High-performance Liquid Chromatography-tandem Mass Research Articles

Serum metabolites and inflammation predict brain functional connectivity changes in Obsessive-Compulsive disorder.

Currently, our understanding of the metabolic and immune pathways involved in obsessive-compulsive disorder (OCD), as well as the precise mechanisms by which metabolism and immunity impact brain activity and function, is limited. This study aimed to examine the alterations in serum metabolites, inflammatory markers, brain activity, and brain functional connectivity (FC) among individuals with OCD and investigate the relationship between these factors. The study included 55 individuals with moderate-to-severe OCD (either drug-naïve or not taking medication for at least eight weeks) and 54 healthy controls (HCs). The High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) technique was used to detect serum metabolites, whereas the enzyme-linked immunosorbent assay (ELISA) was utilized to identify inflammatory markers. The FC of the brain was investigated using resting-state functional magnetic resonance immaging(rs-fMRI). The findings demonstrated that individuals with OCD exhibited significant alterations in 11 metabolites compared to HCs. In particular, 10 of these metabolites exhibited an increase, while one metabolite displayed a decrease. Additionally, individuals with OCD experienced a marked elevation in the levels of five inflammatory factors (TNF-α, IL-1β, IL-2, IL-6, and IL-12). Rs-fMRI analysis revealed that individuals with OCD exhibited atypical FC in various brain regions, such as the postcentral gyrus, angular gyrus, and middle temporal gyrus. These specific brain areas are closely associated with sensory-motor processing, cognitive control, and emotion regulation. Further stepwise multiple regression analysis revealed that serum metabolite levels, particularly phosphatidylcholine, and inflammatory markers such as IL-1β could predict alterations in brain FC among individuals diagnosed with OCD. In summary, this study uncovered that individuals with OCD exhibit alterations in serum metabolites, inflammatory markers, brain activity, and FC. The findings suggest that these metabolites and inflammatory markers might play a role in the development and progression of OCD by affecting brain activity and the FC of neural networks.

Gut microbiota interacting with vitamin D but not anandamide might contribute to the pathogenesis of preeclampsia: a preliminary study

IntroductionPreeclampsia (PE) is a pregnancy-specific multisystem disorder and a leading cause of maternal and perinatal mortality globally. Despite numerous studies highlighting the potential roles of gut microbiota, anandamide (AEA), and Vitamin D (VitD) in PE, none have established them as reliable biomarkers for predicting disease onset. Moreover, their interactions in late-stage pregnancy women remain poorly understood.MethodsThirty-four preeclamptic patients (called PE group) and thirty-nine matched healthy late-pregnant women (called LP group) were involved in this case-control study. Fecal samples, which were used to acquire the diversity and composition of gut microbiota, were analyzed by 16S rRNA gene sequencing. Plasma AEA concentrations and serum VitD levels were determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively.ResultsIn this study, β diversity but not α diversity significantly differed between the LP and PE groups. Compared with the LP group, the relative abundances of Prevotella, Erysipelotrichaceae_UCG-003, and Dorea were increased dramatically in the PE group, whereas the relative abundances of Subdoligranulum, Parabacteroides, Bacteroides were significantly decreased in the PE group. Furthermore, women with PE had a substantially lower plasma level of AEA and a marked decrease in serum VitD compared to normal late-pregnant women. Lastly, although the serum level of AEA was not significantly correlated with VitD or any of the top 6 marker genera, VitD was significantly negatively correlated with the relative abundance of Dorea, a novel finding in this context.DiscussionThe gut microbiota profile of the PE group was significantly different from that of the LP group. Although no significant correlations were identified between the plasma AEA levels and serum VitD levels or any of the top 6 identified marker genera, a significant negative correlation was observed between VitD and Dorea, indicating VitD and gut microbiota have the potential to be combined targets for early diagnosis and management of PE.

Urinary concentrations of eighteen environmental phenols and the associations with feeding pattern in infants in the first 6 months of life.

