Search Results (71)

Background: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency. Methods: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach’s applicability in putative biomarker discovery. Results: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness. Conclusions: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential. Full article

Compound Glycyrrhizin tablets (CGTs) are a combination of glycyrrhizin, glycine and methionine. Glycine and methionine have relatively high polarity and lack chromophore; therefore, it is difficult to simultaneously determine the various components using traditional reversed-phase chromatography and ultraviolet detectors. In addition, it is even more challenging to obtain a comprehensive and systematic impurity profiling for the CGTs. In this study, an ion-pair high-performance liquid chromatography (HPLC)–charged aerosol detection (CAD) method was established to determine the content of glycyrrhizin, glycine and methionine. The impurities of CGTs were also identified using mass spectrometry. By optimizing the content of trifluoroacetic acid (TFA) in the mobile phase and optimizing the CAD parameter settings, the developed method was verified in accordance with the guidelines outlined in ICH Q2 (R2). The results indicated that the method demonstrated high accuracy and sensitivity. Glycine, methionine and glycyrrhizin all showed a good linear relationship within the labeled range of 50–200%, and the average recoveries of the three components were 97.62–100.6%. The impurity detection was quantified via the principal component control method. The limit of detection (LOD) method showed an equivalent to 0.05% of the glycyrrhizin in CGTs, approximately 12.5 ng. The ion-pair HPLC–CAD method developed in this study simultaneously determined the content of the main component and the impurities of CGTs, without necessitating derivatization. This has provided a research basis for further improving the quality standards of CGTs. Full article

Chiral analysis is becoming increasingly important across various scientific fields, including chemistry, pharmaceuticals, biosciences, and more recently, metabolomics. In this context, a high-resolution and high-throughput method was developed for the simultaneous determination of the enantiomeric ratio (er) of seven pairs of amino acid (AA) enantiomers (Arg, Gln, His, Met, Pro, Tyr, and Trp) using flow injection analysis coupled with ion mobility-mass spectrometry (FIA-IM-MS) technology. Specifically, the Single Ion Mobility Monitoring (SIM²) mode on a TIMS-Tof^TM instrument enabled the rapid relative quantification of chiral compound mixtures. A linear model accurately described the relationship between enantiomeric ratio and IM-MS response for Arg, Gln, and Pro enantiomers, as evidenced by high R² values and unbiased residuals. In contrast, non-linear trends were observed for His, Tyr, and Trp, where a quadratic model significantly improved the fit. However, the linear model was retained for Met, despite an R² of about 0.98, due to its comparable performance and simplicity. Measurement accuracy was confirmed with very good recovery rates for er values of 0.95 and 0.99 across all AAs. Finally, the potential of the FIA-SIM²-MS approach in chiral analysis was demonstrated, particularly its ability to provide a reliable and efficient high-throughput tool for accurate er determination. Full article

Electrospray mass spectrometry off-line profiling monitored the recovery of targeted indole alkaloids from a fortified crude extract of Catharanthus roseus (790 mg) using semi-preparative high-performance countercurrent chromatography (HPCCC) fractionation. Visualization of selected single-ion traces projected the HPCCC molecular weight elution profile. Experimental partition-ratio values K_D and peak widths for detected metabolites were determined. Structural characterization of metabolites and co-elution effects were monitored in the scan range m/z 100–2000. In this study, the biphasic solvent system containing n-hexane–n-butanol–water with 0.5% ion-pair reagent trifluoro-acetic acid [1:1:2, v/v/v] was used based on partition ratio K_D-value liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) analysis prediction. The monitoring of target ions resulted in the isolation of six major concentrated indole alkaloids (akuammicine, catharanthine, perivine, vindoline, vindorosine, and 19R-vindolinine), which were fully elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Full article

