Mass Spectrometry Measurements Research Articles

Probing the Intramolecular Folding of Nucleic Acids with Native Ion Mobility Mass Spectrometry: Strategies and Caveats.

The goal of native mass spectrometry is to obtain information on noncovalent interactions in solution through mass spectrometry measurements in the gas phase. Characterizing intramolecular folding requires using structural probing techniques such as ion mobility spectrometry. However, inferring solution structures of nucleic acids is difficult because the low-charge state ions produced from aqueous solutions at physiological ionic strength get compacted during electrospray. Here we explored whether native supercharging could produce higher charge states that would better reflect solution folding, and whether the voltage required for collision-induced unfolding (CIU) could reflect preserved intramolecular hydrogen bonds. We studied pH-responsive i-motif structures with different loops, and unstructured controls. We also implemented a multivariate curve resolution procedure to extract physically meaningful pure components from the CIU data and reconstruct unfolding curves. We found that the relative unfolding voltages reflect to some extent, but not always unambiguously, the number of intramolecular hydrogen bonds that were present in solution. Reaching phosphate charging densities over 0.25 makes it easier to discriminate between structures, and the use of native supercharging agents is thus essential. We also uncovered several caveats in data interpretation: (1) when different structures (for example the i-motif with and without hairpin) unfold via different pathways, the unfolding voltages do not necessarily reflect the number of hydrogen bonds, (2) unstructured controls also undergo unfolding, and the base composition influences the unfolding voltage, (3) changing the solution pH also unexpectedly changed the unfolding voltage, and (4) the ion mobility patterns become more complicated when two structures are present simultaneously, such as an i-motif and a harpin, because of opposite effects on the collision cross section upon activation.

Electron‐Induced Radiolysis of Water Ice and the Buildup of Oxygen

AbstractIrradiation by energetic ions, electrons, and UV photons induces sputtering and chemical processes (radiolysis) in the surfaces of icy moons, comets, and icy grains. Laboratory experiments, both of ideal surfaces and of more complex and realistic analog samples, are crucial to understand the interaction of surfaces of icy moons and comets with their space environment. This study shows the first results of mass spectrometry measurements from porous water ice regolith samples irradiated with electrons as a representative analogy to water‐ice rich surfaces in the solar system. Previous studies have shown that most electron‐induced O radiolysis products leave the ice as and and that can be trapped under certain conditions in the irradiated ice. Our new laboratory experiments confirm these findings. Moreover, they quantify residence times and saturation levels of in originally pure water ice. O may also be released from the water ice by irradiation, but the quantification of the released O is more difficult and the total amount is sensitive to the electron flux and energy.

The effects of sample preparation on the interpretation of pyrolysis-based organic matter analysis in immature oil shale

Oil shale as well as shale oil and shale gas are significant energy resources with huge reserves present in different parts of the world. Various geochemical proxies have been applied to assess the petroleum potential of oil shales with samples pre-treated in various ways, e.g. as whole rock or demineralized sample or as solvent extracted rock/kerogen. In this respect, it is important to understand and quantify, how achieved geochemical parameters are influenced by pre-treatment. In this study, a systematic comparison is presented based on a study on i) whole rock, ii) extracted whole rock, iii) kerogen concentrate, and iv) extracted kerogen concentrate obtained after solvent extraction of demineralized shales. In total, seven immature, organic matter-rich samples from the Miocene lacustrine sediments of the Nördlinger Ries impact crater, Germany, were pretreated in this way leading to overall 28 samples. A set of elemental analysis (C, H, N), Rock-Eval pyrolysis, and Curie Point-pyrolysis–gas chromatography–mass spectrometry measurements were performed on these pretreated samples. Mineral matter removal leads to significant increase of total organic carbon, but also thermally evaporable and pyrolytically cracked organic matter (Rock-Eval S1 and S2 peaks). To some extent, labile organic matter represented in the original S2 peak can be destructed by mineral removal with hydrochloric and hydrofluoric acid, as shown by elevated values of PI [S1/(S1 + S2)] after demineralization. The organic matter type tends to be more petroleum-prone with raised hydrogen index (HI) and aliphaticity values after demineralization, while Rock-Eval Tmax values commonly applied as parameters for thermal maturity tend to decrease, though not for all samples.Reduced TOC, S1, S2 and PI values of extracted samples suggest that both thermally evaporable and additionally some non-evaporable but soluble organic matter hidden in the original S2 peak can be lost after solvent extraction. Increased values of HI, H/C and N/C of extracted samples indicate more oil-prone organic matter types than unextracted samples. Typical maturity-related parameters such as aliphaticity and Rock-Eval Tmax decrease. Smectite and zeolites, which are abundant in the samples, are suggested to protect organic matter to some extent against solvent extraction, influencing a variety of geochemical proxies and the occurrence of metal cation-containing minerals. Zeolite invigorates the protection effect.

