Antibody glycan quality predicted from CHO cell culture media markers and machine learning.

N-glycosylation can have a profound effect on the quality of mAb therapeutics. In biomanufacturing, one of the ways to influence N-glycosylation patterns is by altering the media used to grow mAb cell expression systems. Here, we explore the potential of machine learning (ML) to forecast the abundances of N-glycan types based on variables related to the growth media. The ML models exploit a dataset consisting of detailed glycomic characterisation of Anti-HER fed-batch bioreactor cell cultures measured daily under 12 different culture conditions, such as changes in levels of dissolved oxygen, pH, temperature, and the use of two different commercially available media. By performing spent media quantitation and subsequent calculation of pseudo cell consumption rates (termed media markers) as inputs to the ML model, we were able to demonstrate a small subset of media markers (18 selected out of 167 mass spectrometry peaks) in a Chinese Hamster Ovary (CHO) cell cultures are important to model N-glycan relative abundances (Regression - correlations between 0.80-0.92; Classification - AUC between 75.0-97.2). The performances suggest the ML models can infer N-glycan critical quality attributes from extracellular media as a proxy. Given its accuracy, we envisage its potential applications in biomaufactucuring, especially in areas of process development, downstream and upstream bioprocessing.

Methamphetamine detection using portable capillary electrophoresis coupled with a swab-based extraction device.

Methamphetamine (MA) is one of the most virulent illicit drugs that can be synthesized from household materials leading to its prevalent trafficking and local manufacturing in clandestine drug laboratories (clan labs). The significant problems of tracing MA in clan labs and monitoring drug abusers lie in the lag time between sample collection and analysis and the number of tests done. Capillary electrophoresis (CE) is a rapid separation technique amenable to miniaturization and field testing. Herein, we developed a simple transient isotachophoretic (tITP)-CE method to detect MA and its precursor pseudoephedrine (PSE) in clan labs and non-invasive biological fluids. The method was implemented on the ETD-100, a commercial fully automated portable CE instrument with an integrated swab-based extraction system. Within 2 min of insertion of the swab, MA and PSE were automatically extracted with a leading electrolyte (LE) and then separated on covalently modified capillaries. The ETD-100 showed a limit of detection (LOD) and quantification (LOQ) of MA 0.02 and 0.05 µg/swab and 0.02 and 0.06 µg/swab of PSE, with an enhancement factor of 118 and 328, respectively, when compared to a normal non-tITP injection. The intra and inter-day relative standard deviation in terms of migration time were in the range of 0.75-1.93 % for both MA and PSE and were 2.0-2.4 % for both MA and PSE peak height. The method was demonstrated with the detection of spiked MA and PSE on different household materials as well as in non-invasive biological fluids with a recovery above 60 %.

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.

Enantiomeric separation of nefopam and cathinone derivatives using a supramolecular deep eutectic solvent as a chiral selector in capillary electrophoresis.

The present study investigates the utilization of a supramolecular deep eutectic solvent (SUPRADES), consisting of sulfated-ß-cyclodextrin (S-ß-CD) and citric acid (CA), as a chiral selector (CS) in capillary electrophoresis for the enantiomeric separation of nefopam (NEF) and five cathinone derivatives (3-methylmethcathinone [3-MMC], 4-methylmethcathinone [4-MMC], 3,4-dimethylmethcathinone [3,4-DMMC], 4-methylethcathinone [4-MEC], and 3,4-methylendioxycathinone [MDMC]). A significant improvement in enantiomeric separation of the target analytes was observed upon the addition of S-ß-CD-CA to the background electrolyte (BGE), leading to a baseline separation of all analytes. In particular, the optimum percentage of S-ß-CD-CA, added to the BGE, was determined to be 0.075% v/v for NEF (Rs = 1.5) and 0.050% v/v for three out of five cathinone derivatives (Rs = 1.5, 1.6, and 2.4 for 3-MMC, 4-MEC, and 3,4-DMMC, respectively). In the case of 4-MMC and MDMC, a higher percentage of the CS, equal to 0.075% and 0.10% v/v, respectively, was required to achieve baseline separation (Rs = 1.5, 1.9 for MDMC and 4-MMC, respectively). The outcomes of the present study highlight the potential effectiveness of using SUPRADES as a CS in electrophoretic enantioseparations.

Atypical applications of transverse diffusion of laminar flow profiles methodology for in-capillary reactions in capillary electrophoresis.

