Journal of Mass Spectrometry

The exposure to chemical pesticides is one of the world's concerns, especially in Vietnam, which is becoming a key player in global agriculture. The chronic long-term exposure to pesticides, especially organophosphorus groups poses increased health risks such as cancer and related diseases due to their hazardous metabolites. The characterization of urinary pesticides is essential to understand the pesticide exposure patterns. Therefore, this research aims to develop a liquid chromatography–tandem mass spectrometry procedure for the quantification of six dialkyl phosphate metabolites of organophosphorus pesticides based on simulated human urine containing dialkyl phosphates and urine samples of organophosphorus pesticides-exposed farmers in An Giang province of Vietnam. As a result, a highly sensitive procedure with a negative ion atmospheric pressure chemical ionization source, fosfomycin as internal standard and multireaction monitoring, was successfully validated in compliance with international guidelines for simultaneous quantitative determination of six dialkyl phosphates in human urine samples. Molecular and fragmented ions for quantification were consistent with the standard spectrum. The linear ranges of DMP, DEP, DMTP, DETP, DMDTP, and DEDTP were 5.29–1000.58, 5.10–1000.19, 5.10–1000.20, 5.06–1000.11, 5.06–1000.11, 5.30–1000.60, and 5.06–1000.12 ng/mL, respectively. The validation results showed that the selectivity, intraday and interday precision and accuracy, matrix effect, carry over, dilution, and stability of all the analytes were in the acceptable range. In total, 383 spot urine samples from people working with pesticides were satisfactorily analyzed by the proposed procedure. Over 80% of farmers were detected with at least one organophosphate metabolite, especially DEDTP with high concentrations, up to 5015.0 ng/mL, which alerts the high likelihood of pesticide exposure in the community of rural areas in Vietnam.

Danzhi Jiangtang capsule (DJC) is a traditional Chinese medicine prescription that has been clinically used to treat Type 2 diabetes mellitus and its complications. However, research on the chemical compounds present in DJC remains limited. In this study, an analytical strategy based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was developed for the rapid and systematic characterization of chemical compounds in DJC. Firstly, a DJC self-built database was established, and UPLC-Q-TOF/MS was applied for comprehensive profiling of DJC's chemical compounds. Then, R language combined with MZmine was used for data preprocessing to construct the ion information list and extract effective data. Finally, the compounds were identified by multiple data processing techniques (multiple-point screening mass defect filtering [MDF], extracted ion chromatogram [EIC], neutral loss filter [NLF], diagnostic fragment ion filtering [DFIF], and direct identification method [including retention time, fragment behavior and reference substances]). Eventually, 137 compounds were characterized from DJC, including 19 monoterpenoids, 26 triterpenoids, 8 flavonoids, 12 iridoids, 7 phenylethanoid glycosides, 8 acetophenones, 23 organic acids, 2 violet ketones, 13 cyclic peptides, 8 alkaloids, 2 fatty acids, and 9 other compounds. Among these, 16 compounds were verified using reference substances. The study indicated that the analytical strategy established in this study effectively supports the in-depth study of DJC's chemical constituents and provides essential data for subsequent in vivo studies.

Pulmonary fibrosis (PF) is a chronic and progressive lung disease with fatal consequences. The study of PF is challenging due to the complex mechanism involved, the need to understand the heterogeneity and spatial organization within lung tissues. In this study, we investigate the metabolic heterogeneity between two forms of lung fibrosis: idiopathic pulmonary fibrosis (IPF) and silicosis, using advanced spatially-resolved metabolomics techniques. Employing high-resolution mass spectrometry imaging, we spatially mapped and identified over 260 metabolites in lung tissue sections from mouse models of IPF and silicosis. Histological analysis confirmed fibrosis in both models, with distinct pathological features: alveolar destruction and collagen deposition in IPF, and nodule formation in silicosis. Metabolomic analysis revealed significant differences between IPF and silicosis in key metabolic pathways, including phospholipid metabolism, purine/pyrimidine metabolism, and the TCA cycle. Notably, phosphocholine was elevated in silicosis but reduced in IPF, while carnitine levels decreased in both conditions. Additionally, glycolytic activity was increased in both models, but TCA cycle intermediates showed opposing trends. These findings highlight the spatial metabolic heterogeneity of PF and suggest potential metabolic targets for therapeutic intervention. Further investigation into the regulatory mechanisms behind these metabolic shifts may open new avenues for fibrosis treatment.

