Determination of α-methylenecyclopropylglycine in Shahi and China litchi cultivars at three different maturity stages: A quantitative study using liquid chromatography tandem mass spectrometry.

This study presents the contents of α-methylenecyclopropylglycine, a potentially toxic amino acid, in the peel, pulp and seed fractions of two well-known litchi varieties, namely Shahi and China, over a span of three harvest-seasons. For analysing α-methylenecyclopropylglycine, an LC-MS/MS-based method was validated. The method-accuracies fell within 75-110 % (RSD, <15 %) at 0.1 mg/kg (LOQ) and higher levels. A comparative evaluation of the results in peel, pulp and seed at 30 days before harvest (DBH), 15-DBH, and edible-ripe stage revealed that α-methylenecyclopropylglycine content increased as the litchi seeds grew towards maturity, regardless of the cultivar. In arils, at maturity, the concentration of α-methylenecyclopropylglycine ranged from not-detected to 11.7 µg/g dry weight. The Shahi cultivar showed slightly higher α-methylenecyclopropylglycine content in comparison to China litchi. This paper presents the first known analysis of combined seasonal data on different fruit components at various growth stages for the two chosen litchi cultivars grown in India.

Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting.

BACKGROUND AND AIMS: Current laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. MATERIALS AND METHODS: DART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. RESULTS: DART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. CONCLUSION: In this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

Evidence to support cultivated fruiting body of Ophiocordyceps sinensis (Ascomycota)'s role in relaxing airway smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiocordyceps sinensis (O. sinensis) is a genus of Ascomycete fungus that is endemic to the alpine meadows of the Tibetan Plateau and adjoining Himalayas. It has been used traditionally as a tonic to improve respiratory health in ancient China as well as to promote vitality and longevity. Bioactive components found in O. sinensis such as adenosine, cordycepin, 3-deoxyadenosine, L-arginine and polysaccharides have gained increasing interest in recent years due to their antioxidative and other properties, which include anti-asthmatic, antiviral, immunomodulation and improvement of general health. AIM OF THE STUDY: This study's primary aim was to investigate the effect of a cultivated fruiting body of O. sinensis strain (OCS02®) on airways patency and the secondary focus was to investigate its effect on the lifespan of Caenorhabditis elegans. MATERIALS AND METHODS: A cultivated strain, OCS02®, was employed and the metabolic profile of its cold-water extract (CWE) was analysed through liquid chromatography-mass spectrometry (LC-MS). Organ bath approach was used to investigate the pharmacological properties of OCS02® CWE when applied on airway tissues obtained from adult male Sprague-Dawley rats. The airway relaxation mechanisms of OCS02® CWE were explored using pharmacological tools, where the key regulators in airway relaxation and constriction were investigated. For the longevity study, age-synchronised, pos-1 RNAi-treated wild-type type Caenorhabditis elegans at the L4 stage were utilised for a lifespan assay. RESULTS: Various glycopeptides and amino acids, particularly a high concentration of L-arginine, were identified from the LC-MS analysis. In airway tissues, OCS02® CWE induced a significantly greater concentration-dependent relaxation when compared to salbutamol. The relaxation response was significantly attenuated in the presence of NG-Nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and several K+ channel blockers. The longevity effect induced by OCS02® CWE (5 mg/mL and above) was observed in C. elegans by at least 17%. CONCLUSIONS: These findings suggest that the airway relaxation mechanisms of OCS02® CWE involved cGMP-dependent and cGMP-independent nitric oxide signalling pathways. This study provides evidence that the cultivated strain of OCS02® exhibits airway relaxation effects which supports the traditional use of its wild O. sinensis in strengthening respiratory health.

Catalytic detoxification of mitoxantrone by graphitic carbon nitride (g-C3N4) supported Fe/Pd bimetallic nanoparticles.

