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Thin-layer chromatography (TLC) hyphenated to bioassays is a modern tool used for discovery of biologically active compounds from complex mixtures. The first bioautographic assay for detecting laccase inhibitors on a TLC plate was developed in this study. The on-plate reaction of laccase with colourless ABTS that renders the blue ABTSâ+ radical was optimised. Combination of the enzymatic TLC-assay with a control TLC-assay, wherein ABTSâ+ radical is chemically generated and then applied on the TLC, allowed to differentiate between the pure laccase inhibitor sodium azide and radical scavengers such as gallic and kojic acids. The limit of detection and quantification for the method were 54.9 and 166 ng of sodium azide respectively. The methodology was applied successfully to a recently discovered laccase inhibitor chemotype: hydrazones. A model hydrazone was compared with several hydrazones synthesized for this study. For the first time, laccase inhibitors separated on a TLC plate can be detected individually.
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Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins.
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Hemoglobin (Hb) disorders are among the most prevalent inherited diseases. Despite a limited number of involved genes, these conditions represent a broad clinical and prognostic spectrum. The menu of laboratory tests is extensive. From widely available modalities, for example, complete blood count to rather sophisticated molecular technologies, the investigation of Hb disorders recapitulates an increasing complexity of laboratory workup in other medical fields. This review highlights a current state of biochemical and molecular investigation of Hb disorders and offers a glimpse on technologies that are yet to be fully embraced in clinical practice.
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Hemoglobinopatias , Talassemia , Humanos , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/genética , Talassemia/diagnóstico , Talassemia/genéticaRESUMO
In this study, we propose a novel strategy utilizing deep eutectic solvents (DESs) as both the extraction solvent and dispersing liquid, with nanometer zinc oxide (ZnO) serving as the adsorbent. This method incorporates ultrasound-assisted matrix solid phase dispersion (UA-MSPD) for the extraction of six active components (salidroside, echinacoside, acteoside, specnuezhenide, nuezhenoside G13, and oleanolic acid) from Ligustri Lucidi Fructus samples. The extracts were then analyzed using high-performance liquid chromatography equipped with a diode array detector. The effects of various parameters such as dispersant dosage, DESs volume, grinding time, ultrasonication duration, and eluent volume on extraction recovery were investigated and optimized using a central composite design under response surface methodology. The optimized conditions yielded detection limits ranging from 0.003 to 0.01 mg/g and relative standard deviations of 8.7% or lower. Extraction recoveries varied between 93% and 98%. The method demonstrated excellent linearity for the analytes (R2 ≥ 0.9997). The simple, green, and efficient DESs/ZnO-UA-MSPD technique proved to be rapid, accurate, and reliable for extracting and analyzing the six active ingredients in Ligustri Lucidi Fructus samples.
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Ligustrum , Extração em Fase Sólida , Ondas Ultrassônicas , Óxido de Zinco , Extração em Fase Sólida/métodos , Óxido de Zinco/química , Ligustrum/química , Solventes Eutéticos Profundos/química , Cromatografia Líquida de Alta Pressão , Frutas/química , Extratos Vegetais/química , Extratos Vegetais/análise , Tamanho da Partícula , Solventes/químicaRESUMO
Aroma is a crucial indicator of hop quality. This study analyzed the differences in aroma compound composition among six hop varieties from three regions: North America, Europe, and Asia. Descriptive analysis and sensomic approaches including gas chromatography-olfactometry/aroma extract dilution analysis, odour activity value calculation and aroma recombination were used for the detailed characterization and comparative analysis of hop aroma. A total of 55 aroma-active compounds were identified. Among them, linalool, geraniol, ß-myrcene, 2-undecanone, and methyl decanoate contributed significantly to hop aroma. Orthogonal partial least squares discriminant analysis revealed that, except for the SAAZ and XinYuan hops with some similarities in their aroma composition, the remaining hops exhibited unique aroma characteristics. A total of 16 compounds, including methyl 5-methylhexanoate and (E)-ß-farnesene, were identified as differentiating aroma compounds in the six hop samples. This study enriches the knowledge on hop flavour with different origins and provides valuable insights into its application.
