Analytical and Bioanalytical Chemistry最新文献

Dalbavancin, unlike other peptides administered through the IV route, is highly influenced by subtle environmental shifts. These shifts may alter the primary structure and trigger immunogenic reactions leading to reduced efficacy and safety. Hence, there is a critical need to investigate the factors influencing dalbavancin. In this respect, the behavior of dalbavancin under heat and light was studied. Eight degradation products were identified after the thermal and photolytic exposure of dalbavancin. Using tandem mass spectrometric studies (MSⁿ), the degradation products and mechanistic pathways for the construction of these degradation products were identified. The formation of four isomeric degradation products was especially identified under thermal stress conditions. Further elucidation and characterization of isomeric degradation products were processed through 3D energy minimization approaches. Glycan and the modified N-terminus were translated as plausible isomeric soft spots altering the primary structure of dalbavancin. In photolytic conditions under visible light, four degradation products were identified, where epoxidation and hydroxylation were identified as the major mechanistic pathways. Especially under UV light, dalbavancin was adsorbed on the surfaces of the storage containers. Through FE-SEM investigations, the phenomenon of self-assembly for the adsorbed particles was identified. Further EDAX studies confirmed the surface interaction where chlorine, a key distinguishing element of dalbavancin, was identified on the surfaces exposed to UV light. These results collectively suggest dalbavancin is highly vulnerable to environmental conditions altering its primary structure and emphasizing the need for proper storage conditions during handling, usage, and the development of similar therapeutics.

The abnormally high expression of glutathione (GSH) in hepatocellular carcinoma (HCC) cells significantly contributes to chemotherapy resistance by neutralizing reactive oxygen species (ROS). To address this problem, we developed a nanocomposite probe (micelle@PDA@MnO₂ NPs) which was composed of free radical-labeled micelles as the core, polydopamine (PDA) as the inner shell and MnO₂ nanosheets as the outer shell. In the HCC microenvironment, the over-expressed GSH triggered the decomposition of the MnO₂ shell, releasing Mn²⁺ ions, and subsequently, the acidic condition and over-expressed carboxylesterase 2 (CES2) worked together to mediate the controlled release of nitroxide free radicals (i.e., TEMPONE) from the micelle core. With the help of dual-signal electron spin resonance (ESR) detection, it was found that much more free radicals and Mn²⁺ were released in hepatoma cells than in normal liver cells, rendering the proposed nanotherapeutic platform specific to HCC and a good candidate for free radical therapy and chemodynamic therapy (CDT). Moreover, the therapy featured an intelligent self-amplified property: the abundant GSH in the tumor microenvironment rapidly activated the decomposition of the nanocomposite and triggered the release of free radical TEMPONE and Mn²⁺, and TEMPONE and Mn²⁺ further amplified the oxidative stress to induce tumor apoptosis via ROS generation and GSH depletion. Finally, in combination with three therapy modes (Mn²⁺-mediated CDT, the oxidative stress enhancement effect of TEMPONE, and PDA-based photothermal therapy), significant therapeutic effects including facilitation of HepG2 cell apoptosis and inhibition of tumor growth in the HCC mouse model were achieved.

The thiopurines 6-mercaptopurine and 6-thioguanine (TG) are analogs of guanine and are used in the treatment of hematological malignancies and immune-mediated inflammatory diseases. The mechanism of action includes the incorporation of TG into DNA (DNA-TG) in competition with natural guanine. DNA-TG can undergo S⁶-methylation (DNA-MeTG), and O⁶-methylguanine methyltransferase can remove alkyl groups from O⁶-alkylguanine, but is less effective with DNA-MeTG, which favors S⁶-MeTG·T mismatching. If unrecognized by the post-replicative mismatch repair system (MMR), this can lead to an increased mutational load with the formation of neoepitopes, which can potentially increase tumor cell recognition by the immune system. The exact role of DNA-MeTG in the antileukemic efficacy of thiopurines remains to be elucidated. In this work, an LC-MS/MS method for the quantitation of 6-thio-2'-deoxyguanosine (dTG) and its methylated metabolite 6-methylthio-2'-deoxyguanosine (dMeTG) in genomic DNA using enzymatically synthesized isotope-labeled internal standards 6-thio-2'-deoxyguanosine-¹³C₂¹⁵N and 6-methylthio-2'-deoxyguanosine-D₃ is presented. Purified DNA was enzymatically digested into nucleosides, from which dMeTG and dTG could be quantified with LC-MS/MS and normalized to the amount of 2'-deoxyguanosine in the DNA. The applicability of the method was demonstrated in Jurkat cells treated with TG, where dMeTG and dTG could be quantified in samples containing less than 2 µg DNA. dMeTG could also be detected in patient samples, although in low amounts and primarily in samples with high DNA-TG levels. The developed method for the quantitation of dMeTG and dTG can be used in further studies to investigate the role of DNA-MeTG in the mechanism of action of thiopurines, including its antileukemic efficacy and effects on acquired mutations.