Environmental phenols are a group of typical endocrine disruptors, and widely detectable in human breast milk and infant formulas. However, exposures data are scarce in early infancy, a particularly sensitive period to environmental pollutants exposures. We aimed to prospectively assess urinary concentrations of eighteen environmental phenols in infants from birth to 6 months of age and their associations with feeding patterns (breastfeeding, formula and mixed-feeding). This study included 197 mother-infant pairs. Urinary concentrations of six parabens (PBs), seven bisphenols, four benzophenones (BPs) and triclosan were measured in infants at the ages of 3 days, 42 days, 3 and 6 months by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Multivariable linear regression models were applied to evaluate the associations between infant feeding types and urinary phenol concentrations in log10-transformed scale. The detection rates of bisphenol A and S, methyl 4-hydroxybenzoate (MeP), ethyl 4-hydroxybenzoate (EtP), and propyl 4-hydroxybenzoate (PrP) were persistently high (ranged 48.7%-100.0%) across all ages (3 days, 42 days, 3 and 6 months). The detection rates of bisphenol P, B and AP were high (ranged 76.9%-95.9%) at age 3 days, and lower (14.4%-88.0%) at later ages. Compared to exclusive breastfeeding, mixed feeding was associated with 0.32 (95% CI: 0.01, 0.62, p<0.05) higher log (BPA) (μg/g creatinine) at 42 days, 0.69 (0.03, 1.35) higher log (TCS) (μg/g creatinine) at 3 months, 0.54 (0.08, 1.00) higher log (EtP) (μg/g creatinine) at 6 months; formula feeding was associated with 1.30 (0.59, 2.00) and 0.91 (0.22, 1.60) higher log (BPB) (μg/g creatinine) at the age of 42 days and 3 months respectively, 1.19 (0.003-2.37) higher log (BPP) and 0.95 (0.27-1.64) higher log (BPS) (μg/g creatinine) at 3 months. In conclusion, exposures to BPA and its analogs, and parabens (MeP, EtP, and PrP) were extensive in early postnatal life of infants. Breastfeeding might be a safer form of feeding for infants against exposure to some phenols.

Exploring the link between serum betaine levels and hyperuricemia risk in middle-aged and older adults: insights from a prospective cohort study.

Betaine is connected to various cardio-metabolic outcomes, yet its relationship with hyperuricemia remains uncertain. We aimed to longitudinally investigate the association between serum betaine levels and the risk of hyperuricemia in middle-aged and older adults. This study utilized data from the Guangzhou Nutrition and Health Study (GNHS). Participants were enrolled between 2008 and 2010, with follow-ups conducted every three years, comprised an analysis sample of 2204 adults aged 40-75. Baseline serum betaine levels were assessed using high-performance liquid chromatography-tandem mass spectrometry. Serum uric acid (SUA) levels were measured at baseline and every three years thereafter. Linear mixed-effects models (LMEMs) and generalized estimating equations (GEEs) were employed to examine the longitudinal association between serum betaine levels and SUA levels, as well as hyperuricemia risk, respectively. LMEMs indicated that compared to individuals in the lowest quartile (Q1) of serum betaine levels, those in the highest quartile (Q4) exhibited the lowest SUA levels (Q4 vs. Q1: β -8.19, 95% CI -16.32 to -0.06, P-trend = 0.023). Each standard deviation increase in betaine (16.5 µmol/L) was associated to SUA levels decrease of -3.28 (-6.10, -0.45). Results from the GEE model suggested a 19% reduction in the odds of hyperuricemia (OR = 0.81; 95% CI: 0.68, 0.95, P-trend = 0.051) in the Q4 group compared to the Q1 category. Our findings highlight a negative association between serum betaine levels and SUA levels, as well as the risk of hyperuricemia in middle-aged and older adults, which is more pronounced in individuals with better dietary quality. This trial was registered at clinicaltrials.gov as NCT03179657. Website: https://clinicaltrials.gov/ct2/show/NCT03179657.

Facile synthesis of hierarchically flower-like hollow covalent organic frameworks for enrichment and metabolic analysis of benzophenone derivatives in mouse serum.

Benzophenone derivatives (BPs), as synthetic chemicals widely used in personal care products, have drawn increasing attention due to their potential health risks. However, monitoring BPs in biological samples remains challenging due to their complex matrices and the deficiency in sensitivity and selectivity in current methods. Herein, a method combining hierarchically flower-like hollow covalent organic frameworks (HFH-COFs) with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for the enrichment and detection of BPs in serum samples. The HFH-COFs were synthesized at room temperature and employed as an adsorbent due to their advantageous properties. The as-prepared HFH-COFs exhibited high specific surface area (2286.53 m²/g), excellent chemical stability, and good thermal stability, making them ideal for efficient enrichment applications. Under optimized experimental conditions, five BPs were effectively enriched and quantified by HPLC-MS/MS in the range of 50.0-5000.0 ng/L with good linearity (r > 0.9992). The limit of detections was 0.5-10.0 ng/L. Furthermore, HFH-COFs showed high enrichment factors even over multiple adsorption-desorption cycles. This study proposed a reliable and efficient method for monitoring endocrine-disrupting compounds in complex biological samples and highlighted the potential of HFH-COFs as a superior adsorbent material for BP.

