Isotope Ratio Mass Spectrometry Research Articles

Анализ отношений стабильных изотопов легких элементов в отдельных компонентах мёда

Honey possesses excellent sensory and nutritional properties, which makes it a valuable food ingredient. However, the same qualities make it one of the most often adulterated products in the world. Constant violations of processing technology threaten the beekeeping industry. In this regard, new authenticity criteria are a popular area of honey studies. The article introduces a method for establishing ratios of light stable isotopes in honey. The study featured 36 samples of honey of various geographical origins and botanical profiles, as well as five samples of sugar syrups from various raw materials. The quantitative profiles were obtained using a Delta Advantage V isotope mass spectrometer (USA – Germany) with additional Flash IRMS and Conflo IV modules. The experiment involved the ratios of stable carbon isotopes in honey δ13C (gross) and nitrogen δ15N in its protein fraction, as well as the values of δ13C, δ18O, and δ2H of ethanol isolated from fermented honey. The values of δ13C (gross) and δ13C of honey proteins made it possible to calculate the amount of added sugar of corn and cane origin. Exogenous sugars in the amounts of 6.5% and 18% cane sugar were detected in two samples. Isotope mass spectrometry was able to identify honey samples with exogenous sugars of C4-plant origin. However, the method failed to detect sugar-containing substances from C3-type plants. The δ18O index demonstrated some prospects as an identification criterion for sugars from C3-plants in honey. Nitrogen isotope ratios in honey proteins proved to be an efficient tool for determining honey authenticity and an additional criterion for identifying bee products. The research resulted in a patent (RU2809285C1) for a new method of determining exogenous sugar-containing substances in honey

Establishment of a method for cultivating tomatoes using the method of isotope mass spectrometry

Establishment of a method for cultivating tomatoes using the method of isotope mass spectrometry

Improving the Determination of Carbon Isotope Ratios of Endogenous Steroids Found in Human Serum.

The determination of serum concentrations of testosterone (T) and 4-androstenedione (A4) was implemented into the steroidal module of the Athlete Biological Passport in 2023. Monitoring T, A4, and the ratio of T/A4 in a longitudinal manner enables the detection of the misuse of low-dose T administrations especially in female athletes, whereas urinary markers of the steroid profile may not be influenced significantly. In contrast to the urinary steroid profile, knowledge on confounding factors regarding serum concentrations of T and A4 is yet comparably scarce, and corroborating exogenous sources of the target analytes by isotope ratio mass spectrometry (IRMS) is desirable. In a recent study, it was demonstrated that carbon isotope ratios (CIRs) of serum steroids can be determined if analyte concentrations permit. The therein-employed method utilized 2D-GC/IRMS, and only a limited number of potential endogenous reference compounds were available. The here-presented approach uses complementary analyte purification strategies, addressing previous limitations. A high-performance liquid chromatography cleanup was developed and fully validated for serum steroids in order to enable all doping control laboratories to potentially implement this method alongside existing protocols for urinary steroids. Besides the already-investigated endogenous steroids cholesterol, dehydroepiandrosterone sulfate, androsterone sulfate, and epiandrosterone sulfate, two additional steroids were included in the test menu, namely, pregnenolone sulfate and 5-androstene-3β,17β-diol sulfate. Serum steroid concentrations down to 25 ng/mL were found to allow robust CIR determinations, and a reference population encompassing 124 male and female athlete samples was investigated to enable the calculation of population-based thresholds for all relevant steroid combinations.

Isotopic Composition of C, N, and S as an Indicator of Endometrial Cancer.

The metabolic pathway of cancerous tissue differs from healthy tissue, leading to the unique isotopic composition of stable isotopes at their natural abundance. We have studied if these changes can be developed into diagnostic or prognostic tools in the case of endometrial cancer. Measurements of stable isotope ratios were performed using isotope ratio mass spectrometry for nitrogen, carbon, and sulfur isotopic assessment. Uterine tissue and serum samples were collected from patients and the control group. At a natural abundance, the isotopic compositions of all three of the studied elements of uterus cancerous and healthy tissues are different. However, no correlation of the isotopic composition of the tissues with that of serum was found. Differences in the isotopic composition of the tissues might be a potential prognostic tool. However, the lack of a correlation between the differences in the isotopic composition of the tissues and serum seems to exclude their application as diagnostic biomarkers, which, however, might be possible if a position-specific isotopic analysis is performed.

