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: 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.

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.

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: 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.

RATIONALE: In stable isotope mass spectrometry, isotope values are normalized to internationally recognized reference scales using certified reference materials and working standards. Numerous techniques exist for performing this normalization, but these methodologies need to be experimentally assessed to compare their impact on reproducibility of isotope results. METHODS: We tested normalization methods by the number of standards used, their matrix, their isotope range, and whether normalization required extrapolating beyond the isotope range. Using 8 certified reference materials and 5 working standards on a ThermoFinnigan Delta-V IRMS and Elementar VisION IRMS for nitrogen and carbon isotope composition via solid combustion with an elemental analyzer, we computed every possible isotope normalization (n=6272). Additionally, we assessed how sample matrix impacted linearity effects on both instruments. RESULTS: Normalizations composed of three or four reference materials had better performance than one-point and two-point methods, especially when the normalization was matrix-mixed or extrapolated, and normalizations with an isotope range greater than 15‰ were more accurate under these conditions. Normalizations that were matrix-matched and were not extrapolated exhibited the highest accuracy. Linearity effects were found to exceed instrument precision by two orders of magnitude irrespective of sample matrix and were not predicted by reference gas diagnostics. CONCLUSIONS: To maximize interlaboratory comparability of isotope results, operators of EAIRMS systems should use at least 3 calibration standards to construct their normalizations, select standards with a large isotope range to avoid extrapolation, and match the matrix of their standards to their samples to the best extent possible.

Rationale: Diaryliodonium salts are useful electrophilic reagents in organic chemistry, finding extensive applications in arylations and photo-induced polymerizations. However, the comprehensive mechanistic investigations, particularly concerning the mass spectrometric behaviors of diaryliodonium salts, are relatively scarce in the literatures. Methods: Diaryliodonium salts could be readily ionized in ESI-MS to give [Ar 1-I +-Ar 2], and the high-resolution ESI-MS/MS experiments were conducted to investigate their gas-phase chemical reactions. Results: Investigations on ESI-MS/MS of [Ar 1-I +-Ar 2] revealed two major fragmentation patterns: 1) Reductive elimination resulting the diaryl coupling product ion [Ar 1-Ar 2] +• by the loss of I. 2) Generating aryl cations [Ar 1] + or [Ar 2] + through cleavage of the C–I bonds. We unrevealed that the introduction of NO 2 into Ar 2 of [Ar 1-I +-Ar 2] could lead to an unexpected fragmentation ion [Ar 1O] + in MS/MS, arising from an O-atom transfer process from NO 2 to Ar 1. Particularly, when NO 2 was ortho-positioned to the iodine in Ar 2, the [Ar 1O] + sometimes exhibited dominant behavior. Conclusions: Comprehensive ESI-MS/MS studies and theoretical calculations provided strong support for the O-atom transfer mechanistic pathway: [Ar 1-I +-( o-NO 2-Ar 2)] initially underwent a Smiles rearrangement to the intermediate [Ar 1-O-( o-NO-Ar 2I)] +, which subsequently dissociated to [Ar 1O] + or [ o-NO-Ar 2I] +•. Herein, we proposed an unexpected ” ortho-effect” in the gas-phase fragmentation reaction of [Ar 1-I +-( o-NO 2-Ar 2)], in which the crucial determinant factor for the aryl migration was identified as the Smiles rearrangement reaction.

