Liquid Chromatography-tandem Mass Spectrometry Research Articles

Placental Methylglyoxal in Preeclampsia: Vascular and Biomarker Implications.

Preeclampsia is a multifaceted syndrome that includes maternal vascular dysfunction. We hypothesize that increased placental glycolysis and hypoxia in preeclampsia lead to increased levels of methylglyoxal (MGO), consequently causing vascular dysfunction. Plasma samples and placentas were collected from uncomplicated and preeclampsia pregnancies. Uncomplicated placentas and trophoblast cells (BeWo) were exposed to hypoxia. The reactive dicarbonyl MGO and advanced glycation end products (Nε-(carboxymethyl)lysine [CML], Nε-(carboxyethyl)lysine [CEL], and MGO-derived hydroimidazolone [MG-H]) were quantified using liquid chromatography-tandem mass spectrometry. The activity of GLO1 (glyoxalase-1), that is, the enzyme detoxifying MGO, was measured. The impact of MGO on vascular function was evaluated using wire/pressure myography. The therapeutic potential of the MGO-quencher quercetin and mitochondrial-specific antioxidant mitoquinone mesylate (MitoQ) was explored. MGO, CML, CEL, and MG-H2 levels were elevated in preeclampsia-placentas (+36%, +36%, +25%, and +22%, respectively). Reduced GLO1 activity was observed in preeclampsia-placentas (-12%) and hypoxia-exposed placentas (-16%). Hypoxia-induced MGO accumulation in placentas was mitigated by the MGO-quencher quercetin. Trophoblast cells were identified as the primary source of MGO. Reduced GLO1 activity was also observed in hypoxia-exposed BeWo cells (-26%). Maternal plasma concentrations of CML and the MGO-derived MG-H1 increased as early as 12 weeks of gestation (+16% and +17%, respectively). MGO impaired endothelial barrier function, an effect mitigated by MitoQ, and heightened vascular responsiveness to thromboxane A2. This study reveals the accumulation of placental MGO in preeclampsia and upon exposure to hypoxia, demonstrates how MGO can contribute to vascular impairment, and highlights plasma CML and MG-H1 levels as promising early biomarkers for preeclampsia.

Pharmacokinetics of Anti-Rheumatic drugs Methotrexate and Tofacitinib with its metabolite M9 in Rats by UPLC-MS/MS.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA). The clinical efficacy and safety of an administered tofacitinib, either monotherapy or in combination with conventional synthetic disease-modifying anti-rheumatic drugs, mainly methotrexate (MTX), have been evaluated. The high plasma concentration with delayed medicine clearance may affect the liver and/or kidney functions. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC- MS/MS) method for the quantitative analysis of methotrexate, tofacitinib, and metabolite M9 in plasma of Sprague Dawley (SD) rats was developed, and its effectiveness was validated as well. Methotrexate, tofacitinib, M9 and fedratinib (internal standard, IS) were separated by gradient elution. The chromatography was performed on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 μm) column with the mobile phases of acetonitrile and 0.1% formic acid aqueous solution with different proportions at the flow rate of 0.30 mL/min. In the positive ionization mode, the analyzes were detected using a Xevo TQ-S triple quadrupole tandem mass spectrometer, with the following mass transition pairs: m/z 313.12 → 148.97 for tofacitinib, m/z 329.10 → 165.00 for M9 and m/z 455.12 → 308.05 for methotrexate. The obtained results manifested good calibration linearity over the ranges of tofacitinib at 0.1-100 ng/mL, M9 at 0.05-100 ng/mL, and methotrexate at 0.05-100 ng/mL. The lower limit of quantifications (LLOQs) of methotrexate, tofacitinib and M9 were 0.05 ng/mL, 0.1 ng/mL and 0.05 ng/mL, respectively. Intra-day and inter-day accuracy values were confirmed with a range of -6.3% to 12.7%, while intra-day and inter-- day precision values were ≤14.4%. Additionally, recoveries were greater than 86.5% for each compound without significant matrix effects. The currently established analytical method exhibited great potential for the evaluation of plasma concentrations of methotrexate, tofacitinib and M9 simultaneously, greatly reducing the detection time, which would serve as a supplementary role in formulating dose decisions to achieve personalized treatment, identify drugs that cause adverse reactions and finally, to assess drug-drug interactions on clinical studies.

