Common Reagents Research Articles

Non-destructive viability assessment of cancer cell spheroids using dynamic optical coherence tomography with trypan blue validation

3D cell cultures are widely used in biomedical research for the recapitulation of in vivo microenvironments. Viability assessment and monitoring of these intricate conformations remain an open problem as standard cell viability protocols based on colorimetry or microscopy are not directly applicable to intact 3D samples. Optical coherence tomography (OCT) has been explored extensively for subsurface structural and quasi-functional analysis of 3D cell cultures and tissue. Recent studies of dynamic OCT as a source of cellular contrast have found qualitative associations with necrosis in cell spheroids, suggesting potential as a viability marker. We present empirical and validated evidence for dynamic OCT as a quantitative indicator of cell viability in 3D cultures. We analysed over 240 MCF-7 cancer cell spheroids with dynamic OCT and corresponding viability measurements using the trypan blue exclusion assay. Significant effects of common reagents dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) on OCT readouts were noted. We proposed a regression-based OCT brightness normalisation technique that removed reagent-induced OCT intensity biases and helped improve correspondence to the viability assay. These results offer a quantitative biological foundation for further advances of dynamic OCT as a novel non-invasive modality for 3D culture monitoring.

Reactivities of N-Nitrosamines against Common Reagents and Reaction Conditions.

The knowledge of the reactivity of N-nitrosamines (NSAs) with common organic reagents in synthesis is essential in determining their presence in pharmaceutical products, if formed and retained during synthesis. In this study, we carried out a comprehensive survey of the Reaxys database for all reactions in which the NSA functional group is consumed. Very different reactivities for different classes of NSAs, e.g., N,N-dialkylnitrosamines and N,N-diphenylnitrosamine, were identified, suggesting substrates which should be included in any future reactivity screening. A classification of NSAs based on their reactivities, and corresponding reagents and transformations, was drawn up based on the data. Furthermore, the survey identified missing areas in the reported reactivities of NSAs with different reagents. This led to an experimental reactivity screening of 8 commercial NSAs with common synthetic reagents in the Mirabilis tool for purge assessment. The results showed Na2S2O4 in 1 M aqueous NaOH at 50 °C to be highly effective at destroying NSAs without damaging other organic compounds.

An europium complex assembled by a flexible derivative of imidazole-4,5-dicarboxylic acid as a fluorescence turn-off chemosensor to detect Cr(VI) anions

A new europium(III) CP [Eu(HBBIDA)(H2O)]n (1) (H4BBIDA = 1,1′-(butane-1,4-diyl)bis(1H-imidazole-4,5-dicarboxylic acid) was successfully constructed by hydrothermal synthesis method and detailed characterization was performed. Crystal structure analysis reveals that CP 1 presents a 2D layered structure composed of cage-like SBUs with a TiS2 topological network. CP 1 can maintain framework integrity in an air environment below 260 °C, as well as in a certain pH range of acid-base aqueous solutions and common organic reagents, showing better thermal and chemical stability. Fluorescence measurements show that CP 1 exhibited excellent sensing activities toward CrO42− and Cr2O72− anions through fluorescence quenching, and their average quenching Ksv values of 3.99 × 103 M−1 (CrO42−) and 4.36 × 103 M−1 (Cr2O72−) and the LOD of 3.30 × 10−6 M (CrO42−) and 3.02 × 10−6 M, which makes it very suitable for the detection of hexavalent chromium anions contaminants in aqueous solutions. Furthermore, possible sensing mechanisms have been studied and discussed in detail, and the results show that static photocompetitive absorption should be the primary cause for the fluorescence quenching of the sensor.

The Pre- and Post-Column Derivatization on Monosaccharide Composition Analysis, a Review.

