CE-MS-Based Clinical Metabolomics of Human Plasma.

Capillary electrophoresis coupled to mass spectrometry (CE-MS) has emerged as a powerful analytical technique with significant implications for clinical research and diagnostics. The integration of information from CE and MS strengthens confidence in the identification of compounds present in clinical samples. The ability of CE to separate molecules based on their electrophoretic mobility coupled to MS enables the accurate identification and quantification of analytes, even in complex biological matrices such as human plasma.Here, we present a detailed protocol for an untargeted metabolomics study using CE-MS and its application in a study on human plasma from patients suffering Long COVID syndrome. The protocol ranges from sample preparation to biological interpretation, detailing a workflow enabling the analysis of cationic and anionic compounds, metabolite identification, and data processing.

Electrophoresis-Correlative Data-Independent Acquisition (Eco-DIA) Improves the Sensitivity of Mass Spectrometry for Limited Proteome Amounts.

Capillary zone electrophoresis (CE) combines high separation power, scalability, and speed to limited proteome analyses by mass spectrometry (MS). However, compressed separation in CE challenges the duty cycle of tandem MS, even during data-independent acquisition (DIA). To help remedy this limitation, we introduce the concept of electrophoresis-correlative (Eco) data acquisition for CE-MS. We recognize CE electrospray ionization (ESI) to sort peptide ions into reproducible mass-to-charge (m/z) vs migration time (MT) trends in the solution phase, before subsequent ionization and m/z analysis. We proposed that such a correlation can be leveraged to improve the economy of data acquisition. We test this hypothesis using DIA frames that are tailored to the observed m/z-MT trends. The resulting Eco-DIA method substantially improves the bandwidth utilization of tandem MS during CE-MS. In proof-of-principle studies, Eco-DIA identified and quantified ∼38% more proteins from 1 ng of the HeLa proteome digest compared to the classical DIA, without the assistance of a project-specific tandem MS spectral library. Eco-DIA was able to quantify ∼51% more proteins with <10% coefficient of variation vs the control DIA approach. Based on label-free quantification, the proteins that were exclusively measured by Eco-MS occupied the lower dynamic range of the detected proteome concentration, revealing sensitivity enhancement. In addition to marking the inception of Eco-MS, this work lays the foundation for the development of next-generation data acquisition strategies that leverage electrophoretic ion sorting for high-sensitivity proteomics.

A simple and efficient approach for preparing cationic coating with tunable electroosmotic flow for capillary zone electrophoresis-mass spectrometry-based top-down proteomics.

BACKGROUND: Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has become a valuable analytical technique in top-down proteomics (TDP). CZE-MS/MS-based TDP typically employs separation capillaries with neutral coatings (i.e., linear polyacrylamide, LPA). However, issues related to separation resolution and reproducibility remain with the LPA-coated capillaries due to the unavoidable non-specific protein adsorption onto the capillary wall. Cationic coatings can be critical alternatives to LPA coating for CZE-MS/MS-based TDP due to the electrostatic repulsion between the positively charged capillary inner wall and proteoform molecules in the acidic separation buffer. Unfortunately, there are only very few studies using cationic coating-based CZE-MS/MS for TDP studies. RESULTS: In this work, we aimed to develop a simple and efficient approach for preparing separation capillaries with a cationic coating, i.e., poly (acrylamide-co-(3-acrylamidopropyl) trimethylammonium chloride [PAMAPTAC]) for CZE-MS/MS-based TDP. The PAMAPTAC coating-based CZE-MS produced significantly better separation resolution of proteoforms compared to the traditionally used LPA-coated approach. It achieved reproducible separation and measurement of a simple proteoform mixture and a complex proteome sample (i.e., a yeast cell lysate) regarding migration time, proteoform intensity, and the number of proteoform identifications. The PAMAPTAC coating-based CZE-MS enabled the detection of large proteoforms (≥30 kDa) from the yeast cell lysate reproducibly without any size-based prefractionation. Interestingly, the mobility of proteoforms using the PAMAPTAC coating can be predicted accurately using a simple semi-empirical model. SIGNIFICANCE: The results render the PAMAPTAC coating as a valuable alternative to the LPA coating to advance CZE-MS-based TDP towards high-resolution separation and highly reproducible measurement of proteoforms in complex samples.