Multiomics approaches enable a comprehensive characterization of complex biological systems by simultaneously investigating multiple molecular layers. Generating multiple omics datasets from a single sample is crucial to minimize biological variability and ensure cross-layer consistency, which is critical for robust downstream data analysis. However, existing workflows often require adaptation to the specific experimental context and instrumental setup. This study systematically compared two established protocols for the simultaneous extraction of metabolites, lipids, and proteins from HepG2 cells: (i) a biphasic extraction with subsequent overnight protein digestion from the interphase pellet, and (ii) a monophasic extraction involving on-bead protein digestion. For the monophasic approach, we further investigated the effects of bead size and digestion conditions. Metabolomics samples were analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry; lipidomics and proteomics samples were analyzed by nano-scale liquid chromatography coupled with ion mobility separation and high-resolution tandem mass spectrometry. Each method was evaluated in terms of total feature count, selectivity, reproducibility, handling complexity, and overall performance. While neither protocol was optimal across all criteria, the monophasic extraction using paramagnetic beads with shortened incubation time proved to be the most reproducible, efficient, and cost-effective solution for in-house multiomics workflows in HepG2 cells.

Biogenic amines are low-molecular-weight nitrogenous compounds naturally present in various foods, whose accurate quantification is essential for quality control and safety monitoring. This study presents a novel and sustainable method for the determination of biogenic amines (BAs) in food samples, particularly vinegar, by integrating molecular modeling with experimental optimization. Molecular docking was used to evaluate the interactions between biogenic amines and both native cyclodextrin monomers (α-, β-, and γ-CD) as well as their polymers crosslinked with citric acid. These insights guided the rational selection of β-cyclodextrin crosslinked with citric acid (p-β-CDCA) as a promising green adsorbent for BAs extraction. A dispersive solid-phase microextraction (d-SPME) procedure was then developed and optimized based on multivariate methodology, identifying the optimum extraction conditions as 20 mg of adsorbent, 8 min of desorption time, and a sample solution pH of 8.0. Under these conditions, high enrichment factors of 368-380 were obtained. The method demonstrated excellent analytical performance, including recoveries of 91.6-95.8%, LOQs of 0.45-0.62 ng mL^-1, intermediate precision of 7.3-9.5%, and a linear range from LOQ to 500.0 ng mL^-1. The developed protocol was successfully applied to determine tryptamine, histamine, and putrescine in both homemade and commercial vinegar samples. These results highlight the potential of combining in silico modeling and d-SPME to develop green, cost-effective, and highly sensitive analytical methods for ultra-trace analysis in food quality assessment.

Acylcarnitines are important intermediates in fatty acid metabolism, shuttling acyl groups into mitochondria for β-oxidation and energy production. As biomarkers, their concentrations are utilized to diagnose metabolic and cardiovascular diseases, insulin resistance, and neurodegenerative disorders. However, in-depth structural characterization of acylcarnitines is limited by conventional collision-induced dissociation (CID), which yields fragments predominantly from the carnitine headgroup with minimal information regarding the fatty acyl chains. In this case, we employed a novel quadrupole time-of-flight (QToF) mass spectrometer, the Sciex ZenoTOF 8600, with CID and electron-induced dissociation (EID) to carry out in-depth analysis of acylcarnitines detected in National Institute of Standards and Technology (NIST) SRM 1950 reference plasma. With increased sensitivity and reduced accumulation times (95 ms) on the 8600 platform, we were able to confidently annotate 35 acylcarnitines, including isomeric species and functional groups such as hydroxylations and double bonds. EID provided comprehensive structural information, enabling the discrimination of isomers such as valeryl-, isovaleryl-, and 2-methylbutyryl-carnitine (Car 5:0), as well as the positioning of methyl branching via diagnostic fragments. Hydroxylated species, such as Car 16:0;3OH, were confirmed by the presence of diagnostic ions and matched to reference standards by retention time. Moreover, EID enabled the localization of double bonds within unsaturated species (e.g., Car 18:1, Car 18:2) via fragmentation patterns that are indicative of unsaturation positions, following established lipid fragmentation mechanisms. This work demonstrates that EID offers significant advancements for the structural elucidation of acylcarnitines, delivering enhanced sensitivity and deeper insights into isomeric and functional diversity.

