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In this work, multiple extraction thermal desorption (METD), as a sample introduction method for GC, was developed. This technique was used for the determination of residual solvents (RS) in modified cellulose, because it is practically impossible to dissolve or distribute it uniformly in water and common organic solvents. Moreover, METD facilitates the optimization of the desorption time and it is more sensitive to quantify trace level volatiles in insoluble material, compared to direct dynamic desorption (DDD). In addition, METD provides diagnostic information about the sample-sorbent interaction. Three solvents (methanol, ethanol and tert-butanol) were determined in two types of modified cellulose (dialdehyde cellulose (DAC) and DAC-ethylenediamine (DAC-EDA)). It was shown that good linearity over a wide concentration range was achieved. The limits of detection (LOD) and limits of quantification (LOQ) for the different solvents ranged from 0.1 to 0.3⯵g and from 0.3 to 0.9⯵g per tube, respectively. Accuracy of the METD method was verified by using an alternative method based on the decomposition of the modified celluloses by Trichoderma reesei cellulase, followed by headspace-trap-GC (HS-trap-GC). The results obtained from the two validated methods were found to be similar (relative deviation < 17.0â¯%). However, the developed METD-GC method is preferable for the analysis of RS in modified cellulose since it does not require sample pretreatment and possesses higher sensitivity.
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Over the past few years, there has been growing interest in developing new methods of embryo quality assessment to improve the outcomes of assisted reproductive technologies in the medical field. Raman microscopy as an increasingly promising analytical tool has been widely used in life sciences, biomedicine and "omics" to study molecular, biochemical components, living cells and tissues due to the label-free and non-destructive nature of the imaging technique. This paper reviews the analytical capability of Raman microscopy and applications of Raman spectroscopy technology mainly in reproductive medicine. The purpose of this review is to introduce the Raman spectroscopy technology, application and underlying principles of the method, to provide an intact picture of its uses in biomedical science and reproductive medicine, to offer ideas for its future application, verification and validation. The focus is on the application of Raman spectroscopy in the reproductive medicine field, including the application in gametes, embryos and spent embryo culture media.
Assuntos
Medicina Reprodutiva , Análise Espectral Raman , Análise Espectral Raman/métodos , Humanos , Medicina Reprodutiva/métodos , Técnicas de Reprodução Assistida , Animais , Células Germinativas , Embrião de MamíferosRESUMO
Monitoring the levels of amino acids (AAs) in biological cell cultures provides key information to understand the regulation of cell growth and metabolism. Saccharomyces cerevisiae can naturally excrete AAs, making accurate detection and determination of amino acid levels within the cultivation medium pivotal for gaining insights into this still poorly known process. Given that most AAs lack ultraviolet (UV) chromophores or fluorophores necessary for UV and fluorescence detection, derivatization is commonly utilized to enhance amino acid detectability via UV absorption. Unfortunately, this can lead to drawbacks such as derivative instability, labor intensiveness, and poor reproducibility. Hence, this study aimed to develop an accurate and stable hydrophilic interaction liquid chromatography-tandem mass spectrometry analytical method for the separation of all 20 AAs within a short 17-min run time. The method provides satisfactory linearity and sensitivity for all analytes. The method has been validated for intra- and inter-day precision, accuracy, recovery, matrix effect, and stability. It has been successfully applied to quantify 20 AAs in samples of yeast cultivation medium. This endeavor seeks to enhance our comprehension of amino acid profiles in the context of cell growth and metabolism within yeast cultivation media.