Review of Applications of ß-Cyclodextrin as a Chiral Selector for Effective Enantioseparation.

The significance and necessity of separating enantiomers in food, pharmaceuticals, pesticides, and other samples remains constant and unrelenting. The successful chiral separation usually includes the application of a chiral auxiliary compound, known also as a chiral selector (CS), that forms complexes with enantiomers of different physicochemical properties, enabling efficient separation. While both native and substituted cyclodextrins (CDs) are commonly used as CSs, ß-CD is undoubtedly the most popular one among them. This review includes recent advancements in the application of ß-CD as a CS. While the theoretical background behind the enantioseparation is also part of this work, the main emphasis is put on the factors that affect the efficacy of this process such as temperature, pH, solvent, and the choice of other additives. Also, the different analytical methods: Nuclear Magnetic Resonance (NMR) spectroscopy, Capillary Electrophoresis (CE), fluorescence spectroscopy (FS), High-Performance Liquid Chromatography (HPLC), Isothermal Titration Calorimetry (ITC), and UV-vis spectroscopy, used for enantioseparation with the aid of ß-CD as CS, are thoroughly compared. Also, since some of the chiral compounds have been studied in the context of their enantioseparation more than once, those works are compared and critically analyzed. In conclusion, while ß-CD can be in most cases used as CS, the choice of the experimental conditions and method of analysis is crucial to achieve the success.

[Recent advances in capillary electrophoresis-mass spectrometry analysis of |trace biomolecules].

In recent years, various trace bioanalysis methods have been developed, including single-cell transcriptome analysis methods. As the sample volume and amount of biomolecules contained therein are extremely limited, development of new single-cell analysis methods require extremely high-level techniques. It is necessary to design an appropriate analysis system that integrates a highly sensitive detection system and a pretreatment protocol for minimizing sample loss, where separation method is especially important for analyzing diverse mixtures of biomolecules. Among them, capillary electrophoresis (CE) can separate biomolecules in nanoliter-scale solutions with high resolution, making it highly compatible with trace samples such as single cells. By combining with highly sensitive nano-electrospray ionization-mass spectrometry (MS), it is possible to detect nanomolar to sub-nanomolar biomolecules, which can be further improved by using online sample preconcentration methods. These highly sensitive analytical techniques have made it possible to analyze trace amounts of metabolites, proteins, lipids, etc. This review paper summarizes the research on CE-MS trace bioanalysis that has been reported to date, with a focus on single-cell analysis.

Imaged capillary isoelectric focusing method development for charge variants of high DAR ADCs.

BACKGROUND: Charge heterogeneity is a critical quality attribute for therapeutic biologics including antibody-drug conjugates (ADCs). Developing an ion exchange chromatography (IEX) or an imaged capillary isoelectric focusing (icIEF) method for ADCs with high drug-to-antibody ratio (DAR) is challenging because of the increased hydrophobicity from the payload-linker, DAR heterogeneity, and payload-linker instability. A sub-optimal method can be poorly stability-indicating due to the inability to discern contributions from charge and size variants conjugated with different number of drugs/payloads. Systematic strategy and guidance on charge variant method development is highly desired for high DAR ADCs with various complex structures. RESULTS: This work encompasses the development and optimization of icIEF methods for high DAR ADCs of various DAR values (4-8) and payload linker chemistry. Method optimization focuses on improving resolution and stability indicating capabilities and differentiating contributions from the protein and payload-linker. Types, proportion, and combination of solubilizers and carrier ampholytes, as well as focusing parameters were interrogated. Our findings show that the structural units of the linker, the DAR, and the payload chemistry prescribe the selection of buffer, solubilizer, and ampholyte. We demonstrate that a stronger denaturant or solubilizer is needed for high DAR ADCs with polyethylene glycol (PEG)-containing linker structure compared to peptide linker. For unstable payload-linker, buffer system enhances sample stability which is vital to method robustness. In addition, a longer isoelectric focusing time is necessary for an ADC than its corresponding antibody to reach optimal focusing. SIGNIFICANCE: To the best of our knowledge, this is the first comprehensive study on icIEF method development for charge variant determination of high DAR ADCs with unique physicochemical properties.