Synthesis, characterization, and bioactivity of Cu(II), Fe(II), Co(II), Ni(II), and Mn(II) complexes with benzilmonoximethiocarbohydrazide-O-methoxybenzaldehyde: experimental and computational insights.

In this study, a novel ligand, benzilmonoximethiocarbohydrazide-O-methoxybenzaldehyde (HBMToMB), was synthesized and subsequently complexed with Cu(II), Fe(II), Co(II), Ni(II), and Mn(II) ions. The metal complexes were comprehensively characterized using techniques such as NMR, IR, Mass Spectrometry, UV-Vis, elemental analysis (CHNS), and magnetic susceptibility measurements. The complexes exhibited superior antibacterial and antifungal activity compared to the free ligand. In addition, cytotoxicity was evaluated using the brine shrimp lethality bioassay, demonstrating significant activity. Computational studies, including molecular docking, DFT, and ADMET analysis, provided further insights into the compounds' binding affinities and electronic properties. These findings underscore the potential of these metal complexes as promising candidates for therapeutic applications, particularly in antimicrobial and anticancer therapies.

Hydrophilic Modification of Macroscopically Hydrophobic Mineral Talc and Its Specific Application in Flotation.

Sodium diethyldithiocarbamate (DDTC), a common collector used to enhance the hydrophobicity of minerals in froth flotation, nevertheless weakens the hydrophobicity of the talc surface. To rationalize this anomaly, the interactions of a hydrophobic alkyl group and hydrophilic mineralophilic group (-NCS2-) of heteropolar surfactant DDTC, and a water molecule with the talc (001) surface, were investigated. Herein, DFT simulations found that the talc (001) surface features natural hydrophobicity determined by the competition between adhesion (surface water) and cohesion (water-water interactions). The interaction of the hydrophobic alkyl group of DDTC with the talc surface is more favorable compared to that of the -NCS2- group and H2O, favoring the hydrophilic modification of the talc surface. Additionally, adsorption isotherms, time-of-flight secondary ion mass spectrometry (ToF-SIMS), microflotation tests, and contact angle measurements also indicate that the differences in adsorption orientation of the heteropolar surfactant DDTC on the talc surface enhance the hydrophilicity of the talc surface, leading to a decreased recovery of the talc. This study provides crucial surface chemistry evidence for the selective adsorption of heteropolar surfactants and contributes to the understanding of the mechanism for the efficient flotation separation of molybdenite from talc.

An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS).

The crucial role of steroid hormones in health and diseases merits their high-throughput, accurate and affordable measurements in biological specimens. Despite advances in analytical methods, sensing and quantifying steroid hormones remains challenging. Immunoassays offer excellent sensitivity but are inherently labour-intensive, costly, and prone to false positives. Mass spectrometry (MS) has been increasingly utilised, with the main hurdle being the isobaric tendencies of similar analytes, which complicates their separation and accurate quantification. This study compares ultrahigh-performance supercritical fluid chromatography separation (UHPSFC) and ultra-high-performance liquid chromatography (UHPLC) for MS detection. It optimises the column chemistry, temperature, and pressure to provide an operational protocol for the resolution and quantification of analytes. It presents the systematic characterisation of UHPSFC-MS performance by investigating spiked blood samples using Solid-Phase Extraction (SPE) and describes the matrix effects associated with MS measurements. Although both separation methods showed adequate resolution, specificity, and retention time, UHPSFC-MS was superior for five out of seven columns tested. With added high-throughput capacities, UHPSFC-MS, thus, offers an optimal solution for the analysis of steroid hormones for research, medical chemistry, and clinical diagnostics.

