RATIONALE:Lepidoceras peruvianum Kuijt is an understudied hemiparasitic species endemic to the Peruvian Andes that lacks comprehensive phytochemical characterization. Investigating its metabolome is important for understanding its chemotaxonomic relevance and potential as a source of bioactive compounds. METHODS:Leaves and fruits of L. peruvianum were subjected to untargeted metabolomic profiling using high-resolution UHPLC-ESI-Orbitrap-MS/MS in positive and negative ionization modes. Metabolites were annotated based on accurate mass measurements, isotopic patterns, collision-induced dissociation (CID) fragmentation data, and spectral matching with the Global Natural Products Social Molecular Networking (GNPS), METLIN, and MassBank databases. Structural elucidation employed diagnostic fragmentation pathways, including retro-Diels-Alder (RDA), heterocyclic ring fission (HRF), quinone methide (QM), and benzofuran-forming (BFF) cleavages. Antioxidant activity was evaluated using DPPH●, ABTS●+, and FRAP assays, while antimicrobial activity and toxicity were assessed through antibacterial testing and the Artemia salina lethality assay. RESULTS:Leaves contained 34 metabolites, mainly flavan-3-ols, proanthocyanidins, flavonols, lignans, and isoquinoline alkaloids, whereas fruits were characterized by anthocyanins, organic acids, and polar lipids. Total phenolic content was higher in leaves (292.4 mg GAE/g) than in fruits (216.8 mg GAE/g), corresponding to stronger antioxidant activity. Leaves extracts exhibited greater antibacterial activity against Staphylococcus aureus, while fruit extracts showed stronger effects against Gram-negative bacteria. LC50 values in the A. salina assay were 181.8 μg/mL for leaves and 475.9 μg/mL for fruits. CONCLUSIONS:This study provides the first comprehensive metabolomic characterization of L. peruvianum, revealing organ-specific chemical diversity and notable bioactive properties. The findings demonstrate the utility of HRMS/MS fragmentation analysis for metabolite annotation and identify this endemic species as a promising source of bioactive natural products.
RATIONALE:Stable nitrogen (δ15N) and sulfur (δ34S) isotopes within the metabolically inert organic matrix of teleost otoliths provide high-fidelity archives for reconstructing animal life histories. However, low organic content limits the spatial resolution of conventional bulk isotopic analysis, leaving biomineralization kinetics and the temporal integration of these dietary signals poorly quantified. We utilized nanoscale secondary ion mass spectrometry (NanoSIMS) to overcome these limitations and determine intracrystalline isotopic dynamics. METHODS:Isotopic pulses were established by feeding juvenile Mozambique tilapia (Oreochromis mossambicus) 15N-enriched algae and diets supplemented with L-methionine-34S, utilizing Alizarin Red S fluorescent marking for precise temporal referencing. High-resolution and in situ NanoSIMS analysis was applied to quantify the temporal distribution of the labeling signals within the otolith organic matrix. A first-order kinetic model was used to estimate isotopic dynamics half-lives (t50%). RESULTS:NanoSIMS analysis revealed rapid isotopic changes across both assimilation and depuration phases (t50% = 0.5-13.9 days). Both δ15N and δ34S exhibited statistically indistinguishable kinetics, reflecting synchronized metabolic routing where dietary signals are deposited directly into the otolith without being buffered by internal tissue reserves. Crucially, the dietary matrix modulated assimilation; despite comparable dietary δ34S proportions, otolith sulfur incorporation and signal attenuation were significantly faster in fish fed algae-based diets than in those fed eel-meal-based diets. This demonstrates the preferential utilization of exogenous labeled methionine when the base diet is naturally deficient in this essential amino acid. CONCLUSIONS:High-resolution NanoSIMS profiling demonstrates that the otolith organic matrix archives dietary signals governed by rapid assimilation pathways rather than strict thermodynamic equilibrium. This analytical approach establishes a crucial mechanistic basis and experimental framework for utilizing intracrystalline organic isotopes as precise indicators in trophic ecology.