In the current scenario, herbal raw materials are identified via morphotaxonomy, microscopic pharmacognosy, or DNA barcoding. However, these methods do not reveal their chemical integrity, while plant raw materials play a crucial role in the quality of plant-based medicine. To overcome this limitation, we used a mass spectrometry-based method to identify 30 botanicals. This assay followed a standard operating procedure (SOP) from sample preparation to the reference library's mass spectrum fingerprint (MSFP) search. The MS1 score showed a similarity index between the input data and the reference mass spectrum. A more than 50% MS1 score was the critical threshold for accurately identifying botanicals based on their chemical integrity. Interestingly, the analysis of 30 different plant species yielded no false results. The results were 100% accurate and selective for tested botanical samples. However, we found that the standard deviation of analytical assays and biological replicates was ± 3.5 and ± 6.3 (MS1 score) for all analyzed samples, respectively. Intraspecies variability showed MS1 scores > 50% ± 10, whereas interspecies variability was observed with MS1 scores < 50% ± 10. The MS1 score was observed, dependent on the plant species, ranging from 53.00% (± 2.65) to 89.76% (± 4.08). In addition, the method was tested to see how seasonal and geographical changes affected search results. The MS1 score changed by less than 15%. We simultaneously created a chemical barcode (unique molecular weight sequence) for each plant species to validate search results and ensure the reliable identification of botanicals.

Sulfides are ubiquity in atmosphere and can convert to be H2SO3/H2SO4, which could affect the inflammatory responses induced by ozone. However, the mixing effect and mechanism of H2SO3/H2SO4 and ozone at molecular-level on the lung surface is still indefinable. Herein, using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) monolayer as a model, effects of H2SO4/H2SO3 on interfacial ozonolysis of phospholipids were explored. Both H2SO4 and H2SO3 could decrease ozonolysis efficiencies of POPG and showed a remarkable concentration dependence. The main components of H2SO4 and H2SO3 in investigated system and their effects on POPG ozonolysis were separately explored, and the mechanism was proposed. The observed decrease of ozonolysis efficiencies resulted from POPG hydrolysis induced by H+ and reactive activity of HSO3 − and SO3 2− towards ozone. The hydrolysis of POPG could provide oleic acids, which further lowered the ozonolysis efficiency. In addition, the efficiency of POPG ozonolysis in H2SO3 case was lower than that in H2SO4 case, and the self-sacrificing oxidation of HSO3 − and SO3 2− by ozone was responsible for this process. Considering extra phospholipids in epithelial lining fluid of lung, the short-term or low concentration exposure of H2SO4/H2SO3 was thought to trigger the self-protection of lung. However, the long-term or high concentration exposure would lead to irreversible damage.