PSIX-13 Methionine requirements across the life cycle of working Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Abstract Methionine is a limiting amino acid in canine diets, but few requirement studies have been conducted across life stages in large-breed dogs. The objective of this study was to determine the minimum requirement (MR) for methionine (Met) in Labrador retrievers at 4 life stages using the indicator amino acid (AA) oxidation (IAAO) technique. In total, 24 dogs were used: 6 puppies aged 10 to 14 wk, 6 puppies aged 14 wk to 9 mo, 6 adults aged 2 to 5 yr, and 6 seniors aged over 8 yr in feeding studies to evaluate the changing requirements of methionine (Met) as canines age. After 2-d adaptation to Met-adequate basal diet (Met = 0.56% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of Met ranging from deficient to sufficient (final Met content in experimental diets was 0.1982, 0.2386, 0.279, 0.49, 0.525, 0.56% dry matter, with indispensable amino acids formulated at 1.6x NRC values. Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C]phenylalanine based on body weight was first given, followed by [1-13C]Phe doses every 30 min spanning a 4-h period), and breath samples were collected via respiration mask every 30 min. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total CO2 production measured via indirect calorimetry and isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for Met were estimated to be 0.84 ± 0.05 g/1000 kcal (mean ± 2SD) for puppies 10 to 14 wk, 0.78 ± 0.16 g/1,000kcal (mean ± 2SD) for puppies 14 wk to -9 mo, 0.68 ± 0.19 g/1,000kcal (mean ± 2SD) for adults 2 to 5 yr, and 0.62 ± 0.17 g/1,000kcal (mean ± 2SD) for seniors. This research provides important information to assist in accurate formulation of diets that contain appropriate methionine for large breed dogs at all life stages.

PSLBI-17 Total sulfur amino acid requirements across the canine life cycle in Labrador Retrievers using the indicator amino acid oxidation (IAAO) technique

Abstract Methionine and cysteine are the principal sulfur-containing amino acids that are incorporated into proteins, although homocysteine and taurine also have important physiological roles in canines. As limited studies have been conducted for total sulfur-containing amino acids (TSAA) requirements across the canine life cycle, our objective was to determine the minimum requirements (MR) for TSAA at 4 life stages using the indicator amino acid oxidation (IAAO) technique. Labrador Retrievers (n = 24) were used in feeding studies to evaluate the changing requirements of TSAA as dogs mature: 6 puppies aged 10 to 14 wk, 6 puppies >14 wk to 9 mos, 6 adults 2 to 5 yr (yr), and 6 seniors > 8yr. Following a 2-d adaptation to a TSAA-adequate basal diet (TSAA = 1.12% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of TSAA ranging from deficient to sufficient (final TSAA content in experimental diets were 0.41, 0.46, 0.51, 0.98, 1.05, and 1.12% dry matter, with indispensable amino acids formulated at 1.6x NRC values). The test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was included (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 spanning 4 h). Breath samples were collected via a respiration mask every 30 min. Total CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for TSAA were estimated to be 1.55 ± 0.069 g/1,000 kcal (mean ± 2 SD) for puppies 10 to 14 wk, 1.53 ± 0.21 g/1,000 kcal (mean ± 2 SD) for puppies 14 wk to 9 mo, 1.4 ± 0.30 g/1000 kcal (mean ± 2 SD) for adults 2 to 5 yr, and 1.27 ± 0.23 g/1,000 kcal (mean ± 2 SD) for seniors. These results may contribute to the formulation of diets that meet the age-appropriate requirements of TSAA for large-breed dogs at all life stages.