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As vastly modified on secreted proteins, N-glycosylation is found on milk proteins and undergo dynamic changes during lactation, characterizing milk protein glycosylation would benefit the elucidation of glycosylation pattern differences between samples. However, their low abundance required specific enrichment. Herein, through rational design and controllable synthesis, we developed a novel multi-functional polymer for the isolation of protein glycosylation. It efficiently separated glycopeptides from complex background inferences with mutual efforts of hydrophilic interaction chromatography (HILIC), metal ion affinity and ion exchange. By fine-tuning Ca2+ as regulators of aldehyde hyaluronic acid (HA) conformation, the grafting density of HA was remarkably improved. Moreover, grafting Ti4+ further enhanced the enrichment performance. Application of this material to characterize bovine milk and colostrum proteins yields 479 and 611 intact glycopeptides, respectively. Comparative analysis unraveled the distinct glycosylation pattern as well the different distribution of glycoprotein abundances between the two samples, offering insights for functional food development.
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Imazethapyr is the most common herbicide used for weed management in pulses. A field trial was carried out with imazethapyr 10% SL formulation at 100 and 150 g a.i./ha application rates, as pre-and post-emergence, to study dissipation of imazethapyr in soil, persistence in urdbean plant, terminal residues in urdbean grains and effect on soil microbes. An acetate buffered- quick, easy, cheap, effective, rugged, and safe (QuEChERS) method in combination with high-performance liquid chromatography (HPLC) was validated for imazethapyr residue analysis. The half-life of imazethapyr in soil ranged from 15.12 to 18.02 days. The residues of imazethapyr persist up to 60 days in soil and up to 7-15 days in urdbean plant. Residues were not detected in grains at the time of harvest. Persistence of imazethapyr residues in soil significantly impact soil microbial populations depending on herbicide application rates and timing.
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Herbicidas , Ácidos Nicotínicos , Resíduos de Praguicidas , Microbiologia do Solo , Poluentes do Solo , Solo , Vigna , Herbicidas/análise , Poluentes do Solo/análise , Vigna/química , Ácidos Nicotínicos/análise , Resíduos de Praguicidas/análise , Solo/química , Cinética , Cromatografia Líquida de Alta Pressão , Meia-VidaRESUMO
BACKGROUND: The integration of positron emission tomography (PET) and magnetic resonance imaging (MRI) holds promise for advancing diagnostic imaging capabilities. The METRICS project aims to develop cyclotron-driven production of 52Mn for PET/MRI imaging. RESULTS: Using the 52Cr(p,n)52Mn reaction, we designed chromium metal targets via Spark Plasma Sintering and developed a separation procedure for isolating 52Mn. Labeling tests were conducted with traditional chelators (i.e. S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid) and the 1.4-dioxa-8-azaspiro[4.5]decane-8- carbodithioate ligand to produce radioactive complexes suitable for PET/MRI applications. Our methodology yielded high-quality 52Mn suitable for PET radiopharmaceuticals and PET/MRI imaging. Preliminary studies on phantom imaging using microPET and clinical MRI demonstrated the efficacy of our approach. CONCLUSIONS: The developed technology offers a promising avenue for producing 52Mn and enhancing PET/MRI imaging capabilities. Further in vivo investigations are warranted to evaluate the potential advantages of this hybrid imaging technique.
