Sourcing in chemical forensic science refers to the attribution of a sample to a specific source using a characteristic signature. It relies on the identification of chemical attribution signatures (CAS), including chemical markers such as residual synthetic precursors, impurities, reaction by-products and degradation products, or even metabolites. Undertaking CAS for chemical threat agents (CTA) can be used to provide an evidentiary link between the use of a given chemical and its precursor(s) to support forensic investigations. Organophosphorus compounds, a class of nerve agents can be produced by different, more or less complex synthesis routes that can lead to specific CAS. Chlorpyrifos (CPF), an organophosphorus pesticide was selected as model compound. To assess the specificity of impurity markers originated from a chemical synthesis, untargeted fingerprints of crude CPF from different synthesis pathways were analyzed as a first use-case using metabolomics-based trace discovery strategies. Seven different CPF synthesis routes were considered and their crude mixtures were analyzed with a minimal sample preparation. Analyses were performed on a trapped ion mobility spectrometry (TIMS) coupled to liquid chromatography (LC) and high-resolution mass spectrometry (HRMS). Chemometrics analyses were conducted with multivariate methods to extract discriminating features (i.e. relevant impurities), annotate and identify them. Then, unknown samples were analyzed in blind conditions without any information of the synthesis pathway employed. The aim is to validate the methodology seeking some discriminating impurities identified in the first section to attribute and classify them according to the synthesis route.

Rationale: Aerodyne Tunable Infrared Laser Differential Absorption Spectrometer (TILDAS) allows for the measurement of carbonate-clumped (∆ 638) and stable oxygen isotope ratios (δ 628) without the mass interference of 17O, and understanding acid fractionation factors during phosphoric acid digestion is essential for inter-laboratory data comparison. Here, we present δ 628 and ∆ 638 ratios in CO 2 generated during phosphoric acid digestion of a reference carbonate at different temperatures. Methods: Carrara Marble (MAR-J1) calcite is digested with ⁓104% phosphoric acid using a) Break Seal method is a modified version of a McCrea-type reaction vessel where complete equilibration is achieved between product CO 2 and H 2O generated from the digestion and b) Individual Acid Bath (IAB) where the samples are digested with fresh aliquot of acid each time and the product CO 2 and H 2O simultaneously frozen in a U-trap connected to the reaction chamber by liquid N 2. Results: The regression equations for acid fractionation of δ 628 are: a) Break Seal Method: 1000 ln α (dig. temp- 25℃) = (0.532 ± 0.030) × 10 6/T 2 + (-6.185 ± 0.308) ; R 2 = 0.98. b) IAB: 1000 ln α (dig. temp- 25℃) = (0.359 ± 0.027) × 10 6/T 2 + (-4.114 ± 0.247) ; R 2 = 0.97. The regression equations for acid fractionation of ∆ 638 are: a) Break Seal Method: 1000 ln α (dig. temp - 25℃) = (0.0189 ± 0.0017) × 10 6/T 2 + (-0.2151 ± 0.0174) ; R 2 = 0.95. b) IAB: 1000 ln α (dig. temp - 25℃) = (0.0529 ± 0.0041) × 10 6/T 2 + (-0.6072 ± 0.0356) ; R 2=0.97. Conclusion: The shallow slope of the IAB method for δ 628 and the steeper slope for ∆ 638 indicate minimal resetting of the isotope composition of product CO 2 due to re-equilibration or interaction with free H 2O molecules. This observation enables inter-laboratory comparisons of δ 628 and ∆ 638 in calcite.

Rationale: The study aims to explore the degradation products of gimeracil via forced degradation analysis, necessitating the development of an analytical method for liquid chromatography mass spectrometry (LC-MS). Methods: Gimeracil was subjected to various stress conditions in accordance with International Conference on Harmonization guidelines, including acidic, alkaline, oxidative, photolytic, and thermal conditions. The analytical method was optimized to effectively separate gimeracil from its potential degradation products. Results: Under acidic, alkaline, photolytic, and thermal conditions, minimal degradation of gimeracil was observed. However, oxidative stress led to significant degradation, resulting in the identification of fourteen previously unreported degradation products. Structural elucidation of these products was achieved through orbitrap HRMS (High Resolution Mass Spectrometry) and HRMS/MS spectra analysis, with pathways for the formation of various daughter ions provided for some degradation products. Conclusion: Forced degradation analysis revealed the susceptibility of gimeracil to oxidative stress, leading to the generation of numerous degradation products. The developed LC-MS method proved effective in separating gimeracil from its degradation products, enabling comprehensive structural elucidation and identification.

