Monosaccharide Analysis Research Articles

Analytical method development and validation for monosaccharide profiling in Lignosus rhinocerotis using rP-HPLC

Lignosus rhinocerotis is rich in polysaccharide with diverse ­bioactivities. This study developed a pre-column derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method for analyzing monosaccharides in Lignosus rhinocerotis polysaccharides (LRP). LRP underwent hydrolysis, derivatization, and separation on a Cosmosil 5C18-MS-II column at 254 nm. Baseline separation of eight standard monosaccharides was achieved within 45 min. Calibration curves, precision, and accuracy were validated. Quantitative analysis revealed LRP as a heteropolysaccharide containing mannose, ribose, rhamnose, glucose, galactose, xylose, and arabinose, with 100.28-111.02% recovery. This optimized RP-HPLC offers a simple, reproducible, and accurate tool for LRP monosaccharides analysis, facilitating in understanding its structure-function relationship.

Simultaneous determination of naphthalimide-labelled monosaccharides in P. cyrtonema Hua. polysaccharides utilizing the HPLC-UV technique.

4-Amidogen-1,8-naphthalimide (ANA), a novel pre-column derivatization reagent, has been successfully synthesized and utilized for the highly sensitive analysis of monosaccharides. ANA reacts with the reducing carbonyl groups of saccharides, facilitating monosaccharide detection. The resulting monosaccharide derivatives were meticulously investigated using High-Performance Liquid Chromatography (HPLC) coupled with ultraviolet (UV) spectroscopy. The derivatization process was conducted in an environmentally friendly solvent mixture of EtOH and AcOH at a constant temperature of 100 °C for a duration of 4 hours across all monosaccharides. After derivatization, the separated adducts were eluted through a ZORBAX SB-C8 column within a mere 22 minutes at a flow rate of 0.8 mL min-1, with detection occurring at a wavelength of 416 nm. By employing this derivatization technique, the comprehensive analysis of the monosaccharide composition in P. cyrtonema Hua. polysaccharides (PCPs) following strong acid hydrolysis was achieved. The monosaccharide profiles of PCPs predominantly consist of glucose (Glc), galactose (Gal), mannose (Man), xylose (Xyl), and galacturonic acid (GalA), with a molar ratio of Glc : Gal : Man : Xyl : GalA of 20.26 : 12.76 : 24.24 : 41.91 : 0.83. The LOD values for the six sugars ranged from 27.37 to 84.37 μg mL-1. The validated HPLC method, characterized by its exceptional precision and accuracy, has successfully facilitated both rapid qualitative and quantitative determination of PCPs, thereby establishing a robust quality control protocol for P. cyrtonema Hua.

Deepening the Role of Pectin in the Tissue Assembly Process During Tomato Grafting.

Cell walls play essential roles in cell recognition, tissue adhesion, and wound response. In particular, pectins as cell-adhesive agents are expected to play a key role in the early stages of grafting. To test this premise, this study focused on examining the dynamics of the accumulation and degree of methyl-esterification of pectic polysaccharides at the graft junctions using tomato autografts as an experimental model. Monosaccharide analysis showed a marked increase in homogalacturonan from 25% to 32 or 34% at the junction zones early after grafting. In addition, a decrease in the degree of homogalacturonan methyl-esterification up to 38% in the scion and 64% in the rootstock was observed in the first few days after grafting, accompanied by an increase in pectin methyl-esterase activity of up to 20-30% in the tissues surrounding the graft junction. These results shed light on the role of homogalacturonan in grafting and reinforce the key function of pectin as one of the most relevant cell wall components during the grafting process.