Rationale: A novel laser ablation-isotope ratio mass spectrometry (LA-IRMS) method for in situ sulfur isotope analysis of sulfides has been developed. Instead of the in situ reaction applied by the traditional laser microprobe, the analyte gas preparation in this method is separated temporally and spatially from the laser ablation, resulting in improved precision and accuracy. Methods: Our LA-IRMS system combines an ultraviolet (UV) LA system, an elemental analyzer (EA), a custom-built cryogenic concentration system, a continuous-flow interface and an IRMS. The sulfide aerosol particles generated from LA were transferred by a helium carrier gas from the ablation cell into the reaction tube and were converted into SO 2. Then SO 2 was enriched in two cold traps and was finally introduced into the ion source of an IRMS through a continuous-flow interface. Results: We measured three synthetic and four natural sulfide reference materials to test the performance of this method. Precisions of ±0.25-±0.64‰ (1SD, n=5) for δ 34S values can be obtained at a spot size of 64~80 μm. Measured values and their known true values for these sulfur isotope reference materials showed good linear relationship (R 2=0.998~0.9995) with slope of approaching unity (1.0509~1.1313). Conclusions: Data from the measurement of reference materials showed that the precision and accuracy of our method was satisfactory. This method is a powerful tool for in situ sulfur isotope measurement of sulfides, and can be further applied to in situ carbon and oxygen isotope analyses.

Advances in sulfur isotope measurement techniques have led to increased analytical precision. However, accurate measurement of 36S remains a challenge, hindering research such as that focused on microbial metabolic processes. This difficulty arises partly from isobaric interferences of 36SF 5 + at m/z = 131 amu, namely 186WF 4 2+ and 12C 3F 5 + which lead to scale compression. Our study develops an interference-free four-sulfur isotope measurement method using the high-resolution mass spectrometer Panorama. Panorama’s peak scan showed that the relative intensity of 186WF 4 2+ was initially 9.4% of 36SF 5 + but was reduced to 1.5% through tuning, while the 12C 3F 5 + relative intensity dropped from 74% to 40% after flushing with air and continued to decrease over time. Theoretically, an unresolved isobaric interference with 2% relative intensity could cause a 1‰ underestimation in a sample with a real δ 36S value of +60‰. We analyzed three IAEA sulfur isotope standards. Recommended 36S values we obtained relative to IAEA-S-1 are Δ 36S IAEA-S-2 = 1.238 ± 0.040‰, and Δ 36S IAEA-S-3 = -0.882 ± 0.030‰. For cases where the interferences cannot be removed, we offer a calibration method to correct for the scale compression effect. This involves bracketing two or more IAEA standards in the sample measurement sequence and then extending the measured IAEA δ 36S values by a calibration factor to match the values reported in this study. Applying this calibration factor to the samples effectively corrects the scale compression effect.

RATIONALE: A general N-glycoproteomics analysis pipeline has been established for characterization of mutation-related gain-of-glycosylation (GoG) at intact N-glycopeptide molecular level, generating comprehensive site and structure information of N-glycosylation. METHODS: This study focus on mutation-originated gain-of-glycosylation using mass spectrometry-based N-glycoproteomics analysis workflow. In brief, GoG intact N-glycopeptide databases were built, consisting of 2,701 proteins (potential GoG N-glycosites and amino acids derived from MUTAGEN, VARIANT and VAR_SEQ in UniProt) and 6,709 human N-glycans (≤50 sequence isomers per monosaccharide composition). We employed the site- and structure-specific N-glycoproteomics workflow utilizing intact N-glycopeptides search engine GPSeeker to identify GoG intact N-glycopeptides from parental breast cancer stem cells (MCF-7 CSCs) and adriamycin-resistant breast cancer stem cells (MCF-7/ADR CSCs). RESULTS: With the criteria of spectrum-level FDR control of ≤1%, we identified 88 and 96 GoG intact N-glycopeptides corresponding to 38 and 36 intact N-glycoproteins from MCF-7 CSCs and MCF-7/ADR CSCs, respectively. Among KEGG annotation of GoG N-glycoproteins, DNA polymerase eta (POLH), serine-protein kinase ATM (ATM) and cellular tumor antigen p53 (P53) were enriched in platinum drug resistance signal pathway. ATM, P53 and G2 and S phase-expressed protein 1 (GTSE1) were associated with p53 signaling pathway. CONCLUSIONS: The integration of site- and structure-specific N-glycoproteomics approach, conjugating with GoG characterization, provides a universal workflow for revealing comprehensive N-glycosite and N-glycan structure information of gain-of-glycosylation. The analysis of mutation-originated gain-of glycosylation can be extended GoG characterization to all the other N-glycoproteome systems including complex clinical tissues and body fluids.