Relationship between plasma polymyxin B concentrations and acute kidney injury in critically ill elderly patients: Findings from a prospective study.

The objective of this study was to determine the relationship between the plasma polymyxin B concentration and renal function in elderly patients. This prospective, case-control, observational study included elderly patients who received polymyxin B therapy and were divided into an acute kidney injury (AKI) group and a non-AKI group based on their renal function. We monitored the pharmacokinetics and pharmacodynamics of polymyxin B, including the minimum plasma concentration (Cmin), mean blood drug concentration at steady state (Css,avg), and area under the concentration-time curve across 24 h at steady state (AUCss,24h) in both study groups. The plasma polymyxin concentration was determined using high-performance liquid chromatography-tandem mass spectrometry. The loading doses, Cmin, Css,avg, and AUCss,24h were significantly higher in the AKI group than in the non-AKI group (p < 0.05). Receiver-operating characteristic curve analysis showed that the optimal cutoff values for predicting AKI were 2.94 mg/L for Cmin, 4.14 mg/L for Css,avg, and 99.35 mg·h/L for AUCss,24h, with corresponding sensitivities and specificities ranging from 78.57% to 82.14%. Monitoring plasma polymyxin B concentrations is essential in elderly patients. Keeping the Cmin below 2.94 mg/L, the Css,avg below 4.14 mg/L, and the AUCss,24h below 99.35 mg h/L may help prevent AKI in this population.

Real-world Plasma Exposure of Nirmatrelvir/Ritonavir in Chinese Hospitalized Patients With COVID-19: A Multicenter Retrospective Study.

Nirmatrelvir/ritonavir is licensed for the treatment of mild-to-moderate coronavirus disease (COVID-19) in patients at an increased risk of progression to severe disease. However, data on the real-world plasma exposure to nirmatrelvir/ritonavir remain limited, particularly in Chinese patients. This study aimed to assess the nirmatrelvir/ritonavir trough concentration (Ctrough) and identify its critical factors in hospitalized Chinese patients treated with nirmatrelvir/ritonavir 300 mg/100 mg twice daily over a 5-day course. A high-performance liquid chromatography-tandem mass spectrometry assay was developed and validated to measure the nirmatrelvir/ritonavir Ctrough. Correlation analyses were performed to identify the variables influencing nirmatrelvir/ritonavir Ctrough. Among the 110 patients, 100% had plasma concentrations above the antiviral in vitro 90% effective concentration. The median Ctrough of nirmatrelvir was 4.55 mcg/mL (15.6× 90% effective concentration), ranging from 0.65 to 12.44 mcg/mL. Nirmatrelvir Ctrough in normal and mild renal impairment cohorts were comparable (4.09 ± 1.97 mcg/mL and 4.57 ± 2.21 mcg/mL) but significantly increased in the moderate renal impairment cohort (6.41 ± 2.31 mcg/mL). Sex, age, and obesity were not significantly associated with nirmatrelvir exposure. Nirmatrelvir Ctrough was high in Chinese patients with COVID-19, and therapeutic drug monitoring should not be routinely recommended, except in patients with renal impairment.

Contamination Characterization, Toxicological Properties, and Health Risk Assessment of Bisphenols in Multiple Media: Current Research Status and Future Perspectives

Bisphenols (BPs) are ubiquitous environmental endocrine disruptors that cause various human health hazards and pollute water, soil, and the atmosphere to varying degrees. Although various studies have investigated the pollution characteristics and health hazards of BPs in different media, a systematic review of BPs in the broader environmental context is still lacking. This study highlights the pollution characteristics, detection methods, and risk assessment status of BPs by combining relevant studies from both domestic and international sources, and their environmental distribution characteristics are summarized. The results show that BP pollution is a widespread and complex global phenomenon. Bisphenol A (BPA) remains the predominant component of BPs, which can damage the nervous and reproductive systems. At present, high-performance liquid chromatography–tandem mass spectrometry, high-performance liquid chromatography, and liquid chromatography–tandem mass spectrometry are the main detection methods used for BPs. BPs can also damage the reproductive system, leading to germ cell apoptosis and ovarian damage. Future research should focus on expanding the BP testing repertoire, advancing rapid detection techniques, elucidating toxic mechanisms, conducting comprehensive safety assessments, and developing systematic health risk assessment methods. These efforts will provide a scientific foundation for preventing and controlling emerging pollutants.