RATIONALE:Acid-related disorders, including gastro-oesophageal reflux disease (GERD), peptic ulcers and Helicobacter pylori infection, affect millions worldwide. Vonoprazan fumarate, a potassium-competitive acid blocker, provides rapid and sustained acid suppression. Understanding its degradation behaviour is essential for ensuring drug stability, supporting formulation development and guiding quality control. METHODS:Vonoprazan fumarate was subjected to acidic and basic hydrolysis, oxidation, thermal and photolytic stress to evaluate its degradation behaviour. Degradation products were separated by reversed-phase high-performance liquid chromatography (HPLC) using a gradient program optimised for mobile-phase composition and flow rate, thereby enabling the chromatographic resolution of closely eluting degradation products. Structural characterisation was performed using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) based on accurate-mass measurements and diagnostic MS/MS dissociation patterns. The method was validated for linearity, precision, accuracy, selectivity and robustness. RESULTS:Nine degradation products were detected under stress conditions: Basic hydrolysis yielded degradation product (DP)-1 and DP-8; acidic stress produced DP-1, DP-2 and DP-4; oxidation generated DP-1, DP-2, DP-7 and DP-8; and photolysis formed DP-1 to DP-6, DP-8 and DP-9. Three degradation products exhibited identical protonated molecules and closely comparable MS/MS dissociation patterns to the parent drug but differed in chromatographic retention times, with their relative distribution varying in solution- and solid-state upon UV exposure. CONCLUSIONS:This study provides a comprehensive evaluation of the forced degradation behaviour of vonoprazan fumarate by RP-HPLC and characterisation of its degradation products using LC-QTOF-MS/MS, providing insights into its degradation pathways and supporting stability assessment. Comparative solution- and solid-state photostability studies revealed distinct degradation profiles, providing further insight into the photodegradation behaviour of vonoprazan fumarate.
RATIONALE:Tiletamine, an animal tranquilizer increasingly misused by humans, lacks comprehensive metabolic characterization in authentic biological matrices. To date, tiletamine phase II metabolism remains poorly characterized. This study investigated the metabolic fate of tiletamine in human urine and hair by identifying novel metabolites and elucidating biotransformation pathways relevant to forensic toxicological monitoring. METHODS:Authentic human urine (n = 3) and hair (n = 2) samples from individuals with documented tiletamine exposure were analyzed using liquid chromatography coupled with Q Extractive HF hybrid quadrupole-Orbitrap high-resolution mass spectrometry (LC-QE-HF-MS). Metabolites were detected and identified based on full-scan MS and data-dependent MS/MS (ddMS2) fragmentation patterns. Structural elucidation, including the assignment of hydroxylation sites, was achieved through a diagnostic fragment ion analysis. RESULTS:A total of 14 urinary metabolites were identified, 11 of which (4 phase I and 7 phase II) were previously unreported. Hydroxylation was the predominant phase I process, whereas glucuronidation predominated among phase II reactions. Methylation and glucuronidation were identified as previously unreported metabolic pathways. In hair samples, tiletamine and four metabolites, including three reduced metabolites and T1, were identified for the first time. In addition, three metabolic transformations, namely, reduction, hydroxylation, and methylation, were newly identified in this matrix. Notably, T1 exhibited substantially higher signal intensities than all other metabolites across all urine samples. CONCLUSIONS:This study substantially expands the known urinary metabolome by identifying novel phase I and II metabolites. The consistently high abundance of T1 in urine suggests that it is a promising urinary biomarker for monitoring tiletamine use. These findings broaden the range of analytical targets available for toxicological screening and confirmational analysis, thereby improving the detection and monitoring of tiletamine exposure.