Polo-like kinase 1 (Plk1) is a serine/threonine kinase involved in regulating the cell cycle. It is activated by aurora kinase B along with the cofactors Borealin, INCE, and survivin. Plk1 is involved in the development of resistances to chemotherapeutics such as doxorubicin, Taxol, and gemcitabine. It has been shown that patients with higher levels of Plk1 have lower survival rates. Onvansertib is a competitive ATP inhibitor for Plk1 in clinical trials for the treatment of tumors and has recently entered a trial for the treatment of KRAS mutant colorectal cancers (CRCs). In this study, we conducted an untargeted liquid chromatography–mass spectrometry (LC–MS) proteomics study as well as an untargeted lipidomics analysis of HCT 116 spheroids treated with onvansertib over a 72-h treatment time-course experiment. Mass spectrometry imaging (MSI) showed that onvansertib begins to accumulate most prominently after 12 h of treatment and continues to accumulate through 72 h. Proteomic results displayed alterations to cell cycle control proteins and an increasing abundance of aurora kinase B and Borealin. The proteomics data also showed alterations to many lipid metabolism enzymes. The MSI lipidomics data indicated alterations to phosphatidylcholine lipids, with many lipids increasing in abundance over time or increasing until 12 h of onvansertib treatment and decreasing after that time point. In summary, these results suggest that onvansertib is causing cells within the spheroid to halt at a certain phase of the cell cycle in accordance with previous literature. Our findings suggest the S phase is likely interrupted, with observed alterations in cell cycle control proteins and PC lipid abundance.

Oral fluid sampling offers advantages over other biological matrices, mainly due to its noninvasive procedure avoiding privacy intrusion. The fully automated sample preparation procedure is based on salting-out assisted liquid–liquid extraction (SALLE) combined with high-efficiency LC-MS/MS methods for both screening and confirmation of 37 drugs and incorporates novel features enabling direct injection of acetonitrile extracts into an innovative chromatographic system. The methods' drug panel includes opioids, benzodiazepines, benzodiazepine-like drugs, cannabinoids, and stimulants. A full method validation was performed using OF/buffer from Greiner Bio-ONE International and Quantisal saliva collection devices. The validation included assessments of linearity, sensitivity, precision, accuracy, extraction recovery, matrix effects, process efficiency, stability, and carryover. All compounds demonstrated linearity across the concentration range 1–25 ng/mL, with R 2 ≥ 0.99. Both methods' limit of detection ranged between 0.001 and 0.03 ng/mL, and the limit of quantification ranged between 0.02 and 0.09 ng/mL. Precision was ≤ 14.8% for screening and ≤ 8.5% for the confirmation method. Accuracy was ± 13.6% for screening and ± 8.7% (except at 0.5 and 1 ng/mL, where it was ± 25.3% and ± 17.6%, respectively) for the confirmation method. Extraction recoveries ranged from 40.0% to 95.1%, except for hydromorphone (27.4%) and morphine (34.4%). Although matrix effects were observed for a large number of compounds to varying degrees, they were largely compensated for by the use of deuterium- and 13C-labeled internal standards (IS). IS-corrected overall process efficiency ranged from 100.7% to 119.1% with precision (CV%) ≤ 10.8% for all compounds. Spiked calibrators and QC samples in OF were stable in autosampler for up to 72 h and in the freezer for 3 days. Methanol working solutions were stable for 6 months. No significant carryover was observed. The methods have been successfully implemented in the routine analysis of approximately > 1000 samples per month since March 2024.

Direct analysis of aromatic glycosidic precursors in plants has posed an analytical challenge for decades. Traditional techniques, such as SPE-GC/MS, primarily provided information on volatile aglycones released through hydrolysis. However, the application of high-resolution mass spectrometry combined with liquid chromatography has enabled the direct analysis of intact glycosides without the need for derivatization or hydrolysis. Advances in soft ionization methods, such as DART, offer a novel approach to exploring the hidden aromatic potential in grapes without chromatographic separation. In this work, we present a novel and rapid method for screening aromatic glycosidic precursors in white grapes using high-resolution mass spectrometry (Orbitrap) combined with the soft ionization DART method with nitrogen plasma. Optimization of N2-DART ionization parameters, including grid voltage, gas temperature, and Dip-it sampler speed, performed on selected synthetic glycosidic precursors, allowed the establishment of characteristic ionization patterns and evaluation of 15 groups of glycosidic precursors. The results from the profiling analysis using the N2-DART-Orbitrap-MS method are comparable to those obtained by HPLC/Orbitrap-MS method. This new analytical approach, N2-DART-Orbitrap-MS, reduces drastically analysis time by eliminating the need for chromatographic separation when screening glycoside precursors, uses a convenient Dip-it tips for sampling. It also allows for deeper exploration of ionization using nitrogen plasma, applied for the first time in the analysis of glycoside precursors, demonstrating the applicability of this method for the rapid characterization and screening of glycosidically bound aroma compounds in plants.

