Gas chromatography-mass spectrometry (GC-MS) is a cornerstone technique for analyses of semi-volatile compounds, constantly evolving to meet new challenges. The adoption of Time-of-flight mass spectrometers (TOF MS) has enhanced GC-MS with faster spectral acquisition and broader mass coverage, enabling rapid GC separations and even faster two-dimensional GCxGC analyses. Multi-reflecting TOF MS (MRT) further improves resolution compared to traditional TOF MS, facilitating the detection of minor analytes within complex matrices. This paper presents a significant improvement in MRT sensitivity and dynamic range achieved through a strong ion beam compression within the ion guide and the use of a conventionally oriented orthogonal accelerator. Sensitivity is improved to 100,000 ion/pg at a resolution of R=25,000, enabling the detection of low fg analyte quantities within 3 μg of matrix at 10 ng/s matrix fluxes. The demonstrated linear dynamic range exceeding 6 orders of magnitude potentially enables even deeper exploration of complex mixtures, which is crucial for ultra-trace analyses and metabolomic studies.

not-yet-known not-yet-known not-yet-known unknown An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of three ephedrine psychoactive substances in water. By optimizing chromatography conditions, mass spectrometry parameters, and solid-phase extraction (SPE) conditions (including the SPE column, sample pH, washing, and elution), the optimal treatment conditions were determined. Poly-Sery MCX was selected as the extraction column, with samples loaded at pH 3. A 4 mL solution of 2% formic acid in water was used as the eluent, and a 5 mL solution of 5% ammonia in acetonitrile was used to elute the target compounds. The best results were obtained when the residue was redissolved in acetonitrile after nitrogen evaporation. The method exhibited a good linear relationship in the range of 0-50.00 μg/L, with determination coefficients (R 2) greater than 0.9990. The detection limit and quantitation limit were 0.05-0.10 μg/L and 0.20-0.50 μg/L, respectively. Recovery rates of the target compounds in blank sewage at three different concentration levels ranged from 92.37% to 106.31%, with relative standard deviations (RSD) of 0.77-4.83% (n=7). This method has been successfully applied to the analysis of surface water and domestic sewage, demonstrating stable treatment effects and practical significance for detecting ephedrine psychoactive substances in water.

Rationale: Helium (He) and energy shortages have dramatically increased prices and reduced their availability of these commodities. The use of three combustion reactions per acquisition of carbon and nitrogen isotope ratios saved 50% He and energy used in elemental analysis/isotope ratio mass spectrometry (EA/IRMS). This approach should be tested for sulfur isotope analyses. Mathods: A method was developed to measure sulfur isotope ratios ( d 34S values) in the SO 2 produced from three sequential combustion reactions in a single EA/IRMS acquisition. The combustion cycles involved capsules of the same or different materials. Two other developments are presented: a system for simultaneous extraction of chromium-reducible sulfur from four sediments or rock samples and the connections between the SO 2 reference gas and nitrogen cylinders for purging residual gases at the end of the EA/IRMS sequences. Results: The 3×EA/IRMS- d 34S method was validated with replicate analyses of international reference materials and laboratory standards with a wide range of mineralogical compositions and d 34S values. It was used for d 34S measurements of CRS-pyrites from Archean black shales and Swiss lake sediments. The accuracy and precision of the 3×EA/IRMS values were essentially matched those obtained by conventional EA/IRMS, with good agreement between the mean ± SD values and the recommended values and their Conclusions: Compared with the conventional EA/IRMS method. the proposed method provides accurate and precise sulfur isotope compositions of sulfate and sulfide samples while saving approximately 50% of the He, energy, SO 2 reference gas, O 2, and analysis time and cost. Notably, 3×EA/IRMS provided two d 34S values unaffected by potential memory effects.

Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine’s metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS). For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. The method was validated to meet the criteria for application in a bioanalytical laboratory. Injection of 2 μL was done in GC/MS, and SIM (selected ion monitoring) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) and were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and for COT 9.743 min. The duration of the GC-MS analysis was 20 minutes. The method showed a linear dynamic range from 0.5 to 50 ng/mL. Finally, the method was applied to the analysis of sixty clinical pediatric samples obtained from Aristotle University’s pediatric clinic to check for possible exposure to smoke.