Capillary electrophoresis (CE) is a powerful separation technique offering quick and efficient analyses in various fields of bioanalytical chemistry. It is characterized by many well-known advantages, but one, which is perhaps the most important for this application field, is somewhat overlooked. It is the possibility to perform chemical and biochemical reactions at the nL scale inside the separation capillary. There are two basic formats applicable for this purpose, heterogeneous and homogeneous. In the former, one reactant is immobilized onto a particle or monolithic support or directly on the capillary wall, and the other is injected. In the latter, the reactant mixing inside a capillary is based on electromigration or diffusion. One of the diffusion-based methodologies, termed Transverse Diffusion of Laminar Flow Profiles, is the subject of this review. Since most studies utilizing in-capillary reactions in CE focus on enzymes, which are being continuously and exhaustively reviewed, this review covers the atypical applications of this methodology, but still in the bioanalytical field. As can be seen from the demonstrated applications, they are not limited to reactions, but can also be utilized for other biochemical systems.

Comparison between capillary electrophoresis and fluorescence anisotropy competitive immunoassay for glucagon.

Glucagon plays a crucial role in regulating glucose homeostasis; unfortunately, the mechanisms controlling its release are still unclear. Capillary electrophoresis (CE)- and fluorescence anisotropy (FA)-immunoassays (IA) have been used for online measurements of hormone secretion on microfluidic platforms, although their use in glucagon assays is less common. We set out to compare a glucagon-competitive IA using these two techniques. Theoretical calibration curves were generated for both CE- and FA-IA and results indicated that CE-IA provided higher sensitivity than FA-IA. These results were confirmed in an experiment where both assays showed limits of detection (LOD) of 30 nM, but the CE-IA had ∼300-fold larger sensitivity from 0 to 200 nM glucagon. However, in online experiments where reagents were mixed within the device, the sensitivity of the CE-IA was reduced ∼3-fold resulting in a higher LOD of 70 nM, whereas the FA-IA remained essentially unchanged. This lowered sensitivity in the online CE-IA was likely due to poor sampling by electroosmotic flow from the high salt solution necessary in online experiments, whereas pressure-based sampling used in FA-IA was not affected. We conclude that FA-IA, despite lowered sensitivity, is more suitable for online mixing scenarios due to the ability to use pressure-driven flow and other practical advantages such as the use of larger channels.

Implementation of Modern Therapeutic Drug Monitoring and Lipidomics Approaches in Clinical Practice: A Case Study with Colistin Treatment.

Nowadays, lipidomics plays a crucial role in the investigation of novel biomarkers of various diseases. Its implementation into the field of clinical analysis led to the identification of specific lipids and/or significant changes in their plasma levels in patients suffering from cancer, Alzheimer's disease, sepsis, and many other diseases and pathological conditions. Profiling of lipids and determination of their plasma concentrations could also be helpful in the case of drug therapy management, especially in combination with therapeutic drug monitoring (TDM). Here, for the first time, a combined approach based on the TDM of colistin, a last-resort antibiotic, and lipidomic profiling is presented in a case study of a critically ill male patient suffering from Pseudomonas aeruginosa-induced pneumonia. Implementation of innovative analytical approaches for TDM (online combination of capillary electrophoresis with tandem mass spectrometry, CZE-MS/MS) and lipidomics (liquid chromatography-tandem mass spectrometry, LC-MS/MS) was demonstrated. The CZE-MS/MS strategy confirmed the chosen colistin drug dosing regimen, leading to stable colistin concentrations in plasma samples. The determined colistin concentrations in plasma samples reached the required minimal inhibitory concentration of 1 µg/mL. The complex lipidomics approach led to monitoring 545 lipids in collected patient plasma samples during and after the therapy. Some changes in specific individual lipids were in good agreement with previous lipidomics studies dealing with sepsis. The presented case study represents a good starting point for identifying particular individual lipids that could correlate with antimicrobial and inflammation therapeutic management.

Development of automated proteomic workflows utilizing silicon-based coupling agents.