Optimal transport for automatic alignment of untargeted metabolomic data.

Untargeted metabolomic profiling through liquid chromatography-mass spectrometry (LC-MS) measures a vast array of metabolites within biospecimens, advancing drug development, disease diagnosis, and risk prediction. However, the low throughput of LC-MS poses a major challenge for biomarker discovery, annotation, and experimental comparison, necessitating the merging of multiple datasets. Current data pooling methods encounter practical limitations due to their vulnerability to data variations and hyperparameter dependence. Here, we introduce GromovMatcher, a flexible and user-friendly algorithm that automatically combines LC-MS datasets using optimal transport. By capitalizing on feature intensity correlation structures, GromovMatcher delivers superior alignment accuracy and robustness compared to existing approaches. This algorithm scales to thousands of features requiring minimal hyperparameter tuning. Manually curated datasets for validating alignment algorithms are limited in the field of untargeted metabolomics, and hence we develop a dataset split procedure to generate pairs of validation datasets to test the alignments produced by GromovMatcher and other methods. Applying our method to experimental patient studies of liver and pancreatic cancer, we discover shared metabolic features related to patient alcohol intake, demonstrating how GromovMatcher facilitates the search for biomarkers associated with lifestyle risk factors linked to several cancer types.

Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).

Characterised intron retention profiles in muscle tissue of idiopathic inflammatory myopathy subtypes

ObjectivesIdiopathic inflammatory myopathies (IIMs) are a group of heterogeneous autoimmune diseases. Intron retention (IR) serves as an important post-transcriptional and translational regulatory mechanism. This study aims to identify changes in...

Differential analysis of aqueous humor metabolomics between presenile cataract and senile cataract

Objective: To explore the differences in metabolites and metabolic pathways in the aqueous humor between patients with presenile cataracts and senile cataracts. Methods: This metabolomic study was conducted at Tianjin Medical University Eye Hospital from August 2020 to September 2022. Eight patients with presenile cataracts (8 eyes) and 8 patients with senile cataracts (9 eyes) were included. Data were collected, including age, gender, preoperative uncorrected visual acuity, intraocular pressure, lens dysfunction index, and axial length. Aqueous humor and anterior capsule tissue samples were obtained during cataract surgery. Metabolites in the aqueous humor were detected using Liquid Chromatography-Mass Spectrometry in a non-targeted approach. The principal component analysis, differential analysis, clustering analysis, and correlation analysis were performed to identify differentially expressed metabolites. These metabolites were ranked based on the fold change (FC). The receiver operating characteristic (ROC) curve analysis and metabolic enrichment analysis were used to identify differential pathways and potential biomarkers for presenile cataracts. Immunohistochemistry was conducted on anterior capsule tissues, and pyruvate levels were measured by colorimetry to validate metabolomic results. Results: Patients with presenile cataracts included 7 males and 1 female, with a mean age of (37.50±4.90) years. Patients with senile cataracts were 7 males and 1 female, with a mean age of (73.44±5.22) years. Except for age, there were no significant differences in baseline data (P>0.05). A total of 347 differential metabolites were identified, 10 of which were potential biomarkers for presenile cataract according to the ROC curve analysis (all P<0.05), including propoxycaine (log2FC=7.26), 2-methyl-2, 3, 4, 5-tetrahydro-1, 5-benzodiazepine-4-ketone (log2FC=6.35), l-pyroglutamic acid (log2FC=-1.72), leanly-proline (log2FC=-0.77), and choline (log2FC=-0.56) in the positive ion mode, and N-phenylacetyl glutamine (log2FC=-1.84), pyruvate (log2FC=1.07), ascorbic acid (log2FC=0.92), pseudouracil nucleoside (log2FC=-0.68), and palmitic acid (log2FC=-0.51) in the negative ion mode. The metabolic enrichment analysis identified 72 differential pathways (32 cationic and 40 anionic), with significant differences in glutathione metabolism, cysteine and methionine metabolism, glycolysis or gluconeogenesis, pyruvate metabolism, and the citric acid cycle (P<0.05). The experimental validation showed reduced lactate dehydrogenase and increased pyruvate levels in patients with presenile cataracts (P<0.05). Conclusions: Pyruvate and nine other metabolites may serve as potential biomarkers for presenile cataracts. Pathways involving glutathione metabolism, cysteine and methionine metabolism, glycolysis or gluconeogenesis, pyruvate metabolism, and the citric acid cycle are notably dysregulated in patients with presenile cataracts.

