Rapid Analytical Method Research Articles

Accurate and highly sensitive detection of Alzheimer's disease-related extracellular vesicles via förster resonance energy transfer

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-β (1-42) (Aβ42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aβ42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EVMPFAP assay, could specifically measure the Aβ42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EVMPFAP assay was 1.4 × 103 particles mL−1 and the linear range was from 104 to 108 particles mL−1. EVMPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EVMPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD.

From laboratory to industrial use: Understanding process variation during enzymatic protein hydrolysis with dry film fourier-transform infrared spectroscopy

Industries are continuously looking for dedicated sensor solutions for evaluating the chemical composition of products that can provide valuable insights into process control, process optimization, and product quality. A rapid and robust analytical method, Fourier-Transform Infrared (FTIR) spectroscopy, holds significant potential in this regard as it provides detailed compositional values of food nutrients. The main aim of the present study was to use dry film FTIR spectroscopy as an analytical tool to characterize products from an industrial enzymatic protein hydrolysis process and link this to understand industrial process variations. For this purpose, 463 protein hydrolysate samples were obtained from an industrial enzymatic protein hydrolysis plant. In the same period, systematic variations in process parameters such as raw material composition, enzyme type, and water addition, were performed. All samples were analyzed using dry film FTIR spectroscopy. Two subsets containing 200 and 68 hydrolysate samples were chosen for measuring average molecular weight (AMW) and collagen content respectively, providing reference data for constructing calibration models based on partial least squares regression (PLSR). The percentage of low molecular weight constituents was derived from the molecular weight distribution of size exclusion chromatograms of protein hydrolysates and also used in the modeling. Calibration models for the prediction of AMW, low molecular weight constituents, and collagen content were obtained with a good fit and lower estimation errors. Principal component analysis (PCA) of protein hydrolysates' FTIR spectra effectively differentiated process variations (enzyme types and raw materials) without extensive reference analysis. One-factor analysis of variance (ANOVA) tests was used to observe the impact of process variation on product quality. FTIR proved to be a sensitive method for liquid protein analysis and process control with a significant potential in process optimization approaches. To the authors’ knowledge, this is the first time dry film FTIR spectroscopy has been used to evaluate an industrial bioprocess with designed process variations.

Nile red staining for rapid screening of plastic-suspect particles in edible seafood tissues

Concerns regarding microplastic (MP) contamination in aquatic ecosystems and its impact on seafood require a better understanding of human dietary MP exposure including extensive monitoring. While conventional techniques for MP analysis like infrared or Raman microspectroscopy provide detailed particle information, they are limited by low sample throughput, particularly when dealing with high particle numbers in seafood due to matrix-related residues. Consequently, more rapid techniques need to be developed to meet the requirements of large-scale monitoring. This study focused on semi-automated fluorescence imaging analysis after Nile red staining for rapid MP screening in seafood. By implementing RGB-based fluorescence threshold values, the need for high operator expertise to prevent misclassification was addressed. Food-relevant MP was identified with over 95% probability and differentiated from natural polymers with a 1% error rate. Comparison with laser direct infrared imaging (LDIR), a state-of-the-art method for rapid MP analysis, showed similar particle counts, indicating plausible results. However, highly variable recovery rates attributed to inhomogeneous particle spiking experiments highlight the need for future development of certified reference material including sample preparation. The proposed method demonstrated suitability of high throughput analysis for seafood samples, requiring 0.02–0.06 h/cm2 filter surface compared to 4.5–14.7 h/cm with LDIR analysis. Overall, the method holds promise as a screening tool for more accurate yet resource-intensive MP analysis methods such as spectroscopic or thermoanalytical techniques.Graphical

Selection of texture-associated biomarkers in chilled and iced grass carp (Ctenopharyngodon idella) fillets via DIA-based proteomics

Consumers care about the texture of fresh fish flesh, but a rapid quantitative analytical method for this has not been properly established. In this study, texture-associated biomarkers were selected by DIA-based proteomics for possible future application. Results indicated a significant decline in texture and moisture characteristics with extended storage under chilled and iced conditions, and flesh quality was categorized into three intervals. A total of 8 texture-associated biomarkers were identified in the chilled storage group, and 3 distinct ones in the iced storage group. Biomarkers were further refined based on their expression levels. Isobutyryl-CoA dehydrogenase, mitochondrial and [Phosphatase 2A protein]-leucine-carboxy methyltransferase were identified as effective texture-associated biomarkers for chilled fish, and Staphylococcal nuclease domain-containing protein 1 for iced fish. This study provided suitable proteins as indicators of fresh fish flesh texture, which could help establish a rapid and convenient texture testing method in future studies.

