Liquid Chromatography-mass Spectrometry Method Research Articles

A simple ultraperformance liquid chromatography mass spectrometry method for the determination of cortisol level in sea bass plasma

A simple, precise, and rapid ultra-performance liquid chromatography method for cortisol quantification in sea bass plasma, based on LC-ESI-UHR-QqTOF-MS, was optimised using tolperisone as internal standard. Sea bass plasma samples containing cortisol were spiked with tolperisone as internal standard and extracted with acetonitrile. Samples were vortexed and centrifuged at 15,000g for 10 min at 4 °C. The final extract, diluted in ammonium formate (1:1), was injected in the LC-ESI-UHR-QqTOF-MS system for analysis. Mass spectrometry acquisition parameters were set using positive ionisation mode over a m/z range from 150 to 2200. Cortisol and tolperisone were detected and quantified at m/z 363.21 and 246.18, respectively, in an Auto MS (MS/MS) mode. The detection and quantification limits of cortisol in sea bass plasma were 0.01 and 0.02 μg/mL, respectively. The mean extraction recovery of cortisol was of 99.1 ± 4.0 %. The within- and between-day precision presents a relative standard deviation (RSD) below 2.9 % and 5.3, respectively. Furthermore, the effect of four different diets (fish feed 1–4) in the basal and stress-induced plasma cortisol levels of sea bass was also assessed.

In-Depth Host Cell Protein Analysis and Viral Protein Impurity Monitoring in Adeno-Associated Virus-Based Gene Therapy Products Using Optimized Wide Window Data-Dependent Acquisition Method.

Compared to other protein therapeutics, there is currently limited knowledge about the residual host cell proteins (HCPs) in adeno-associated virus (AAV)-based gene therapy products. This is primarily due to the lack of a robust and sensitive mass spectrometry-based method for HCP analysis in AAV samples. Existing liquid chromatography-mass spectrometry methods used for analyzing HCPs in therapeutic monoclonal antibodies (mAbs) often cannot be directly applied to AAVs, due to some unique characteristics of AAV samples encountered during their development such as limited sample availability/protein concentration and the presence of surfactants. In this study, we have developed a novel workflow for robust and in-depth HCP analysis of AAV samples by combining wide-window data-dependent acquisition for improved low-abundance HCP detection with single-pot, solid-phase-enhanced sample preparation (SP3) for low-input sample preparation. Using this newly developed method, we were able to detect more than 650 HCPs in a commercial AAV1 sample with a high quantitative reproducibility. This represents a greater than 5-fold increase in HCP protein identification compared to an in-solution digestion method followed by traditional data-dependent acquisition. Similar benefits can also be achieved for other AAV serotypes that were produced internally and purified through different processes. The detection limit of this method is as low as 0.06 ng/mL, enabling more comprehensive HCP coverage in AAV samples. Moreover, for the first time, we have identified several process-related viral proteins, such as Rep 78 and E4. These proteins need to be closely monitored during AAV process development as they may present a greater risk for immunogenicity compared to HCPs that are derived from human HEK293 cells.

Overcoming Challenges in Oligonucleotide Therapeutics Analysis: A Novel Nonion Pair Approach.

Oligonucleotide therapeutics (OT) have emerged as promising drug modality for various intractable diseases. Recently, liquid chromatography-mass spectrometry (LC-MS) has been commonly employed for characterizing and quantifying OT in biological samples. Traditionally, the ion pairing-reverse phase (IP-RP) LC-MS method has been utilized in OT bioanalyses; however, this approach is associated with several limitations, including the memory effect and ion suppression effect of IP reagents. Therefore, this study aimed to develop a new RP-LC-MS method that eliminates the need for IP reagents. Our investigation revealed that ammonium bicarbonate was essential for the successful implementation of this nonIP-RP-LC-MS-based bioanalysis of OT. Moreover, the developed method demonstrated high versatility, accommodating the analysis of various natural or chemically modified oligonucleotides. The sensitivity of the method was further assessed using reconstituted plasma samples (the lower limit of quantification in this experiment was 0.5-1 ng/mL). In summary, the developed nonIP-RP-LC-MS method offers an easy, reliable, and cost-effective approach to the bioanalysis of OT.