Structural and genetic relationship of the O-antigens of the type strains <I>Azospirillum agricola</I> CC-HIH038 and <I>Azospirillum doebereinerae</I> GSF71

Lipopolysaccharide was isolated from cells of the type strain of rhizobacteria Azospirillum agricola CC-HIH038Т by phenol extraction. O-specific polysaccharide was obtained by mild acid hydrolysis of lipopolysaccharide followed by chromatographic fractionation. On the basis of monosaccharide analysis, including determination of absolute configurations, 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the O-specific polysaccharide repeating unit of A. agricola CC-HIH038T was elucidated: →3)-α-L-Rhap2Ac-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-β-D-GlcpNAc6Ac-(1→ , which is structurally related to A. doebereinerae GSF71T . Based on the analysis of full-genome sequencing data for strains A. agricola CC-HIH038T and A. doebereinerae GSF71T the O-specific polysaccharide biosynthesis loci were identified, which were characterized by a similar organization and a high level of gene homology, confirming the common structure of the O-antigens of these strains.

Negative effect and removal of trace amounts of 1,3-dialkylimidazolium ionic liquids in samples from biorefineries

Ionic liquids (ILs), based on 1,3-dialkylimidazolium cations, are frequently used solvent components or auxiliaries for various types of biomass in biorefinery approaches. Unless washing and sample preparation have been carried out very carefully, analytical samples often contain residual traces of such ionic liquids. These residues can compromise the quality of physicochemical analyses, as was demonstrated for monosaccharide analysis after hydrolysis by gas chromatography, high-performance thin-layer chromatography, or ion chromatography (IC), and even damage analytical equipment, such as gas chromatographic capillaries or IC electrodes. We suggest a simple procedure—short stirring with solid elemental sulfur adsorbed on alumina as the scavenger—as a pretreatment of the analytical samples to remove interfering imidazolium IL traces. The chemistry underlying this pretreatment is the conversion of the 1,3-dialkylimidazolium cation to the corresponding, water-insoluble, neutral, volatile 1,3-dialkylimidazole-2-thiones. Given the negative effect of imidazolium IL impurities, the minor extension of the sample preparation by one short additional step appears to be a small price to pay for an unperturbed and instrument-safe analysis.Graphical abstract

Unveiling a novel exopolysaccharide produced by Pseudomonas alcaligenes Med1 isolated from a Chilean hot spring as biotechnological additive

Exopolysaccharides (EPSs), a constitutive part of bacterial biofilm, act as a protecting sheath to the extremophilic bacteria and are of high industrial value. In this study, we elucidate a new EPS produced by thermotolerant (growth from 34–44 °C) strain Pseudomonas alcaligenes Med1 from Medano hot spring (39.1 °C surface temperature, pH 7.1) located in the Central Andean Mountains of Chile. Bacterial growth was screened for temperature tolerance (10–60 °C) to confirm the thermotolerance behaviour. Physicochemical properties of the EPS were characterized by different techniques: Scanning Electron Microscopy- Energy Dispersive X-ray Spectroscopy (SEM-EDS), Atomic Force Microscopy (AFM), High-Performance Liquid Chromatography (HPLC), Gel permeation chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), and Thermogravimetric analysis (TGA). Whole genome of P. alcaligenes Med1 has also been studied in detail to correlate the structural and functional characteristics with genomic insight. The EPS demonstrated amorphous surface roughness composed of evenly distributed macromolecular lumps composed of mainly carbon and oxygen. The monosaccharide analysis has shown the presence of glucose, galactose, and mannose sugars at different ratios. TGA revealed the high thermal stability (315.3 °C) of the polysaccharide. The GPC has shown that Med1 is a low molecular weight polysaccharide (34.8 kDa) with low PI. The 2D-NMR linkage analysis suggests a diverse array of glycosidic bonds within the exopolysaccharide structure. The functional properties of the EPS were evaluated for food industry applications, specifically for antioxidant (DPPH, FRAP an H2O2). Extracted Med1 EPS revealed significant emulsification activity against different food grade vegetative oils (Coconut oil, Corn oil, Canola oil, Avocado oil, Sunflower oil, Olive oil, and Sesame oil). The highest 33.9% flocculation activity was observed with 60 mg L−1 EPS concentration. It showed water-holding (WHC) of 107.6% and oil-holding (OHC) capacity of 110.8%. The functional EPS produced by Pseudomonas alcaligenes Med1 from Central Andean Chilean hot spring of central Chile can be a useful additive for the food-processing industry.