Automated methods for enzyme immobilization via 4-triethoxysilylbutyraldehyde (TESB) derived silicone-based coupling agents were developed. TESB and its oxidized derivative, 4-triethoxysilylbutanoic acid (TESBA), were determined to be the most effective. The resulting immobilized enzyme particles (IEPs) displayed robustness, rapid digestion, and immobilization efficiency of 51 ± 8%. Furthermore, we automated the IEP procedure, allowing for multiple enzymes, and/or coupling agents to be fabricated at once, in a fraction of the time via an Agilent Bravo. The automated trypsin TESB and TESBA IEPs were shown to rival a classical in-gel digestion method. Moreover, pepsin IEPs favored cleavage at leucine (>50%) over aromatic and methionine residues. The IEP method was then adapted for an in-situ immobilized enzyme microreactor (IMER) fabrication. We determined that TESBA could functionalize the silica capillary's inner wall while simultaneously acting as an enzyme coupler. The IMER digestion of bovine serum albumin (BSA), mirroring IEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as little as 15 min. Overall, our findings underscore the potential of both IEP and IMER methods, paving the way for automated analysis and a reduction in enzyme waste through reuse, thereby contributing to a more cost-effective and timely study of the proteome. SIGNIFICANCE: This research introduces 4-triethoxysilylbutyraldehyde (TESB) and its derivatives as silicon-based enzyme coupling agents and an automated liquid handling method for bottom-up proteomics (BUP) while streamlining sample preparation for high-throughput processing. Additionally, immobilized enzyme particle (IEP) fabrication and digestion within the 96-well plate allows for flexibility in protocol where different enzyme-coupler combinations can be employed simultaneously. By enabling the digestion of entire microplates and reducing manual labor, the proposed method enhances reproducibility and offers a more efficient alternative to classical in-gel techniques. Furthermore, pepsin IEPs were noted to favor cleavage at leucine residues which represents an interesting finding when compared to the literature that warrants further study. The capability of immobilized enzyme microreactors (IMER) for rapid digestion (in as little as 15 min) demonstrated the system's efficiency and potential for rapid proteomic analysis. This advancement in BUP not only improves efficiency, but also opens avenues for a fully automated, mass spectrometry-integrated proteomics workflow, promising to expedite research and discoveries in complex biological studies.

Phaseolus coccineus L. Landraces in Greece: Microsatellite Genotyping and Molecular Characterization for Landrace Authenticity and Discrimination.

Phaseolus coccineus L. is a highly valuable crop for human consumption with a high protein content and other associated health benefits. Herein, 14 P. coccineus L. landraces were selected for genetic characterization: two Protected Geographical Indication (PGI) landraces from the Prespon area, namely "Gigantes" ("G") and "Elephantes" ("E"), and 12 additional landraces from the Greek Gene Bank collection of beans (PC1-PC12). The genetic diversity among these landraces was assessed using capillary electrophoresis utilizing fluorescence-labeled Simple Sequence Repeat (SSR) and Expressed Sequence Tag (EST); Simple Sequence Repeat (SSR) is a molecular marker technology. The "G" and "E" Prespon landraces were clearly distinguished among them, as well as from the PC1 to PC12 landraces, indicating the unique genetic identity of the Prespon beans. Overall, the genetic characterization of the abundant Greek bean germplasm using molecular markers can aid in the genetic identification of "G" and "E" Prespon beans, thus preventing any form of fraudulent practices as well as supporting traceability management strategies for the identification of authenticity, and protection of the origin of local certified products.

Identification of Novel Hb Guiyang [HBA2: c.151C > A α2 50 (CE8) His- Asn] and Phenotype- Genotype Correlation of Abnormal Hemoglobins in Guizhou, Southwest China.

Purpose: To analyze the composition of abnormal hemoglobin and the relationship between genotype and phenotype by screening abnormal hemoglobin in a subpopulation of Guizhou, China. Patients and Methods: Routine blood evaluation, capillary electrophoresis of hemoglobin, and mutation of α - and ß - thalassemia genes were evaluated in 19,976 individuals for thalassemia screening in Guizhou. Sanger sequencing of HBA1, HBA2 and HBB genes was performed in samples with abnormal bands or unexplained increases of normal bands. The types of abnormal hemoglobin were obtained by sequence analysis. Results: Abnormal hemoglobin was detected in 84 individuals (detection rate, 0.42%). Ten types each of α and ß globin chain variants were detected, including most commonly Hb E, Hb New York and Hb Port Phillip. In this study, the abnormal Hb Mizuho was identified for the first time in a Chinese population, and a novel abnormal hemoglobin Hb Guiyang (HBA2: c.151C > A) was detected for the first time. Except for Hb Mizuho, other abnormal hemoglobin heterozygotes without thalassemia or iron deficiency had no significant hematological changes. Conclusion: This study enriched the molecular epidemiological data of abnormal hemoglobin in Guizhou, China and provided reference data for genetic counseling and prenatal diagnosis of abnormal hemoglobin.