RATIONALE:Mass spectrometry imaging (MSI) generates high-dimensional spatial-spectral data that requires efficient computational methods for tissue classification and candidate biomarker feature extraction. Deep learning offers a promising approach, yet the interpretability of model predictions and identification of biologically relevant spectral features remain challenging. METHODS:A comprehensive computational pipeline was developed for automatic tissue layer classification of a public mouse urinary bladder MSI dataset. Building upon prior work that compared manual tissue layer labels with those automatically generated via spectral preprocessing, t-SNE, and hierarchical clustering, in this study we separately use each type of class label to train convolutional neural networks (CNNs) for supervised classification. Gradient-weighted Class Activation Mapping (Grad-CAM) and SHapley Additive exPlanations (SHAP) were employed to compute layer-specific summed importance scores to evaluate each mass spectral feature and extract class-discriminative features. RESULTS:On the mouse urinary bladder MSI dataset, both manual labels and cluster-derived labels (t-SNE + hierarchical clustering) enabled the CNN model to achieve training accuracies exceeding 0.9 for classifying three tissue layers. Interpretability methods successfully identified discriminative m/z features, including known lipids such as SM(34:1) (m/z 741.54) and PC(34:1) (m/z 798.54), consistent with previously reported biological markers. Compared to the intensity values, the importance scores of the top class-discriminative features generated by both interpretability methods exhibited a sharper contrast and superior ability to delineate tissue-layer-specific distributions in their ion images. Furthermore, evaluation on an independent colorectal cancer dataset yielded a test accuracy of 0.758, suggesting that cross-patient generalizability varied across different data sources. CONCLUSIONS:This study presented an effective deep learning framework for accurate and interpretable tissue classification in MSI data. The CNN modeling and deep learning interpretability provided a robust approach for both automated segmentation and biological discovery, facilitating the identification of spatially resolved metabolic features in tissue sections.
RATIONALE:As a classic multiorigin herbal medicine, the precise authentication of Curcumae Radix (Yujin) is crucial for ensuring market standardization and clinical efficacy. Given the complexity of herbal materials, single-method identification often lacks the resolution required to distinguish between closely related species or handle degraded with significant DNA degradation. METHODS:A two-dimensional analytical strategy was applied to 24 batches of Curcumae Radix samples. This approach combined ITS2 DNA barcoding for genetic identification with headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) to analyze volatile organic compound (VOC) profiles. The resulting HS-GC-IMS data were processed using chemometric tools, including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to compare and differentiate the decoction pieces. RESULTS:The results demonstrated that while ITS2 DNA barcoding effectively identified Curcuma kwangsiensis S. G. Lee et C. F. Liang, it failed to discriminate between the closely related species Curcuma longa L. and Curcuma phaeocaulis Val. and was ineffective for DNA-degraded samples of Curcuma wenyujin Y. H. Chen et C. Ling. Conversely, HS-GC-IMS successfully established distinct VOC fingerprints for each source. When coupled with chemometric analysis, this method pinpointed specific interspecific discriminatory biomarkers that allowed for clear differentiation where genetic barcoding fell short. CONCLUSION:By integrating stable genetic information with volatile chemical fingerprints, this study establishes a complementary two-dimensional identification system. This strategy provides an innovative and reliable solution for the botanical authentication and quality control of Curcumae Radix, offering a broadly applicable paradigm for the precise identification of other complex herbal materials.
RATIONALE:Ganjianglingzhu decoction (GJLZD), a classical traditional Chinese medicine (TCM) formula, has been widely used in the treatment of low back pain. However, its chemical composition and underlying mechanisms remain insufficiently elucidated. METHODS:The chemical profile of GJLZD was comprehensively characterized by UPLC-Q-TOF-MS/MS. Representative bioactive constituents were subsequently selected for target prediction using the PharmMapper and SwissTargetPrediction databases. Overlapping targets between compound-related targets and low back pain-related targets retrieved from the GeneCards database were subjected to protein-protein interaction (PPI) analysis, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, as well as molecular docking and molecular dynamics (MD) simulation. RESULTS:Based on MS/MS fragmentation patterns and literature comparison, a total of 58 constituents were tentatively characterized from GJLZD. These mainly included flavonoids, alkaloids, phenolic acids, and terpenoids. We identified 466 overlapping targets between the active components and low back pain. PPI analysis highlighted SRC, PIK3CA, and AKT1 as the major hub targets. GO and KEGG enrichment analyses suggested that these targets were primarily involved in signal transduction, inflammatory regulation, and metabolism. The enriched pathways included VEGF, FcεRI, and AGE-RAGE signaling. Molecular docking revealed that neoglycyrol showed the strongest binding affinity to AKT1, with a binding energy of -7.93 kcal/mol. MD simulation further confirmed the stability of the neoglycyrol-AKT1 complex over 100 ns. CONCLUSIONS:This study systematically characterized the chemical constituents of GJLZD and elucidated its potential mechanisms in treating low back pain. The results suggest a multicomponent and multi-target mode of action. The integrated strategy combining UPLC-Q-TOF-MS/MS with network pharmacology and molecular simulations proved valuable for investigating the pharmacological basis of TCM formulas. Further experimental validation is still needed to confirm these findings.