Monosaccharide composition and glycosidic linkages analysis by ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry—Case study of plant polysaccharides

Monosaccharide composition and glycosidic linkages analysis are essential for the structural characterization and biological activity research of polysaccharides. To simplify the analysis steps and improve detection efficiency, this study developed monosaccharide compositions and glycosidic linkages detection methods based on UPLC-QqQ-MS/MS, and established a plant polysaccharide glycosidic linkages library. Furthermore, the detailed analysis process of monosaccharide compositions and glycosidic linkages was presented through a plant polysaccharide (Chinese bayberry wine polysaccharide, CPW) example. The results showed that the monosaccharide analysis method could identify and quantify 20 monosaccharides within 13 min by 6 chromatograms. By this method, 7 monosaccharides were detected in CPW, which was consistent with previous results obtained using ion chromatography. Meanwhile, the UPLC-QqQ-MS/MS based method could analyze 112 glycosidic linkages of 9 plant monosaccharides within 30 min, and the results were distributed on 11 chromatograms. According to the approach, 44 different glycosidic linkages were identified in CPW. Based on Congo red assay and molecular weight as well as our findings of monosaccharide composition and glycosidic linkages, we proposed that CPW may consist of two distinct sugar chains: One composed of 1–2,4-Rha, 1–3,4-GalA, and 1-F-Ara, and the other composed of 1–3,4-Gal, 1–4-Gal, 1–3,4,6-Gal, and 1-F-Ara.

Low‐field nuclear magnetic resonance relaxometry: a new approach for monosaccharide identification in sugar solutions

SummaryMonosaccharides such as glucose, fructose, and galactose are the building blocks of oligosaccharides and act as the major energy sources in our body. These three monosaccharides are the most prominent in food processing and widely consumed analytes, found in fruits or milk. Although several analytical methods are available to identify and quantify sugar solutions, their drawbacks urge the search for better possible alternatives. A simple, rapid, and efficient method for identifying monosaccharides (ᴅ‐fructose, ᴅ‐galactose, and ᴅ‐glucose) was investigated using low‐field nuclear magnetic resonance (LF‐NMR), exploring the applicability and reliability of this machine. The transverse (T2) relaxation curve was analysed to distinguish monosaccharides from each other. The increasing monosaccharide concentration in the sugar solution causes leftward shifts in signals, indicating a gradual reduction in the mobility of water molecules. The addition of sugar generated a secondary peak, which assisted in identifying monosaccharides. Notably, fructose exhibited distinct behaviour from that of glucose and galactose. The regression coefficient consistently exceeded 0.98, indicating the reliability of this experiment. LF‐NMR encountered challenges in differentiating stereo‐isomeric glucose and galactose. However, quantification of monosaccharides from known binary mixtures (water and one monosaccharide) is possible by preparing standard curves of different concentrations of sugars. These standard curves can be either T2 or the total surface area of the peaks as a function of sugar concentration. The study concludes that LF‐NMR has potential for the qualitative and quantitative analysis of monosaccharides in pure solutions or powdered samples. It is hoped that the rapid and easy way of monosaccharide identification by simple analysis of peaks obtained from NMR spectra will enhance its accessibility for users. However, it also acknowledges limitations in applying this method to complex systems (mixtures of different sugars in a solution).