The overuse of antibiotics and antitumor drugs has resulted in more and more extensive pollution of water bodies with organic drugs, causing detrimental ecological effects, which have attracted attention towards effective and sustainable methods for antibiotics and antitumor drug degradation. Here, the hybrid nanomaterial (g-C3N4@Fe/Pd) was synthesized and used to remove a kind of both an antibiotic and antitumor drug named mitoxantrone (MTX) with 92.0% removal efficiency, and the MTX removal capacity is 450 mg/g. After exposing to the hybrid material the MTX aqueous solution changed color from dark blue to lighter progressively, and LC-UV results of residual solutions show that a new peak at 3.0 min (MTX: 13.2 min) after removal by g-C3N4@Fe/Pd appears, with the simultaneous detection of intermediate products indicating that g-C3N4@Fe/Pd indeed degrades MTX. Detailed mass spectrometric analysis suggests that the nuclear mass ratio decreased from 445.2 (M+1H) to 126.0 (M+1H), 169.1 (M+1H), 239.2 (M+1H), 267.3 (M+1H), 285.2 (M+1H), 371.4 (M+1H) and 415.2 (M+1H), and the maximum proportion (5.63%) substance of all degradation products (126.0 (M+1H)) is 40-100 times less toxic than MTX. A mechanism for the removal and degradation of mitoxantrone was proposed. Besides, actual water experiments confirmed that the maximum removal capacity of MTX by g-C3N4@Fe/Pd is up to 492.4 mg/g (0.02 g/L, 10 ppm).

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.

Identifying an Abnormal Phosphorylated Adaptor by Viral Kinase Using Mass Spectrometry.

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

BRF1 promotes the odontogenic differentiation of dental pulp stem cells in pulpitis by inducing autophagy.

Objective: While post-transcriptional modifications play a pivotal role in the autophagy regulation, studies on dental pulp disease are limited. This study investigated the effect of BRF1 on autophagy in inflamed pulp tissue and human dental pulp stem cells (hDPSCs). Methods: Immunohistochemical analysis was used to examine BRF1 expression, autophagy levels, and dentinogenic markers in normal and inflamed pulp. The presence of autophagosomes was observed by transmission electron microscopy. Primary hDPSCs were treated with 1 µg/mL lipopolysaccharide (LPS) for different lengths of time. The expression of BRF1 and autophagy makers was determined by Western blotting. BRF1 knockdown and 3 MA treatment were employed to assess changes in autophagy and dentinogenic differentiation. Double immunofluorescence staining was performed to co-localize BRF1 with LC3B in pulp tissue. Results: The expressions of BRF1, LC3, DMP1, and DSP were significantly elevated in the inflamed pulp. LPS enhanced the protein production of IL-6, BRF1, LC3, and Beclin-1 from 6 h to 24 h after the treatment. BRF1 knockdown reduced the ratio of LC3-II/LC3-I and the differentiation ability of hDPSCs, while 3 MA inhibited LPS-mediated dentinogenic differentiation. Double-labeling revealed that BRF1 co-localized with LC3B in inflamed pulp. Conclusion: This study demonstrated that BRF1 promoted autophagy activation and odontogenic differentiation in pulpitis.

Metabolic profiling and spatial metabolite distribution in wild soybean (G. soja) and cultivated soybean (G. max) seeds.

Wild soybeans retain many substances significantly reduced or lost in cultivars during domestication. This study utilized LC-MS to analyze metabolites in the seed coats and embryos of wild and cultivated soybeans. 866 and 815 metabolites were identified in the seed extracts of both soybean types, with 35 and 10 significantly differing metabolites in the seed coat and embryos, respectively. The upregulated metabolites in wild soybeans are linked to plant defense, stress responses, and nitrogen cycling. MALDI-MSI results further elucidated the distribution of these differential metabolites in the cotyledons, hypocotyls, and radicles. In addition to their role in physiological processes like growth and response to environmental stimuli, the prevalent terpenoids, lipids, and flavonoids present in wild soybeans exhibit beneficial bioactivities, including anti-inflammatory, antibacterial, anticancer, and cardiovascular disease prevention properties. These findings underscore the potential of wild soybeans as a valuable resource for enhancing the nutritional and ecological adaptability of cultivated soybeans.

LC-ESI-MS/MS method validation for simultaneous quantification of FDA-approved anticancer agents futibatinib and binimetinib in rat plasma: Insights from preclinical pharmacokinetics.

This study investigates the combination of FGFR inhibitor futibatinib (FTB) and MEK inhibitor binimetinib (BTB) for KRASmt NSCLC therapy. An analytical method was developed and validated for measuring FTB and BTB concentrations in rat plasma, adhering to USFDA guidelines. Using liquid-liquid extraction on 45-µL plasma samples, a 6.5-min run time was achieved. The linear calibration curve ranged from 2 to 100 ng/mL. Intra-day and inter-day accuracy ranged between 92.06% and 100.08%. Four blank injections post high-concentration samples resolved significant carryover. Extraction recoveries averaged 92.06% to 102.37% across concentrations. No significant endogenous interference was detected in blank plasma. The LLOQ for both drugs was 2.0 ng/mL. Selectivity, matrix effects, stability, and dilution integrity met the acceptance criteria. The method assessed FTB and BTB interaction potential in combination therapy at 5 mg/kg. The findings provide essential pharmacokinetics insights for future clinical trials.