Rapid and simultaneous determination of ultrashort-, short- and long- chain perfluoroalkyl substances by a novel liquid chromatography mass spectrometry method

Per- and Polyfluoroalkyl Substances (PFAS) are a group of persistent organic pollutants that have received considerable attention from public and regulatory groups. Due to regulations of long-chain PFAS, the use of short-chain and ultrashort-chain PFAS is rapidly growing. Thus, there is an urgent need to develop quantitative methods for determining PFAS with different chain lengths in various environmental matrices. This study introduces an innovative liquid chromatography-mass spectrometry (LC-MS) system combining large volume injection (LVI) and online solid phase extraction (SPE). This system incorporates three columns: a reverse-phase (RP) column, a weak anion exchange (WAX) trap column, and a hybrid HILIC/ion-exchange (HILIC/IE) column, controlled by two valves. With valve switching, ultrashort-chain PFAS that are not retained by the RP column are enriched by the trap column, while other PFAS are separated by the RP column. The trapped ultrashort PFAS are then transferred to the HILIC/IE column for further separation. The LVI significantly enhances the method's sensitivity, allowing for rapid and simultaneous determination of ultrashort-, short- and long- chain PFAS in aqueous samples. The matrix effects from various environmental samples were evaluated, and the results indicate that this unique LC-MS method is suitable for analyzing all chain-length PFAS in various matrices, including surface water, sewage effluent, and seawater. Finally, this novel LC-MS method was applied to quantify PFAS in various water samples.

Ultra-performance liquid chromatography-mass spectrometry methods for the determination of the residual quantities of ramipril and hydrochlorothiazide for controlling the cleaning of equipment

Monitoring the completeness of equipment cleaning is essential to prevent cross-contamination of medicinal products. Therefore, it is necessary to develop fast and sensitive methods for studying residual quantities of active ingredients on the surfaces of technological equipment. The aim of the work was to develop and validate analytical methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry method. Materials and methods. In the study, standard samples of ramipril (USP RS) and hydrochlorothiazide (USP RS), as well as class A reagents, were used. Samples were analysed on a liquid chromatograph with an MS detector (Agilent 6420 and Waters Xevo TQD ACQUITY). We used the Kinetex C18 column (2.1 mm × 30 mm × 1.7 μm); mobile phase – 0.1% formic acid solution in deionised water – Acetonitrile (ratio 73:27 for the determination of ramipril and 91.5:8.5 for the determination of hydrochlorothiazide); mobile phase rate of 0.4 mL/min for the determination of ramipril and 0.35 mL/min for the determination of hydrochlorothiazide; column temperature 45 °C for the determination of ramipril and 40 °C for the determination of hydrochlorothiazide, ionisation mode - electric spray in positive mode; The detection parameters are the mode of registration of the daughter ion 417 → 234 m/z for the determination of ramipril and 298 → 281 m/z for the determination of hydrochlorothiazide. Results and discussion. Methods for the determination of ramipril and hydrochlorothiazide in wash waters by ultra-performance liquid chromatography–mass spectrometry have been developed. The developed methods have sufficient linearity, correctness and precision. The sensitivity of the techniques was confirmed at the level of 0.0026 μg/ml. The techniques can be used in the concentration range of 0.0026 – 0.0255 μg/ml Conclusions. Analytical methods for determining ramipril and hydrochlorothiazide in wash waters have been developed and validated.

Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Comparison of Four Rapid N-Glycan Analytical Methods and Great Application Potential in Cell Line Development

N-glycan, a critical quality attribute (CQA) of monoclonal antibodies, profoundly impacts potency, immunogenicity, safety, and clinical efficiency. Therefore, N-glycan should be monitored and controlled during development. The conventional 2-AB method is considered the gold standard for N-glycan analysis, which is labor intensive and time consuming. Moreover, its sample requirement is relatively high and cannot be met in early-stage development, including cell line development. In this study, we compared the performance of four rapid analytical methods in N-glycan analysis of mAbs, including the rapid 2-AB method, reduction method, off-line IdeS digestion method, and two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) method. Our results showed that these four rapid analytical methods could provide comparable N-glycan data. Moreover, these four rapid methods shortened the testing time for the conventional 2-AB method from days to just minutes. They also reduced the sample requirement for the conventional 2-AB method from milligrams to micrograms. Among these four rapid methods, the 2D-LC-MS method demonstrated great potential for applications in time-consuming cell line development because it required less testing time and a lower sample requirement.

Establishment of a UPLC-MS method for quantitative analysis of tryptophan-kynurenine metabolism in IBS-D model rats

Background and Aims Abnormalities in tryptophan (TRP) metabolism induce abdominal pain and intestinal motility disorders. The study of TRP metabolism in diarrhea-predominant-irritable bowel syndrome (IBS-D) is important for the prevention, diagnosis, and treatment of this disease. In this study, a rapid and reliable ultra performance liquid chromatography-mass spectrometry (UPLC-MS) method was established to quantify tryptophan-kynurenine (TRP-Kyn) metabolism in the colon of a rat model with IBS-D. Methods The proteins were precipitated by methanol, chromatographically separated on a Welch Ultimate® Polar RP column with a gradient elution for 12 min, and detected by high-resolution tandem mass spectrometry. Pure water were used as an alternative mechanism for standard calibration, and the stable structural analog 2-Cl-Phe was used as an internal standard. Results Within a certain range, the r of TRP, kynurenine (Kyn) and quinolinic acid (QA), kynurenic acid (KA) are greater than 0.99, were found to be accurate and precise. The metabolism of TRP was significantly up-regulated along the Kyn pathway in the IBS-D model rats and normalized after treatment with pivacurium bromide. Conclusion This study investigates the mechanisms of IBS-D gastrointestinal dysfunction from the perspective of colonic TRP metabolism, and also provides new directions for the diagnosis and therapeutic approach of this disease.

The genetics and epidemiology of N- and O-immunoglobulin A glycomics

BackgroundImmunoglobulin (Ig) glycosylation modulates the immune response and plays a critical role in ageing and diseases. Studies have mainly focused on IgG glycosylation, and little is known about the genetics and epidemiology of IgA glycosylation.MethodsWe generated, using a novel liquid chromatography-mass spectrometry method, the first large-scale IgA glycomics dataset in serum from 2423 twins, encompassing 71 N- and O-glycan species.ResultsWe showed that, despite the lack of a direct genetic template, glycosylation is highly heritable, and that glycopeptide structures are sex-specific, and undergo substantial changes with ageing. We observe extensive correlations between the IgA and IgG glycomes, and, exploiting the twin design, show that they are predominantly influenced by shared genetic factors. A genome-wide association study identified eight loci associated with both the IgA and IgG glycomes (ST6GAL1, ELL2, B4GALT1, ABCF2, TMEM121, SLC38A10, SMARCB1, and MGAT3) and two novel loci specifically modulating IgA O-glycosylation (C1GALT1 and ST3GAL1). Validation of our findings in an independent cohort of 320 individuals from Qatar showed that the underlying genetic architecture is conserved across ancestries.ConclusionsOur study delineates the genetic landscape of IgA glycosylation and provides novel potential functional links with the aetiology of complex immune diseases, including genetic factors involved in IgA nephropathy risk.