Improved Data Acquisition Settings on Q Exactive HF-X and Fusion Lumos Tribrid Orbitrap-Based Mass Spectrometers for Proteomic Analysis of Limited Samples.

Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Xylitol is prothrombotic and associated with cardiovascular risk.

The pathways and metabolites that contribute to residual cardiovascular disease risks are unclear. Low-calorie sweeteners are widely used sugar substitutes in processed foods with presumed health benefits. Many low-calorie sweeteners are sugar alcohols that also are produced endogenously, albeit at levels over 1000-fold lower than observed following consumption as a sugar substitute. Untargeted metabolomics studies were performed on overnight fasting plasma samples in a discovery cohort (n = 1157) of sequential stable subjects undergoing elective diagnostic cardiac evaluations; subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on an independent, non-overlapping validation cohort (n = 2149). Complementary isolated human platelet, platelet-rich plasma, whole blood, and animal model studies examined the effect of xylitol on platelet responsiveness and thrombus formation in vivo. Finally, an intervention study was performed to assess the effects of xylitol consumption on platelet function in healthy volunteers (n = 10). In initial untargeted metabolomics studies (discovery cohort), circulating levels of a polyol tentatively assigned as xylitol were associated with incident (3-year) major adverse cardiovascular event (MACE) risk. Subsequent stable isotope dilution LC-MS/MS analyses (validation cohort) specific for xylitol (and not its structural isomers) confirmed its association with incident MACE risk [third vs. first tertile adjusted hazard ratio (95% confidence interval), 1.57 (1.12-2.21), P < .01]. Complementary mechanistic studies showed xylitol-enhanced multiple indices of platelet reactivity and in vivo thrombosis formation at levels observed in fasting plasma. In interventional studies, consumption of a xylitol-sweetened drink markedly raised plasma levels and enhanced multiple functional measures of platelet responsiveness in all subjects. Xylitol is associated with incident MACE risk. Moreover, xylitol both enhanced platelet reactivity and thrombosis potential in vivo. Further studies examining the cardiovascular safety of xylitol are warranted.

CSF sphingolipids are correlated with neuroinflammatory cytokines and differentiate neuromyelitis optica spectrum disorder from multiple sclerosis

BackgroundThere is a need for biomarkers of disease progression and therapeutic response in multiple sclerosis (MS). This study aimed to identify cerebrospinal fluid (CSF) lipids that differentiate MS from other...

AI-driven discovery of blood xenobiotic biomarkers in neovascular age-related macular degeneration using iterative random forests.

To investigate the xenobiotic profiles of patients with neovascular age-related macular degeneration (nAMD) undergoing anti-vascular endothelial growth factor (anti-VEGF) intravitreal therapy (IVT) to identify biomarkers indicative of clinical phenotypes through advanced AI methodologies. In this cross-sectional observational study, we analyzed 156 peripheral blood xenobiotic features in a cohort of 46 nAMD patients stratified by choroidal neovascularization (CNV) control under anti-VEGF IVT. We employed Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) for measurement and leveraged an AI-driven iterative Random Forests (iRF) approach for robust pattern recognition and feature selection, aligning molecular profiles with clinical phenotypes. AI-augmented iRF models effectively refined the metabolite spectrum by discarding non-predictive elements. Perfluorooctanesulfonate (PFOS) and Ethyl β-glucopyranoside were identified as significant biomarkers through this process, associated with various clinically relevant phenotypes. Unlike single metabolite classes, drug metabolites were distinctly correlated with subretinal fluid presence. This study underscores the enhanced capability of AI, particularly iRF, in dissecting complex metabolomic data to elucidate the xenobiotic landscape of nAMD and environmental impact on the disease. The preliminary biomarkers discovered offer promising directions for personalized treatment strategies, although further validation in broader cohorts is essential for clinical application.