Rational Design of Highly Selective Sialyllactose-Imprinted Nanogels.

We describe a facile method to prepare water-compatible molecularly imprinted polymer nanogels (MIP NGs) as synthetic antibodies against target glycans. Three different phenylboronic acid (PBA) derivatives were explored as monomers for the synthesis of MIP NGs targeting either α2,6- or α2,3-sialyllactose, taken as oversimplified models of cancer-related sT and sTn antigens. Starting from commercially available 3-acrylamidophenylboronic acid, also its 2-substituted isomer and the 5-acrylamido-2-hydroxymethyl cyclic PBA monoester derivative were initially evaluated by NMR studies. Then, a small library of MIP NGs imprinted with the α2,6-linked template was synthesized and tested by mobility shift Affinity Capillary Electrophoresis (msACE) to rapidly assess an affinity ranking. Finally, the best monomer o-acrylamido PBA was selected for the synthesis of polymers targeting both sialyllactoses. The resulting MIP NGs display an affinity constant ≈ 106 M-1 and selectivity towards imprinted glycans. This general procedure could be applied to any non-modified carbohydrate template possessing a reducing end.

Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA. METHODS: A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probe amplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing. RESULTS: A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. No difference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations. CONCLUSIONS: The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce the time frame for diagnosis, facilitating early intervention and preventing birth defects.

Selection of structure-induced aptamer targeting small molecule based on capillary sieving electrophoresis.

In this study, a structure-induced aptamer targeting small molecules was selected using capillary sieving electrophoresis (CSE). CSE was conducted using a capillary filled with a background solution containing hydroxypropyl cellulose as a sieving matrix to separate the aptamer candidates by changing their structures via complexation. Before aptamer selection, the original random-sequence DNA library was used to create structure-not-preorganized DNA sub-library containing straight-chain-like structures using CSE. Next, a structure-induced aptamer targeting L-tyrosinamide was selected from the prepared sub-library. Six aptamer candidates were selected, one of which showed a binding ability comparable to that of the reported L-tyrosinamide aptamer and selectivity toward the analogs. These results indicated that the proposed method can be applied to select structure-induced aptamers that target small molecules.

Comparison of aqueous and non-aqueous capillary electrophoresis for the determination of four benzalkonium chloride homologues in compound chemical disinfectants.

A new method was established for the simultaneous analysis of four homologous benzalkonium chlorides (dodecyldimethylbenzyl ammonium chloride, tetradecyldimethylbenzyl ammonium chloride, hexadecyldimethylbenzyl ammonium chloride, and octadecyldimethylbenzyl ammonium chloride) in compound chemical disinfectants using non-aqueous capillary electrophoresis (CE) based on a micellar electrokinetic chromatography mode with direct ultraviolet detection. The separation was performed on an uncoated fused quartz capillary with a total length of 60.2 cm and a diameter of 25 µm. The separation buffer consisted of a mixture of methanol/acetonitrile (60:40, v/v) containing 70 mmol/L sodium acetate, 60 mmol/L trifluoroacetic acid and 20 mmol/L sodium dodecyl sulfate. The sample buffer was a methanol solution containing only 2 mmol/L trifluoroacetic acid. The separation voltage was set at 8 kV with a working current of approximately 2.3 µA. The detection wavelength was 214 nm. Under optimal conditions, the limit of detection and limit of quantification for these four benzalkonium chlorides (BACs) were 1.0 mg/L and 5.0 mg/L, respectively. Good linearities were observed in the concentration ranges from 5.0 to 100.0 mg/L, with correlation coefficients above 0.999 for all compounds. The recoveries of these four BACs ranged from 92.5 % to 109.1 % with relative standard deviations below 4.7 %. With the new method, all four BACs could be analyzed in a single injection. In contrast, the aqueous CE method in the National Standard GB/T 26369-2020 only allowed for the simultaneous analysis of the first three homologous. The new method demonstrated the improved peak shape compared to the aqueous CE method and then was successfully applied to the analysis of 19 commercially available samples, such as object table disinfectants, hand sanitizers, and disinfectant wipes, which claimed to contain quaternary ammonium compound. The results obtained using the new method were compared with those of the aqueous CE of the National Standard Method, and no statistically significant differences were observed. The new method is simple in pre-treatment and provides accurate results, making it highly suitable for routine analysis.

