAs of the two groups suggested different FAs fates depending on the diet.

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.

Nitrogen use efficiency, partitioning, and storage in cool climate potted Pinot Noir vines

Optimal timing of nitrogen (N) fertilizer to promote N use efficiency (NUE) in cool climate wine regions is largely unknown. One year-old potted Vitis vinifera L. (Vitaceae) cv. Pinot Noir vines were subjected to soil applied 15N-labelled calcium nitrate (5.5 atom%) fertilizer at four application timings (budbreak to fruit set, fruit set to veraison, veraison to harvest, or spread evenly across the growing season) over two growing seasons (2017/18 and 2018/19) at a rate of 12 g N vine−1. Annual vine parts (leaves and clusters) were collected at commercial harvest in both 2018 and 2019 and the remaining vine was destructively harvested during winter dormancy. Vines were separated into organs (leaves, clusters, shoot, trunk, and roots) and organ dry matter was determined before samples were ground into a fine powder to determine total N percentage and 15N atom percentage using flash combustion isotope ratio mass spectrometry (varioPYRO cube coupled to Isoprime100 mass spectrometer). Compared to the average values obtained from the other treatments, the veraison to harvest treatment significantly reduced whole vine dry matter by 39.1% and vine NUE by 42.1%. Yield was also reduced by 12.9% compared to the budbreak to fruit set treatment. NDF, defined here as the proportion of N within the vine (or vine organ) that was derived from fertilizer N, and overall vine total N content was lower in the veraison to harvest treatment due to the reduction of root and shoot NDF content. This suggests that for young, actively growing vines in cool climates, limiting N applications until veraison is inefficient and ineffective. No significant differences in NUE, N partitioning or N storage were observed between the other fertilizer N application timings.

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.

Hydrocarbon sedimentary organic matter composition from different water-type floodplain lakes in the Brazilian Amazon

Amazon rivers and flooded areas can be categorized into three types of water: white, black, and clear. These categories present unique physical–chemical characteristics that influence the characteristics and distribution of sedimentary organic matter (SOM). A geochemical assessment of the short sedimentary cores of four floodplain lakes within the Brazilian Amazon basin was performed. Two black, one white, and one clear-water floodplain lakes were selected to evaluate the variability of OM composition based on aliphatic biomarker and carbon isotopic ratios, as proxies for an enhanced methane cycle, anoxia, and to determine the origin of predominant aliphatic biomass. Gas chromatography was coupled to flame ionization detection (GC–FID), mass spectrometry (GC–MS) and combustion-isotope ratio mass spectrometry (GC–C–IRMS). These were used to determine aliphatic biomarker ratios, for quantification, and to measure carbon isotopic compositions. The TOC content was inversely proportional to the expected OM productivity; one black water (low OM productivity) presented the highest TOC content, and white water (high productivity) presented the lowest. Also, clear water lake (intermediate productivity) presented TOC content between the other lakes. Based on n-alkane proxies and profiles, as well as bacterial and plant-derivative terpene distribution, we observed a mostly terrigenous contribution for the black and clear water lakes (allochthonous SOM), as well as a prevalence of aquatic organisms in white water lake (autochthonous SOM). Only in the most superficial section of the white water lake, was intense OM degradation substantiated with n-alkanes showing an even-short-chain predominance, indicative of OM cycling. Also, for one black water lake, we detected depleted n-alkane and diploptene δ13C values (–42‰) associated with methanotrophic archaea and higher relative concentrations of des-A-triterpenes commonly associated with anoxic sedimentation. Based on these data, the influence of water-type on the SOM composition was verified. The approach helps to understand the application of n-alkane proxies to differentiate Amazonian environments.

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.

Fatty acids stable carbon isotope fractionation in the bovine organism. A compound-specific isotope analysis through gas chromatography combustion isotope ratio mass spectrometry

The contribution of dietary fatty acids to the quality of the meat and their path through the bovine organism is currently the subject of a lot of research. Stable isotope ratio analysis represents a powerful tool for this aim, one that has not been studied in depth yet. In this work, for the first time, the carbon isotopic ratios of six fatty acids (myristic 14:0, palmitic 16:0, stearic 18:0, oleic 18:1n-9, linoleic 18:2n-6 and linolenic 18:3n-3 acids) in different matrixes (diet, rumen, duodenal content, liver and loin) were analysed through gas chromatography combustion isotope ratio mass spectrometry. Moreover, the quantification of the single fatty acids was carried out, providing important information supporting the carbon isotopic ratio results. The variation in the concentration of the fatty acids in the different matrices depends on the chemical modifications they undergo in the sequential steps of the metabolic path. GC-C-IRMS turned out to be a powerful tool to investigate the fate of dietary fatty acids, providing information about the processes they undergo inside the bovine organism.

The Carbon Isotope Ratios of Serum Amino Acids in Combination with Participant Characteristics can be Used to Estimate Added Sugar Intake in a Controlled Feeding Study of US Postmenopausal Women

Background:The carbon isotope ratio (CIR) is a proposed biomarker for added sugar (AS) intake in the United States; however, because the CIR is also associated with meat intake in most populations the need for specificity remains. The CIR of amino acids (AAs) has the potential to differentiate sugars from meat intakes, because essential AAs must derive from dietary protein whereas certain nonessential AAs can be synthesized from sugars. Objectives:We tested whether serum CIR-AAs in combination with participant characteristics could meet a prespecified biomarker criterion for AS intake in the Nutrition and Physical Activity Assessment Study Feeding Study (NPAAS-FS) of the Women’s Health Initiative, a population in which the whole-serum CIR was not associated with AS intake. Methods:Postmenopausal women (n = 145) from Seattle, WA, were provided with individualized diets that approximated their habitual food intakes for 2 wk. Dietary intakes from consumed foods were characterized over the feeding period using the Nutrition Data System for Research. The CIR of 7 AAs–Ala, Gly, Val, Leu, Ile, Pro, and Phe–were measured in fasting serum collected at the end of the 2-wk feeding period, using gas chromatography–combustion isotope ratio mass spectrometry. Biomarker models were evaluated using regressionR2 = 0.36 as a major biomarker criterion, based on the benchmarkR2values of well-established recovery biomarkers in the NPAAS-FS. Results:AS intake was associated with CIR-Ala (ρ = 0.32;P< 0.0001). A model of AS intake based on CIR-Ala, CIR-Gly, CIR-Ile, smoking, leisure physical activity, and body weight met the biomarker criterion (R2 = 0.37). Biomarker-estimated AS intake was not associated with meat or animal protein intake. Conclusions:Results support serum CIR-AAs in combination with participant characteristics as potential biomarkers of AS intake in US populations, including those with low AS intake.

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.

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.

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.