Alkali metals have been used to degrade SF 6 in liquid ammonia. The products include metal fluorides. In this study, we reacted K − and Ag − with SF 6 in a triple quadrupole mass spectrometer. The atomic metal anions were formed by in-source collision-induced dissociation (CID) of their respective oxalate salts as previously described by our group. The only two reaction products observed were SF 6 − and SF 5 −. At low collision energy, the latter was deduced to be formed via an abstraction by the metal of F from SF 6 − formed by electron transfer in the encounter complex between the metal anion and neutral SF 6. As the collision energy was increased, there was evidence of a CID contribution to SF 5 − directly from SF 6 −.

The unimolecular reactions of protonated α- and β-pinene ions were explored with a combination of collision-induced dissociation tandem mass spectrometry and theory. The two main dissociation reactions in each ion lead to the losses of neutral propene and isobutene. The difference observed between the two ions is a greater relative abundance of propene loss in the case of protonated a-pinene. Both ions were found to dissociate over the same minimum energy reaction pathway, the only difference being the site of initial protonation in the two ions. a-Pinene preferentially protonates at the bridging carbon, while b-pinene can only significantly protonate at the exocyclic double bond. This leads to a lower appearance energy for loss of isobutene, and thus relatively greater m/z 81 fragment ion abundance for b-pinene.

The complexation of mercury (Hg) with dissolved organic matter (DOM) is a pivotal factor influencing transformations, transport, and bioavailability of Hg in aquatic environments. However, identifying these complexes poses a significant challenge because of their exceedingly low concentrations and presence of coexisting ions. In this study, mercury-dissolved organic matter (Hg-DOM) complexes were isolated through solid phase extraction (SPE) from Hg-humic acid suspensions and complexes were putatively identified using ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Dissolved organic carbon (DOC) and total Hg analysis before and after SPE showed an increase in DOC:Hg ratio. The DOC:Hg ratio was lower in extracts from cartridges with silica structure bonded with hydrocarbon chains (C18) than priority pollutant cartridges (PPL) cartridges at circumneutral pH, indicating that C18 was more effective at extracting DOM complexed Hg. These results were confirmed with FTICR-MS analysis, where two Hg-DOM complexes were putatively identified from PPL extracts as opposed to nine from C18 (Winnow score >75%). In addition, C 8H 13HgN 2O 2S, a molecular formula with a m/z ratio of 403.04, was identified across three separate extractions using a C18 cartridge, suggesting that the complexes were preserved during extraction and, presumably, electrospray ionization. These results highlight the effectiveness of the methodology developed in this study - SPE coupled with FTICR-MS for isolating and identifying Hg-DOM complexes. This approach allows for the exploration of the elemental and structural composition of Hg-DOM complexes, which affects Hg speciation, bioavailability, and transformations in aquatic ecosystems.

RATIONALE: Understanding plant defense mechanisms against pathogens is critical for agricultural productivity and crop protection. This study focuses on the quantification of camalexin and scopoletin, essential phytoalexins in Arabidopsis thaliana, using advanced mass spectrometry techniques. Accurate measurement of these compounds can provide insights into plant resistance and support agricultural research. METHODS: Camalexin and scopoletin were quantified using matrix-assisted laser desorption ionization high-resolution mass spectrometry (MALDI-HRMS). The matrix and solvent conditions were optimized to enhance sensitivity and accuracy. MS/MS experiments confirmed the identification with high mass accuracy (mass error < 5 ppm). RESULTS: The method demonstrated high linearity for scopoletin (R 2 = 0.9992) and camalexin (R 2 = 0.9987) over concentration ranges of 0.16-5 µM and 0.31-5 µM, respectively. The limits of detection (LOD) were 0.16 µM for camalexin and 0.04 µM for scopoletin, while the limits of quantification (LOQ) were 0.31 µM for camalexin and 0.16 µM for scopoletin. The average relative standard deviation was 1.43% for scopoletin and 2.46% for camalexin, with average relative errors of 3.91% and 4.11%, respectively. CONCLUSIONS: This study presents a precise, and accurate method for the quantification of key phytoalexins in Arabidopsis thaliana. The developed MALDI-HRMS approach significantly contributes to the understanding of plant defense mechanisms and offers potential applications in agricultural and biotechnological research.