BACKGROUND:Chronic obstructive pulmonary disease (COPD) is a leading cause of death, underscoring the need for improved therapies. Xuanfei Heji (XFHJ), a hospital-prepared herbal formula, has been used clinically in the treatment of COPD. However, its mechanisms remain unclear. METHODS:XFHJ constituents were profiled using UHPLC-HRMS. COPD was induced in rats by intratracheal lipopolysaccharide instillation and cigarette smoke exposure. Treatment effects were assessed using pulmonary function, lung histopathology, and proinflammatory cytokines. Untargeted serum metabolomics and fecal 16S rRNA gene sequencing were performed; associations among differential metabolites, microbial taxa, and inflammatory markers were evaluated using Spearman's rank correlation analysis. RESULTS:Chemical profiling tentatively identified 374 constituents. XFHJ improved pulmonary function and attenuated lung histopathological injury and inflammation. Tryptophan and glycerophospholipid metabolism were the principal treatment-associated pathways. XFHJ also altered gut microbial diversity and composition, with enrichment of potentially beneficial taxa such as Bifidobacterium, Roseburia, and several Clostridia-related taxa. Treatment-responsive taxa correlated positively with indole-related metabolites, which correlated inversely with pulmonary inflammatory markers. CONCLUSIONS:XFHJ exhibited significant therapeutic effects on COPD rats, and its mechanism may be correlated with regulating the intestinal microbiota structure and metabolic profiles of COPD rats, thereby attenuating lung histopathological injury and pulmonary inflammation.
RATIONALE:Peptides and oligonucleotides are increasingly used as therapeutic agents, making the identification and structural elucidation of their metabolites a critical step in drug development. Liquid chromatography-mass spectrometry (LC-MS/MS) is the primary analytical technique for this purpose; however, extensive backbone fragmentation generates highly complex MS/MS spectra, making spectral interpretation challenging and limiting the exploitation of the structural information contained in MS/MS fragmentation data. METHODS:This study introduces two fragment-based metrics, fragment coverage and complete fragment coverage, to quantify structural coverage derived from MS/MS fragmentation at metabolite and parent compound levels. Their performance was evaluated across 33 LC-MS/MS experiments comprising peptide and oligonucleotide datasets. Ranking analysis based on mean average precision (MAP) assessed discrimination between true and false metabolite assignments. Additionally, a fragment viewer was developed to map MS/MS fragment ions onto structures, facilitating visualization and spectral interpretation. RESULTS:A total of 255 metabolites were identified, including 159 true metabolites and 96 false positives. True metabolites consistently exhibited higher fragment coverage and complete fragment coverage values than false positives. This trend was supported by ranking analysis using mean average precision (MAP), where complete fragment coverage achieved the highest performance (0.9737), followed by fragment coverage (0.9676), MassMetaSite score (MMS score) (0.9390), and isotopic similarity (0.9090). Two representative case studies demonstrated the applicability of the proposed metrics. CONCLUSIONS:The proposed fragment coverage metrics enable quantitative assessment of MS/MS fragmentation, providing complementary information to support manual interpretation of LC-MS/MS data in MetID workflows. Together with the fragment viewer tool, these approaches facilitate more efficient and interpretable analysis of complex MS/MS spectra in macromolecules.