Anodized CuO-based reusable non-enzymatic glucose sensor as an alternative method for the analysis of pharmaceutical glucose infusions: a cyclic voltammetric approach.

Analyzing pharmaceutical products is a quality control requirement in a production facility. This study presents a CuO electrode-based reusable non-enzymatic sensor as an alternative method for rapid analysis of glucose levels in glucose infusions. CuO is extensively employed as an electrode material in non-enzymatic glucose sensors. Conventionally, these electrodes are fabricated using chemical synthesis of CuO followed by immobilization to the electrode substrate. In contrast, here, Cu metal was mechanically modified to create a grooved surface, followed by electrochemical anodization and subsequent annealing process to grow a seamless CuO layer in situ with enhanced catalytic activity. The morphology of the electrodes was characterized using scanning electron microscopy (SEM) and X-ray diffractometry (XRD). The direct electrocatalytic activity of the developed CuO-modified electrode towards glucose oxidation in alkaline media was investigated by cyclic voltammetry in detail. The CuO-modified electrode commenced the oxidation process around 0.10V vs. Ag pseudo-reference electrode, demonstrating a significant reduction in the overvoltage for glucose oxidation compared to the bare Cu electrode. The sensor is capable of detecting glucose at low oxidation potentials such as 0.2V with a sensitivity value of 0.37 µA ppm-1, a wide linear range (80-2300ppm), limit of quantification (LOQ) of 1ppm, greater repeatability, 1% precision, 3% bias, a short response time (80s), good reproducibility and excellent reusability (196 consecutive attempts). The enhanced performance and cost-effectiveness make this sensor a promising alternative method for product analysis in glucose injection solutions.

Development of a rapid quantitative method of analysis of carbohydrate fatty acid ester reaction mixtures using 1H qNMR

Two new analytical methods, applying absolute 1H qNMR, were developed to monitor product yield and quantify unreacted carbohydrate and fatty acid reactants, in the synthesis of carbohydrate fatty acid esters (CFAE). These methods provide a mass balance of the crude reaction mixtures and diversify the analytical screening and quantitation approaches available within the synthesis of these molecules. Both methods were validated for the model reaction of methyl α-d-glucopyranoside (MAG) and lauric acid (LA) to form the mono ester product, methyl 6-O-dodecanoyl-α-d-glucopyranoside. Analysis in CD3OD by 1H qNMR, with fumaric acid (FA) as an internal standard (IS), allowed monitoring of all reaction components. Alternatively, using CDCl3 and (E)-stilbene as IS enabled the analysis of CFAE and fatty acid. Parameters calculated for method validation included specificity and selectivity, linearity, accuracy, intermediate precision, limit of detection (LOD), limit of quantification (LOQ) and robustness. Both methods provided excellent linearity with R2 > 0.997. The accuracy, precision, and robustness of the method in CD3OD was <2 % uncertainty making it suitable for complete reaction analysis. The method completed in CDCl3 resulted in accuracy, intermediate precision, and robustness of <5 %, except for accuracy in the lowest levels of concentration (>5 %). For all related analytes in the CD3OD and CDCl3 methods, the LOD and LOQ were determined to ensure applicability for the intended use in the assessment of reaction crude composition. Finally, the system suitability was assessed in a scaled lipase catalysed CFAE synthetic reaction. The determined qNMR product yields were verified against isolated purified product yields with <5 % uncertainty.

Simultaneous Analysis of 272 Pesticides in Agricultural Products by the QuEChERS Method and Gas Chromatography with Tandem Mass Spectrometry.

