Pulsed Amperometric Detection Research Articles

Development and characterization of microchip electrophoresis pulsed amperometric detector-based soil pesticide analyser

The present study involved the fabrication and testing of a Microchip electrophoresis (MCE) device for pulse amperometry based detection of pesticides from their mixture. We were able to separate and then quantify three distinct types of insecticides, namely Chlorpyrifos, Imidacloprid, and Fipronil using on chip MCE followed by pulsed amperometric detection. All these results were obtained with an inhouse developed potentiostat cum controller unit with a detection time of only 15 min, employing a minimal sample size of 2 μL without any preconcentration or extraction procedure. The limit of detection (LOD) was calculated as 42.69 μM, 62.61 μM, and 71.14 μM or 14.96, 16.0 and 31.09 ppm, respectively for Chlorpyrifos, Imidacloprid, and Fipronil and their respective migration times as 536 ± 6.3 s, 484 ± 1.7 s, and 604 ± 3.5 s (n = 14). The sensitivity of detection was determined as 0.03 nA/μM for Chlorpyrifos, 0.0265 nA/μM for Imidacloprid, and 0.035 nA/μM for Fipronil. In addition, the efficacy of the produced microchip was confirmed by analysing soil extract spiked with known pesticides concentrations while the recovery percentage, representing a ratio of calculated concentration to spiked concentration multiplied by hundred was found as 84.3% (±9.4%) (n = 9). Thus, integrating microchip technology with the developed analytical instruments presents significant promise for practical field applications and the analysis of diverse analytes by way of creating a library where the migration coefficient and peak detection current are needed for any analyte which can be made cationic or anionic using a suitable buffer.

Relationship between dietary fiber content and prebiotic potential of polysaccharides from the seaweeds of the North west coast of India

The macroalgae are a sustainable bioresource that can be harnessed for their functional food and nutraceutical applications. This study characterized the biochemical composition and bioactive potential of natural biological macromolecules, such as macroalgal polysaccharides extracted using a green, aqueous extraction process. The in-vitro antioxidant and antiglycemic activity of these polysaccharides were evaluated using model, free radical and antiglycemic compounds. The prebiotic potential of macroalgal polysaccharides were analysed based on their ability to promote the growth of two potential probiotic bacteria Lactobacillus acidophilus and L. bulgaricus and suppress the growth of enteric bacteria, Escherichia coli. Among the polysaccharides studied, the brown algal polysaccharide MPS8 MPS9 and MPS10 exhibited good antioxidant, antiglycemic and prebiotic activity. Based on infrared spectroscopy, the functional groups sulfation and carboxylation were identified in potential polysaccharides. The monosaccharide composition in the bioactive polysaccharides was determined using High Performance Anion Exchange Chromatography Pulse Amperometric detector (HPAEC-PAD). These bioactive polysaccharides were fractionated using ion exchange chromatography to purify it and further characterized using gel permeation chromatography and NMR spectroscopy. The results these polysaccharides are mainly composed of fucose and glucose which is due to the fucoidan and laminarin, respectively. Such macromolecules with high dietary fiber content and bioactivity are in global demand as functional food, nutraceutical and pharmaceutical formulations.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Use of human Caco-2 cells and HPAE-PAD for α-glucosidase assay

To measure α-glucosidase activity, rat intestinal acetone powder is commonly used as a source of α-glucosidase, and the mutarotase-glucose oxidase (GOD) methods commonly used to quantitate glucose produced by enzymatic hydrolysis of the substrates. In this study, we compared human Caco-2 cell extracts with rat intestinal acetone powder extracts. We also compared high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with the mutarotase-GOD method. The sensitivity of HPAE-PAD was higher than that of mutarotase-GOD. The glucose concentration quantified by HPAE-PAD was similar to that quantified using the mutarotase-GOD method. In the maltase reaction, 1-deoxynojirimycin (1-DNJ) exerted a more potent inhibitory effect on human enzymes than on rat enzymes. This order was reversed during the sucrase reaction. These results suggested that the combined use of Caco-2 cell extracts and HPAE-PAD is advantageous for use in α-glucosidase-related basic research.