Quantitative analysis of mixed lipid nanostructures in rat skin by HPLC-MS

Abstract Liquid chromatography-mass spectrometry (LC-MS) is a very sensitive technique for determining small concentrations of drugs in fixed dose combinations or even those deposited in skin layers. Therefore, an LC-MS method was applied for determining the drugs under investigation, namely, clioquinol (CLIO), tolnaftate (TOL), and betamethasone (BETA) in Quadriderm® cream and mixed lipid nanostructures (MLNs) prepared in laboratory in the presence of potential interferents, and was applied as a dermato-kinetic study in rat’s skin. The separation was achieved within 4.5 min by using C18 column as a stationary phase and the mobile phase used were 20% phase A composed of 0.1% formic acid (v/v) and 80% phase B composed of 0.1% formic acid in acetonitrile (v/v), coupled with triple quadrupole mass spectrometer. MLNs were prepared and characterized to be compared with the conventional commercially available Quadriderm® cream. The proposed method was accurate and precise with a linearity range of 0.2–20.0 µg·mL−1 for BETA, and 0.5–400.0 µg·mL−1 for CLIO and TOL and a better bioavailability of the new formulation was obtained ensuring the capability of the nanoparticles to accumulate the drugs within the skin layers. In conclusion, the LC-MS method was accurate and precise for the determination of the three drugs under investigation in cream dosage form and skin tissues.

Mechanism investigation of anti-NAFLD of Shugan Yipi Granule based on network pharmacology analysis and experimental verification

As a classical traditional Chinese patent medicine, Shugan Yipi Granule is widely used in China to treat non-alcoholic fatty liver disease (NAFLD) recently. Our previous study confirmed that Shugan Yipi Granule are effective in NAFLD. However, its underlying mechanism is still unknown. This study aims to investigate the mechanism of Shugan Yipi Granule on NAFLD based on network pharmacology prediction, liquid chromatography-mass spectrometry (LC-MS) analysis and in vitro verification. We obtained the active ingredients and targets of Shugan Yipi Granule and NAFLD from 6 traditional Chinese medicine databases, and the crucial components and targets screened by protein-protein interaction (PPI) network were used for molecular docking. Plasma metabolomics of NAFLD patients treated with Shugan Yipi Granule for one month was analyzed using LC-MS methods and MetaboAnalyst 4.0 to obtain significant differential metabolites and pathways. Finally, free fatty acid (FFA) induced HepG2 cells were treated with different concentrations of quercetin and kaempferol, then oil red o (ORO) and triglyceride (TG) level were tested to verify the lipid deposition of the cell. Network pharmacology analysis showed that the main active ingredients of Shugan Yipi Granule include quercetin, kaempferol and other 58 ones, as well as 188 potential targets. PI3K/Akt signaling pathway was found to be the most relevant pathway for the treatment of NAFLD. Non-targeted metabolomics showed that quercetin and kaempferol were significantly up-regulated differential metabolites and were involved in metabolic pathways such as thyroid hormone signaling. In vitro results showed that quercetin, kaempferol were effective in reducing lipid deposition and TG content by inhibiting cellular fatty acid uptake. Ultimately, with the network pharmacology and serum metabolomics analysis, quercetin and kaempferol were found to be the important active ingredients and significantly up-regulated differential metabolites of Shugan Yipi Granule against NAFLD, which we inferred that they may regulate NAFLD through PI3K/Akt signaling pathway and thyroid hormone metabolism pathway. The in vitro experiment verification results showed that quercetin and kaempferol attenuated the lipid accumulation and TG content by inhibiting the fatty acid uptake in the FFA-induced HepG2 cell. Current study provides the necessary experimental basis for subsequent in-depth mechanism research.

Acid Hydrolytic Degradation Profiling of Ezetimibe: Identification, Isolation, and Structural Elucidation of Its Degradants

ABSTRACTEzetimibe (EZE) is a dyslipidemic agent used to treat hyperlipidemia along with statins and diet changes. To ascertain the drug's degradation profile, stress tests were conducted on it in accordance with International Council for Harmonization recommendations. An ultrahigh‐performance liquid chromatography–mass spectrometry method was developed and validated for the identification and quantification of EZE and its degradants. Using preparative high‐performance liquid chromatography, all impurities were isolated, and their structures were characterized thoroughly using advanced spectral techniques. The drug was relatively stable in basic, peroxide, photolytic, and thermal conditions; however, five degradants were observed in acid degradation. Among the five degradants, three degradation products eluted as Peak‐1, Peak‐2, and Peak‐3 which were found to be novel impurities that were not reported previously. However, the remaining two impurities were identified as Peak‐4 and Peak‐5, despite the insufficient information available. The present study has focused on the isolation of these five acid degradation products and the structural confirmation of these degradants by highly efficient spectral techniques. Moreover, the possible degradation mechanism of the azetidinone ring in the presence of acid has been explained, which might be helpful in determining the quality and purity of EZE and similar pharmaceutical compounds.