Development, synthesis, and application of magnetic layered double hydroxides (Fe3O4@SiO-LDH/DS-) as an efficient adsorbent for the removal of tetracyclines from milk samples.

This work presents the development, synthesis, and application of a layered double hydroxide (LDH) coupled to magnetic particles for the removal of antibiotics as tetracyclines (TC´s): tetracycline (TC), chlortetracycline (CT), oxytetracycline (OT), and doxycycline (DT) from milk samples. The LDH synthesis conditions, reaction time (30-90 min), molar ratios Mg2+/Al3+ (7:1-1:7), interlayer anion (NO3-, Cl-, CO32-, and dodecyl sulphate (DS-)) were evaluated. Under synthesis conditions (reaction time of 30 min, Mg2+/Al3+ molar ratio of 7:1, and DS- as interlayer anion), the LDH was coupled in a magnetic solid phase microextraction (MSPµE) methodology. At the optimal extraction conditions (pH 6, 5 min of contact time, 10 mg of adsorbent), a removal percentage of 99.0 % was obtained for each tetracycline. FTIR, TGA, SEM, and adsorption isotherms were employed to characterize the optimal adsorbent. Each experiment was corroborated by large-volume sample stacking capillary electrophoresis (LVSS-CE). The adsorbent was applied directly to positive milk samples (previously tested) for TC´s removal.

Analytical quality by design-based development of a capillary electrophoresis method for Omeprazole impurity profiling.

Omeprazole (OME) is a proton pump inhibitor used to treat gastroesophageal reflux disease associated conditions. The current study presents an Analytical Quality by Design-based approach for the development of a CE method for OME impurity profiling. The scouting experiments suggested the selection of solvent modified Micellar ElectroKinetic Chromatography operative mode using a pseudostationary phase composed of sodium dodecyl sulfate (SDS) micelles and n-butanol as organic modifier in borate buffer. A symmetric three-level screening matrix 37//16 was used to evaluate the effect of Critical Method Parameters, including Background Electrolyte composition and instrumental settings, on Critical Method Attributes (critical resolution values, OME peak width and analysis time). The analytical procedure was optimized using Response Surface Methodology through a Central Composite Orthogonal Design. Risk of failure maps made it possible to define the Method Operable Design Region, within which the following optimized conditions were selected: 72 mM borate buffer pH 10.0, 96 mM SDS, 1.45 %v/v n-butanol, capillary temperature 21 °C, applied voltage 25 kV. The method was validated according to ICH guidelines and robustness was evaluated using a Plackett-Burman design. The developed procedure enables the simultaneous determination of OME and seven related impurities, and has been successfully applied to the analysis of pharmaceutical formulations.

[Progress of highly reproducible capillary electrophoresis].

Following rapid developments in capillary electrophoresis (CE), this technology has become an established analytical technique owing to its microscale characteristics, high speed, high efficiency, and versatility. However, the challenges of poor peak stability and/or reproducibility have consistently hindered its wider applications. CE has long been used as a measurement tool for plotting signal intensities versus the migration time; however, the migration time is not an independent variable in CE, but is affected by many direct and indirect parameters, including capillary (length, diameter, and inner surface properties), electric field (or voltage, current, and/or power), temperature, and running buffer (electrolytes, additives, solvents, and their concentration, buffering pH, etc.). These intricacies render the acquisition of reproducible electropherograms difficult. Various studies ranging from those on the early stages of CE development to those on the exploration of three important strategies have been conducted to address this issue. In the first strategy, the CE conditions, especially those parameters that can maintain a stable electro-osmotic flow, are strictly controlled and stabilized to significantly improve peak repeatability. In the second strategy, either the peak position is corrected using internal standards or the peak time is converted into other variables, such as electrophoretic mobility, to offset or eliminate some unstable factors, thereby improving the repeatability and even reproducibility of the peaks; this strategy is useful when plotting signals versus the migration time ratio, correlated migration time, effective mobility, or temperature-correlated mobility. In the third strategy, a new methodology called highly reproducible CE (HRCE) is established using theoretical studies to explore better principles for real-time CE with the aim of the complete removal of the challenge from the root. This strategy includes the development of novel methods that plot electropherograms based on weighted mobility, migrated charge, charge density, or partial differential molar charge density. Similar to ordinary CE approaches, this strategy can also draw electropherograms based on the ratios of these properties. As theoretically predicted, these novel methods can offset or resist changes in critical CE conditions (mainly electric field strength, capillary length and diameter, and/or some buffer parameters such as concentration). Our experimental results demonstrate that given certain prerequisites, a new set of methods can produce highly reproducible electropherograms. This review focuses on the theoretical basis and advancements of HRCE, and elucidates the link between electrophoretic migration/peak expression theories and their impact on reproducibility. Studies on the transformation of time-scale electropherograms in the CE literature are summarized and analyzed in general. However, this review does not directly discuss research on and progress in improving CE repeatability or reproducibility through instrument upgrades, parameter optimization, or practical method refinements.

