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.

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.

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.

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipelines.

Untargeted metabolomics is a powerful profiling tool for the discovery of possible biomarkers of disease onset and progression. Analytical pipelines applying liquid chromatography (LC) and mass spectrometry (MS)-based methods are widely used to survey a broad range of metabolites within various metabolic pathways, including organic acids, amino acids, nucleosides, and lipids. Accurate and complete identification of putative metabolites is an ongoing challenge in untargeted metabolomics studies. Highly sensitive instrumentation can result in the detection of adduct and fragment ions that form reproducibly and contain identifiable ions that are difficult to distinguish from metabolic pathway intermediates, which may result in false-positive identification. At concentrations as low as 10 µM, free fatty acids have been found to form homo- and heterodimers in untargeted metabolomics pipelines that resemble the lipid class fatty acid esters of hydroxy fatty acids (FAHFAs), resulting in misidentification. This chapter details a protocol for LC-MS-based untargeted metabolomics using hydrophilic interaction chromatography (HILIC) that specifically aids in distinguishing artifactual fatty acid dimers from endogenous FAHFAs.

LC-MS/MS Quantification of Endocannabinoids in Tissues.

Endocannabinoids (ECBs) are lipid-derived endogenous molecules with important physiological roles such as regulation of energy balance, immunity, or neural development. Quantitation of ECBs helps better understand their physiological role and modulation of biological processes. This chapter presents the simultaneous quantification of 14 ECBs and related molecules in the brain, liver, and muscle, as well as white and brown adipose tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The dynamic range of the method has been tuned to cover the endogenous concentrations of these analytes given the fact that they are endogenously present at different orders of magnitude. Specifically, three groups are established: 0.5-5000 ng/mL for 2-oleoyl- and 2-linoleoylglycerol and arachidonic acid, 0.05-500 ng/mL for 2-arachidonoylglycerol, and 0.0005-0.5 ng/mL for anandamide, palmitoyl-, palmitoleoyl-, stearoyl-, oleoyl-, linoleoyl-, alpha-linolenoyl-, dihomo-gamma-linolenoyl-, docosahexaenoyl-, and pentadecanoylethanolamide.

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.

Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 µm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

Correlating plasma protein profiles with symptomatology and treatment response in acute phase and early remission of major depressive disorder.

Objectives: This study aimed to explore the relationship between plasma proteome and the clinical features of Major Depressive Disorder (MDD) during treatment of acute episode. Methods: In this longitudinal observational study, 26 patients hospitalized for moderate to severe MDD were analyzed. The study utilized Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) alongside clinical metrics, including symptomatology derived from the Montgomery-Åsberg Depression Rating Scale (MADRS). Plasma protein analysis was conducted at the onset of acute depression and 6 weeks into treatment. Analytical methods comprised of Linear Models for Microarray Data (LIMMA), Weighted Correlation Network Analysis (WGCNA), Generalized Linear Models, Random Forests, and The Database for Annotation, Visualization and Integrated Discovery (DAVID). Results: Five distinct plasma protein modules were identified, correlating with specific biological processes, and uniquely associated with symptom presentation, the disorder's trajectory, and treatment response. A module rich in proteins related to adaptive immunity was correlated with the manifestation of somatic syndrome, treatment response, and inversely associated with achieving remission. A module associated with cell adhesion was linked to affective symptoms and avolition, and played a role in the initial episodes and treatment response. Another module, characterized by proteins involved in blood coagulation and lipid transport, exhibited negative correlations with a variety of MDD symptoms and was predominantly associated with the manifestation of psychotic symptoms. Conclusion: This research points to a complex interplay between the plasma proteome and MDD's clinical presentation, suggesting that somatic, affective, and psychotic symptoms may represent distinct endophenotypic manifestations of MDD. These insights hold potential for advancing targeted therapeutic strategies and diagnostic tools. Limitations: The study's limited sample size and its naturalistic design, encompassing diverse treatment modalities, present methodological constraints. Furthermore, the analysis focused on peripheral blood proteins, with potential implications for interpretability.