Polysaccharides, as common metabolic products in organisms, play a crucial role in the growth and development of living organisms. For humans, polysaccharides represent a class of compounds with diverse applications, particularly in the medical field. Therefore, the exploration of the monosaccharide composition and structural characteristics of polysaccharides holds significant importance in understanding their biological functions. This review provides a comprehensive overview of extraction methods and hydrolysis strategies for polysaccharides. It systematically analyzes strategies and technologies for determining polysaccharide composition and discusses common derivatization reagents employed in further polysaccharide studies. Derivatization is considered a fundamental strategy for determining monosaccharides, as it not only enhances the detectability of analytes but also increases detection sensitivity, especially in liquid chromatography (LC), capillary electrophoresis (CE), and gas chromatography (GC) techniques. The review meticulously examines pre-column and post-column derivatization techniques for monosaccharide analysis, categorizing them based on diverse detection methodologies. It delves into the principles and distinctive features of various derivatization reagents, offering a comparative analysis of their strengths and limitations. Ultimately, the aim is to provide guidance for selecting the most suitable derivatization approach, taking into account the structural nuances, biological functions, and reaction dynamics of polysaccharides.

Continued harmonization of the international normalized ratio across a large laboratory network: Evidence of sustained low interlaboratory variation and bias after a change in instrumentation.

Our objective was to maintain low interlaboratory variation and bias in international normalized ratio (INR) results following a network change in instrumentation and reagents, using a process of ongoing standardization and harmonization. Network-wide standardization to new common instrument and reagent platforms followed by network-wide application of a simple novel process of verification of international sensitive index and mean normal prothrombin time values for each new lot of prothrombin time (PT) reagent that does not require use of World Health Organization reference thromboplastin or INR calibration/certified plasma. The network transitioned from mechanical hemostasis detection instruments with associated PT reagent (Diagnostica Stago; NeoPTimal) to optical detection (ACL TOPs) with associated PT reagent (Werfen; RecombiPlasTin 2G). Comparing 3 years of data for each situation, the network (n=27 laboratories) maintained low INR variability and bias relative to general mechanical and optical groups and other laboratories. Harmonized support for patient management of vitamin K antagonists such as warfarin was continuously maintained in our geography, with potentially positive implications for other coagulation laboratories and geographies. For the United States in particular, paucity of US Food and Drug Administration-cleared INR certified plasmas potentially compromises INR test accuracy; our novel approach may provide workable alternatives for other laboratories/networks.

Increase in cathepsin K gene expression in Duchenne muscular dystrophy skeletal muscle.

Dystrophinopathy is caused by alterations in the dystrophin gene. The severe phenotype, Duchenne muscular dystrophy (DMD), is caused by a lack of dystrophin in skeletal muscles, resulting in necrosis and regenerating fibers, inflammatory cells, and muscle fibrosis. Progressive muscle weakness is a characteristic finding of this condition. Here, we encountered a rare case of a 10-year-old patient with asymptomatic dystrophinopathy with no dystrophin expression and investigated the reason for the absence of muscle weakness to obtain therapeutic insights for DMD. Using RNA-seq analysis, gene expression in skeletal muscles was compared among patients with asymptomatic dystrophinopathy, three patients with typical DMD, and two patients without dystrophinopathy who were leading normal daily lives. Cathepsin K (CTSK), myosin heavy chain 3 (MYH3), and nodal modulator 3-like genes exhibited a >8-fold change, whereas crystallin mu gene (CRYM) showed a <1/8-fold change in patients with typical DMD compared with their expression in the patient with asymptomatic dystrophinopathy. Additionally, CTSK and MYH3 expression exhibited a >16-fold change (P < 0.01), whereas CRYM expression showed a <1/16-fold change (P < 0.01) in patients with typical DMD compared with their expression in those without dystrophinopathy. CTSK plays an essential role in skeletal muscle loss, fibrosis, and inflammation in response to muscles injected with cardiotoxin, one of the most common reagents that induce muscle injury. Increased CTSK expression is associated with muscle injury or necrosis in patients with DMD. The lack of muscle weakness in the patient with asymptomatic dystrophinopathy might be attributed to the low CTSK expression in the muscles. To the best of our knowledge, this is the first report to demonstrate that CTSK expression was significantly higher in the skeletal muscles of patients with DMD with a typical phenotype than in those without dystrophinopathy.