Pharmacokinetics of anti-Mycobacterium avium-intracellulare disease drugs in silkworms

Pulmonary Mycobacterium avium-intracellulare complex (MAC) disease is a typical non-tuberculous mycobacterial infection. The incidence of pulmonary MAC is increasing worldwide. This study aimed to clarify the pharmacokinetic parameters of anti-pulmonary MAC disease drugs in silkworms. The pharmacokinetic parameters investigated included maximum concentration, area under the concentration–time curve, total clearance, and volume of distribution at steady-state. In addition, protein-binding rates, fat body transferability, and drug-drug interactions were examined. Antibiotic concentrations were measured using a validated high-performance liquid chromatography-mass spectrometry method. Among the antibiotics investigated, amikacin was not eliminated from silkworms during the 48-h observation period. In contrast, dose-proportional pharmacokinetics were observed in silkworms for all antibiotics tested, except for amikacin. Protein-binding rates in hemolymph for clarithromycin, azithromycin, rifampicin, ethambutol, and amikacin were 39.6 ± 3.0%, 39.5 ± 4.3%, 76.3 ± 3.2%, 20.9 ± 4.2%, and 73.1 ± 4.7%, respectively (mean ± standard deviation). The distribution of antibiotics in the fat bodies of silkworms was related to drug lipophilicity. No drug-drug interactions were observed in the silkworms. The pharmacokinetics of these drugs in silkworms differed significantly from those in humans. Therefore, while it is challenging to predict the pharmacokinetics of these drugs in humans based on silkworm data, the silkworm infection model has facilitated a comprehensive assessment of the relationship between antibiotic exposure and efficacy.

Sustainable and Stability Indicating Liquid Chromatography–Mass Spectrometry Method for the Quantitation of Delafloxacin in Pharmaceutical Formulations

ABSTRACTA sensitive, precise, accurate, and green analytical HPLC–ESI–MS method for the quantification of delafloxacin and its degradation products in pharmaceutical dosage forms has been optimized and validated. The best separation was achieved with isocratic elution, the mobile phase is composed of a mixture of 0.1% trifluoroacetic acid in water and acetonitrile 65:35 (v/v), the flow rate is 0.5 mL min−1, and the column used is Kinetex Core‐Shell C18 (250 × 4.6 mm, 5 µm). Forced degradation studies were performed to prove that the method indicates stability. The pharmaceutical substance is prone to oxidative (H2O2 3%), acidic (HCl 0.1 M), and basic (0.1 M) conditions. Delafloxacin proved to be susceptible to acidic (HCl 0.1 M), basic (0.1 M), and oxidative (H2O2 3%) conditions. The proposition of the structures of degradation products has been based on the MS spectrum and the known reactivity of delafloxacin in the oxidative medium. The validation of the analytical method was carried out following the International Conference on Harmonization guidelines. The method was validated in terms of specificity, precision, linearity, and accuracy. The limits of detection and quantification of delafloxacin are, respectively, 0.005 and 0.017 µg mL−1.

Tire-derived contaminants 6PPD and 6PPD-Q: Analysis, sample handling, and reconnaissance of United States stream exposures

