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Summary: Antibiotic resistance is now deemed a worldwide problem that puts public health at risk. The potential of Murraya (Murraya koenigii (L.) Spreng. and Murraya paniculata (L.) Jacq.) leaves and stems as antibacterial agents against multidrug‐resistant Acinetobacter baumannii (MDRAB) was assessed in our study. First, screening was performed by disc diffusion assay, and minimum inhibitory concentration values were then determined as compared to tigecycline. A. baumnii mouse model of infection was established to substantiate the antibacterial activity of Murraya species. Results revealed high antimicrobial activity for stem of both plants where leaves showed moderate to weak activity. Phytochemical characterisation revealed the identification of 129 metabolites belonging to different classes of compounds viz. coumarins, carbazole alkaloids, flavonoids, phenolic acids, and miscellaneous. In vivo data from the animal model supported the high efficiency of M. paniculata stems as promising extract for lead candidates against MDRAB pulmonary infections. Inhibition of its essential MurF (UDP‐N‐acetylmuramoyl‐tripeptide‐D‐alanyl‐d‐alanine ligase) protein has been reported as a potential target for broad‐spectrum drugs. In silico results after molecular docking to MurF from Acinetobacter baumannii (PDB ID: 4QF5) showed competitive binding mode to ATP ligand at the active site predicting antibacterial activity of the tested compounds. Furthermore, chlorogenic acid, caffeic acid, sinapic acid, feruloyl agmatine, and mahanimbidine were detected as the key discriminatory metabolites correlated with antibacterial activity. To our knowledge, this is the first in vivo anti‐MDRAB study for the investigated plant. Murraya plants have enormous possibility for the discovery of novel bioactive compounds which could combat against resistant microorganisms. [ABSTRACT FROM AUTHOR]

Invasive fungal aspergillosis is a leading cause of morbidity and mortality in many species including avian species such as common ravens (Corvus corax). Methods were developed for mass spectral determination of voriconazole in raven plasma as a means of determining pharmacokinetics of this antifungal agent. Without further development, GC/MS/MS (gas chromatography-tandem quadrupole mass spectrometry) proved to be inferior to LC/MS/MS (liquid chromatography-tandem quadrupole mass spectrometry) for measurement of voriconazole levels in treated raven plasma owing to numerous heat-induced breakdown products despite protection of voriconazole functional groups with trimethylsilyl moieties. LC/MS/MS measurement revealed in multi-dosing experiments that the ravens were capable of rapid or ultrarapid metabolism of voriconazole. This accounted for the animals' inability to raise the drug into the therapeutic range regardless of dosing regimen unless cytochrome P450 (CYP) inhibitors were included. Strategic selection of CYP inhibitors showed that of four selected compounds including cimetidine, enrofloxacin and omeprazole, only ciprofloxacin (Cipro) was able to maintain voriconazole levels in the therapeutic range until the end of the dosing period. The optimal method of administration involved maintenance doses of voriconazole at 6 mg/kg and ciprofloxacin at 20 mg/kg. Higher doses of voriconazole such as 18 mg/kg were also tenable without apparent induction of toxicity. Although most species employ CYP2C19 to metabolize voriconazole, it was necessary to speculate that voriconazole might be subject to metabolism by CYP1A2 in the ravens to explain the utility of ciprofloxacin, a previously unknown enzymatic route. Finally, despite its widespread catalog of CYP inhibitions including CYP1A2 and CYP2C19, cimetidine may be inadequate at enhancing voriconazole levels owing to its known effects on raising gastric pH, a result that may limit voriconazole solubility. [ABSTRACT FROM AUTHOR]

Abstract Acinetobacter baumannii is without a doubt one of the most problematic bacteria causing hospital-acquired nosocomial infections in today's healthcare system. To solve the high prevalence of multi-drug resistant (MDR) in A. baumannii, we investigated one of the medicinal plants traditionally used as antibacterial agent; namely Murraya koenigii (L.) Sprengel. The total methanolic extracts of seeds and pericarps were prepared and their anti-bacterial activity was assessed using the agar diffusion method and minimum inhibitory concentration (MIC) was then calculated as compared to tigecycline. Then, an in-vivo murine model was established which confirmed the promising activity of M. koenigii seeds in demonstrating anti-bacterial and anti-inflammatory actions. The histopathological study of lungs, scoring of pulmonary lesions, counting of bacterial loads after infection by multi-drug resistant A. baumannii all provided evidence to support these findings. LC–MS/MS profiling coupled to molecular networking and chemometrics detected the presence of carbazole alkaloids, and coumarins as dominate metabolites of the active seed extracts. Positively correlated metabolites to antibacterial potential were 6-(2ʹ,3ʹ-dihydroxy-3-methylbutyl)-8-prenylumbelliferone, scopoline, and 5-methoxymurrayatin. An in-silico study was also performed on the crystal structure of MurF from A. baumannii (PDB ID: 4QF5), the studied structures of the mentioned extracts revealed good docking interaction at the active site suggestive of competition with the ATP ligand. These collective findings suggest that extracts of Murraya koenigii (L.) Sprengel seed is a novel prospective for the discovery of drug candidates against infections caused by MDR A. baumannii.

A sensitive and specific bioanalytical method was required to measure the exposure of a LAGA-mutated surrogate mouse IgG2a monoclonal antibody in mouse plasma, but the lack of highly specific reagents for the LAGA mutant hindered the development of a ligand-binding assay. Equally problematic is that no sensitive unique tryptic peptides suitable for quantitative mass spectrometric analysis could be identified in the mIgG2a complementarity-determining regions. To overcome these challenges, a trypsin alternative pepsin, an aspartic protease, was systematically investigated for its use in digesting the mutated mIgG2a antibody to allow generation of signature peptides for the bioanalytical quantification purpose. After a series of evaluations, a rapid one-hour pepsin digestion protocol was established for the mutated Fc backbone. Consequently, a new pepsin digestion-based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was successfully developed to support the mouse pharmacokinetic (PK) sample analysis. In brief, robust and reproducible C-terminal cleavage of both leucine and phenylalanine near the double mutation site of the mutated mIgG2a was accomplished at pH ≤2 and 37°C. Combined with a commercially available rat anti-mIgG2a heavy-chain antibody, the established immunoaffinity LC/MS/MS assay achieved a limit of quantitation of 20 ng/mL in the dynamic range of interest with satisfactory assay precision and accuracy. The successful implementation of this novel approach in discovery PK studies eliminates the need for tedious and costly generation of specific immunocapturing reagents for the LAGA mutants. The approach should be widely applicable for developing popular LAGA mutant-based biological therapeutics.

Glycosylation changes in cancer proteins have been associated with malignant transformation. However, techniques for analyzing site-specific glycosylation changes in target proteins obtained from clinical tissue samples are insufficient. To overcome these problems, we developed a targeted N-glycoproteomic approach consisting of immunoprecipitation, glycopeptide enrichment, LC/MS/MS and structural assignment using commercially available analytical software followed by manual confirmation. This approach was applied to the comparative site-specific glycosylation analysis of lysosome-associated membrane glycoprotein 1 (LAMP1) between breast cancer (BC) tumors and normal tissues adjacent to tumors. Extensive determination of glycan heterogeneity from four N-glycosylation sites (Asn84/103/249/261) in LAMP1 identified 262 glycoforms and revealed remarkable diversity in tumor glycan structures. A significant increase in N-glycoforms with multiple fucoses and sialic acids at Asn84/249 and high-mannose-type glycans at Asn103/261 were observed in the tumor. Principal component analysis revealed that tumors of different subtypes have independent distributions. This approach enables site-specific glycopeptide analysis of target glycoprotein in breast cancer tissue and become a powerful tool for characterizing tumors with different pathological features by their glycan profiles. [ABSTRACT FROM AUTHOR]

