{"title":"RH-RPA: A Rapid and Highly Sensitive Assay for Nucleic Acid Detection Based on RNase HII Combined with Recombinase Polymerase Amplification","authors":"Yuting Jin, Yangnan Fu, Qingyang Liu, Suhang Li, Yingzhou Zeng, Lijuan Fu, Yongyou Zhang","doi":"10.1021/acs.analchem.4c06578","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06578","url":null,"abstract":"Currently, RPA-exo and RPA-nfo are the primary methods for RPA/RT-RPA probe assays, both of which have been widely applied to the detection of various targets. However, RPA-nfo exhibits lower sensitivity compared with the exo probe method, while RPA-exo lacks the capability for equipment-free visualization inherent to RPA-nfo. Both of the approaches mentioned above limit the broader application of RPA/RT-RPA probe assays. To address those limitations, we have developed a novel recombinase polymerase amplification (RPA) combined with an <i><i>E. coli</i></i> RNase HII assay (RH-RPA). This approach supports both fluorescence signal detection and lateral-flow strip readouts. Due to the high efficiency and specificity of <i><i>E. coli</i></i> RNase HII in recognizing and cleaving targets, this method serves as a rapid and accurate molecular diagnostic platform. Under the fluorescence detection mode, RH-RPA achieves a limit of detection as low as 10 copies per reaction for both DNA and RNA within 20 min. Additionally, the lateral-flow strip mode enables the detection of as few as 5 copies per reaction of nucleic acids within 20 min. In clinical sample analysis, the RT RH-RPA demonstrated 100% accuracy in detecting the influenza A virus, underscoring its reliability in practical diagnostics. These findings highlight the stable specificity, rapid performance, high sensitivity, and cost-effectiveness of the RH-RPA methods, showcasing their potential as promising tools for point-of-care nucleic acid detection.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"15 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143872940","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"SNAr-Based Labeling of Proteins with Trityl Radicals Enables High-Precision, High-Sensitivity, and Long-Range Distance Measurement","authors":"Yande Gao, Bin-bin Pan, Yurui Leng, Xi-wei Wang, Shuai Li, Peng Deng, Ying Ma, Yuguang Song, Xun-cheng Su, Yin Yang, Yangping Liu","doi":"10.1021/acs.analchem.4c06851","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06851","url":null,"abstract":"Distance measurement using pulsed dipolar electron paramagnetic resonance spectroscopy (PD-EPR) coupled with site-directed spin labeling is a powerful approach to study the structure and dynamics of biomolecules in nearly native environments. However, the accuracy and sensitivity of the distance measurement in cellular systems is often limited by long, flexible, and/or reduction-sensitive linker of spin labels and their low biostability. Herein, we report the first class of aryl-linked trityl spin labels in which pyrimidine and pyridine methylsulfones (FPS1–2) act as protein tagging groups and are directly conjugated to the trityl moiety. FPS1 underwent a specific nucleophilic aromatic substitution (SNAr) reaction with cysteine (Cys) as its free form or in glutathione (GSH) and proteins to produce stable C–S conjugation. The resulting conjugates experienced negligible hydrolysis after a long-term measurement and exhibited high biostability to the commonly used reducing agents and also in cell lysates, which is in contrast to the conjugates of the maleimide spin label, CT02MA. PD-EPR studies on the FPS1-labeled immunoglobulin G-binding protein (GB1) and second-mitochondrion-derived activator of caspases (Smac) demonstrated that FPS1 enables highly sensitive and long-range distance measurement in the proteins. Importantly, due to its short and rigid aryl linker, the use of FPS1 provides significantly narrower distance distributions than those from the maleimide spin labels CT02MA and OXMA. Moreover, using FPS1, the longest distance measurement (∼6.01 nm) so far in all of the trityl-labeled protein samples has also been achieved in the FPS1-labeled Smac. Overall, this work demonstrates that the SNAr-based aryl groups can be ideally united into the skeleton of trityl radicals, and the resulting spin labels enable sensitive, precise, and ultralong distance measurements in proteins under diverse conditions.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"22 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hao Chen, Wen-Qiang Zu, Yue-Ru Zhou, Shuang-Long Wang, Wen-Li Yuan, Song Qin, Ling He, Guo-Hong Tao