Assuntos
Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Saccharomyces cerevisiae , Espectrometria de Massas em Tandem , Aminoácidos/metabolismo , Aminoácidos/análise , Espectrometria de Massas em Tandem/métodos , Saccharomyces cerevisiae/metabolismo , Cromatografia Líquida , Cromatografia Líquida de Alta Pressão/métodosRESUMO
In this study, a liquid chromatographic method was developed for the fast determination of lincomycin, polymyxin and vancomycin in a preservation solution for transplants. A Kinetex EVO C18 (150 × 4.6 mm, 2.6 µm) column was utilized at 45 °C. Gradient elution was applied using a mixture of mobile phases A and B, both including 30 mM phosphate buffer at pH 2.0 and acetonitrile, at a ratio of 95:5 (v/v) for A and 50:50 (v/v) for B. A flow rate of 1.0 mL/min, an injection volume of 20 µL and UV detection at 210 nm were used. A degradation study treating the three antibiotics with 0.5 M hydrochloric acid, 0.5 M sodium hydroxide and 3% H2O2 indicated that the developed method was selective toward lincomycin, polymyxin, vancomycin and their degradation products. Other ingredients of the preservation solution, like those from the cell culture medium, did not interfere. The method was validated with good sensitivity, linearity, precision and accuracy. Furthermore, lincomycin, polymyxin and vancomycin were found to be stable in this preservation solution for 4 weeks when stored at -20 °C.
Assuntos
Lincomicina , Polimixinas , Vancomicina , Lincomicina/análise , Vancomicina/análise , Polimixinas/análise , Cromatografia Líquida/métodos , Soluções para Preservação de Órgãos , Antibacterianos/análise , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodosRESUMO
As a pivotal enzyme that regulates dephosphorylation in cell activities and participates in the insulin signaling pathway, protein tyrosine phosphatase 1B (PTP1B) is considered to be an important target for the therapy of diabetes. In this work, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE), and used for screening and evaluating the inhibition effect of Traditional Chinese Medicine on PTP1B. Response Surface Methodology was used for optimizing the conditions of analysis. After method validation, the enzyme kinetic study and inhibition test were performed. As a result, the IC50 of PTP1B inhibitors â £ and â ©â § were consistent with reported values measured by a conventional method. It was found that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. showed prominent inhibition on the activity of PTP1B, which were stronger than the positive controls. Meanwhile, on top of the excellent advantages of CE, the whole analysis time is less than 2â¯min. Thus, the results demonstrated that a fast and efficient screening method was successfully developed. This method could be a powerful tool for screening inhibitors from complex systems. It can also provide an effective basis for lead compound development in drug discovery.
Assuntos
Medicamentos de Ervas Chinesas , Eletroforese Capilar , Hipoglicemiantes , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Humanos , Astragalus propinquus/química , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/análise , Eletroforese Capilar/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/análise , Hipoglicemiantes/farmacologia , Cinética , Medicina Tradicional Chinesa/métodos , Morus/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismoRESUMO
Antimicrobials, particularly antibiotics, are among the most common classes of drugs reported as substandard and falsified (SF) in developing countries. Therefore, it is important to develop simple and affordable analytical methods for the quality control of antimicrobial medicines. In this study, a liquid chromatographic method with ultraviolet detection (LC-UV) was developed and validated for the screening and quantification of 13 antimicrobial medicines and one beta-lactamase inhibitor in pharmaceutical formulations. LC separation was carried out on a Kinetex C18 column (150â¯mm × 4.6â¯mm, 2.6⯵m) with gradient elution. The mobile phase consisted of mixtures of acetonitrile-water-10â¯mM phosphate buffer pH 3.5 at ratios of 3:92:5, v/v/v for mobile phase A and 50:45:5, v/v/v for mobile phase B with a flow rate of 0.5â¯mL/min. The screening method was intended for confirmation of the identity of the actives and validated for specificity and robustness, whereas the quantification method (using only a different detection wavelength) was further validated in terms of linearity, accuracy, sensitivity and precision (repeatability, intermediate precision). For all compounds, the method was found to be linear (r2 > 0.999), precise (%RSD < 1%), accurate (% recovery of 98-102%), sensitive, specific and robust. The developed LC method was successfully applied for the identification and assay of 12 antimicrobial samples from Ethiopia. Among the 12 samples analyzed, one (8.3%) product was confirmed to be falsified.