Simultaneously performing Taylor dispersion analysis with fluorimetry, photometry, and contactless conductometry at the same detection window.

Taylor dispersion analysis (TDA) is a rapid and precise method for determining the hydrodynamic radius (RH) of various substances. We present a versatile TDA system with a flow-through sample injection device, two compact 3-in-1 detectors, and a high-voltage power supply. The 3D-printed detectors combine fluorimetry (FD), photometry (AD@255 nm), and contactless conductometry (C4D) in a single head, enabling simultaneous detection at one capillary window. Using bovine serum albumin (BSA) as a model analyte, we compare TDA with different detection methods. BSA labeled with fluorescein isothiocyanate (FITC) is analyzed in both pulse mode and capillary electrophoresis (CE) TDA. FD and AD detection yield similar RH values, except when FITC binds with small ions in the buffer. In phosphate buffer, C4D underestimates RH values by approximately 18 % due to BSA self-association. In Tris-based buffers, C4D values are 87%-96 % of AD values in pulse mode. With CE-TDA using Tris-CHES buffer, no statistical difference is found across all detections. The system is also applied to CE-TDA of various compounds, particularly charged saccharides. CE-TDA improves the accuracy of TDA results from C4D. We demonstrate the resolution of mixed C4D-TDA signals with assistance from FD and AD signals, successfully resolving gluconate peaks fully covered by another compound. The versatile system with 3-in-1 detection offers a powerful tool for TDA of mixtures and enhances sample throughput.

Using Variable Data-Independent Acquisition for Capillary Electrophoresis-Based Untargeted Metabolomics.

Capillary electrophoresis coupled with tandem mass spectrometry (CE-MS/MS) offers advantages in peak capacity and sensitivity for metabolic profiling owing to the electroosmotic flow-based separation. However, the utilization of data-independent MS/MS acquisition (DIA) is restricted due to the absence of an optimal procedure for analytical chemistry and its related informatics framework. We assessed the mass spectral quality using two DIA techniques, namely, all-ion fragmentation (AIF) and variable DIA (vDIA), to isolate 60-800 Da precursor ions with respect to annotation rates. Our findings indicate that vDIA, coupled with the updated MS-DIAL chromatogram deconvolution algorithm, yields higher spectral matching scores and annotation rates compared to AIF. Additionally, we evaluated a linear migration time (MT) correction method using internal standards to accurately align chromatographic peaks in a data set. Postcorrection, the data set exhibited less than 0.1 min MT drifts, a difference mostly equivalent to that of conventional reverse-phase liquid chromatography techniques. Moreover, we conducted MT prediction for metabolites recorded in mass spectral libraries and metabolite structure databases containing a total of 469,870 compounds, achieving an accuracy of less than 1.5 min root mean squares. Our platform provides a peak annotation platform utilizing MT information, accurate precursor m/z, and the MS/MS spectrum recommended by the metabolomics standards initiative. Applying this procedure, we investigated metabolic alterations in lipopolysaccharide (LPS)-induced macrophages, characterizing 170 metabolites. Furthermore, we assigned metabolite information to unannotated peaks using an in silico structure elucidation tool, MS-FINDER. The results were integrated into the nodes in the molecular spectrum network based on the MS/MS similarity score. Consequently, we identified significantly altered metabolites in the LPS-administration group, where glycinamide ribonucleotide, not present in any spectral libraries, was newly characterized. Additionally, we retrieved metabolites of false-negative hits during the initial spectral annotation procedure. Overall, our study underscores the potential of CE-MS/MS with DIA and computational mass spectrometry techniques for metabolic profiling.

Capillary Electrophoresis-Laser Induced Fluorescence Method Development and Validation for Quantification of Nine Gangliosides-Application to Analysis of Cell Lines of CNS Origin.

Gangliosides are sialic acid-containing glycosphingolipids that play an essential role in many biological and pathophysiological processes. They are present in high amounts in the central nervous system and their abnormal metabolism or expression has been observed in many diseases. We have developed and validated a sensitive capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the separation and quantification of oligosaccharides digested from nine gangliosides of high biological relevance. APTS was used for the labeling of the glycans. Reverse polarity CE was performed for the separation of the labeled glycans bearing negative charges. The optimized background electrolyte is a 15 mM lithium acetate buffer with pH of 5 containing 5% w/v linear polyacrylamide, which allows for the separation of all nine gangliosides. Validation parameters including linearity, precision, and accuracy were evaluated. LOQ and LOD were in the nM range, comparable to those of LC-MS techniques. The method was used to identify and quantify the ganglioside pattern of glioblastoma and neuroblastoma cell lines. The presented method is a valuable tool for further investigations aiming at understanding the role of gangliosides in various neurological diseases or CNS tumors.