Assessment of acupuncture's effectiveness in treating gulf war illness using laser induced breakdown spectroscopy and inductively coupled plasma mass spectrometry

In an effort to contribute to define whether Gulf War Illness (GWI) has physiological aspects, we evaluated the effectiveness of acupuncture treatment by measuring blood plasma metal levels and comparing them to the McGill pain scores of 22 patients. We analyzed the blood plasma samples of 22 GWI patients who underwent 2 acupuncture treatments per week. We analyzed the blood metal levels of these patients at baseline and after 2 and 4 months of treatment with acupuncture using laser-induced breakdown spectroscopy (LIBS). Principal component analysis (PCA) loadings of the LIBS data revealed that emission lines of sodium, potassium, magnesium, and calcium were key features differentiating the two types of blood plasma samples. We also conducted inductively coupled plasma mass spectrometry (ICP-MS) measurements on the same samples and compared the findings to the McGill pain scores of the patients. Levels of sodium, potassium, magnesium, phosphorus, iron, copper, and zinc for all 22 patients were measured using ICP-MS. We observed after 2 and 4 months of acupuncture treatment, the Pearson correlation coefficient of the McGill pain score and the concentrations of phosphorus (P), and zinc (Zn), exhibit a moderate positive correlation with changes in the McGill pain score, with Pearson correlation coefficients of 47 % (p = 0.026) for the 0–2-month period, 45 % (p = 0.038) for the 0–4-month period, and 42 % (p = 0.05) for the 0–2-month period, respectively. Sodium (Na) exhibits a nearly moderate correlation, with a Pearson coefficient of 39 % and a p-value of 0.07. Our result suggests a potential link between element concentrations, acupuncture treatment and pain response.

Dendritic Molecular Baskets for Selective Binding of Toxic Methotrexate.

We describe the preparation, assembly, recognition characteristics, and bioactivity of dendritic basket 612-. This novel cavitand has a deep aromatic pocket with three (S)-glutamic acid dendrons at the rim to amplify water solubility and prevent self-association. 1H NMR spectroscopy, calorimetry (ITC), and mass spectrometry (ESI-MS) measurements validate the formation of an inclusion complex between 612- and anticancer drug methotrexate (MTX2-) in water (Kd = 9.2 μM). To identify the docking pose, a comparison of computed (DFT and MM) and experimental 1H NMR chemical shifts suggests that MTX2- folds inside 612- (π···π), forming HBs with the peptidic dendrons while anchoring (C-H···π) to the aromatic pocket through its N-methyl group. In consequence, 612- selectively binds MTX2- in competition with structurally similar folic acid and leucovorin (reversal poisoning agent). While the host is biocompatible (HEK293; IC50 > 150 μM) and produces inclusion complex [MTX⊂6]14- in cell media, it experiences limitation in pharmacokinetic sequestration of MTX2- as dihydrofolate reductase's affinity to the drug is suggested to prevail over that of 612-. Nonetheless, considering the basket's biocompatibility, tunability, and chemoselectivity, it stands as the leading candidate in the pursuit of an effective abiotic antidote for methotrexate poisoning.

Dendritic Molecular Baskets for Selective Binding of Toxic Methotrexate

We describe the preparation, assembly, recognition characteristics, and bioactivity of dendritic basket 612‐. This novel cavitand has a deep aromatic pocket with three (S)‐glutamic acid dendrons at the rim to amplify water solubility and prevent self‐association. 1H NMR spectroscopy, calorimetry (ITC), and mass spectrometry (ESI‐MS) measurements validate the formation of an inclusion complex between 612‐ and anticancer drug methotrexate (MTX2‐) in water (Kd = 9.2 μM). To identify the docking pose, a comparison of computed (DFT and MM) and experimental 1H NMR chemical shifts suggests that MTX2‐ folds inside 612‐ (π···π), forming HBs with the peptidic dendrons while anchoring (C−H···π) to the aromatic pocket through its N‐methyl group. In consequence, 612‐ selectively binds MTX2‐ in competition with structurally similar folic acid and leucovorin (reversal poisoning agent). While the host is biocompatible (HEK293; IC50 > 150 μM) and produces inclusion complex [MTX⊂6]14‐ in cell media, it experiences limitation in pharmacokinetic sequestration of MTX2‐ as dihydrofolate reductase’s affinity to the drug is suggested to prevail over that of 612‐. Nonetheless, considering the basket’s biocompatibility, tunability, and chemoselectivity, it stands as the leading candidate in the pursuit of an effective abiotic antidote for methotrexate poisoning.