Elevated plasma testosterone concentrations from males on testosterone replacement therapy are mitigated with pathogen reduction technology.

Donors on testosterone replacement therapy (TRT) may require frequent whole blood donation due to erythrocytosis, but FDA guidelines prevent the transfusion of plasma-based products from these donors. This study surveyed TRT donor testosterone levels in whole blood components and evaluated a possible mitigation strategy with a pathogen reduction technology using UVA light and compound adsorption device (CAD) steps. Whole blood from male TRT donors and controls were processed into red blood cells and plasma components. Free and total testosterone were measured in 78 TRT donors and 48 controls by high-performance liquid chromatography-tandem mass spectrometry. Pathogen reduction (INTERCEPT Blood System) on pooled plasma components (n = 10) with supraphysiologic testosterone were sampled: before treatment, after UVA illumination, and after CAD incubation. TRT donors had 3.8 and 3.9 times more free testosterone in plasma and red blood cell supernatant, respectively, and 2.3 times more total testosterone in both components than controls (p < .0001). Two controls and 33 TRT donors had supraphysiologic testosterone. The CAD incubation reduced the mean free and total testosterone by 88% (571.72-73.8 pg/mL) and 84% (1498.61-240.59 ng/mL), respectively (p = .0065), but UVA light had no effect (p > .9999). TRT donors had significantly higher testosterone levels than controls. The CAD step in the pathogen reduction process abrogated supraphysiologic testosterone in plasma at or below the reference range. Studies validating testosterone removal from plasma can support the transfusion of pathogen-reduced plasma and platelets from TRT donors.

Licorice and dried ginger decoction inhibits inflammation and alleviates mitochondrial dysfunction in chronic obstructive pulmonary disease by targeting siglec-1.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease. Licorice and dried ginger decoction (LGD) is traditional Chinese medicine prescription with multiple effects. Glycyrrhetinic acid (GA) is the main bioactive components of LGD, which has been proven to have a relieving effect on various inflammatory diseases. Siglec-1 is a cell surface sialoadhesin and has been confirmed to be overexpressed in COPD and facilitate inflammatory reaction. This study is aimed to probe the interaction between LGD, GA, and siglec-1. Cigarette smoke (CS) combined with lipopolysaccharide (LPS) treatment was utilized to construct a COPD rat model. Cigarette smoke extract (CSE) was utilized to induce alveolar macrophage NR8383 to construct a COPD cell model. HE staining was applied for measuring histopathological changes of COPD rats. Enzyme-linked immunosorbent assay (ELISA), reverse transcription real-time polymerase chain reaction (RT-qPCR), and western blot were applied for testing the concentrations and expressions of proinflammatory factors. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS) analysis was utilized to determine the combination between siglec-1 and GA. JC-1 assay was utilized to evaluate mitochondrial function. Reactive oxygen species (ROS) production was tested by dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. LGD treatment notably alleviated lung injury and inflammatory response in COPD rats. In CSE-induced cells, LGD treatment suppressed the contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8. Sialic-acid-binding Ig-like lectin 1 (Siglec-1) expression induced by CS was decreased after LGD treatment. Furthermore, we proved that GA could target siglec-1 to regulate the inflammatory response in COPD rats and cells. Additionally, GA could reduce ROS production and alleviate mitochondrial dysfunction to suppress COPD progression. LGD inhibits inflammation and alleviates mitochondrial dysfunction in COPD by targeting siglec-1.