Metabolic Pathway of Osilodrostat in Equine Urine Established through High-resolution Mass Spectrometric Characterization for Doping Control.

OBJECTIVE: Osilodrostat, used to treat Cushing's disease, exhibits an anabolic effect, leading to its classification as a prohibited substance in horseracing and equestrian sports. This study reports the characterization of osilodrostat metabolites in horse urine and elucidates its metabolic pathways for the first time for doping control purposes. METHODS: Osilodrostat was administered nasoesophageally to four thoroughbreds (one gelding and three mares) at a dose of 50 mg each. Potential metabolites were extensively screened via our developed generic approach employing differential analysis to identify metabolites. Specifically, high-resolution mass spectral data were compared between pre- and post-administration samples on the basis of criteria of fold-changes of peak areas and their P values. Potential metabolite candidates were further identified through mass spectral interpretations using product ion scan data. RESULTS: A total of 37 metabolites were identified after comprehensive analysis. Osilodrostat was predominantly metabolized into a mono-hydroxylated form M1c (~40%) alongside osilodrostat glucuronide M2 (~17%). Given their longest detection time (2 weeks after administration) and the identification of several conjugates of osilodrostat and M1c, including a novel conjugate of riburonic acid, we recommend monitoring both osilodrostat and M1c after hydrolysis during the screening stage. However, only osilodrostat can be used for confirmation because of the availability of a reference material. CONCLUSION: It is advisable to screen for both osilodrostat and its mono-hydroxylated metabolite M1c to effectively monitor horse urine for the potential misuse or abuse of osilodrostat. For suspicious samples, confirmation of osilodrostat using its reference material is required.

Hibiscus schizopetalus boosts wound healing via restoring redox balance and hindering inflammatory responses in rats: Insights on metabolome profiling and molecular docking.

Hibiscus species (Malvaceae) possess a plethora of appealing pharmacological activities with an extended history of customary use in diverse medical conditions. The present study aimed at comparing the metabolomic analyses of three Hibiscus species native to Egypt, namely H. tiliaceus, H. schizopetalus extract (HSE), and H. rosa-sinensis, alongside identifying a promising natural wound healing candidate. Chemical profiling of the leaf extracts was achieved via UPLC-ESI/MS/MS-guided analysis that resulted in the tentative identification of a total of 48 secondary metabolites pertaining to phenolic acids, flavonoids, anthocyanins, fatty acids, and fatty amides. Remarkably, in vitro studies revealed that HSE exhibited the topmost wound healing activity. Subsequently, HSE was formulated into hydro- and nanogel (1% w/v) formulations for further assessing its efficacy in the wound excision model. HSE-nanogel demonstrated a significant in vivo wound contraction activity alongside improving histopathological abnormalities. Mechanistically, HSE-nanogel upregulated the wound antioxidant status through increasing the levels of reduced glutathione (GSH) and catalase activity. Moreover, HSE-nanogel suppressed the wound inflammatory responses by diminishing the expressions of NF-ĸB, TNF-α, and IL-6. Molecular docking studies were performed on HSE's major constituents using CDOCKER, which further supported the in vivo findings. Collectively, HSE nanogel exhibits notable aptitude as a wound-healing agent, warranting further clinical appraisal.

Free thyroid hormone: Methods and standardization.

Free thyroid hormone (FTH) serves as the preferred indicator for the clinical assessment of thyroid function, mainly encompassing free thyroxine and free triiodothyronine. The immunoassay commonly employed in the clinical setting exhibits certain unresolvable deficiencies. The results of over 5,500 clinical laboratories for FTH from China in 2024 demonstrated that the outcomes of immunoassay were not comparable, with robust CVs calculated in accordance with ISO 13528 ranging from 13.82% to 21.42%. Establishing reference methods is an important tool to achieve accurate and comparable results of free hormones. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) holds a distinct advantage in the precise detection of small molecules, and two reference methods for free thyroxine based on LC-MS/MS are included in the JCTLM list. This article conducts a comprehensive review of the detection methods and standardization of FTH. It presents the metabolism of thyroid hormones, the significance of detection, the techniques, and application examples of free thyroid hormone assays, and deliberates on the current status, prospects, and recommendations for the standardization of FTH assays. Immunoassay and LC-MS/MS, as significant techniques for FTH detection, are predominantly emphasized in the case references. Ultrafiltration and equilibrium dialysis, which are utilized to separate FTH, are also addressed. This article aims to discuss the status quo of FTH detection and clarify the advantages of LC-MS/MS in FTH detection, propose that LC-MS/MS can be utilized as an auxiliary validation method or alternative method in clinical applications, and offer suggestions for the standardization of testing results.