A Sterically Accessible Monomeric Stibine Oxide Activates Organotetrel(IV) Halides, Including C-F and Si-F Bonds.

Phosphine oxides and arsine oxides are common laboratory reagents with diverse applications that stem from the chemistry exhibited by these monomeric species. Stibine oxides are, in contrast, generally dimeric or oligomeric species because of the reactivity-quenching self-association of the highly polarized stiboryl (Sb=O/Sb+-O-) group. We recently isolated Dipp3SbO (Dipp = 2,6-diisopropylphenyl), the first example of a kinetically stabilized monomeric stibine oxide, which exists as a bench-stable solid and bears an unperturbed stiboryl group. Herein, we report the isolation of Mes3SbO (Mes = mesityl), in which the less bulky substituents maintain the monomeric nature of the compound but unlock access to a wider range of reactivity at the unperturbed stiboryl group relative to Dipp3SbO. Mes3SbO was found to be a potent Lewis base in the formation of adducts with the main-group Lewis acids PbMe3Cl and SnMe3Cl. The accessible Lewis acidity at the Sb atom results in a change in the reactivity with GeMe3Cl, SiMe3Cl, and CPh3Cl. With these species, Mes3SbO formally adds the E-Cl (E = Ge, Si, C) bond across the unsaturated stiboryl group to form a 5-coordinate stiborane. The biphilicity of Mes3SbO is sufficiently potent to activate even the C-F and Si-F bonds of C(p-MeOPh)3F and SiEt3F, respectively. These results mark a significant contribution to an increasingly rich literature on the reactivity of polar, unsaturated main-group motifs. Furthermore, these results highlight the utility of a kinetic stabilization approach to access unusual bonding motifs with unquenched reactivity that can be leveraged for small-molecule activation.

Green and Versatile High-Throughput Microwell Oxidation-Based Spectrophotometric Methods for Determination of Galidesivir in Capsules.

Galidesivir (GDV) is a promising new antiviral drug for the potent and safe treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form. The aim of this study was the development of versatile green and simple microwell spectrophotometric methods (MW-SPMs) for the determination of GDV in its bulk form and capsules. Three MW-SPMs were developed involving the oxidation of GDV by ammonium metavanadate (AMV), chromium trioxide (CTO), and potassium iodate (PIO) in an acid medium. The reactions were carried out in 96-well plates at room temperature and the absorbances of chromogenic reaction products were measured by an absorbance microplate reader at 780, 595, and 475 nm for AMV, CTO, and PIO, respectively. Variables influencing the reactions were carefully investigated and optimized. Linear relations with excellent correlation coefficients (0.9991-0.9997) were found between the absorbances and GDV concentrations in the range of 25-500 µg/mL. The LODs were ≥8.3 µg/mL. The accuracy and precision of the three MW-SPMs were confirmed by recovery and replicate analysis, respectively. The recovery values were 98.6-101.2% and the RSDs were ≤1.02%. The proposed MW-SPMs were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision. The greenness of the MW-SPMs was confirmed by three comprehensive metric tools. The proposed MW-SPMs combined the inherent advantages of microwell-based analysis and the use of common laboratory reagents for the reactions involved. These advantages include high-throughput, ready automation, reduced sample/reagent volume, precise measurements, and versatility. The advantages of the use of common laboratory reagents include availability, consistency, compatibility, safety, and cost-effectiveness. Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Mining flotation reagents: Quantitative and robust analysis of metal-xanthate complexes in water