Tear metabolomics reveals novel potential biomarkers in epithelial herpes simplex keratitis

BackgroundHerpes simplex keratitis (HSK) is a recurrent inflammatory disease of cornea primarily initiated by type I herpes simplex virus infection of corneal epithelium. However, early diagnosis of HSK remains challenging due to the lack of specific biomarkers. This study aims to identify biomarkers for HSK through tear metabolomics analysis between HSK and healthy individuals.MethodsWe conducted a cross-sectional study enrolling 33 participants. Tear samples were collected from one eye of 18 HSK patients and 15 healthy volunteers using Schirmer-strips. Tear metabolomic profiling was performed using high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS). Metabolites were quantified and matched against entries in the human metabolome database (HMDB) and small molecule pathway database (SMPDB) to identify metabolites and metabolic pathways, respectively. Metabolic differences between HSK and control group were determined using multivariate statistical analysis.ResultsA total of 329 metabolites were identified, of which 18 were significantly altered in HSK patients. Notably, 12 metabolites were significantly increased, and 6 were significantly decreased in HSK patients. The changed metabolites were enriched in these pathways: arginine and proline metabolism, phospholipid biosynthesis, alpha linolenic acid and linoleic acid metabolism, retinol metabolism. To assess the potential utility of tear biomarkers, a predictive model was developed combining 4 metabolites (AUC = 0.998 [95%CI: 0.975, 1]): D-proline, linoelaidic acid, plantagonine, and phosphorylcholine.ConclusionsOur study establishes that HSK has a distinctive metabolomic profile, with 4 key elements maybe emerging as potential biomarkers for diagnostic purposes. These findings may provide novel insights into early and rapid diagnosis of HSK.

Pharmacokinetics and bioequivalence evaluation of two oral formulations of topiramate tablets in healthy Chinese male volunteers under fasting and fed conditions.

Topiramate is an antiepileptic drug (AED) that is effective in treating various types of epilepsy. This study evaluated the bioequivalence and safety of two topiramate tablets in healthy Chinese subjects under fasting and fed conditions. We designed an open-label, randomized, single-dose, two-period, crossover trial protocol. Two independent trials were conducted, each including 26 volunteers. In the fed trial, subjects were randomly assigned to two groups (1:1 ratio) to receive either 100mg of test formulation or reference formulation of topiramate. After a 21-day washout period, crossover administration was implemented. The fasting trial design was similar to that of the fed trial. Plasma concentrations of topiramate were determined using a validated method (high-performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS). Pharmacokinetic (PK) parameters such as maximum plasma drug concentration (Cmax), time to reach maximum concentration (Tmax), and the area under the plasma concentration-time curve from time 0 to 72h (AUC0-72) were calculated using a non-compartment model to evaluate the bioequivalence of two formulations. The safety of volunteers was monitored during the entire study. In the fasting trial, 90% confidence intervals (CIs) of geometric mean ratios (GMRs) of the test to reference formulations were 101.26-112.94% for Cmax and 98.50-102.69% for AUC0-72, all within the recognized bioequivalence range of 80.00-125.00%. In the fed trial, 90% CIs of GMRs of the test to reference formulations were 92.08-101.54% for Cmax and 95.81-99.79% for AUC0-72, both were within the bioequivalence range of 80.00-125.00%. Research findings indicated that the two topiramate formulations were bioequivalent and had a favorable safety profile.

Bimetallic metal-organic framework based dispersive solid phase extraction followed by using a carbon dot solution as the elution solvent; application in the extraction of imidacloprid and acetamiprid from pepper samples.

This study focuses on the dispersive solid phase extraction technique for the efficient extraction and enrichment of imidacloprid and acetamiprid from pepper samples. A synthesized sorbent was used for this purpose. Once the target analytes were adsorbed, the sorbent was separated using a centrifuge and the analytes were desorbed using a carbon dot solution. After centrifugation, a portion of the eluent was injected into a high-performance liquid chromatography-tandem mass spectrometry system for analysis of the analytes. Several parameters affecting the performance of the method were investigated, such as the amount of sorbent, the type and volume of elution solvent, pH, and so on. By optimizing these parameters, the method showed favorable results under the following conditions: a sample solution volume of 5 mL, a sorbent amount of 10 mg, vortexing for 2 min, 150 μL of carbon dot solution as the elution solvent, a pH of 10, and 3 min of agitation during the desorption step. Notably, this optimized method exhibited high extraction recoveries ranging from 67% to 78% as well as low detection limits (0.44 μg L-1 and 0.32 μg L-1) and quantification limits (1.4 μg L-1 and 1.0 μg L-1) for imidacloprid and acetamiprid, respectively. To validate the effectiveness of the method, four pepper samples were successfully analyzed and no analyte was detected in any of them using this approach. Overall, the developed DSPE method represents a reliable and sensitive technique for the extraction and analysis of the studied pesticides in pepper samples.