A novel approach to the dual sugar test for the assessment of intestinal epithelium permeability in response to exertional heat stress and nutritional intervention

An acute increase in intestinal epithelium permeability is induced by prolonged exertion in the heat, resulting in the translocation of pathogenic bacteria and endotoxins from the lumen into the circulation, causing a systemic inflammatory response and debilitating symptoms(1). Acute exercise-induced gastrointestinal syndrome mimics chronic health conditions with which an impaired intestinal barrier function is associated, including coeliac disease, inflammatory bowel disease, diabetes, Alzheimer’s and liver diseases(2). Intestinal epithelium permeability is typically assessed using a dual sugar absorption test, by administering a drink containing non-metabolisable sugars (e.g. lactulose [L] and L-rhamnose [R]) that can enter the circulation by paracellular translocation when the epithelium is compromised, and are subsequently excreted and measured cumulatively in the urine(3). We aimed to demonstrate that our recently developed ion chromatography protocol(4) can be used to accurately quantify L/R ratio in the plasma of participants exercising in hot ambient conditions and to determine the impact of nutritional intervention on intestinal epithelium permeability. Further, we hypothesised that measuring L/R in plasma collected at intervals during the post-exercise recovery period would reveal more information about intestinal permeability compared to previously published cumulative urine L/R data(3). Endurance-trained participants completed a set of randomised crossover studies, consisting of 2 h running at 60% V˙O2max in temperate, warm and hot ambient conditions (n = 8) and/or in the heat while consuming water, carbohydrate or protein (n = 9). The dual sugar solution was ingested at 90 min of exercise and blood was sampled at 0, 1, 2 and 4 h post-exercise. Plasma sugars were quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and L/R ratios were compared by two-way repeated measures ANOVA with Tukey’s multiple comparisons. Plasma L/R increased immediately post-exercise in the heat (0.15 ± 0.11) compared with temperate (0.06 ± 0.04, p<0.001) and warm (0.09 ± 0.08, p<0.01) conditions, while consuming glucose before and during exercise alleviated this (0.02 ± 0.02, p<0.001), and this novel information was otherwise missed when measuring urine L/R. Consuming glucose or whey protein hydrolysate during exercise attenuated intestinal permeability from exertional heat stress throughout recovery, with the mean plasma L/R over 4 h reduced from 0.11 ± 0.05 to 0.04 ± 0.03 (p<0.001) and 0.06 ± 0.04 (p<0.01) with glucose and protein, respectively. We recommend using the dual sugar test with quantification of plasma sugars at intervals by HPAEC-PAD to maximise intestinal permeability data collection in exercise gastroenterology research and beyond, as this gives additional acute response information compared to urinary measurements. Our approach can be employed to investigate and develop personalised nutrition strategies that prevent intestinal hyperpermeability during exertional heat stress. This has implications for athlete performance and safety, and can also build upon occupational health and safety practices and inform chronic disease management.

Investigating the effect of polyphenols from nuts on human carbohydrate digestion in vitro

Recent studies have documented the importance of postprandial hyperglycaemia in the incidence of chronic diseases, including type 2 diabetes. Inhibition of digestive enzymes, including membrane-bound brush-border α-glucosidases, leads to slowed carbohydrate digestion and absorption, and reduced postprandial glycemia. Nuts are widely eaten around the world and have the potential to inhibit α-glucosidases through their content of polyphenols and other bioactive compounds. According to our recent systematic review(1), no study has investigated the inhibitory effects of nut extracts on human α-glucosidase activities. Almost all studies in this area have been conducted on yeast α-glucosidase, with only a few using rat α-glucosidase. While there is no sequence homology between yeast and human α-glucosidase, there is 74% to 78% sequence homology between rat and human α-glucosidases(1). The lack of studies on the effect of bioactive compounds from nuts on human α-glucosidases, along with the growing attention to nuts as an important component of a healthy diet with the potential to reduce the risk of chronic diseases(2), highlights the need for research to evaluate the inhibitory effect of nut extracts on human α-glucosidases. The aim of the current study is to explore the inhibitory effect of extracts from nuts on human carbohydrate digestive enzymes. Walnuts and almonds were ground and defatted with hexane, extracted in 80% (v/v) acetone, and further purified using solid-phase extraction to obtain phenolic-rich extracts. The Folin–Ciocalteu assay was used to approximate the polyphenol content of the samples. Following our recently published detailed protocol(3), cell-free extracts from human intestinal Caco-2/TC7 cells were used as a source of α-glucosidase in enzyme inhibition assays, with sucrose, maltose and isomaltose as substrates and appropriate controls. The assay products were quantified using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Glucose production in the presence of various concentrations of phenol-rich nut extracts was compared using a one-way ANOVA and half-maximal inhibitory concentration (IC50) values were calculated. The Folin–Ciocalteu data demonstrate that walnut extracts comprise a relatively high polyphenol content, with 18.1 ± 0.23 mg (epigallocatechin gallate [EGCG] equivalent) per gram of fresh weight, while almond extracts contain 0.87 ± 0.03 mg EGCG equivalent/g of fresh weight. The walnut phenolic-rich extract dose-dependently inhibited human intestinal sucrase and maltase activities (both p<0.01), with IC50 values of 1.67 mg/mL and 2.84 mg/mL, respectively. We demonstrate that phenolic-rich walnut extracts can inhibit human α-glucosidases in vitro and therefore walnuts may contribute to slowing carbohydrate digestion in humans. As such, we plan to assess the effects of walnuts on postprandial glycaemia in vivo.