The aim of this study is to develop a rapid and accurate method for simultaneous analysis of multi-residue pesticides and conduct pesticide monitoring in agricultural products produced by the production and distribution stage in Korea. The representative agricultural products were selected as brown rice, soybean, potato, mandarin, and green pepper and developed using gas chromatography with tandem mass (GC-MS/MS) for the analysis of 272 pesticide residues. The experimental samples were extracted by the QuEChERS-EN method and then cleaned up by using d-SPE, including MgSO4 and primary secondary amine (PSA) sorbents. The established method was validated in accordance with Codex CAC-GL/40, and the limit of quantitation (LOQ) was determined to be 0.01 mg/kg. A total of 243 pesticides satisfied the guidelines in five samples at three levels with values of 60 to 120% (recovery) and ≤45% (coefficient of variation, CV). The remaining 29 pesticides did not satisfy the guidelines, and these pesticides are expected to be used as a screening method for the routine inspection of agricultural products. As a result of analyzing 223 agricultural products in South Korea by applying the simultaneous analysis method, none of the detected levels in the samples exceeded the standard values based on maximum residue limits (MRLs). The developed method in this study will be used to inspect residual pesticides in agricultural products, and it is anticipated to contribute to the distribution of safe agricultural products to consumers.

Electrochemical Sensing Device for Carboplatin Monitoring in Proof-of-Concept Drug Delivery Nanosystems.

(1) Background: Carboplatin (CBP) is a chemotherapeutic drug widely used in the treatment of a variety of cancers. Despite its efficiency, CBP is associated with side effects that greatly limit its clinical use. To mitigate these effects, CBP can be encapsulated in targeted delivery systems, such as liposomes. Ensuring the adequate loading and release of CBP from these carriers requires strict control in pharmaceutical formulation development, demanding modern, rapid, and robust analytical methods. The aim of this study was the development of a sensor for the fast and accurate quantification of CBP and its application on proof-of-concept CBP-loaded nanosomes. (2) Methods: Screen-printed electrodes were obtained in-lab and the electrochemical behavior of CBP was tested on the obtained electrodes. (3) Results: The in-lab screen-printed electrodes demonstrated superior properties compared to commercial ones. The novel sensors demonstrated accurate detection of CBP on a dynamic range from 5 to 500 μg/mL (13.5-1350 μM). The method was successfully applied on CBP loaded and released from nanosomes, with strong correlations with a spectrophotometric method used as control. (4) Conclusions: This study demonstrates the viability of electrochemical techniques as alternative options during the initial phases of pharmaceutical formulation development.

Developing a multidisciplinary medication review and deprescribing intervention in primary care for older people living with frailty

Abstract Introduction Reducing overprescribing in primary care through targeted structured medication reviews (SMRs) is a national priority. Shared decision-making and a multidisciplinary team (MDT) approach to SMR are recommended for successful deprescribing (stop/reduce inappropriate medications) among older people, specifically those with frailty. Aim To develop a multidisciplinary medication review and deprescribing intervention in primary care for older people with frailty. Methods The intervention development involved two stages and was informed by the Medical Research Council framework for complex intervention. Stage one involved intervention planning and included: 1) a realist review of 28 articles that identified 35 context-mechanism-outcome (CMO) configurations for successful MDT medication review and deprescribing in primary care[1] and 2) a qualitative study with 26 primary care healthcare professionals (HCPs) including GPs, pharmacists, and advanced clinical practitioners, and 13 older people with polypharmacy and their informal carers recruited from two general practices within Wessex. Using the person-based approach, drawing on the COM-B model for behaviour change, and a normalisation process theory lens, the findings of these two studies informed the development of intervention’s guiding principles. Stage two involved intervention development based on six co-design online workshops with key stakeholders (n=25), including primary care HCPs recruited from across the South region, patient and carer representatives, and the research management team. The intervention guiding principles were presented in these workshops and possible intervention content, format and delivery were proposed, discussed and refined through an iterative process. Workshop data were analysed drawing on a rapid analysis method and a final version of the intervention was agreed. Results The final version of the complex intervention consisted of five main components based on findings from stages one and two: 1) Proactive identification of patients with frailty and polypharmacy for targeted SMR using the electronic frailty index embedded in primary care health systems and the UK national polypharmacy prescribing comparators, 2) HCPs preparation to conduct SMR through targeted education on deprescribing and identifying and prioritising high-risk medications for deprescribing using evidence-based deprescribing tool(s), 3) Preparing and educating patients and carers about the purpose of SMR and reasons for potentially stopping or changing medications (patient leaflet sent prior to SMR), 4) Conducting a person-centred SMR that includes in-person, phone or virtual appointments tailored to patient and carer needs and preferences, involving other MDT members based on their expertise, documenting and sharing any agreed changes with patients and other staff members, and 5) Tailored follow-up plans that allow continuity of care and management and include highlighting specific signs and symptoms for patients and carers to monitor following medication changes, and arranging further contact through text, phone or in-person follow-up appointment. Conclusion The use of multiple research methods has led to the development of a complex MDT medication review and deprescribing intervention for older people with frailty and drawing on behaviour and implementation theories could potentially maximise the intervention’s feasibility, acceptability and successful implementation. A mixed-method study in primary care is currently underway to test the feasibility and acceptability of intervention implementation, in preparation for a full trial.