Xanthates, common mining flotation reagents, strongly bind thiophilic metals such as copper (Cu), lead (Pb), cadmium (Cd), and zinc (Zn) and consequentially change their bioavailability and mobility upon their discharge into the environment. However, accurate quantification of the metal-xanthate complexes has remained elusive. This study develops a novel and robust method that realizes the accurate quantification of the metal-xanthate complexes resulted from single and multiple reactions of three typical xanthates (ethyl, isopropyl, and butyl xanthates) and four thiophilic metals (Cu, Pb, Cd, and Zn) in water samples. This method uses sulfur (S2-) dissociation, followed by tandem solid phase extraction of C18 + PWAX and subsequent LC-MS/MS analysis. It has a wide linearity range (1–1000 μg/L, R2 ≥ 0.995), low method detection limits (0.002–0.036 μg/L), and good recoveries (70.6–107.0 %) at 0.01–10 mg/L of xanthates. Applications of this method showed ubiquitous occurrence of the metal-xanthate complexes as the primary species in flotation wastewaters, which the concentrations were 4.6–28.9-fold higher than those previously determined. It is the first quantitative method established for the analysis of metal-xanthate complexes in water samples, which is of great importance to comprehensively understand the fate and risks of xanthates in the environment.

Synthesis of itaconyl dichloride with high purity and its application to polycondensation

Itaconyl dichloride (IC) was prepared using an industrially viable method to guarantee sufficiently high purity for polycondensation. Itaconic acid, a bio-based dicarboxylic acid with an acrylic skeleton, generates anhydride during chlorination by common reagents such as thionyl chloride and oxalyl chloride, resulting in poor yields and purity. In contrast, the combination of dichloromethyl alkyl ether and a Lewis acid catalyst was effective because it converted the anhydride intermediate into IC. Although melt polymerization of IC and aliphatic diols resulted in low conversions, interfacial polymerization with bisphenol Z afforded the corresponding semi-aromatic polyesters containing itaconic skeletons.

Facile synthesis of pyrimidine substituted-1,3,4-oxadiazole, 1,3,4-thiadiazole and 1,2,4-triazole derivatives and their antimicrobial activity correlated with molecular docking studies

Organosilicon reagent-trimethylsilyl isothiocyanate, a common reagent for the development of 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles was reported by the reaction with heteroaromatic acid hydrazides under conventional and ultrasonication methodologies in a polar protic solvent, ethanol under appropriate conditions. Molecular docking studies were performed with the X-ray crystal structures of the target molecules (PDB ID: 2w9s and PDB ID: 6f0e). The docking results indicated that H-bond interactions and hydrophobic interactions were responsible for the inhibition of protein. All 15 synthesized molecules showed excellent binding energy than the standard drugs. The compounds 3a, 3b, 3e, 5a, 5b, 5e and 7a exhibited low MIC values against S. aureus whereas 3a, 3b, 5a, 5b and 7a against B. subtilis. The compounds 5a, and 5b also displayed low MIC values against P. aeruginosa. The MBC of these compounds is 2×MIC, and is equal to standard drug, Chloramphenicol. The compounds 3a, 3b, 3e, 5a, 5b, 5d and 5e showed low MIC values against A. niger whereas 3a, 5a, 5b, 7a, 7b and 7e against P. Chrysogenum. The MFC is 2×MIC and is equal to standard drug, Fluconazole. The structure-activity relationship of the compounds revealed that unsubstituted, methyl and nitro substituted compounds showed greater antimicrobial activity than those with chloro and bromo substituents. [Display omitted]

Photoreduction of Trifluoromethyl Group: Lithium Ion Assisted Fluoride-Coupled Electron Transfer from EDA Complex.