Acinetobacterbaumannii is without a doubt one of the most problematic bacteria causing hospital-acquired nosocomial infections in today's healthcare system. To solve the high prevalence of multi-drug resistant (MDR) in A.baumannii, we investigated one of the medicinal plants traditionally used as antibacterial agent; namely Murrayakoenigii (L.) Sprengel. The total methanolic extracts of seeds and pericarps were prepared and their anti-bacterial activity was assessed using the agar diffusion method and minimum inhibitory concentration (MIC) was then calculated as compared to tigecycline. Then, an in-vivo murine model was established which confirmed the promising activity of M.koenigii seeds in demonstrating anti-bacterial and anti-inflammatory actions. The histopathological study of lungs, scoring of pulmonary lesions, counting of bacterial loads after infection by multi-drug resistant A.baumannii all provided evidence to support these findings. LC–MS/MS profiling coupled to molecular networking and chemometrics detected the presence of carbazole alkaloids, and coumarins as dominate metabolites of the active seed extracts. Positively correlated metabolites to antibacterial potential were 6-(2ʹ,3ʹ-dihydroxy-3-methylbutyl)-8-prenylumbelliferone, scopoline, and 5-methoxymurrayatin. An in-silico study was also performed on the crystal structure of MurF from A.baumannii (PDB ID: 4QF5), the studied structures of the mentioned extracts revealed good docking interaction at the active site suggestive of competition with the ATP ligand. These collective findings suggest that extracts of Murrayakoenigii (L.) Sprengel seed is a novel prospective for the discovery of drug candidates against infections caused by MDR A.baumannii. [ABSTRACT FROM AUTHOR]

Mentha rotundifolia is a member of the Lamiaceae family and is distributed mainly in traditional medicine in Algeria and the Mediterranean locality. This study aimed to demonstrate how the extraction technique can determine the type of active ingredients obtained, as well as their potential pharmacological and therapeutic effects. The methanolic extracts were obtained by maceration and Soxhlet apparatus. The study of the chemical composition was determined by colourimetric methods and liquid chromatography–mass spectrometry (LC–MS/MS); the antioxidant activity was evaluated in vitro by three tests: DPPH test, ferrous ion chelating test and reducing power test. The antimicrobial effect of extracts was evaluated by agar diffusion assay against six microbial strains. The results demonstrate that the best yield of extraction, Total phenolic and flavonoid contents, and antioxidant activity were found to be higher in the extract obtained by Soxhlet than those obtained by maceration. The phytochemical screening tests identified polyphenols, flavonoids, tannins, terpenoids, and quinones, and anthraquinones and saponins were absent in both extracts. The LC–MS/MS revealed the presence of several phenolic compounds, the predominant in both extracts was rosmarinic acid. The antimicrobial activity shows that both extracts have no effect. In conclusion, this study reveals that extraction by Soxhlet is more suitable than extraction by maceration for this plant.

Antimicrobial drugs and coccidiostat compounds are commonly used in poultry farming. These compounds are subsequently excreted and released into the environment via broiler litter (BL) and can re-enter the food chain as fertilizer or animal feed. Such residue in animal feed can encourage the appearance of antibiotic-resistant bacteria as well as toxicity. Most analytical methods used to identify and quantitate these drug residues are traditional, and are specific to some antimicrobials and present limitations in assessing complex matrixes like BL. The aim of this study was to develop a multi-residue analytic method for assessing 30 antimicrobial drugs and coccidiostats associated with BL. We investigated the presence and the effects of biotic stack treatment on the degradation of drug residue in BL. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) were replaced by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) clean-up steps and detected by liquid chromatography mass spectrometry (LC/MS/MS). Results show that a wide spectrum of residues were detected from 0.4 to 8.9 mg kg−1. Following lab-scale stacking treatment, tilmicosin and eight coccidiostats persisted in BL (26–100%). This research supports the need for better understanding, regulation, and management of the use of BL that might carry a high risk of residue drugs. [ABSTRACT FROM AUTHOR]

Increased acceptance of cannabis containing the psychoactive component, Δ9‐tetrahydrocannabinol (THC), raises concerns about the potential for impaired drivers and increased highway accidents. In contrast to the "breathalyzer" test, which is generally accepted for determining the alcohol level in a driver, there is no currently accepted roadside test for THC in a motorist. There is a need for an easily collectible biological sample from a potentially impaired driver coupled with an accurate on‐site test to measure the presence and quantity of THC in a driver. A novel breath collection device is described, which includes three separate sample collectors for collecting identical A, B, and C breath samples from a subject. A simple one‐step ethanol extraction of the "A" breath collector sample can be analyzed by UHPLC/selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS) to provide qualitative and quantitative determination of THC in breath sample in less than 4 min for samples collected up to 6 h after smoking a cannabis cigarette. SIM LC/MS bioanalyses employed d3‐THC as the stable isotope internal standard fortified in negative control breath samples for quantitation including replicates of six calibrator standards and three quality control (QC) samples. Subsequent confirmation of the same breath sample in the B collectors was then confirmed by a reference lab by LC/MS/MS analysis. Fit‐for‐purpose bioanalytical validation consistent with pharmaceutical regulated bioanalyses produced pharmacokinetic (PK) curves for the two volunteer cannabis smokers. These results produced PK curves, which showed a rapid increase of THC in the breath of the subjects in the first hour followed by reduced THC levels in the later time points. A simpler single‐point calibration curve procedure with calibrators and QC prepared in ethanol provided similar results. Limitations to this approach include the higher cost and operator skill sets for the instrumentation employed and the inability to actually determine driver impairment. [ABSTRACT FROM AUTHOR]

Background: Lasmiditan is an agonist of 5HT1F receptor, acute migraine is treated and oral dose is safe and effective. No analytical methods were reported on Lasmiditan Drug. Objectives: The goal of the current project is to create a quick, effective, and reproducible LC/MS/MS method for the estimation of Lasmiditan. The mobile phase was used Acetonitrile: 0.1% formic acid (70:30) in the developed method, and it was running down a C18 column (SP) with dimensions of 150 mm, 4.6 mm i.d., and 3.5 µm at a rate of 1 mL/minute. The [M+H]+ ion m/z ratio for the substance was observed in the mass spectrum was 378.24. The drug had a retention time of 2.3 minutes. The linearity range was found to be in between 12.50 ng/mL and 75 ng/mL. The regression coefficient value was found to be 0.999, which is good satisfactory value for linearity. The LOD and LOQ for baseline measurement and detection were 0.66 ng/mL and 2.22 ng/mL, respectively; the method was shown good accuracy and precision levels, and it was proved through the assay values. The method was proved as robust after deliberated changes in parameters of flow rate (±0.2mL) and organic phase (±3 mL) in mobile phase composition and values were found to be 99.50%-100.73% & 99.10%- 101.83% respectively. Conclusion: The LC/MS/MS method is more advanced and sensitive to determine the drugs at nanograms level. Hence developed method used for the regular assay. This is more sensitive, selective, and rapid for identification and quantification of LMT in the bulk and Pharmaceutical formulation. Keywords: Lasmiditan, LC/MS/MS, Acetonitrile, Formic acid. [ABSTRACT FROM AUTHOR]