The ion collision cross section (CCS) is closely related to the structural and physical conformation of compounds, making it an ideal parameter for constructing databases. In recent years, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has gained widespread application in CCS measurements thanks to its ultra-high mass resolution and accuracy. Due to the collisions between ions and neutral molecules within the FT-ICR MS cell, the image current decays. Based on this feature, the ion CCS can be precisely calculated by applying corresponding collision models and algorithms. A new time-frequency analysis method is introduced: the flipping-filtering method based on the Levenberg–Marquardt algorithm. Before filtering, the data undergoes flipping and extension, effectively mitigating the issue of signal point waste commonly associated with traditional filtering techniques. This approach ensures a smooth and uninterrupted envelope of the image current signal while significantly enhancing the accuracy of decay factor and ion CCS measurements. Compared to the linewidth correction method and the line shape fitting method, this method exhibits superior noise resistance and resolution capabilities, serving as a valuable adjunct to ion CCS measurement techniques.

Because the manual interpretation of ESI-MS n fragmentation spectra is time-consuming and usually requires expert knowledge, automated annotation is often sought. The fragmentation software ChemFrag enables the annotation of MS n spectra by combining a rule-based fragmentation and a semiempirical quantum chemical approach. In this study, the rule set was extended by 31 cleavage rules and 12 rearrangement rules and used for the interpretation of ESI(+)-MS n spectra of antibiotics, pesticides, and natural products as well as their structural analogs. The fragmentation pathways predicted by ChemFrag for compounds such as 17β-estradiol were confirmed by a comparison with pathways published in other studies. In addition, the annotations were compared with those of the programs MetFrag and CFM-ID , for example, with regard to the number and intensity of annotated fragment ions. Our experiments show that ChemFrag provides reliable and in some cases chemically more realistic annotations for the fragment ions of the investigated compounds. Thus, ChemFrag is a helpful addition to the established in silico methods for the interpretation of ESI(+)-MS n spectra.

Mitapivat is a novel, first-in-class, allosteric activator of pyruvate kinase enzyme. It has been approved by the US FDA in February 2022 for disease modifying treatment of haemolytic anaemia in adults. In the current study, the in vivo metabolites of mitapivat in the rat model were identified using quadrupole-time-of-flight mass spectrometry. A total 20 metabolites were identified, out of which nine metabolites were found to be novel and reported first time in the literature. The study also further refined the chemical structures of some of the reported metabolites. Oxidation, N-dealkylation, oxidation followed by dehydrogenation, and hydrolysis were the major Phase I metabolic pathways of mitapivat. The chief Phase II metabolism pathway was glucuronide conjugation of oxidised and amide hydrolysed metabolites of mitapivat.

Matrix-assisted laser desorption/ionization–imaging mass spectrometry (MALDI–IMS) is applied in drug discovery and development. A high-throughput quantitative MALDI–IMS methodology was developed to confirm whether epertinib is superior to lapatinib in penetrating brain metastases using intraventricular injection mouse models (IVMs) of human EGFR2 (HER2)-positive breast or T790M–EGFR-positive lung cancer cells. A simple calibration curve was prepared for each compound via spotting standard solutions without using blank tissue sections or blank tissues onto the same glass slide as the epertinib or lapatinib brain section samples. Quantitative MALDI–IMS was performed via coating a glass slide with a MALDI matrix solution containing each internal standard solution. The samples of calibration curve and brain section were analyzed using a linear ion trap mass spectrometer with a MALDI ion source. Epertinib and lapatinib responses were strongly linear, with a wide dynamic range and low variation (relative standard deviation [RSD] < 20%) among the individual concentrations. Epertinib and lapatinib were sufficiently extracted from brain sections after oral administration in a breast cancer IVM. The quantitative MALDI–IMS results revealed that the epertinib concentrations administered to the brain sections in the lung cancer IVM were similar to those measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Quantitative MALDI–IMS, owing to its high reproducibility and throughput, is useful for selecting drug candidates in the early stages of discovery and development, enabling efficient and rapid screening of candidate compounds as well as an understanding of the mechanisms of drug efficacy, toxicity, and pharmacokinetics/pharmacodynamics.