Photoinduced single-electron reduction is an efficient method for the mono-selective activation of the C-F bond on a trifluoromethyl group to construct a difluoroalkyl group. We have developed an electron-donor-acceptor (EDA) complex mediated single-electron transfer (EDA-SET) of α,α,α-trifluoromethyl arenes in the presence of lithium salt to give α,α-difluoroalkylarenes. The C-F bond reduction was realized by lithium iodide and triethylamine, two common feedstock reagents. Mechanistic studies revealed the generation of a α,α-difluoromethyl radical by single-electron reduction and defluorination, followed by the radical addition to alkenes. Lithium salt interacted with the fluorine atom to promote the photoinduced reduction mediated by the EDA complex. Computational studies indicated that the lithium-assisted defluorination and the single-electron reduction occurred concertedly. We call this phenomenon fluoride-coupled electron transfer (FCET). FCET is a novel approach to C-F bond activation for the synthesis of organofluorine compounds.

Development of Lab-Scale Continuous Stirred-Tank Reactor as Flow Process Tool for Oxidation Reactions Using Molecular Oxygen.

The use of sustainable oxidants is of great interest to the chemical industry, considering the importance of oxidation reactions for the manufacturing of chemicals and society's growing awareness of its environmental impact. Molecular oxygen (O2), with an almost optimal atom efficiency in oxidation reactions, presents one of the most attractive alternatives to common reagents that are not only toxic in most cases but produce stoichiometric amounts of waste that must be treated. However, fire and explosion safety concerns, especially when used in combination with organic solvents, restrict its easy use. Here, we use state-of-the-art 3D printing and experimental feedback to develop a miniature continuous stirred-tank reactor (mini-CSTR) that enables efficient use of O2 as an oxidant in organic chemistry. Outstanding heat dissipation properties, achieved through integrated jacket cooling and a high surface-to-volume ratio, allow for a safe operation of the exothermic oxidation of 2-ethylhexanal, surpassing previously reported product selectivity. Moving well beyond the proof-of-concept stage, we characterize and illustrate the reactor's potential in the gas-liquid-solid triphasic synthesis of an endoperoxide precursor of antileishmanial agents. The custom-designed magnetic overhead stirring unit provides improved stirring efficiency, facilitating the handling of suspensions and, in combination with the borosilicate gas dispersion plate, leading to an optimized gas-liquid interface. These results underscore the immense potential that lies within the use of mini-CSTR in sustainable chemistry.

Metal-organic framework-based aptasensor utilizing a novel electrochemiluminescence system for detecting acetamiprid residues in vegetables

The work was based on N-(4-Aminobutyl)-N-ethylisoluminol (ABEI)-functionalized Fe-MIL-101 and gold nanoparticles (AuNPs) as sensing materials, and an electrochemiluminescence (ECL) aptasensor was constructed for detecting acetamiprid. As a metal-organic framework (MOF) material, Fe-MIL-101, was renowned for its unique three-dimensional network structure and efficient catalytic capability. ABEI, a common ECL reagent, was widely applied. ABEI was introduced into the Fe-MIL-101 structure as a luminescence functionalization reagent to form Fe-MIL-101@ABEI. This approach avoided limitations on the loading capacity of luminescent reagents imposed by modification and encapsulation methods. With character of excellent catalytic activity and ease of bioconjugation, AuNPs offered significant advantages in biosensing. Leveraging the reductive properties of ABEI, AuNPs were reduced around Fe-MIL-101@ABEI, resulting in the modified luminescent functionalized material denoted as Fe-MIL-101@ABEI@AuNPs. An aptamer was employed as a recognition element and was modified accordingly. The aptamer was immobilized on Fe-MIL-101@ABEI@AuNPs through gold-sulfur (Au–S) bonds. After capturing acetamiprid, the aptamer induced a decrease in the ECL signal intensity within the ABEI-hydrogen peroxide (H2O2) system, enabling the quantitative detection of acetamiprid. The aptasensor displayed remarkable stability and repeatability, featured a detection range of 1×10−3-1×102 nM, and had a limit of detection (LOD) of 0.3 pM (S/N=3), which underscored its substantial practical application potential.

Expanding horizons in conceptual density functional theory: Novel ensembles and descriptors to decipher reactivity patterns.