Peak-Based Machine Learning for Plastic Type Classification in Time-of-Flight Secondary Ion Mass Spectrometry.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurement data and machine learning were used in this work to classify six different types of plastics. In order to take into account the characteristics of the measurement data, the local maxima of the measurement data were first examined in a preprocessing step. Several machine learning methods were then implemented to create a model that could successfully classify the plastics. To visualize the data distribution, we applied a dimensionality reduction method, namely, principal component analysis. Finally, to distinguish between the six types of plastics, we conducted an ensemble analysis using four tree-based algorithms: decision tree, random forest, gradient boosting, and LIGHTGBM. This approach can identify the feature importance of plastic samples and allow the inference of the chemical properties of each plastic type. In this way, ToF-SIMS data could be utilized to successfully classify plastics and enhance explainability.

Novel Imine Complexes Incorporating ZrO(II), VO(II), and Pd(II) Cations: Synthesis, DFT Calculation, Molecular Docking, Antimicrobial, and Antitumor Activity

ABSTRACTNovel imine complexes of ZrO(II), VO(II), and Pd(II) have been synthesized and described using IR, TGA, UV/Vis, and 1HNMR methods, along with powder X‐ray diffraction (XRD), elemental (CHN) analysis, mass spectrometry, molar conductance, and magnetic susceptibility measurements. Conductivity measurements indicate that none of the complexes function as electrolytes. Magnetic susceptibility data confirm the paramagnetism of the VO(II) complex, while ZrO(II) and Pd(II) complexes exhibit diamagnetic properties. Various analyses suggest that divalent cations favor a 1:1 metal‐to‐ligand ratio. Biological studies demonstrated that all complexes possess significant antimicrobial activity against a range of bacterial and fungal strains. We also observed that the antimicrobial potency of the metal complexes follows the order: ZrO(II) > Pd(II) > VO(II) > H2A, with the ZrO(II) complex showing the highest inhibition zone (28 mm) against Gram‐negative bacteria. The antifungal activity against Aspergillus flavus increases in the order VO(II) > ZrO(II) > Pd(II) > H2A. Additionally, the complexes exhibit high cytotoxicity against carcinoma cell lines, including the HepG‐2, when compared with the clinically proven vinblastine. Density functional theory (DFT) calculations were performed to elucidate the equilibrium geometry of the imine ligand and its complexes at the B3LYP level of theory, using the Gaussian 09 program with 6‐311G++(dp) basis set for C, H, O, N, and Cl atoms, and lanl2dz for Pd, V, and Zr atoms. Finally, docking studies revealed potential binding modes at the active sites of bacterial and fungal receptors.

Laser ablation inductively coupled plasma mass spectrometry measurements for high-resolution chemical ice core analyses with a first application to an ice core from Skytrain Ice Rise (Antarctica)

Abstract. Conventional methods of inorganic impurity analysis do not provide high enough depth resolution for many scientific questions in ice core science. In this study, we present a setup of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for high-resolution glacier ice impurity analysis to the sub-millimetre scale. This setup enables ice core chemical impurity analysis to a depth resolution of ∼182 µm while consuming only very small amounts of ice. The system performs simultaneous analysis of sodium, magnesium and aluminium incorporated in the ice matrix. In this case study within the framework of the WACSWAIN (WArm Climate Stability of the West Antarctic Ice Sheet in the last INterglacial) project, our method is applied to a selection of samples from the Skytrain ice core (West Antarctica) over a total length of 6.7 m consisting of about 130 single samples. The main goal of this study is to use the new LA-ICP-MS method to extract meaningful climate signals on a depth resolution level beyond the limits of continuous-flow analysis (CFA). A comparison between low-resolution CFA data and the high-resolution LA-ICP-MS data reveals generally good agreement on the decimetre scale. Stacking of parallel laser measurements together with frequency analysis is used to analyse the high-resolution LA-ICP-MS data at millimetre resolution. Spectral analysis reveals that despite effects of impurity accumulation along ice crystal grain boundaries, periodic concentration changes in the Skytrain ice core on the millimetre scale can be identified in ice from 26.8 ka (kiloyears before present, i.e. 1950 CE). These findings open new possibilities for climate data interpretation with respect to fast changes in the last glacial period and beyond, for example within the Beyond EPICA oldest-ice project.

