Analytical and Bioanalytical Chemistry最新文献

The determination of biomarkers is a significant field of analytical chemistry research under continuous evolution that contributes to enhance diagnostics and enable more personalized medicine. Celiac disease is a systemic autoimmune disorder caused by the ingestion of gluten (Glu) proteins found in various cereals. Currently, the only effective way to prevent and manage potential complications is through a strict gluten-free diet (GFD). However, both intentional and unintentional dietary transgressions can occur, often leading to persistent symptoms and ineffective treatment. In this scenario, the development of analytical strategies to detect biomarkers of gluten intake and monitor adherence to a GFD is of significant interest. Herein, we present an analytical strategy based on high-performance thin-layer chromatography (HPTLC) combined with colorimetric detection to estimate 3,5-dihydroxycinnamic acid (3,5-DHCA) as selective biomarker of Glu intake in urine. The approach combined Fast Blue (FB)-doped polydimethylsiloxane (PDMS) membrane with colorimetric HPTLC (RP-C18) giving rise to a selective method to isolate 3,5-DHCA response in urine samples. Detection by visual inspection, image analysis, and spectroscopic response was evaluated and compared. Analytical parameters were estimated showing a good sensitivity (limit of detection (LOD) ≤ 0.8 mg L^-1) and precision, relative standard deviation (RSD) values < 7%. Analysis of urine samples of celiac patients and control patients was performed, and recovery studies showed satisfactory values (R > 80%). The preliminary results indicated correlation between Glu intake and positive 3,5-DHCA responses. This study demonstrated that FB-doped PDMS membranes-HPTLC is a promising tool for detecting dietary transgressions to the GFD by visual inspection, and subsequent quantitative analysis by image analysis and spectroscopic techniques. Hence, the proposed analytical method contributes to the advance of knowledge about celiac disease, which still remains an important challenge to our society.

Internal quality control (IQC) is essential for ensuring the accuracy of results in clinical trials. However, there is a significant shortage of commercial quality control materials for IQC in flow cytometry. This study aimed to develop cost-effective in-house-made whole blood materials from clinically discarded samples to serve as IQC in clinical flow cytometry analysis. Discarded clinical whole blood samples were collected to prepare red blood cell (RBC) suspensions through density centrifugation. White blood cell (WBC) suspensions were prepared using made in-house (MiH) lysing buffer, followed by fixation with the MiH fixative solution. The in-house-made whole blood materials were then prepared by mixing the RBC and WBC suspensions. These mixtures were stored under controlled temperature conditions to ensure long-term stability. These materials are intended for use in internal quality control (IQC) for clinical flow cytometry analysis. The recovery rate of RBC suspensions achieved through density centrifugation was 95%. Different blood type RBC suspensions were effectively preserved in Alsever's solution via plasma washing and re-mixing. The average viability of MiH WBC suspensions was 97%, with a recovery rate of 84%, both significantly higher than those observed with ACK (p < 0.001). Among the in-house-made whole blood material samples, sample a1-which included plasma, Alsever's solution, RBC suspension, WBC suspension, and Proclin 300-exhibited the best stability in flow cytometry, demonstrating stable expression of cell antigens for over 5 months. The in-house-made whole blood materials proved to be cost-effective and suitable for use in IQC for clinical flow cytometry analysis.

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism, and are typically present at low levels in therapeutic monoclonal antibody (mAb) and other recombinant biopharmaceutical products. Firstly, a high-pH-low-pH "two-dimensional" reversed-phase nano-LC-MS/MS label-free quantification (2D nano-LC-MS/MS LFQ) method with a robust stability (CV% < 20%) was developed. Subsequently, this study developed economical hexamer ligand (HWRGWV) magnetic beads (HLMB), which aim to improve the sensitivity and reliability of HCP detection and has an IgG antibody-binding capacity similar to that of Protein A. In turn, the HCP study based on 2D nano-LC-MS/MS, a comparison between the removal and the reservation of the main antibody components was developed, and the qualitative and quantitative results were compared among HLMB/Protein A depletion and other two pretreatment processes. Results of this study indicated that after using HLMB/ProA material for antibody primary component removal, the content of HCPs in the elution buffer (associated or co-purified with the antibody) was significantly higher than that in the flow-through. In total, 22 kinds of HCPs were co-identified in HLMB eluent and ProA eluent, in which most of the HCPs exhibited weak alkalinity, while the sensitivity of HCPs identified with a low molecular weight (ranging from 13 to 21 kDa) was as low as 0.1 ppm. Together, this study established an economical and effective approach to comprehensively evaluate HCPs in antibodies, while also globally presenting the impact of major antibody components on the qualitative and quantitative analyses of HCPs.