Quantitative analysis of fucose in fucoidans from Sargassum spp. in Jeju Island, South Korea using 3-methyl-1-phenyl-5-pyrazolone derivatization and RP-HPLC-UV method

Sargassum species are representative brown algae found worldwide, with 27 species native to the coast of Jeju Island in South Korea. Fucoidans are derived from brown algae and exhibit a variety of biological activities. Analysis of the monosaccharide composition is necessary for an in-depth study of fucoidans. However, quantitative analysis of fucoidans with complex structures according to species and habitat is insufficient. Furthermore, complex pre-processing is often required to analyze sugars, making it less economical. This study aimed to validate high-performance liquid chromatography (HPLC)-UV analysis based on 3-methyl-1-phenyl-5-pyrazolone (PMP) derivatization of monosaccharides. The proposed method had a short running time and could be repeated economically using UV detection. The detected PMP-fucose was confirmed to be uninterfered by comparing the analytical chromatograms of the internal standard solution. This analytical method was characterized for system suitability, precision, accuracy, linearity, limit of detection (LOD), and limit of quantification (LOQ), demonstrating satisfactory results. The quantification was verified by comparison with the quantitative results of commercial fucoidan obtained using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). Furthermore, the antioxidant effects of algal polysaccharides from Sargassum spp. isolated on Jeju Island was evaluated using a zebrafish model. Polysaccharides from Sargassum coreanum (SCpol) and Sargassum autumnale (SApol) showed remarkable free radical scavenging activities. In addition to the results obtained in the zebrafish experiments, all species-derived polysaccharides showed antioxidant potential. In conclusion, this study demonstrates a useful analytical method using HPLC-UV and PMP derivatives for the effective determination of monosaccharide contents, including fucose, in Sargassum spp.

Separation of All Classes of Carbohydrates by HPAEC-PAD

High performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is a potential method of choice for the analysis of carbohydrates.

Label-Free Liquid Chromatography-Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan.

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography-mass spectrometry (LC-MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC-MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1-2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.

Evaluation of a novel stationary phase for the separation of lactose and isomers in lactose-free products using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD)

Lactose intolerance is a widespread condition, which prevents a large number of people from consuming dairy products as a part of their daily diet. It is estimated that an average of 65% of the global population is suffering from lactose intolerance. The global market for 'lactose-free' dairy products is rapidly growing and the criteria for 'lactose-free' labelled products are becoming stricter. To check the lactose contents in these products there is a need for fast, sensitive, and selective analytical method. A method is presented for fast and sensitive determination of lactose and its isomers using High-Performance Anion Exchange Chromatography in combination with Pulsed Amperometric Detection (HPAEC-PAD). The use of a new anion-exchange column, SweetSep™ AEX200, which is a strong anion-exchange column with highly monodisperse 5µm particles, allowed the separation of all compounds of interest in less than 8min with high resolution. A variety of dairy products were analyzed to demonstrate the versatility of the method.