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High-throughput proteomics has become an exciting field and a potential frontier of modern medicine since the early 2000s. While significant progress has been made in the technical aspects of the field, translating proteomics to clinical applications has been challenging. This review summarizes recent advances in clinical applications of high-throughput proteomics and discusses the associated challenges, advantages, and future directions. We focus on research progress and clinical applications of high-throughput proteomics in breast cancer, bladder cancer, laryngeal squamous cell carcinoma, gastric cancer, colorectal cancer, and coronavirus disease 2019. The future application of high-throughput proteomics will face challenges such as varying protein properties, limitations of statistical modeling, technical and logistical difficulties in data deposition, integration, and harmonization, as well as regulatory requirements for clinical validation and considerations. However, there are several noteworthy advantages of high-throughput proteomics, including the identification of novel global protein networks, the discovery of new proteins, and the synergistic incorporation with other omic data. We look forward to participating in and embracing future advances in high-throughput proteomics, such as proteomics-based single-cell biology and its clinical applications, individualized proteomics, pathology informatics, digital pathology, and deep learning models for high-throughput proteomics. Several new proteomic technologies are noteworthy, including data-independent acquisition mass spectrometry, nanopore-based proteomics, 4-D proteomics, and secondary ion mass spectrometry. In summary, we believe high-throughput proteomics will drastically shift the paradigm of translational research, clinical practice, and public health in the near future.
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BACKGROUND: Post-mortem toxicology constantly deals with the research of reliable alternative matrices to be applied in case of highly damaged corpses (such us carbonized, skeletonized, human remains, etc.). Teeth represent a promising alternative matrix since dental tissues are endowed by different features, resistance and stability after death. SCOPE: Since scant literature reported on the pharmacokinetics and mechanism of incorporation of xenobiotics into dental tissues, this pilot research aims to investigate whether in the pulp can be detected the same substances found in blood in drug related death cases. Secondly, the study is addressed to disclose the possible deposit of drugs in dental hard tissues (dentine and/or enamel), thus contributing to reconstruct the drug abuse history (timing, e.g.). MATERIALS AND METHODS: The study experimented with a novel method to separately analyse dental enamel, dentin, and pulp, applied to 10 teeth collected during autopsies of drug-related deaths along with blood and hair samples for classic toxicological analyses. Each tooth was prepared by "pulverization technique" and then analysed by gas chromatography paired with mass spectrometry (GC-MS) and ultra high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC/HR-MS) for searching cocaine, opiates, and metabolites. The results were then compared with those obtained from blood and hair samples. RESULTS: Preliminary results demonstrated that teeth differ from any other classic matrix (blood and hairs) since the qualitative correspondence of the detected substances between pulp and blood as well as dental hard tissues and hair suggests that they can be useful in post-mortem evaluation as a unique matrix for both acute and chronic assumptions of drugs. The mechanism of accumulation of substances in mineralized dental tissues emerged the most significant result, being influenced by the type of molecule and the method of assumption. The main limitation of this study is the limited availability of the sample and the absence of anamnestic information of the time, rates and method of drug assumption during life. Further research is necessary to systematically investigate the distribution of different substances within the different tissues of the tooth.
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Per- and polyfluoroalkyl substances (PFASs) have attracted increasing attention due to their persistence in the environment and potential adverse effects on human and animal health. The detection and quantification of PFASs in livestock could substantially contribute to monitoring their presence within the food chain. We developed a targeted quantification method for 34 PFASs in livestock serum by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). We used protein precipitation for serum sample extraction and accurate mass measurement of targeted PFAS compounds for quantification. We validated the method with various analytical parameters, achieving accuracy of 70-120% and precision of <20%. The method also demonstrated good analytical sensitivity, with a limit of detection of <0.051 ng/mL and a limit of quantification of <0.175 ng/mL. When applying the developed method to actual serum samples from a variety of livestock, we successfully identified and quantified various PFASs in different livestock species. Our method has the potential to be a valuable tool for veterinary laboratory analysis of PFAS contamination in livestock.