[Highly sensitive detection of fluorescent whitening agents in flour using sheathless capillary electrophoresis-electrospray ionization-tandem mass spectrometry].

Fluorescent whitening agents (FWAs) are dyes that emit visible blue or blue-purple fluorescence upon ultraviolet-light absorption. Taking advantage of light complementarity, FWAs can compensate for the yellow color of many substances to achieve a whitening effect; thus, they are used extensively in various applications. FWAs are generally stable, but their presence in the environment can lead to pollution and accumulation in the body through the food chain. Recent studies have revealed that some types of FWAs, such as coumarin-based FWAs, may exhibit photo-induced mutagenic effects that can trigger allergic reactions in humans and even pose carcinogenic risks. Hence, the development of an accurate and highly sensitive method for detecting FWAs in food-related samples is a crucial endeavor. Owing to the high polarity and structural similarity of FWAs, the accurate determination of these substances in complex food samples requires an analytical method that offers both efficient separation and sensitive detection. Capillary electrophoresis (CE) exhibits essential features such as high separation efficiency, short analysis times, very small sample injection requirements, minimal use of organic solvents, and simple operation. Thus, it is often used as an effective alternative to liquid chromatographic techniques. Over the past few decades, electrospray ionization mass spectrometry (ESI-MS) has been utilized as a highly sensitive and accurate detection method in numerous chemical analytical fields because it enables the analysis of molecular structures. By combining the high separation efficiency of CE with the high sensitivity of ESI-MS, a powerful tool for identifying and quantifying trace amounts of FWAs in food samples may be obtained. In this study, we present a method based on sheathless CE coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the simultaneous detection of six trace FWAs in flour. In the proposed method, the CE separation device is directly coupled to the mass spectrometer through a sheathless interface without the need for a sheath liquid for electric contact, thereby avoiding the dilution of the analytes and improving detection sensitivity. Various conditions that could affect extraction recovery, separation efficiency, and detection sensitivity were evaluated and optimized. The FWAs were effectively extracted from the sample matrix with reduced matrix effects by ultrasonic-assisted extraction at a temperature of 30 ℃ for 20 min using CHCl3-MeOH (3∶2, v/v) as the extraction solvent. The extract was centrifuged, dried under N2, and reconstituted in CHCl3-MeOH (1∶4, v/v) for subsequent analysis. During the detection process, the CE device was coupled to the ESI-MS/MS instrument via a highly sensitive porous spray needle, which served as the sheathless electrospray interface. The target FWAs were scanned in positive-ion mode (ESI+) to ensure the stability and intensity of the obtained signals. Additionally, multiple-reaction monitoring (MRM) mode and MS/MS analysis were used to simultaneously quantify the six targets with high selectivity. The developed sheathless CE-ESI-MS/MS method detected the FWAs with high sensitivity over wide linear ranges with low method limits of detection (0.04-0.67 ng/g). The recoveries of the six target FWAs at three spiked levels were between 77.5% and 97.2%, with good interday (RSD≤11.5%) and intraday (RSD≤10.2%) precision. Analyses of the six target FWAs in eight commercial flour samples were performed using this method, and four positive samples were identified. These results demonstrate that the proposed CE-ESI-MS/MS method is a promising strategy for the determination of trace FWAs in complex food sample matrices with efficient separation and high sensitivity.

Investigation of on-line electrokinetic enrichment strategies for capillary electrophoresis of extracellular vesicles.

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 108 EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 1010 EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.