Assuntos
Anti-Infecciosos , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodos , Anti-Infecciosos/análise , Controle de Qualidade , Cromatografia Líquida/métodos , Espectrofotometria Ultravioleta/métodos , Limite de Detecção , Antibacterianos/análiseRESUMO
A thermal desorber (TD) can be used in different ways to introduce samples in a gas chromatographic (GC) system. Besides its conventional use where the collected analytes are released from the sorbent in the sample tube, direct dynamic desorption (DDD) is an interesting option where a solid sample is put directly in the TD tube. However, since no sorbent is used for the sample, proper calibration is not straightforward. This issue was investigated in the present work using offline liquid calibration (OLC) and inline liquid calibration (ILC). Unexpectedly, ILC yielded a lower response than OLC. This could be related to the adsorption kinetics of the analytes and water on the cold trap of the TD. More insight was gained performing double injection ILC experiments with toluene as diluent for the analytes and injecting water before or after the toluene solution. This revealed a clear influence of the diluent. The influence of water was further explored applying two cold trap temperatures (4 °C and -30 °C). Inserting a LiCl trap in the TD tube to capture the water was found to be an effective solution for the problem. Finally, quantitative aspects of this approach were demonstrated.
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Temperatura Baixa , Água , Calibragem , Cromatografia Gasosa/métodos , Água/química , ToluenoRESUMO
In this study, methods for analyzing inorganic ions and carbohydrates in cardioplegia and nephroplegia solutions were developed and validated using ion chromatography with both conductivity and pulsed amperometric detection. The inorganic ions such as sodium, potassium, and calcium were separated by a cation-exchange column with 27 mM methanesulfonic acid as mobile phase at 0.5 mL/min. The anion (chloride) and carbohydrates (mannitol and glucose) were analyzed by an anion-exchange column using a mobile phase of 20 mM sodium hydroxide at 1.0 mL/min. The methods showed a high sensitivity for all analytes, with quantification limits from 0.0002 to 0.06 mg/L. Good linearities between the peak areas and concentrations were found for all analytes within the selected concentration range (R2 > 0.999). Relative standard deviation values for repeatability and interday precision were 0.1%-1.0% and 0.7%-1.6%, respectively. The accuracy was validated by determining the percentage recovery, which was between 98.0% and 101.3% for all analytes, indicating good accuracy of the methods. The robustness was verified by using an experimental design. Finally, real samples were analyzed to determine the content of the analytes. All assay values were between 96.8% and 102.5%.
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Carboidratos , Glucose , Cromatografia por Troca Iônica/métodos , Carboidratos/análise , Ânions , Parada Cardíaca InduzidaRESUMO
Different CE techniques have been used to analyze erythropoietin. These techniques have been shown to be effective in differentiating and quantifying erythropoietin isoforms, including natural and recombinant origins. This review provides a comprehensive overview of various capillary electrophoresis-based techniques used for the analysis of erythropoietin isoforms. The importance of erythropoietin in clinical practice and the necessity for the accurate analysis of its isoforms are first discussed. Various techniques that have been used for erythropoietin isoform analysis are then described. The main body of the review focuses on the different capillary electrophoresis-based methods that have been developed for erythropoietin isoform analysis, including capillary zone electrophoresis and capillary isoelectric focusing. The advantages and drawbacks of each method as well as their applications are discussed. Suggestions into the future directions of the area are also described.
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Eritropoetina , Eletroforese Capilar , Focalização Isoelétrica Capilar , Isoformas de ProteínasRESUMO
The significance of branched-chain amino acids in diseases was clearly shown over the years. This review aims to describe the available techniques for their analytical determination. The article provides examples of the use of various analytical methods. The methods are divided into two categories: derivatization and non-derivatization approaches. Separation is achieved through different chromatography or capillary electrophoresis techniques and can be combined with different detectors such as flame ionization, ultraviolet, fluorescence, and mass spectrometry. It compares the application of various derivatization reagents or detection as such for different detectors.