A sensitive immunosensor via Pd@Au0.85Pd0.15 in situ electrocatalysis generating H2O2 for quenching electrochemiluminescence of Ir(pbi)2(acac)@Ti3C2Tx MXene-PVA

The establishment of rapid target analysis methods for cytokeratin fragment antigen 21-1 (CYFRA 21-1) is urgently needed. [Ir(pbi)2(acac)] (pbi = 2-(4-bromophenyl)-1-hydrogen -benzimidazole, acac = acetylacetonate) as traditional electrochemiluminescence (ECL) luminophores has been confined due to its non-negligible dark toxicity and poor water solubility leading to poor biocompatibility and electrical conductivity as an organic molecule. Hence, to overcome this limitation, [Ir(pbi)2(acac)] can be effectively loaded on the polyvinyl alcohol hydrogel modified Ti3C2Tx MXene surface (Ir@Ti3C2Tx-PVA) as sensing platform which can emit high ECL signals. Then, a quenching strategy was proposed to fabricate an ECL sandwich immunosensor using H2O2 as quencher molecules which can generated by Pd@Au0.85Pd0.15. Especially, the generation of O2 to H2O2 can be achieved through a two-electron (2e–) reaction pathway by Pd@Au0.85Pd0.15, to overcome the restriction that the H2O2 was virtually impossible to label or immobilize on the non-enzyme nanomaterials. The proposed ECL assay achieves a response to CYFRA 21-1 within the range of 0.1 pg/mL–100 ng/mL, with a detection limit of 8.9 fg/mL (S/N = 3). This work provided a feasible tactic to seek superior-performance ECL luminophore and quencher consequently set up a novel means to makeup ultrasensitive ECL biosensor, which extended the utilization potential of Ir(pbi)2(acac) in ECL assays.

Ultrasensitive Aptasensor for α-Amatoxin Detection Based on the DNA Tetrahedral Nanostructure Triggering Rolling Circle Amplification and Signal Amplification.

Poisonous mushrooms containing α-amatoxin can be lethal, making it imperative to develop a rapid and sensitive detection method for α-amatoxin. Utilizing the DNA tetrahedral structure as its foundation, the aptamer allows controlled density and orientation. Consequently, we designed aptamer tetrahedral functionalized magnetic beads that specifically target α-amanitin to release complementary DNA (C-DNA) strands. These strands were then employed as primers to initiate rolling circle amplification (RCA) with fluorescent dyes. The combination of SYBR Green I detection probes facilitated the amplification of the detection signal, enhancing the detection sensitivity of the aptasensor. The calculated detection limit was determined to be 3 ng/mL, a magnitude lower than that of other aptasensors by 2 orders of magnitude. The aptasensor integrates the advantages of high sensitivity and specificity, offering a simple and reliable rapid detection method for α-amanitin analysis.