Streptomyces avermitilis MICNEMA2022: a new biorational strain for producing abamectin as an integrated nematode management agent.

BACKGROUND: Abamectin (ABA) is considered a powerful insecticidal and anthelmintic agent. It is an intracellular product of Streptomyces avermitilis; is synthesized through complicated pathways and can then be extracted from mycelial by methanol extraction. ABA serves as a biological control substance against the root-knot nematode Meloidogyne incognita. This investigation is intended to reach a new strain of S. avermitilis capable of producing ABA effectively. RESULTS: Among the sixty actinobacterial isolates, Streptomyces St.53 isolate was chosen for its superior nematicidal effectiveness. The mycelial-methanol extract of isolate St.53 exhibited a maximum in vitro mortality of 100% in one day. In the greenhouse experiment, the mycelial-methanol extract demonstrated, for the second-stage juveniles (J2s), 75.69% nematode reduction and 0.84 reproduction rate (Rr) while for the second-stage juveniles (J2s), the culture suspension demonstrated 75.38% nematode reduction and 0.80 reproduction rate (Rr). Molecular identification for St.53 was performed using 16 S rRNA gene analysis and recorded in NCBI Genbank as S. avermitilis MICNEMA2022 with accession number (OP108264.1). LC-MS was utilized to detect and identify abamectin in extracts while HPLC analysis was carried out for quantitative determination. Both abamectin B1a and abamectin B1b were produced and detected at retention times of 4.572 and 3.890 min respectively. CONCLUSION: Streptomyces avermitilis MICNEMA2022 proved to be an effective source for producing abamectin as a biorational agent for integrated nematode management.

Evaluation of the cytotoxic activity of chemically characterized propolis originating from different geographic regions and vitamin D co-supplementation against human ovarian cancer cells.

Ovarian cancer is the second most common and lethal gynecologic malignancy. Among natural product-based therapy, the honeybee products, particularly propolis, serve a valuable source contributing directly to human nutrition and health.In the present study, we determined the chemical composition of different types of propolis originating from Egypt, Germany and France using liquid chromatography-tandem mass spectrometry. The compounds identified belong to different metabolite classes, including flavonoids, cinnamic acid, chalcones, terpenoids, phenolic lipids, stilbenes, phenolic compounds, carbohydrates, vitamins, coumarins, polyprenylated benzophenone, benzoic acids, fatty acid methyl ester, and coumaric acid, and their derivatives. The most active extract is from France then Egypt and Germany.Afterwards, we treated the human ovarian cancer cells, OVCAR4, with different concentrations (1-400 µg/mL) of variable propolis types supplemented or not with vitamin D (0.0015-0.15 µg/mL) in order to evaluate the efficacy and the cytotoxic activities of our local P as compared to other types collected from different geographic regions. Importantly, the combinatorial treatment of OVCAR4 cancer cells with propolis and vitamin D in the same concentration ranges resulted in enhanced cell viability inhibition. Furthermore, such co-supplementation with vitamin D inhibits predominately the proliferative activity of cell population with the French propolis type as manifested by Ki67 expression, while it reduces considerably its expression, particularly with the German type, followed by the Egyptian one.Nowadays, scientists are interested by natural products which have risen to the forefront of drug discovery. Chemically characterized propolis showing cell viability inhibition and antiproliferative potential seems a valuable extract for further consideration as anti-carcinogenic agent.

Uptake and translocation of nanoplastics in mono and dicot vegetables.