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Aminoácidos de Cadeia Ramificada , Cromatografia , Espectrometria de Massas/métodos , Indicadores e Reagentes , Eletroforese Capilar/métodosRESUMO
A sensitive, accurate and precise liquid chromatography (LC) method for the simultaneous determination of ceftazidime and pyridine in human plasma has been developed and validated. Acetonitrile (ACN) was employed to precipitate the proteins in the plasma samples. Chromatographic separation was performed with a Kinetex® C18 (150 mm × 3 mm, 2.6 µm) column with gradient elution. Ammonium formate 20 mM and ACN were mixed in a ratio of 98:2 (v/v) for mobile phase A and 85:15 (v/v) for mobile phase B. Both were adjusted to pH 4.5 with formic acid. The flow rate was 0.4 mL/min. UV detection was performed at 254 nm. Calibration curves were linear in the range from 0.3 to 225 µg/mL for ceftazidime and from 0.2 to 10 µg/mL for pyridine with correlation coefficients ≥ 0.999. Within- and between-run precision and accuracy were satisfactory with coefficients of variation (CV) ≤ 8.0% and deviations ≤ 7.0%, respectively. The method fulfilled all validation criteria prescribed by the European Medicines Agency guidelines. Next, it has been used successfully to analyze plasma samples of patients who received ceftazidime under intermittent and continuous administration. With intermittent administration, the concentration of the antibiotics reached a peak and then dropped quickly, which may be below the minimal inhibitory concentration (MIC). With continuous administration, the concentration of the antibiotics remained stable over 24 h, certainly above the MIC. Although the same tendency in ceftazidime concentration changes over time was observed, a difference in concentration amongst the patients was noticeable. The concentration of pyridine in plasma was negligible.
Assuntos
Antibacterianos , Ceftazidima , Piridinas , Humanos , Antibacterianos/análise , Antibacterianos/sangue , Antibacterianos/química , Ceftazidima/análise , Ceftazidima/sangue , Ceftazidima/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Preparações Farmacêuticas , Piridinas/análise , Piridinas/sangue , Piridinas/química , Reprodutibilidade dos TestesRESUMO
In this study, a ligand fishing method was developed to screen potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors from coffee extracts by immobilization of IDO1 enzyme on amino-modified magnetic nanoparticles combined with UHPLC-Q-TOF-MS/MS analysis. Parameters including enzyme concentration, immobilization time, the pH of glutaraldehyde and the amount of magnetic nanoparticles were optimized. The results indicated that immobilized IDO1 could be reused 5 times and was stable during storage for 7 days. Several IDO1 ligands were captured by incubating immobilized IDO1 with coffee extract, of which 10 showed an obvious difference comparing to non-conjugated bare nanoparticles. In vitro inhibitory activity was further performed by CE analysis, in which ferulic acid and chlorogenic acid had better IDO1 inhibitory activity, with IC50 value of 113.7 µM and 307.5 µM. These results demonstrate that this method provides an effective platform for identifying and screening IDO1 inhibitors from natural products.
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Tyrosine kinases have been intensively investigated as drug targets for several decades, since they regulate many cellular processes including cell growth, differentiation, and proliferation. Indeed, the deregulation of tyrosine kinases has been confirmed to play a vital role in the pathophysiology of many diseases. During the last few years, varieties of techniques have been developed to search for new tyrosine kinase inhibitors for cancer therapy, such as traditional filtration binding assay, scintillation proximity assay and some high-throughput screening methods. In this review, we describe the basic rules, merits and demerits, and application of a number of general and advanced technologies. The purpose of this review is to provide an insight into the numerous assays to achieve the exploration of new tyrosine kinase inhibitors.