Proximity labeling (PL) proteomics has emerged as a powerful tool to capture both stable and transient protein interactions and subcellular networks. Despite the wide biological applications, PL still faces technical challenges in robustness, reproducibility, specificity, and sensitivity. Here, we discuss major analytical challenges in PL proteomics and highlight how the field is advancing to address these challenges by refining study design, tackling interferences, overcoming variation, developing novel tools, and establishing more robust platforms. We also provide our perspectives on best practices and the need for more robust, scalable, and quantitative PL technologies.

In this study, the components of Moringa oleifera (Lam) seed were extracted using an ultrasonic-assisted extraction technique with 70% methanol solution as the solvent. The antioxidant properties of the extract were evaluated through its scavenging abilities against DPPH. and ABTS. The antiproliferative effects of this extract on breast cancer MDA-MB-231, colon cancer SW480, leukemia HL-60, liver cancer SMMC-7721, and lung cancer A549 were assessed using the MTS assay. The distribution of MA, D-trehalose, and rbGlu in the heart, kidney, liver, and spleen of mice in various intervals was visually investigated using MALDI-IMS. In particular, the metabolic pathways of rbGlu were further elucidated. The result shows that rbGlu is metabolized to rbot-Glu in mice within 1 h. This approach further substantiates the use of MALDI-MSI technique in situ for studying the pharmacological mechanisms of bioactive component in natural products.

The fragmentation pathways for amines dissolved in methanol (CH3OH) or deuterated methanol (CD3OD) have been investigated by high-resolution accurate mass gas chromatography mass spectrometry (HRAM-GCMS) or GC-Orbitrap. Primary and secondary amines used in this study were 1,3-dimethylamylamine (1,3-DMAA) and ephedrine hydrochloride (Eph), respectively. For isotopic labeling experiment, 1S, 2R (+) ephedrine-D3 hydrochloride (D3-Eph) was used. Under splitless injection mode at an inlet temperature of 250°C, formaldehyde and its deuterated form were generated from CH3OH and CD3OD, respectively. This was evidenced by the oxonium ions generated from each solvent. When 1,3-DMAA was dissolved in CH3OH or CD3OD, distinct separation between the unreacted amine and condensation product fragments was observed, specifically methylene-imine (M + 12) and deuteromethylene-imine (M + 14) artifacts. More complex condensation patterns for Eph and D3-Eph were observed, attributed to the labile hydrogen/deuterium exchange and gradual deuteration from CH3OH to CD3OD. The fragmentation pathways were supported by the presence of oxazolidine intermediates before forming smaller condensation product fragments. Despite their close retention time and mass, the HRAM data distinguished the isobaric unreacted amine and condensation product fragments produced by Eph and D3-Eph in the coeluting region.

No abstract is available for this article.