Detection of IDH mutation in glioma by desorption electrospray ionization (DESI) tandem mass spectrometry

Desorption electrospray ionization (DESI) tandem mass spectrometry (MS) is used to assess mutation status of isocitrate dehydrogenase (IDH) in human gliomas. Due to the diffuse nature of gliomas, total gross resection is not normally achieved during surgery, leading to tumor recurrence. The mutation status of IDH has clinical significance due to better prognosis in IDH-mutant patients. The mutant IDH converts alpha-ketoglutaric acid (α-KG) into 2-hydroxyglutarate (2HG), which accumulates abnormally in cells. Immunohistochemical staining (IHC) and genetic testing, the gold standards, are incompatible with intraoperative applications but DESI tandem mass spectrometry (MS/MS) can be used to assess the mutation status of IDH enzyme from tissue intraoperatively. Here, on off-line evaluation is made of the performance of two different types of mass spectrometers in characterization of IDH mutation status. The intensity of 2HG is measured against glutamate (Glu), an intrinsic reference molecule, in both tandem MS measurements. In both cases using DESI clear separation between IDH-mutant (mut) and IDH-wildtype (wt) samples (p < 0.0001) is observed, despite the short analysis time. Due to the higher detection sensitivity, multiple reaction monitoring experiments using a triple quadrupole show slightly better performance compared to product ion MS/MS performed on a simple linear ion trap. Both DESI-MS platforms are capable of providing information on IDH mutation status, which might in future be used at the time of surgery to support decision-making on resection regions, especially at tumor margins.

Tridentate N-donor Schiff base metal Complexes: Synthesis, Characterization, computational Studies, and assessment of biomedical applications in cancer Therapy, Helicobacter pylori Eradication, and COVID-19 treatment

A unique Schiff base ligand (BCA), 4-(2-((1E,2E)-1-(2-(p-tolyl)hydrazono)propan-2-ylidene)hydrazinyl)quinazoline, was synthesized and complexed with Mn [II], Co [II], Cu [II, and Cd [II] ions, with 1,10-Phenanthroline (PHN) as an auxiliary ligand. The unique Schiff base ligand BCA and its metal complexes were characterized using multiple analytical techniques, including elemental analysis, UV–visible spectroscopy, FT-IR, mass spectrometry, and conductometric measurements. These methods provided detailed insights into the compounds’ structural and electronic properties. Density Functional Theory (DFT) computations complemented the experimental data, elucidating electronic configuration stability, HOMO-LUMO energy gaps, chemical hardness, and dipole moments. The computational results confirmed the distorted octahedral structure of the complexes. Antimicrobial testing against various bacterial and fungal strains revealed that the Cd (II) complex exhibited the highest activity, with inhibition zones ranging from 19.7 ± 0.6 mm to 35.0 ± 1.0 mm, surpassing the activity of standard antibiotics in some cases. Notably, the compounds displayed significant efficacy against Helicobacter pylori, with the Cd (II) complex showing an inhibition zone of 32.67 ± 0.58 mm and a minimum inhibitory concentration (MIC) of 7.8 μg/ml. Anticancer properties were evaluated against MCF-7 (Breast carcinoma) cells, with the [(BCA)(PHN)Cd(H2O)].2Cl·2H2O complex demonstrating superior activity (IC50 = 29.97 μg/ml) compared to the free ligand (IC50 = 183.96 μg/ml) and other metal complexes. Notably, the Cd(II) complex showed reduced cytotoxicity towards normal VERO cells, suggesting potential selectivity for cancer cells. Molecular docking simulations evaluated the complexes’ binding interactions with protein targets associated with Helicobacter pylori, MCF cancer cells, and the COVID-19 virus. The main objective of this research was to design and synthesize a unique Schiff base ligand and its new corresponding Ternary metal complexes and then investigate their potential medical applications. Specifically, the study aimed to evaluate the compounds’ effectiveness as antibiotics, antitumor agents, and possible treatments for COVID-19.

8415 Rapid Generation Of An In Vivo Loss-Of-Function Genetic Model Of sdhb Zebrafish: Functional And Metabolic Characterization