RATIONALE:The investigation of lignocellulosic biomass by atmospheric-pressure chemical ionization (APCI) mass spectrometry (MS) has revealed significant issues for carbohydrate ionization, particularly during biomass pyrolysis analyses. Unequal ionization of carbohydrates and significant fragmentation have been reported. The introduction of suitable dopants into the ion source shows promise for limiting fragmentation. METHODS:To investigate these ionization disturbances, both direct infusion (DI) and modified direct insertion probe (DIP) APCI coupled with a Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) were employed. Solvents with increasing proton affinity (PA) were used to assess solvent PA influence in DI-APCI ionization processes. The DIP system was modified to enable the introduction of an inert gas stream passing through a dopant-rich atmosphere prior to entering the ion source. RESULTS:A significant PA difference between the solvent and the analyte promotes carbohydrate dissociation after their ionization. Experiments without solvent and with deuterated compounds showed that carbohydrate ionization and dissociation may be self-induced by water formed during carbohydrate dehydration. The introduction of ammonia in the ion source promoted the [M + NH4]+ adduct formation, reducing ionization-induced dissociation and increasing the intensity of the carbohydrate signal by at least one order of magnitude in DI-APCI. The method was successfully applied to both standards and cellulose pyrolysis samples. CONCLUSIONS:The dopant-assisted APCI (dAPCI) approach appears highly promising for the rapid assessment of biomass fast pyrolysis, particularly when highly oxygenated compounds are key markers. Careful selection of dopants and solvents is essential in carbohydrate analysis to preserve analyte structural integrity during ionization.
RATIONALE:Ketoconazole (KCZ) is a clinically essential antifungal and potent CYP3A4 inhibitor. Its gas-phase fragmentation under positive electrospray ionization tandem mass spectrometry (ESI-MS/MS) remains poorly characterized, undermining metabolite profiling and structural identification of related azoles. METHODS:We systematically mapped the fragmentation pathways of protonated KCZ using a dual-platform strategy combining quadrupole time-of-flight collision-induced dissociation (QTOF-CID) and Orbitrap higher-energy collisional dissociation (HCD) across a 10-60-eV collision energy range. Mechanistic assignments and protonation sites were supported by density functional theory (DFT) calculations at B3LYP/6-311++G(d,p) level in order to quantify thermodynamic barriers and dissociation energies. RESULTS:Our study identified three distinct dissociation pathways: (1) piperazine ring opening driven by ketene loss (A1, m/z 489.1453) and alkylamine eliminations; (2) 1,3-dioxolane ring cleavages (e.g., C10, m/z 277.1547); and (3) radical-driven dissociation at the ether linker, yielding an imidazole radical cation (A28, m/z 82.0531, C4H6N2 •+). DFT calculations established a clear thermodynamic hierarchy, where even-electron ions (e.g., A1, ΔG ≈ 19 kcal mol-1) prevail at low energies, whereas the radical cation (A28, ΔG ≈ 86 kcal mol-1) is only accessible at ≥ 40 eV. HCD conditions selectively promoted odd-electron ion formation, revealing platform-dependent fragmentation behavior. CONCLUSIONS:This work delivers the first comprehensive, computationally validated fragmentation map for KCZ, directly linking MS/MS observations to thermodynamic predictions and providing a robust reference for high-confidence identification of synthetic imidazole antifungals.
RATIONALE:The proteolytic behavior of chymosins during cheese ripening impacts cheese texture, flavor, and quality. Especially, the specific hydrolysis of αS1-casein to αS1-I-casein is relevant for early cheese texture development, and this activity varies between chymosin variants. Accurate profiling of αS1-casein and αS1-I-casein during cheese ripening is crucial for understanding these differences between chymosins and the impact on cheese quality. METHODS:Intact-protein liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was employed to monitor the hydrolysis of αS1-casein to αS1-I-casein during mozzarella cheese ripening. Cheese samples, produced with both bovine and camel chymosin variants, were collected at multiple ripening stages. Protein extraction, separation, and quantification were performed using optimized LC-HRMS protocols to assess hydrolysis kinetics and phosphorylation variants. RESULTS:LC-HRMS analysis revealed that the tested bovine chymosins displayed significantly higher rates of αS1-casein hydrolysis to αS1-I-casein compared to camel chymosins throughout mozzarella cheese ripening. Distinct phosphorylation variants were confidently detected and quantified. Kinetic profiles demonstrated consistent proteolytic activity differences between coagulants. CONCLUSIONS:LC-HRMS allows tracking of αS1-casein hydrolysis kinetics throughout mozzarella cheese ripening. This approach not only highlights the pronounced and faster proteolytic activity of bovine chymosins compared to camel chymosins, resulting in more rapid αS1-I-casein formation, but also facilitates the monitoring of phosphorylation variants present in the cheese. These findings emphasize the enzyme-specific effects on cheese texture and quality, demonstrating the importance of advanced protein analysis for comprehensively understanding cheese production and maturation processes.
