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We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2–5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms, and exploits an efficient combination of hydrophilic interaction liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labeled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species, including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants. [ABSTRACT FROM AUTHOR]

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

Oxytocin and cortisol (OXY and CORT) are hormones related to stress, cognitive, and social behaviors. Their detection is relevant to epidemiological studies aimed at investigating the effects of stressor factors on human life. The aim of this study was to develop and validate an assay for the measurement of OXY and CORT in saliva samples using liquid chromatography/tandem mass spectrometry (LC-MS/MS) in the presence of deuterated analogs. A 500 mL aliquot of oral fluid, obtained by the centrifugation of a chewed swab, was purified by solid-phase extraction. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive electrospray ionization, and then quantified using a triple-quadrupole mass detector in multiple-reaction monitoring mode. The limits of quantification and the linear dynamic ranges were 2.0 × 10−3 and 0.5 nmol/L, and up to 1.0 × 10−1 and 20 nmol/L for OXY and CORT, respectively. Inter- and intra-run precision, expressed as relative standard deviation, was <7%, and accuracy was within 93–104% of the theoretical concentrations. The evaluation of matrix effects showed that the use of internal standards controlled sources of bias. The high sensitivity of the method allowed the quantification of OXY and CORT in the salivary samples of both adults and children: levels of CORT ranged from 0.6 to 18.5 nmol/L, while OXY levels were two orders of magnitude lower (from 1.7 × 10−3 to 1.1 × 10−2 nmol/L). To our knowledge, this is the first method that can analyze, in the same chromatographic run, both hormones in saliva samples. [ABSTRACT FROM AUTHOR]

Objectives We compared tacrolimus concentrations obtained by the more recently US Food and Drug Administration–approved tacrolimus assay (CMIA) on the Alinity i analyzer (Abbott Laboratories) with a liquid chromatography/tandem mass spectrometry (LC-MS/MS)–based method from 2 reference laboratories. We also investigated the correlation between the CMIA tacrolimus and Elecsys tacrolimus assays. Methods Tacrolimus concentrations were measured in EDTA whole blood by the chemiluminescent microparticle immunoassay (CMIA) using the Alinity i analyzer, and then 2 aliquots were sent to 2 reference laboratories, both using ascomycin as the internal standard for the LC-MS/MS method. Results The total precision of the CMIA tacrolimus assay was excellent. When tacrolimus concentrations obtained by the LC-MS/MS method from reference laboratory A were plotted on the x-axis and corresponding CMIA values were plotted on the y-axis, the following regression equation was observed: y = 0.9721x + 1.005 (r = 0.95), indicating no significant bias with the CMIA. However, when tacrolimus values obtained from reference laboratory B were used for comparison, the regression equation was y = 0.7664x + 1.775 (r = 0.93), indicating significant negative bias with the CMIA. When we compared tacrolimus concentrations obtained by reference laboratories A and B, we observed positive bias with tacrolimus concentrations obtained by reference laboratory B. However, tacrolimus concentrations obtained by both CMIA and Elecsys immunoassays were comparable. Conclusions Because of good correlation of tacrolimus concentrations using the CMIA and LC-MS/MS from reference laboratory A, our long-term reference laboratory for drug analysis, we concluded that the CMIA on the Alinity i can be used for therapeutic drug monitoring of tacrolimus. [ABSTRACT FROM AUTHOR]

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein–protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus–host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines. [ABSTRACT FROM AUTHOR]

Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC–MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa (Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter) and others never detected in these media (Asaia and Bombella, among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry. [ABSTRACT FROM AUTHOR]

Flavonoids are a prominent group of plant phenolics (polyphenol group) extensively present in the human diet and associated with health promotion. Flavonoids are essential in various pharmaceutical, nutraceutical, and cosmetic applications. To collect and categorize these compounds, we comprehensively assessed High-performance liquid chromatography (HPLC) techniques published to analyze flavonoids in various matrices. Most documented methods employed liquid chromatography with ultraviolet detection (HPLC-UV) and tandem mass spectrometry (LC-MS/MS) methods. Among chromatographic techniques, reversed-phase liquid chromatography remains the predominant and efficient method for separating various analytes. This review explores the primary chromatographic methods used to separate and quantify flavonoids in herbs, fruits, and biological samples. This article examines recent literature on applying liquid chromatography with different detection methods for analyzing flavonoids. To examine recent literature on applying liquid chromatography with different detection methods for analyzing flavonoids. It offers valuable insights to researchers as they prepare to develop quantification methods. [ABSTRACT FROM AUTHOR]

Diquat (DQ), paraquat (PQ), glufosinate (GLU), and glyphosate (GLYP) are commonly used herbicides that have been confirmed to be toxic to humans. Rapid and accurate measurements of these toxicants in clinical practice are beneficial for the correct diagnosis and timely treatment of herbicide-poisoned patients. The present study aimed to establish an efficient, convenient, and reliable method to achieve the simultaneous quantification of DQ, PQ, GLU, and GLYP in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without using derivatization or ion-pairing reagents. DQ, PQ, GLU, and GLYP were extracted by the rapid protein precipitation and liquid-liquid extraction method and then separated and detected by LC-MS/MS. Subsequently, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, extraction recovery, matrix effect, dilution integrity, and stability were evaluated to validate the method based on the FDA criteria. Finally, the validated method was applied to real plasma samples collected from 166 Chinese patients with herbicide poisoning. The results showed satisfactory linearity with low LOD (1 ng/mL for DQ and PQ, 5 ng/mL for GLU, and 10 ng/mL for GLYP, respectively) and low LOQ (5 ng/mL for DQ and PQ, 25 ng/mL for GLU and GLYP, respectively). In addition, the precision, accuracy, extraction recovery, and stability of the method were acceptable. The matrix effect was not observed in the analyzed samples. Moreover, the developed method was successfully applied to determine the target compounds in real plasma samples. These data provided reliable evidence for the application of this LC-MS/MS method for clinical poisoning detection. [ABSTRACT FROM AUTHOR]

Context Reliable estradiol (E2) reference intervals (RIs) are crucial in pediatric endocrinology. Objectives This study aims to develop a sensitive ultra-performance liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method for E2 in serum, to establish graphically represented RI percentiles and annual RIs for both sexes, and to perform a systematic literature comparison. Methods First, a UPLC-MS/MS method for E2 was developed. Second, graphically represented RI percentiles and annual RIs covering 0-18 years were computed (cohort of healthy children [1181 girls and 543 boys]). Subsequently, RIs were compared with published data by systematic searches. Results Lower limit of quantification was 11 pmol/L, indicating high sensitivity. Estradiol first peaked during mini-puberty in both sexes (girls up to 192 pmol/L; boys up to 225 pmol/L). As could be expected, girls showed higher pubertal E2 (up to 638 pmol/L). However, boys' RIs (up to 259 pmol/L) overlapped considerably. We found 4 studies in the literature that also used LC-MS/MS to determine E2 and published RIs for the complete pediatric age range. Reference intervals varied considerably. Pre-pubertal and pubertal phases were present in all studies. Higher E2 during the time of mini-puberty in both sexes was documented in 3 studies including ours. Conclusions Variability of RIs for E2 between studies illustrates the importance of laboratory-specific RIs despite using a LC-MS/MS reference method. In boys, the striking E2 peak during mini-puberty as well as high pubertal E2 without phenotypic estrogenization in regular male puberty indicates that the role of E2 in children and, especially in boys, requires better functional understanding. [ABSTRACT FROM AUTHOR]