Development of an advanced analytical technique for detecting multiple pesticide residues in vegetables through liquid chromatography tandem mass spectroscopy (LC-MS/MS).

A comprehensive LC-MS/MS method, which employs Positive Electrospray Ionization (PEI) and Multiple Reaction Monitoring (MRM) was developed for the simultaneous determination of 35 pesticides belonging to various chemical classes in tomato, brinjal, chili, and okra samples. Extraction was facilitated using a modified QuEChERS method, which allows efficient sample analysis in a single run. Calibration curves for each pesticide exhibited linearity within the concentration range of 0.0025 to 0.1 µg mL-1, with correlation coefficients ranging from 0.993 to 0.999. Mean recoveries at five fortification levels (0.01 to 0.5 µg kg-1) ranged from 80 to 90%, demonstrating satisfactory precision (RSD < 20%). The matrix effects, mitigated through an optimized cleanup process, were observed within the range of 6.42% to 19.52%. The developed method having the limit of quantification of 0.01 mg kg-1 for all 35 pesticides, proved to be highly sensitive and rapid for multi-residue estimation in diverse vegetable samples. Subsequently, the method was used to analyze the market samples from Varanasi, India, which revealed the presence of pesticides like Chlorpyrifos, Chlorantraniliproleand Indoxacarb in tomato, brinjal, chili and okra. Therefore, the method could be considered as a robust tool for monitoring pesticide residues in vegetables, aiding in quality assessment and regulatory compliance in the agriculture sector.

Influence of sex and functional status on the value of serum steroid profiling in discriminating adrenocortical carcinoma from adrenocortical adenoma.

Background: It is challenging for clinicians to distinguish adrenocortical carcinoma (ACC) from benign adrenocortical adenomas (ACA) in their early stages. This study explored the value of serum steroid profiling as a complementary biomarker for malignancy diagnosis of ACC other than diameter and explored the influence of sex and functional status. Methods: In this retrospective study, a matched cohort of patients diagnosed with either ACC or ACA based on histopathology was meticulously paired in a 1:1 ratio according to sex, age, and functional status. Eight serum steroids including 11-deoxycortisol, 11-deoxycorticosterone, progesterone, androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17-hydroxyprogesterone, and estradiol, were quantified by liquid chromatography tandem mass spectrometry. We conducted a comparative analysis of the clinical characteristics and serum steroid profiles of patients with ACC and ACA, with further subgroup analysis. Results: The study included 31 patients with ACC and 31 matched patients with ACA. Patients with ACC exhibited significantly larger tumor diameters, lower body mass index (BMI), and higher levels of 11-deoxycortisol, progesterone, and androstenedione than those with ACA. 11-deoxycortisol was the only valuable index for discriminating ACC from ACA, regardless of functional status and sex. Progesterone, DHEA, and DHEAS levels were higher in the functional ACC group than in the non-functional ACC group. Female ACC patients, especially in postmenopausal female exhibited higher levels of androstenedione than male patients. The area under the curve of tumor diameter, 11-deoxycortisol, and BMI was 0.947 (95% CI 0.889-1.000), with a sensitivity of 96.8% and specificity of 90.3%. Conclusion: Serum steroid profiling serves as a helpful discriminative marker for ACC and ACA, with 11-deoxycortisol being the most valuable marker. For other steroid hormones, consideration of sex differences and functional status is crucial.

Analysis of tetrahydroisoquinolines formed after simultaneous consumption of ethanol and amphetamine or its derivatives by LC-MS/MS in human blood, brain, and liver tissue.