RATIONALE:Norvancomycin (NVCM) hydrochloride belongs to a group of glycopeptide antibiotics and exhibits strong effects against Gram-positive cocci and bacilli. The safety of patients' lives is directly related to the quality of drugs. The impurities play often an important role in this process. This study aimed to investigate the impurity profile of NVCM hydrochloride. METHODS:To identify the impurities, a simple and sensitive ultrahigh performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS) method was developed. An ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) was used. The flow rate was set at 0.2 mL/min. Mobile phase (A) consisted of 10 mmol/L ammonium formate (pH 9.0) and mobile phase (B) was methanol. Gradient elution was performed as follows: 0-5 min, 15% B; 5-25 min, 15% B to 20% B; 25-40 min, 20% B to 70% B. Stress degradation experiments were conducted to check the specificity of the method. RESULTS:Thirteen impurities and two degradation products were characterized using UHPLC-QTOF-MS. CONCLUSIONS:Based on MS/MS spectral data and exact mass measurements, the chemical structures of the impurities were elucidated. This study provided a reference method for the quality control of NVCM hydrochloride.
RATIONALE:Diphenylamine (DPAH) derivatives, widely used as antioxidants, interact with oxygen molecules (or ozone) to form radicals at the amine group. Although the antioxidation mechanism involves radical processes, separating these radicals using chromatography is challenging. METHODS:Rapid oxidation of DPAH was achieved through ozonation in an acetone solution, and the oxidation products, including radical and neutral species, were analyzed using direct APCI-MS. The analysis was performed using a single quadrupole mass spectrometer in both positive- and negative-ion modes. The types and yields of the oxidation products were analyzed according to the ozonation time. Energy differences based on the oxidation product size were compared to explain their favorability for formation. RESULTS:The major oxidation products included (DPA• + nO) and (DPA• + nO - H). They were detected as [DPA• + nO + H]+ and [DPA• + nO]+ in the positive-ion mode, respectively, and observed as [DPA• + nO - H]- and [DPA• + nO - 2H]- in the negative-ion mode, respectively. The abundance of oxidized DPAH products increased significantly until (DPA• + 2O) and (DPA• + 2O - H) and then notably decreased as the number of oxygen atoms increased. CONCLUSION:No significant differences in energies were observed for the consecutive addition of oxygen atoms to (DPA• + nO) and (DPA• + nO - H) with n = 1-6. Steric hindrance significantly influenced the number of oxygen atoms adducted to DPAH. The reasonable maximum number of oxygen atoms adducted to DPAH is five, considering thermodynamic favorability and steric hindrance.
RATIONALE:Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vitamin D3 metabolites is analytically challenging because these compounds are structurally similar, strongly matrix-associated, and have poor ionization efficiency. These limitations are particularly important for low-abundance metabolites such as 1,25(OH)2D3. This study systematically compared extraction and derivatization strategies to develop a simplified workflow for simultaneous quantification of 25(OH)D3, 3-epi-25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3 in plasma. METHODS:Five sample-preparation approaches-simple protein precipitation, liquid-liquid extraction, salting-out assisted liquid-liquid extraction, solid-phase extraction, and supported liquid extraction-were evaluated under comparable conditions. Two derivatization reagents, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) and 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS), were compared. The selected workflow was validated according to bioanalytical method validation criteria. RESULTS:Supported liquid extraction using ethyl acetate/methyl tert-butyl ether (1:1, v/v) provided the best overall balance of signal intensity, reproducibility, simplicity, and suitability for high-throughput processing. DAPTAD derivatization produced a substantially higher MS response than FMP-TS and was selected for the final method. The optimized SLE-DAPTAD LC-MS/MS workflow showed good linearity (r2 > 0.99), acceptable accuracy and precision, recovery of 72.5%-104%, and limits of quantification of 0.08 ng/mL for 3-epi-25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3, and 0.88 ng/mL for 25(OH)D3. CONCLUSIONS:The optimized SLE-DAPTAD LC-MS/MS workflow provides a simple, sensitive, and automation-compatible approach for simultaneous quantification of four vitamin D3 metabolites in plasma. The method is positioned as an analytical workflow for method development and low-volume plasma analysis, rather than as a fully standardized routine diagnostic assay for endogenous 1,25(OH)2D3 measurement.
