Analytical and Bioanalytical Chemistry最新文献

A hydrophilic interaction liquid chromatography-data-independent acquisition mass spectrometry (HILIC-DIA-MS) workflow was developed for simultaneous targeted semi-quantification of polar peptides and untargeted profiling of both peptides and other polar compounds in complex food matrices. The method uses a zwitterionic HILIC column optimized for separation of short polar peptides that are challenging to retain on reversed-phase columns. Many of these peptides contain charged amino acids and contribute to basic taste modalities such as umami and saltiness. Both targeted peptide analysis and comprehensive untargeted profiling were achieved by applying DIA-MS detection. This data acquisition mode was shown to be reproducible and sensitive while enabling retrospective data processing. High-resolution MS1 scans (60.000 FWHM), combined with fast MS2 scans and DIA mass windows of 15 m/z yielded highly repeatable and selective LC-MS profiles, allowing differentiation of structural isomers (e.g., alpha-glutamyl (umami) and gamma-glutamyl (kokumi)). The method was validated using taste-relevant dipeptides, demonstrating low detection limits (0.1-0.9 µM), good intra-day and inter-day precision, and high recovery (96%) in commercial soy sauce and yeast extract matrices. The workflow was further applied to the relative quantification of peptides and the untargeted profiling of characteristic molecular features in cheese, ham, and extracts from dried food ingredients. The integration of targeted and untargeted analyses demonstrates the suitability of HILIC-DIA-MS for comprehensive characterization of polar compounds in food systems.

The opioid crisis remains a significant public health concern, necessitating the development of sensitive and reliable analytical methods for drug detection. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of fentanyl, buprenorphine, oxycodone, morphine, tramadol, and tapentadol in plasma and oral fluid. The method was validated according to FDA guidelines, assessing selectivity, linearity, precision, accuracy, matrix effect, extraction efficiency, stability, carryover, and dilution integrity. The lower limits of quantification (LLOQs) were established at 0.1 ng/mL for fentanyl, 1.2 ng/mL for tramadol, and 0.6 ng/mL for the remaining opioids, demonstrating high sensitivity. The method exhibited excellent precision and accuracy, with coefficients of variation below 15% for intra-day, inter-day, and intermediate precision analyses. Extraction efficiencies exceeded 90% for most analytes, and matrix effects remained within acceptable limits. Real-world application to authentic plasma and oral fluid samples confirmed the method's robustness and reliability. Oral fluid concentrations were detectable across all target opioids, although plasma-oral fluid ratios showed some compound-dependent variability. These findings highlight the potential of oral fluid as a non-invasive complementary matrix to plasma for opioid monitoring, with relevant implications for forensic toxicology and clinical drug monitoring.

Virgin olive oil quality assessment is critical for consumer protection and industry regulation. Fraudulent activities involving virgin olive oil are common worldwide. In Brazil, recent nationwide inspections have revealed recurrent cases of fraud, including the sale of adulterated or mislabeled olive oils. As a result, the Brazilian federal agency has suspended the commercialization of numerous brands and seized large quantities of products that failed to meet the official quality requirements. This study presents an automated analytical workflow for classifying Brazilian virgin olive oils based on their volatile organic compound (VOC) profiles. Headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) were used to analyze 215 certified virgin olive oil samples, categorized as either defective (VOO, virgin and LVOO, lampante virgin) or non-defective (EVOO, extra virgin), and 21 undisclosed oils (UNKN). Chemometric modeling using partial least squares-discriminant analysis (PLS-DA) was applied to the resulting data, which comprised 108 recurring VOCs. The PLS-DA model successfully differentiated between the two quality classes (defective and non-defective) with a predictive accuracy of 91% based on the external validation set. Key chemical markers driving the classification were identified. Non-defective oils were characterized by higher levels of C₅ and C₆ aldehydes and alcohols from the lipoxygenase (LOX) pathway, such as (E)-2-hexenal, associated with positive green and fruity notes. Conversely, defective oils showed higher levels of compounds such as nonanal and acetic acid, linked to rancidity and other off-flavors. Finally, the model was used to predict the quality of 21 previously undisclosed samples. This instrumental approach demonstrates a powerful and reliable alternative for forensic analysis of olive oils, generating models that can be interpreted in sensory terms.