Foodborne pathogens, a major cause of foodborne illness due to their high virulence, pose a serious threat to public health. Consequently, identification of foodborne pathogens is essential for the prevention and treatment of foodborne infections. Consequently, there is an immediate need to establish a highly specific and precise approach for the concurrent detection of several foodborne pathogens. Herein, we developed a DNAzyme-based self-protecting dual-response nanoprobe for the simultaneous detection of two foodborne pathogens. The technique utilizes nanostructures to achieve logical signal input and output. In the presence of the target pathogen, the pathogen binds to the arch probe and releases the activation chain, which in turn activates a strand-displacement reaction and DNAzyme for signal amplification, producing different output signals to complete the simultaneous detection of multiple pathogens. The limits of detection for E. coli O157:H7 and S. typhimurium were determined to be 3.7 cfu/mL and 3.2 cfu/mL, with a measurement response time of 2 h. This approach enables ultrasensitive, specific, and simultaneous detection of two foodborne pathogens and is applicable for identifying foodborne pathogens in actual biological samples. The fluorescence detection of foodborne pathogens with a three-way Y-probe and DNAzyme coupling represents a novel approach for the concurrent identification of several foodborne diseases.

The US Environmental Protection Agency (EPA) uses non-targeted analysis (NTA) to characterize potential risks associated with environmental pollutants and anthropogenic materials. NTA is used throughout EPA's Office of Research and Development (ORD) to support the needs of states, tribes, EPA regions, EPA program offices, and other outside partners. NTA methods are complex and conducted via myriad instrumental platforms and software products. Comprehensive standards do not yet exist to guide NTA quality assurance/quality control (QA/QC) procedures. Furthermore, no single software tool meets EPA's needs for QA/QC review and documentation. Considering these factors, ORD developed "INTERPRET NTA" (Interface for Processing, Reviewing, and Translating NTA data) to support liquid chromatography (LC) high-resolution mass spectrometry (HRMS) NTA experiments. For purposes of NTA QA/QC, INTERPRET NTA (1) calculates data quality statistics related to accuracy, precision, and reproducibility; (2) produces interactive visualizations to facilitate quality threshold optimization; and (3) outputs comprehensive documentation for inclusion in official reports and research publications. INTERPRET NTA has additional functionality to facilitate rapid chemical identification and risk-based prioritization. The current article describes only the QA/QC elements of INTERPRET NTA's MS¹ workflow, which are demonstrated using published data from a de facto water reuse study. INTERPRET NTA, in its current form, exists primarily to meet the needs of EPA and its partners, but a public release is planned. Workflows, terminology, and outputs of INTERPRET NTA provide a focal point for necessary discussions on the harmonization of NTA QA/QC practices.

Macrocyclic peptides (MCPs) have remained a compelling modality in drug discovery and development, with many successful marketed drugs. Their unique molecular structure and ADME properties have posed bioanalytical challenges that cannot be fully addressed with conventional small molecule LC-MRM assays. In this work, we developed and optimized a high-throughput discovery bioanalytical strategy for MCPs with 16 marketed MCP drugs. By evaluating ten different sample extraction methods based on the recovery and matrix effect, we identified that the protein precipitation extraction with MeOH/ACN (1/1 v/v) with 0.5% FA outperformed the other sample extraction methods, achieving 80% recovery for 80% of the MCP drugs and 90% matrix effect for 90% of the MCP drugs. By assessing the sensitivity of the targeted-selected ion monitoring (t-SIM) and parallel reaction monitoring (PRM) on the Orbitrap HRMS and comparing with the conventional LC-MRM, we concluded that the t-SIM provided comparable sensitivity with MRM (LOQ at 1~3 ng/mL for the majority of the MCP drugs), with the extra benefits of minimal method development and high post-acquisition flexibility in data processing. The optimized bioanalytical strategy was applied to various biological matrices and displayed performance that met the quantitation requirements for discovery bioanalysis.

Therapeutic drug monitoring for immunosuppressants is a widely conducted global practice. Traditionally, the pretreatment of whole blood involves the use of metal ions combined with organic solvents. However, this method requires multiple reagent additions, repeated opening, closing, and vortexing of vials, and it also leads to heavy metal pollution. Given the typically large sample volumes, optimizing this process is crucial for increasing throughput, reducing the workload of clinical staff, and lowering costs. We discovered that treating whole blood with a 60 to 75% acetonitrile (ACN) solution effectively releases tacrolimus, sirolimus, and cyclosporine A while simultaneously precipitating protein. This allowed us to significantly simplify the pretreatment process to just adding 65% ACN solution containing internal standards, manually shaking for 20 s, and centrifuging for 2 min. The resulted supernatant can then be directly analyzed by mass spectrometry. Method validation demonstrated that the new approach can accurately quantify tacrolimus in the range of 0.64 to 37.5 ng/ml, cyclosporine A at 12 to 976 ng/ml, and sirolimus at 0.99 to 43.4 ng/ml. A comparison of paired samples showed the new method to be perfectly consistent with the classical method, with 293 out of 300 results deviating by no more than ± 20%. This study has greatly simplified the workflow, increased throughput, and resolved environmental concerns for therapeutic drug monitoring of immunosuppressants, including tacrolimus, sirolimus, and cyclosporine A, in whole blood samples. The proposed method is a viable replacement for existing protocols and deserves to be adopted in all clinical laboratories with relevant practical needs globally.