RATIONALE:According to the mass spectrometry (MS) design principles, the ions generated in an atmospheric pressure ion source must pass through a narrow-bore capillary in order to maintain the needed system vacuum pressure. The ion transmission efficiency through the inlet capillary is one of the key factors directly influencing the analytical performance of MS. Its design and operating parameters, including inner diameter, length, temperature, and those related to the ion source operation, can alter ion diffusion losses, gas flow dynamics, and space-charge effects both around and inside the inlet capillary, thereby influencing ion sampling efficiency and transmission efficiency to the mass analyzer. METHODS:A comprehensive simulation of ion transmission efficiency through an MS inlet capillary has been performed in this study by adjusting the important design and operating parameters. RESULTS:The simulation results show that the ion transmission efficiency through the inlet capillary is positively correlated with the inner diameter of the capillary and negatively correlated with the length of the capillary. Specifically, under the conditions of a capillary diameter of 0.8 mm and a temperature of 573.15 K, the ion transmission efficiency through a 10-cm-long capillary can reach 90%. The efficiency decreases with a reduction in the inner diameter or an increase in the length. When the temperature exceeds 573.15 K, the flow field inside the capillary transits from laminar to turbulent flow, leading to a sharp decline in ion transmission efficiency. For every 100-K increase in temperature beyond this point, the ion transmission efficiency decreases by 10%-20%. CONCLUSIONS:The main results from this study have provided crucial design guidelines, emphasizing the use of larger diameters, moderate lengths, and strict temperature control below the turbulent transition threshold to maximize ion transmission and overall instrument sensitivity for MS instruments.
RATIONALE:The comprehensive quality control of chiral pharmaceuticals like mitiglinide necessitates simultaneous assessment of chemical impurities and enantiomeric purity, yet conventional workflows address these separately, leading to inefficiency. This study develops a comprehensive analytical strategy to overcome this challenge for the anti-diabetic drug mitiglinide. METHODS:For chiral analysis, an online heart-cutting two-dimensional liquid chromatography-high-resolution mass spectrometry (2D-LC-HRMS) method was developed. First, impurity profiling of mitiglinide was accomplished using a one-dimensional reversed-phase LC-HRMS (1D-LC-HRMS) method. Subsequently, the 2D-LC-HRMS system achieved enantiomer separation by online coupling of a C18 column (first dimension) with a polysaccharide-based chiral column (second dimension), with the separated analytes detected by an Orbitrap mass spectrometer. RESULTS:1D-LC-HRMS identified five major impurities, structurally characterizing four, with the main component accounting for only 49.12% of the total integrated peak area (relative abundance by EIC peak area normalization, not absolute purity). The 2D-LC-HRMS method achieved effective enantiomer separation. A consistent third minor chromatographic peak was observed across six replicate analyses, which is tentatively assigned as a potential diastereomeric impurity based on stereochemical interpretation of the chromatographic behavior; confirmatory evidence is required for definitive identification. CONCLUSIONS:This work successfully establishes a comprehensive strategy that efficiently consolidates impurity profiling and chiral purity assessment for mitiglinide. It provides a reliable, more informative approach for the quality control of complex chiral pharmaceuticals.