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During the last years, there has been an increasing research interest in the analysis of biological fluids requiring non-invasive sampling for biomedical and clinical applications. In this work, we have focused on the nasal exudate with the aim of investigating the potential use of this fluid to know the role of iron in stroke and also for diagnosis. Potential differences in the nasal exudate, collected in swabs, from diagnosed hemorrhagic stroke, ischemic stroke, and control groups were investigated with regard to total iron by inductively coupled plasma-mass spectrometry, iron fractionation studies by size exclusion chromatography together with post-column isotope dilution analysis, and four proteins containing iron (ferritin, transferrin, lactoferrin, and ferroportin) with ELISA kits. All these analyses represent an analytical challenge, considering the rather limited amount of sample (10-40 mg) available, being the nasal exudate extracted from the swab with 300 µL 10 mM Tris/HCl, pH = 7.4. Studies to obtain reliable analytical information, such as the blank contribution of the sampling step, evaluation of the extraction efficiency of the nasal exudate from the swab, and normalization strategies for data treatment, have been carried out. Results showed that despite the limited number of investigated samples, fractionation studies as well as the concentrations of ferritin and ferroportin obtained with ELISA kits showed a differential behavior between the different cohorts.
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The request for novel hyphenated instruments and techniques, capable of affording exhaustive information and results, is a focus continuously watched out. In this context, the present work aimed at the development of an integrated system combining gas chromatographic (GC) separation with mass spectrometry (MS) and (solid deposition) Fourier transform infrared spectroscopy (FTIR) detection. An external transfer line was designed in the lab for the parallel coupling of the two detectors, in such a way to obtain complementary analytical information consisting of an MS spectrum, an IR spectrum and linear retention indices (LRI), within a single analysis. The instrument performance was demonstrated for the analysis of a commercial mixture consisting of 139 hydrocarbons, comprising linear, branched, unsaturated and aromatic compounds. A 100-m poly(dimethylsiloxane) column was employed for the separation, and the outlet flow was split 95:5 between the IR and MS detectors using two uncoated capillaries. The IR spectra were acquired from solid deposits on a zinc selenide disc (-90 °C), over a spot (detector area) of about 0.1 mm2, in the range of 4000-700 cm-1 and at a resolution of 4 cm-1. Final identification of the separated compounds by a library search was achieved by excluding incorrect results, sequentially using a three-filter approach (85% similarity against reference MS and IR library spectra and ±10 LRI unit tolerance). Based on these preliminary results, the GC-MS/sd-FTIR system is a promising tool for the characterization of complex matrix constituents, for which identification is cumbersome, by using only one detection technique.
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Cigars, treasured for their rich aromatic profiles, occupy a notable segment in the global consumer market. The objective of this study was to characterize the volatile aroma compounds that shape the flavor profiles of six distinct varieties of Great Wall cigars, contributing to the understanding of cigar aroma analysis. Utilizing HS-GC-IMS and sensory evaluation, the study discerned the aroma profiles of GJ No. 6 (GJ), Animal from the Chinese zodiac (SX), Range Rover No. 3 Classic (JD), Miracle 132 (QJ), Sheng Shi No. 5 (SS), and Red 132 (HS) cigars. The analysis uncovered a spectrum of characteristic aromas, including tobacco, creaminess, cocoa, leather, baking, herbaceous, leathery, woodsy, and fruity notes. A total of 88 compounds were identified, categorized into 11 chemical classes, with their quantities varying among the cigars in a descending order of QJ, JD, GJ, SS, HS, and SX. 24 compounds, such as 2-heptanone, n-butanol, 2,6-dimethylpyrazine and 2-furfuryl methyl sulfide were considered as key differential components. The volatile components were effectively differentiated using principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and cluster analysis, revealing correlations between sensory attributes, key components, and electronic nose (E-nose). This research introduces a novel method for analyzing volatile aroma components in cigars, offering insights to enhance cigar quality and to foster the development of new products with unique aroma profiles.