Rationale: The Δ 36S standard deviation (SD) measured in a conventional isotope-ratio mass spectrometer (IRMS) such as MAT 253 is at c.a. 0.1‰ to 0.3‰. At this precision, it is difficult to resolve the origin of non-mass-dependent sulfur (NMD-S) isotope fractionation in the tropospheric sulfate aerosol and in Martian meteorites or small deviations from the canonical mass-dependent fractionation laws. Interfering ions (originated from fluorination and/or mass spectrometer ion source itself) with m/z at 131 of 36SF 5 + by the community as the cause of the poor precision, but the exact ion species has not been identified or confirmed. Methods: Here we examined the potential interfering ions by using the Thermo Scientific MAT 253 Ultra, a high-resolution (mass resolving powers up to 40,000) stable isotope-ratio mass spectrometer, to measure the SF 6 working gas and SF 6 gases converted from IAEA-S1 Ag 2S reference materials via a fluorination system. Results: We found that there is a resolvable peak to the right of the 36SF 5 + peak for both the SF 6 working gas and SF 6 generated by the fluorination system. The peak is identified as the 12C 3F 5 + ion, generated inside the instrument during the ionization process. By minimizing the presence of carbon-bearing compounds (e.g., organic matter in sample or helium gas, glue or o-ring in sample tubes, or carbon-bearing gases inside the mass spectrometer), we were able to achieve a Δ 36S SD of 0.046‰ (n=8) for SF 6 zero-enrichment and 0.069‰ (n=8) for overall measurement start from sliver sulfide IAEA-S1. Conclusions: Minimizing the presence of carbon-bearing compounds and avoiding the interfering signals from 36SF 5 + via MAT 253 Ultra high‐resolution isotope-ratio mass spectrometer, we can improve Δ 36S measurement precision by 2 to 5 folds, which helps to open new territories for research using quadruple sulfur isotope composition.

RATIONALE: Urine contains free and conjugated steroids. Total and free steroids were comprehensively quantified; UPLC-MS/MS based on Li adduction allowed for detecting thirteen 3-OH-containing steroids, two of which were detected in human urine for the first time. METHODS: Free urinary steroids were isolated by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. The free and total steroids were separately analyzed by UPLC-MS/MS with and without introduction of Li + solution. The steroids were quantified by two standard curves created using product ion transitions derived from MH + and [M+Li] +. RESULTS: Two groups of human urine, male and female urine, were analyzed. The absolute amount of each steroid was determined based on creatinine levels. The differences between the male and female groups are clearly attributable to sex steroids. 7-OH P5 and 7-OH DHEA were, for the first time, quantified in the total steroids of female urine, and the latter was identified in both female and male urine. CONCLUSIONS: By combining UPLC-MS/MS based on lithium ion incorporation with conventional UPLC-MS/MS, a total of 29 steroids were identified in human urine containing two newly found steroids.

Rationale: Increasing demand for fish and seafood means that traceability of marine products is becoming ever more important for consumers, producers and regulators. Highly complex and globalised supply networks create challenges for verifying the claimed catch region. Atlantic cod is one of the most commercially important species in the northeast Atlantic. Several regional fisheries supply cod into the trade network, of which some are at more risk of overexploitation than others. Tools allowing retrospective testing of spatial origin for traded cod products would significantly assist sustainable harvesting of wild fish, reducing incentives for illegal fishing and fraud. Methods: Here we investigate whether stable isotope ratios of carbon, nitrogen and sulfur in muscle tissue can be used to identify the catch region of Atlantic cod ( Gadus morhua). We measured the isotopic composition of muscle tissue from 377 cod from ten known catch regions across the Northeast Atlantic and Northeast Arctic, and then applied three different assignment methods to classify cod to their region of most likely origin. The assignment method developed was subsequently tested using independent known-origin samples. Results: Individual cod could be traced back to their true origin with an average assignment accuracy of 70-79% and over 90% accuracy for certain regions. Assignment success rates comparable to those using genetic techniques were achieved when the same origin regions were selected. However, assignment accuracy estimated from independent samples averaged c25% overall. Conclusion: Stable isotope techniques can provide effective tools to test for origin in Atlantic cod. However not all catch regions are isotopically distinct. Stable isotopes could be used in conjunction with genetic techniques to result in higher assignment accuracy than could be achieved using either method independently. Assignment potential can be estimated from reference datasets, but estimates of realistic assignment accuracy require independently collected data.