Cystatin C Use for CKD Detection in the Veterans Health Administration System: A Qualitative Study of Barriers and Facilitators

Rationale & ObjectiveThe measurement of cystatin C has been recommended to enhance chronic kidney disease (CKD) detection and risk stratification in clinical practice. This study gathered insights into the perceptions and experiences of clinical staff regarding the use of cystatin C in CKD detection within the Veterans Health Administration (VHA) System. Study DesignA qualitative approach was employed to explore barriers and facilitators of clinical staff regarding the use of cystatin C in CKD detection within the VHA system. The Organizational Theory of Implementation Effectiveness informed the development of a semi-structured interview guide. Setting & ParticipantsHealthcare providers, nurses, and clinical pharmacists from the VHA systems in San Francisco, San Diego, and Houston were interviewed between October 2021 and May 2022. Analytical ApproachParticipant responses from individual interviews were analyzed by a multidisciplinary team using rapid qualitative analysis methods. ResultsFourteen in-depth interviews were conducted across the three VHA systems. Ten of eleven providers worked in primary care. Five key barriers to using cystatin C for CKD detection were identified. These included: lack of patient awareness of CKD testing, lack of provider awareness about cystatin C, knowledge barriers about cystatin C testing, unclear roles and ownership of CKD detection, and lack of clinic support to enhance CKD detection. Suggested interventions to overcome these barriers included educational and training programs, improved clinic workflows, and electronic health record aids to support CKD detection and use of cystatin C. LimitationsThe results may not be generalizable to other healthcare systems outside the VHA. ConclusionsFindings indicate a need for targeted interventions such as educational and training programs, improved clinical workflows, and electronic health record aids to address barriers limiting the use of cystatin C in clinical practice for enhanced CKD detection.

Quality study of animal-derived traditional Chinese medicinal materials based on spectral technology: Calculus bovis as a case.

Calculus bovis (C. bovis) is a typical traditional Chinese medicine (TCM) derived from animals, which has a remarkable curative effect and high price. Rapid identification of C. bovis from different types was realized based on spectral technology, and a rapid quantitative analysis method for the main quality control indicator bilirubin was established. We conducted a supervised and unsupervised pattern recognition study on 44 batches of different types of C. bovis by five spectral pretreatment methods. Three variable selection methods were used to extract the essential information, and the partial least squares regression (PLSR) quantitative model of bilirubin by near-infrared (NIR) spectroscopy was constructed. The partial least squares discriminant analysis (PLS-DA) model could achieve 100% accuracy in identifying different types of C. bovis. The R2 of the NIR quantitative model was 0.979, which is close to 1, and the root mean square error of calibration (RMSEC) was 2.3515, indicating the good prediction ability of the model. The study was carried out to further improve the basic data of quality control of C. bovis and help the high-quality development of TCM derived from animals.

Development and Validation of a Capillary Zone Electrophoresis-Tandem Mass Spectrometry Method for Simultaneous Quantification of Eight β-Lactam Antibiotics and Two β-Lactamase Inhibitors in Plasma Samples.

Monitoring plasma concentrations of β-lactam antibiotics is crucial, particularly in critically ill patients, where variations in concentrations can lead to treatment failure or adverse events. Standardized antimicrobial regimens may not be effective for all patients, especially in special groups with altered physiological parameters. Pharmacokinetic/pharmacodynamic (PK/PD) studies highlight the time-dependent antibacterial activity of these antibiotics, emphasizing the need for personalized dosing. Therapeutic drug monitoring (TDM) is essential, requiring rapid and accurate analytical methods for precise determination of drugs in biological material (typically plasma or serum). This study presents a novel capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) method designed for the simultaneous quantification of five penicillin antibiotics, two cephalosporins, one carbapenem, and two β-lactamase inhibitors in a single run. The method involves a simple sample pretreatment-precipitation with organic solvent-and has a run time of 20 min. Optimization of CZE separation conditions revealed that 20 mM ammonium hydrogen carbonate (NH4HCO3) serves as the optimal background electrolyte (BGE). Positive electrospray ionization (ESI) mode, with isopropyl alcohol (IP)/10 mM ammonium formate water solution (50/50, v/v) as the sheath liquid, was identified as the optimal condition for MS detection. Method validation according to the Food and Drug Administration (FDA) guideline for development of bioanalytical methods demonstrated satisfactory selectivity, linearity, recovery, robustness, and stability. The method's practicality was evaluated using the Blue Applicability Grade Index (BAGI), yielding a score of 77.5. Moreover, the greenness of the proposed method was evaluated by two commonly used metric tools-Analytical GREEnness (AGREE) and Green Analytical Procedure Index (GAPI). The developed CZE-MS/MS method offers a practical and reliable approach for quantifying a broad spectrum of β-lactam antibiotics in plasma. Its ability to simultaneously quantify multiple analytes in a single run, coupled with a straightforward sample pretreatment, positions it as a valuable and prospective tool for TDM in critically ill patients.