{"title":"Catalytic Strategy for Chemical Analysis of Volatile Iodine with the Assistance of Machine Learning","authors":"Hao Chen, Wen-Qiang Zu, Yue-Ru Zhou, Shuang-Long Wang, Wen-Li Yuan, Song Qin, Ling He, Guo-Hong Tao","doi":"10.1021/acs.analchem.4c06653","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06653","url":null,"abstract":"A strategy of catalytic chemical detection (CCD) with the assistance of a machine learning (ML) approach was proposed and evaluated in this work. In the CCD method, the target analyte acts as the catalyst of the detection reaction rather than traditional reactants. The detection of a typical environmental contaminant-volatile iodine was selected as an example to establish the general routine in designing CCD. One major obstacle lies in the complex of manual selection of detection reaction, especially considering that more than 650,000 related reactions were exhibited in SciFinder database. Traditional workflow is time-consuming and material-consuming; therefore, the ML approach with descriptors directly related to CCD was employed. The reaction of indoles and aromatic aldehydes to bis(indolyl)methanes was screened out with the ML approach. After preliminary experiments, the screened reaction for iodine detection achieved desirable sensitivity, specificity, and recognizability simultaneously. The fabricated sensor devices were practicable for portable detection in real gas samples with a low concentration. This work provides a practical example of chemical analysis based on catalytic strategy and exemplifies the powerful application for the ML method in chemistry through the introduction of original descriptors.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"33 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866234","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Guojuan Yan, Meihan Guo, Ruiju Yang, Xiaoxiao Li, Chenlei Gu, Ke Wang, Yiming Liu, Mingxuan Gao, Chengzhi Huang, Hongyan Zou
{"title":"Copper Vacancy-Doped Cu2-xSe Activating Nanozyme Sensor Arrays for Multiprotein Discrimination and Cancer Screening","authors":"Guojuan Yan, Meihan Guo, Ruiju Yang, Xiaoxiao Li, Chenlei Gu, Ke Wang, Yiming Liu, Mingxuan Gao, Chengzhi Huang, Hongyan Zou","doi":"10.1021/acs.analchem.5c00078","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00078","url":null,"abstract":"Given the complexity and interrelated nature of biological activities, acquiring multitarget information is crucial for accurate cancer diagnosis. In this study, we developed a cross-reactive sensor array using peroxidase-like nonstoichiometric copper selenide nanoparticles (Cu<sub>2-x</sub>Se), which featured varying copper vacancies, for multiplex protein detection and cancer screening. The Cu<sub>2-x</sub>Se nanoparticles demonstrated outstanding peroxidase-like activity that can be modulated to varying degrees in the presence of multiple proteins with varying isoelectric points and compositions, generating fingerprint outputs. By integrating pattern recognition method principal component analysis with linear discriminant analysis (PCA–LDA), the sensor array effectively discriminated among six proteins and was further applied to cells and clinical samples, attaining 100% accuracy in differentiating cancer patients from healthy individuals, as well as in identifying specific cancer types, namely, liver and prostate cancers. Moreover, as a proof-of-concept, the partial least-squares regression (PLSR) approached the accurate detection of AFP and PSA within the range of 0.1–2.0 μg/L in the complex biological matrix. These results highlighted the practical potential of multitarget sensing and clinical diagnosis by the nanozyme-based chemical nose strategies.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"5 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866237","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Longyu Yi, Yuqi Liu, Chao Wan, Shunji Li, Mengfan Zhou, Fukang Qi, Han Xie, Xin Wang, Yixiao Su, Wei Du, Xiaojun Feng, Yiwei Li, Bi-Feng Liu, Peng Chen