Cannabinoid molecules are the family of molecules that bind to the cannabinoid receptors (CB1 and CB2) of the human body and cause changes in numerous biological functions including motor coordination, emotion, and pain reception. Cannabinoids occur either naturally in the Cannabis Sativa plant or can be produced synthetically in the laboratory. The need for accurate analytical methods for analyzing cannabinoid molecules is of considerable current importance due to demands for detecting illegal cannabinoids and for monitoring the manufacture of popular, non-illegal cannabinoid products. Mass spectrometry has been shown to be an optimum technique for identifying cannabinoids. In this work, we perform Higher Collisional Dissociation (HCD) mass spectrometric measurements on an Orbitrap Fusion Tribrid Mass Spectrometer to measure the collision-energy-dependent molecular fragmentation pathways of a group of key cannabinoids and their metabolites (cannabidiol, Δ9-Tetrahydrocannabinol, 11-Hydroxy-Δ9-tetrahydrocannabinol, 11-nor-9-Carboxy-Δ9-tetrahydrocannabinol, cannabidiolic acid, tetrahydrocannabinolic acid), along with two synthetic cannabinoids (JWH-018 and MDMB-FUBINACA). This is the first time that cannabinoid molecules have been studied using energy-resolved HCD methods. We identified a number of common, primary fragmentation pathways, including loss of water, loss of other small neutral molecule units (e.g., butene), and rupture of the central C-C bond that links the aromatic and alkyl ring groups. Quantum chemical calculations are presented to provide insights into preferred protonation sites and to characterize isomerization of protonated open-ring cannabinoids (e.g., [CBDA + H]⁺) into closed-ring analogues (e.g., [THCA + H]⁺). A key result to emerge from our study is that energy-resolved HCD measurements are particularly valuable in identifying isomerization, since the isobaric pairs of molecular ions studied here (e.g., [CBDA + H]⁺ and [THCA + H]⁺) are associated with identical HCD profiles indicating that isomerization of one structure into the other has occurred during the electrospray–mass spectrometry process. This is an important result as it will have general applicability to other tautomeric ions and thus demonstrates the application of energy-resolved HCD as a tool for identifying tautomerization proclivity. Full article

Kefir, a fermented milk product produced using kefir grains, is a symbiotic consortium of bacteria and yeasts responsible for driving the fermentation process. In this study, an in-depth analysis of kefir’s lipid profile was conducted, with a focus on its phospholipid (PL) content, employing liquid chromatography with high-resolution mass spectrometry (LC-HRMS). Nearly 300 distinct polar lipids were identified through hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization (ESI) and Fourier-transform orbital-trap MS and linear ion-trap tandem MS/MS. The identified lipids included phosphatidylcholines (PCs), lyso-phosphatidylcholines (LPCs), phosphatidylethanolamines (PEs) and lyso-phosphatidylethanolamines (LPEs), phosphatidylserines (PSs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The presence of lysyl-phosphatidylglycerols (LyPGs) was identified as a key finding, marking a lipid class characteristic of Gram-positive bacterial membranes. This discovery highlights the role of viable bacteria in kefir and underscores its probiotic potential. The structural details of minor glycolipids (GLs) and glycosphingolipids (GSLs) were further elucidated, enriching the understanding of kefir’s lipid complexity. Fatty acyl (FA) composition was characterized using reversed-phase LC coupled with tandem MS. A mild epoxidation reaction with meta-chloroperoxybenzoic acid (m-CPBA) was performed to pinpoint double-bond positions in FAs. The dominant fatty acids were identified as C18:3, C18:2, C18:1, C18:0 (stearic acid), C16:0 (palmitic acid), and significant levels of C14:0 (myristic acid). Additionally, two isomers of FA 18:1 were distinguished: ∆9-cis (oleic acid) and ∆11-trans (vaccenic acid). These isomers were identified using diagnostic ion pairs, retention times, and accurate m/z values. This study provides an unprecedented level of detail on the lipid profile of kefir, shedding light on its complex composition and potential nutritional benefits. Full article

To characterize the trace-element characteristics of chrysoberyl, we studied twenty-six chrysoberyl samples from various localities by using laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS), photoluminescence (PL), and ultraviolet–visible–near-infrared (UV–Vis–NIR) spectroscopy. Chemical analysis has confirmed the existence of trace elements, including Fe, Ti, Ga, Sn, B, Cr, and V. The phenomenon of ionic isomorphic substitution frequently occurs at lattice sites within chrysoberyl. Notably, the isomorphic substitution of Al³⁺ in octahedral sites is significant, with the primary substituting elements being Fe, Ti, Cr, V, Ga, and Sn. The PL spectra of chrysoberyl samples exhibit sharp peaks at 678 and 680 nm, which are attributed to Cr³⁺, even in samples in which the Cr concentration is below the detection limit of LA-ICP-MS. This demonstrates the high-sensitivity feature of PL spectroscopy. The UV–Vis–NIR spectra of chrysoberyl samples consistently exhibit a band at 440 nm, and strong double narrow bands near 367 nm and 375 nm are observed. These spectral features are associated with Fe³⁺ chromophores—specifically, Fe³⁺-Fe³⁺ pairs or clusters and Fe³⁺ ions, respectively. By combining LA–ICP–MS analysis and PL mapping on a sample exhibiting color zoning, it has been found that the darker sections contain a higher concentration of Cr compared to the lighter sections, while the concentrations of other elements remain largely consistent. In other words, subtle variations in Cr concentration may be the underlying cause of color zoning in chrysoberyl. Full article