The objective of the study was to determine the total phenolic and total flavonoid contents (TPC and TFC, respectively), as well as the solvent-partitioned fractions, of the leaf extract of Cassia fistula. Using DPPH, TAC, and FRAP tests, the in vitro antioxidant properties of crude extract and its ethyl acetate fraction were investigated. Additionally, C. fistula chemical profiling was accomplished using LC-ESI-MS/MS. Along with that, the effectiveness of crude extract (200 and 400 mg/kg) in treating male rats with ulcerative colitis (UC) induced by acetic acid was assessed. According to the findings, the ethyl acetate fraction had the highest concentrations of TPC (12.36 1.46 mg GAE/100 mg) and TFC (5.12 0.64 mg QE/100 mg), as well as impressive in vitro antioxidant activity with IC50 values of DPPH (12.7 g/mL), FRAP (4.87 0.71 mM Fe+2/g), and TAC (55. Fifty-five chemicals, predominantly phenolics (catechins, flavonoids, anthraquinones, chalcones, and phenolic acids), were detected by the LC/MS/MS. The anti-UC effect of C. fistula was dose-dependent. The hematological parameters, liver biomarkers, oxidative stress, histopathology, and immunohistochemistry revealed that prophylactic administration of crude extract to colitis rats ameliorated all the previous biomarkers. However, when the crude extract was administered on established colitis, it facilitated the recovery of the inflamed mucosa and showed better results than the protection mode. The phytoconstituents of C. fistula that were discovered here by LC/MS/MS were examined by molecular docking studies for their binding affinities towards COX-2 and caspase-3 proteins to highlight the anti-inflammatory and antiapoptotic activities of the plant. The Emodin compound displayed the highest binding affinity for COX-2 proteins, while isorhamnetin was discovered to have the best binding associations with the essential amino acids of caspase-3. Procyanidin B2 interestingly shows potent interactions with important amino acids of two targets. This study has made it possible to utilize C. fistula in the treatment of UC and has clarified its mechanism of action.

End-stage renal disease (ESRD) is a rapidly increasing health problem, and every year, about 2 million ESRD cases are reported worldwide. Hemodialysis (HD) is the vital renal reinstatement therapy for ESRD, and HD patterns play a crucial role in patients' health. Plasma metabolomics is the potential approach to understanding the HD process, effectiveness, and patterns. The lack of protein vitality is a primary problem for HD patients, and the quantities of amino acids intracellularly and in the blood are considered to be a symbolic index of protein metabolism and nutrition conditions. In the current study, LC/MS/MS and GC/MS methods were developed for 29 targeted plasma metabolites and validated as per ICH bioanalytical method validation M10 guidelines. The 29 metabolites included 20 proteinogenic amino acids and nine other related metabolites. The methods were employed to measure the absolute quantities (µM) of the targeted metabolites in HD patients (n=60) before and after dialysis (PRE-HD and POST-HD), and compared with the healthy control (HC) group (n=60). Phenylacetylglutamine was found to be higher in both PRE-HD (72.88±14.5 µM) and POST-HD (26.62±7.9 µM), when compared to HC (1.61±0.6 µM). On the other hand, glutamic acid was lower in PRE-HD (14.90±6.5 µM), and POST-HD (13.6±6.1 µM) than that of HC (245.4±37.8 µM). The dialytic loss was found to be 52–45% for arginine, lysine, and histidine, while it was 38–26% for glycine, cysteine, proline, alanine, threonine, glutamine, valine, and methionine. The dialytic loss was low (≤12%) for aspartic acid, glutamic acid, asparagine, leucine, tyrosine, tryptophan, and isoleucine. Graphical abstract adapted from mass spectrometry templates by Biorender.com retrieved from https://app.biorender.com/biorender-templates. [ABSTRACT FROM AUTHOR]

Phthalates are used as plasticizers and additives in food products and personal care items. The conducted studies have demonstrated the negative effects of phthalates on human health. Five phthalates esters (dibutyl phthalate (DBP), benzyl butylphthalate (BBP), di(2- ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), and diisodecyl phthalate (DIDP)) were investigated in 30 different types of tea and herbal infusions using liquid chromatography-mass spectrometry (LC/MS/MS). This study demonstrated the presence of phthalates in black tea, green tea, and herbal infusions consumed in Turkey. Among the analyzed tea samples, DBP was the most common, detected in 19 out of 30 samples (ranging from 5.78 to 44.99 µg/kg). DEHP was the second most frequently detected phthalate, found in 14 out of 30 samples (ranging from 5.65 to 60.83 µg/kg). The other detected phthalate was BBP, which was found in 5 out of 30 samples (ranging from 9.35 to 44.95 µg/kg). [ABSTRACT FROM AUTHOR]

Abstract Background The yellow jasmine flower (Jasminum humile L.) is a fragrant plant belonging to the Oleaceae family with promising phytoconstituents and interesting medicinal uses. The purpose of this study was to characterize the plant metabolome to identify the potential bioactive agents with cytotoxic effects and the underlying mechanism of cytotoxic activity. Methods First, HPLC–PDA-MS/MS was used to identify the potential bioactive compounds in the flowers. Furthermore, we assessed the cytotoxic activity of the flower extract against breast cancer (MCF-7) cell line using MTT assay followed by the cell cycle, DNA-flow cytometry, and Annexin V-FITC analyses alongside the effect on reactive oxygen species (ROS). Finally, Network pharmacology followed by a molecular docking study was performed to predict the pathways involved in anti-breast cancer activity. Results HPLC–PDA-MS/MS tentatively identified 33 compounds, mainly secoiridoids. J. humile extract showed a cytotoxic effect on MCF-7 breast cancer cell line with IC50 value of 9.3 ± 1.2 µg/mL. Studying the apoptotic effect of J. humile extract revealed that it disrupts G2/M phase in the cell cycle, increases the percentage of early and late apoptosis in Annexin V-FTIC, and affects the oxidative stress markers (CAT, SOD, and GSH-R). Network analysis revealed that out of 33 compounds, 24 displayed interaction with 52 human target genes. Relationship between compounds, target genes, and pathways revealed that J. humile exerts its effect on breast cancer by altering, Estrogen signaling pathway, HER2, and EGFR overexpression. To further verify the results of network pharmacology, molecular docking was performed with the five key compounds and the topmost target, EGFR. The results of molecular docking were consistent with those of network pharmacology. Conclusion Our findings suggest that J. humile suppresses breast cancer proliferation and induces cell cycle arrest and apoptosis partly by EGFR signaling pathway, highlighting J. humile as a potential therapeutic candidate against breast cancer.

Several compounds with different physical properties are present in foods, biological components, and environmental samples, and there are cases in which these must be analyzed simultaneously. However, it is difficult to extract compounds with different physical properties from the same sample using a single method. In the present study, we examined the optimal conditions for the QuEChERS extraction of several kinds of compounds from orange juice using design of experiments (DoE) and response surface methodology (RSM) to determine the optimal ratio of organic solvent to sodium chloride. We determined the optimal extraction conditions, which were within the design space, using 100% tetrahydrofuran (THF) as the extraction organic solvent and NaCl:MgSO4 = 75:25 as the salt. The developed LC/MS/MS method using QuEChERS extraction achieved specific detection and precise quantification. Finally, we measured the polyphenols, sterols, and carotenoids in citrus juice using the optimized QuEChERS extraction method before LC/MS/MS analysis. Most of the analytes were quantifiable in orange juice. The optimized method achieved ease of operation, the extraction of analytes from food samples in a short time (within 30 min), minimization of analytical residues, and reliability. The DoE and RSM approach may contribute to better optimization of the extraction conditions for the lowest number of experiments. [ABSTRACT FROM AUTHOR]