The determination of an equilibrium dissociation constant (K_d) alongside the maximum binding capacity (B_max) of protein-ligand interactions is essential in understanding binding affinities and binding capacities which play a major role in the design and development of new therapeutic molecules. Rapid and accurate determination of these biophysical parameters (K_d and B_max) in noncovalent protein-ligand interactions is desirable in the modern drug discovery process. Previously, we demonstrated the detection of noncovalent protein-ligand complexes by infrared-assisted matrix desorption electrospray ionization-mass spectrometry (IR-MALDESI-MS). Here, we report the first determination by IR-MALDESI-MS of biophysical parameters from noncovalent protein-ligand interactions using a ligand titration approach. Unlike conventional electrospray ionization-mass spectrometry (ESI-MS), IR-MALDESI-MS enables analysis in < 13 s per ligand concentration emphasizing its speed, automation, and sensitivity. Native mass spectrometry by IR-MALDESI was performed on carbonic anhydrase II (CAH) incubated with sulfanilamide (SLFA), a known inhibitor. Coupled with other published studies, these results demonstrate that IR-MALDESI has strong potential as a high-throughput screening (HTS) technique for rapidly and accurately determining the K_d and B_max of protein-ligand complexes.

A novel bioanalytical method combining surfactant-enhanced emulsification liquid-liquid microextraction (SE-LLME) with sweeping micellar electrokinetic chromatography-tandem mass spectrometry (MEKC-MS/MS) was developed and validated for therapeutic drug monitoring (TDM) of alpelisib (ALP) and fulvestrant (FUL) in human plasma. This method addresses the need for sensitive, selective quantification in patients with PIK3CA-mutated, HR+/HER2- breast cancer. Sample preparation involved protein precipitation followed by SE-LLME using pentadecafluorooctanoic acid (PFOA) and chloroform, yielding recoveries greater than 88.4% for ALP and 78.7% for FUL. Optimised MEKC conditions were 50 mM ammonium pentadecafluorooctanoate at pH 9.75 with 25% methanol, 30 kV separation voltage, 30 °C capillary temperature, and 100 mbar additional pressure during the analysis. Sweeping preconcentration significantly enhanced sensitivity-109-fold for ALP and 11.2-fold for FUL. The method was validated per ICH guidelines, demonstrating excellent linearity ( $r\ge$ 0.9963) across the calibration ranges (200-2000 ng/mL for ALP, 10-100 ng/mL for FUL), accuracy (mean biases -10.3% to 7.5%), and precision (RSD < 12.6%). Despite notable matrix effects for ALP, consistency across six different plasma sources (RSD ≤ 12.7%) ensured reliability. Analytes were stable under benchtop, autosampler, freeze-thaw, and long-term conditions (bias ≤ 11.1%). No carry-over was detected, and dilution integrity was confirmed (bias ≤ 2.7%). Application to patient samples validated the method's clinical relevance, with measured concentrations aligning with the expected ones. This is the first capillary electrophoresis method for ALP in biological matrices and the only method for simultaneous TDM of ALP and FUL, offering a robust, cost-effective, and eco-friendly alternative to traditional chromatographic approaches.

Herein , an electrochemical/photoelectrochemical (EC/PEC) dual-mode sensing platform based on carbon dots/liquid exfoliated graphene (CDs/LEG) nanocomposites was developed in this work. The CDs/LEG heterogeneous effectively exploited the excellent photoelectric activity of CDs and the high conductivity of LEG. Ferrocene-labelled aptamer (Fc-Apt) and methylene blue-labelled complementary DNA (MB-cDNA) were introduced as dual-signal probes. Upon aflatoxin B1 (AFB₁) binding, it competitively displaced MB-cDNA, restoring Fc-Apt hairpin conformation and causing MB to move away from the electrode surface while Fc approached it. This conformational change significantly increased the I_Fc to I_MB peak current ratio (I_Fc/I_MB), enhancing the EC signals; meanwhile, the aptamer complex immobilized on the electrode surface reduced the PEC signals through spatial site-blocking and light-shielding effects. The dual-mode responded synergistically to form a unique self-validation mechanism, which showed excellent linearity over the AFB₁ concentration range of 0.01-100 ng/mL, with detection limits (LOD) of 1.356 pg/mL (EC mode) and 0.087 pg/mL (PEC mode).

Human epidermal growth factor receptor 2 (HER2) is a key biomarker associated with breast cancer, and its reliable detection using effective diagnostic tools is clinically significant. Here, we developed an electrochemical aptasensor based on an electroactive labeling strategy for the detection of HER2 using differential pulse voltammetry (DPV). A nickel nanofoam/gold nanoparticles (AuNPs)/silver nanoparticles (AgNPs) nanocomposite was employed as a novel electrode modifier synthesized via a simple, low-cost, and reproducible method. In this design, AuNPs enabled stable aptamer immobilization, while AgNPs acted as electroactive labels, enhancing the current response through a synergistic effect that could not be achieved with either nanoparticle alone. The newly developed aptasensor demonstrated high sensitivity and good reproducibility for HER2 detection. The aptasensor showed a wide linear range of 0.001-100 ng mL^-1, a very low limit of detection (LOD) of 0.30 pg mL^-1, and a limit of quantification (LOQ) of 1.0 pg mL^-1, indicating its significant potential for clinical diagnostic applications.