Simple Summary: Antibiotics are emerging contaminants found in several matrices of environmental, animal, and human origin. This high occurrence constitutes a major global threat, which is also responsible for the development of antimicrobial resistance. Therefore, it is vitally important to develop new, simple, and reliable methods by which to quantify them in several matrices. In this work, the mussel matrix was chosen as the sentinel organism; a new analytical method for the determination of two commonly used macrolides in mussels harvested along the Sardinian coasts was developed. None of the tested macrolides were found in the sampling sites at concentrations quantifiable by the developed method. These results can be useful for competent authorities evaluating the food safety of mussels (as concerns the studied antibiotics) and pollution derived from these drugs in the marine environment. Contaminants of emerging concern (CECs) are compounds found in several environmental compartments whose ubiquitous presence can cause toxicity for the entire ecosystem. Several personal care products, including antibiotics, have entered this group of compounds, constituting a major global threat. It is essential to develop simple and reliable methods by which to quantify these contaminants in several matrices. In this work, mussels were chosen as sentinel organisms to assess environmental pollution and the safety of bivalve mollusk consumption according to the "One Health perspective". A liquid chromatographic tandem mass spectrometry method (LC-MS/MS) was developed for the quantification of two macrolides, erythromycin (ERY) and azithromycin (AZI), in mussels. This new method was validated according to international guidelines, showing high selectivity, good recoveries (>60% for both of them), sensitivity, and precision. The method was successfully applied for ERY and AZI research in mussels farmed along the Sardinian coasts (Italy), demonstrating itself to be useful for routine analysis by competent authorities. The tested macrolides were not determined in the analyzed sites at concentrations above the limits of detection (LODs). These results demonstrate the food safety of mussels (as concerns the studied antibiotics) and a negligible amount of pollution derived from these drugs in the studied area. [ABSTRACT FROM AUTHOR]

Background: Amivantamab is a monoclonal bispecific anti-EGFR-MET antibody used to treat non-small cell lung cancer. There were no published methods using a liquid chromatographic—tandem mass spectrometric approach to develop and validate a feasible, novel, and thoroughly validated method for quantifying amivantamab in rat plasma. Results: The liquid–liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile–ammonium formate buffer (40:60) as a mobile phase on waters, alliance e-2695 model high-pressure liquid chromatographic system having Agilent eclipse C18, 150 mm × 4.6 mm, 3.5 µm column. The overall runtime was 6 min at a 1.0 ml/min flow rate. The method showed significant sensitivity and acceptable linearity over the 5.00–100.00 ng/ml concentration range. Accuracy was proved by mean percent recovery ranging from 98.03 to 99.99%. The intraday precision coefficient of variation (%) ranged between 0.31 and 5.43. Also, the findings such as Cmax, tmax, AUC0− t, AUC0− ∞, and half-life values of amivantamab showed that the technique was helpful for pharmacokinetic studies. Conclusions: All the validated parameters were found to be within the acceptable range. The validated method was found to be simple, accurate, precise, and reproducible and hence can be used for the routine analysis of amivantamab, such as in-process quality control by liquid chromatographic—tandem mass spectrometry. [ABSTRACT FROM AUTHOR]

Many dietary supplements claim the ability to enhance sports performance and to improve the fitness of the consumers. Occasionally, along with legal ingredients, illicit compounds may be added without being labelled, leading to unintended doping. Hence, the aim of this study was to develop an analytical method to determine a set of 12 polar (logDpH=7 from −2.0 to +0.3) compounds including diuretics, stimulants, β2-agonists, methylxanthines, and sweeteners. Hydrophilic interaction liquid chromatography was chosen as separation strategy, coupled with tandem mass spectrometry. The instrumental method was optimized using a two-step design of experiments (DoE). Firstly, a Plackett–Burman (PB) DoE was performed to identify the more influencing variables affecting peak areas and chromatographic resolution among temperature, water percentage in the mobile phase, and flow rate, as well as type and concentration of buffers. Secondly, a D-optimal DoE was set, considering only the most significant variables from the PB-DoE results, achieving a deeper understanding of the retention mechanism. Sample processing by salt-assisted liquid–liquid extraction was studied through DoE as well, and the whole method showed recoveries in the range 40–107% and procedural precision ≤11% for all analytes. Finally, it was applied to real samples, in which the four methylxanthines and two artificial sweeteners were detected and quantified in the range of 0.02–192 mg g−1. These values were compared to the quantities declared on the DS labels, when possible. Furthermore, a sequence of MS/MS scans allowed detection of a signal in one of the samples, structurally similar to the β2-agonist clenbuterol. [ABSTRACT FROM AUTHOR]

Women have been found to be at a higher risk of morbidity and mortality from type 2 diabetes mellitus (T2DM) and asthma. a-Glucosidase inhibitors have been used to treat T2DM, and arachidonic acid 15-lipoxygenase (ALOX15) inhibitors have been suggested to be used as treatments for asthma and T2DM. Compounds that inhibit both enzymes may be studied as potential treatments for people with both T2DM and asthma. This study aimed to determine potential anti-diabetic and anti-inflammatory bioactive hits from Coriaria intermedia Matsum. stem and Dracontomelon dao (Blanco) Merr. & Rolfe bark. A bioassay-guided fractionation framework was used to generate bioactive fractions from C. intermedia stem and D. dao bark. Subsequently, dereplication through ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and database searching was performed to putatively identify the components of one bioactive fraction from each plant. Seven compounds were putatively identified from the C. intermedia stem active fraction, and six of these compounds were putatively identified from this plant for the first time. Nine compounds were putatively identified from the D. dao bark active fraction, and seven of these compounds were putatively identified from this plant for the first time. One putative compound from the C. intermedia stem active fraction (corilagin) has been previously reported to have inhibitory activity against both a-glucosidase and 15-lipoxygenase-1. It is suggested that further studies on the potential of corilagin as an anti-diabetic and anti-inflammatory treatment should be pursued based on its several beneficial pharmacological activities and its low reported toxicity. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus–host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development. [ABSTRACT FROM AUTHOR]