RATIONALE: Glycol-based antifreeze liquids, commonly composed of ethylene glycol or propylene glycol, have important uses in automotive cooling, but they should be handled with care due to their toxicity, ethylene glycol is highly toxic to humans and animals. A fast, accurate, precise and robust method was developed for simultaneous quantification of 7 most important glycols and their isomers. METHODS: Glycols were analyzed from diluted sample solution of coolants using gas-chromatography coupled with mass spectrometry in single ion monitoring mode. RESULTS: The method was developed and validated for 7 individual glycols (ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, propylene glycol, dipropylene glycol and tripropylene glycol). Limits of detection (1-2 µg/mL) and limit of quantification (10 µg/mL) obtained were appropriate. The present method was applied for determination of glycols in 10 different anti-freeze liquids commercially available on Romanian market, proving to be reliable. CONCLUSIONS: A method that requires only a two-step dilution of anti-freeze samples combined with direct liquid injection GC-MS was validated for the simultaneous quantification of 7 glycols (and their isomers) in 10 different types of anti-freeze liquids. The results obtained in the validation procedure proved that the GC-MS method is sensitive and precise for quantification of glycols.

Rationale. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein’s conformational dynamics across varied states, such as heat-denatured vs. native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions, if high-energy collision-induced dissociation (HCD) MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans then electron transfer low-energy collision-induced dissociation (EThcD) MS/MS scans of the precursor identified the modified asparagine residue and the glycan’s mass. As in traditional HDX-MS the identified glycopeptides were then analyzed at the MS level in samples labeled with D 2O. Results. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Conclusion. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

RATIONALE: Helium (He) is used in cutting-edge research in industry and science as a carrier and/or ionization gas. The global He shortages have dramatically increased its prices and reduced its availability, to what comes together the current energy crisis. A strategy is proposed to save He and energy in elemental analysis/isotope ratio mass spectrometry (EA/IRMS). METHODS: A method for analysis of carbon and nitrogen isotope composition (δ13C and δ15N values) is proposed using two or three sequential combustion reactions in a single EA/IRMS acquisition. The methods (hereafter called 2×EA/IRMS and 3×EA/IRMS) use the time needed for the peak center and reference gas (CO2 or N2) pulses to measure δ13C or δ15N in two or three capsules with samples or standards. The combustion cycles can be replicate analyses of the same or different material. RESULTS: The methods were validated with replicate analysis of C and N in RMs and laboratory standards of a broad range of biological and geological matrices, δ13C and δ15N values, and C/N molar ratios. The accuracy and precision of the 2×EA/IRMS and 3×EA/IRMS values were15 essentially the same, with good agreement between the mean ± 1 SD values from RMs and standard analyses and the recommended or accepted values and their uncertainties. CONCLUSIONS: The proposed methods save He, save energy, save reference gases (CO2, N2), and O2, while reducing the analysis and instrumental times by ca. 50 %.

RATIONALE: The multi-reflection time-of-flight mass spectrograph (MRTOF-MS) is a complex nonlinear system with dozens of variables that are impossible to determine in theory. Numerical analysis is the only method to determine a solution. Therefore, a numerical simulation is applied with a modified Nelder–Mead simplex (MNMS) algorithm for optimizing voltage configurations. METHODS: Ion trajectories for injection and confinement are simulated using the software SIMION 8.1. The goal of optimization is to find a more suitable configuration for the electric field. This task becomes more challenging as the number of variables, the complexity of the objective function, and the accuracy of the variable intervals increase. A simplex search algorithm was used to perform the optimization process. We modified the searching algorithm by incorporating a variable transformation to ensure that the variables have smooth boundaries. Additionally, we introduced a dedicated benchmark to facilitate global searches. RESULTS: By iteratively using the MNMS algorithm, a total of eight electrodes have been optimized, resulting in a smaller beam size and more efficient ion transport. CONCLUSIONS: The MNMS algorithm is effectively for optimizing nonlinear MRTOF-MS system. It improves the adaptability and globality of the original algorithm, making it applicable for the numerical analysis of complex mass spectrometry systems and problems in engineering.