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A design procedure for the separation of charge variants of a monoclonal antibody (mAb) was developed, which was based on the coupling of cation-exchange chromatography (CEX) and anion-exchange chromatography (AEX) under high loading conditions. The design of the coupled process was supported by a dynamic model. The model was calibrated on the basis of band profiles of variants determined experimentally for the mAb materials of different variant compositions. The numerical simulations were used to select the coupling configuration and the loading conditions that allowed for efficient separation of the mAb materials into three products enriched with each individual variant: the acidic (av), main (mv) and basic (bv) one. In the CEX section, a two-step pH gradient was used to split the loaded mass of mAb into a weakly bound fraction enriched with av and mv, and a strongly bound fraction containing the bv-rich product. The weakly bound fraction was further processed in the AEX section, where the mv-rich product was eluted in flowthrough, while the av-rich product was collected by a step change in pH. The choice of flow distribution and the number of columns in the CEX and AEX sections depended on the variant composition of the mAb material. For the selected configurations, the optimized mAb loading density in the CEX columns ranged from 10 to 26 mg mL-1, while in the AEX columns it was as high as 300 or 600 mg mL-1, depending on the variant composition of the mAb material. By proper selection of the loading condition, a trade-off between yield and purity of the products could be reached.
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Alcoholization is an integral part of tobacco processing and volatile compounds are key to assessing tobacco alcoholization. In this study, a total of 154 volatiles from nine categories were determined by gas chromatography-ion mobility spectrometry (GC-IMS) from four grades of tobacco, of which 114 were better identified. And then, the dynamic trends of volatile compounds with significant changes in tobacco alcoholization were analyzed. The relevant volatiles with the alcoholization indices (AIs) (R > 0.8) were screened as indicators of tobacco alcoholization. Cinnamyl isobutyrate, linolenic acid alcohol, propanoic acid-M and propanoic acid-D in all tobacco samples were highly correlated with the AIs and tended to increase during the alcoholization process. In addition, linear discriminant analysis (LDA), back-propagation neural network (BPNN) and random forest (RF) classifiers were constructed for discrimination of tobacco AIs. Three classifiers trained with a combination of 20 volatiles achieved satisfactory results with area under the curve (AUC) of 0.95 (LDA), 0.94 (BPNN) and 0.97 (RF), respectively. The RF classifier gained optimal accuracy of 100 % and 96.1 % for the training and test sets, respectively. The study confirmed that GC-IMS can be used to characterize the changes of volatile compounds in tobacco during alcoholization and combined with machine learning to achieve the determination of AIs. The results of the study may provide a new means for the tobacco industry to monitor the alcoholization process and determine the degree of alcoholization.
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Volatile organic compounds (VOCs) are continuously emitted into the atmosphere from natural and anthropogenic sources and rapidly spread from the atmosphere to different environments. A large group of VOCs has been included in the class of air pollutants; therefore, their determination and monitoring using reliable and sensitive analytical methods represents a key aspect of health risk assessment. In this work, an untargeted approach is proposed for the evaluation of the exposure to volatile organic compounds of workers in an engine manufacturing plant by GC-MS measurements, coupled with solid-phase microextraction (SPME). The analytical procedure was optimized in terms of SPME fiber, adsorption time, desorption time, and temperature gradient of the chromatographic run. For the microextraction of VOCs, the SPME fibers were exposed to the air in two different zones of the manufacturing factory, i.e., in the mixing painting chamber and the engine painting area. Moreover, the sampling was carried out with the painting system active and running (system on) and with the painting system switched off (system off). Overall, 212 compounds were identified, but only 17 were always present in both zones (mixing painting chamber and engine painting area), regardless of system conditions (on or off). Finally, a semi-quantitative evaluation was performed considering the peak area value of the potentially most toxic compounds by multivariate data analyses.