The environmental ubiquity of tire and road wear particles (TRWP) underscores the need to understand the occurrence, persistence, and environmental effects of tire-related chemicals in aquatic ecosystems. One such chemical is 6PPD-quinone (6PPD-Q), a transformation product of the tire antioxidant 6PPD. In urban stormwater runoff 6PPD-Q can exceed acute toxicity thresholds for several salmonid species and is being implicated in significant coho salmon losses in the Pacific Northwest. There is a critical need to understand the prevalence of 6PPD-Q across watersheds to identify habitats heavily affected by TRWPs. We conducted a reconnaissance of 6PPD and 6PPD-Q in surface waters across the United States from sites (N = 94) with varying land use (urban, agricultural, and forested) and streamflow to better understand stream exposures. A rapid, low-volume direct-inject, liquid chromatography mass spectrometry method was developed for the quantitation of 6PPD-Q and screening for 6PPD. Laboratory holding times, bottle material, headspace, and filter materials were investigated to inform best practices for 6PPD-Q sampling and analysis. Glass bottles with PTFE-lined caps minimized sorption and borosilicate glass fiber filters provided the highest recovery. 6PPD-Q was stable for at least 5 months in pure laboratory solutions and for 75 days at 5 °C with minimal headspace in the investigated surface water and stormwaters. Results also indicated samples can be frozen to extend holding times. 6PPD was not detected in any of the 526 analyzed samples and there were no detections of 6PPD-Q at agricultural or forested sites. 6PPD-Q was frequently detected in stormwater (57%, N = 90) and from urban impacted sites (45%, N = 276) with concentrations ranging from 0.002 to 0.29 μg/L. The highest concentrations, above the lethal level for coho salmon, occurred during stormwater runoff events. This highlights the importance of capturing episodic runoff events in urban areas near ecologically relevant habitat or nursery grounds for sensitive species.

A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

Integrating metabolomics and bioinformatics to reveal the mechanism of Epimedium-induced liver injury.

Epimedium is a traditional Chinese medicine with a wide range of clinical applications; however, there have been numerous reports of adverse reactions in recent years. The most common side effect of Epimedium is liver injury. In this study, the liquid chromatography-mass spectrometry (LC-MS) method has been established to study the components of Epimedium and to identify the components absorbed into the blood of rats. Bioinformatics was used to screen out potential toxic components, and the integrating metabolomics method was used to explore the molecular mechanism of Epimedium-induced liver injury. The chemical constituents of Epimedium were identified by LC-MS, and 62 compounds were obtained, including 57 flavonoids, four organic acids and one alkaloid. The toxicity network of "Epimedium-component-target-liver injury" was constructed using bioinformatics research methods, and then the key hepatotoxic component icaritin was identified. Integrating metabolomics was used to investigate the changes in the metabolic profile of L-02 cells with different durations of icaritin administration compared with the control group, and 106 different metabolites were obtained. A total of 14 potential biomarkers significantly associated with cell survival were screened by Pearson correlation analysis combined with the L-02 cell survival rate. Our study preliminarily revealed the mechanism of hepatotoxicity induced by Epimedium.

Separating and identifying multiple structural isomers of 3-hydroxy-3-(3′-hydroxyphenyl)propanoic acid (3,3′-HPHPA)

3-hydroxy-3-(3′-hydroxyphenyl)propanoic acid (CID: 102959) (3,3’-HPHPA) is one of several phenolic molecules that are generated by a process involving gut microbiota and have been implicated in autism and schizophrenia. The structural isomers of 3,3′-HPHPA have been challenging to separate chromatographically. Accordingly, their distribution across brain and other compartments in humans and experimental animals remains poorly characterized. We describe a modified liquid chromatography mass spectroscopy method for the detection and separation of seven commercially available structural isomers of 3,3′-HPHPA. We used the method to screen different compartments in the mouse and demonstrate its utility in identifying multiple structural isomers and conjugates of 3,3′-HPHPA in biological matrices. Characterizing the diversity and complexity of this family of structural isomers will advance studies of their bioactivity and clinical relevance.