Abstract Disclosure: S. Parisien-La Salle: None. F. Nobilleau: None. J. Lamontagne: None. S. Éric: None. I. Bourdeau: None. Introduction: Animal research has been limited in pheochromocytomas and paragangliomas (PPGLs). Recently, Dona et al., described a sdhb mutated zebrafish model by introducing a 13 bp frameshift mutation at the sdhb exon1—intron1 boundary into the zebrafish sdhb gene that recapitulated features of PPGLs. Objective: To generate a new model of mutated sdhb zebrafish to further investigate the underlying metabolic and biochemical perturbations in vivo. Methods: We took advantage of the genetic accessibility of zebrafish embryo to generate loss-of-function model for sdhb using CRISPR/Cas9 technology. Three guide RNAs were designed and microinjected in one-cell stage zebrafish embryos to target the coding sequence of zebrafish sdhb gene. In order to control for the stress brought on by injections, we injected a group of zebrafish with Cas9 endonuclease as a control. At five days post fertilization, mutant and control zebrafish were flash frozen and sent for liquid chromatography mass spectrometry measurements of Krebs cycle metabolites and normetanephrines/metanephrines. All results were normalized to the control groups consisting of wild-type or Cas9 injected larvae. Sdhb expression was analyzed by RT-qPCR in sdhb mutants and wild-type zebrafish larvae. We also monitored survival and heart rate in sdhb mutant zebrafish, compared to wild-type and Cas9. Results: Firstly, we confirmed the significant reduction of sdhb expression in CRISPR-injected larvae compared to wild-type larvae at 5 dpf (0.0613 vs 1.000 p&amp;lt;0.0001). Our metabolic panel showed that succinate was up to 26 times more elevated in sdhb mutants than in wild-type fish extracts (sdhb: 26.51 p&amp;lt;0.0001). Lactate (sdhb: 5.27 p&amp;lt;0.0001), leucine (sdhb: 2.88 p&amp;lt;0.0001), arginine (sdhb: 1.50 p=0.0034) and HMG-CoA (sdhb: 1.36 p=0.0033) were also higher in sdhb mutants. Whereas aspartate (sdhb: 0.244 p&amp;lt;0.0001) and oxidized gluthiatone (sdhb: 0.64 p=0.0011) were significantly lower in sdhb mutants than in wild-types. In regards to functionality, normetanephrine and metanephrine levels were at least four folds higher in sdhb mutants than in Cas9 injected larvaes (4.06 p&amp;lt;0.0001 and 3.42 p&amp;lt;0.0001 respectively). Interestingly, this difference was more noticeable in the fish bathing medium than in larvae tissue extracts, suggesting an increased hormonal excretion (normetanephrines: 4.81 p&amp;lt;0.0001 and metanephrines: 9.40 p&amp;lt;0.0001). Finally, sdhb mutated zebrafish presented with a higher heart rate (148 vs 133 p&amp;lt;0.0001) and reduced lifespan (p&amp;lt;0.0001) when compared to the wild-type/Cas9 group. Conclusion: These findings confirm that the zebrafish is an ideal model for studying PPGLs and their associated genetic defects. Our approach allows the in vivo assessment of a large metabolic panel, opening avenues on new potential clinical metabolic biomarkers in a loss-of-function of the SDHB gene. Ref: Dona M, et al. Endocr Relat Cancer. 2021 Presentation: 6/1/2024

B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry

Abstract Background Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. Methods After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column. Results Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Conclusions The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.

O2-Dependence of reactions of 1,2-dimethoxyethanyl and 1,2-dimethoxyethanylperoxy isomers

Reaction mechanisms of R˙ and ROO˙ radicals derived from low-temperature oxidation of 1,2-dimethoxyethane (CH3O(CH2)2OCH3) were investigated using speciation from multiplexed photoionization mass spectrometry (MPIMS) measurements via Cl-initiated oxidation, in conjunction with electronic structure calculations. The experiments were conducted at 5 bar, from 450 K – 650 K, and O2 concentrations from 1 · 1014 cm–3 – 6 · 1018 cm–3 to probe the effects on competing reaction channels of 1,2-dimethoxyethanyl (R˙) and 1,2-dimethoxyethanylperoxy (ROO˙) isomers. Several species were detected with photoionization spectral fitting – ethene, formaldehyde, methyl vinyl ether, and 2-methoxyacetaldehyde – and, as determined by electronic structure calculations, may form via unimolecular decomposition of 1,2-dimethoxyethanyl or 1,2-dimethoxyethanylperoxy. O2-dependent yield ratios show that the formation pathways for all species undergo a competition between O2-addition and unimolecular decomposition. Adiabatic ionization energies were also calculated and utilized along with exact mass determinations to infer contributions for other species derived exclusively from first- and second-O2-addition, including 1,2-dimethoxyethene, cyclic ethers, and dicarbonyls.In addition to species formed from conventional low-temperature oxidation pathways, an important conclusion is derived from the detection of species produced from an O2-addition step involving ĊH2CH2OCH3 (R˙′), which forms via prompt dissociation of the primary 1,2-dimethoxyethanyl radical (ĊH2O(CH2)2OCH3). Species derived from R˙′ + O2 – 1,3-dioxolane and methyl acetate – were detected at [O2] = 1.2 · 1017 cm–3 and formed on timescales parallel to the main R˙ + O2 reactions. In addition, ion signal at m/z 106 was detected and increased with O2 concentration from which connections are drawn to ketohydroperoxides produced by Q˙′OOH + O2. Detection of such species indicate that β-scission of 1,2-dimethoxyethanyl is sufficiently facile such that timescales of R˙′ + O2 compete with conventional R˙ + O2 pathways.