Flavonoids are a class of polyphenolic secondary metabolites found in plants. Due to their ubiquity in our daily dietary intake and their major anti-oxidative, anti-inflammatory and anti-mutagenic activities, they have been a major focus of wide-ranging research for the past two decades. Mass spectrometry combined with liquid chromatography is one of the most popular techniques for the analysis of flavonoids. In this study, high-resolution accurate mass electrospray tandem mass spectrometry was used to study 30 flavonoids in both positive and negative ionisation modes. From the data obtained, common losses were summarised and compiled. Dominating neutral losses were tabulated. The radical loss of CH₃· was observed in flavonoids containing methoxy groups and three key diagnostic product ions were identified. These were m/z 153 (indicative of two OH groups on ring A) m/z 167 (indicative of one OH and one methoxy group on ring A) and m/z 151 (a flavanol, with no ketone oxygen but two OH groups on ring A). These will be useful in structural elucidation of unknown flavonoids and flavonoid metabolites. Energy breakdown graphs were utilised to distinguish between three pairs of structural isomers, and to help rationalise proposed fragmentation pathways. Lastly, a competition of loss of CH₃· and methane was reported for rhamnetin and isorhamnetin in the negative ion mode for the first time. Proposed fragmentation pathways were given to rationalise the differences in peak intensities for this competitive process. Full article

In the present work, we report an update and extension of the previous ion-pair formation study of Hubers, M.M.; Los, J. Chem. Phys. 1975, 10, 235–259, noting new fragment anions from time-of-flight mass spectrometry. The branching ratios obtained from the negative ions formed in K + SF₆ collisions, in a wide energy range from 10.7 up to 213.1 eV in the centre-of-mass frame, show that the main anion is assigned to SF₅⁻ and contributing to more than 70% of the total ion yield, followed by the non-dissociated parent anion SF₆⁻ and F⁻. Other less intense anions amounting to <20% are assigned to SF₃⁻ and F₂⁻, while a trace contribution at 32u is tentatively assigned to S⁻ formation, although the rather complex intramolecular energy redistribution within the temporary negative ion is formed during the collision. An energy loss spectrum of potassium cation post-collision is recorded showing features that have been assigned with the help of theoretical calculations. Quantum chemical calculations for the lowest-lying unoccupied molecular orbitals in the presence of a potassium atom are performed to support the experimental findings. Apart from the role of the different resonances participating in the formation of different anions, the role of higher-lying electronic-excited states of Rydberg character are noted. Full article

Bispecific antibodies (BsAbs) can bind to two different antigens, enabling therapeutic concepts that cannot be achieved with monoclonal antibodies. Immuno-competent mice are essential for validating drug discovery concepts, necessitating the development of surrogate mouse BsAbs. In this study, we explored the potential of FAST-Ig^TM, a previously reported BsAb technology, for mouse BsAb production. We investigated charge-based orthogonal Fab mutations to facilitate the correct assembly of heavy and light chains of mouse antibodies and employed knobs-into-holes mutations to facilitate the heterodimerization of heavy chains. We combined five anti-CD3 and two anti-HER2 antibodies in mouse IgG1 and IgG2a subclasses. These 20 BsAbs were analyzed using mass spectrometry or ion exchange chromatography to calculate the percentages of BsAbs with correct chain pairing (BsAb yields). Using FAST-Ig, 19 out of the 20 BsAbs demonstrated BsAb yields of 90% or higher after simple protein A purification from transiently expressed antibodies in Expi293F cells. Importantly, the mouse BsAbs maintained their fundamental physicochemical properties and affinity against each antigen. A Jurkat NFAT-luciferase reporter cell assay demonstrated the combined effects of epitope, affinity, and subclasses. Our findings highlight the potential of FAST-Ig technology for efficiently generating mouse BsAbs for preclinical studies. Full article

Centruroides possanii is a recently discovered species of “striped scorpion” found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD₅₀ to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field. Full article