The excessive production and use of plastics increase the release of micro- and nanoplastics (MNPs) into the environment. In recent years, research has focused on the occurrence of MNPs in air, soil and water. Nevertheless, there is still a lack of knowledge regarding MNPs in plants. To determine the load, translocation of MNPs and their effects on metabolism, pak choi, tomato, radish and asparagus have been exposed with fluorescent-labelled poly(methyl methacrylate) or polystyrene (PS) MNPs. The entry of nanoparticles (NPs) of various sizes (100-500 nm) and surface modifications (unmodified, COOH or NH2) into plants has been demonstrated using confocal laser scanning microscopy (CLSM). The translocalization from root to shoot and the accumulation of NP in the intercellular spaces were regardless of the surface modification. In addition, metabolomics was used to evaluate metabolic changes induced by MNPs in pak choi. Changes in phenolic compounds, phytohormone derivatives and other classes of compounds known to be triggered by various environmental stresses have been identified. The present study demonstrates the uptake and translocalization of MNPs in edible parts of vegetables and may pose a hazard for humans.

Analysis of Unfolded Protein Response Activation in Colon Adenocarcinoma Epithelial Cells: A Proteomic Study.

PURPOSE: High throughput technologies have identified molecular patterns in colorectal cancer (CRC) cells, aiding in modeling responses to anti-cancer treatments. The different responses observed depend on the type of cancer, the tumour grade and the functional programme of the cancer cells. Recent studies suggest that the unfolded protein response (UPR), autophagy and apoptosis could be involved in treatment resistance mechanisms by interacting with the tumour microenvironment (TME). EXPERIMENTAL DESIGN: We analysed by LC-MS/MS the proteome of two representative colon adenocarcinoma epithelial cell lines from different tumour grades (CCL-233 and CCL-221) at the basal state or after the UPR induction. RESULTS: Cell lines expressed a different proteome on about 10% of their total proteins identified, especially on UPR, autophagy and apoptosis pathways proteins at basal state. After UPR induction, the proteome of the cells was modified with a greater adaptive response to cellular stress in CCL-221 cells where the UPR was strongly activated at the basal state. CONCLUSIONS AND CLINICAL RELEVANCE: CRC cell lines at different tumour grades expressed different functional programmes at the proteomic level and were characterised by different responses to the UPR induction. This study suggests that baseline cancer cell stress status could have an impact on the efficiency of cancer therapies.

Efficacy and Safety of Different Preemptive Analgesia Measures in Pain Management after Laparoscopic Cholecystectomy: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.

INTRODUCTION: The purpose of this systematic review and network meta-analysis was to evaluate the efficacy and safety of different preemptive analgesia measures given before laparoscopic cholecystectomy (LC) for postoperative pain in patients. METHODS: We conducted a comprehensive search in databases including PubMed, Web of Science, Embase, and the Cochrane Library up to March 2024, and collected relevant research data on the 26 preemptive analgesia measures defined in this article in LC surgery. Outcomes included postoperative Visual Analogue Scores (VAS) at different times (2, 6, 12, and 24 h), opioid consumption within 24 h post-operation, time to first rescue analgesia, incidence of postoperative nausea and vomiting (PONV), and incidence of postoperative headache or dizziness. RESULTS: Forty-nine articles involving 5987 patients were included. The network meta-analysis revealed that multimodal analgesia, nerve blocks, pregabalin, and gabapentin significantly reduced postoperative pain scores at all postoperative time points and postoperative opioid consumption compared to placebo. Tramadol, pregabalin, and gabapentin significantly extended the time to first rescue analgesia. Ibuprofen was the best intervention for reducing PONV incidence. Tramadol significantly reduced the incidence of postoperative headache or dizziness. Subgroup analysis of different doses of pregabalin and gabapentin showed that compared to placebo, pregabalin (300 mg, 150 mg) and gabapentin (600 mg, 300 mg, and 20 mg/kg) were all more effective without significant differences in efficacy between these doses. Higher doses increased the incidence of PONV and postoperative headache and dizziness, with gabapentin 300 mg having a lower adverse drug reaction (ADR) incidence. CONCLUSIONS: Preemptive analgesia significantly reduced postoperative pain intensity, opioid consumption, extended the time to first rescue analgesia, and decreased the incidence of PONV and postoperative headache and dizziness. Multimodal analgesia, nerve blocks, pregabalin, and gabapentin all showed good efficacy. Gabapentin 300 mg given preoperatively significantly reduced postoperative pain and ADR incidence, recommended for preemptive analgesia in LC. TRIAL REGISTRATION: PROSPERO CRD42024522185.

Molecular fingerprint by omics-based approaches in saliva from patients affected by SARS-CoV-2 infection.