{"title":"Programmable Manually Powered Microfluidics for Rapid Point-of-Care Diagnosis of Urinary Tract Infection","authors":"Longyu Yi, Yuqi Liu, Chao Wan, Shunji Li, Mengfan Zhou, Fukang Qi, Han Xie, Xin Wang, Yixiao Su, Wei Du, Xiaojun Feng, Yiwei Li, Bi-Feng Liu, Peng Chen","doi":"10.1021/acs.analchem.5c00847","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00847","url":null,"abstract":"Point-of-care testing (POCT) for urinary tract infection (UTI) holds significant importance in the field of disease prevention and control, as well as the advancement of personalized precision medicine. However, conventional methods for detecting UTIs continue to face challenges such as time-consuming and labor-intensive detection processes, and reliance on specialized equipment and personnel rendering them unsuitable for point-of-care applications, especially in resource-limited areas. Here, we propose a novel flexible programmable manually powered microfluidic (FPM) for rapid point-of-care diagnosis of UTIs. For the first time, the proposed FPMs was achieved through a combined strategy of laser printing, cutting, and laminating, with the entire process completed in under 15 min at a cost of less than $0.5, which effectively circumvent the traditionally time-consuming and labor-intensive soft lithography techniques. By incorporating a modular structure-based design concept, we successfully developed various types of portable FPMs with functionalities including parallel pumping, simultaneous releasing, quantitative dispensing, sequential releasing, cyclic motion of multiple liquids and concentration gradient generating. As a proof-of-concept demonstration, we initially employed a high-throughput parallel dispensing design to analyze six urinary biochemical markers within 1 min, presenting potential applicability for future at-home testing. We then integrated a manually powered concentration gradient generator with spatial confinement signal enhancement to enable rapid phenotypic antimicrobial susceptibility testing (AST) within three to 5 h, while achieving clinical diagnostic accuracy rates of up to 95.56%. Therefore, our proposed FPMs eliminate the need for external pumps or actuators and could serve as an affordable hand-held POCT tool for UTI diagnosis. Moreover, in resource-poor areas, they have potential utility as robust POCT devices addressing diverse rapid detection needs.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"31 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Huan Ma, Hui Wang, Ru Sun, Wen-Juan Gan, Jian-Feng Ge
{"title":"Facile Way to Differentiate Normal and Cancerous Tissues via Polarity-Sensitive Fluorescent Probes Based on 1,6-Naphthyridine Derivatives","authors":"Huan Ma, Hui Wang, Ru Sun, Wen-Juan Gan, Jian-Feng Ge","doi":"10.1021/acs.analchem.5c00787","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00787","url":null,"abstract":"Cancer is a genetic disorder caused by the long-term interaction of many factors, which has become the most important factor to take away human health; therefore, it is essential to develop a more efficient and sensitive cancer detection technology. This study developed two polarity sensitive probes <b>1a</b> and <b>1b</b> based on a 1,6-naphthyridine moiety linked to different targeting groups by vinyl as the π bridge. As the solvent polarity decreased, the emission wavelength of probes <b>1a</b> and <b>1b</b> experienced a blue shift, resulting in a significant enhance in fluorescence intensity by 135-fold and 53-fold, respectively, and a good linear relationship between <i>F</i><sub>max</sub> of probes <b>1a</b> and <b>1b</b> and Δ<i>f</i> was established with high correlation coefficients. Furthermore, probes <b>1a</b> and <b>1b</b> exhibited large Stokes shifts, high photostability, and low cytotoxicity, successfully targeting intracellular lipid droplets and mitochondria. Fluctuation in polarity was detected by real-time changes in fluorescence intensity of probes in lipid droplets and mitochondria. Moreover, probe <b>1b</b> was capable of real-time monitoring mitochondrial polarity during starvation or rapamycin-induced autophagy. It was worth standing out <b>1a</b> and <b>1b</b> could distinguish normal cells from cancer cells, and then the probes also were successfully applied for imaging to differentiate between human normal tissues and cancerous tissues, with the fluorescence intensity of malignant tumor tissues being 15.4–19.9 folds higher than that of normal tissues and 5.3–7.2 times higher than that of benign tumor tissues. Therefore, this research offers potential applications for cancer diagnosis.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"46 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yanwen Liu, Su Li, Xinmeng Wang, Xiya Liu, Juan Wang, Zhihong Liu