Rationale: Ion chromatography combined with inductively-coupled plasma mass spectrometry is an ideal tool for measuring low concentrations of anionic species such as phosphite; however, the high concentration of chloride and other anions in natural solutions may negatively impact chromatographic separation and data quality. Method: We developed an on-line mechanism of removing chloride from the sample within the ion chromatograph, using an additional valve and a separation column that transfers chloride to waste while phosphite and most other anions are retained. We installed this system in a coupled IC-ICPMS system (ICS6000 and Element 2 in medium resolution mode) and determined linearity and detection limits. In addition, we measured phosphorous species by NMR for comparison as an alternative method for phosphite determination. Results: Chloride was fully removed from the samples while phosphite was retained and could be analysed by IC-ICPMS. Concentrations could be measured down to 0.003 µmol/L and possibly less with good linearity over the explored range (up to 1.615 µmol/L; r 2 = 0.999). In contrast, the detection limit by NMR was 6.46 µmol/L. Conclusions: The on-line removal mechanism works well for simplifying sample matrices. It removes the need for costly pre-analytical sample treatment with OnGuard columns. We confirm that IC-ICPMS is the most powerful technique for quantifying phosphite in natural solutions. The new Cl-removal method may also be applicable to analyses of other anions.

RATIONALE: The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. MALDI-MS and MS/MS are useful for differentiating these isobaric species. METHODS: Four cross-linked peptides with thiosuccinimide linkers were synthesized. Analogs with the linker that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then generated by incubating the samples at neutral or basic pH. All of the cross-linked peptides were purified by rp-HPLC and differentiated by MALDI-MS, -MS/MS and UVPD. RESULTS: A cysteinyl peptide-containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI-MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product gave two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI-MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4-elimination: Cys-SH and maleimide, and dehydro-alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI-MS. The thiazine form gave fragment ions resulting from the cleavage of the newly formed amide bond in the linker that arose from thiazine formation. CONCLUSIONS: The thiosuccinimide (but not thiazine) form of the cross-linked peptide yielded individual constituent peptides in MALDI-MS; MALDI-MS/MS showing specific 1,4-elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclisation for the thiazine form.

RATIONALE: Many chemical processes operate at unsteady state and require a rapid mass analysis of transients with high resolution. An atmospheric pressure interface multi-reflection time-of-flight mass spectrometer (API-MRTOF-MS) has the potential to be a powerful process analytical tool. METHODS: The ion flight path of the API-MRTOF-MS was extended from meters to over one kilometer, and the mass resolution was increased to an ultra-high level. Furthermore, the mass analysis could be done at around ten milliseconds due to the rapidity of TOFMS. The mass analyzer reflects ions via electrostatic mirrors and directs them along a folded flight path, significantly increasing flight distances and theoretically with no upper limit. The powerful API, equipped with two quadrupoles, makes the API-MRTOF-MS suitable for complex mass spectrometry methods with alternative ion selection schemes. RESULTS: A mass resolution of 116,050 for Cs + ions is achieved within a total time-of-flight of only 18 ms. An ion transmission efficiency of over 50% was achieved after 600 cycles. CONCLUSIONS: The performance of the API-MRTOF-MS demonstrates that it is exceedingly suitable for high-resolution process analysis, particularly for transient process analysis, due to its fast analysis speed and high-selectivity.