A method was developed and validated for vanillin, ethyl vanillin, and methyl vanillin determination in Thai rice cultivars by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLCMS/MS) under the selected reaction monitoring mode. In rice samples, the limit of detection was 13.3, 11.2 and 5.0 μg/kg for vanillin, ethyl vanillin, and methyl vanillin. A high correlation coefficient was obtained (R² = 0.9999, 0.9999 and 0.9998) within the linear range (0 - 300 μg/L) in the MS method. The recoveries ranging from 90.63 % ± 6.25 to 108.7 % ± 2.35 were obtained in different spiking concentrations. The average RSD% of intra-day for vanillin, ethyl vanillin, and methyl vanillin was 6.90, 5.13 and 2.16 %, and the inter-day were 6.72, 5.30 and 5.64 %, respectively. The developed method was applied to 24 commercial rice samples. Vanillin, were found in 24 samples ranging from 95.2 to 773.2 μg/kg. However, ethyl vanillin and methyl vanillin were not detected under LOD. The developed UPLC-MS/MS method for vanillin, ethyl vanillin and methyl vanillin determination include the merits of simple preparation steps and high sensitivity. [ABSTRACT FROM AUTHOR]

Objectives This study evaluated the suitability and accuracy of the automated Roche Elecsys tacrolimus electrochemiluminescence immunoassay (ECLIA) by comparing it with a current laboratory-developed test by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Methods The tacrolimus ECLIA was evaluated for precision, linearity, interference, and postextraction stability. Accuracy was compared with LC-MS/MS. Results The tacrolimus ECLIA assay is precise, exhibits a measuring range of 0.75 to 30 ng/mL, and is tolerant of significant interferences (plasma indices: hemolysis <2306, icterus <55, lipemia <1427, and biotin <1200 ng/mL). Comparison with LC-MS/MS revealed a 26% proportional bias in patient samples evaluated for tacrolimus concentration (y = 1.26x + 0.08; r 2 = 0.97; Sy/x = 0.94; n = 43) and an absolute mean (SD) bias of 2.2 (1.7) ng/mL. Postextraction studies confirmed that samples were stable for up to 30 minutes under routine laboratory conditions. Conclusions The 2 major challenges for implementation of the tacrolimus ECLIA assay are the postextraction sample stability and the significant proportional bias observed compared with the LC-MS/MS reference method. The 30-minute window for analysis of extracted samples is a practical challenge to the routine workflow of the core laboratory. In addition, disagreement between the immunoassay and LC-MS/MS methods can lead to discordant clinical interpretations and ultimately affect patient care. [ABSTRACT FROM AUTHOR]

Results obtained from rat studies indicate that, even at low concentrations, mercurial species cause harmful effects on the kidneys, by inducing the nephrotic oxidative stress response. In the present work, Hg-associated proteins were identified as possible mercury-exposure biomarkers in rat kidneys exposed to low mercury chloride concentrations for 30 days (Hg-30) and 60 days (Hg-60), using metalloproteomic strategies. The renal proteomic profile was fractioned by two-dimensional electrophoresis and the mercury determinations in kidney samples, protein pellets and protein spots were performed using graphite furnace atomic absorption spectrometry. The characterization of Hg-associated protein spots and the analysis of differentially expressed proteins were performed by liquid chromatography, coupled with tandem mass spectrometry. Eleven Hg-associated protein spots with a concentration range of 79 ± 1 to 750 ± 9 mg kg−1 in the Hg-60 group were identified. The characterization and expression analyses allowed the identification of 53 proteins that were expressed only in the Hg-60 group, 13 "upregulated" proteins (p > 0.95) and 47 "downregulated" proteins (p < 0.05). Actin isoforms and hemoglobin subunits were identified in protein spots of the Hg-60 group, with mercury concentrations in the range of 138 to 750 mg kg−1, which qualifies these proteins as potential mercury-exposure biomarkers. [ABSTRACT FROM AUTHOR]

The number of methods for the analysis of new psychoactive substances (NPS) is continually increasing, and there is no indication that this trend will change in the near future. The constantly growing market of "designer drugs" makes it necessary to develop new methods of their analysis. The aim of this review is to present the multi-component methods of detection and identification of NPS using low-resolution tandem mass spectrometry coupled with liquid chromatography. For this purpose, 36 articles were selected by applying strictly defined search criteria. Due to the large differences in the matrices and physicochemical properties of the analytes, the described research methods are diverse. These differences are visible in sample preparation methods, chromatographic columns, mobile phases, gradients, or additives to mobile phases used. This work collects and organizes the existing information on the subject of NPS screening analysis methods and will be helpful to forensic scientists working on this topic. [ABSTRACT FROM AUTHOR]

The present study verified that it is possible to analyze melengesterol acetate using the existing multi-residue method. Melengestrol acetate was extracted from livestock products using acidic acetonitrile acidified with acetic acid in the presence of n-hexane and anhydrous sodium sulfate. The crude extracts were cleaned up using an octadecylsilanized silica gel cartridge column. Separation by HPLC was performed using an octadecylsilanized silica gel column with linear gradient elution of 0.1 vol％ formic acid and acetonitrile containing 0.1 vol％ formic acid. For the determination of the analyte, tandem mass spectrometry with positive ion electrospray ionization was used. In recovery tests using four livestock products fortified with maximum residue limits levels of melengestrol acetate (0.001–0.02 mg/kg), the truenesses ranged from 82％ to 100％, and the repeatabilities for the entire procedure ranged from 0.5 RSD％ to 5.6 RSD％. In recovery tests using 11 livestock products fortified with 0.0005 mg/kg of melengestrol acetate, the truenesses ranged from 88％ to 99％, and the repeatabilities ranged from 1.3 RSD％ to 5.4 RSD％. The limit of quantification for melengestrol acetate in livestock products was 0.0005 mg/kg. [ABSTRACT FROM AUTHOR]

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g., rat brain, it is essential to know the mean TH concentrations in this tissue under control conditions. In this publication, an online solid-phase extraction (SPE) liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was validated and used to measure TH metabolites (T4, T3, rT3, T2 and T1) in the brains of untreated rats. Data on TH concentrations in the whole brain and separate data from the cerebellum and the cortex are shown. The corresponding samples were gathered from young rats at postnatal days (PND) 4 and 21/22 and from adult rats. The results show inter alia the high accuracy and precision of the method, and LOQs of 0.02 ng/mL were determined for T1, T2 and rT3 and of 0.15 ng/mL for T3 and T4. Technical variability is low, as shown by the relative standard deviations of 7.5–20%. For our rat model, we found that T4, T3 and T2 concentrations rise from PND4 to PND21, whereas the rT3 concentration decreases; as well as there is no statistical difference between TH concentrations in the male and female rat brain. This method is suitable to analyze TH metabolites in the brain and build up a database of historical TH concentrations in control rats. Together, this yields a robust diagnostic tool to detect potentially adverse disturbances of TH homeostasis in the most vulnerable anatomic structure. [ABSTRACT FROM AUTHOR]