{"title":"Support-Free Implantable Photoelectrochemical Hydrogel Fiber Enables Long-Term Monitoring in Free-Behaving Organisms","authors":"Yanwen Liu, Su Li, Xinmeng Wang, Xiya Liu, Juan Wang, Zhihong Liu","doi":"10.1021/acs.analchem.5c01013","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c01013","url":null,"abstract":"The development of long-term and in situ in vivo monitoring techniques is critical for environmental biology, life sciences, and analytical chemistry. However, existing in vivo analysis methods are limited by the complex and large instruments or adverse impacts of rigid implanted substrates on living organisms, making it difficult to achieve continuous in situ detection. Herein, taking advantage of the flexibility and biocompatibility of the hydrogel fiber and solving its instability or opacity problems caused by ionic or polymer conduction for hydrogel fibers, a photoelectrochemical (PEC) hydrogel fiber free of conventional rigid substrate support is successfully prepared and achieves long-term tracking of persistent organic pollutants in free-behaving fish, timely identifying their environmental ecological risks. This support-free PEC fiber exhibits fascinating properties of electrical and light conductivity, flexibility, antifouling ability, and biocompatibility, allowing it to be implanted in vivo for 70 days without experiencing significant loss of sensing performance and causing apparent inflammation and immune responses. Moreover, the fabricated fiber not only achieves in vitro pentachlorophenol detection with high selectivity, low detection limit, good reproducibility, and dual-mode sensing but also realizes in vivo monitoring of pentachlorophenol enriched in fish brain for up to 70 days with satisfactory reliability, unraveling its tempting potential for various in vivo application.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"108 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143862485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Peptide Molecular Siege Machine: Breaking through Mycobacterium tuberculosis’s Cellular Defenses for Precise Detection and Monitoring","authors":"Wenting Cheng, Yuanyuan Miao, Chuang Wang, Yanwen Zu, Zhisong Wu, Yongchen Zhang, Jinlong Li","doi":"10.1021/acs.analchem.4c05763","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05763","url":null,"abstract":"This work introduces a peptide biosensor for detecting <i>Mycobacterium tuberculosis</i> (Mtb). The designed peptide probe exhibits specific affinity toward distinct components of Mtb. First, a peptide sequence is tailored to target hydrophobic long-chain fatty acids in the mycobacterial cell wall. Following this, an electrochemical potential scan releases a peptide sequence aimed at the intracellular molecular chaperones of Mtb. This sequence, upon penetration of the bacterial cell membrane, binds with molecular chaperones, which is crucial for Mtb survival and stress response. The biosensor incorporates complementary peptide sequences to capture chaperone-bound peptides back onto the substrate surface, allowing for their subsequent electrochemical detection. This multistep process enables selective and sequential interactions with Mtb components, minimizing interference from nontarget molecules. By integrating these innovative peptide probes into a wearable substrate using conductive polymer technology, the biosensor achieves high sensitivity and accuracy, offering a promising tool for the real-time monitoring of tuberculosis progression and treatment response.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"42 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866232","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Salman Khan, Sneha Asok, Veda V. Dasari, Sharayu Magar, Bhagyasree Paila, Anil K. Suresh