Antimalarial analysis of pharmaceutical formulations and biological samples by capillary electrophoresis: the state of the art and applications.

Malaria is a serious public health problem, being an endemic disease in 84 countries, mainly in Africa. This review explores the application of capillary electrophoresis (CE) techniques for analyzing antimalarial drugs, highlighting methods from 2000 to 2023 for the analysis of pharmaceutical formulations and human biological samples. The versatility, selectivity, high efficiency, cost-effectiveness, and high analytical frequency of CE techniques have become attractive choices for pharmaceutical analysis, focusing on quality control and impurity analysis applications. The evolution of achiral and chiral electromigration methods has been described based on the features of each mode of separation: capillary zone electrophoresis (CZE), micellar electrokinetic chromatography, microemulsion electrokinetic chromatography, and capillary electrochromatography. As expected, CZE is reported in most articles owing to its compatibility with drug properties and separation mode. However, it is necessary to perform other separation modes for a few drugs that are present in neutral form. After exhaustive research using different databases and statistical analyses, 27 articles using CE techniques for antimalarial drug analysis were found and are mentioned in this review.

Current green capillary electrophoresis and liquid chromatography methods for analysis of pharmaceutical and biomedical samples (2019-2023) - A review.

Separation analytical methods, including liquid chromatography (LC) and capillary electrophoresis (CE), in combination with an appropriate detection technique, are dominant and powerful approaches preferred in the analysis of pharmaceutical and biomedical samples. Recent trends in analytical methods are focused on activities that push them to the field of greenness and sustainability. New approaches based on the implementation of greener solvents, non-hazardous chemicals, and reagents have grown exponentially. Similarly, recent trends are pushed in to the strategies based on miniaturization, reduction of wastes, avoiding derivatization procedures, or reduction of energy consumption. However, the real greenness of the analytical method can be evaluated only according to an objective and sufficient metric offering complex results taking into account all twelve rules of green analytical chemistry (SIGNIFICANCE mnemonic system). This review provides an extensive overview of papers published in the area of development of green LC and CE methods in the field of pharmaceutical and biomedical analysis over the last 5 years (2019-2023). The main focus is situated on the metrics used for greenness evaluation of the methods applied for the determination of bioactive agents. It critically evaluates and compares the demands of the real applicability of the methods in quality control and clinical environment with the requirements of the green analytical chemistry (GAC). Greenness and practicality of the summarized methods are re-evaluated or newly evaluated with the use of the dominant metrics tools, i.e., Analytical GREEnness (AGREE), Green Analytical Procedure Index (GAPI), Blue Applicability Grade Index (BAGI), and Sample Preparation Metric of Sustainability (SPMS). Moreover, general conclusions and future perspectives of the greening procedures and greenness evaluation metrics systems are presented. This paper should provide comprehensive information to analytical chemists, biochemists, and it can also represent a valuable source of information for clinicians, biomedical or quality control laboratories interested in development of analytical methods based on greenness, practicality, and sustainability.

Development of a CZE-MS/MS method with dynamic pH junction sample pretreatment for analysis of kratom psychoactive alkaloids in urine.