Evaluation of transgalactosylation activity of commercial β-galactosidase from Bifidobacterium bifidum for synthesis of prebiotic oligosaccharides

Galacto-oligosaccharides (GOS) are products of transgalactosylation reaction of β-galactosidase when lactose is used as the substrate. These carbohydrates are considered prebiotics which stimulate beneficial effects to human health. In the present work, Saphera, a commercial preparation of β-galactosidase from Bifidobacterium bifidum, was biochemically characterised for production of GOS. Using o-nitrophenyl-β-D-galactopyranoside (oNPG) as the substrate, optimal activity for the enzyme was found to be at pH 6.0 and at 45°C. Ten (10) mM each of either Na+ or K+ enhanced enzyme activity by 10%, while Cu2+, Zn2+, Fe2+, and EDTA showed inhibitory effect on the enzyme activity. When incubated in 50 mM sodium phosphate and pH 6.5, the enzyme was found to have half-life time of 136 ± 6 and 2.1 ± 0.2 h at 30 and 50°C, respectively. The hydrolysis activity of the enzyme predominated when the initial lactose concentration used was 5% (w/v). When initial lactose concentration was increased to 20% (w/v), maximum GOS yield obtained was 10% (w/w) achieved at 86% lactose conversion. Analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the major oligosaccharides produced by the enzyme were 3’-galactosylglucose, 3’-galactosyllactose, 3’-galactobiose, and allolactose, thus showing that this enzyme would prefer to form β-(13)- and β-(16)-linked GOS. Despite its predominantly hydrolytic activity, Saphera synthesised prebiotic GOS which could be interesting to dairy industry.

Using Analytical Quality by Design to improve analytical method development in vaccines quality control: Application to an optimized quantitative high-performance anion-exchange chromatographic method

Analytical quality by design (AQbD) is an enhanced approach for the development of analytical methods. AQbD has received much industrial interest, being the subject of several recently published draft guidelines. This article demonstrates the application of AQbD to determine the quantity of non-adsorbed polysaccharide polyribosyl ribitol phosphate (PRP) and percentage of depolymerized PRP in a commercial hexavalent liquid vaccine, and establishment of an analytical control strategy (ACS). The quantification method developed is high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection, preceded by ultracentrifugation (sample preparation) for separation of the depolymerized polysaccharide from the native adsorbed polysaccharide. The first step was to develop the analytical target profile (ATP) which defines the purpose of the analytical measurement as well as the development scope. As a second step, risk assessment tools were used for identification and ranking of the critical method variables (CMVs) which have a potential impact on method performance if not controlled.Based on a multivariate Design of Experiments (DoE) approach, a proposed method operational design region (MODR) was determined for seven CMVs. Finally, the ACS was established from the understanding of the analytical method and the robustness study. This article focuses on robust and operational ranges of critical parameters linked to the ultracentrifugation and chromatographic steps for depolymerized polysaccharide content control. The design space proposed for CMVs corresponds to the ranges that ensure a product that complies with the previously established precision criteria (±2% equivalent to ± 10 % around the product criterion, which is 20 % for depolymerized polysaccharide control limit).The following design space was established from the DoE statistical modeling for ultracentrifugation critical parameters: [483,000–520,000] g for speed, [11–19]°C for temperature, [29–34] minutes for duration, and from extemporaneous to 8 min for holding time before supernatant recuperation after the ultracentrifugation. For chromatographic critical parameters, the MODR is [2–6] psi for mobile phase helium pressure, [0–7] days for mobile phase storage time, and [0–3] days for samples storage time in the autosampler at 5 °C.Methods optimized using the AQbD approach provide strong justifications during regulatory filing for the selection of analytical CMVs, and for the ACS to be applied during the lifecycle management of the method.

PSIII-1 Effect of Cannabidiol Extraction on the Cell Wall Carbohydrate Composition of Hemp Inflorescences