Maternal pathological conditions such as infections and chronic diseases, along with unexpected events during labor, can lead to life-threatening perinatal outcomes. These outcomes can have irreversible consequences throughout an individual's entire life. Urinary metabolomics can provide valuable insights into early physiological adaptations in healthy newborns, as well as metabolic disturbances in premature infants or infants with birth complications. In the present study, we measured 180 metabolites and metabolite ratios in the urine of 13 healthy (hospital-discharged) and 38 critically ill newborns (admitted to the neonatal intensive care unit (NICU)). We used an in-house-developed targeted tandem mass spectrometry (MS/MS)-based metabolomic assay (TMIC Mega) combining liquid chromatography (LC-MS/MS) and flow injection analysis (FIA-MS/MS) to quantitatively analyze up to 26 classes of compounds. Average urinary concentrations (and ranges) for 167 different metabolites from 38 critically ill NICU newborns during their first 24 h of life were determined. Similar sets of urinary values were determined for the 13 healthy newborns. These reference data have been uploaded to the Human Metabolome Database. Urinary concentrations and ranges of 37 metabolites are reported for the first time for newborns. Significant differences were found in the urinary levels of 44 metabolites between healthy newborns and those admitted at the NICU. Metabolites such as acylcarnitines, amino acids and derivatives, biogenic amines, sugars, and organic acids are dysregulated in newborns with bronchopulmonary dysplasia (BPD), asphyxia, or newborns exposed to SARS-CoV-2 during the intrauterine period. Urine can serve as a valuable source of information for understanding metabolic alterations associated with life-threatening perinatal outcomes. [ABSTRACT FROM AUTHOR]

Introduction: Steroid measurements are important for diagnosis and monitoring of many conditions and treatment regiments; however, due to structural and chemical similarities amongst steroids, these analyses are challenging, even for highly specific techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential mobility spectrometry (DMS) has the potential to improve these analyses by providing an orthogonal and complementary separation technique. Methods: Initially, the potential for DMS to improve signal-to-noise ratio (S/N) and reduce interference was tested by comparing chromatograms acquired with and without DMS when performing measurements of six different steroids. Subsequently, a full clinical validation of cortisol and cortisone in urine was performed with the LC-DMS-MS/MS method. Results and Discussion: DMS significantly reduced interferences observed in the chromatograms and boosted S/N by between 1.6 and 13.8 times. Additionally, DMS improved the agreement between quantifier/qualifier fragment ion results for cortisol and cortisone as indicated by the increase in R2 from approximately 0.81 to 0.98. All validation studies met acceptance criteria and we observed exceptional analytical performance in terms of precision, with % CVs less than 8%. Conclusions: DMS improved the specificity of the steroid measurements by reducing interferences and improving S/N. The validation studies prove that these benefits did not come at the expense of other aspects of analytical performance. This study indicates that DMS has the potential to benefit not just clinical measurements of challenging analytes, but many clinical LC-MS/MS analyses.

The occurrence of pesticide residues, their metabolites and degradation products in 49 edible mushrooms (fresh, dried, canned and frozen) was investigated. Using QuEChERS extraction, followed by liquid and gas chromatography coupled with tandem mass spectrometry, the samples collected from Czech markets were tested for 427 analytes. A total of 21 pesticide residues, their metabolites and pesticide synergists were found in quantifiable concentrations. The most frequently detected pesticide residues included prochloraz and its metabolites, metrafenone and carbendazim. Two mushroom samples were found to be in violation of the maximum residue level. In addition, the fate of 13 pesticides during the processing of fresh mushrooms was investigated. The processing factors calculated from the data ranged from 6.0 to 12.3 for drying and from 0.3 to 1.3 for deep-freezing. The results showed that most of the pesticide residues tested were stable under the conditions of mushroom processing. [ABSTRACT FROM AUTHOR]

Infigratinib, a protein kinase inhibitor employed in the therapeutic management of cholangiocarcinoma, was subjected to various stress conditions, including hydrolytic (acidic and alkaline), oxidative, photolytic, and thermal stress, in accordance with the rules established by the International Council for Harmonization. A cumulative count of five degradation products was observed. The application of the Quality by Design principle was utilized in the development of a rapid and specific separation method for Infigratinib and its degradation products. The methodology employed in this study was derived from an experimental design approach, which was utilized to examine the critical process parameters associated with chromatographic systems. The reversed-phase high-performance liquid chromatography technique, employing a C18 column and a mobile phase composed of a gradient mixture of 25 mM ammonium acetate buffer at pH 6.0 and acetonitrile, successfully facilitated the chromatographic separation. The methodology was expanded to include the utilization of UPLC-quadrupole tandem mass spectrometry in order to conduct a comprehensive analysis of the structural properties and characterize the degradation products. Overall, five degradation products were found in different stress conditions. The method was verified at certain working points, wherein a linearity range (5.0–200.0 µg/mL) was developed and other parameters such as accuracy, repeatability, selectivity, and system suitability were evaluated. Finally, the toxicity and mutagenicity of Infigratinib and its degradation products were predicted using in silico software, namely DEREK Nexus® (version 6.2.1) and SARAH Nexus® (version 3.2.1). Various toxicity endpoints, including chromosomal damage, were predicted. Additionally, two degradation products were also predicted to be mutagenic. [ABSTRACT FROM AUTHOR]

Background: Cynomorium songaricum Rupr. has long been used as an anti-inflammatory, antidepressant, and anti-aging agent in traditional Chinese medicine in Asia. Its ethyl acetate extract (ECS) has been identified as the main antioxidant component with neuroprotective and estrogen-like effects. However, the potential of ECS in treating depression has not been explored yet. Methods: We identified the primary metabolites in ECS in this study using liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Network analysis was used to find the potential targets and pathways associated with the anti-neuroinflammatory depression action of the ECS. In addition, we established a corticosterone (CORT)-induced depression mouse model to assess ECS's antidepressant effects by monitoring various behavioral changes (e.g., sucrose preference, forced swimming, tail suspension, and open field tests) and biochemical indices of the hippocampus, and validating the network analysis results. Significant pathways underwent verification through western blotting based on network analysis prediction. Results: Our study demonstrates that ECS possesses significant antidepressant activity. The LC-MS/MS analysis of ECS identified 30 main metabolites, including phloridzin, phlorizin, ursolic acid, and naringenin, as well as other flavonoids, terpenoids, and phenolic acids. These metabolites were found to be associated with 64 candidate target proteins related to neuroinflammatory depression from the database, and ten hub proteins were identified through filtration: CXCL8, ICAM1, NOS2, SELP, TNF, IL6, APP, ACHE, MAOA and ADA. Functional enrichment analyses of the candidate targets revealed their primary roles in regulating cytokine production, inflammatory response, cytokine activity, and tumor necrosis factor receptor binding. In vivo, ECS improved hippocampal neuroinflammation in the mouse model. Specifically, ECS reduced the expression of inflammatory factors in the hippocampus, inhibited M1 microglial cell polarization, and alleviated depression through the regulation of the NF-κB-NLRP3 inflammation pathway. Conclusion: Based on experimental and network analysis, this study revealed for the first time that ECS exerted antidepression effect via anti-neuroinflammation. Our research provides valuable information on the use of ECS as an alternative therapeutic approach for depression. [ABSTRACT FROM AUTHOR]