Conceptual density functional theory (CDFT) and the quantum reactivity descriptors stemming from it have proven to be valuable tools for understanding the chemical behavior of molecules. This article is presented as being intrinsically of dual character. In a first part, it briefly reviews, in a deliberately didactical way, the main ensembles in CDFT, while the second half presents two additional ensembles, where the chemical hardness acts as a natural variable, and their respective reactivity descriptors. The evaluation of these reactivity descriptors on common organic chemical reagents are presented and discussed.

Development of Green and High-Throughput Microwell Spectrophotometric Methods for Determination of Galidesivir in Bulk Drug and Dosage Forms Based on Simple Oxidimetric Reactions With Inorganic Agents.

Galidesivir hydrochloride (GDV) is a new potent and safe antiviral drug used for the treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk or dosage form. The objective of this study was the investigation of oxidation reactions of GDV with five inorganic oxidizing reagents and the employment of the reactions in the development of five green microwell spectrophotometric methods (MW-SPMs) with simple procedure and high throughputs for determination of GDV in its bulk and dosage forms (capsules). The reactions were carried out in 96-well plates, and the absorbances of reaction solutions were measured by an absorbance microplate reader. Variables influencing the reactions were carefully investigated and optimized. Under the refined optimum conditions, Beer's law with excellent correlation coefficients (0.9992-0.9997) was followed in GDV concentrations in a general range of 5-700 µg/mL, and the limits of detection were ≥1.8 µg/mL. All validation parameters of all methods were acceptable. The methods were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision; the recovery percentages were 98.6-101.2 ± 0.58-1.14%. The greenness of MW-SPMs was evaluated by three comprehensive metric tools, which demonstrated the adherence of MW-SPMs to the principles of the green analytical chemistry (GAC) approach. The proposed MW-SPMs combined the advantages of microwell-based practice and the use of common laboratory reagents for the analysis. The advantages of microwell analysis were the high throughput, readily available for semi-automation, reduced samples/reagents volume, precise measurements, and versatility. The advantages of using common laboratory reagents were the availability, consistency, compatibility, safety, and cost-effectiveness. Overall, the proposed MW-SPMs are versatile, valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Reduction of Pro-Inflammatory Markers in RAW264.7 Macrophages by Polyethylenimines.

The physiological problem of chronic inflammation and its associated pathologies attract ongoing attention with regard to methods for their control. Current systemic pharmacological treatments present problematic side effects. Thus, the possibility of new anti-inflammatory compounds with differing mechanisms of action or biophysical properties is enticing. Cationic polymers, with their ability to act as carriers for other molecules or to form bio-compatible materials, present one such possibility. Although not well described, several polycations such as chitosan and polyarginine, have displayed anti-inflammatory properties. The present work shows the ubiquitous laboratory transfection reagent, polyethylenimine (PEI) and more specifically low molecular weight branched PEI (B-PEI) as also possessing such properties. Using a RAW264.7 murine cell line macrophage as an inflammation model, it is found the B-PEI 700Da as being capable of reducing the production of several pro-inflammatory molecules induced by the endotoxin lipopolysaccharide. Although further studies are required for elucidation of its mechanisms, the revelation that such a common lab reagent may present these effects has wide-ranging implications, as well as an abundance of possibilities.