BACKGROUND: Kratom is a herbal substance belonging to the group of new psychoactive substances. It contains psychoactive indole alkaloids mitragynine and 7-hydroxymitragynine. At low doses, they act as psychostimulants and at higher doses they mediate an opioid-like effect. The increasing misuse of kratom requires the development of analytical methods that will accurately and reliably identify and quantify its psychoactive alkaloids in biological samples. Therefore, the development of effective, precise, and reliable green analytical methods that are easy to implement in practice is of great importance. On-line combination of capillary zone electrophoresis with tandem mass spectrometry (CZE-MS/MS) seems to be a promising solution. RESULTS: We present a novel green approach based on capillary zone electrophoresis - tandem mass spectrometry (CZE-MS/MS) method with on-line dynamic pH junction sample pretreatment to identify and determine mitragynine and 7-hydroxymitragynine in urine samples. The separation was performed in a background electrolyte composed of 100 mM formic acid (pH 2.39). The dynamic pH junction was ensured by injection of a short plug of 12.5 % NH4OH before the sample. Under optimal conditions, the developed method was validated and parameters such as linearity (r2 > 0.99), precision (2.2-8.7 %), accuracy (89.2-102.5 %) or stability of the sample (86.6-114.7 %) met the defined FDA guideline criteria (%RSD and %RE values where within ±15 %). Introduction of a simple in-capillary preconcentration strategy based on dynamic pH junction enabled significant improvement in analytical signal intensity and also the applicability of the method. Applying the presented approach, high sensitivity was achieved as indicated by limit of detection values, which were 0.5 ng mL-1 and 2 ng mL-1 for mitragynine and 7-hydroxymitragynine, respectively. Greenness of the proposed approach was confirmed by the AGREE metrics (score 0.63). The application potential of the developed method was successfully verified using blinded urine model samples. SIGNIFICANCE: For the first time a fully validated CZE-MS/MS method for kratom alkaloids determination was introduced. The presented novel method is a cheaper and more ecological alternative to conventionally used chromatographic techniques what was clearly confirmed by its greenness evaluation and comparison with previously published liquid chromatography (LC) approaches. In-capillary sample pretreatment (dynamic pH junction) has been demonstrated to be an effective and fast tool in bioanalysis, minimizing the number of pretreatment steps and the manipulation with the sample. Moreover, LOD values comparable to those obtained by LC methods were recorded. High potential for the implementation of this approach into the toxicology environment in the near future is expected.

Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis.

BACKGROUND: N-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. RESULTS: In this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. SIGNIFICANCE AND NOVELTY: In conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Development of a dispersive solid phase microextraction method based on the application of MgAl, NiAl, and CoAl-layered double hydroxides for the efficient removal of α- and ß-naphthol isomers from water samples by capillary electrophoresis.

The present work describes a quick, simple, and efficient method based on the use of layered double hydroxides (LDH) coupled to dispersive solid phase micro-extraction (DSPME) to remove α-naphthol (α-NAP) and ß-naphthol (ß-NAP) isomers from water samples. Three different LDHs (MgAl-LDH, NiAl-LDH, and CoAl-LDH) were used to study how the interlayer anion and molar ratio affected the removal performance. The critical factors in the DSPME procedure (pH, LDH amount, contact time) were optimized by the univariate method under the optimal conditions: pH, 4-8; LDH amount, 5 mg; and contact time, 2.5 min. The method can be successfully applied in real sample waters, removing NAP isomers even in ultra-trace concentrations. The large volume sample stacking (LVSS-CE) technique provides limits of detections (LODs) of 5.52 µg/L and 6.36 µg/L for α-naphthol and ß-naphthol, respectively. The methodology's precision was evaluated on intra- and inter-day repeatability, with %RSD less than 10% in all cases. The MgAl/Cl--LDH selectivity was tested in the presence of phenol and bisphenol A, with a removal rate of >92.80%. The elution tests suggest that the LDH MgAl/Cl--LDH could be suitable for pre-concentration of α-naphthol and ß-naphthol in future works.

Rapid identification of antibody impurities in size-based electrophoresis via CZE-MS generated spectral library.

Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.

[On-line enrichment and separation of glycoprotein by capillary electrophoresis based on pH response coating column].