RATIONALE: SLAP is one of the two calibration materials for the isotopic water scale. By consensus the established δ 18O value is -55.5‰, although there are firm indications that δ 18O SLAP is significantly more negative. The real δ 18O SLAP value as such does not influence the isotopic water scale, however knowledge of the size of isotopic scale contraction in stable isotope measurements is vital for second order isotopes. In this study quantification of δ 18O SLAP with respect to δ 18O VSMOW is described. METHODS: SLAP-like water was quantitatively mixed with highly 18O enriched water to mimic VSMOW. The 18O concentration was determined using an electron ionization quadrupole mass spectrometer. The isotopic composition of the SLAP-like and VSMOW-like waters were measured with an optical spectrometer, alongside real VSMOW and SLAP. RESULTS: This study resulted in a much more depleted δ 18O value for SLAP than expected. The averaged outcome of 7 independent experiments is δ 18O SLAP -56.33 ± 0.03‰. There is a large discrepancy between the actual isotopic measurements of even the most carefully operating groups and the true δ 18O value. CONCLUSIONS: Although this finding as such does not influence the use of the VSMOW-SLAP scale, it raises the intriguing question what we actually measure with our instruments, and why even a fully corrected measurement can be so far off. Our result has consequences for issues like the transfer of δ 18O from and to the VPDB scale, various fractionation factors, and the Δ 17O. The absolute 18O abundance for SLAP was determined at 1.88798 (43) x 10 -3 based on the absolute 18O abundance of VSMOW and the presented δ 18O SLAP in this paper.

The traditional van Krevelen diagram, the nominal oxidation of organic carbon, and several aromaticity indexes generally considered C, H, O, N, P, and S factors, or just C, H, and O. However, these evaluated methods may provide incorrect interpretation for high-resolution mass data of organics containing halogen elements. Therefore, we adapted the van Krevelen diagram and the other indexes with halogen elements and verified with 2235 model organics. The results demonstrated that the modified van Krevelen diagram and the adjusted indexes were more accurate for the analysis of mass data, either in the compound classification or in the evaluation of oxidation state of organic matter.

Rationale: Many insect species undertake multi-generational migrations in the Afro-tropical and Palearctic ranges, and understanding their migratory connectivity remains challenging due to their small size, short life span and large population sizes. Hydrogen isotope ( δ 2H) can be used to reconstruct the movement of dispersing or migrating insects, but applying δ 2H for provenance requires a robust isotope baseline map (i.e., isoscape) for the Afro-Palearctic. Methods: We analysed the δ 2H in the wings ( δ 2H wing) of 142 resident butterflies from 56 sites across the Afro-Palearctic. The δ 2H wing values were compared to the predicted local growing-season precipitation δ 2H values ( δ 2H GSP) using a linear regression model to develop an insect wing δ 2H isoscape. We used multivariate linear mixed models and high-resolution and time-specific remote sensing climate and environmental data to explore the controls of the residual δ 2H wing variability. Results: A strong linear relationship was found between δ 2H wing and δ 2H GSP values (r 2=0.53). The resulting isoscape showed strong patterns across the Palearctic but limited variation and high uncertainty for the Afro-tropics. Positive residuals of this relationship were correlated with dry conditions for the month preceding sampling whereas negative residuals were correlated with more wet days for the month preceding sampling. High intra-site δ 2H wing variance was associated with lower relative humidity for the month preceding sampling and higher elevation. Conclusion: The δ 2H wing isoscape is applicable to trace butterflies, moths and other terrestrial herbivorous insects that migrate across the Afro-Palearctic range but has limited geolocation potential in the Afro-tropics. The spatial analysis of uncertainty using high-resolution climatic data demonstrated that many African regions with highly variable evaporation rates and relative humidity have δ 2H wing values that are less related to δ 2H GSP values. Increasing geolocation precision will require new modeling approaches using more time-specific environmental data and/or independent geolocation tools.