ABSTRACT Rationale Postblast analyses of military and terrorist events will benefit from the capacity to learn more about the explosive materials used in an event. Here stable isotope ratio analyses (δ 13 C, δ 15 N) can provide additional information to complement identification of the explosive components. Methods Controlled detonations using different types of military‐grade explosives were conducted in 55‐gal barrels. Additionally, soil analyses were conducted following Mark‐84 field detonations. Swab materials and soils were purified to analyze explosive compounds using established HPLC and IRMS techniques. Results Explosive materials were recovered in the residues of TNT (aromatic‐based explosive) and RDX (nonaromatic explosive) detonations in barrel experiments. Explosive residues were not recovered from PETN (nonaromatic explosive). Postblast δ 13 C and δ 15 N values of TNT residues were similar to δ values in the source explosive, suggesting minimal enrichment in δ residues. While δ 15 N values of RDX in postblast residues were also similar to preblast source values, postblast RDX δ 13 C values were enriched by almost 2‰ relative to the preblast explosive. Similar patterns were observed in HMX, RDX, and NT recovered from soils following Mark‐84 detonations. Conclusions δ 13 C and δ 15 N values can be effectively measured on explosive compounds recovered in residues following detonations. Residue δ 13 C and δ 15 N values can be linked to δ values of undetonated explosive compounds. Additional field studies should be conducted to verify these results.
ABSTRACT Rationale Direct analysis in real time (DART) is an open–air ion source that does not require a sample preparation step. Only a few studies have focused on the negative‐ion formation processes for low–polarity molecules. In this study, an ω‐iodo oligo (vinylidene fluoride) telomer of known composition was investigated to elucidate the involved DART ionization mechanisms. Methods A DART ionization source was coupled to a Fourier transform ion cyclotron resonance mass spectrometer (FT–ICR MS), whose high m/z measurement accuracy enabled the unambiguous assignment of molecular formulas to the detected ions. Tandem MS experiments were performed to confirm these assignments. Data interpretation and visualization were carried out using Kendrick plots. Results Numerous VDF telomer distributions were identified, each attributed to C 6 F 13 (C 2 H 2 F 2 ) n I or C 6 F 13 (C 2 H 2 F 2 ) n H, either in their deprotonated form or forming adducts or associations with I − , O 2 •− , NO 2 − , NO 3 − , HCO 2 − , HCO 3 − , CO 3 •− , and HCO 4 − . The [M + HCO 3 ] − and [M + HCO 4 ] − anions were the most abundant, highlighting the crucial role of carbonate–linked anions in the DART ionization of PVDF. This observation was further supported by neutral losses corresponding to H 2 CO 3 and H 2 CO 4 in tandem mass spectrometry experiments. Furthermore, the oxidative properties of HCO 4 − were emphasized. These results demonstrated the need to reinterpret data obtained in a previous DART‐MS study on PVDF samples with unknown end groups. Conclusions The complex and diverse ionization processes observed in the negative DART ionization of fluorinated telomers underline the need for careful data interpretation. The uncommon [M + HCO 3 ] − and [M + HCO 4 ] − adducts were found to play a significant role in the ionization of iodinated PVDF.
ABSTRACT Rationale Secondary ion mass spectrometry (SIMS) has been widely applied to 26 Al– 26 Mg dating of the earliest Solar System solids. Both 16 O − and 16 O 2 − primary beams have been used for such analyses, yet the influence of primary ion beam species on secondary ion useful yields and matrix effects has not been systematically evaluated. A better understanding of these effects is essential for improving the accuracy and precision of 26 Al– 26 Mg dating. Methods For standard materials used in 26 Al– 26 Mg dating, we determined sputter rates, useful yields, and matrix effects on the relative sensitivity factors (RSFs) of 27 Al/ 24 Mg and the instrumental mass fractionations (IMFs) of Mg isotopes under the 16 O − and 16 O 2 − primary beam conditions using SIMS. The sputter rates were evaluated using a rastered beam, whereas the other parameters were obtained under the actual analytical conditions employed for 26 Al– 26 Mg dating. Results The 16 O 2 − primary beam condition generally provided higher useful yields than the 16 O − condition, except for anorthite and melilite analyzed under low ion current conditions. Variations in RSFs among samples of the same mineral species but with different compositions were either larger or smaller between the 16 O − and 16 O 2 − conditions, depending on the mineral species, whereas IMF variations tended to be slightly larger under the 16 O 2 − condition than under the 16 O − condition. Conclusions Our results highlight the necessity of carefully evaluating useful yields, along with the behaviors of RSF and IMF, when optimizing analytical conditions for SIMS‐based 26 Al– 26 Mg dating, as well as for other isotopic systems that can be analyzed using SIMS.