PSLBI-18 Isoleucine requirements of young adult and senior Labrador retrievers

Abstract Age-appropriate diets that meet the unique nutritional needs of aging are in greater demand as senior dogs represent an increasing percentage of the pet population. Studies targeting the specific amino acid requirements of the aging canine are important because they may inform dietary formulations for this specific life stage. In this study, the indicator amino acid oxidation (IAAO) technique was used to determine the minimum requirements for isoleucine (Iso) in young adult and senior Labrador retrievers. After 2-d adaptation to Iso-adequate basal diet (Iso = 0.608% dry matter), dogs underwent individual IAOO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of isoleucine ranging from deficient to sufficient (final Iso content in experimental diets were 0.244, 0.285, 0.326, 0.532, 0.57 and 0.608% dry matter). Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 min spanning a 4 h period), and breath samples were collected via respiration mask every 30 min. CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for isoleucine were 898 ± 71 mg/1,000 kcal ME (mean ± 2 SD) for young adults and 885 ± 91 mg/1,000 kcal ME (mean ± 2 SD) in senior dogs, which is slightly greater than the National Research Council’s minimum requirement. As the pet food industry offers more specialized diets for aging canines, updating the amino acid requirements for such animals is increasingly important.

Authentication of premium Asian rice varieties: Stable isotope ratios and multi-elemental content for the identification of geographic fingerprints

Over 90 percent of the world's rice is produced and consumed in the Asia-Pacific region. Varieties such as Thai Jasmine rice and Paw San (or “Myanmar pearl rice”) are globally recognised as premium, while more local high-grade varieties include the Indonesian Ciherang and Inpari. Being able to trace the origin of these products has become necessary, since they are marketed at relatively higher prices compared to other cultivars, and they often become the target of fraudulent activities. In this work, we aimed to identify variables that could distinguish the premium-producing regions within each country, by Isotope Ratio Mass Spectrometry (IRMS) and Inductively Coupled Plasma- Mass Spectrometry (ICP-MS). Low-Level Data Fusion (LLDF) followed the analysis of more than 300 authentic samples, and (O)PLS-DA models yielded very high accuracy values. The most important geo-differentiating variables (VIP>1.4) were identified as: δ13C, δ18O, δ2H, δ34S, Co, Rb, Cu, Ba and Zn.

Chicken meal is not an appropriate reference protein for estimating protein quality of ingredients used in extruded diets intended for dogs.

The indicator amino acid oxidation (IAAO) method has been used to determine metabolic availability (MA) of amino acids in feedstuffs for pigs, humans, and preliminarily for cats. Peas are a commonly used protein source in grain-free extruded dog diets. However, peas have a poor sulfur amino acid (AA) ratio (methionine (Met):cysteine) with Met being the first limiting AA. Furthermore, little is known about the MA of Met in peas fed to dogs. Therefore, our objective was to compare the MA of Met in peas to chicken meal (CM), as a gold-standard reference protein. The study was done as a replicated 5 x 5 complete Latin square design. Ten neutered male mixed-breed dogs (1.5 years old; 26.0kg ±2.4kg body weight; BW) fed to maintain ideal BW received all dietary treatments: BAS: lamb-based diet (deboned lamb and lamb meal) providing Met at 50% of its requirement (0.27g/100g DM), CHK: CM and lamb-based diet, and PEA: ground dried pea and lamb-based diet both providing Met at 68% of its requirement (0.35 and 0.37g/100g DM, respectively). Two other treatments were created by blending BAS with PEA (BAP) and the BAS with CHK (BAC) to create diets with Met at 59% of requirement (0.32 and 0.31g/100g DM, respectively). This resulted in three graded levels of Met for both CM and peas to allow for a slope-ratio assay approach to quantify MA with the BAS diet as the common first point. All other AAs were provided to meet at least 120% of the AAFCO recommendations for adult dogs. The BAS diet, with supplemental DL-Met, was fed for a 2-wk wash-in period. After 2 days of diet adaptation IAAO was performed. Dogs were fed 13 small meals where meal 6 contained a priming dose (9.4mg/kg BW) of L-[1-13C]-phenylalanine (Phe; 99%) as well as a constant dose (2.4mg/kg BW) in meals 6-13. Breath samples were collected and enrichment of 13CO2 was measured using isotope-ratio mass spectrometry to calculate the rate of Phe oxidation (F13CO2 umol/kg BW/h). Oxidation was analyzed via SAS using proc GLIMMIX with dog and period as random effects, and diet, %Met, and their interaction as fixed effects. Unexpectedly, the slope of Phe oxidation, in response to increasing Met intake, from CM was 31% of that of peas, indicating a lower MA for Met in CM as compared to peas. This finding may be due to damage of AAs during rendering. At this time, CM in extruded diets is not an acceptable reference protein to determine MA of AAs in dogs and the MA of Met from peas cannot be confidently assessed.