Multi-tracer evidence of hydrology and primary production controls on dissolved organic matter composition and stability in the semi-arid aquatic continuum

Autochthonous dissolved organic matter (Auto-DOM) produced by a biological carbon pump using dissolved inorganic carbon (DIC) from carbonate weathering plays an important role in carbon cycling within inland waters. However, little is known regarding how environmental conditions impact the composition and fate of organic matter, especially in surface waters of the semi-arid Loess Plateau, which is enriched in DIC by significant carbonate weathering. To obtain novel insight, we combined hydrochemistry, isotopic composition (δ2H, δ18O, and δ13CDIC), Rayleigh fractionation model, optical spectroscopy (absorbance and fluorescence), and Fourier transform ion cyclotron resonance mass spectrometry measurements to elucidate how primary production and hydrology impact the composition of DOM and the stability of the resulting Auto-DOM throughout the river–reservoir–wetland aquatic continuum of the Bahe River in the carbonate-mineral-rich semi-arid Loess Plateau where carbonate weathering is significant. The Rayleigh fractionation model results indicated that watershed DIC is primarily consumed through aquatic primary production rather than CO2 degassing. Further investigation revealed that primary production and evaporation co-occurred in this watershed. With the enrichment of the stable water isotope δ2H, the relative abundances of the allochthonous compounds decreased and the relative abundances of the autochthonous substances increased, suggesting that the terrestrial signal of riverine DOM decreased while autochthonous production increased along the flow pathway. In addition, associations between optical and molecular characteristics among DOM samples from different water bodies revealed that the stability ratio (Fmax(C2/(C2 + C4))) of Auto-DOM to the ratio of carboxylic-rich alicyclic molecules showed a consistent trend, suggesting that phytoplankton-derived and biomineralized C2 compounds are potentially recalcitrant DOM in inland waters. We conclude that hydrology and primary production affect the source, composition, and, potentially, the stability of DOM in DIC-enriched surface waters of the semi-arid Loess Plateau, which may lead to a more humic-like DOM composition in inland water and export this lower bioavailability DOC to the ocean in the long term.

Automated Assignment of 15N And 13C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of 15N and 13C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli, a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13C and 15N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15N and 13C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [15N,13C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [15N,13C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae.

Structural characterization of nitazene analogs using electron ionization-mass spectrometry (EI-MS)

Nitazene analogs are among the most recent and potent additions to the novel synthetic opioid (NSO) market, and new analogs continue to emerge. Seized drug analysis commonly utilizes gas chromatography-electron ionization-mass spectrometry (GC-EI-MS), so it is therefore imperative to understand how nitazene analogs behave under EI-MS conditions, and how substitution at various sites on the molecule may impact the resulting EI mass spectra. This study characterizes the EI fragmentation behavior of 20 representative nitazene analogs that contain differing substitutions and proposes rational mechanisms to explain the observed behavior.A general EI fragmentation pathway for nitazene analogs was proposed, with the most common nitazene fragment ions being observed at m/z 86, m/z 107, m/z 58, and m/z 77. Characteristic ions were determined for different substitution groups, enabling the identification of diethyl, desethyl, pyrrolidine, and piperidine substitutions at the amine moiety, and different alkoxy chain lengths at the aromatic ring of the benzyl group. Mechanisms for the formation of these characteristic ions were proposed with the aid of isotopically labeled standards and high-resolution mass spectrometry measurements. To help with the interpretation of EI mass spectra for nitazene analogs, decision trees were developed that encompass the characteristic fragment ions observed for substitutions to the amine moiety and benzyl group, with additional criteria provided for substitutions to the benzimidazole moiety. This study summarizes the fragmentation patterns and characteristic fragment ions in the EI mass spectra of 20 representative nitazene analogs, which will aid the seized drug community in identifying novel nitazene analogs.