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Carbon-13 isotopomics of triacylglycerol (TAG) fatty acids or free fatty acids in biological matrices holds considerable potential in food authentication, forensic investigations, metabolic studies, and medical research. However, challenges arise in the isotopic analysis of short- and medium-chain (C4 to C10) fatty acid methyl esters (SMCFAMEs) through gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The high volatility of these esters results in losses during their preparation, leading to isotopic fractionation. Moreover, the methoxy group added to acyl chains requires the correction of δ13C values, thereby increasing the uncertainty of the final results. Analyzing free fatty acids (FFAs) addresses both issues encountered with SMCFAMEs. To achieve this objective, we have developed a new protocol enabling the isotopomics of individual fatty acids (FAs) by GC-C-IRMS. The same experiment also provides the FA profile, i.e., the relative percentage of each FA in the TAG hydrolysate or its concentration in the studied matrix. The method exhibited high precision, as evidenced by the repeatability and within-lab reproducibility of results when tested on TAGs from both animal and vegetal origins. Compared to the analysis of FAMEs by GC-C-IRMS, the current procedure also brings several improvements in alignment with the principles of green analytical chemistry and green sample preparation. Thus, we present a two-in-one method for 13C-isotopomic and metabolomic biomarker quantitation within quasi-universal TAG compounds, encompassing the short- and medium-acyl chains.
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Introduction: Animal studies suggest that adolescent exposure to Δ9-tetrahydrocannabinol (Δ9-THC), the intoxicating constituent of cannabis, causes lasting functional alterations in brain and other organs. Those studies often neglect the impact that age- and sex-dependent differences in the distribution and metabolism of the drug might exert on its pharmacological effects. Here, we provide a comparative analysis of Δ9-THC pharmacokinetics in adolescent and adult female mice, which identify significant dissimilarities in distribution and metabolism of Δ9-THC between females of these age groups. Materials and Methods: We administered Δ9-THC (5 mg/kg, intraperitoneal) to adolescent (37-day old) and young adult (70-day old) female mice and quantified Δ9-THC and its first-pass metabolites-11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)-in plasma and brain tissue using liquid chromatography/tandem mass spectrometry. Results: Maximal plasma concentrations of Δ9-THC were 8 times higher in adolescent than adult female mice. Conversely, brain concentrations and brain-to-plasma ratios were 25-50% higher in adults than adolescents. Concentrations of Δ9-THC metabolites were higher in plasma but lower in brain of adolescent compared to adult female mice. Conclusions: The results identify multiple age-dependent differences in the pharmacokinetic properties of Δ9-THC in female mice, which might influence the pharmacological response to the drug.
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OBJECTIVE: To develop a method which is used for rapid determination of 16 kinds of polycyclic aromatic hydrocarbons(PAHs) and 16 kinds of phthalates(PAEs) in tap water by stirring rod adsorption extraction(SBSE) combined with gas chromatography-mass spectrometry(GC-MS). METHODS: The twister mixing rod coated with polydimethylsiloxane(PDMS) and ethylene glycol-polydimethylsiloxane(EG-silicone) was used to enrich analyte from 50 mL tap water. The twister mixing rod coated with EG-silicone was directly placed into the sample bottle containing 50 mL of tap water, while fixing the PDMS stir bar on the inner wall of the sample bottle and immersing it in the liquid. Add 5%(W/V) sodium chloride to the sample bottle, followed by adding 5% methanol. Stir at room temperature for 2 hours for extraction. Next, remove the mixing stick and dry its surface. The pre-prepared SBSE was analyzed by TD-GC/MS, with the optimized thermal desorption conditions: desorption temperature 275 â, desorption time 15 min, cryofocusing temperature-40 â. RESULTS: Regression equations revealed acceptable linearity(correlation coefficients >0.986) across the working-standard range from 200-2000 ng/L for the 32 analytes. The limits of detection(LODs)were further evaluated were from 1.13-121 ng/L. With the optimized pretreatment method, the spiked recoveries of tap samples(200 and 2000 ng/L)were in the range of 62.5%-98.4% with the relative standard deviations(RSDs) of 3.5%-25.3%. CONCLUSION: The established method can realize the rapid detection of high throughput in the laboratory, it is simple, convenient to operate, and the extraction and analysis time is short.