Background. The extensive use of carbamate pesticides in agriculture has increased serious public concern regarding the environment and food safety. Carbamate pesticides have been reported to cause deleterious effects on the immune system and fertility issues in both humans and animals. A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of carbamate residues in vegetables has been developed and validated with quantification levels respecting the action levels established for the maximum residue limits (MRL) according to the European Union, World Health Organization (WHO), and Vietnam. Materials and methods. In the proposed method, the pretreatment protocol was shortened to reduce time, and chemical usage was also minimized to decrease cost. The validation was performed according to the Commission Implementing Regulation (EU) 2021/808, which includes linearity, limits of detection (LOD), limits of quantification (LOQ), recovery, and repeatability. Results. Based on the LC-MS/MS experiments, the matrix-matched calibration method showed good performance in the concentration from 5 to 200 µg/kg for vegetable samples, with a correlation coefficient of R2 value of > 0.996. The limits of quantification for all carbamate pesticides were 5 µg/kg. Recoveries of all carbamate pesticides were from 91 to 109% and repeatabilities (RSD) were < 10%. Conclusion. The validation data indicate the developed LC/MS/MS method is suitable for the determination of carbamate pesticides in vegetables. [ABSTRACT FROM AUTHOR]

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L‐dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter‐day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L‐dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight. [ABSTRACT FROM AUTHOR]

BackgroundTreatment monitoring of drug-resistant tuberculosis (DR-TB) in resource-limited settings is challenging. We developed a multi-analyte assay for eleven anti-TB drugs in small hair samples as an objective metric of drug exposure.MethodsSmall hair samples were collected from participants at various timepoints during directly observed RR-TB treatment at an inpatient tertiary referral facility in South Africa (DR-TB cohort). We assessed qualitative determination (i.e., detection above limit of detection) of bedaquiline, linezolid, clofazimine, pretomanid, levofloxacin, moxifloxacin, pyrazinamide, isoniazid, ethambutol, ethionamide, and prothionamide in an LC-MS/MS index panel assay against a reference standard of inpatient treatment records. Because treatment regimens prior to hospitalization were not available, we also analyzed specificity (for all drugs except isoniazid) using an external cohort of HIV-positive patients treated for latent TB infection with daily isoniazid (HIV/LTBI cohort) in Uganda.ResultsAmong the 57 DR-TB patients (58% with pre-XDR/XDR-TB; 70% HIV-positive) contributing analyzable hair samples, the sensitivity of the investigational assay was 94% or higher for all drugs except ethionamide (58.5, 95% confidence interval [CI], 40.7-99.9). Assay specificity was low across all tested analytes within the DR-TB cohort; conversely, assay specificity was 100% for all drugs in the HIV/LTBI cohort.ConclusionsHair drug concentrations reflect long-term exposure, and multiple successive regimens commonly employed in DR-TB treatment may result in apparent false-positive qualitative and falsely elevated quantitative hair drug levels when prior treatment histories within the hair growth window are not known.

Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front‐ and back‐end of MS‐based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple‐quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments. [ABSTRACT FROM AUTHOR]

To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). The established method is precise (intra-day CV: 1.4–6.3%; inter-day CV: 1.3–15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61–121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: −5.6 to 7.4%), are stable in thymol (variation: −8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism. [ABSTRACT FROM AUTHOR]

In this study, the presence of 11-nor-Δ9-carboxy tetrahydrocannabinol (THC-COOH) in postmortem fluid obtained from the chest cavity (FCC) of postmortem cases collected from drug-related fatalities or criminal-related deaths in Jeddah, Saudi Arabia, was investigated to evaluate its suitability for use as a complementary specimen to blood and biological specimens in cases where no bodily fluids are available or suitable for analysis. The relationships between THC-COOH concentrations in the FCC samples and age, body mass index (BMI), polydrug intoxication, manner, and cause of death were investigated. Methods: Fifteen postmortem cases of FCC were analyzed using fully validated liquid chromatography-positive-electrospray ionization tandem mass spectrometry (LC-MS/MS). Results: FCC samples were collected from 15 postmortem cases; only THC-COOH tested positive, with a median concentration of 480 ng/mL (range = 80–3010 ng/mL). THC-COOH in FCC were higher than THC-COOH in all tested specimens with exception to bile, the median ratio FCC/blood with sodium fluoride, FCC/urine, FCC/gastric content, FCC/bile, FCC/liver, FCC/kidney, FCC/brain, FCC/stomach wall, FCC/lung, and FCC/intestine tissue were 48, 2, 0.2, 6, 4, 6, 102, 11, 5 and 10-fold, respectively. Conclusion: This is the first postmortem report of THC-COOH in the FCC using cannabinoid-related analysis. The FCC samples were liquid, easy to manipulate, and extracted using the same procedure as the blood samples. The source of THC-COOH detected in FCC could be derived from the surrounding organs due to postmortem redistribution or contamination due to postmortem changes after death. THC-COOH, which is stored in adipose tissues, could be a major source of THC-COOH found in the FCC. [ABSTRACT FROM AUTHOR]