Novel glucomannan-like polysaccharide from Lycium barbarum L. ameliorates renal fibrosis via blocking macrophage-to-myofibroblasts transition

The macrophage to myofibroblasts transition (MMT) has been reported as a newly key target in renal fibrosis. Lycium barbarum L. is a traditional Chinese medicine for improving renal function, in which its polysaccharides (LBPs) are the mainly active components. However, whether the role of LBPs in treating renal fibrosis is related to MMT process remain unclear. The purpose of this study was to explore the relationship between the regulating effect on MMT process and the anti-fibrotic effect of LBPs. Initially, small molecular weight LBPs fractions (LBP-S) were firstly isolated via Sephadex G-100 column. Then, the potent inhibitory effect of LBP-S on MMT process was revealed on bone marrow-derived macrophages (BMDM) model induced by TGF-β. Subsequently, the chemical structure of LBP-S was elucidated through monosaccharide, methylation and NMR spectrum analysis. In vivo biodistribution characteristics studies demonstrated that LBP-S exhibited effectively accumulation in kidney via intraperitoneal administration. Finally, LBP-S showed a satisfactory anti-renal fibrotic effect on unilateral ureteral obstruction operation (UUO) mice, which was significantly reduced following macrophage depletion. Overall, our findings indicated that LPB-S could alleviate renal fibrosis through regulating MMT process and providing new candidate agents for chronic kidney disease (CKD) related fibrosis treatment.

The Pre- and Post-Column Derivatization on Monosaccharide Composition Analysis, a Review.

Polysaccharides, as common metabolic products in organisms, play a crucial role in the growth and development of living organisms. For humans, polysaccharides represent a class of compounds with diverse applications, particularly in the medical field. Therefore, the exploration of the monosaccharide composition and structural characteristics of polysaccharides holds significant importance in understanding their biological functions. This review provides a comprehensive overview of extraction methods and hydrolysis strategies for polysaccharides. It systematically analyzes strategies and technologies for determining polysaccharide composition and discusses common derivatization reagents employed in further polysaccharide studies. Derivatization is considered a fundamental strategy for determining monosaccharides, as it not only enhances the detectability of analytes but also increases detection sensitivity, especially in liquid chromatography (LC), capillary electrophoresis (CE), and gas chromatography (GC) techniques. The review meticulously examines pre-column and post-column derivatization techniques for monosaccharide analysis, categorizing them based on diverse detection methodologies. It delves into the principles and distinctive features of various derivatization reagents, offering a comparative analysis of their strengths and limitations. Ultimately, the aim is to provide guidance for selecting the most suitable derivatization approach, taking into account the structural nuances, biological functions, and reaction dynamics of polysaccharides.

Pre-column Derivative HPLC and LC–Orbitrap-MS Analysis of Monosaccharides and Non-polysaccharides in Polygonati Rhizoma

Pre-column Derivative HPLC and LC–Orbitrap-MS Analysis of Monosaccharides and Non-polysaccharides in Polygonati Rhizoma

Comparison of kink-band structures and specificities of cell wall polysaccharides in modern and ancient flax fibres

Flax (Linum usitatissimum L.) is a plant of industrial importance, its fibres being presently used for high-value textile applications, composite reinforcements as well as natural actuators. Human interest in this fibre-rich plant dates back several millennia, including to Ancient Egypt where flax was used extensively in various quotidian items. While the recent technical developments of flax fibres continue to diversify through scientific research, the historical use of flax also has rich lessons for today. Through careful examination of ancient Egyptian and modern flax fibres, this study aims to conduct a multi-scale characterization from the yarn to the fibre cell wall scale, linking differences in structure and polysaccharide content to the mechanical performance and durability of flax. Here, a multi-scale biochemical study is enriched by scanning electron microscopy and nanomechanical investigations. A key finding is the similarity of cellulose features, crystallinity index and local mechanical performances between ancient and modern fibres. Biochemically speaking, monosaccharides analysis, deep-UV and NMR investigations demonstrate that ancient fibres exhibit less pectins but a similar hemicellulosic content, especially through uronic acids and galactose, suggesting the sensitivity of these non-crystalline components.

