A document by Colette LaMonica Kelly. Click on the document to view its contents.

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.

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

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: 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: 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.

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: 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.

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

Rationale: IMS has been widely used for the on-site detection of explosives. Air sampling method is applicable only when the concentration of explosive vapor is considerably high in the air, but vapor pressures of common explosives such as TNT, RDX, and PETN are very low. A test method for analyzing the vapor detection efficiency of explosives with low vapor pressure via IMS was developed using artificial vapor and collection matrices. Methods: Artificial explosive vapor was produced by spraying an explosive solution in acetone. Fifteen collection matrices of various materials with woven or nonwoven structure were tested. Two arrangements of horizontal and vertical positions of the collection matrices were employed. Explosive vapor collected in the matrix was analyzed through IMS. Results: Only three collection matrices of stainless steel mesh (SSM), polytetrafluoroethylene sheet (PFS), and lens cleansing paper (LCP) showed the TNT and/or RDX ion peaks at explosive vapor concentration of 49 ng/L. There was no collection matrix to detect PETN vapor at lower than 49 ng/L. For the PFS, TNT and RDX were detected at 49 ng/L vapor concentration. For the LCP, TNT and RDX were detected at vapor concentrations of 14 and 49 ng/L, irrespectively. Conclusions: The difference in the explosive vapor detection efficiencies could be explained by the adsorption and desorption capabilities of the collection matrices. The proposed method can be used for evaluating the vapor detection efficiency of hazardous materials with low vapor pressure.

Extraction protocols and liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry methods (LC-MS/MS) for the measurement of five lipid categories, namely; glycerophospholipids, glycerolipids, sphingolipids, sterol lipids and fatty acyls in human plasma are described here. Step-by-step instructions are provided for the liquid/liquid extraction methods, including solution preparation and the non-endogenous lipid internal standards used. All instrumental conditions, chromatography columns and solutions required for the LC-MS and LC-MS/MS methods are also detailed.

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.

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.

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.

Within the framework of radioactive waste management, the evolution of the gaseous composition inside the underground repository structures must be understood. Indeed, chemical and microbial reactions and exchanges occur between the rock, the structures and the air. These processes are studied in the Andra’s Meuse/Haute-Marne Underground Research Laboratory’s (URL). Recently, a gas monitoring station “Flair soil™” has been designed to monitor the gaseous composition inside this URL. This station is composed by two analyzers: a quadrupole mass spectrometer (QMS) which allows to follow the evolution of several gases and an infrared laser spectroscope (Picarro) providing simultaneous measurements of CH4, CO2 and CO. Thus, a multivariate calibration method for the quantitative detection of interfering and non-interfering gases in a nitrogen matrix has been developed for the QMS. The MS was calibrated from pure gases in a nitrogen matrix, with known concentrations and ion currents obtained from the measurement of each species of gas. This method uses matrix calculations to calculate the relative concentrations of an unknown gas mixture from the ion currents measured directly in the MS. The test gases T1 and T2 were used to assess the accuracy of the method. Daily ion currents are corrected from theoretical ion currents obtained from calibration coefficients and test gas concentrations. Some gases are less well quantified due to their low concentration in the test gases and interferences on the measured masses. One of the motivations of this study lay in developing an advanced measurement tool allowing to be low in sensitivity, and thus to improve the detection and quantification of gases at low concentrations. The objective of this paper is to propose an analytical method to measure the gaseous composition with several molecules. This method was able to detect hydrocarbons, noble gases, sulfides, greenhouse gases, oxygen, hydrogen, nitrogen in the same mixture.

The researcher will introduce the way of effective liquid chromatography tandem mass spectrometric method (HPLC-MS) which is quick, sensitive, and alternative to determine the (E)-3-(3,4-dihydroxyphenyl) -2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A1) and rosmarinic acid (RA) of the plasma in rats. The analyses were divided into a C18 column (1.9 μm, 2.1 mm × 100 mm) with a security guard C18 column (5 μm, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry with an electrospray ionization (ESI) ion-source generates ions. With Pseudoephedrine hydrochloride being a standard, the sample pretreatment is relevant to the one-step protein precipitation with isopropanol: ethyl acetate (v/v, 20:80). This method presented a linear relationship within ranges of the concentration of 5–750 ng/ml for RA and A1. Relative standard deviations (RSD) in daily courses stood less than 15% and the relative errors (RE) registered within 15%. The means adopted in this research makes the RA’s and A1’s unambiguous quantification and identification and in vivo possible. And this study can be the first one to focus on determining the A1 and RA of rat plasma with administration orally. The results served as a significant basis to evaluate the medicine’s applications in the clinic.

Chlorinated aromatics and alkanes have important use value because of their flame retardancy，but they need to be monitored when used in recycled pulp. This paper report the use of palladium acetate/ Activated carbon activated by nitric acid as an online catalyst in the determination of chlorinated aromatics and chlorinated alkanes in recycled paper products with Gas Chromatography-Tandem Mass Spectrometry method, by which significantly improve the sensitivity of the method and dramatically lower the detection limits.