Clinical expression of coronavirus disease 2019 (COVID-19) infectionis widely variable including fatal cases and patients with mild symptoms and a rapid resolution. We studied saliva from 63 hospitalized COVID-19 patients and from 30 healthy controls by integrating large-scale proteomics, peptidomics and targeted metabolomics to assess the biochemical alterations following the infection and to obtain a set of putative biomarkers useful for noninvasive diagnosis. We used an untargeted approach by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomics and peptidomics analysis and targeted LC-multiple reaction monitoring/MS for the analysis of amino acids. The levels of 77 proteins were significantly different in COVID-19 patients. Among these, seven proteins were found only in saliva from patients with COVID-19, four were up-regulated and three were down-regulated at least five-folds in saliva from COVID-19 patients in comparison to controls. The analysis of proteins revealed a complex balance between pro-inflammatory and anti-inflammatory proteins and a reduced amount of several proteins with immune activity that possibly favours the spreading of the virus. Such reduction could be related to the enhanced activity of endopeptidases induced by the infection that in turn caused an altered balance of free peptides. In fact, on a total of 28 peptides, 22 (80%) were differently expressed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and control subjects. The multivariate analysis of such peptides permits to obtain a diagnostic algorithm that discriminate the two populations with a high diagnostic efficiency. Among amino acids, only threonine resulted significantly different between COVID-19 patients and controls, while alanine levels were significantly different between COVID-19 patients with different severity. In conclusion, the present study defined a set of molecules to be detected with a quick and easy method based on mass spectrometry tandem useful to reveal biochemical alterations involved in the pathogenesis of such a complex disease. Data are available via ProteomeXchange with identifier PXD045612.

Establishment of LC-MS/MS Methodology for the Determination of Rapamycin Concentration in Rat Whole Blood.

BACKGROUND: The establishment and validation of methods for testing biological samples are crucial steps in pharmacokinetic studies. Currently, several methodological reports have been published on the detection of rapamycin plasma concentrations. OBJECTIVE: The objective of this study was to explore an effective method for detecting rapamycin in rat whole blood biological samples. METHOD: In this study, we designed a rapid, sensitive, and specific liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) methodology for detecting rapamycin in rat whole blood biological samples. We comprehensively validated the specificity, linear range, lower limit of quantification (LLOQ), precision, accuracy, recovery, and stability of this method. RESULTS: The findings of this study confirmed the successful implementation of LC-MS/MS for the detection of rapamycin, demonstrating its sensitivity, specificity, and reliability in quantitative analysis. This method ensures the accuracy and reliability of subsequent study data through our validated LC-MS/MS approach. CONCLUSION: The results demonstrated the successful implementation of an LC-MS/MS method for sensitive, specific, and reliable quantitative analysis of rapamycin in rat whole blood samples. This method ensures the accuracy and reliability of subsequent study data. SIGNIFICANCE: The importance of this study lies in the successful establishment of a rapid, sensitive, and specific LC-MS/MS method for detecting rapamycin concentration in rat whole blood, ensuring the accuracy and reliability of subsequent research data. This provides a crucial tool and foundation for further understanding the metabolism and pharmacological effects of rapamycin in vivo, aiding in the advancement of drug research and clinical applications in related fields.

A selective, sensitive and fast LC-MS/MS method for cabotegravir quantification in rat plasma and pharmacokinetic investigations.

This study presents a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying cabotegravir (CAB) in rat plasma. A novel, sensitive, and rapid LC-MS/MS method has been developed and validated. Furthermore, protein precipitation technique allowed us to lowered the limit of quantification (LOQ) to nanogram levels, allowing detection of smaller CAB amounts in plasma samples. A review of scientific literature reveals that this method is superior than published methods in terms of runtime, sensitivity, wide linearity, and cost, using LC-MS/MS to quantify CAB in biological samples. CAB reached its maximum concentration (Cmax) of 78.401 µg/mL in rat plasma at 1.50 h (Tmax). Linearity was evaluated across 0.05-1000 µg/mL for CAB using five calibration curves with at least nine standards each with r2 > 0.9997. The intra- and inter-day precision and accuracy results were below 15% and acceptable as per Food and Drug Administration (FDA) guidelines. Stability of compounds were established in a battery of stability studies, that is, benchtop, autosampler, and long-term storage stability as well as freeze thaw cycles. The validated method can be used as a routine method to support pharmacokinetic studies.