The combination of sulfadoxine (SDO) with trimethoprim (TMP) is widely used in veterinarian medicine. The aim of the present study was to compare excretion profiles and detection time windows of SDO and TMP in plasma and urine by means of a validated quantitative method. Eight horses received a single intravenous (i.v.) dose of 2.7 mg TMP and 13.4 mg SDO per kg bodyweight. Plasma and urine samples were collected up to 15 and 70 days post‐administration, respectively. While urine samples underwent an enzymatic hydrolysis, plasma samples were proteolysed before further analysis. After solid‐phase extraction, samples were analysed by liquid chromatography/electrospray ionisation tandem mass spectrometry in positive ionisation mode. The applied multiple reaction monitoring (MRM) method allowed the detection of SDO and TMP with a lower limit of detection of 0.03 ng/mL in plasma and 0.2 (SDO) and 0.4 ng/mL (TMP) in urine, respectively. In the present study, detection times for SDO were 15 days in plasma and 49 days in urine, respectively. TMP was detected for up to 7 days in plasma and up to 50 days in urine, respectively. The detection via the TMP metabolite 3‐desmethyl‐trimethoprim was possible for 70 days in urine. Detection times of the other confirmed metabolites N4‐acetylated sulfadoxine, hydroxytrimethoprim, trimethoprim‐1‐oxide and trimethoprim‐3‐oxide were significantly lower. In order to postulate reasonable screening limits (SLs) to control specific withdrawal times, a Monte Carlo simulation was performed for SDO. The proposed SL of 10 ng/mL SDO in blood and 300 ng/mL urine corresponds to a detection time of 4 days. [ABSTRACT FROM AUTHOR]

Background: The yellow jasmine flower (Jasminum humile L.) is a fragrant plant belonging to the Oleaceae family with promising phytoconstituents and interesting medicinal uses. The purpose of this study was to characterize the plant metabolome to identify the potential bioactive agents with cytotoxic effects and the underlying mechanism of cytotoxic activity. Methods: First, HPLC–PDA-MS/MS was used to identify the potential bioactive compounds in the flowers. Furthermore, we assessed the cytotoxic activity of the flower extract against breast cancer (MCF-7) cell line using MTT assay followed by the cell cycle, DNA-flow cytometry, and Annexin V-FITC analyses alongside the effect on reactive oxygen species (ROS). Finally, Network pharmacology followed by a molecular docking study was performed to predict the pathways involved in anti-breast cancer activity. Results: HPLC–PDA-MS/MS tentatively identified 33 compounds, mainly secoiridoids. J. humile extract showed a cytotoxic effect on MCF-7 breast cancer cell line with IC50 value of 9.3 ± 1.2 µg/mL. Studying the apoptotic effect of J. humile extract revealed that it disrupts G2/M phase in the cell cycle, increases the percentage of early and late apoptosis in Annexin V-FTIC, and affects the oxidative stress markers (CAT, SOD, and GSH-R). Network analysis revealed that out of 33 compounds, 24 displayed interaction with 52 human target genes. Relationship between compounds, target genes, and pathways revealed that J. humile exerts its effect on breast cancer by altering, Estrogen signaling pathway, HER2, and EGFR overexpression. To further verify the results of network pharmacology, molecular docking was performed with the five key compounds and the topmost target, EGFR. The results of molecular docking were consistent with those of network pharmacology. Conclusion: Our findings suggest that J. humile suppresses breast cancer proliferation and induces cell cycle arrest and apoptosis partly by EGFR signaling pathway, highlighting J. humile as a potential therapeutic candidate against breast cancer. [ABSTRACT FROM AUTHOR]

The effect of gender and body mass index (BMI) on the pharmacokinetics of rivastigmine was studied in Egyptian human subjects using new bio-analytical validated LC/MS/MS method. In this study, Rivastigmine was estimated in human plasma using Escitalopram as an internal standard (IS). Rivastigmine and Escitalopram were extracted from human plasma samples by liquid–liquid extraction using diethyl ether (DEE)–dichloromethane (DCM) (70:30, v/v). Chromatographic separation was performed on a reversed phase C18 INERTSIL ODS column using 0.05% aqueous formic acid, acetonitrile in the ratio (50:50, v/v) as a mobile phase. Multiple reaction monitoring (MRM) was applied and operated by positive mode electrospray ionization. A significant difference between male and female Cmax (maximum plasma concentration) (P  = 0.0205; CL = 95.4) was found using Mann–Whitney U test. Also, a moderate negative correlation was found between BMI and Tmax (time to peak plasma concentration) using spearman rho test. The calculated results confirm the difference of Rivastigmine pharmacokinetics between male and female subjects. Furthermore, it indicates that Rivastigmine dose adjustment may be necessary. The method was applied for the estimation of pharmacokinetic parameters in volunteers (n  = 26, 17 male and 9 female) and the effects of gender and BMI were investigated. [ABSTRACT FROM AUTHOR]

Introduction: In the advanced stage of chronic kidney disease (CKD), electrolytes, fluids, and metabolic wastes including various uremic toxins, accumulate at high concentrations in the patients' blood. Hemodialysis (HD) is the conventional procedure used worldwide to remove metabolic wastes. The creatinine and urea levels have been routinely monitored to estimate kidney function and effectiveness of the HD process. This study, first from in Indian perspective, aimed at the identification and quantification of major uremic toxins in CKD patients on maintenance HD (PRE-HD), and compared with the healthy controls (HC) as well as after HD (POST-HD). Objectives: The study mainly focused on the identification of major uremic toxins in Indian perspective and the quantitative analysis of indoxyl sulfate and p-cresol sulfate (routinely targeted uremic toxins), and phenyl sulfate, catechol sulfate, and guaiacol sulfate (targeted for the first time), apart from creatinine and urea in PRE-HD, POST-HD, and HC groups. Methods: Blood samples were collected from 90 HD patients (both PRE-HD and POST-HD), and 74 HCs. The plasma samples were subjected to direct ESI-HRMS and LC/HRMS for untargeted metabolomics and LC-MS/MS for quantitative analysis. Results: Various known uremic toxins, and a few new and unknown peaks were detected in PRE-HD patients. The p-cresol sulfate and indoxyl sulfate were dominant in PRE-HD, the concentrations of phenyl sulfate, catechol sulfate, and guaiacol sulfate were about 50% of that of indoxyl sulfate. Statistical evaluation on the levels of targeted uremic toxins in PRE-HD, POST-HD, and HC groups showed a significant difference among the three groups. The dialytic clearance of indoxyl sulfate and p-cresol sulfate was found to be < 35%, while that of the other three sulfates was 50–58%. Conclusion: LC-MS/MS method was developed and validated to evaluate five major uremic toxins in CKD patients on HD. The levels of the targeted uremic toxins could be used to assess kidney function and the effectiveness of HD. [ABSTRACT FROM AUTHOR]

Panax notoginseng (Burk) F. H. Chen is a traditional Chinese medicinal and edible plant. However, Panax notoginseng flower (PNF) is rarely used. Therefore, the purpose of this study was to explore the main saponins and the anti-inflammatory bioactivity of PNF saponins (PNFS). We explored the regulation of cyclooxygenase 2 (COX–2), a key mediator of inflammatory pathways, in human keratinocyte cells treated with PNFS. A cell model of UVB-irradiation-induced inflammation was established to determine the influence of PNFS on inflammatory factors and their relationship with LL–37 expression. An enzyme-linked immunosorbent assay and Western blotting analysis were used to detect the production of inflammatory factors and LL37. Finally, liquid chromatography–tandem mass spectrometry was employed to quantify the main active components (ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, and notoginsenoside R1) in PNF. The results show that PNFS substantially inhibited COX–2 activity and downregulated the production of inflammatory factors, indicating that they can be used to reduce skin inflammation. PNFS also increased the expression of LL-37. The contents of ginsenosides Rb1, Rb2, Rb3, Rc, and Rd in PNF were much higher than those of Rg1, and notoginsenoside R1. This paper provides data in support of the application of PNF in cosmetics. [ABSTRACT FROM AUTHOR]

Antimicrobial drugs and coccidiostat compounds are commonly used in poultry farming. These compounds are subsequently excreted and released into the environment via broiler litter (BL) and can re-enter the food chain as fertilizer or animal feed. Such residue in animal feed can encourage the appearance of antibiotic-resistant bacteria as well as toxicity. Most analytical methods used to identify and quantitate these drug residues are traditional, and are specific to some antimicrobials and present limitations in assessing complex matrixes like BL. The aim of this study was to develop a multi-residue analytic method for assessing 30 antimicrobial drugs and coccidiostats associated with BL. We investigated the presence and the effects of biotic stack treatment on the degradation of drug residue in BL. Liquid-liquid extraction (LLE) and solid phase extraction (SPE) were replaced by Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) clean-up steps and detected by liquid chromatography mass spectrometry (LC/MS/MS). Results show that a wide spectrum of residues were detected from 0.4 to 8.9 mg kg−1. Following lab-scale stacking treatment, tilmicosin and eight coccidiostats persisted in BL (26–100%). This research supports the need for better understanding, regulation, and management of the use of BL that might carry a high risk of residue drugs.