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

An optimize adaptable method for determining the monosaccharide composition of pectic polysaccharides

Pectic polysaccharides are considered the highly complex natural plant polysaccharides which plays a vital role in plant tissue structure and human health. Detailed characterization of the monosaccharide composition can provide insights into the pectic polysaccharide structure. Nevertheless, when analyzing the monosaccharides of pectic polysaccharide, it is crucial to address the issue of incomplete hydrolysis that can occur due to the formation of acid-induced precipitates. Based on above, the main purpose of this article is to provide an optimized method for monosaccharide analysis of pectic polysaccharides through acid hydrolysis optimization using high-performance anion exchange chromatography (HPAEC) The results indicate that reducing the sample concentration to 0.5 mg/mL effectively reduces the acid gelling phenomenon and promotes the complete hydrolysis of pectin polysaccharides. The optimized parameters for acid hydrolysis involve 110 °C for 6 h in 2 M TFA. Furthermore, the consistency of this method is assessed, along with its ability to analyze pectin polysaccharides from various fruits. This hydrolysis approach holds promise for enabling accurate quantification of monosaccharide composition in pectic polysaccharides.

The Role of Grifola frondosa Polysaccharide in Preventing Skeletal Muscle Atrophy in Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a burgeoning public health challenge worldwide. Individuals with T2DM are at increased risk for skeletal muscle atrophy, a serious complication that significantly compromises quality of life and for which effective prevention measures are currently inadequate. Emerging evidence indicates that systemic and local inflammation stemming from the compromised intestinal barrier is one of the crucial mechanisms contributing to skeletal muscle atrophy in T2DM patients. Notably, natural plant polysaccharides were found to be capable of enhancing intestinal barrier function and mitigating secondary inflammation in some diseases. Herein, we hypothesized that Grifola frondosa polysaccharide (GFP), one of the major plant polysaccharides, could prevent skeletal muscle atrophy in T2DM via regulating intestinal barrier function and inhibiting systemic and local inflammation. Using a well-established T2DM rat model, we demonstrated that GFP was able to not only prevent hyperglycemia and insulin resistance but also repair intestinal mucosal barrier damage and subsequent inflammation, thereby alleviating the skeletal muscle atrophy in the T2DM rat model. Additionally, the binding free energy analysis and molecular docking of monosaccharides constituting GFP were further expanded for related targets to uncover more potential mechanisms. These results provide a novel preventative and therapeutic strategy for T2DM patients.

Metabolite Compounds of Euglena sp. on Mass Cultivation System under MgCl2 and CaCl2 Salt Stress

Euglena sp. is rich in primary and secondary metabolite compounds. This cosmopolitan organism can survive extreme conditions such as salt stress because it can increase cell growth rate and metabolism. This mass cultivation research uses an open pond system that is very vulnerable to contamination, so variations in salinity of MgCl2 and CaCl2 are used as treatments because they minimize contamination. The research examines the correlation of salt types to cell growth rate, biomass, monosaccharide profile, paramylon, pigment, and lipid of Euglena sp. Research methods include preparation, medium making, mass cultivation, and measurement of test parameters. Data were analyzed through One-Way ANOVA followed by DMRT, with a 5% confidence level. Based on the research result, the highest value of biomass and paramylon content and productivity at CaCl2 treatment with consecutive values 0.013 ± 0.001, 0.002 ± 0.000, 9.556 ± 0.070, and 0.149 ± 0.019. Monosaccharides analysis produced in the form of sucrose, glucose, fructose, and arabinose amounted to < 10 ppm. CaCl2 treatment produced the highest sucrose of 6251 ppm. The highest chlorophyll-a and carotenoid pigments are located at CaCl2 treatment, respectively (1.698 ± 0.051)b and (0.500 ± 0.032)b. At the same time, the salt treatment has a significant effect on lipid content. To summarize, almost all metabolite compounds were highest in CaCl2 treatment. This research is expected to contribute to developing the Euglena sp. mass cultivation system as a source of bioenergy and biomaterials.