Mimotopes of the porcine epidemic diarrhea virus (PEDV) spike protein (SP) were screened from a Fv-antibody library, and the neutralizing activity of screened mimotopes was analyzed using plaque assay and docking simulation. The screened Fv-antibody (mimotopes) corresponded to the variable region of the heavy chain of immunoglobulin G, composed of three complementarity-determining regions (CDRs) and four framework regions. The Fv-antibody library was constructed by randomizing the amino acid sequence of CDR3 and expressed on the outer membrane of E. coli using auto-display technology. Monoclonal anti-PEDV SP antibody was used as probes to screen Fv-antibodies mimicking PEDV SP from the library. Two screened Fv-antibodies (mimotopes of PEDV SP) with binding affinity to the monoclonal antibody were expressed as soluble proteins, and their binding affinity was estimated using a surface plasmon resonance biosensor. The neutralizing activity of PEDV SP mimotopes to prevent PEDV infection was calculated using a plaque assay based on the cytopathic effect. Additionally, molecular docking simulations were performed to examine the interactions of PEDV SP mimotopes with ACE2 receptor as well as APN which had been considered infection-related receptors for coronavirus.

Meat adulteration poses significant social concerns, including the infringement of consumer rights, potential risks of food allergies, and conflicts with religious dietary practices. Therefore, a pressing need exists for the creation of swift and highly sensitive techniques to verify the authenticity of meat products. A portable and quantitative detection assay for pork identification in food was established by integrating a personal glucose meter (PGM) with a heparin-mediated one-pot RPA/Cas12a (called hRPA/SCas12a-PGM). In the hRPA/SCas12a-PGM assay, the target DNA sequence initiates the heparin-enhanced RPA/Cas12a reaction in a single tube, subsequently activating Cas12a's DNase function. Once activated, Cas12a cleaves sucrase-labeled DNA probes immobilized on the electrode, which are labeled with sucrase. Sucrase-labeled DNA fragments are released into the solution through this cleavage process. Sucrase then catalyzes the conversion of sucrose into glucose, producing a quantifiable signal that is detected by the PGM. The integrated system exhibited a broad dynamic range (10 pg/μL to 100 ng/μL), a low detection limit (10 pg/μL), and strong specificity against non-target samples. The hRPA/SCas12a-PGM assay provides a powerful, field-deployable solution for meat authenticity testing, offering significant potential for application in food safety surveillance and regulatory enforcement.

Examining the changes in microRNA expression in vivo enhances the understanding of their tumor-associated functions and facilitates clinical assays based on their roles as disease markers. However, due to the low abundance and high homology, their detection remains challenging. Herein, we developed an exponential amplification method by combining a symmetric dumbbell-type probe (SDTP) and toehold-initiated hyperbranched rolling circle amplification (HRCA) to obtain high sensitivity and selectivity. The toehold region of the dumbbell-type probe specifically binds to the target, triggering a toehold-mediated strand displacement reaction and thus activating SDTP into a circular structure. In the presence of phi29 DNA polymerase, primer P2, and dNTPs, the HRCA reaction proceeds continuously using the circular structure as a template, enabling exponential amplification of let-7a. When the concentration of let-7a ranges from 1.00 fmol·L^-1 to 1.00 nmol·L^-1, the fluorescence intensity obtained by this method exhibits a linear correlation with the logarithm of let-7a concentration (unit: pmol·L^-1), with a detection limit of 214 amol·L^-1. This method can distinguish homologous miRNAs with single-base mismatches. Serum spiking recovery experiments verified the accuracy of the method in actual serum sample detection; meanwhile, the simplicity was demonstrated by the detection of let-7a in different cell lysates without RNA extraction. Therefore, our work exhibited a simple, sensitive, and specific detection of miRNAs in biological samples, which holds promise as a potential tool for miRNA analysis in clinical testing.