Quantitative determination of tetracyclines in medicated feed for food-producing animals by HPLC–DAD

Tetracyclines are a group of antibiotic substances largely administered through medicated feed to control diseases in food-producing animals. Fine dosing of antibiotics contained in medicated feed is crucial for the success of the treatment as well as minimising potential threats such as the spread of antimicrobial resistance and the transfer of antibiotic residues in food. A rapid analytical method based on HPLC with diode array detection (HPLC–DAD) was developed to quantify oxytetracycline, chlortetracycline and doxycycline in medicated feed. The reported method underwent in-house validation and was found to be suitable for the quantification of three target tetracyclines within the concentration range of 40–1000 mg kg−1 in official routine analysis. The method was applied to 103 official samples in the framework of the Italian National Plan on animal feed during the years 2021–2023 and nine non-compliant concentrations were identified in swine and fish feed samples.

Simultaneous trace mass and quantity of airborne microplastics based on electromagnetic heating-pyrolysis-mass spectrometry, taking surgical face masks as an example

Microplastics (MPs) in the atmosphere have attracted global concern due to their potential threats to human health. However, the current rapid analytical method for airborne MPs is insufficient, which hinders a comprehensive understanding of their environmental risks. In recent years, the disposable face masks have emerged as a new source of airborne MPs. There are significant knowledge gaps regarding MPs from mask, including detection methods, formation mechanisms, and environmental abundance. This study presented a novel analytical approach for the rapid detection of airborne MPs using the electromagnetic heating-pyrolysis-mass spectrometry (Eh-Py-MS). To validate this method, surgical face masks were employed as an illustrative example. The mass-related quantification of MPs was realized based on the Eh-Py-MS. By employing a conversion model of mass, size, and density, simultaneous assessment of both mass and quantity of MPs was realized. The recovery rates were in the range of 86.6%–111.6% and the precision (RSD) was 8.4%, which was sufficient for rapid quantification. Results showed that the simultaneous tracing of mass and quantity of airborne microplastics could be successfully achieved through the developed method. Furthermore, the formation mechanism of MPs in surgical masks under UV exposure was also investigated to address the knowledge gap of masks MPs. Results indicated that surgical masks could be a neglected source of airborne microplastics in the environment. This study aims to provide valuable insights for the rapid assessment of airborne MPs in the environment.

Acidic Derivatization of Thiols Using Diethyl 2-Methylenemalonate: Thiol-Michael Addition Click Reaction for Simultaneous Analysis of Cysteine and Cystine in Culture Media Using LC-MS/MS.

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7kb. The limit of detection of the method is 0.1ng/µL for the supercoiled and linear isoforms and 0.5ng/µL for the open circular isoform, with a maximum loading capacity of 10-15ng/µL. The turnaround time is 1min/sample, and the volume requirement is 10µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Modified QuEChERS method combined with ultra performance liquid chromatography-tandem mass spectrometry for detection of cyclopiazonic acid in feeds