A capillary column coated with 3-aminophenylboronic acid (APBA)-functionalized gold nanoparticles (AuNPs@APBA) was prepared via electrostatic self-assembly. The coated column exhibited anti-nonspecific adsorption of glycoproteins, enabling selective online enrichment during capillary electrophoresis (CE). First, gold nanoparticles (AuNPs) were synthesized using the sodium citrate reduction method. Then, APBA was self-assembled electrostatically on the surface of the AuNPs to obtain AuNPs@APBA. This nanomaterial was bonded to the inner wall of a capillary through ion adsorption to produce a AuNPs@APBA-coated capillary column. Glycoproteins were adsorbed via bond formation with boric acid groups under alkaline conditions (pH 8) to generate borate esters. Under acidic conditions (pH 3), the borate esters dissociated to release the glycoproteins, thereby achieving the selective online enrichment and separation of glycoproteins. The AuNPs and AuNPs@APBA were characterized using Fourier transform infrared spectroscopy, and their sizes and Zeta potentials were determined. In addition, the electroosmotic flow (EOF) of the AuNPs@APBA-coated capillary column was measured. The results showed that the surface of the AuNPs was successfully modified with APBA and that AuNPs@APBA was adsorbed on the inner wall of the capillary. The peak area of ovalbumin (OVA) on the AuNPs@APBA-coated column was 26.46 times higher than that on a bare column via conventional electrophoresis. In contrast, the peak area of bovine serum albumin (BSA) only increased by 8.47 times, indicating that the AuNPs@APBA coated column selectively enriched glycoproteins. Evaluation of the reproducibility and stability of this method revealed that the AuNPs@APBA coated capillary column could be used continually for 33-67 h. The relative standard deviations (RSDs) of the peak areas for intra-day (n=5) and inter-day (n=6) analyses were 2.2% and 3.0%, respectively. The developed method was successfully applied to enrich glycoproteins in a 1×106-fold diluted egg white sample. Glycoproteins were not detected using conventional electrophoresis on the bare column, whereas the AuNPs@APBA-coated capillary column effectively enriched and separated glycoproteins, resulting in a peak area of 10469 mAU·ms. Furthermore, the entire enrichment and separation process was completed within 3 min. This new online enrichment and separation method for glycoproteins has the advantages of low sample consumption, simple operation, and high separation efficiency.

Detection of N-desethyl etonitazene in a drug checking sample: Chemical analysis and pharmacological characterization of a recent member of the 2-benzylbenzimidazole "nitazene" class.

The emergence of 2-benzylbenzimidazole "nitazene" opioids is stirring up the recreational synthetic opioid market. Many nitazene analogues act as potent agonists at the µ­opioid receptor (MOR), as demonstrated in various in vitro and in vivo studies. Severe intoxication and overdose deaths associated with nitazene analogues are increasingly being reported. Nitazene opioids are classified as a public health threat, stressing the need for close monitoring of new developments on the recreational drug market. This study reports on the detection of N-desethyl etonitazene in a sample handed in by a recreational drug user at a Swiss drug checking service in August 2023. The person bought the sample through an internet source where it was stated to contain isotonitazene. Chemical analyses were conducted to characterize the sample, i.e. nuclear magnetic resonance (NMR), capillary electrophoresis (CE), and high-resolution mass spectrometry (HRMS). The sample was additionally investigated using two different in vitro MOR activation assays. NMR and high-performance liquid chromatography (HPLC) coupled to HRMS confirmed the presence of N-desethyl etonitazene at a high purity and in the absence of isotonitazene and etonitazene. N-Desethyl nitazene analogues have been detected before as metabolites of isotonitazene and etonitazene. However, as first seen with N-desethyl isotonitazene, they are now emerging as standalone drugs. The applied bioassays demonstrated increased efficacy and approximately 6-9-fold higher potency of N-desethyl etonitazene at MOR compared to fentanyl. N-Desethyl etonitazene showed EC50 values of 3.35 nM and 0.500 nM in the ß-arrestin 2 recruitment and Aequoscreen® assays, respectively. The opioid activity present in the collected sample was additionally evaluated using the bioassays and showed good overlap with the reference standard, in line with the analytical purity assessment. This demonstrates the potential of these bioassays to provide a rapid opioid activity assessment of authentic samples. The emergence of other N-desethyl nitazene analogues must be considered during forensic and clinical toxicology casework, to avoid misclassification of intake of such analogues as metabolites. Finally, drug checking services enable the close monitoring of market developments and trends and are of great value for early warning and harm reduction purposes.

Impact of haemoglobin variants on the diagnostic sensitivity of glycated haemoglobin (HbA1c) assay methodologies in sub-Saharan Africa: a laboratory-based method validation study.