Assuntos
Bioensaio , Ensaios de Triagem em Larga Escala , Ciclo Celular , Proteínas Tirosina Quinases , Inibidores de Proteínas Quinases/farmacologia , TirosinaRESUMO
Tetracyclines (TCs) are a family of broad-spectrum antibiotics. During the manufacturing process or storage, epimerization of tetracyclines could occur, leading to 4-epimers which are nearly inactive. From an analytical point of view, isomers are often difficult to distinguish. Previously, four pairs of TCs (oxytetracycline, tetracycline, doxycycline, chlortetracycline and their respective 4-epimers) were differentiated by mass spectrometry (MS) through protonated ions. However, they do not follow common rules and so it is still quite difficult to differentiate between them. In order to solve this, the four pairs were differentiated in the current study by collision induced dissociation (CID) spectra of the alkali adduct ions, including lithium, sodium and potassium. In the spectra of the sodium adducts, all studied tetracyclines showed a tendency to form [M+Na-NH3]+ ions, while the 4-epimers liked to form [M+Na-NH3-H2O]+ ions. Meanwhile, energy resolved mass spectrometry (ERMS) showed that all four 4-epimers' sodium adducts had the tendency to fragment at higher energy points. In the CID spectra of lithium adducts of TCs, a similar trend was observed for three pairs, except for doxycycline. For potassium adducts, the fragmentation was found to be less discriminative. As was derived from the 3D model, the four pairs all interact with the alkali metal through the dimethyl amino group at the C-4 position. The lithium adduct species also bound through the hydroxyl group at the C-5 position. If the TCs did not have a hydroxyl group at the C-5 position, they bound with the hydroxyl group at the C-6 position. For the same TC, with an increase of the diameter of the metal ion, the loss of H2O decreased gradually. As sodium adduct ions are common during the ionization process, TCs and their 4-epimers could be differentiated rapidly by ERMS of the sodium adduct ions.
Assuntos
Lítio , Metais Alcalinos , Espectrometria de Massas por Ionização por Electrospray/métodos , Tetraciclinas/química , Doxiciclina , Metais Alcalinos/química , Íons/química , Sódio/química , Antibacterianos , PotássioRESUMO
Natural products are an important source of major compounds in drug discovery. Currently, rapid screening and identification of bioactive compounds is a challenge due to the complicated chemical composition of natural products. Affinity screening methods based on liquid chromatography coupled with mass spectrometry have seen increasing interest in the past few years. In this review, the various strategies are classified into off-line and on-line modes. The principles and applications of these screening methods such as magnetic nanoparticles, affinity solid-phase extraction, immobilized liposome chromatography and cell membrane chromatography are introduced. This review covers the research advances of LC-MS based screening methods from 2019 to mid 2022, discussing their advantages and disadvantages, and providing an outlook for the future of this field.
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Produtos Biológicos , Lipossomos , Avaliação Pré-Clínica de Medicamentos , Cromatografia Líquida , Espectrometria de Massas/métodos , Produtos Biológicos/químicaRESUMO
A new, simple and sensitive ion chromatography (IC) method for the determination of sodium, potassium, magnesium, calcium and chloride in a parenteral nutrition (PN) solution was developed and validated. Before sample analysis, a sample pretreatment by calcination was applied which could totally remove interference from other constituents of the PN solution. Methanesulfonic acid (MSA) and sodium hydroxide were used as the mobile phase for the determination of cations and anions, respectively. The calibration curves showed good correlation between analyte peak area and concentration (r2 > 0.999). Detection limits ranged from 0.0001 to 0.02 mg/L and quantification limits from 0.0002 to 0.06 mg/L. Relative standard deviation (RSD) values for repeatability and inter-day precision did not exceed 1.0% and the recoveries for all analytes were between 99.1−101.1%. The robustness was verified by using an experimental design.
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Cloretos , Soluções de Nutrição Parenteral , Ânions/análise , Cátions/análise , Cromatografia por Troca Iônica/métodosRESUMO
Parabens, bisphenols, and triclosan are used in many baby products, including pacifiers. However, the migration through oral saliva will result in a potential health risk. The present study proposes a sensitive and simple method for the analysis of these chemicals in saliva simulants by solid phase microextraction (SPME) with amino-functionalized microporous organic network (MON-NH2) coated fiber. The MON-NH2 showed an excellent adsorption ability for phenolic compounds. The adsorption isotherm fitted the Langmuir isotherm model and the adsorption kinetics followed the pseudo second-order model. The developed SPME method exhibited wide linear ranges (0.005-500 µg/L), good linearity, low limits of quantitation (0.005 µg/L), great recoveries (87.0-112.5 %), and excellent precision (RSD < 8.3 % for intra-day and RSD < 13.7 % for inter-day). Mathematical models based on Fick's second law were applied to predict migration from pacifiers into saliva simulants and a good fit between theoretical and experimental migration results was found. The daily exposure assessment results indicated that these chemicals in pacifiers do not pose unacceptable health risks to infants. However, exposure risks still should be monitored and appropriate precautions are still needed to protect infants from exposure to these chemicals.