The significance of glycans in various biological processes has been widely acknowledged. Quantitative glycomics is emerging as an important addition to clinical biomarker discovery, as it helps uncover disease-associated glycosylation patterns that are valuable for diagnosis, prognosis, and treatment evaluation. Compared to glycoproteomics and other established omics approaches, quantitative glycomics exhibits greater methodological diversity and it encounters various challenges in automation and standardization. Nonetheless, numerous advancements have been made in this field over the past 5 years. Here, we have reviewed recent progress in analytical methods and software to improve mass spectrometry-based quantitative glycomics primarily on N- and O-glycosylation. The discussion is organized into four sections: stable isotopic labeling, isobaric labeling, label-free, and fluorescence labeling strategies, with a particular emphasis on quantitative data interpretation. Novel derivatization methods and advanced techniques have been developed for high-throughput and highly sensitive glycan quantification with high accuracy. However, due to variations in glycan derivatization and difficulties in structural identification, most glycomic quantification methods are tailored to specific applications, and manual inspection is frequently necessary for precise data interpretation. Therefore, further advancements in glycan sample preparation, structural characterization, and automated data interpretation are essential to facilitate comprehensive and accurate quantification across a wide array of glycans.

This study explores the potential of using liposomal electrokinetic chromatography as a ranking method for the rapid and simultaneous evaluation of drug-membrane interactions of a larger group of substances and assessing their sensitivity to tissue-specific parameters, namely pH, temperature, and lipid composition. We used a group of nine model drug substances to manifest how molecules could be classified for the relative sensitivity of drug-membrane interactions to pH and temperature. We observed that increasing the amount of liposomes in the background electrolyte significantly affected the separation kinetics of various active pharmaceutical ingredients, altering their mobility and/or peak shapes. Experiments with liposomes from bovine liver and heart tissue extracts revealed different interactions based on the lipid composition. Canagliflozin, which initially showed no electrophoretic mobility, migrated toward the anode in the presence of negatively charged liposomes. Mobility of positively charged substances, ambroxol and maraviroc, was suppressed by the interactions with liposomes. Their peaks also exhibited significant tailing. The effect on the separation of negatively charged compounds was significantly weaker. A small change in mobility was observed only in the case of deferasirox. We also examined the effect of temperature during separation, and we observed that increased temperature generally enhanced effective mobility due to lower electrolyte viscosity and increased lipid bilayer fluidity. Lastly, we tested the effect of sodium phosphate buffer pH (ranging from 6.0 to 8.0) with 4% liposomes on drug-liposome interactions. However, the effects were complex due to changes in API ionization and liposome surface charge, complicating the distinction between pH effects and liposome presence on API behavior. Our findings emphasize the significance of liposome composition, temperature, and pH in studying the interactions of liposomes with drugs, which is crucial for optimizing liposome-based drug delivery systems.

Nitroaromatics are a significant concern due to their high explosiveness and potential for water pollution. Optical waveguide sensing technology has been employed in the detection of nitroaromatics, leveraging its advantages of affordability, high sensitivity, reusability, and effective detection results. However, most current optical waveguide sensors operate on the principle of cumulative refractive index change, which necessitates extended detection times. Additionally, although many optical waveguide sensors are reusable, they often require complex and time-consuming post-processing steps for device recovery, and their detection performance significantly degrades after multiple uses, thus limiting their practical applications. In this work, we developed an evanescent field optical waveguide sensor for the detection of nitroaromatics in water, utilizing polymeric optical waveguide materials and D-π-A chromophore molecule. We integrated the sensing molecules into the hydrophobic fluorosilicone resin upper cladding material and employed the evanescent field principle to monitor changes in the optical properties of the surface sensing molecules following their interaction with nitroaromatics. This approach not only prevented contaminant penetration into the sensor, allowing for rapid device recovery, but also facilitated quick quantitative detection. Our sensor demonstrates a detection time of approximately 5 s, a recovery time of about 3 s, and achieves a detection limit of 0.11 ppm, with performance remaining largely intact after several detection cycles.