Mass spectrometry imaging (MSI) enables spatial mapping of chemical distributions across complex surfaces, with broad applications spanning materials analysis, life sciences, and clinical diagnostics. However, conventional ionization sources often suffer from limited ionization efficiency, creating a trade-off between detection sensitivity and spatial resolution. Post-ionization technology addresses this constraint by incorporating a secondary ionization step, significantly enhancing ionization yield and establishing itself as a leading approach for advancing mass spectrometry techniques. In this study, we have independently developed a vacuum ultraviolet-laser desorption post-ionization mass spectrometry (VUV-LDPI-MS) system, which significantly enhances detection sensitivity through the implementation of post-ionization technology. By integrating low-photon-energy long-wavelength laser desorption with the soft-ionizing capability of VUV light, this technique enhances the detection of chemical species within biological tissues-including both exogenous drugs and endogenous metabolites. The method achieved a theoretical detection limit of 8.3 pg/spot for methylene blue (MB), while markedly reducing substrate effects and sample background interference. Imaging results revealed that endogenous compounds closely aligned with zebrafish tissue anatomy, whereas the spatial distribution of exogenous MB strongly correlated with optical images. Successful application to zebrafish tissue sections enabled clear visualization of both MB and intrinsic metabolites. VUV-LDPI-MSI offers superior ionization efficiency with minimal sample preparation, presenting a robust alternative to conventional LDI-MS approaches and holding considerable promise for future technological development.

The advancement of microfluidics has enabled a wide range of biochemical and biological applications, such as high-throughput drug testing or point-of-care diagnostics, and has also enabled dielectrophoretic applications. Dielectrophoresis (DEP) is based on the movement of polarizable particles in a non-uniform electric field. Implementing insulator-based dielectrophoresis (iDEP) in microfluidic systems has provided a new dimension for the precise manipulation of biomolecules. However, iDEP has been hampered due to the often cumbersome and expensive microfabrication methods that are required, especially for sub-µm analytes, including biomolecules, since extremely large electric field gradients are needed to achieve successful iDEP manipulation. In recent years, 3D printing has drawn attention in microfluidics, alleviating several issues with cleanroom-based fabrication methods. Among the 3D printing repertoire, two-photon polymerization (2PP) is a novel 3D printing technique that offers unique capabilities with unprecedented resolution compared to standard stereolithography. Here, we report the first iDEP-based manipulation of biomolecules, namely, λ-DNA and Phycocyanin, within a completely 3D-printed microfluidic device realized with 2PP printing. iDEP microfluidic devices with different post geometries were 3D-printed and developed with a gap resolution down to 2 µm using the IP-S photoresist. Furthermore, sub-micrometer spatial resolution was achieved down to 800 nm using the IP-Dip photoresist. Additionally, a numerical model was developed to determine the electric field gradients, DEP trapping force, and infer the associated polarizability and DEP characteristics of the analytes. This 3D printing technology may offer impactful potential for rapid prototyping of novel iDEP microdevices and the opportunity to explore iDEP for various biomolecular applications in the future.

Globoid cell leukodystrophy (GLD) is a genetic neurodegenerative disease caused by mutations in galactosylceramide β-galactosidase (GALC) that results in the accumulation of the cytotoxic sphingolipid, psychosine. As psychosine is a biomarker specific to GLD, identifying the most afflicted regions of the nervous system can assist in better understanding the disease mechanism. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) and parallel reaction monitoring were utilized to elucidate the spatial distribution of the psychosine analyte and confirm the identity of the ion in a sagittal section of a GALC-deficient mouse brain. The presence of the psychosine was increased in specific anatomical regions of the brain responsible for the bodily functions that are impaired by GLD (cerebellum and brain stem). Several electrospray solvent additives (dopants) have enhanced the detection of various analyte types but with little success in enhancing the detection of sphingolipids. This study investigates the usefulness of ammonium fluoride electrospray doping in the positive ion mode for lipidomic IR-MALDESI MSI analysis.

Stroke is the second most common cause of death in the world and a leading cause of disability. Ischemic stroke is the most common type of stroke (~87%), necessitating research into effective treatments. Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) found in the central nervous system (CNS) that contains labile sulfate groups which, upon loss, leads to inaccurate structural annotations. Variable sulfation patterns have been implicated in several neurological diseases. Additionally, CS-GAG analysis is challenging due to labile sulfate groups and the presence of positional isomers. These isomers must be distinguished to develop effective targeted therapies. Currently, glycan mass spectrometry imaging (MSI) lacks soft ionization sources which impedes intact analysis of the labile sulfate modifications. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is a soft ambient ionization technique capable of preserving labile species without chemical derivatization. In this work, IR-MALDESI with parallel reaction monitoring (PRM) was used to energetically resolve and characterize intact mono-sulfated CS-GAG positional isomers in healthy and ischemic stroke brain. Our results revealed that both positional isomers were upregulated in the stroke brain and their relative abundance remained constant across the tissue.