{"title":"Feeling of an Eye When It Meets the Unseen “Nano”","authors":"Salman Khan, Sneha Asok, Veda V. Dasari, Sharayu Magar, Bhagyasree Paila, Anil K. Suresh","doi":"10.1021/acs.analchem.5c00035","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00035","url":null,"abstract":"Nanomedicine is increasingly being utilized in addressing various eye ailments and holds immense potential in rectifying ocular diseases; however, the interactions between nanomedicines and their route of administration via tear fluid remain poorly understood. When nanoparticles are introduced into the tear fluid, a layer of protein corona is formed on their surface that not only influences the properties and biological fate of nanoparticles but also potentially interferes with the function of endogenous proteins. To investigate the interactions between gold nanoaprticles (AuNPs) and tear fluid, focusing on the physicochemical changes of the particles, and to quantitatively and qualitatively identify the key proteins involved in the corona formation, we employed label-free techniques for material and biophysical characterizations along with proteomic analyses and mass spectrometry. The AuNPs remained stable without forming aggregates, showing only an ∼31 nm increase in hydrodynamic diameter after interacting with tear fluid. Notably, their overall zeta potential increased significantly from −12 to −23 eV due to the supplemented charge by the adsorbed proteins. Proteomic analysis and liquid chromatography/mass spectrometry (LC-MS/MS) identified 31 proteins that were bound with the nanoparticles from a total of 174 proteins that were detected in the tear fluid. Bioinformatic classification revealed an enrichment of specific proteins essential for ocular health; proteins such as clusterin, lactotransferrin, adenosine triphosphate (ATP) synthase, lysozyme, alpha enolase, keratin, apolipoprotein, and epidermal growth factor receptor (EGFR) with pivotal roles in anti-inflammatory, immune response, cell adhesion, cellular organization, plasminogen activation, cell signaling, stress response, and corneal epithelial homeostasis. Overall, our study provides an unresolved comprehensive map of the tear protein corona landscape and its impact on nanoparticle behavior in the tear fluid. These insights must be considered and are valuable for designing safer and more effective nanomedicines for the treatment of various eye diseases.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"42 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jiří Kohoutek, Juan I. Sánchez-Avila, Marie Smutná, Petr Janků, Jana Klánová, Klára Hilscherová
{"title":"Determination of Thyroid Hormones and 11 Metabolites in the Human Serum Using a Simple Derivatization Strategy and Analysis by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry","authors":"Jiří Kohoutek, Juan I. Sánchez-Avila, Marie Smutná, Petr Janků, Jana Klánová, Klára Hilscherová","doi":"10.1021/acs.analchem.5c00714","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00714","url":null,"abstract":"Many analytical methods for thyroid hormone (TH) determination lack sensitivity and/or specificity. The thyroid hormone metabolites (THMs) are usually not measured at all. This study describes the development of sensitive high-throughput analytical methods for determining the total concentration and free fraction of TH and THM in the human serum. For the analysis of the TOTAL fraction, we employed protein precipitation and anionic exchanger solid-phase extraction. For the FREE fraction, ultrafiltration and salt-out liquid partitioning were used. Derivatization using dansyl chloride was employed to enhance the sensitivity of HPLC-ESI-MS/MS analysis. Both protocols were validated according to the European Analytical Guidelines (2002/657/EC). We obtained very good recoveries (73–115%) and precision. Interday coefficients of variation (CVs) for most of the analytes ranged from 1.2 to 16.4%. The sensitivity was excellent with detection limits in the sub ppt range for the majority of TH and THM. A significant enhancement in sensitivity (>10 fold) was achieved through derivatization. The applicability was proved on a set of samples from pregnant women enrolled in the CELSPAC cohort (<i>n</i> = 120). Our TH reference ranges are in good agreement with those reported in the literature. The methods also allowed us to quantify the levels of 11 THM, including some previously undetected THM in total and free fractions, and proved to be suitable for high-throughput routine TH and THM analyses. Our approach offers an important advancement in thyroid hormone analysis. To the best of our knowledge, it is for the first time that data for T1A and T2A as well as for free THM levels in the human serum are published in the literature. Moreover, our study also brings the first information about the levels of most of the THM in pregnant women.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"5 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}