Proteomic profiling of human dental enamel affected by molar incisor hypomineralisation of different clinical severity grades: an in vitro study.

The aim of this study was to explore the potential to profile and distinguish varying clinical severity grades of MIH, compared to normal enamel, using proteomics. Liquid chromatography-mass spectrometry analyses were conducted on enamel samples of extracted teeth, from 11 children and adolescents, spanning an age range of 6-18years. Enamel powder samples were collected from extracted, third molars (n = 3) and first permanent molars diagnosed with MIH (n = 8). The MIH tooth samples were categorized into subgroups based on clinical severity grade. The data were statistically analyzed using ANOVA and Welch's t test. Teeth affected by MIH exhibited a diverse array of proteins, each with different functions related to dental enamel, distinguishing them from their normal enamel counterparts. The application of microdissection combined with LC-MS techniques has revealed the potential to discern unique proteomic profiles among MIH-affected teeth, characterized by varying clinical severity grades. Both analyzed MIH groups displayed consistent trends in the presentation of biological processes, including underabundance of proteins primarily associated with cell organization and biogenesis. Furthermore, proteins linked to cell death were overabundant in both MIH groups. Proteomics enabled the detection and differentiation of various proteins across different clinical severity grades of MIH.

Transcriptomics and metabolomics reveal hypothalamic metabolic characteristics and key genes after subarachnoid hemorrhage in rats.

Subarachnoid hemorrhage (SAH) is a serious hemorrhagic event with high mortality and morbidity. Multiple injurious events produced by SAH can lead to a series of pathophysiologic processes in the hypothalamus that can severely impact patients' life. These pathophysiologic processes usually result in physiologic derangements and dysfunction of the brain and multiple organs. This dysfunction involved multiple dimensions of the genome and metabolome. In our study, we induced the SAH model in rats to obtain hypothalamic tissue and serum. The samples were subsequently analyzed by transcriptomics and metabolomics. Next, the functional enrichment analysis of the differentially expressed genes and metabolites were performed by GO and KEGG pathway analysis. Through transcriptomic analysis of hypothalamus samples, 263 up-regulated differential genes, and 207 down-regulated differential genes were identified in SAH groups compared to Sham groups. In the KEGG pathway analysis, a large number of differential genes were found to be enriched in IL-17 signaling pathway, PI3K-Akt signaling pathway, and bile secretion. Liquid chromatography-mass spectrometry metabolomics technology was conducted on the serum of SAH rats and identified 11 up-regulated and 26 down-regulated metabolites in positive ion model, and 1 up-regulated and 10 down-regulated metabolites in negative ion model. KEGG pathways analysis showed that differentially expressed metabolites were mainly enriched in pathways of bile secretion and primary bile acid biosynthesis. We systematically depicted the neuro- and metabolism-related biomolecular changes occurring in the hypothalamus after SAH by performing transcriptomics and metabolomics studies. These biomolecular changes may provide new insights into hypothalamus-induced metabolic changes and gene expression after SAH.

A novel standard-free detection of adulteration method for sildenafil derivatives in dietary supplements.

The rapid and accurate detection of illegal adulteration of chemical drugs into dietary supplements is a big challenge in the food chemistry field. Detection of compounds without a standard reference is even more difficult; however, this is a common situation. Here in this study, a novel "standard-free detection of adulteration" (SFDA) method was proposed and phosphodiesterase-5 inhibitor derivatives were used as an example to figure out the possibility and reliability of this SFDA method. After analysis by quadrupole coupled time of flight-tandem mass spectrometry detection and multivariable statistics, six common fragment ions were chosen to indicate whether adulteration was present or not, while 20 characteristic fragment ions indicated whether adulteration was by nitrogen-containing heterocycles or by anilines. Furthermore, the quantitative methods were conducted by high-performance liquid chromatography-tandem mass spectrometry. In a word, this strategy allows for a quick determination of dietary supplement adulteration without any need for standard materials, improving the efficacy of food safety testing.

Epitope-Shared Hapten Enhancing Antibody Polyreactivity for Simultaneous Immunochromatography of Oxyphenisatin Adulterants.