The tandem mass spectrometry (MS/MS) approach employing an ion trap mass analyzer (IT) was evaluated in isomers recognition. The proposed approach consists of sole, simple, and rapid liquid chromatographic separation (HPLC) without requiring resolution between the analytes. Then, the MS/MS properties were optimized to solve the signal assignment using post-processing data elaboration (LEDA). The IT-MS/MS experiment uses the same site, helium as collision gas, and different time steps to modify the applied conditions on the studied ions. Nevertheless, helium cannot ensure the quick energization of the precursor ion due to its small cross-section. Then, different combinations between excitation amplitude (ExA) and excitation time (ExT) were tested to achieve the activation of the fragmentation channels and the formation of the MS/MS spectrum. Usually, the IT-MS/MS acquisition cycle is longer for other multistage instruments, decreasing the frequency of sample data collection and influencing the chromatographic profile. To solve these problems, two time segments were set up, and the elution conditions were optimized with a compromise between peaks distinction and run time reduction. The developed HPLC-MS/MS method was checked and applied to analyze a series of human plasma samples spiked with an equimolar mixture of pair of isomers. Full article

The processes of the sublimation and thermal decomposition of the 1-ethyl-3-methylimidazolium hexafluorophosphate ionic liquid (EMImPF₆) were studied by a complex approach including Knudsen effusion mass spectrometry, IR and NMR spectroscopy, and quantum chemical calculations. It was established that the vapor over the liquid phase primarily consists of decomposition products under equilibrium conditions. Otherwise, the neutral ion pairs are the only vapor components under Langmuir conditions. To identify the nature of the decomposition products, an experiment on the distillation of the ionic liquid was performed and the collected distillate was analyzed. It was revealed by the IR and NMR spectroscopy that EMImPF₆ decomposes to substituted imidazole-2-ylidene (C₆N₂H₁₀PF₅) and HF. The measured vapor pressure of C₆N₂H₁₀PF₅ reveals a very low activity of the decomposition products (<10⁻⁴) in the liquid phase. The absence of a significant accumulation of decomposition products in the condensed phase makes it possible to determine the enthalpy of sublimation of the ionic liquid assuming its unchanged activity. The thermodynamics of the EMImPF₆ sublimation was studied by Knudsen effusion mass spectrometry. The formation enthalpy of EMImPF₆ in the ideal gas state was found from a combination of the sublimation enthalpy and formation enthalpy of the ionic liquid in the condensed state. The obtained value is in good agreement with those calculated by quantum chemical methods. Full article

Poly-diallyldimethylammonium (PDADMAC) chloride is a flocculant agent extensively used in water clarification treatments. Commercial polyelectrolyte formulations often contain residual monomer (DADMAC), reaction by-products and other reactants as contaminants that could potentially affect human health. In the present study, we optimized an analytical method based on reversed-phase ion-pair chromatography coupled with mass spectrometry for quantifying ultra-trace levels of DADMAC, without preliminary treatments, in drinking water distributed by the Palermo aqueduct. The method was validated in terms of accuracy (recovery between 80 to 120%), precision (RSD < 10%), linearity range (from 0.5 to 15 µg/L), limit of quantification and limit of detection (LOQ 0.42 µg/L and LOD 0.12 µg/L). DADMAC was detected in all analysed water samples and the concentration ranged from 1.2 to 3.8 µg/L whit a mean value of 1.7 µg/L. To the best of our knowledge, this paper represents the first study concerning DADMAC concentration in water samples collected in Italy and, in this context, this paper can be considered very interesting when comparing future data on DADMAC monitoring in water. Moreover, this is one of a few cases where DADMAC was found in drinking water. Full article

The evaporation/decomposition behavior of the ionic liquid 1-butyl-3-methylimidazolium chloride (BMImCl) was studied with various techniques, such as thermogravimetry (TG), Knudsen effusion mass loss (KEML), and Knudsen effusion mass spectrometry (KEMS), in order to investigate the competition between the simple evaporation of the liquid as gaseous ion pairs (NIP: neutral ion pair) and the thermal decomposition releasing volatile species. TG/DSC experiments were carried out from 293 to 823 K under both He and N₂ flowing atmospheres on BMImCl as well as on BMImNTf₂ (NTf₂: bis(trifluoromethylsulfonyl)imide). Both ionic liquids were found undergoing a single step of mass loss in the temperature range investigated. However, while the BMImNTf₂ mass loss was found to occur in different temperature ranges, depending on the inert gas used, the TG curves of BMImCl under helium and nitrogen flow were practically superimposable, thus suggesting the occurrence of thermal decomposition. Furthermore, KEML experiments on BMImCl (in the range between 398 and 481 K) indicated a clear dependence of the unit area mass loss rate on the effusion hole diameter, an effect not observed for the ILs with NTf₂ anion. Finally, KEMS measurements in the 416–474 K range allowed us to identify the most abundant species in the vapor phase, which resulted in methyl chloride, butylimidazole, butyl chloride, and methylimidazole, which most probably formed from the decomposition of the liquid. Full article