Fipronil (FIP) is a phenylpyrazole insecticide that, along with neonicotinoid insecticides, is regularly used worldwide. Photodegradation of FIP in aqueous systems is thought mainly to involve the reaction of desulfinylation to give fipronil desulfinyl (FIP-desulfinyl); however, little is known about further degradation reactions. We investigated FIP photodegradation by analyzing photodegradation products by liquid chromatography and liquid chromatography high-resolution tandem mass spectrometry using an Orbitrap instrument. A wide range of products, including dechlorinated compounds, was detected, and the structures were identified. FIP-desulfinyl has previously been found to be an important and persistent FIP photodegradation product; however, we also found that FIP-desulfinyl was photochemically decomposed to a didechlorinated product via a monodechlorinated product. The main photodegradation pathway was probably similar to that of ethiprole, which has a similar skeleton. The photodegradation rate constant was 22.6 times lower for FIP-desulfinyl (0.00372 min−1) than FIP (0.0839 min−1). The photodegradation rate constant was lower for the newly found didechlorinated product (0.001 min−1 or below) than FIP-desulfinyl, suggesting that the product is persistent in aquatic environments and could be an important indicator of long-term FIP contamination. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the present study was to establish a liquid chromatography- tandem mass spectrometry (LC-MS/MS) method for the determination of SHR9146, a novel IDO1/TDO dual inhibitor, in mouse plasma and tissues, and to apply it to investigate the preclinical plasma pharmacokinetics and tissue distribution of SHR9146 in mice. Methods: Samples were spiked with deuterated SHR9146-d4 as an internal standard and pretreated by protein-precipitation extraction with methanol. Chromatographic separation was performed on a Venusil ABS C18 column (150 × 4.6 mm, 5 mm) by isocratic elution with 10 mM ammonium acetate buffer containing 0.1% formic acid solution and methanol as mobile phases. MS detection was conducted in positive electrospray ionization with multiple reaction monitoring at m/z 444.1/229.4 for SHR9146 and m/z 448.4/229.2 for the internal standard. Results: The method showed good linearity in the calibration range from 0.05 to 50.0 mg/mL. Precisions (intra- and inter-run) were in the range from 0.5% to 5.1%, and accuracies (RE) were between -3.0% and 4.4% for all the concentration levels. SHR9146 was stable in all the tested bio-samples with recoveries >90%. Pharmacokinetic parameters were obtained by non-compartmental analysis. SHR9146 has a half-life of 0.713 h when IV-injected, with CL 12 mL/min/kg and Vd 0.666 L/kg. After oral dosing from 20 to 80 mg/kg, Cmax (range from 8.751 to 12.893 mg/mL) and AUC0-t (range from 15.606 to 69.971 mg·h/mL) of SHR9146 showed dose proportionality. Other post-oral pharmacokinetic parameters in plasma were as follows (n=6): Tmax 0.79 ± 0.36 h, t1/2 1.586 ± 0.853 h, CL 19.8 ± 0.9 mL/min/kg, Vd 3.427± 1.617 L/kg, and absolute bioavailability of 54.2% ± 12.6% (range from 40.2% to 64.7%). In addition, SHR9146 was found to be absorbed rapidly and distributed widely and mainly in the stomach, adrenal gland, liver, and lung. Conclusion: The method was simple, sensitive, accurate, and specific and was successfully applied for the preclinical pharmacokinetic and tissue distribution study of SHR9146 in mice. The results showed that SHR9146 had doseindependent kinetics in mice via oral administration and was absorbed rapidly and distributed widely. The study provides a good basis for further drug development assessment. [ABSTRACT FROM AUTHOR]

Six sartan substances including telmisartan, losartan, valsartan, irbesartan, olmesartan, and candesartan were selected in this work. A simple and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated to simutaniously detect six drugs. Linearity, LOD, and LOQ were satisfied for routine quantitative and qualitative detection. The linearity ranged from 2 to 1000 ng/mL with the determination coefficient more than 0.98. The intra-day and inter-day precisions were all below 27.5%. All the recoveries were above 80%, and matrix effects were between 25 and 120%. Profiling detection of six sartan substances in human urine was also developed after administration. Irbesartan urinary metabolic pathway was firstly investigated by liquid chromatography quadruple time-of-flight mass spectrometry using full scan and targeted tandem mass spectrometry techniques. Fifteen urinary metabolites of irbesartan were identified and characterized, and ten of them were firstly reported. [ABSTRACT FROM AUTHOR]

In this study, 135 samples of cocoa beans collected in the Amazon and Atlantic Forest regions of Brazil were analysed to evaluate the possible co-occurrence of 34 mycotoxins. The results indicate that 42% of the cocoa samples exhibited quantifiable levels for 11 mycotoxins: aflatoxins (AFs) B1, B2 and G1; ochratoxin A; citrinin; cyclopiazonic acid; tenuazonic acid; paxilline; sterigmatocystin; zearalenone and fumonisin B2. Of the samples, 18% exhibited the co-occurrence of up to six mycotoxins. No toxins belonging to the groups of trichothecenes or ergot alkaloids were detected. Contingency analysis of the incidence of mycotoxins did not show significant differences between the two regions evaluated. Seven samples were contaminated with AFs, while only one contained ochratoxin A above 10 μg kg−1. The accuracy of the method was evaluated by proficiency testing for ochratoxin A, where satisfactory Z-scores were obtained. [ABSTRACT FROM AUTHOR]

Background: Preclinical studies showed that mesenchymal stem cells (MSCs) ameliorate tau phosphorylation, amyloid-beta accumulation, and inflammation in Alzheimer's disease (AD) mouse models via secretion of neurotrophic factors and cytokines. We aimed to identify CSF biomarkers that can be used to predict or monitor the response to MSCs in patients with AD. Methods: AD patients were injected with human umbilical cord blood-MSCs (n = 22) or placebo (n = 12). The cerebrospinal fluid (CSF) samples were collected at baseline, one day after the first injection, and one day after the third injection. The patients injected with MSCs were classified into good responder (GR) or poor responder (PR) groups based on the rate of changes in the ratio of total-tau and phosphorylated-tau in the CSF. We selected three typical participants in each group, and their CSF protein levels were analyzed using liquid chromatography/tandem mass spectrometry (LC–MS/MS). Results: In the LC–MS/MS analysis, 1,667 proteins were identified. Eleven proteins showed significant differences between the typical GR and PR at baseline. Based on their significance level and known functions, two proteins, reticulocalbin-3 (RCN3) and follistatin-related protein 3 (FSTL3), were selected as potential biomarkers to predict MSC response. A total of 173 proteins showed significant change one day after the third injection compared to the baseline in typical GR. We excluded 45 proteins that showed significant change after the third injection compared to the baseline in the typical PR. Based on their significance level and known function, four proteins, scrapie-responsive protein 1 (SCRG1), neural proliferation differentiation and control protein (NPDC1), apolipoprotein E (ApoE), and cystatin C (CysC), were selected as potential biomarker to monitor MSC response. Additionally, functional analysis revealed that the increased CSF proteins after the third injection compared to the baseline in the typical GR were associated with synaptogenesis. Conclusions: This study identified two proteins (RCN3 and FSTL3) that may be potential biomarkers for predicting MSC response and four proteins (SCRG1, NPDC1, ApoE, CysC) that may be potential biomarkers for monitoring MSC response in patients with AD. Further studies are needed to validate our results. Trial registration Clinical Trials.gov, NCT02054208. Registered on 4 February 2014. Samsung Medical Center IRB File No.2017-04-025. Registered on 20 June 2017. [ABSTRACT FROM AUTHOR]