The effects of the simultaneous consumption of amphetamine or amphetamine derivatives and alcohol have not yet been adequately clarified, particularly concerning potential condensation products resulting from the endogenous reaction between these substances and their metabolites (e.g., acetaldehyde, a metabolite of ethanol). In this study, we developed an LC-MS/MS method employing liquid-liquid extraction for the qualitative detection of some relevant condensation products belonging to the class of tetrahydroisoquinolines and their derivatives in human blood, brain, and liver samples. This includes the analysis of the substrates amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, as well as the condensation products 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, 1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline, and N-methyl-1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline. Therefore, the reference standards of the mentioned tetrahydroisoquinolines were synthesized in advance and the method was validated with regard to the question of the qualitative detection of these compounds. The validation parameters included selectivity, specificity, limit of detection, lower limit of quantification, recovery, matrix effects, and stability for blood, brain, and liver samples. Following the analysis of human blood and post-mortem tissue samples, evidence of the condensation product 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline originating from the interaction between amphetamine and acetaldehyde was identified in two liver samples. On the contrary, no evidence of this or other tetrahydroisoquinolines was found in the remaining tissue and serum samples.

Metabolome and Transcriptome Profiling Reveals Age-Associated Variations in Meat Quality and Molecular Mechanisms of Taihe Black-Bone Silky Fowls.

To explore the changes in meat quality and molecular mechanisms during the growth and development of Taihe black-bone silky fowl, this study employed liquid chromatography-mass spectrometry (LC-MS/MS) metabolomics to elucidate the dynamic changes of key differential metabolites (DMs) affecting meat quality, indicating that chicken at D120 had higher levels of ω-3 polyunsaturated fatty acids (PUFAs), creatine, anserine, and homocarnosine, while D150 had the most stachydrine and D210 had the most acylcarnitines. Additionally, D120 and D180 had more umami and sweet compounds. Furthermore, key metabolic pathways influenced by age included purine metabolism, the pentose phosphate pathway, nicotinate and nicotinamide metabolism, and taurine and hypotaurine metabolism. Transcriptomic identified differential expression genes (DEGs) are predominantly enriched in focal adhesion, the TGF-ß signaling pathway, and the MAPK signaling pathway. Integrated metabolomics and transcriptomics revealed complex regulatory networks of DEGs and DMs in key metabolic pathways. This research enhanced our understanding of the biology of Taihe black-bone silky fowl meat quality, revealing possible biomarkers.

Pharmacokinetics and related gender difference studies of four active components of Codonopsis Pilosula by LC-MS/MS determination.

ETHNOPHARMACOLOGICAL RELEVANCE: Codonopsis pilosula (C. pilosula), commonly known as Dangshen in Chinese, had been used to regulate the immune, digestive, and circulatory systems of human. The reported pharmacokinetic studies on C. pilosula are mainly limited to in vivo profile studies of a single component. It has not been detected simultaneously the in vivo pharmacokinetic profiles of multiple active components as well as related gender difference after oral dosing of the extraction of C. pilosula. AIM OF THE STUDY: This study aims to reveal the pharmacokinetic characteristics of the four main active components of C. pilosula after oral dosing of its extraction in rats, and to explain the gender differences in absorption and metabolism. MATERIALS AND METHODS: The plasma pharmacokinetic characteristics of four main active components of C. pilosula was explored using the established LC-MS/MS method after oral dosing of the extraction of C. pilosula in male and female rats. In vitro intestinal pouch permeability and liver microsome metabolic stability were also observed to classify the possible mechanism of gender difference existed in the pharmacokinetic profiles of the four active components in rats. RESULTS: Four effective components were absorbed quickly in rats after oral administration of alcoholic extract of C. pilosula (1.36 g/mL, equivalent to 2 g/mL as crude drug), and their exposure order was as follows: Atractylenolide III > Lobetyolin > Tangshenoside I > Syringin. The exposure (AUC) and peak concentration (Cmax) of Atractylenolide III in female rats were much higher than those in male rats, indicating a significant gender difference in pharmacokinetics of Atractylenolide III between female and male animals. With the help of the rat model of intestinal sac in vitro, it was found that Lobetyolin was a hypertonic compound, and both Tangshenoside I and Syringin were compounds with medium permeabiltiy. Notably, the Papp of Atractylenolide III was 3.3×10-6 cm/s in male rat intestinal sac assay, while that was 10×10-6 cm/s in female rat intestinal sac model, showing a significant gender difference in intestinal permeability (P<0.05). After the addition of NADPH, the four compounds were reduced in a time-dependent manner, suggesting that CYP450s could catalyze their metabolism. After incubation, the remaining content of Atractylenolide III in the liver microsomes of male and female rats was 27% and 57%, respectively, suggesting slower metabolic rate of in female rat liver microsomes. CONCLUSION: A simple, efficient and reliable LC-MS/MS method for the simultaneous determination of four active index components of C. pilosula, Lobetyolin, Tangshenoside I, Atractylenolide III and Syringin, in rat plasma was established and verified. This method was successfully applied in the pharmacokinetic study after single oral administration of the alcoholic extract of C. pilosula in rats. Gender difference was observed in the pharmacokinetic profile of Atractylenolide III in rats. Intestinal absorption and liver metabolism might be two key factors that resulted in the gender difference in exposure and pharmacokinetics of Atractylenolide III in rats. This study provides supportive data for clinical rational application of C. pilosula in individualized medication therapy.