Rationale: Sex estimation by analysis of amelogenin peptides in archaeological and fossil material has recently been gaining great traction within the fields of archaeology and palaeontology. Current widely used proteomic amelogenin sex estimation methods are hindered by relatively long mass spectrometric run times, or targeting peptides specific to human amelogenin proteins. Untargeted, high-throughput amelogenin sexing would be invaluable for a range of applications, from sex estimation of remains at mass grave sites to broadening the application of rapid amelogenin sexing to non-hominin species for husbandry and evolutionary studies. Methods: A new acid etch amelogenin analysis protocol followed by Evosep-LC-TIMS-TOF mass spectrometry is presented, providing global peptide data through rapid mass spectrometric methods in under 20 minutes per sample (including sample preparation, mass spectrometric acquisition and data processing). Furthermore, this method is applied to a preliminary study of both modern and archaeological Bos taurus remains, alongside archaeological human remains, showing the potential of straightforward application of this rapid amelogenin sexing method to a range of taxa. Results: Application of the developed Evosep-LC-TIMS-TOF mass spectrometry shows the novel acid etch approach improved peptide counts. Furthermore, rapid untargeted mass spectrometry using the Evosep-LC-TIMS-TOF gave comparable peptide counts to conventional long untargeted methods, while maintaining similar, or faster, acquisition times to those reported in methods targeting specific human amelogenin peptides. Conclusions: Rapid, untargeted Evosep-LC-TIMS-TOF mass spectrometry was successfully implemented in sex estimation of modern and archaeological material from Bos taurus and human teeth. This demonstrates an advancement in low-cost, high-throughput amelogenin sex estimation, for both human and zooarchaeological applications.

Rationale: These experiments were conducted to measure the diversity of organo-U(IV) and U(III) ions created using multidimensional tandem MS and collision-induced dissociation of halogen-substituted U VIO 2-phenide complexes [UO 2(C 6H 3FX)] +, X = Cl, Br, or I. Methods: Samples of UO 2(O 2C-C 6H 3FX) 2 were prepared by digesting UO 3 with appropriate halogen-substituted carboxylic acids in deionized water. Solutions for ESI were created by diluting the digested sample in 50:50 H 2O/CH 3OH. Precursor ions for multiple-stage tandem MS were generated by electrospray ionization (ESI). Multiple-stage collision-induced dissociation (He collision gas) in a linear quadrupole ion trap mass spectrometer was used to prepare species such as [U IVFX(C≡CH)] + and U IIIF(C≡CH)] + for subsequent study of ion-molecule reactions with adventitious neutrals in the ion trap. Results: Multiple-stage CID of the [UO 2(C 6H 3FX)] +, X= Cl, Br, or I, complexes caused removal of both “yl” oxo ligands from of the UO 2 2+ moiety to create ions such as [U IVFX(C≡CH)] + and [U IIIFX] +. For [U IVFXC≡CH] + and [U IIIFC≡CH] + products, hydrolysis to generate [U IVFX(OH)] + and [U IIIF(OH)] +, with concomitant loss of HC≡CH, was observed. When X = Cl and Br, elimination of HX was competitive with loss of HC≡CH. CID of [UO 2(C 6H 3FBr)] + and [UO 2(C 6H 3FI)] + caused reductive elimination of the respective halogen radicals to generate interesting organo-U(III) species such as [U IIIF(C≡CH)] + and [U IIIC 2] +. Conclusions: The use of “preparative” tandem mass spectrometry and a suite of halogen substituted benzoic acid ligands can be used to remove both oxo ligands of UO 2 2+ and generate a group of homologous organo-U(IV) and organo-U(III) ions for studies of intrinsic reactivity.

The analysis of natural abundance isotopes in biogenic N 2O molecules can give precious information such as the nature of their precursor. However, many uncertainties exist and further validations are necessary to confirm this method as a reliable tracer of biogeochemical cycles. In particular, current methodologies (such as the isotopocule map approach) can only estimate the combined contributions of several processes at once. In this study, we aimed to develop a new methodology capable of individually discriminating the main sources of N 2O production in soil by combining natural abundance isotopes with the use of a 15N tracer ( 15N Gas Flux method). To achieve this, we conducted parallel laboratory incubations of an agricultural soil, during which we optimized the denitrification conditions through increase of moisture and amendment of nitrate; where this nitrate was either labelled or unlabeled with 15N atoms. A new linear system combined with Monte Carlo simulation enabled the determination of N 2O source partitioning, where bacterial denitrification was identified as the dominant process (87.6%), compared to fungal denitrification (9.4%), nitrification (1.5%) and nitrifier denitrification (1.6%). This new system has been compared to a recently developed stable isotope modelling tool applying Bayesian statistics (FRAME). The results agreed generally well at the exception of lower bacterial denitrification (80%) and higher nitrifier-denitrification (9%) contributions found with the FRAME model. This new approach provides a perspective for a wider application, potentially enabling the source partitioning of nitrous oxide emissions in agroecosystems.

Rationale: Chemical pre-treatment is an important methodological step aimed to remove exogenous materials introduced to archaeological tooth enamel through various diagenetic pathways prior to carbon and oxygen isotope analysis. Concerningly, some of these pre-treatment methods (NaClO, H 2O 2 and CH 3COOH) have been shown to cause significant changes to enamel chemical properties and stable isotope values. This study aims to re-examine the effects of commonly used pre-treatment protocols applied to the enamel structure on bioapatite δ 13C and δ 18O values, as well as investigate patterns related to site burial context and sample preservation, as indicated by pre-screening using Fourier Transform Infrared (FTIR) spectroscopy. Methods: Modern and archaeological samples were subjected to ten commonly used pre-treatment protocols using NaClO, H 2O 2 and CH 3COOH at various time intervals. Preservation status and diagenetic alteration prior to and after treatment were investigated using Attenuated Total Reflectance- Fourier Transform Infrared Spectroscopy (ATR-FTIR). δ 13C and δ 18O values were measured before and after the treatment to determine if treatments induced isotopic shifts. Results: The results show that all pre-treatment protocols caused shifts in δ 13C and δ 18O values in the range of 0‰ to ±1.5‰ in both archaeological and modern samples. Most treated samples also shown increased crystallinity, likely indicating sample recrystallisation. We argue that these changes are indicative of more than just removal of contamination and diagenetic alterations, but also of dissolution and/or restructuring of the enamel carbonate leading to changes to the in vivo isotope signal. Conclusions: We recommend careful consideration of sample burial context and possible routes of diagenesis when choosing a pre-treatment protocol. Use of H 2O 2 and NaClO to remove organic matter from samples is discouraged as it incurs unwanted changes to the enamel chemical properties and isotope values. We also recommend use of only short acetic acid treatment protocols to avoid possible recrystallisation during prolonged contact.