A new method named "Stability Indicating LC-MS/MS" was developed to make analysis on acalabrutinib and this method was stable for this anticancer drug. In acid, basic and oxidation the drug was shown instability on these stress conditions. Acalabrutinib was used to form four degradation products and the developed method was used to separate based on "Zorbax C18 column (150 mm x 4.6 mm, 5 µm)" and for this separation the technique isocratic elution was used with the flow rate of 1.0 mL/min. It was found that the new developed method was valid as per the guidelines of ICH. A pathway known as fragmentation of drug was founded initially to identify the formed degradation products and in this identification LC-MS/MS method was used and also fragmentation studies were used. After the identification, isolation of the degradation products was done and (1H-NMR) was subjective for this process. The environment of basic, acid and oxidative were used to obtain the degradation products and these were isolated by the advanced techniques known as acid degradation product (DP1) of acalabrutinib with a molecular mass of 390.200 Da, empirical formula C21H19N5O3 with name as (S)-4-(8-amino-3-(1-(but-2-ynoyl) pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl) benzoic acid". 442.100 Da is the molecular mass of base degradation product (DP2), C24H23N7O2 is the empirical formula with name as "(S)-4-(3-(1-ace tylpyrrolidin-2-yl)-8-aminoimidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamide". 498.400 Da is the molecular mass of another acalabrutinib base degradation product (DP3), C27H27N7O3 is the empirical formula which is known as "(S)-4-(8-amino-3-(1-(3-methoxybut-2-enoyl) pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamide". 482.200 Da is the molecular mass of oxidation degradation product (DP4), C26H23N7O3 is the empirical formula which is known as "2-(4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl) benzamido) pyridine-1-oxide". [ABSTRACT FROM AUTHOR]

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New all-oral regimens for rifampin-resistant tuberculosis (RR-TB) are being scaled up globally. Measurement of drug concentrations in hair assesses long-term drug exposure. Delamanid (DLM) is likely to be a key component of future RR-TB treatment regimens, but a method to describe its quantification in hair via liquid chromatography-tandem mass spectrometry (LC-MS/MS) has not previously been described. We developed and validated a simple, fast, sensitive, and accurate LC-MS/MS method for quantifying DLM and its metabolite DM-6705 in small hair samples. We pulverized and extracted two milligrams of hair in methanol at 37 °C for two hours, and diluted 1:1 with water. A gradient elution method eluted DLM, DM-6705, and the internal standard OPC 14714 within 3 min, bringing overall analysis time to 5.5 min. The method has limits of detection (LOD) of 0.0003 ng/mg for DLM and 0.003 ng/mg for DM-6705. The established linear dynamic ranges are 0.003-2.1 ng/mg and 0.03-21 ng/mg for DLM and DM-6705, respectively. Eleven of 12 participant hair samples had concentrations within DLM's linear dynamic range, while all 12 samples had concentrations within the quantifiable range for DM-6705. The ranges of concentrations observed in these clinical samples for DLM and DM-6705 were 0.004-0.264 ng/mg hair and 0.412-12.041 ng/mg hair respectively. We demonstrate that while DLM was detected in hair at very low levels, its primary metabolite DM-6705 had levels approximately 100 times higher. Measuring DM-6705 in hair may accurately reflect long-term adherence to DLM-containing regimens for drug-resistant TB.

Human milk contains oxylipins involved in infant development. Although oxylipins have been identified in whole or skim milk, their localization within human milk cream, cell, and skim fractions is not known. This study determined the distribution of free and esterified oxylipins in cream, cell, and skim fractions of human milk. Out of 72 oxylipins probed by mass-spectrometry, 42, 29, and 41 oxylipins (free or bound) were detected in cream, cell, and skim fractions, respectively. Over 90% of free and bound oxylipins were derived from linoleic acid in all milk fractions. Other oxylipins were derived from n-6 arachidonic acid and dihomo-gamma-linolenic acid, and n-3 alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. Free oxylipins were more abundant in skim milk (59.9% of total oxylipins) compared to cream and cell pellet, whereas esterified oxylipins were most abundant in milk cream and cell pellets (74.9-76.9%). The heterogenous distribution of oxylipins in different fractions of human milk may regulate the guided release of these bioactive signaling molecules within infants.

Background: Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by loss of immune tolerance to platelet autoantigens leading to excessive destruction and insufficient production of platelets. Method: Quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed to detect the differentially expressed proteins in bone marrow samples from active ITP patients and normal controls. Result: Our bioinformatic analysis identified two upregulated proteins (ORM1 and vWF) and two downregulated proteins (PPBP and SPARC) related to immune function. The four proteins were all found to be related to the tumor necrosis factor (TNF) -α signalling pathway and involved in the pathogenesis of ITP in KEGG pathway analysis. Conclusion: Bioinformatics analysis identified differentially expressed proteins in bone marrow that are involved in the TNF-α signalling pathway and are related to the activation of immune function in ITP patients. These findings could provide new ideas for research on the loss of immune tolerance in ITP patients. [ABSTRACT FROM AUTHOR]

• A highly accurate and precise candidate Reference Measurement Procedure (cRMP) was developed for CDC Clinical Standardization Programs (CDC CSP) free thyroxine (FT4) measurements. • The FT4 cRMP was based on equilibrium dialysis (ED) and isotope dilution liquid chromatography – tandem mass spectrometry (ID-LC-MS/MS) procedures. • The accuracy and precision of the cRMP were validated via an interlaboratory comparison study with excellent agreement between four independent laboratories. • The low limit of detection of cRMP provided sufficient sensitivity to determine FT4 for patients with hypothyroidism. Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays. [ABSTRACT FROM AUTHOR]

Epilepsy is one of the most prevalent neurological conditions and antiepileptic drugs are the mainstay of epilepsy treatment. High variation in pharmacokinetic profiles of several antiepileptic drugs highlights the importance of therapeutic drug monitoring to estimate pharmacokinetic properties and consequently individualize drug posology. In this work, a simple, rapid and robust liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of carbamazepine and its metabolite carbamazepine-10,11-epoxide, gabapentin, levetiracetam, lamotrigine, oxcarbazepine and its metabolite mono-hydroxy-derivative metabolite, phenytoin, topiramate, and valproic acid in human plasma for therapeutic drug monitoring. d6-Levetiracetam, d4-gabapentin and d6-valproic acid were used as internal standards. After addition of internal standards along with two-step protein precipitation and dilution sample preparation, plasma samples were analyzed on a C18 column using a gradient elution in 5 min without interference. The calibration curves were linear over a 100-fold concentration range, with determination coefficients (r2) greater than 0.99 for all analytes. The limit of quantification was 0.5 μg mL−1 (0.1 μg mL−1 for oxcarbazepine, 2 μg mL−1 for levetiracetam, and 10 μg mL−1 for valproic acid) with precision and accuracy ranging from 3% to 9% and from 94% to 112%, respectively. Intra- and inter-day precision and accuracy values were within 15% at low, medium and high quality control levels. No significant matrix effect was observed in the normal, hemolyzed, lipemic, and hyperbilirubin blood samples. This method was successfully used in the identification and quantitation of antiepileptic drugs in patients undergoing mono- or polytherapy for epilepsy. [ABSTRACT FROM AUTHOR]