Rationale Na v1.1, 1.2 and 1.6 are transmembrane proteins acting as voltage gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Methods Unique peptides for Na v1.1, Na v1.2 and Na v1.6 were selected as quantotropic peptides for each protein and used for their quantification in plasma membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry. Results Na v 1.1, 1.2 and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1±0.2, 6.4±1.2 and 4.0±0.6 fmole/µg membrane protein respectively. Na v1.2 showed the highest expression, with approximately 3 times higher (p<0.003) in rodents than in human at 3.05±0.57, 3.35±0.56 in mouse and rat brains and 1.09±0.27 fmole/µg in human, respectively. Both Na v1.1 and 1.6 expressions were much lower than Na v1.2, with approximately 40% less expression in human Na v1.1 when compared with rodents Na v1.1 at 0.49±0.1 (mouse), 0.43±0.3 (rat), and 0.28±0.04 (human); while Na v1.6 was approximately 60% less expression in human when compared to rodents at 0.27±0.09 (mouse), 0.26±0.06 (rat) and 0.11±0.02 (human) fmole/µg membrane proteins. Conclusions MRM was used to quantify sodium channels Na v1.1, 1.2 and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mouse, rat, and human. We found significant differences in the expression of these channels in mouse, rat, and human brains. Na v expression ranking among the three species was Na v1.2 >> Na v1.1> Na v1.6, with the human brain expressing much lower concentrations overall in comparison to rodents.

Rationale: Trimetazidine and its metabolites are prohibited substances in sports. With a growing number of adverse findings in human athletes, it is crucial to develop doping control strategies that include screening for trimetazidine in animal sports. This study aims to detect and characterize trimetazidine and its metabolites for doping control in camel racing. Methods: Camel urine and plasma samples were collected from four healthy animals following a single oral dose of trimetazidine. invitro studies utilized camel liver cells. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) techniques were employed for the extraction of trimetazidine metabolites from plasma and urine matrices, the metabolites were analyzed using a thermo Orbitrap exploris LCMS system with optimized settings to achieve maximum sensitivity and accurate mass measurements. Results: Comprehensive metabolite profiling of trimetazidine in camels revealed the identification of seven phase I and five phase II metabolites. Phase I metabolites were primarily formed through delakylation, while phase II metabolites were dominated by glucuronide conjugation of demethylated trimetazidine. The findings provided insights into the distinct metabolic pathways and biotransformation patterns of trimetazidine in camels under the experimental conditions. Conclusion: The developed method enables detection and characterization of trimetazidine and its metabolites in camels. The identified metabolites have the potential to serve as biomarkers for trimetazidine abuse in camel racing .this study provides valuable insights into the metabolism of trimetazidine in camels.

A document by Simone Moretti. Click on the document to view its contents.

13C-Breath testing is increasingly used in physiology and ecology research because of what it reveals about the different fuels that animals oxidize to meet their energetic demands. Here we review the practice of 13C-breath testing in humans and other animals and describe the impact that contamination that ambient/background CO 2 in the air can have on the accuracy of 13C-breath measurements. We briefly discuss physical methods to avoid sample contamination as well as the Keeling plot approach that researchers have been using for the past two decades to estimate δ 13C from breath samples mixed with ambient CO 2. Unfortunately, Keeling plots are not suited for 13C breath testing in common situations where 1) a subject’s VCO 2 is dynamic 2) ambient [CO 2] may change, 3) a subject is sensitive to hypercapnia, or 4) in any flow-through indirect calorimetry system. As such, we present a mathematical solution that addresses these issues by using information about the instantaneous [CO 2] and the δ 13CO 2 of ambient air as well as the diluted breath sample to back-calculate the δ 13CO 2 in the CO 2 exhaled by the animal. We validate this approach by titrating a sample of 13C-enriched gas into an air stream and demonstrate its ability to provide accurate values across a wide range of breath and air mixtures. Researchers can now instantaneously calculate the δ 13C of alveolar gas of humans or animals in real time without having to scrub ambient CO 2 or rely on estimated values.

A document by Chaohui Qiu. Click on the document to view its contents.