Aim: Cupressus torulosa (family Cupressaceae), widely distributed in the north western Himalayan region of India, is a coniferous aromatic tree with various traditional uses of its aerial parts. Its needles have been used for anti-inflammatory, anticonvulsant, antimicrobial, and wound-healing properties. Antioxidants are pivotal in mitigating oxidative stress, a contributing factor to various diseases and aging processes. Employing a diverse array of analytical techniques, ranging from TPC, TFC to in vitro, in vivo assays and UPLC-QTOF-MS the research assesses the antioxidant potential of the Cupressus torulosa. Mаteriаls аnd methods: Employing а diverse аrrаy of аnаlyticаl techniques, rаnging from TPC, TFC to in vitro, in vivo аnd ex- vivo аssаys аnd UPLC-QTOF-MS the reseаrch аssesses the аntioxidаnt potentiаl of the plаnt. The initiаl screenings reveаl promising аntioxidаnt properties of the extrаct, with vаlues of 55.28± 57.07*,52.27± 19.50 in SGOT, SGPT (IU/L) аssаys respectively when compаred to positive control glibenclаmide. With subsequent frаctionаtion into butаnol аnd ethyl аcetаte frаctions. The ethyl аcetаte frаction exhibited pаrticulаrly notаble аctivity with lower vаlue of EC50 vаlue of 85.6 μg/mL in DPPH аssаy offering insights into the mechаnisms аnd effectiveness of these compounds in scаvenging free rаdicаls аnd mitigаting oxidаtive dаmаge. Results аnd Conclusion: Moreover, the chemicаl composition of аctive frаctions undergoes chаrаcterizаtion utilizing UHPLC-QTOF-MS, fаcilitаting the identificаtion of 34 metаbolites identified in negаtive mode with 10 key аntioxidаnt compounds аnd the elucidаtion of their frаgmentаtion pаtterns. These findings underscore the potentiаl of C. torulosа аs а bountiful reservoir of bioаctive аntioxidаnts, emphаsizing their significаnce in phаrmаceuticаl аnd nutrаceuticаl аpplicаtions. This reseаrch mаkes а substаntiаl contribution towаrds identifying potent аntioxidаnt compounds, emphаsizing the need for future efforts in isolаting аnd clinicаlly testing them. It enhаnces our understаnding of nаturаl аntioxidаnts аnd their criticаl role in enhаncing heаlth аnd аddressing oxidаtive-relаted conditions.

This study addresses three unmet needs: 1) development of widely accessible native mass spectrometry (MS) methods and 2) native top-down mass spectrometry (TDMS) methods for high flow sources on an Agilent Q-TOF mass spectrometer, and 3) confirming suitability of structures for lossless ion manipulation (SLIM) ion mobility (IM) MS methods for analysis of native intact proteins. To decrease the cost and training barrier to performing MS, TDMS, and IM-MS analysis of native proteins, we have developed methods for nanospray, microspray, and heated high flow electrospray ionization sources. SLIM offers the highest full-scan IM resolution available, but commercial instrumentation has not previously been applied to native MS. Our optimized methods extend the upper m/z limit beyond the manufacturer’s default IM mode upper mass limit of 4000 m/z to 7000 m/z, which allows for intact protein and native MS analysis of a variety of proteins, including native MS of intact IgG antibodies. Using the robust and facile Agilent Jet Spray (AJS) source, we demonstrate the application of native protein MS to amyotrophic lateral sclerosis (ALS)-associated Cu/Zn-superoxide dismutase proteoforms, characterizing a novel conformer and quaternary structural changes.

We describe a workshop which prompts chemistry students in the final two years of secondary school to apply their understanding of modern analytical chemistry techniques to a ‘real world’ example. The scenario used is that of a forensic science laboratory that has been asked to determine the structure of an illicit compound, Revisomed (methamphetamine) being sold as a revision aid, and seized by police. Over the course of an hour, the students use a combination of infrared (IR) spectroscopy, liquid chromatography (LC), high resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy data to determine the structure of Revisomed from first principles. The bulk of the workshop is spent reviewing MS data and using m/z, the isotope pattern, elemental composition and product ion data to reach three plausible isomeric structures for Revisomed which are then distinguished by NMR spectroscopy. More broadly, the workshop focusses on the use of the scientific method and the concept that ‘no ideas are bad’ when exploring hypotheses. We describe the structure of the workshop, and our experience delivering it to a local academy over the last 9 years.