Introduction: the utility of glycated haemoglobin (HbA1c) for the diagnosis and monitoring of diabetes in sub-Saharan Africa is uncertain due to limited data on the performance of the available HbA1c assay methods in this population, which has a high prevalence of haemoglobin variants. We aimed to compare the diagnostic accuracy of the major HbA1c methodologies (Boronate Affinity, Capillary Electrophoresis, High Performance Liquid Chromatography, Immunoassay) in an African population, and assess the impact of the common haemoglobin variant HbAS (sickle cell trait). Methods: whole blood samples were obtained from 182 individuals living with type 2 diabetes in Uganda. HbA1c values for each method were compared to average glucose measured over 14 days by continuous glucose monitoring (CGM). To determine concordance, the three HbA1c assay methods were compared to the capillary electrophoresis method. Results: there was a strong correlation between CGM average glucose levels and all four HbA1c methodologies (r=0.81-0.89) which did not differ in those with and without HbAS (present in 37/182 participants). The presence of HbAS did not alter the relationship between HbA1c and CGM glucose for any assay (p for interaction >0.2 for all methods). Diagnostic accuracy for CGM average glucose thresholds of 7 and 10mmol/L was similar across methods (area under the receiver operating characteristic curve 0.80-0.84 and 0.76-0.84 respectively). The maximum bias between the HbA1c assay methodologies was 2 mmol/mol (2.07%). Conclusion: all major HbA1c technologies offer accurate and comparable HbA1c measurement even in this population with high prevalence of haemoglobin variants.

Enantioseparation of six profenoid drugs by capillary electrophoresis with bovine serum albumin-modified gold nanoparticles as quasi-stationary phases.

Profenoid drugs are a kind of common non-steroidal anti-inflammatory drugs and their chiral enantiomers often have huge differences in pharmacological activities. In this work, a novel chiral separation system by capillary electrophoresis (CE) was constructed using gold nanoparticles (AuNPs) functionalized with bovine serum albumin (BSA) as a quasi-stationary phase (QSP), and the enantioseparation of six profenoid drugs was efficiently accomplished. Under optimal chromatographic conditions, the enantioseparation performance of the AuNP@BSA-based chiral separation system was greatly improved compared with that of free BSA (Resolutions, Ibuprofen: 0.89 â†’ 8.15; Ketoprofen: 0 â†’ 10.02; Flurbiprofen:0.56 â†’ 9.83; Indoprofen: 0.88 â†’ 13.83; Fenoprofen: 0 â†’ 15.21; Pyranoprofen: 0.59 â†’ 5.34). Such high Rs are exciting and satisfying and it is in the leading position in the reported papers. Finally, through molecular docking, it was also found that the difference in binding energy between BSA and enantiomers was closely related to the resolutions of CE systems, revealing the chiral selection mechanism of BSA. This work significantly improves the CE chiral separation performance through a simple strategy, providing a simple and efficient idea for the chiral separation method.

Analysis of nanomaterial biocoronas in biological and environmental surroundings.

A biomolecular coating, or biocorona, forms on the surface of engineered nanomaterials (ENMs) immediately as they enter biological or environmental systems, defining their biological and environmental identity and influencing their fate and performance. This biomolecular layer includes proteins (the protein corona) and other biomolecules, such as nucleic acids and metabolites. To ensure a meaningful and reproducible analysis of the ENMs-associated biocorona, it is essential to streamline procedures for its preparation, separation, identification and characterization, so that studies in different labs can be easily compared, and the information collected can be used to predict the composition, dynamics and properties of biocoronas acquired by other ENMs. Most studies focus on the protein corona as proteins are easier to monitor and characterize than other biomolecules and play crucial roles in receptor engagement and signaling; however, metabolites play equally critical roles in signaling. Here we describe how to reproducibly prepare and characterize biomolecule-coated ENMs, noting especially the steps that need optimization for different types of ENMs. The structure and composition of the biocoronas are characterized using general methods (transmission electron microscopy, dynamic light scattering, capillary electrophoresis-mass spectrometry and liquid chromatography-mass spectrometry) as well as advanced techniques, such as transmission electron cryomicroscopy, synchrotron-based X-ray absorption near edge structure and circular dichroism. We also discuss how to use molecular dynamic simulation to study and predict the interaction between ENMs and biomolecules and the resulting biocorona composition. The application of this protocol can provide mechanistic insights into the formation, composition and evolution of the ENM biocorona, ultimately facilitating the biomedical and agricultural application of ENMs and a better understanding of their impact in the environment.