Contaminated Characteristics Variation in Different Aquaculture Modes: A Case Study in Northern China

Various aquaculture modes have been developed to satisfy the growing demands of aquatic products. The contaminated characteristics may distribute along with the aquaculture modes, threatening the ecological environment to varying degrees. Herein, the five most common aquaculture modes (small-scale intensive mode, extensive free-range mode, concentrated contiguous mode, funnel-type mode, and recirculating aquaculture system) were selected to study the contaminated characteristics (including nine kinds of water quality parameters and eight kinds of antibiotics) in Henan Province, a province in northern China, and analysed using high-performance liquid chromatography tandem secondary mass spectrometry (HPLC–MS/MS). The funnel-type mode, as a unique mode developed in Henan Province, appears highest in nutrient content, wherein TN and TP concentrations reach 29.28 mg/L and 2.20 mg/L, respectively. The small-scale intensive mode has the highest average antibiotic concentration in five different aquaculture modes, with a concentration of 502 ng/L. Overall, the most abundant antibiotic was quinolones (QNs), followed by sulfonamides (SAs), chloramphenicols (CAs), and tetracyclines (TCs). Pearson correlation analysis showed that ENR had a strong positive correlation with TN, TP, and Zn, indicating the enrofloxacin (ENR) may have existed as the addictive in aquaculture feed. Moreover, the risk quotient (RQ) analysis indicated that ENR posed a medium to high risk, highlighting the importance of antibiotics man-agement in aquaculture. This work provides theoretical guidance for the formulation of aquaculture water pollutant control of different aquaculture modes.

Challenges and Insights in Absolute Quantification of Recombinant Therapeutic Antibodies by Mass Spectrometry: An Introductory Review.

This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer and inflammatory, infectious, and autoimmune diseases. We trace their development from hybridoma technology and the first murine mAbs in 1975 to today's chimeric and fully human mAbs. With increasing commercial relevance, the absolute quantification of mAbs, traceable to an international standard system of units (SI units), has attracted attention from science, industry, and national metrology institutes (NMIs). Quantification of proteotypic peptides after enzymatic digestion using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has emerged as the most viable strategy, though methods targeting intact mAbs are still being explored. We review peptide-based quantification, focusing on critical experimental steps like denaturation, reduction, alkylation, choice of digestion enzyme, and selection of signature peptides. Challenges in amino acid analysis (AAA) for quantifying pure mAbs and peptide calibrators, along with software tools for targeted MS data analysis, are also discussed. Short explanations within each chapter provide newcomers with an overview of the field's challenges. We conclude that, despite recent progress, further efforts are needed to overcome the many technical hurdles along the quantification workflow and discuss the prospects of developing standardized protocols and certified reference materials (CRMs) for this goal. We also suggest future applications of newer technologies for absolute mAb quantification.

Levels and Profile of Tetrodotoxins in Spawning Cephalothrix mokievskii (Palaeonemertea, Nemertea): Assessing the Potential Toxic Pressure on Marine Ecosystems.

The ribbon worms of the closely related species Cephalothrix simula, Cephalothrix cf. simula, and Cephalothrix mokievskii, representing the C. simula species complex, possess high concentrations of tetrodotoxin (TTX) and its analogues in all developmental stages from eggs to adults. It has recently been suggested that the eggs and larvae of these animals can be a source of tetrodotoxins (TTXs) for other aquatic organisms. In the current study, TTXs in mature and post-spawning individuals and in the eggs of C. mokievskii were identified using high-performance liquid chromatography-tandem mass spectrometry. For the first time, the quantity and profile of TTXs that these nemerteans released into the environment during spawning were estimated. We showed that the spawning C. mokievskii females released significant amounts of TTX and 5,6,11-trideoxyTTX with their eggs; these levels were sufficient for the potential toxification of marine bioresources. The issues surrounding the monitoring of TTXs in commercial marine animals, and collecting at the sites of the spawning of nemerteans from the C. simula species complex, are discussed.

Ceftriaxone-associated dysbiosis decreases voriconazole bioavailability by upregulating intestinal P-glycoprotein expression through activation of the Nrf2-mediated signalling pathway.