Rationale: Impurities refer to any substances that affect the purity of pharmaceuticals. Controlling impurities has always been a significant concern during drug development. Impurities impact the drug’s purity, diminish the efficacy of pharmaceuticals, and alter their appearance, physical, and chemical properties. Additionally, impurities can compromise the stability of pharmaceuticals and elevate their toxic and side effects. Methods: Impurity source analysis is the basis of drug impurity control. To clarify the source of impurity can optimize the synthesis process, prescription process, packaging and storage conditions of the pharmaceuticals, and control the impurity within a reasonable limit to achieve the ultimate goal of impurity research. Results: Analysis method is a means to obtain impurity information, and diversified analysis methods are also possible for effective control of different types of impurities. At present, there are relatively many quantitative studies on impurities, but there are still some challenges for the structure analysis of impurities, especially trace impurities. Conclusion: The research progress of drug impurity control and evaluation from the sources of various drug impurities and impurity analysis techniques was reviewed in this article, with the aim of providing references for the related research.

Stable isotopes of the water molecule have emerged as powerful tracers of the sources and trajectories of water leading to precipitation, at different spatial and temporal scales. However, the high cost of commercially available rain samplers for isotopic analysis, have made using them for high spatial resolution networks and for studies being conducted in developing countries prohibitively expensive. We have designed a low-cost, simple, and robust rain sampler capable of sampling precipitation for isotopic analysis on the event and monthly scale, based on the existing designs provided in the literature. The event rain samplers were tested to determine the minimum amount of rainfall to minimize isotopic fractionation, both from post-sampling evaporation and equilibration. These new rain samplers will enable isotopic sampling of precipitation at high spatial resolutions. All the instructions for constructing and using these samplers are made openly accessible to the scientific community so they can easily be repeated and adapted to the needs of each project. This open access and low-cost methodology will help democratize the use of isotopes for hydrological studies in developing countries.

Rationale: The structures of organometallic complexes determine their stable functioning in product performance. Electrospray ionization-mass spectrometry (ESI-MS) is used in studying metal complexes, but it exhibits limitations in analyzing labile organometallics. Therefore, identifying a method of detecting unstable organometallics and evaluating their stabilities is necessary, providing a theoretical basis for material selection and performance evaluation. Methods: The standard complexes Zn(BTZ) 2, Fe(acac) 3, and Sn(Oct) 2 were analyzed using nanoelectrospray ionization-quadrupole orbitrap MS (nanoESI-MS) and compared with ESI-MS. Alkylamine-Ag + complexes were analyzed using nanoESI and collision-induced dissociation MS/MS (CID-MS/MS). Breakdown plots of the ion relative abundances against collision energies expressed in terms of the center-of-mass were constructed according to the obtained product ion spectra. Quantum chemical calculations based on density functional theory were performed to calculate the binding energies between the alkylamines and Ag +. Results: The molecular ions of the three standard complexes were only detected using nanoESI-MS, which confirmed the suitability of soft nanoESI for use in detecting unstable organometallics. The gas-phase stabilities of the amine-Ag + complexes, as estimated using the breakdown plots constructed by plotting the data obtained via nanoESI and CID-MS/MS, were consistent with the established theories, previous studies, and binding energies calculated using computational methods. Conclusions: NanoESI-MS is suitable for detecting labile organometallics and enables the structural analyses of unknown organometallic additives. A novel approach based on nanoESI and CID-MS/MS was developed to determine the gas-phase stabilities of organometallics, enabling their quantification and comparison and providing a technical basis for product improvement, which is essential in developing industrial materials.