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific protein phosphatase that regulates a variety of synaptic proteins, including NMDA receptors (NAMDRs). To better understand STEP's effect on other receptors, we used mass spectrometry to identify the STEP61 interactome. We identified a number of known interactors, but also ones including the GluA2 subunit of AMPA receptors (AMPARs). We show that STEP61 binds to the C termini of GluA2 and GluA3 as well as endogenous AMPARs in hippocampus. The synaptic expression of GluA2 and GluA3 is increased in STEP-KO mouse brain, and STEP knockdown in hippocampal slices increases AMPAR-mediated synaptic currents. Interestingly, STEP61 overexpression reduces the synaptic expression and synaptic currents of both AMPARs and NMDARs. Furthermore, STEP61 regulation of synaptic AMPARs is mediated by lysosomal degradation. Thus, we report a comprehensive list of STEP61 binding partners, including AMPARs, and reveal a central role for STEP61 in differentially organizing synaptic AMPARs and NMDARs.

Membrane transport proteins are fundamental components of blood-tissue barriers and affect the absorption, distribution and elimination, and interactions of many of the drugs commonly used in veterinary medicine. A quantitative, simultaneous measurement of these proteins across dog tissues is not currently available, nor is it possible with current immune-based assays such as western blot. In the present study, we aimed to develop a sensitive and specific liquid chromatography tandem-mass spectrometry (LC/MS/MS) based quantitation method that can simultaneously quantitate 14 ATP-binding cassette transporters. We applied this method to a panel of normal canine tissues and compared the LC/MS/MS results with relative messenger RNA (mRNA) abundance using quantitative real-time polymerase chain reaction (qRT-PCR). Our LC/MS/MS method is sensitive, with lower limits of quantitation ranging from 5 to 10 fmol/μg of protein. We were able to detect and/or quantitate each of the 14 transporters in at least one normal dog tissue. Relative protein and mRNA abundance within tissues did not demonstrate a significant correlation in all cases. The results presented here will provide for more accurate predictions of drug movement in dogs through incorporation into physiologically based pharmacokinetic (PBPK) models; the method described here has wide applicability to the quantitation of virtually any proteins of interest in biologic samples where validated canine antibodies do not exist.

Ceftobiprole is a novel β-lactam antibiotic, active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant S. aureus and penicillin-resistant Streptococcus pneumoniae. To artificially generate potential degradation products (DPs) of ceftobiprole that may be formed under relevant storage conditions, acidic, alkaline, oxidative, photolytic and thermolytic stress tests were performed in both solution and solid state. A novel selective HPLC method was developed for the separation of ceftobiprole from its DPs and synthesis by-products (SBPs) using Kinetex Biphenyl column, ammonium acetate buffer pH 5.8 and acetonitrile. The kinetic studies demonstrated the low stability of ceftobiprole in alkaline solution, in the presence of an oxidising agent and under irradiation with near UV. In the solid state, ceftobiprole underwent oxidation when the powder was irradiated with visible light and UV. Based on mass spectroscopic analysis, 13 new structural formulas of SBPs and DPs were proposed, along with molecular formulas for three other DPs obtained in solution and four oxidative DPs characteristic of solid-state degradation. [ABSTRACT FROM AUTHOR]

Honeybees play a key in the environmental ecosystem. Unfortunately, this bio indicator species is affected by multiple stressors, such as the exposure to plant protection products could have a greater impact on colony health and their hives products qualities. Monitoring contaminants residues in honey help to avoid risks to human health. This study aimed to investigate the presence of different classes of pesticides residues in organic honey samples from different production area (Ain-Defla, Djelfa, Ghardaia), using different analytical methods (LC/MS/MS; GC/MS), basing a modified version of the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, And Safe). Pesticides residues in honey were detected very low from areas choosing. The limit of detection LOD and limit of quantification LOQ values for the analytes determined by GC/MS/MS were 0.0010-0.005 mg/kg. In conclusion, the monitoring pesticide residues in local honey does not compromise the consumer's health. [ABSTRACT FROM AUTHOR]

In this study, we developed an easily operable quantification method for 21 plant-derived alkaloids in human serum by automatic sample preparation and liquid chromatography–tandem mass spectrometry. We designed to perform parallel sample preparation by a developed apparatus, which increased sample throughput. We conducted an automatic sample preparation through de-proteinization with 0.1% formic acid in methanol and achieved recovery rates of 89–107% (2.0–14% RSD) for all targeted analytes, demonstrating its high repeatability. The method validation results were satisfactory as follows: the linearity (r2) of each calibration curve ranged from 0.978 to 1.000; the inter- and intra-day accuracies were 89.0–125% and 82.1–110%, respectively; the inter- and intra-day precisions were below 13% and 10%, respectively. Additionally, the lower limits of detection and quantification were 0.0044–0.047 and 0.013–0.14 ng/mL, respectively. Finally, the developed method was applied to pseudo-protoveratrine A poisoning serum and pseudo-colchicine poisoning serum, which were prepared by diluting acute-poisoning mice serum with human serum. Our method successfully quantitated protoveratrine A (0.15–0.25 ng/mL) and colchicine (4.8–6.0 ng/mL). Thus, our method is essential for prompt clinical treatment and critical care on patient in acute intoxication cases caused by plant-derived alkaloids. [ABSTRACT FROM AUTHOR]

The environmental presence and drinking water contamination of per- and polyfluoroalkyl substances (PFAS) have been reported since the early 2000s. This study seeks to develop a liquid chromatography/tandem mass spectrometry analytical method for the simultaneous determination of 21 perfluoroalkyl acids (PFAAs) in drinking water to support future regulations in Japan. Inter-laboratory tests were conducted in 16 laboratories using different instrument to verify the applicability of the developed method for a wide range of drinking water samples in Japan. Recovery tests of PFAA-fortified tap water samples obtained in each laboratory were conducted at set points of 1 and 10 ng/L. Calibration curve linearity, trueness (recovery), repeatability (RSDr), and reproducibility (RSDR) at these analyte concentrations were calculated using data obtained from the recovery tests. The trueness, RSDr, and RSDR of most PFAA analytes were satisfactory when the recoveries were corrected by 13C-PFAA extraction standards with similar recovery to the corresponding PFAS analytes. The developed analytical method is valid for the quantification of the target PFAAs in drinking water. However, satisfactory PFAA quantification requires recovery adjustment using surrogates with similar recovery characteristics to the PFAA analytes.