The effects of fingerprinting agents on the stable isotope ratios of polyethylene films

Traditional forensic chemical comparisons of polymeric materials are often affected by fingerprinting agents that enhance and visualise finger-marks. This can inhibit detailed analysis used to provide an association or discrimination between two samples. In this study, we have demonstrated that the method for polymer comparisons using isotope ratio mass spectrometry analysis is not affected by fingerprinting matter on the surface in contrast to other forensic instrumental tests. A selection of resealable bags was analysed for carbon and hydrogen isotope ratio values of bags that had been subject to various fingerprinting agents and compared to values of untreated bags. The results showed no significant difference between samples that had been fingerprinted from those that were untreated.

Pretreatment method for oxygen stable isotope ratio analysis of the sugar-rich fraction in fruit juice via isotope ratio mass spectrometry.

The oxygen stable isotope ratio (δ18O) of the sugar-rich fraction of fruit juice is important as a tracer of the geographical origin of raw material. This study sought to minimize the inter-day variation of δ18O attributable to the influence of water to accurately monitor geographical origin labeling. Two drying devices (freeze dryer and vacuum oven) were compared. Then, two humidity levels (normal and low humidity) at which the samples were placed after drying were compared. The low-humidity environment was constructed using a glove bag and pure argon gas. δ18O was measured using thermal conversion elemental analyzer/isotope ratio mass spectrometry. Improvements were made to the measurement method based on aforementioned analyses results, and the performance of the initial and improved methods was compared. δ18O of juice dried in a vacuum oven was 3.30‰ lower than that of juice dried in a freeze dryer. Moreover, δ18O of juice samples exposed to normal humidity was 3.74‰ lower than that of samples exposed to low humidity. The combined inter-day and intra-day standard deviation was reduced from 1.20‰ in the initial method to 0.42‰ in the improved method. This study describes a pretreatment method for δ18O measurement in the sugar-rich fraction of fruit juice with less inter-day variation, and it will be useful for monitoring geographical origin labeling.

Simultaneous observation of 2H and 13C enrichment of methyl phosphonic acid via Orbitrap-IRMS with applications to nerve agent forensics

Quantification of the stable isotopes within a compound aids forensic investigations as it provides a fingerprint which can determine that compound's source substrates, synthetic route, and possible mechanisms of degradation. Previous stable isotope studies have explored 13C and 2H measurements of the sarin precursors methylphosphonic dichloride (DC) and methylphosphonic difluoride (DF) as forensic signatures. However, these measurements required different sample preparations and measurement techniques. Orbitrap isotope ratio mass spectrometry (Orbitrap-IRMS) is a developing technique which can characterize multiple stable isotopes simultaneously. Here, we apply Orbitrap-IRMS to simultaneously observe the 13C and 2H content of methylphosphonic acid (MPA), the hydrolysis product of DC and DF, which can be used as a proxy for the isotopic content of DC and DF. Our method requires 20 min analyses and consumes ≈60 nmol of sample, with precisions of ≈0.9 ‰ (13C) and ≈3.6 ‰ (2H). We apply our method to both commercially acquired MPA and MPA obtained from the hydrolysis of commercially acquired DC. We validate our methods via comparison to elemental-analyzer isotope ratio mass spectrometry (EA-IRMS). The combined 13C and 2H measurement creates a more robust forensic tool than either isotope individually. Our results demonstrate the viability of Orbitrap-IRMS for chemical forensic measurements.

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.