The purpose of this study was to investigate the effect of intestinal dysbiosis on the bioavailability of voriconazole and to explore any underlying mechanisms. Sprague-Dawley rats were randomly divided into two groups: a normal control group and a ceftriaxone-associated dysbiotic group. The composition of the intestinal flora was examined using 16S rRNA sequencing analysis. Voriconazole concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry. Outer membrane vesicles (OMVs) of microbes from the different groups were prepared for in vitro study in Caco-2 cells. The Nrf2 pathway and its related proteins involved in modifying P-glycoprotein (P-gp) expression were clarified by a series of immunoblot analyses. The diversity and richness of intestinal bacteria, especially the abundance of gram-negative bacteria, were significantly decreased after ceftriaxone treatment. The AUC(0-t) and Cmax of voriconazole were reduced, and greater voriconazole clearance were noted in the dysbiotic group. An in vivo study also indicated that the expression of P-glycoprotein was significantly increased after ceftriaxone treatment, which may be due to the absence of gram-negative bacteria in the intestine. Finally, in vitro findings in Caco-2 cells treated with OMVs from the ceftriaxone-associated dysbiotic group suggested that Nrf2 translocation into the nucleus induced high expression of P-gp. OMVs from intestinal bacterial in the ceftriaxone-associated dysbiotic group induced high P-gp expression by regulating the Nrf2 signalling pathway, which led to an in vivo reduction in the bioavailability of voriconazole due to ceftriaxone-associated dysbiosis.

Current advances in the analysis of free RNA modified nucleosides by high performance liquid chromatography-tandem mass spectrometry

Post-transcriptional ribonucleic acid (RNA) modifications play crucial roles in regulating gene expression, with both eukaryotic and prokaryotic RNA exhibiting more than 170 distinct and ubiquitous modifications. RNA turnover generates numerous free nucleosides, including unmodified nucleosides and a variety of modified ones. Unlike unmodified nucleosides, modified nucleosides are not further degraded or used in the salvage-synthesis pathway owing to a lack of specific enzymes, which leads to the cytosolic accumulation or cellular efflux of modified nucleosides. These modified nucleosides can act as signaling molecules that regulate downstream pathways once transported to the extracellular space; alternatively, they are metabolized in the bloodstream and excreted in urine. Metabolized modified nucleosides are altered by cellular stress responses and mediate abnormal physiological states. Changes in the urinary and blood levels of modified nucleosides associated with cancer can serve as biomarkers for disease. Therefore, identifying and accurately quantifying nucleosides is vital for understanding RNA degradation and associated patterns of nucleoside metabolism. Such analyses are helpful when studying the biological functions and potential clinical applications of modified nucleosides. In this regard, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) offers significant advantages in terms of sensitivity, selectivity, and efficiency, and has been widely used to analyze DNA and RNA nucleosides/nucleotides and their analogues. Multiple MS detection patterns and quantification methods have been established to detect nucleosides in biological samples, including cultured cells, urine, blood, and tissue samples. However, the development of an accurate HPLC-MS/MS method faces several challenges. Firstly, the presence of a complex biological matrix that contains macromolecules, small molecules, and salts can interfere with analysis. Salts and co-eluting substances in the extraction solution often affect mass-spectrometric responses for target analytes. Secondly, various nucleosides are present in vastly different abundances, with contents varying by up to four orders of magnitude; hence, accurately quantifying multiple nucleosides in a single assay is challenging. Thirdly, N-glycosidic bonds are favorably cleaved in most nucleosides during MS to produce the same characteristic fragment ions, which are often accompanied by nucleobases. This tendency poses challenges for distinguishing structural isomers and mass-analogs of modified nucleosides by MS. Post-transcriptional chemical modifications include methylation, hydroxylation, sulfur/oxygen substitution, and side-chain additions. Developing a unified method for simultaneously screening modified nucleosides is difficult owing to biochemical diversity; consequently, there is a need for advanced HPLC-MS/MS method capable of accurately quantifying such nucleosides. This review summarizes the development and applications of LC-MS technologies for analyzing endogenous nucleosides, covering sample preparation, chromatographic-separation and mass-spectrometric-detection conditions, and the development of quantification methods. Additionally, we discuss applications aimed at detecting and quantifying RNA-derived modified nucleosides in biological samples. The applications of HPLC-MS/MS technology are highlighted, the regulation and function of free modified nucleosides are discussed, and the potential functions of modified nucleosides as disease biomarkers for clinical applications are introduced.