Aberrant glycosylation is a prominent feature of cancer, that can be used as targets to improve the existing cancer biomarkers, and help to assess metastasis risks, and therapeutic effects. We developed a targeted O-glycoproteomics method using serum specimens, and evaluated its utility in identifying advanced colorectal cancer (CRC) markers. To this end, we combined consecutive lectin affinity purification using Maclura pomifera lectin (MPL), jacalin, and Sambucus nigra lectin, which have affinities for the following O-glycans, that have received attention as cancer-related antigens, Tn (GalNAc-Ser/Thr), Sialyl Tn (Siaα2-6GalNAc-Ser/Thr), T (Galβ1-3GalNAc-Ser/Thr), Sialyl T (Siaα2-3Galβ1-GalNAc-Ser/Thr), and di-Sialyl T (Siaα2-3Galβ1-3[Siaα2-6] GalNAc-Ser/Thr), with a unique O-glycoproteomics approach. A total of 2,068 O-glycoforms derived from 265 proteins were identified in healthy individuals and patients with advanced CRC, of which 44 CRC-specific O-glycoforms were extracted. Particularly, five glycoproteins with T, Sialyl T, and di-Sialyl T antigens in specific peptide regions were evaluated quantitatively and statistically. We found that fibulin-2 (FBLN2) (aa330-349)/T antigen (area under the curve [AUC] = 0.92); macrophage colony-stimulating factor 1 (CSF1) (aa370-395)/(T + di-Sialyl T) (AUC = 0.94); macrophage mannose receptor 1 (MRC1) (aa1083-1101 and aa1215-1229)/T (AUC = 0.96 and 0.99); fibrinogen alpha chain (FGA) (aa354-367, aa511-527 and aa559-573)/Sialyl T (AUC = 0.98, 0.90 and 0.94); and complement component C7 (C7) (aa692-701)/di-Sialyl T (AUC = 1.00), can have high diagnostic efficacy to strategically predict advanced CRC groups. Hence, they could be promising markers for detection of advanced CRC, and provide new clinical test indicators along with lectins, such as MPL and jacalin. Our O-glycoproteomics platform provides a novel tool and resource, for researchers and clinicians seeking to better understand and treat advanced CRC.

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Arsenic is a widespread natural metalloid element. Long-term chronic exposure to arsenic can lead to different degrees of liver injury. Although the etiology of this disease is well known, to date, the underlying mechanism of arsenic-induced liver injury remains unclear, and no specific treatment exists because of the complexity of arsenic. In the present study, potential biomarkers and metabolic pathways in the livers of Wistar rats treated with arsenic for 24 weeks were investigated using an integrated metabolic approach with an LC-Orbitrap Q Exactive™ HF-X mass spectrometer. Markedly increased liver levels of arsenic, alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bile acid (TBA) were detected in the arsenic treatment groups (P < 0.05). Furthermore, histopathological examination of liver tissues showed obviously swollen, loose cytoplasm and increased necrosis in the arsenic treatment groups compared with those in the control group (P < 0.05). Metabonomics results showed that 109 metabolites (variable importance in the projection (VIP) > 1; fold change > 2 or < 0.5; P adjusted < 0.05) changed significantly after exposure to arsenic and included 71 upregulated metabolites and 38 downregulated metabolites. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that 6 metabolic pathways with statistical significance—phenylalanine metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), thiamine metabolism, and vitamin B6 metabolism—were selected, and 13 differential metabolites were detected to be involved in regulating these metabolic pathways. The present study could help identify potential biomarkers and their functions, as well as metabolic pathways, likely providing evidence for the early diagnosis, prevention, and mechanistic study of arsenism. [ABSTRACT FROM AUTHOR]

Respiratory viruses have led to many deaths and hospitalizations per year in the world. The influenza virus is one of the most important respiratory viruses. Recently, metabolic studies in viral infections have been widely studied by scientists. Metabolomics states the metabolites present in a living organism under certain conditions. In this study, peripheral blood mononuclear cells were spinoculated using a virus produced by the Madin‐Darby canine kidney cell culture system, and cells were harvested following spinoculation by the influenza virus. Isolation of peripheral blood mononuclear cells was performed by Ficoll‐Paque density gradient centrifugation. Metabolites were extracted using organic and water approaches. Metabolic profiling was performed by a nontargeted technique using liquid chromatography with tandem mass spectrometry. Multivariate analysis methods were used to determine the main variables. the metabolic pathways involved were determined using databases. Results of the present study showed changes in biosynthesis pathways such as lipids, polyamines, catecholamines, and vitamins. Findings also showed that it is possible to explain the process of inflammation caused by the influenza virus by studying the metabolism of immune cells. [ABSTRACT FROM AUTHOR]

Nitrosamines are known to be a group of probable human carcinogens that are generally formed by the reaction of secondary and tertiary amines, amides, carbamates, or derivatives of urea with nitrite or other nitrogenous agents. Nitrites are common nitrosating impurities that have been reported in many excipients at part per million levels. Guidelines from FDA and EMA have identified potential nitrosamine impurities that could theoretically be present in drug products. As part of the guidelines, it's crucial to have an established method to screen drug substances and products. Furthermore, drug products containing buffers/salts and polymeric excipients were challenging to test as current literature methods would not retain early-eluting nitrosamine impurities long enough to bypass the salts/excipients from the mass spectrometer ion source using a converter switch.An improved analytical method, aligned with the FDA and EMA guidance, using HPLC/MS/MS, was developed by our lab to address the above unmet need. The method demonstrates an excellent sensitivity and linearity range (0.2–200 ng/mL). It was also shown that the method tolerates much higher organic solvents percentage as diluent. Interested readers can use the method as a starting point for further optimization for analyzing their specific drug substances and drug products.

BackgroundHepatocellular carcinoma (HCC) is one of the most common primary liver tumors in humans and dogs. Excessive adrenocortical hormone exposure may cause steroid hepatopathy, which may develop into HCC. In our previous study, hyperadrenocorticism (HAC) was a highly concurrent disease in dogs with HCC. Therefore, this study hypothesized that adrenal steroid alterations might be involved in hepatocarcinogenesis and aimed to specify the relationship between HAC and HCC in dogs.Materials and methodsThis study included 46 dogs brought to the Hokkaido University Veterinary Teaching Hospital between March 2019 and December 2020. Owners gave their signed consent for blood collection on their first visit. A total of 19 steroids (14 steroids and 5 metabolites) in the baseline serum of 15 dogs with HCC, 15 dogs with HAC, and 10 dogs with both diseases were quantitatively measured using the developed liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method.ResultsIn each group, 11 steroids were detected higher than 50%. The detection rate of steroid hormones did not significantly differ between the groups (p > 0.05). Principle component analysis (PCA) showed that the steroid profiles of the three groups were comparable. Median steroid hormone concentrations were not significantly different between the study diseases (p > 0.05).ConclusionThe developed LC/MS/MS was useful for measuring steroid hormones. Although it was clear that HAC was concurrent in dogs with HCC, none of the serum steroids was suggested to be involved in HCC.

Several compounds with different physical properties are present in foods, biological components, and environmental samples, and there are cases in which these must be analyzed simultaneously. However, it is difficult to extract compounds with different physical properties from the same sample using a single method. In the present study, we examined the optimal conditions for the QuEChERS extraction of several kinds of compounds from orange juice using design of experiments (DoE) and response surface methodology (RSM) to determine the optimal ratio of organic solvent to sodium chloride. We determined the optimal extraction conditions, which were within the design space, using 100% tetrahydrofuran (THF) as the extraction organic solvent and NaCl:MgSO4 = 75:25 as the salt. The developed LC/MS/MS method using QuEChERS extraction achieved specific detection and precise quantification. Finally, we measured the polyphenols, sterols, and carotenoids in citrus juice using the optimized QuEChERS extraction method before LC/MS/MS analysis. Most of the analytes were quantifiable in orange juice. The optimized method achieved ease of operation, the extraction of analytes from food samples in a short time (within 30 min), minimization of analytical residues, and reliability. The DoE and RSM approach may contribute to better optimization of the extraction conditions for the lowest number of experiments.