Elemental and stable isotopic signatures for dynamic traceability of genuine and "bathing" cultured Yangcheng Eriocheir sinensis crabs

This study examines whether the "bathing" culture of Chinese mitten crabs Eriocheir sinensis outside the Yangcheng Lake area converges with the origin characteristics of crabs from Yangcheng Lake and Kunshan Ponds. Inductively coupled plasma mass spectrometry (ICP-MS) and stable isotope ratio mass spectrometry (IRMS) were used to analyze mineral elements and stable isotopes in crabs at various stages of "bathing". The results indicated a dynamic transformation in the origin characteristics of "bathed" crabs; however, full convergence with genuine crabs was not achieved. Notably, disparities persisted between genuine crabs and "bathed" crabs. The combination of mineral elements and stable isotopes exhibited a discrimination accuracy of 95 % and 81.3 % between genuine crabs and "bathed crabs" from Kunshan Ponds and the Yangcheng Lake during each period of “bathing", respectively, which was higher than that using either method alone. This suggests limitations in altering original characteristics through "bathing". The combination of mineral elements and stable isotopes provides promising avenues for origin identification and understanding changes in crab characteristics during the "bathing" process. These findings highlight the complexities of crab culture practices and have implications for quality control and origin authentication in the crab industry.

Synthesis and Characterization of Metallic (Fe‐Ni, Fe‐Ni‐Si) Reference Materials for SIMS 34S/32S Measurements

Secondary ion mass spectrometry (SIMS) is often used to determine the sulfur contents and isotope ratios of metallic alloys in meteorites or high‐pressure experimental samples. However, SIMS analyses involve calibration and the determination of instrumental mass fractionation in reference materials with a matrix composition similar to that of the unknown samples. To provide metallic reference materials adapted to S measurements via SIMS, we synthesised a series of twenty‐eight alloys comprising four FeNi(±Si) compositions (Fe95Ni5, Fe90Ni10, Fe80Ni20, and Fe80Ni15Si5) with S contents varying from 100 μg g−1 to 4 g/100g using the “melt spinning” method, which guarantees that the metal alloys are rapidly quenched at ~ 106 K s−1. Sulfur contents were determined at the Service d'Analyse des Roches et Minéraux at the CRPG and absolute δ34S values were determined by multi‐collector ICP‐MS (MC‐ICP‐MS, ThermoScientific Neptune) and isotope ratio mass spectrometry (Thermoscientific Delta V). A δ34S value of 16.01 ± 0.31‰ was consistently obtained using the MC‐ICP‐MS, which was indistinguishable of the δ34S value of the FeS starting material (15.95 ± 0.08‰). It suggests that S did not undergo isotopic fractionation during the melting process. Of fifteen samples containing ≤ 5000 μg g−1 S, SIMS measurements with 15‐μm‐diameter spots were repeatable to within 10% relative (1 standard deviation, 1s) for S contents and 2‰ for δ34S values. However, samples containing > 5000 μg g−1 S showed FeNi–FeS immiscibility, leading to minor dispersion of the S mass fractions and δ34S values. No matrix effect was observed for Fe‐Ni, Si, or S contents in terms of the calibration curves and instrumental mass fractionation. We ultimately recommend eight samples as reliable reference materials for S isotopic measurements by SIMS, which we can share worldwide with other laboratories.

High-temperature liquid chromatography-isotope ratio mass spectrometry methodology for carbon isotope ratio determination of anabolic steroids in urine

BackgroundGas Chromatography Isotope Ratio Mass Spectrometry (GC-C-IRMS) has long been used in routine laboratories to determine the δ13C values of anabolic steroids in urine, differentiating between, e.g., endogenous and synthetic testosterone (T) in sports doping control. Until now, liquid chromatography (LC-IRMS) has not been used. The LC-IRMS setup doesn't allow organic solvents or modifiers in the mobile phase for δ13C determinations. Mid-to non-polar analytes such as steroids can be analysed in water heated to High Temperatures (HT, up to 200 °C) because at 200 °C has a similar polarity as 80/20 methanol/water at ambient temperature. In this work, we developed a method for steroids in urine, extending the application of the LC-IRMS to non-polar analytes in complex matrices. ResultAn HT-LC-IRMS method capable of determining the δ13C values of four steroids (i.e., testosterone (T), 5α-androstane-3α,17β-diol (ααβ), 5β-androstane-3α,17β-diol (βαβ) and pregnanetriol (PT)) in urine was developed and validated. Accuracy ranged from 0.23 ‰ (ααβ and βαβ) to 0.49 ‰ (T), and the detection limit was set at 10 ng mL−1 (T, ααβ+βαβ). The validation data and a comparison of authentic urine samples analysed with HT-LC-IRMS and GC-C-IRMS indicated a comparable performance between HT-LC-IRMS and GC-C-IRMS. SignificanceHT-LC-IRMS can be used to determine δ13C values of anabolic steroids, extending the applicability of both HT-LC and LC-IRMS to non-polar substances determined in a complex matrix in routine laboratory practice.