Xylene versus Isopropanol for Paraffin Wax Processing of Lung Tissue

The microscopic observation of lung tissue is challenging due to its fragile nature. Xylene and isopropanol are common tissue-clearing reagents used before paraffin embedding, yet no studies have compared these two reagents in lung tissue processing. Due to the well-known health risks xylene could introduce to operators, as well as its environmental hazards, it has long been desired that a less harmful alternative to xylene with the same staining effects be introduced. Thus, we systematically assessed the efficacy of isopropanol as a substitution for xylene. Lung tissue obtained from diseased donors and explanted lungs from recipients were processed simultaneously using either xylene or isopropanol prior to paraffin embedding. Scoring of the overall staining quality after H&amp;E staining, along with the ease of sectioning, was compared systematically. Fluorescent staining was performed to explore alveolar morphology and the overall lectin fluorescence signal between groups. To understand differences in antibody staining, the signal-to-noise ratio (SNR) of smooth muscle actin (SMA) and elastin was examined. No difference was observed with regard to ease of sectioning, staining quality, alveolar circularity, alveolar wall thickness or the SNR between slides processed with xylene or isopropanol. This study demonstrated comparable outcomes of isopropanol and xylene in lung tissue processing, suggesting isopropanol as a more favorable, operator- and environment-friendly substitute for xylene with regards to tissue processing.

Coprecipitation as a One-Step Se Separation for Determination of Isotope Ratios Completed with Revised Uncertainty Evaluation.

This study introduces a simplified purification method for analyzing 82Se/78Se isotope ratios in diverse natural samples using hydride generation MC-ICP-MS. Unlike the thiol resin method, which is time-consuming and sensitive to the concentrations of reagents used at individual stages, our proposed alternative is quicker, simpler, and robust. The procedure involves coprecipitation of selenium with iron hydroxide and dissolution in hydrochloric acid. Combining hydride generation and a second cleanup stage achieves sufficient purification for Se isotope ratio measurements. The method is efficient, taking 3-4 h after sample decomposition, utilizing common reagents [HCl, Fe(NO3)3, NH4Cl] without evaporation or clean lab conditions. Results on 82Se/78Se isotope ratios in various matrices are presented, comparing them with literature data. All isotopic results have been subjected to a newly proposed state-of-the-art approach to uncertainty estimation dedicated to isotope ratio measurements. The approach is based on applying Monte Carlo simulations with consideration of different samples' results normalized by the expected value. By doing that, we obtained estimated uncertainty for any Se sample with the influence of particular measurements on the final estimation included. We employ a Monte Carlo simulation-based uncertainty estimation approach for isotope ratio measurements, providing estimated uncertainty for each selenium sample.

Structural elucidation of derivatives of polyfunctional metabolites after methyl chloroformate derivatization by high-resolution mass spectrometry gas chromatography. Application to microbiota metabolites

Metabolomics has become an essential discipline in the study of microbiome, emerging gas chromatography coupled to mass spectrometry as the most mature, robust, and reproducible analytical technique. Silylation is the most widely used chemical derivatization strategy, although it has some limitations. In this regard, alkylation by alkyl chloroformate offers some advantages, such as a rapid reaction, milder conditions, better reproducibility, and the generation of more stable derivatives. However, commercial spectral libraries do not include many of the alkyl derivatives, mainly for polyfunctional metabolites, which can form multiple derivatives. That introduces a huge bias in untargeted metabolomics leading to common errors such as duplicates, unknowns, misidentifications, wrong assignations, and incomplete results from which non-reliable findings and conclusions will be retrieved. For this reason, the purpose of this study is to overcome these shortcomings and to expand the knowledge of metabolites in general and especially those closely related to the gut microbiota through the thorough study of the reactivity of the different functional groups in real matrix derivatized by methyl chloroformate, a common representative alkylation reagent. To this end, a systematic workflow has been developed based on exhaustive structural elucidation, along with computational simulation, and taking advantage of the high sensitivity and high-resolution gas chromatography-mass spectrometry. Several empirical rules have been established according to chemically different entities (free fatty acids, amino acids, polyols, sugars, amines, and polyfunctional groups, etc.) to predict the number of derivatives formed from a single metabolite, as well as their elution order and structure. In this work, some methyl chloroformate derivatives not previously reported as well as the mechanisms to explain them are given. Extremely important is the interconversion of E- and Z- geometric isomers of unsaturated dicarboxylic acids (case of fumaric-maleic and case of citraconic-mesaconic acids), or the formation of cycled derivatives for amino acids, as well as common metabolites, as in the case of serine and cysteine, and many others.