Abstract Since the 2018 Farm Bill, US growers have been allowed to cultivate hemp (Cannabis sativa L.), prompting interest in exploring the use of hemp products in ruminant diets. Extraction of cannabidiol (CBD) oil from hemp biomass generates spent inflorescent material that could be upcycled as a feedstuff, yet little is known about its fiber composition. A recent study measured in vitro true digestibility and fermentation parameters of multiple hemp cultivars, some intact and some CBD-extracted (Altman et al., 2022). Both categories of hemp inflorescence material had similar or greater total digestible nutrients (TDN) than an alfalfa cube control, supporting hemp’s potential as a feedstuff. However, comparisons between non-extracted and CBD-extracted samples could not be made, and the fiber content was only described as neutral detergent fiber (NDF). Fiber composition can significantly impact ruminal digestibility, and in-depth characterizations of cell wall carbohydrate composition can provide further insight into the digestibility of a specific plant-based feed. Therefore, in the present study, we examined the effect of CBD oil extraction on the cell wall carbohydrate composition of the inflorescent material of two hemp cultivars. Intact (Biomass) and CBD-extracted (Spent) inflorescent material from two hemp cultivars, Mary and Zelios, were analyzed. The scell wall monosaccharides of the samples were released via two complementary acid hydrolyses, methanolysis and Saeman (sulfuric acid) hydrolysis, and separated and quantified using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Additionally, the carbohydrate linkage patterns were assessed by generating partially methylated alditol acetates (PMAAs). Student’s unpaired t-tests were run in GraphPad Prism, and no differences (P > 0.05) in monosaccharide concentrations based upon treatment (CBD extraction) or cultivar were found with either hydrolysis method except for the methanolysis glucose concentration between the biomass forms of the two cultivars where Mary Biomass possessed more glucose than Zelios Biomass (P <b0.001). In the Saeman hydrolysates, glucose was the dominant monosaccharide (38.75 ± 1.44 molar %), indicating a large amount of cellulose, supported by the presence of 1,4-Glcp in the linkage analysis. Linkage analysis also revealed the presence of xyloglucans and rhamnogalacturonan Type 1 pectins in all materials. The data showed no effect of CBD oil extraction on the monosaccharide composition, suggesting that the extraction process leaves the fiber components mostly intact, but there may be differences in glucose concentration (and consequently cellulose content) between cultivars of hemp. Future studies may benefit more from investigating the impact of hemp cultivar on fiber digestibility rather than impact of the CBD extraction process.

Electrochemical biosensing based comparative study of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein

In this study, we are reporting an electrochemical biosensor for the determination of three different clones of monoclonal antibodies (mAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant nucleocapsid protein (rN). The nucleocapsid protein was chosen as a system component identifying and discriminating antibodies that occur after virus infection instead of S protein used in serological tests to measure antibodies raised after vaccination and infection. The sensing platform was based on a screen-printed carbon electrode (SPCE) covered with gold nanoparticles (AuNP) and subsequently modified with a self-assembled monolayer (SAM) to ensure the covalent immobilization of the rN. The interaction between the protein and three clones of mAbs against SARS-CoV-2 rN with clone numbers 4G6, 7F10, and 1A6, were electrochemically registered in the range of concentrations. Three techniques, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and pulse amperometric detection (PAD) were used for the detection. A gradual change in the responses with an increase in mAbs concentration for all techniques was observed. To assess the performance of the developed electrochemical biosensor, ‘complexation constant’ (KC), limit of detection (LOD), and limit of quantification (LOQ) were calculated for all assessed clones of mAbs and all used techniques. Our results indicated that DPV possessing higher fitting accuracy illustrated more significant differences in KC constants and LOD/LOQ values. According to the DPV results, 7F10 clone was characterized with the highest KC value of 1.47 ± 0.07 μg/mL while the lowest LOD and LOQ values belonged to the 4G6 clone and equaled 0.08 ± 0.01 and 0.25 ± 0.01 μg/mL, respectively. Overall, these results demonstrate the potential of electrochemical techniques for the detection and distinguishing of different clones of mAbs against SARS-CoV-2 nucleocapsid protein.

An oxygen-insensitive amperometric galactose biosensor based on galactose oxidase co-immobilized with an Os-complex modified redox polymer

Monitoring galactose levels in dairy products can help to prevent severe complications with a hereditary metabolic disease such as galactosemia, a life-threatening disease. The current state of the art requires the development of less expensive, more reliable and specific methods to determine galactose levels in food in a practical way. We report the development and optimization of an amperometric biosensor for determination of galactose in dairy products based on galactose oxidase (GaOx) co-immobilized with an osmium-complex modified redox polymer on glassy carbon electrodes. To attain the maximum catalytic currents based on mediated electron transfer, two Os-complex based polymers with different redox potentials and different enzyme:redox polymer ratios were studied. The optimized GaOx-modified electrode gave a maximum electrocatalytic response of galactose oxidation that was not affected by the presence of O2, indicating fast wiring of the enzyme by the Os-complex modified redox polymer. The biosensor that gave the best analytical parameters for galactose detection was further tested for measuring galactose concentration in lactose-containing and lactose-free milk and yogurt samples under aerobic conditions. The results obtained with the amperometric biosensor were validated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Complete composition analysis of polysaccharides based on HPAEC-PAD coupled with quantitative analysis of multi-components by single marker.

Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. Using rhamnose as an internal reference standard, the contents of the other monosaccharidecomponents in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.

Fructan Catabolism by Rumen Microbiota of Cattle and Sheep

Fructans serve as the primary form of storage carbohydrate in cool-season grasses, but little is known about potential differences in ruminal fermentation of fructans between cattle and sheep. An ex vivo study was conducted to evaluate species differences in fructan catabolism. Buffered media containing ground orchardgrass (Dactylis glomerata L.) substrate was inoculated with uncultivated rumen microbiota obtained from cattle and sheep (n = 4 species−1). Fructan profiles were monitored over the incubation period (8 h; 39 °C) using high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD). In both species, disappearance of long-chain fructans (degree of polymerization [DP] > 8) was evident by 2 h of incubation (p < 0.01), whereas short-chain fructans (DP 4–8) increased from 0 to 2 h prior to subsequent degradation (p < 0.01). However, the overall rate of long-chain fructan catabolism was greater in bovine versus ovine fermentations, particularly between 2 and 4 h (p < 0.01). Additionally, rapid utilization of short-chain fructans occurred from 2 to 4 h in bovine fermentations, but was delayed in ovine fermentations, with substantial degradation occurring only after 4 h of incubation (p < 0.01). These results indicate that rumen microbiota of cattle may have a greater capacity for fructan degradation.

Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF–HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Determination of inorganic ions and carbohydrates in cardioplegia and nephroplegia solutions by ion chromatography.

In this study, methods for analyzing inorganic ions and carbohydrates in cardioplegia and nephroplegia solutions were developed and validated using ion chromatography with both conductivity and pulsed amperometric detection. The inorganic ions such as sodium, potassium, and calcium were separated by a cation-exchange column with 27mM methanesulfonic acid as mobile phase at 0.5mL/min. The anion (chloride) and carbohydrates (mannitol and glucose) were analyzed by an anion-exchange column using a mobile phase of 20mM sodium hydroxide at 1.0mL/min. The methods showed a high sensitivity for all analytes, with quantification limits from 0.0002 to 0.06mg/L. Good linearities between the peak areas and concentrations were found for all analytes within the selected concentration range (R2 >0.999). Relative standard deviation values for repeatability and interday precision were 0.1%-1.0% and 0.7%-1.6%, respectively. The accuracy was validated by determining the percentage recovery, which was between 98.0% and 101.3% for all analytes, indicating good accuracy of the methods. The robustness was verified by using an experimental design. Finally, real samples were analyzed to determine the content of the analytes. All assay values were between 96.8% and 102.5%.

Diabetes Management by Fourth-Generation Glucose Sensors Based on Lemon-Extract-Supported CuO Nanoporous Materials.

Diabetes is a major worldwide health issue, impacting millions of people around the globe and putting pressure on healthcare systems. Accurate detection of glucose is critical for efficient diabetes care, because it allows for prompt action to control blood sugar levels and avoid problems. Reliable glucose-sensing devices provide individuals with real-time information, allowing them to make more educated food, medicine, and lifestyle decisions. The progress of glucose sensing holds the key to increasing the quality of life for diabetics and lowering the burden of this prevalent condition. The present investigation addresses the synthesis of a CuO@lemon-extract nanoporous material using the sol-gel process. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the morphological properties of the composite, which revealed a homogeneous integration of CuO nanoparticles (NPs) on the surface of the matrix. The existence of primarily oxidized copper species, especially CuO, was confirmed by X-ray diffraction spectroscopy (XRD) investigation in combination with energy-dispersive X-ray (EDX) spectroscopy. The CuO@lemon-extract-modified glassy carbon electrode (CuO@lemon-extract GCE) performed well in non-enzymatic electrochemical sensing applications such as differential pulse voltammetry (DPV) and amperometric glucose detection. The electrode achieved a notable sensitivity of 3293 µA mM-1 cm-2 after careful adjustment, with a noticeable detection limit of 0.01 µM (signal-to-noise ratio of 3). The operational range of the electrode was 0.01 µM to 0.2 µM, with potential applied of 0.53 V vs. Ag/AgCl. These findings underscore the CuO@lemon-extract GCE's promise as a robust and reliable platform for electrochemical glucose sensing, promising advances in non-enzymatic glucose sensing (NEGS) techniques.