Position-Specific Oxygen Isotope Analysis in Inositol Phosphates by Using Electrospray Ionization-Quadrupole-Orbitrap Mass Spectrometry.

Conventional isotope-ratio mass spectrometry measurements obscure position-specific isotope distributions in molecular compounds because these measurements require an initial step that converts compounds into simple gases by combustion or pyrolysis. Here, we used electrospray ionization (ESI)-based Orbitrap mass spectrometry to measure oxygen isotope ratios in the phosphate and hydroxyl moieties of inositol phosphates. A thermal hydrolysis experiment was conducted using 18O-labeled water to examine the position-specific oxygen isotope exchange in inositol hexakisphosphate (IP6) as well as its hydrolysis products IP5, IP3, and PO3 fragments. Measurement precisions of the position-specific and molecular-average oxygen isotope values of inositol phosphates were better than ±1.1‰ and ±0.5‰, respectively. Under optimized ionization and Orbitrap parameters, this level of precision was obtained within 30 min of run time at 60 μM initial concentration of inositol phosphate. The ability to measure phosphate-specific oxygen isotopes in inositol phosphate enabled the quantification of isotope exchange, which did not occur in phosphate on IP6, IP5, IP3, and PO3 fragments, meaning that the change in isotopes should have resulted from hydroxyls in the ring. Isotope mass balance calculations corroborated that hydroxyl oxygens are derived from 18O-labeled water. With the observed sensitivity and precision achieved in this study, Orbitrap IRMS proved to be a promising tool for investigating the position-specific oxygen isotopes in organophosphorus compounds. These outcomes open up numerous potential applications that can expand our understanding of phosphorus cycling in the environment.

Probing Biological Nitrogen Fixation in Legumes Using Raman Spectroscopy.

Biological nitrogen fixation (BNF) by symbiotic bacteria plays a vital role in sustainable agriculture. However, current quantification methods are often expensive and impractical. This study explores the potential of Raman spectroscopy, a non-invasive technique, for rapid assessment of BNF activity in soybeans. Raman spectra were obtained from soybean plants grown with and without rhizobia bacteria to identify spectral signatures associated with BNF. δN15 isotope ratio mass spectrometry (IRMS) was used to determine actual BNF percentages. Partial least squares regression (PLSR) was employed to develop a model for BNF quantification based on Raman spectra. The model explained 80% of the variation in BNF activity. To enhance the model's specificity for BNF detection regardless of nitrogen availability, a subsequent elastic net (Enet) regularisation strategy was implemented. This approach provided insights into key wavenumbers and biochemicals associated with BNF in soybeans.

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

Fingerprinting Organofluorine Molecules via Position-Specific Isotope Analysis.

Organofluorine substances are found in a wide range of materials and solvents commonly used in industry and homes, as well as pharmaceuticals and pesticides. In the environment, organofluorine molecules are now recognized as an important class of anthropogenic pollutants. Fingerprinting organofluorine compounds via their carbon isotope ratios (13C/12C) is crucial for correlating molecules with their source. Here we apply a 19F nuclear magnetic resonance spectroscopy (NMR) technique to obtain the first position-specific carbon isotope ratios for a diverse set of organofluorine molecules. In contrast to traditional isotope ratio mass spectrometry, the 19F NMR method provides 13C/12C isotope ratios at each carbon position where a C-F bond is present, and does not require fragmentation or combustion to CO2, overcoming challenges posed by the robust C-F covalent bonds. The method was validated with 2,2,2-trifluoroethanol, and applied to analyze heptafluorobutanoic acid, 5-fluorouracil and fipronil. Results reveal distinct intramolecular carbon isotope distributions, enabling differentiation of chemically identical molecules. Notably, the NMR method accurately analyzes carbon isotopes within target molecules despite impurities. Potential applications include the detection of counterfeit products and drugs, and ultimately pollution tracking in the environment.