Impact of Methotrexate and 7-Hydroxymethotrexate Exposure on Renal Toxicity in Pediatric Non-Hodgkin Lymphoma.

7-Hydroxymethotrexate (7-OHMTX) is the main metabolite in plasma following high-dose MTX (HD-MTX), which may result in activity and toxicity of the MTX. Moreover, 7-OHMTX could produce crystalline-like deposits within the renal tubules under acidic conditions or induce renal inflammation, oxidative stress, and cell apoptosis through various signaling pathways, ultimately leading to kidney damage. The objectives of this study were thus to explore the exposure-safety relationship of two compounds and search the most reliable marker for predicting HDMTX nephrotoxicity. A total of 280 plasma concentration data (140 for MTX and 140 for 7-OHMTX) for 60 pediatric patients with non-Hodgkin lymphoma (NHL) were prospectively collected. Plasma MTX and 7-OHMTX concentrations were determined using a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method. A nonlinear mixed effect model approach was used to build a joint population pharmacokinetic (PopPK) model. After validation, the model estimated the peak concentration (Cmax) and area under the curve within the initial 48 h (AUC0-48h) of the patients after drug administration by Bayesian feedback. The receiver operating characteristic (ROC) curves were generated to identify an exposure threshold associated with nephrotoxicity. A three-compartment chain model (central and peripheral compartments for MTX and central compartment 7-OHMTX) with the first-order elimination adequately characterized the invivo process of MTX and 7-OHMTX. The covariate analysis identified that the aspartate aminotransferase (AST) was strongly associated with the peripheral volume of distribution of MTX. Moreover, the Cmax of MTX and 7-OHMTX showed significant differences (p < 0.0001, p = 0.0472, respectively) among patients with or without nephrotoxicity. Similarly, individuals with nephrotoxicity also exhibited substantially higher ratio of 7-OHMTX to MTX peak concentration and the sum of MTX + 2.25 times the concentration of 7-OHMTX (p < 0.0001, p = 0.0426, respectively). By ROC analysis, the Cmax of MTX and 7-OHMTX had the greatest area under the curve (AUC) values (0.769 and 0.771, respectively). A Cmax threshold of 9.26 μmol/L for MTX or a Cmax threshold of 0.66 μmol/L for 7-OHMTX was associated with the best sensitivity/specificity for toxicity events (MTX: sensitivity = 0.886; specificity = 0.70; 7-OHMTX: sensitivity = 0.886; specificity = 0.70). We demonstrated that the Cmax of MTX and 7-OHMTX were the most reliable markers associated with nephrotoxicity and proposed a Cmax threshold of 9.26 μmol/L for MTX and 0.66 μmol/L for 7-OHMTX as the point with a high risk of nephrotoxicity. Altogether, this study may contribute to crucial insights for ensuring the safe administration of drugs in pediatric clinical practice.

LC/MS/MS detection of uric acid oxidation metabolites to evaluate reactive oxygen and nitrogen species in vivo with application to pseudo-inflammation

Uric acid, a water-soluble antioxidant ubiquitous in human bodily fluids at relatively high concentrations, reacts with a variety of reactive oxygen and nitrogen species. Ours and other previous studies identified allantoin, oxaluric acid, triuret, and 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione as specific metabolites reactive against free radicals, singlet oxygen, peroxynitrite, and hypochlorous anion, respectively. We analyzed human plasma spiked with these products using high-performance liquid chromatography-tandem mass spectrometry. We observed recoveries of 40-110% and coefficients of variance within 7%. Samples remained stable at -80°C for at least 4 weeks, indicating the analytical method is sound. Detection of these metabolites in biological samples enables the identification of each species generated in vivo. We observed changes in the products in human blood during pseudo-inflammation induced by treatment with lipopolysaccharide. Levels of allantoin, oxaluric acid, triuret, and 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione increased after addition of lipopolysaccharide. The formation of singlet oxygen was confirmed by increased formation of Trp-derived cis-hydroxide ([2S,3aR,8aR]-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid) (cis-WOH). We believe our method will aid development of strategies to treat oxidative stress-associated diseases.