Polymer chemistry of disulfide bond reshuffling in whey protein isolate (WPI) molecules initiated by phosphoric acid (P), malic acid (M), citric acid (C), glutamic acid (G), tartaric acid (T), lactic acid (L), acetic acid (A) was investigated. The disulfide crosslinked sites of acidic substance-treated WPI were accurately identified by LC/MS/MS. Acidic substances treatment rendered the cysteine residues of WPI located at α-La (111), (120) and β-Lg (60), (160) active in the disulfide crosslinking reaction. Meanwhile, the number of intermolecular disulfide crosslinked peptides of P-WPI, T-WPI, and C-WPI increased significantly compared to HWPI, with increases of 9, 8, and 8, respectively. The results of SDS-PAGE and size exclusion chromatography (SEC) indicated that acidic substances promoted the formation of inter-/intramolecular disulfide bonds in WPI, resulting in the generation of polymers. Among them, a large amount of α-lactalbumin was involved in the polymer generation, while β-lactoglobulin was involved to a lesser extent. More aggregates were generated in P-WPI, T-WPI and C-WPI. In addition, fluorescence spectroscopy and fourier transform infrared spectroscopy data demonstrated that acidic substance-treatment led to the transition of the WPI secondary structure from an ordered to disordered structure, as well as the disruption of the tertiary structure. This study explained the disulfide cross-linking mechanism of acidic substances-treated WPI, providing theoretical support for the industrial application of acidifier-modified WPI. [Display omitted] • Seven acidic substance induced whey protein aggregation via disulfide bonds. • Disulfide-crosslinked sites of acid-treated WPI were higher than those of WPI. • Phosphoric acid-treated WPI had the highest degree of disulfide cross-linking. [ABSTRACT FROM AUTHOR]

The potential of a laccase from Trametes hirsuta, a hydrolase mixture of amylase, lipase cellulase and protease, was studied for the removal of a set of trace organic contaminants (TrOCs) at 10 µg/L each in a contaminated water. An attempt was also made to develop a combination of cross-linked enzyme aggregates (combi-CLEA) from the two groups of enzymes. After a 24-hour in batch treatments at 22°C and pH 7, compounds such as acetaminophen and levothyroxine showed a decrease in concentration of 97% and 99% respectively, in presence of laccase, while amlodipine, levothyroxine, and trimethoprim were removed at 98%, 99% and 99% respectively, when the contaminated water was treated with the hydrolase mixture. The study of the impact of pH, temperature and contact time revealed that the most favorable temperatures were 22°C and 30°C, but no clear correlation was found between pH and removal in the pH range of 6-8, although high removals were achieved at these pHs. Finally, except few compounds such as acetaminophen, the TrOC removal after 24 h was higher than that obtained in 4 h.

Serum cortisol measurements by chemiluminescence enzyme immunoassay (CLEIA) are widely used to diagnose hypercortisolism (HC) or Cushing's syndrome in dogs. However, they are associated with problems such as the need for multiple blood collections under stressful conditions or cross-reactivity between hormones. Therefore, a less invasive and more accurate diagnostic method is required. This study aimed to develop a urinary steroid profile analysis method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and to evaluate its clinical usefulness. Sixty-five healthy dogs and 38 dogs with suspected HC were included in the study. Using LC/MS/MS, the levels of 11 steroid hormones in the urine were determined. We established the upper limit of the reference interval for each urinary steroid-to-creatinine ratio and evaluated their diagnostic performances. The levels of the five steroid hormones were significantly higher in the 14 dogs with HC than in the 24 dogs with mimicking HC and 65 healthy dogs. The urinary corticosterone-to-creatinine ratio showed the highest diagnostic accuracy (area under the curve, 0.96). A significant correlation was seen between urinary cortisol concentrations measured by LC/MS/MS and CLEIA (rs = 0.88, P <0.001), although the CLEIA measurements were significantly higher than the LC/MS/MS measurements (P <0.001). LC/MS/MS-based urinary steroid profiles are a promising tool for diagnosing canine HC. • Urinary steroid profile analysis method was developed. • Reference interval for each urinary steroid-to-creatinine ratio was established. • Five steroid hormones were significantly higher in dogs with hypercortisolism. • The urinary corticosterone-to-creatinine ratio had the highest diagnostic accuracy. [ABSTRACT FROM AUTHOR]

• A novel LC-MS/MS method was developed for the quantification of 35 drugs in blood. • Analysis targets toxicologically relevant antihypertensive and antiarrhythmic drugs. • Special emphasis was placed on the analysis of post mortem samples. • Validation demonstrated satisfactory results for all analytes. Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples. [ABSTRACT FROM AUTHOR]

Panax notoginseng (Burk) F. H. Chen is a traditional Chinese medicinal and edible plant. However, Panax notoginseng flower (PNF) is rarely used. Therefore, the purpose of this study was to explore the main saponins and the anti-inflammatory bioactivity of PNF saponins (PNFS). We explored the regulation of cyclooxygenase 2 (COX–2), a key mediator of inflammatory pathways, in human keratinocyte cells treated with PNFS. A cell model of UVB-irradiation-induced inflammation was established to determine the influence of PNFS on inflammatory factors and their relationship with LL–37 expression. An enzyme-linked immunosorbent assay and Western blotting analysis were used to detect the production of inflammatory factors and LL37. Finally, liquid chromatography–tandem mass spectrometry was employed to quantify the main active components (ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, and notoginsenoside R1) in PNF. The results show that PNFS substantially inhibited COX–2 activity and downregulated the production of inflammatory factors, indicating that they can be used to reduce skin inflammation. PNFS also increased the expression of LL-37. The contents of ginsenosides Rb1, Rb2, Rb3, Rc, and Rd in PNF were much higher than those of Rg1, and notoginsenoside R1. This paper provides data in support of the application of PNF in cosmetics.

Mitragyna speciosa (Korth.) also known as kratom or ketum has been traditionally used for its diverse medicinal value in Southeast Asia. Despite of its therapeutic value, kratom's safety profile remains deficiently elucidated. Our study aims to characterize the urinary protein profile of regular kratom users to determine its toxic effects on renal functioning. A total of 171 respondents (comprising of n = 88 regular kratom users, and n = 83 healthy controls) were recruited for this study. Urine specimens were collected and analyzed using SDS-PAGE, followed by LC/MS/MS analysis. Our results show albumin is the primary, and most abundant form of protein excreted in kratom user's urine specimens (n = 60/64), indicating that kratom users are predisposed to proteinuria. Kratom users had an elevated urinary protein (with an intensity of 66.7 kDa band), and protein: creatinine ratio (PCR) concentrations relative to healthy controls. However, kratom user's urinary creatinine concentration was found to be in the normal range as the healthy control group. While, kratom users who tested positive for illicit drug use had an elevated urinary albumin concentration. Our preliminary findings indicate that regular consumption of freshly brewed kratom solution over a protracted period (for an average of eleven years) seems to induce proteinuria, suggestive of an early stage of kidney injury. Hence, further studies are urgently needed to confirm our findings, and establish kratom's renal impairing effects. [ABSTRACT FROM AUTHOR]

This study aimed to describe the morphology on oviductal epithelial cells (pOEC) and protein secretions in follicular and luteal phases. Result of proteins band were not different from control group. The fresh secretory proteins of ampulla and isthmus parts pOEC in the follicular phase were 45, 90, 105 kDa, while the fresh secretory fluids from ampulla and isthmus pOEC secretion in luteal phase were 38, 45, 90, 105, 220, >220 kDa. Protein of size 220 and >220 kDa was probably trypsin or protease functioning to stimulate ovulation. Protein sized 105 kDa is glycogen phosphorylase and protein kinase or oviductal glycoprotein is the enzyme for glycogenolysis plays an important role in regulation of glucose levels in the blood vessels. Protein of size 90 KDa was found to be heat shock proteins for inducing cell growth and in vitro fertilization development of spermatozoa. Protein sized 45 kDa is immunoglobulin gamma-chain function on oocyte maturation.