Development of a protocol for fractionating and characterising fibres from lignocellulosic food waste

This study aims to explore an advanced protocol for characterising dietary fibre (DF) fractions to meet the growing demand for accurate and reliable data. Although current enzymatic-gravimetric approaches, e.g., AOAC and Van Soest analysis, provide information about soluble and insoluble DF quantification, they present limitations related to the lack of fractions characterisation. To overcome these limitations, the proposed protocol integrates the official AOAC 991.43 method with the sequential fibre fractionation by exploiting the different resistance of the fibre fractions to acid hydrolysis treatments (TFA and H2SO4), utilising hazelnut shells as a case-study. Each hydrolysed fraction was quantified and characterised through GC–MS analysis of monosaccharides. The data obtained for hemicellulose, cellulose, and lignin fractions were then discussed and compared with the Van Soest method. This approach yields a comprehensive procedure applicable to different food and nutraceutical products, emphasising the importance of DF characterisation for a deeper understanding of their bio-functional properties.

Decoding Polysaccharides from Two Pichia Yeasts and Their Molecular Interaction with Wine Fruity Esters.

This study extensively characterized yeast polysaccharides (YPs) from Pichia fermentans (PF) and Pichia kluyveri (PK), with a specific focus on their structural attributes and their interaction with wine fruity esters in a model wine system. By finely tuning enzymatic reactions based on temperature, pH, and enzyme dosage, an optimal YP yield of 77.37% was achieved, with a specific mass ratio of cellulase, pectinase, and protease set at 3:5:2. There were four YP fractions (YPPF-W, YPPF-N, YPPK-W, and YPPK-N) isolated from the two yeasts. YPPF-N and YPPK-N were identified as glucans based on monosaccharide analysis and Fourier-transform infrared spectroscopy analysis. "Specific degradation-methylation-nuclear magnetic" elucidated YPPF-W's backbone structure as 1,3-linked α-l-Man and 1,6-linked α-d-Glc residues, while YPPK-W displayed a backbone structure of 1,3-linked α-Man residues, indicative of a mannoprotein nature. Isothermal titration calorimetry revealed spontaneous interactions between YPPK-W/YPPF-W and fruity esters across temperatures (25-45 °C), with the strongest interaction observed at 30 °C. However, distinct esters exhibited varying interactions with YPPK-W and YPPF-W, attributed to differences in molecular weights and hydrophobic characteristics. While shedding light on these intricate interactions, further experimental data is essential for a comprehensive understanding of yeast polysaccharides' or mannoproteins' impact on fruity esters. This research significantly contributes to advancing our knowledge of yeast polysaccharides' role in shaping the nuanced sensory attributes of wine.

Methylation-GC-MS/FID-Based Glycosidic Linkage Analysis of Unfractionated Polysaccharides in Red Seaweeds.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species.

Sensitive, precise fingerprint profiling for monosaccharide analysis of Bacillus Calmette-Guérin polysaccharide and nucleic acid isolates

A sensitive and precise HPLC-DAD method with pre-column PMP derivatization was established and validated, for analyzing the polysaccharides in Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN) isolates, after acid hydrolysis. And the HPLC fingerprint profiling was used to analyze its monosaccharide composition. The monosaccharide concentration-peak area calibration curve was of good linearity (R2 > 0.99), over the range of 0.016–0.08 mg/mL for mannose or 0.24–1.20 mg/mL for glucose, with high recovery of 93–105 % for quality control samples. The intra-day RSD values of mannose and glucose concentration were less than 2.5 % and 2.1 %, respectively, and their inter-day RSD values were less than 4.3 % and 2.2 %, respectively, and remained stable for up to 14 days. This method also remained durable against changes in chromatographic parameters, but it's susceptible to the flow rate of mobile phase. Additionally, the method was applied to analyze the content of mannose and glucose in 22 batches BCG-PSN powder and 17 batches BCG-PSN injection. The results showed that the HPLC-DAD fingerprint spectra of all the BCG-PSN powder and BCG-PSN injection samples had a high degree of similarity, with the similar indexes up to 0.999 and 0.998, respectively. The HPLC-DAD method with pre-column PMP derivatization is highly rapid, effective, visual, and accurate for determination of monosaccharide contents. The validated method was successfully applied to the analysis of polysaccharide in both BCG-PSN powder and injection.