Toxicokinetics of α- and β-amanitin in mice following single and combined administrations: Simulating in vivo amatoxins processes in clinical cases

α-Amanitin and β-amanitin, two of the most toxic amatoxin compounds, typically coexist in the majority of Amanita mushrooms. The aim of this study was to use a newly developed ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method to determine the toxicokinetics and tissue distribution of α- and β-amanitin following single or combined oral (po) administration in mice. α-Amanitin and β-amanitin administered at 2 or 10 mg/kg doses showed similar toxicokinetic profiles, except for peak concentration (Cmax). The elimination half-life (t1/2) values of α-amanitin and β-amanitin in mice were 2.4–2.8 h and 2.5−2.7 h, respectively. Both α- and β-amanitin were rapidly absorbed into the body, with times to reach peak concentration (Tmax) between 1.0 and 1.5 h. Following single oral administration at 10 mg/kg, the Cmax was significantly lower for α-amanitin (91.1 μg/L) than for β-amanitin (143.1 μg/L) (p < 0.05). The toxicokinetic parameters of α-amanitin, such as t1/2, mean residence time (MRT), and volume of distribution (Vz/F) and of β-amanitin, such as Vz/F, were significantly different (p < 0.05) when combined administration was compared to single administration. Tissues collected at 24 h after po administration revealed decreasing tissue distributions for α- and β-amanitin of intestine > stomach > kidney > lung > spleen > liver > heart. The substantial distribution of toxins in the kidney corresponds to the known target organs of amatoxin poisoning. The content in the stomach, liver, and kidney was significantly higher for of β-amanitin than for α-amanitin at 24 h following oral administration of a 10 mg/kg dose. No significant difference was detected in the tissue distribution of either amatoxin following single or combined administration. After po administration, both amatoxins were primarily excreted through the feces. Our data suggest the possibility of differences in the toxicokinetics in patients poisoned by mushrooms containing both α- and β-amanitin than containing a single amatoxin. Continuous monitoring of toxin concentrations in patients’ blood and urine samples is necessary in clinical practice.

Adverse events during intravenous fosfomycin therapy in a real-life scenario. Risk factors and the potential role of therapeutic drug monitoring

BackgroundIntravenous fosfomycin (IVFOF) is gaining interest in severe infections. Its use may be limited by adverse events (AEs). Little experience exists on IVFOF therapeutic drug monitoring (TDM) in real-life setting.Patients and methodsRetrospective study of patients receiving IVFOF for > 48 h at Policlinico Hospital (Milan, Italy) from 01/01/2019 to 01/01/2023. AEs associated to IVFOF graded CTCAE ≥ II were considered. Demographic and clinical risk factors for IVFOF-related AEs were analysed with simple and multivariable regression models. The determination of IVFOF TDM was made by a rapid ultraperformance liquid chromatography mass spectrometry method (LC-MS/MS) on plasma samples. The performance of TDM (trough levels (Cmin) in intermittent infusion, steady state levels (Css) in continuous infusion) in predicting AEs ≤ 5 days after its assessment was evaluated.ResultsTwo hundred and twenty-four patients were included. At IVFOF initiation, 81/224 (36.2%) patients were in ICU and 35/224 (15.7%) had septic shock. The most frequent infection site was the low respiratory tract (124/224, 55.4%). Ninety-five patients (42.4%) experienced ≥ 1AEs, with median time of 4.0 (2.0–7.0) days from IVFOF initiation. Hypernatremia was the most frequent AE (53/224, 23.7%). Therapy discontinuation due to AEs occurred in 38/224 (17.0%). ICU setting, low respiratory tract infections and septic shock resulted associated with AEs (RRadjusted 1.59 (95%CI:1.09–2.31), 1.46 (95%CI:1.03–2.07) and 1.73 (95%CI:1.27–2.37), respectively), while IVFOF daily dose did not. Of the 68 patients undergone IVFOF TDM, TDM values predicted overall AEs and hypernatremia with AUROC of 0.65 (95%CI:0.44–0.86) and 0.91 (95%CI:0.79-1.0) respectively for Cmin, 0.67 (95%CI:0.39–0.95) and 0.76 (95%CI:0.52-1.0) respectively for Css.ConclusionsWe provided real world data on the use of IVFOF-based regimens and associated AEs. IVFOF TDM deserves further research as it may represent a valid tool to predict AEs.Key pointsReal world data on intravenous fosfomycin for severe bacterial infections. AEs occurred in over 40% (therapy discontinuation in 17%) and were related to baseline clinical severity but not to fosfomycin dose. TDM showed promising results in predicting AEs.