Mycotoxins are toxic secondary metabolites produced by fungal species that can cause acute, subacute, and chronic toxicity in humans and animals. Thus, these toxins pose a significant threat to health and safety. Owing to the lack of effective antimold measures in the agricultural industry, feed ingredients such as corn, peanuts, wheat, barley, millet, nuts, oily feed, forage, and their byproducts are prone to mold and mycotoxin contamination, which can affect animal production, product quality, and safety. Cyclopiazonic acid (CPA), which is mainly biosynthesized from mevalonate, tryptophan, and diacetate units, is a myotoxic secondary metabolite produced by Penicillium and Aspergillus fungi. CPA is widely present as a copollutant with aflatoxins in various crops. Compared with some common mycotoxins such as aflatoxins, fumonisins, ochratoxins, zearalenones, and their metabolites, CPA has not been well investigated. In the United States, a survey showed that 51% of corn and 90% of peanut samples contained CPA, with a maximum level of 2.9 mg/kg. In Europe, CPA was found in Penicillium-contaminated cheeses as high as 4.0 mg/kg. Some studies have shown that CPA can cause irreversible damage to organs such as the liver and spleen in mice. Therefore, the establishment of a rapid and efficient analytical method for CPA is of great significance for the risk assessment of CPA in feeds, the development of standard limits, and the protection of feed product quality and safety. The QuEChERS method, a sample pretreatment method that is fast, simple, cheap, effective, and safe, is widely used in the analysis of pesticide residues in food. In this study, a modified QuEChERS method combined with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine CPA levels in feeds. The chromatographic separation and MS detection of CPA as well as the key factors affecting the extraction efficiency of CPA, including the type of extraction solvent, type of inorganic salt, and type and dosage of adsorbent, were optimized in detail. During the optimization of the chromatographic-separation step, the acid and salt concentrations of the mobile phase affected the separation and detection of CPA. During the optimization of the QuEChERS method, the addition of a certain amount of acetic acid improved the extraction efficiency of CPA because of its acidic nature; in addition, GCB and PSA significantly adsorbed CPA from the feed extract. Under optimal conditions, the CPA in the feed sample (1.0 g) was extracted with 2 mL of water and 4 mL of acetonitrile (ACN) containing 0.5% acetic acid. After salting out with 0.4 g of NaCl and 1.6 g of MgSO4, 1 mL of the ACN supernatant was purified by dispersive solid-phase extraction using 150 mg of MgSO4 and 50 mg of C18 and analyzed by UPLC-MS/MS. The sample was separated on a Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm) using 2 mmol/L ammonium acetate aqueous solution with 0.5% formic acid and ACN as the mobile phases and then analyzed by positive electrospray ionization in multiple reaction monitoring mode. CPA exhibited good linearity in the range of 2-200 ng/mL, with a high correlation coefficient (r=0.9995). The limits of detection and quantification of CPA, which were calculated as 3 and 10 times the signal-to-noise ratio, respectively, were 0.6 and 2.0 μg/kg, respectively. The average recoveries in feed samples spiked with 10, 100, and 500 μg/kg CPA ranged from 70.1% to 78.5%, with an intra-day precision of less than 5.8% and an inter-day precision of less than 7.2%, indicating the good accuracy and precision of the proposed method. Finally, the modified QuEChERS-UPLC-MS/MS method was applied to the analysis of CPA in 10 feed samples obtained from Wuhan market. The analysis results indicated that the developed method has good applicability for CPA analysis in feed samples. In summary, an improved QuEChERS method was applied to the extraction and purification of CPA from feeds for the first time; this method provides a suitable analytical method for the risk monitoring, assessment, and standard-limit setting of CPA in feed samples.

Properties of processing and data acquisition methods in test methods of chemical analysis

Chemical analysis is a crucial source of information for assessing the state of the environment, evaluating the human body condition, and serving as a diagnostic tool for detecting diseases and more. The use of simple, rapid, and cost-effective analysis methods without the need for specialized laboratory conditions and personnel with specific qualifications, such as test methods, contributes to the effective implementation of well-known techniques for analyzing the chemical composition of environmental objects and for use in the field of healthcare. Scanner indication of the analytical signal in chemical analysis is an objective, fast, and automated method for assessing the color characteristics of colored samples. The use of scanner indication in obtaining the analytical signal eliminates errors associated with subjective evaluation and assists individuals with color vision impairments, speeding up data collection and processing operations. Considering and controlling the light source in scanning systems is crucial to ensuring the stability and accuracy of obtained images under different lighting conditions. To obtain the analytical signal, it is primarily necessary to process the image to obtain the region of interest, which is determined by segmentation, manual methods, and using image processing techniques. The analysis of obtained values is carried out by constructing a linear or nonlinear relationship of color values in the selected color model converted to a single value of the analytical signal to concentration. Most existing systems for scanner detection of the analytical signal in analytical test methods require human involvement in obtaining and interpreting results, limiting the application areas of these methods of analytical signal indication. Further research is focused on creating a reliable method for obtaining the analytical signal using a smartphone camera based on image processing techniques, allowing for the automation of obtaining, processing, and improving the reproducibility of results in chemical test analyses.