Elucidating the effective age for dietary restriction and the key metabolites involved.

Dietary restriction (DR) extends lifespan in various species, but its effect at different ages, especially when started later, is unclear. This study used Caenorhabditis elegans to explore the impact of DR at different ages. Worms were divided into control and DR groups, with daily survival monitored. To confirm the occurrence of DR, the expression of DR-sensitive genes namely acdh-1, pyk-1, pck-2 and cts-1 were determined using RT-qPCR. Liquid chromatography mass spectrometry (LC-MS) was employed to observe the changes in metabolites affected by DR. The results indicated that young worms subjected to mild DR displayed the longest lifespan, highlighting the effectiveness of initiating DR at a young age. Increased expression of acdh-1 and pck-2 suggests activation of beta-oxidation and gluconeogenesis, while decreased cts-1 expression indicates a reduced citric acid cycle, further supporting the observed effects of DR in these worms. Metabolomic results indicated that DR decreased the activity of mechanistic Target of Rapamycin (mTOR) and the synthesis of amino acids namely leucine, tyrosine and tryptophan to conserve energy for cell repair and survival. DR also decreased levels of N-acetyl-L-methionine and S-adenosyl-methionine (SAM) in methionine metabolism, thereby promoting autophagy, reducing inflammation, and facilitating the removal of damaged cells and proteins. In conclusion, initiating dietary restriction early in life extends the lifespan by modulating amino acid metabolism and enhancing the autophagy pathway, thereby maintaining cellular wellbeing.

Evaluation of 4 quantification methods for monitoring 16 antidepressant drugs and their metabolites in human plasma by LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring for antidepressants (ADs) is vital due to the potentially serious consequences and disputes related to medical events. Therefore, we created a quick and convenient analysis way for separation and quantification of ADs. METHODS: To ensure quantitative stability, we divided the 16 ADs or their metabolites into 4 pools (AD1-AD4), considering the hospital frequency that the clinician prescribed, the physicochemical properties of medicines, and the calibration range of selected ADs. After precipitation with methanol, the analytes were eluted for at least 3.5 min on a BEH C18 analytical column by different gradient elution methods. RESULTS: The LLOQ and LOD were 1.25-10 ng/mL and 0.42-5 ng/mL, respectively. High precision (<12 %) and accuracy (87.07-111.47 %) were demonstrated by quality control samples both within and between days. All the compounds were stable at room temperature and within -80 °C. CONCLUSION: The method is of wide clinical and laboratory interest due to simpler sample cleanup, shorter chromatographic run times, and wider calibration range compared to other methods.

Assessment of the in vitro metabolic stability of CEP-37440, a selective FAK/ALK inhibitor, in HLMs using fast UPLC-MS/MS method: in silico metabolic lability and DEREK alerts screening.