Neurotoxic chemicals that pass through the blood-brain barrier (BBB) can influence brain function. Efficient methods to test the permeability of the BBB to specific chemicals would facilitate identification of potentially neurotoxic agents. We report here a simultaneous blood and brain microdialysis in a free-moving mouse to test BBB permeability of different chemicals. Microdialysis sampling was conducted in mice at 3–5 days after implantation of a brain microdialysis probe and 1 day after implantation of a blood microdialysis probe. Therefore, mice were under almost physiological conditions. Results of an intravenous injection of lucifer yellow or uranine showed that the BBB was functioning in the mice under the experimental conditions. Mice were given phenyl arsenic compounds orally, and concentration-time profiles for phenyl arsenic compounds such as diphenylarsinic acid, phenylarsonic acid, and phenylmethylarsinic acid in the blood and brain dialysate samples were obtained using simultaneous blood and brain microdialysis coupled with liquid chromatography-tandem mass spectrometry. Peak area-time profiles for linalool and 2-phenethyl alcohol (fragrance compounds or plant-derived volatile organic chemicals) were obtained using simultaneous blood and brain microdialysis coupled with gas chromatography-mass spectrometry in mice given lavender or rose essential oils intraperitoneally. BBB function was confirmed using lucifer yellow in these mice, and results indicated that the phenyl arsenic compounds, linalool and 2-phenethyl alcohol, passed through the BBB. The present study demonstrates that simultaneous blood and brain microdialysis in a free-moving mouse makes it possible to test the BBB permeability of chemicals when coupled with appropriate chemical analysis methods.

Ceftobiprole is a novel β-lactam antibiotic, active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant S. aureus and penicillin-resistant Streptococcus pneumoniae. To artificially generate potential degradation products (DPs) of ceftobiprole that may be formed under relevant storage conditions, acidic, alkaline, oxidative, photolytic and thermolytic stress tests were performed in both solution and solid state. A novel selective HPLC method was developed for the separation of ceftobiprole from its DPs and synthesis by-products (SBPs) using Kinetex Biphenyl column, ammonium acetate buffer pH 5.8 and acetonitrile. The kinetic studies demonstrated the low stability of ceftobiprole in alkaline solution, in the presence of an oxidising agent and under irradiation with near UV. In the solid state, ceftobiprole underwent oxidation when the powder was irradiated with visible light and UV. Based on mass spectroscopic analysis, 13 new structural formulas of SBPs and DPs were proposed, along with molecular formulas for three other DPs obtained in solution and four oxidative DPs characteristic of solid-state degradation.

In horses, the benzodiazepine diazepam (DIA) is used as sedative for pre‐medication or as an anxiolytic to facilitate horse examinations. As the sedative effects can also be abused for doping purposes, DIA is prohibited in equine sports. DIA is extensively metabolized to several active metabolites such as nordazepam, temazepam and oxazepam (OXA). For veterinarians, taking into account the detection times of DIA and its active metabolites is needed for minimizing the risk of an anti‐doping rule violation. Therefore, a pharmacokinetic study on 6 horses was conducted using a single intravenous (IV) dose of 0.2 mg/kg DIA Plasma and urine samples were collected at specified intervals until 16 and 26 days post‐administration, respectively. Samples were analysed by a sensitive liquid chromatography–electrospray ionization/tandem mass spectrometry method. DIA showed a triphasic elimination pattern in the horse. The mean plasma clearance of DIA was 5.9 ml/min/kg, and the plasma elimination half‐life in the terminal phase was 19.9 h. Applying the Toutain model approach, an effective plasma concentration of DIA was estimated at 24 ng/ml, and irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were computed to 0.047 and 0.1 ng/ml, respectively. The detection time according to the European Horserace Scientific Liaison Committee (EHSLC), that is the time for which observed DIA plasma concentrations of all investigated horses were below the IPC was 10 days. Using Monte Carlo Simulations, it was estimated that concentrations of DIA in plasma would fall below the IPC 18 days after the DIA administration for 90% of horses. However, in the present study, a single administration of DIA could be detected for 24 days in urine via the presence of OXA, its dominant metabolite. [ABSTRACT FROM AUTHOR]

In this work, pesticide residues in 493 fruit and vegetable samples obtained from markets in Turkey were detected after QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction followed by liquid chromatography-tandem mass spectrometry with electron spray ionization (LC-ESI/MS/MS) and gas chromatography-tandem mass spectrometry (GC/MS/MS). Validation of the method was tested based on the European Union SANTE/12682/2019 guidelines. The samples were analyzed to determine the concentrations of 500 pesticide residues. The results indicated that 254 samples of 493 samples contaminated with pesticides, only 22% contained pesticide residues at or below maximum residue limits (MRLs), and 30% contained pesticide residues above MRLs. Chlorpyrifos was the most common pesticide (105 samples) from the detected pesticides; 49 samples were found above to MRLs with concentrations of 0.011–2.001 mg/kg. Among samples, peach (88%), dill (84%), mushroom (83%), arugula (73%), and spinach (72%) were the crops with the higher percentages of pesticide residues. [ABSTRACT FROM AUTHOR]

Reversible acylation of lysine ε-amino groups, e.g., acetylation, succinylation, maronylation, and myristoylation, is involved in basic physiological processes such as metabolism, cell signaling and aging. In this study, we developed a novel enrichment method for acylated peptides without the use of antibodies, in which endogenously acylated peptides are deacylated by recombinant lysine deacylases based on the enzyme-substrate relationship and enriched by N -hydroxysuccinimidyl chemistry for identification of the acylated sites by nanoscale liquid chromatography-tandem mass spectrometric analysis. To demonstrate the validity of this acylomics platform, we used it to identify acylated sites on chemically acylated model protein samples. We also applied it to the nuclei of HeLa cells to identify endogenous acylated sites. [Display omitted] • A novel strategy for acylated peptide enrichment was developed. • Acylated peptides are enriched depending on the KDAC-substrate relationship. • Chemically acylated proteins were used to demonstrate the method validity. • Endogenously acylated sites on histone proteins were identified. [ABSTRACT FROM AUTHOR]

• Vitamin D metabolites were accurately quantified from DBS samples using LC-MS/MS. • The assay discriminated the C-3 epimer from standard vitamin D metabolites. • Haematocrit remains a challenge when using DBS to quantify plasma metabolites. Where conventional blood sampling is challenging, dried blood spots (DBS) provide a practical sample alternative for measuring vitamin D levels. Our study aimed to develop and evaluate a clinical pathology service-based assay suitable for measuring vitamin D in batches of DBS samples collected remote to the testing site. A high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with derivatisation was developed to measure 25-hydroxyvitamin D metabolites (25OHD3, 25OHD2 and 3-epi-25OHD3) in DBS samples. The assay was validated using paired DBS and plasma samples from 37 healthy adults. The assay reproducibly (<11.5% coefficient of variation) quantified 25OHD3 (range 1–300 nmol/L), 25OHD2 (range 2–300 nmol/L) and 3-epi-25OHD3 (range 1–200 nmol/L) in DBS samples. The 25OHD3 metabolite was detected in all DBS samples, 3-epi-25OHD3 in six plasma (range 2.1–6.3 nmol/L) and paired DBS samples, and 25OHD2 was not detected. Concentrations of 25OHD3 were highly correlated between paired samples: capillary DBS and venous plasma (r = 0.92), venous DBS and venous plasma (r = 0.93), and capillary DBS and venous DBS (r = 0.97). Ordinary least squares regression was used to characterise (β = 0.81) and correct the systematic bias in DBS data (compared to paired plasma). Thereafter, Bland-Altman analysis demonstrated robust agreement between sample-methods. This simple and rapid DBS-based LC-MS/MS assay accurately quantified serum vitamin D metabolites using a paired-sample 'bridging strategy' to correct for the inherent sample-method bias. [ABSTRACT FROM AUTHOR]