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Disruptores Endócrinos , Disruptores Endócrinos/análise , Humanos , Chupetas , Parabenos , Reprodutibilidade dos Testes , Saliva/química , Microextração em Fase Sólida/métodosRESUMO
BACKGROUND: Cytochrome P450 4F3 (CYP4F3) is an ω-hydroxylase that oxidizes leukotriene B4 (LTB4), prostaglandins, and fatty acid epoxides. LTB4 is synthesized by leukocytes and acts as a chemoattractant for neutrophils, making it an essential component of the innate immune system. Recently, involvement of the LTB4 pathway was reported in various immunological disorders such as asthma, arthritis, and inflammatory bowel disease. We report a 26-year-old female with a complex immune phenotype, mainly marked by exhaustion, muscle weakness, and inflammation-related conditions. The molecular cause is unknown, and symptoms have been aggravating over the years. METHODS: Whole exome sequencing was performed and validated; flow cytometry and enzyme-linked immunosorbent assay were used to describe patient's phenotype. Function and impact of the mutation were investigated using molecular analysis: co-immunoprecipitation, western blot, and enzyme-linked immunosorbent assay. Capillary electrophoresis with ultraviolet detection was used to detect LTB4 and its metabolite and in silico modelling provided structural information. RESULTS: We present the first report of a patient with a heterozygous de novo missense mutation c.C1123 > G;p.L375V in CYP4F3 that severely impairs its activity by 50% (P < 0.0001), leading to reduced metabolization of the pro-inflammatory LTB4. Systemic LTB4 levels (1034.0 ± 75.9 pg/mL) are significantly increased compared with healthy subjects (305.6 ± 57.0 pg/mL, P < 0.001), and immune phenotyping shows increased total CD19+ CD27- naive B cells (25%) and decreased total CD19+ CD27+ IgD- switched memory B cells (19%). The mutant CYP4F3 protein is stable and binding with its electron donors POR and Cytb5 is unaffected (P > 0.9 for both co-immunoprecipitation with POR and Cytb5). In silico modelling of CYP4F3 in complex with POR and Cytb5 suggests that the loss of catalytic activity of the mutant CYP4F3 is explained by a disruption of an α-helix that is crucial for the electron shuffling between the electron carriers and CYP4F3. Interestingly, zileuton still inhibits ex vivo LTB4 production in patient's whole blood to 2% of control (P < 0.0001), while montelukast and fluticasone do not (99% and 114% of control, respectively). CONCLUSIONS: A point mutation in the catalytic domain of CYP4F3 is associated with high leukotriene B4 plasma levels and features of a more naive adaptive immune response. Our data provide evidence for the pathogenicity of the CYP4F3 variant as a cause for the observed clinical features in the patient. Inhibitors of the LTB4 pathway such as zileuton show promising effects in blocking LTB4 production and might be used as a future treatment strategy.
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Leucotrieno B4 , Mutação de Sentido Incorreto , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450/genética , Elétrons , Feminino , Humanos , Leucotrieno B4/metabolismoRESUMO
Bisphenols and triclosan have been used in various products, and exposure to these chemicals may affect human health. The present study proposes a sensitive method for the determination of bisphenols A, F, S, and triclosan. The fiber was coated by amino/hydroxyl bifunctional microporous organic network and protected by polyvinylidene fluoride hollow fiber membrane for direct immersion solid phase microextraction. The limit of detection was 0.005 µg/L (µg/kg), and the recoveries were in the range of 76.7% to 107.5% (87.4% to 107.6%) for breast milk (infant formula), with intra-day and inter-day precisions <10.5% (7.3%) and 13.6% (8.4%), respectively. Fiber-to-fiber reproducibility of < 9.5% and a lifespan of >100 cycles were obtained. The 95th percentile estimated daily intake of total bisphenols was close to temporary tolerable daily intake for infants fed by human milk, which highlighted the needs for further attention on human exposure to BPA and its substitutes.