Introduction: CEP-37440 was synthesized and supplied by the research and development division of Teva Branded Pharmaceutical Products (West Chester, PA, United States). CEP-37440 represents a newly developed compound that exhibits selectivity inhibition of Focal Adhesion Kinase and Anaplastic Lymphoma Kinase FAK/ALK receptors, demonstrating novel characteristics as an orally active inhibitor. The simultaneous inhibition of ALK and FAK can effectively address resistance and enhance the therapeutic efficacy against tumors through a synergistic mechanism. Methods: The objective of this research was to create an LC-MS/MS method that is precise, efficient, environmentally friendly, and possesses a high level of sensitivity for the quantification of CEP-37440 in human liver microsomes (HLMs). The aforementioned approach was subsequently employed to evaluate the metabolic stability of CEP-37440 in HLMs in an in vitro setting. The validation procedures for the LC-MS/MS analytical method in the HLMs were performed following the bio-analytical method validation guidelines set out by the US-FDA. The AGREE program was utilized to assess the ecological impacts of the current LC-MS/MS methodology. Results and Discussion: The calibration curve linearity was seen in the range of 1-3000 ng/mL. The inter-day accuracy (% RE) exhibited a range of -2.33% to 3.22%, whilst the intra-day accuracy demonstrated a range of -4.33% to 1.39%. The inter-day precision (% RSD) exhibited a range of 0.38% to 3.60%, whilst the intra-day precision demonstrated a range of 0.16% to 6.28%. The determination of the in vitro half-life (t1/2) and moderate intrinsic clearance (Clint) of CEP-37440 yielded values of 23.24 min and 34.74 mL/min/kg, respectively. The current manuscript is considered the first analytical study for CEP-37440 quantification with the application to metabolic stability assessment. These results suggest that CEP-37440 can be categorized as a pharmaceutical agent with a moderate extraction ratio. Consequently, it is postulated that the administration of CEP-37440 to patients may not lead to the accrual of dosages within the human organs. According to in silico P450 metabolic and DEREK software, minor structural alterations to the ethanolamine moiety or substitution of the group in drug design have the potential to enhance the metabolic stability and safety profile of novel derivatives in comparison to CEP-37440.

Development of a rapid and robust hydrop interaction liquid chromatography tandem mass spectrometry method for the detection of 13 endogenous amino acids as well as trimethylamine oxide in serum and tissues of the mice.

This work aimed to establish an HILIC-MS/MS method to simultaneously determine the levels of 13 endogenous amino acids and trimethylamine oxide in the biological samples from the mice. Electrospray ion source was used for the analysis of mass spectrometry. The 20 min separation was applied in a Dikma Inspire Hilic column (2.1 × 100.0 mm, 3 µM). Positive ion mode under an MRM model gave a satisfying response value. The limits of quantitation were evaluated by accuracy from -12.59% to 7.89% and precision from 1.77% to 14.00% as well as acceptable interday and intraday precision, matrix effect, recovery, and stability. Later, the assay was successfully used to measure the concentrations of the determinands in the biological samples. Individual and tissue distribution differences for these metabolites were observable. The amino acids had a consistent highest content in the spleens, while the lowest levels were found in the livers. Alanine was the most abundant amino acid in the serum, and taurine kept the highest content in all of the tissues. Trimethylamine oxide remained low level, especially in the liver samples.

Untargeted Metabolomics Analysis Using FTIR and LC-HRMS for Differentiating Sonchus arvensis Plant Parts and Evaluating Their Biological Activity.

The composition and concentration of compounds in medicinal plants vary based on several factors, including the specific part of the plant being used. These variations in composition and concentration lead to differences in biological activity levels. In this study, we aimed to assess the phytochemical profile of Sonchus arvensis and to investigate the biological activity of different plant parts (roots, stems, and leaves) using a metabolomics approach. We analyzed the plant extracts for total phenolic and flavonoid levels, antioxidant activity, and xanthine oxidase inhibition. We also conducted metabolite profiling using Fourier-transform infrared spectroscopy and liquid chromatography-high resolution mass spectrometry. A total of 17 metabolites were identified (13 in leaves, 10 in stems, and 9 in roots). Principal component analysis effectively differentiated S. arvensis extracts based on differences in plant parts. These findings indicate that the quantity and diversity of metabolites present in the roots, stems, and leaves influence the biological activity of S. arvensis.