Given the significant threat posed by oxyphenisatin adulterants (OPHs) in weight-loss foods, simultaneous analysis of the OPHs is necessary. Herein, four novel haptens based on the general epitope shared among the OPHs were raised by computer-aided chemical modeling prediction, with the expectation of eliciting antibody responses targeting three of the OPHs. One obtained monoclonal antibody (mAb) showed maximal half-inhibitory concentration (IC50) of 0.40-12.11 ng/mL for OPHs. The key interaction forces responsible for the corecognition of the OPHs were revealed by the intrinsic molecular mechanism. The developed immunochromatography (ICA) indicated a detection capability for screening (CCβ) for OPHs estimated to be 5-600 ng/g in jelly, candy tablets, and oral liquid. Furthermore, the analysis of 15 real samples by our method showed a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our research not only presented a rapid approach for identifying OPHs adulteration but also proposed an effective hapten prediction strategy to enhance antibody polyreactivity.

Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia–Reperfusion Injury in Rats

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia–reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli’s four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.Graphical In a 4-VO rat model, 119 urinary proteins demonstrated significant changes associated with MH. MH is enriched in endopeptidase activity, inflammatory response, oxidative stress, etc, and significantly reversed changes in 12 DEPs. FZD1 and B2M are thought to be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

4-Ethylphenyl Sulfate Detection by an Electrochemical Sensor Based on a MoS2 Nanosheet-Modified Molecularly Imprinted Biopolymer.

One of the gut-derived uremic toxins 4-ethylphenyl sulfate (4-EPS) exhibits significantly elevated plasma levels in chronic kidney diseases and autism, and its early quantification in bodily fluids is important. Therefore, the development of rapid and sensitive technologies for 4-EPS detection is of significant importance for clinical diagnosis. In the current work, the synthesis of a molecularly imprinted biopolymer (MIBP) carrying 4-EPS specific cavities only using the biopolymer polydopamine (PDA) and molybdenum disulfide (MoS2) nanosheets has been reported. The fabricated electrode was prepared using screen-printed carbon electrodes on a polyvinyl chloride substrate. The synthesized material was characterized using several techniques, and electrochemical studies were performed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The DPV technique for the electrochemical sensing of 4-EPS using the fabricated sensor (PDA@MoS2-MIBP) determined a sensitivity of 0.012 μA/ng mL/cm2 and a limit of detection of 30 ng/mL in a broad linear range of 1-2200 ng/mL. Also, the interferent study was performed to evaluate the selectivity of the fabricated sensor along with the control and stability study. Moreover, the performance of the sensor was evaluated in the spiked urine sample, and a comparison was made with the data obtained by ultraperformance liquid chromatography-tandem mass spectroscopy.

Urinary Metabolite Diagnostic and Prognostic Liquid Biopsy Biomarkers of Lung Cancer in Non-smokers and Tobacco Smokers.

Non-smokers account for 10-13% of all lung cancer cases in the United States. Etiology is attributed to multiple risk factors including exposure to secondhand smoking, asbestos, environmental pollution, and radon, but these exposures are not within the current eligibility criteria for early lung cancer screening by low-dose computed tomography (LDCT). Urine samples were collected from two independent cohorts comprising 846 participants (exploratory cohort) and 505 participants (validation cohort). The cancer urinary biomarkers, creatine riboside (CR) and N-acetylneuraminic acid (NANA) were analyzed and quantified using liquid chromatography-mass spectrometry to determine if non-smoker cases can be distinguished from sex and age-matched controls in comparison to tobacco smoker cases and controls, potentially leading to more precise eligibility criteria for LDCT screening. Urinary levels of CR and NANA were significantly higher and comparable in non-smokers and tobacco smoker cases as compared to population controls in both cohorts. Receiver Operating Characteristics (ROC) analysis for combined CR and NANA levels in non-smokers of the exploratory cohort resulted in better predictive performance with the area under the curve (AUC) of 0.94, whereas the validation cohort non-smokers had an AUC of 0.80. Kaplan-Meier survival curves showed that high levels of CR and NANA were associated with increased cancer-specific death in non-smokers as well as tobacco smoker cases in both cohorts. Measuring CR and NANA in urine liquid biopsies could identify non-smokers at high risk for lung cancer as candidates for LDCT screening and warrant prospective studies of these biomarkers.