Adequate vitamin K is crucial for optimal health. Although vitamin K detection methods have been established using liquid chromatography–tandem mass spectrometry (LC–MS/MS), some limitations remain. Therefore, we aimed to establish a stable and rapid LC–MS/MS method that can quantify phylloquinone (VK1), menaquinones-4 (MK-4), and menaquinones-7 (MK-7) in serum and cerebrospinal fluid and explore its clinical applications. We developed an LC–MS/MS method with atmospheric pressure chemical ionization to quantify and validate its performance according to Clinical Laboratory and Standard Institution standards (C62-Ed2). Serums from 50 healthy individuals and cerebrospinal fluid from 15 patients were collected for clinical application. Sample preparation involved lipase incubation, protein precipitation with ethanol, and liquid–liquid extraction with hexane/ethyl; optimization was performed for sample preparation and LC separation. Linearity was 50–10 000 pg/mL for VK1, MK-4, and MK-7. The total coefficient of variation (%) for VK1, MK-4, and MK-7 ranged from 8.5% to 10.4%, 8.0% to 10.4%, and 7.0% to 11.1%, respectively. Recovery of VK1, MK-4, and MK-7 was 82.3%–110.6%, 92.3%–110.6%, and 89.5%–117.8%, respectively. VK1 and MK-7 were detected in the serum of all 50 healthy subjects, whereas MK-4 was detected in only 13 (26%) subjects. Approximately 53.3% (8/15) had no detectable vitamin K in their cerebrospinal fluid. The developed method exhibited satisfactory performance and was applicable for detecting VK1, MK-4, and MK-7 in serum and cerebrospinal fluid.

Electrospray ionization (ESI) is often the ionization method of choice, particularly for high-mass polar molecules and complexes. However, when analyzing mixtures of analytes, charge state ambiguities and overlap in mass-to-charge (m/z) can arise from species with different masses and charges. While solution-phase conditions can sometimes be optimized to produce relatively low charge states—thereby reducing charge-state ambiguity and m/z overlap—gas-phase methods offer greater control over charge state reduction. For complex mixtures, however, charge state reduction alone often fails to resolve individual components in the mixture. Incorporating a mass-selection step prior to charge state manipulation can simplify the mixture and significantly improve the separation of the components. This general tandem mass spectrometry approach is referred to here as precursor resolution via ion z-state manipulation (PRIZM). Examples of variations of PRIZM experiments date back roughly 25 years and have involved ion/molecule proton transfer reactions, ion/ion proton transfer reactions, ion/ion electron transfer reactions, electron capture reactions, and multiply-charged ion attachment reactions. This tutorial review describes the PRIZM approach and provides illustrative examples using each of the charge state manipulation approaches mentioned above.

In the present work, we have first applied the fast nonlinear damping identification (NDI) method to nondestructively and simultaneously determine the values of mass, size, and charge of biological micro-objects trapped in a quadrupole Paul trap. Here, we used well-studied Lycopodium clavatum individual spores as a test object. For the nondestructive determination of spore parameters, we analyzed their extended orbit trajectory implemented in the nonlinear regime of viscous friction while trapping in a quadrupole Paul trap. The article discusses the prospects of the method proposed for investigating a wide class of biological objects.

Fungal infections are caused by opportunistic pathogens that can be life threatening and have been growing in prevalence. Many clinically relevant pathogens have resistance to or are developing resistance to the commonly used antifungal treatments. Occidiofungin (OCF) is a unique cyclic lipoglycopeptide with a novel structure that includes noncanonical amino acid in its covalent structure. It exhibits broad spectrum antifungal activity and has activity against drug resistant Candida species. Occidiofungin is a fungicidal compound that has a novel mechanism of action in which it disrupts higher order actin structures. Currently, occidiofungin is being developed for use in treating vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). This study describes the development and application of a bioanalytical method for the quantification of occidiofungin in rabbit plasma. Method development was performed to quantify occidiofungin in rabbit plasma after intravaginal administration of a hydrogel containing occidiofungin. The method was validated with a linear range of 30–15 000 ng/mL in rabbit plasma. Precision, accuracy, calibration curve linearity, and stability of drug in plasma were established in quality controls. Extract stability, matrix effects, and recovery of drug in the extract were also determined. This study supported a repeat dose toxicity study in rabbits to determine occidiofungin pharmacokinetics and toxicokinetics. The pharmacokinetic and toxicokinetic primarily showed plasma concentrations of occidiofungin below the limit of quantification (BLOQ), suggesting that OCF-B does not readily cross the vaginal epithelial membrane.