This study validated an UPLC-MS/MS analysis method to simultaneously detect twelve cannabinoids. The concentration-response relationship for all analyzed cannabinoids was linear with R 2 values > 0.99 using the developed method, the relative standard deviations of recovery for the three levels of spiked samples are between 66.1%~104.1%. The method was used to analyze 43 industrial hemp flower and leaf samples, with the data being statistically analyzed. The analysis results showed that the cannabinoids content varied significantly among different varieties and different pretreatment methods, sample heating treatment led to the decarboxylation of acidic cannabinoids and the mutual conversion of cannabinoids. Based on the statistical analysis of the cannabinoids, hemp from different regions and different varieties were well distinguished by the PLS-DA model, with the main contributing substances being Cannabidiol, Δ 9-tetrahydrocannabinol, and Δ 8-tetrahydrocannabinol.

Rationale: Mass spectrometry (MS) is introduced to high school students in the UK in many pre-university course syllabi. As such, we have identified the use of MS as a key technique that should be taught practically to undergraduates from the outset of their studies. This mini‑review describes how we introduce and develop students’ use of MS throughout our three‑year undergraduate spiral curriculum practical program, using atmospheric pressure chemical ionisation MS (APCI/MS). Methods: We have used an Advion Expression L spectrometer, fitted with an atmospheric solids analysis probe or a Plate Express TLC sampler for sample introduction. Results: We have successfully demonstrated the use of APCI-MS in a range of practicals and experiments covering organic and organometallic chemistries, with large cohorts of students gaining hands‑on instrumental experience in authentic research settings. Conclusions: APCI-MS has proven to be an easy-to-use and valuable addition to our undergraduate practical course. The robustness of the spectrometer enables routine use by large cohorts of students with minimal supervision, and routine maintenance can be carried out by non-specialist technicians. Students can readily process and interpret results for a series of routine analyses, as well as demonstrate uses in problem-solving exercises.

RATIONALE: Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to yield glutamate (Glu) and N-acetyl-aspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess glutamate in a variety of cell and animal disease models. A robust high-throughput LC/MS/MS method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay. METHODS: A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2x30mm) columns. Each LC Channel was run independently, the cycle time was 2 min per channel. Overall throughput was 1-minute per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, batch metadata was automatically paired with raw data during the review process. RESULTS: Two LC sorbents, BEH-Amide and Penta- HILIC, were tested to separate the NAAG cleavage product Glu from isobaric interference and ion suppressants in the bioassay matrix. Early elution of NAAG and NAA on BEH-Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1 picomoles for Glu. The Z-factor of this assay averaged 0.85. Over 36,000 compounds were screened using this method. CONCLUSIONS: A fast gradient dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical screening assay samples was developed and validated. HILIC separation chemistry offers robust performance and unique selectivity for targeted positive mode quantification of Glu, NAA and NAAG.

Rationale:Pesticide isomers are widely available in agricultural production and may vary widely in biological activity, potency, and toxicity. Chromatographic and mass spectrometric analysis of pesticide isomers is challenging due to structural similarities. Methods:Based on liquid chromatography-time of flight mass spectrometry (LC-Q-TOF/MS), identification of cis-trans isomeric pesticides was achieved through retention time, characteristic fragment ions, and relative abundance ratio. The cleavage pathways of six cis-trans isomers were elucidated through collision-induced dissociation (CID) to explain the origins of different fragment ions. The energy-resolved mass spectrometry combined with computational chemical density functional theory in terms of kinetics, thermodynamics, and bond lengths, the specific process of fragments were simulated to explain the reasons for the differences in characteristic fragment ions and abundance ratios. Results:Based on the above study, a high-resolution mass spectrometry method was developed for the separation and analysis of cis-trans isomers of pesticides in traditional Chinese medicine Radix Codonopsis, and six pesticide isomers were distinguished by retention time, product ions, and relative abundance ratios. The limits of quantification of the six pesticides were up to 10 μg/kg, and the linear ranges of them were 10-200 μg/kg, with the coefficients of determination (R 2)＞0.99, which demonstrated the good linearity of the six pesticides. The recoveries of the pesticides at the spiked concentrations of 10 μg/kg, 20 μg/kg, and 100 μg/kg reached 70-120% with the RSDs ≤20%. Conclusions:It was demonstrated that the application of the method was well-suited for accurate qualitative and quantitative analysis for each isomers with different structures which could avoid false negative results caused by ignoring another isomers effectively.