High-throughput mass spectrometry maps the sepsis plasma proteome and differences in patient response.

Sepsis, the dysregulated host response to infection causing life-threatening organ dysfunction, is a global health challenge requiring better understanding of pathophysiology and new therapeutic approaches. Here, we applied high-throughput tandem mass spectrometry to delineate the plasma proteome for sepsis and comparator groups (noninfected critical illness, postoperative inflammation, and healthy volunteers) involving 2612 samples (from 1611 patients) and 4553 liquid chromatography-mass spectrometry analyses acquired through a single batch of continuous measurements, with a throughput of 100 samples per day. We show how this scale of data can delineate proteins, pathways, and coexpression modules in sepsis and be integrated with paired leukocyte transcriptomic data (837 samples from n = 649 patients). We mapped the plasma proteomic landscape of the host response in sepsis, including changes over time, and identified features relating to etiology, clinical phenotypes (including organ failures), and severity. This work reveals subphenotypes informative for sepsis response state, disease processes, and outcome; identifies potential biomarkers; and advances opportunities for a precision medicine approach to sepsis.

Pharmacokinetics and Tissue Distribution of Itampolin A following Intragastric and Intravenous Administration in Rats Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Itampolin A, a natural brominated tyrosine alkaloid isolated from the sponge Iotrochota purpurea, has been shown to have good inhibitory effects in lung cancer cells as a p38α inhibitor. A simple, sensitive, and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been established, validated, and applied to the study of the pharmacokinetics and tissue distribution of itampolin A following intragastric and intravenous administration. Itampolin A and theophylline (internal standard, IS) were extracted by the simple protein precipitation technique using methanol as the precipitating solvent. Chromatographic separation was achieved by using the optimized mobile phase of a 0.1% formic acid aqueous solution and acetonitrile in the gradient elution mode. Itampolin A and IS were detected and quantified using positive electrospray ionization in the multiple reaction monitoring mode with transitions of m/z 863.9 → 569.1 for itampolin A and m/z 181.1 → 124.1 for IS, respectively. The assay exhibited a linear dynamic range of 1-1600 ng/mL for itampolin A in biological samples and the low limit of quantification was 1 ng/mL. Non-compartmental pharmacokinetic parameters indicated that itampolin A was well-absorbed into the systemic circulation and rapidly eliminated after administration. The apparent distribution volume of itampolin A was much higher after intragastric administration than that after intravenous administration. A tissue distribution study showed that itampolin A could be detected in different tissues and maintained a high concentration in the lung, which provided a material basis for its effective application in lung cancer. The pharmacokinetic process and tissue distribution characteristics of imtapolin A were expounded in this study, which can provide beneficial information for the further research and clinical application of itampolin A.

A Comparative Analysis of Two Commonly Used FDA-Approved Immunoassays for Fentanyl Detection.

Given the opioid epidemic, fentanyl screening in urine has become increasingly important. Immunoassays remain the most common screening methodology due to the high throughput and ease of integration into automated chemistry systems. The fentanyl ARK II from Ark Diagnostics is a widely used immunoassay, while a novel fentanyl assay called FEN2 by Lin-Zhi has become available on the Roche platform. Here, we evaluate and compare their performance. Four hundred and thirty-four urine samples were analyzed for fentanyl across the Lin-Zhi FEN2 and ARK II assays on the Cobas c502 platform. Samples were analyzed immediately upon request for drug of abuse screening or frozen for subsequent analysis. For confirmation testing, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with a limit of detection of 1 ng/mL for fentanyl/norfentanyl was used. Any sample with either fentanyl or norfentanyl above the LC-MS/MS cutoff was deemed positive. The ARK II had 11 false negatives and 7 false positives, while the Lin-Zhi FEN2 had 12 false negatives and 2 false positives. This resulted in ARK II having a sensitivity and specificity of 90.4% and 97.8% respectively, while Lin-Zhi FEN2 had a sensitivity and specificity of 89.5% and 99.4%. Both the ARK II and Lin-Zhi FEN2 immunoassays detected fentanyl well. Overall, the Lin-Zhi assay had slightly better specificity than ARK II, in our data set. While some discrepant results were observed between the 2 immunoassay systems, most occurred near the immunoassay detection cutoffs.