Curry leaf is an evergreen herb with culinary, pharmaceutical, and nutraceutical applications. As pesticide residue in curry leaf has garnered significant regulatory attention in recent years, here we report a reliable method, which was validated for the determination of 265 and 225 pesticides using LC-MS/MS and GC-MS/MS, respectively. At first, the sample was comminuted after adding water (1:2). The sample preparation workflow included extraction of 10 g homogenized sample with 10 mL ethyl acetate (+1% acetic acid), cleanup by dispersive solid phase extraction (d-SPE, 50 mg PSA + 50 mg C18 + 10 mg GCB + 150 mg Na2SO4) and the final analysis by tandem mass spectrometry. The cleanup step adeptly removed co-extractives. The method effectively reduced matrix effects and offered an LOQ of 0.01 mg kg−1 for most compounds. The method's accuracy and precision results fulfilled the requirements of SANTE/11312/2021 guidelines at 0.01 mg kg−1 and higher levels of fortification. The accuracy and precision results were comparable for all pesticides. The successful screening of market samples indicates its high extraction efficiency and precision for incurred residue analysis. Due to its robustness and conformity with regulatory criteria, food testing laboratories worldwide can use the method to monitor pesticide levels in curry leaves. [ABSTRACT FROM AUTHOR]

This study developed a simple, rapid, reproducible, and analytical method using liquid chromatography and electrospray ionization (ESI) with tandem mass spectrometry (LC-MS/MS) to simultaneously quantify pentoxifylline (PTX), its pharmacological active metabolites, lisofylline (PTX-M1) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (PTX-M5), and donepezil (DNP) in rat plasma, using PTX-d6 and DNP-d7 as the internal standards. The LC-MS/MS procedure was performed at the ESI interface, operating in positive ionization and multiple reaction monitoring (MRM) modes; the monitoring of transitions comprised m/z 279.3 > 181.1 for PTX, m/z 281.1 > 263.1 > 160.90 for PTX-M1, m/z 267.1 > 249.0 > 220.9 for PTX-M5, m/z 380.3 > 90.9 for DNP, m/z 285.3 > 187.1 for PTX-d6 (IS1), and m/z 387.3 > 98.3 for DNP-d7 (IS2). After plasma protein precipitation (PP) with methanol, chromatographic separation was performed with an Imtakt Cadenza® CD-C18 (100 × 3 mm, 3 µm) column, using an isocratic mobile phase consisting of 0.1% formic acid in water and methanol (20:80, v/v) at a flow rate of 0.2 mL/min. The retention times of DNP, PTX-M5, PTX, and PTX-M1 were 2.24, 2.50, 2.68, and 2.72 min, respectively, with a total run time of 5 min. This method was validated over a linear concentration range of 5–8000, 10–5000, 20–15,000, and 2–500 ng mL−1 for PTX, PTX-M1, PTX-M5, and DNP, respectively, with a high correlation coefficient (r2 ≥ 0.99). The established method was fully validated in terms of selectivity, the lower limit of quantitation, precision, accuracy, recovery, matrix effect, stability, and dilution integrity according to the regulatory guidelines from the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety. The validated method was successfully applied to a pharmacokinetic study on the concurrent administration of DNP and PTX in rats. [ABSTRACT FROM AUTHOR]

Large molecule protein therapeutics have steadily grown and now represent a significant portion of the overall pharmaceutical market. These complex therapies are commonly manufactured using cell culture technology. Sequence variants (SVs) are undesired minor variants that may arise from the cell culture biomanufacturing process that can potentially affect the safety and efficacy of a protein therapeutic. SVs have unintended amino acid substitutions and can come from genetic mutations or translation errors. These SVs can either be detected using genetic screening methods or by mass spectrometry (MS). Recent advances in Next-generation Sequencing (NGS) technology have made genetic testing cheaper, faster, and more convenient compared to time-consuming low-resolution tandem MS and Mascot Error Tolerant Search (ETS)-based workflows which often require ~6 to 8 weeks data turnaround time. However, NGS still cannot detect non-genetic derived SVs while MS analysis can do both. Here, we report a highly efficient Sequence Variant Analysis (SVA) workflow using high-resolution MS and tandem mass spectrometry combined with improved software to greatly reduce the time and resource cost associated with MS SVA workflows. Method development was performed to optimize the high-resolution tandem MS and software score cutoff for both SV identification and quantitation. We discovered that a feature of the Fusion Lumos caused significant relative under-quantitation of low-level peptides and turned it off. A comparison of common Orbitrap platforms showed that similar quantitation values were obtained on a spiked-in sample. With this new workflow, the amount of false positive SVs was decreased by up to 93%, and SVA turnaround time by LC-MS/MS was shortened to 2 weeks, comparable to NGS analysis speed and making LC-MS/MS the top choice for SVA workflow. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the excretion of methylone and its metabolites in sweat following the ingestion of increasing controlled doses of 50, 100, 150 and 200 mg of methylone to twelve healthy volunteers involved in a clinical trial. Methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone (MDC) were analyzed in sweat patches by liquid chromatography–tandem mass spectrometry. Methylone and MDC were detected in sweat at 2 h and reached their highest accumulation (Cmax) at 24 h after the administration of 50, 100, 150 and 200 mg doses. In contrast, HMMC was not detectable at any time interval after each dose. Sweat proved to be a suitable matrix for methylone and its metabolites' determination in clinical and toxicological studies, providing a concentration that reveals recent drug consumption. [ABSTRACT FROM AUTHOR]

A simple and sensitive method for the determination of moenomycin A residues in livestock products using LC-MS/MS was developed. Moenomycin A, a residual definition of flavophospholipol, was extracted from samples with a mixture of ammonium hydroxide and methanol (1 : 9, v/v) preheated at 50°C. The crude extracted solutions were evaporated and purified by liquid--liquid partitioning between a mixture of ammonium hydroxide, methanol and water (1 : 60 : 40, v/v/v) and ethyl acetate. The alkaline layer was taken, and cleaned up using a strong anion exchange (InertSep SAX) solid phase extraction cartridge. The LC separation was performed on an Inertsil C8 column with liner gradient elution using 0.3 vol% formic acid and acetonitrile containing 0.3 vol% formic acid. Moenomycin A was detected using tandem mass spectrometry with negative ion electrospray ionization. Recovery tests were conducted using three porcine samples (muscle, fat and liver) and chicken eggs. Samples were spiked with moenomycin A at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The trueness ranged from 79 to 93% and precision ranged from 0.5 to 2.8%. The limit of quantification (/N≥10) of the developed method is 0.01 mg/kg. The developed method would thus be very useful for regulatory monitoring of flavophospholipol in livestock products. [ABSTRACT FROM AUTHOR]

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 μg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving.