Chromatography-pulsed Amperometric Detection Research Articles

Efficient Biosynthesis of Theanderose, a Potent Prebiotic, Using Amylosucrase from Deinococcus deserti.

The study aimed to develop an efficient bioprocess for the discovery and synthesis of theanderose by using amylosucrase from Deinococcus deserti (DdAS). An unknown trisaccharide produced by DdAS was detected by high-performance anion-exchange chromatography-pulsed amperometric detection and high-performance liquid chromatography-evaporative light scattering detection, purified using medium-pressure liquid chromatography, and identified as theanderose (α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside) through nuclear magnetic resonance and mass spectrometry. DdAS synthesized theanderose with a 25.4% yield (174.1 g/L) using 2.0 M sucrose at 40 °C for 96 h. In an in vitro digestion model, theanderose showed a 6.5% hydrolysis rate over 16 h. Prebiotic efficacy tests confirmed that theanderose significantly enhanced the proliferation of selected Bifidobacterium strains in the culturing medium with theanderose as the main carbon source. Subsequently, fecal fermentation was performed by adding theanderose to the feces of 20 individuals of varying ages to assess its effect on the gut microbiota. Theanderose increased the relative abundance of Bifidobacteriaceae and Prevotellaceae while decreasing the population ratio of Lachnospiraceae and Ruminococcaceae. Conclusively, theanderose displayed excellent prebiotic potential when judged by low digestibility and selective growth of beneficial microbes over harmful microbes.

Pectins, hemicellulose and lignocellulose profiles vary in leaves among different aromatic Vanilla species (Orchidaceae)

The alcohol-insoluble residue in Vanilla dressleri, V. odorata, V. planifolia and V. pompona mature leaves was fractionated into water-, oxalate-, HCl- and NaOH-soluble pectins, hemicellulose and lignocellulose. Neutral sugars and uronic acids of each fraction were determined by high-performance anion exchange chromatography-pulsed amperometric detection. V. dressleri differentiated from the other three species based on the higher amounts of the HCl-soluble pectin fraction and the amount of glucose exceeding 5–7 times the values found in the NaOH-soluble pectin fraction in the other species, as well as a cellulose/hemicellulose ratio of ca. 2/3. Analogously, V. planifolia could also be distinguished by its higher cellulose/hemicellulose ratio of ca. 7. In addition to taxonomical implications, these results might be instrumental for defining adequate conditions for cell wall digestion aiming at protoplast isolation and appropriate media composition for cell wall regeneration during protoplast culture, which has been elusive in the Orchidaceae.

Comprehensive oligosaccharide profiling of commercial almond milk, soy milk, and soy flour

Oligosaccharides are known for several bioactivities on health, however, in sensitive individuals, can cause intestinal discomfort. This study aimed to investigate the oligosaccharide profiles in selected plant-based food products. A quantification method based on high-performance anion-exchange chromatography-pulsed amperometric detection was developed, validated, and used to measure major oligosaccharides. Additional low-abundant oligosaccharides and glycosides were characterized by liquid chromatography-tandem mass spectrometry and glycosidases. The summed concentration of raffinose, stachyose, and verbascose ranged from 0.12–0.19 mg/g in almond milk, 3.6–6.4 mg/g in soy milk, and 74–77 and 4.8–57 mg/g in defatted and full-fat soy four. Over 80 different oligosaccharides were characterized. Novel compounds, 2,3-butanediol glycosides, were identified in almond milk. Low-abundant oligosaccharides represented 25 %, 6 %, and 10 % of total OS in almond milk, soy milk, and soy flour, respectively. The data here are useful to estimate oligosaccharide consumption from dietary intake and facilitate further studies on their bioactivity.

Valorisation Potential of Invasive Acacia dealbata, A. longifolia and A. melanoxylon from Land Clearings.

Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild alkali pretreatments. For example, in A. dealbata stems treated with alkali and P. ostreatus, saccharification yield was 702.3 nmol mg−1, which is higher than the samples treated only with alkali (608.1 nmol mg−1), and 2.9-fold higher than the non-pretreated controls (243.9 nmol mg−1). By characterising biomass and pretreatments, generated data creates value for unused biomass resources, contributing to the implementation of sustainable biorefining systems. In due course, the generated value will lead to economic incentives for landowners to cut back invasive Acacia spp. more frequently, thus reducing excess biomass, which exacerbates wildfire risk.

Alterations of urinary perchlorate levels in euthyroid postpubertal children with autism spectrum disorder

BackgroundPerchlorates ClO4(−) are known environmental and food contaminants that act as inhibitors of iodine uptake by the thyroid gland; however, information concerning their possible association with the development of autism spectrum disorder (ASD) is still missing. The current study is first presenting the alterations in perchlorate urine levels in euthyroid children with ASD. ObjectivesTo examine urinary perchlorates and iodides in euthyroid children diagnosed with ASD, compared to age-, and BMI-matched neurotypical controls, and to verify the association between these two ions in ASD. MethodsIons were determined in 24 h urine samples determined by ion chromatography–conductivity cell detection (IC-CD) and ion chromatography–pulsed amperometric detection (IC-PAD) techniques, respectively, in a total of 130 postpubertal euthyroid children with normal BMI (the mean age 14.46 years, SD = 1.32; the mean BMI 20.6, SD = 1.37), divided into age- and BMI-matched groups of ASD patients and neurotypical, healthy children (control). ResultsThe ASD group presented with significantly higher perchlorate urine levels than the controls (median = 1.05 μg/L, interquartile range(IQR) = 1.5 versus median = 0.09 μg/L, IQR = 0.097, respectively), as well as lower iodide urine levels (median = 100.2 μg/L, IQR = 37 versus median = 156.95 μg/L, IQR = 26.11, respectively). The ASD group presented significantly lower TSH and higher free thyroid hormone (fT4, fT3) levels than the controls. In regression analyses, perchlorate urine levels showed significant positive relationships with normal BMI values and serum TSH, and inverse relationships with serum fT4. Urinary iodide levels showed significant inverse relationships with BMI values. The absence of ASD was associated with decreased odds of perchlorate urine levels (OR = 0.012, 95 % confidence interval [CI] 0.0002−0.76), and increased odds of iodide urine levels (OR = 1.15, 95 %CI 1.05–1.27). ConclusionsASD may have an independent and significant impact on perchlorate as well as iodide levels in urine of euthyroid lean postpubertal children. Perchlorate levels do not appear to be directly associated with iodide levels in euthyroid children.

Combining HPAEC-PAD, PGC-LC-MS, and 1D 1H NMR to Investigate Metabolic Fates of Human Milk Oligosaccharides in 1-Month-Old Infants: a Pilot Study.

A solid-phase extraction procedure was optimized to extract 3-fucosyllactose and other human milk oligosaccharides (HMOs) from human milk samples separately, followed by absolute quantitation using high-performance anion-exchange chromatography-pulsed amperometric detection and porous graphitized carbon-liquid chromatography–mass spectrometry, respectively. The approach developed was applied on a pilot sample set of 20 human milk samples and paired infant feces collected at around 1 month postpartum. One-dimensional 1H nuclear magnetic resonance spectroscopy was employed on the same samples to determine the relative levels of fucosylated epitopes and sialylated (Neu5Ac) structural elements. Based on different HMO consumption patterns in the gastrointestinal tract, the infants were assigned to three clusters as follows: complete consumption; specific consumption of non-fucosylated HMOs; and, considerable levels of HMOs still present with consumption showing no specific preference. The consumption of HMOs by infant microbiota also showed structure specificity, with HMO core structures and Neu5Ac(α2-3)-decorated HMOs being most prone to degradation. The degree and position of fucosylation impacted HMO metabolization differently.

Real-Time Measurement of Cellobiose and Glucose Formation during Enzymatic Biomass Hydrolysis

Enzymatic hydrolysis of lignocellulosic biomass for biofuel production relies on complex multi-enzyme ensembles. Continuous and accurate measurement of the released key products is crucial in optimizing the industrial degradation process and also investigating the activity and interaction between the involved enzymes and the insoluble substrate. Amperometric biosensors have been applied to perform continuous cellobiose measurements during the enzymatic hydrolysis of pure cellulose powders. The oxygen-sensitive mediators used in these biosensors restricted their function under physiological or industrial conditions. Also, the combined measurements of the hydrolysis products cellobiose and glucose require a high selectivity of the biorecognition elements. We employed an [Os(2,2′-bipyridine)2Cl]Cl-modified polymer and cellobiose dehydrogenase to fabricate a cellobiose biosensor, which can accurately and specifically detect cellobiose even in the presence of oxygen and the other main product glucose. Additionally, a glucose biosensor was fabricated to simultaneously measure glucose produced from cellobiose by β-glucosidases. The cellobiose and glucose biosensors work at applied potentials of +0.25 and +0.45 V versus Ag|AgCl (3 M KCl), respectively, and can selectively detect their substrate. Both biosensors were used in combination to monitor the hydrolysis of pure cellulose of low crystallinity or industrial corncob samples. The obtained results correlate with the high-performance liquid chromatography pulsed amperometric detection analysis and demonstrate that neither oxygen nor the presence of redox-active compounds from the lignin fraction of the corncob interferes with the measurements.

Construction of fungi-microalgae symbiotic system and adsorption study of heavy metal ions

Harvesting and immobilization are problem in microalgae water treatment. Here, mycelial pellets (Aspergillus fumigatus) and Synechocystis sp. PCC6803 comprised a fungi-microalgae symbiotic system (FMSS) of hypha-pellet flocculant auxiliary microalgae solidification. Optical and scanning electron microscopy (SEM) revealed that green particles were attached to fungal mycelia. A 98% adsorption and immobilization efficiency of mycelium pellets to microalgae was evident. Under the condition of 108 cells/mL logarithmic phase microalgae and no fungal culture medium, the symbiotic system had higher stability and better Cd(II) adsorption efficiency (98.89%, 37.3 mg g−1). Meanwhile, the symbiotic system prolonged the growth cycle of microalgae in the adsorption. After Cd(II) ion stimulation, the content of total extracellular polymeric substances (EPS) in the FMSS was significantly higher than that in the control group, and the change of extracellular polysaccharide was more obvious. High performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) showed that the monosaccharide composition of extracellular polysaccharides increased. Three dimensional excitation emission matrix (3D-EEM) spectrum analysis showed the peak intensity of protein fluorescence changed with the treatment of heavy metals. FTIR spectra showed that the functional groups (such as OH, CONH) of EPS participated in the adsorption process. The findings showed that the key components of EPS, extracellular polysaccharides and extracellular proteins, played an important role in the symbiotic system to cope with the stress of heavy metal and resist the toxicity of heavy metal. The preliminary data can inform development and utilization of engineering strains and gene transformation.

Development and Validation of an Analytical Method for the Quantification of Arabinose, Galactose, Glucose, Sucrose, Fructose, and Maltose in Fruits, Vegetables, and Their Products

The paper presents a method for the simultaneous determination of arabinose, galactose, glucose, sucrose, fructose, and maltose in fruits, vegetables, and their products using ion chromatography-pulsed amperometric detection on a gold working electrode. Sample preparation involved simple extraction, dilution with water, and filtration before injection into the system. Separation of the analytes was performed with gradient elution on a Thermo Scientific CarboPac PA20 Analytical Column (3.0 × 150 mm) and a Guard column (2 mm × 50 mm) with a mobile phase. The optimized parameters were an eluent concentration of 10 mmol/L, a column temperature of 30 °C, and a flow rate of 0.4 mL/min. In methodological evaluation, parameters (selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), relative standard deviation (RSD), recovery percentages, and CV%) resulted in compliance with reference values at each level of fortification. All validation parameters were acceptable in the characterization of sugars in fruits, vegetables, and their products. After method validation, we analyzed the sugar content in fresh and dried jujube. The method was accurate, reliable, and reproducible and can be used for the determination of arabinose, galactose, glucose, sucrose, fructose and maltose in fruits, vegetables, and their products.

Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids.

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4-N-acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for Shigella sonnei O-antigen, Streptococcus pneumoniae serotype 12F, and Staphylococcus aureus types 5 and 8 capsular polysaccharides.

Comparison of macronutrient content in human milk measured by mid-infrared human milk analyzer and reference methods

ObjectiveThe study aims at evaluating mid-infrared human milk analyzer (HMA) accuracy and precision, in human milk (HM).Study designRöse-Gottlieb, high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), Kjeldahl and amino acid analysis (AA) were selected as references for total fat, lactose and total protein determination.ResultsNo significant difference was observed in lactose content between HMA and HPAEC-PAD. Significant differences were observed in fat and protein content between HMA and reference methods. However, the difference in fat content was lower than 12%, and therefore within the variability declared by supplier. For protein determination, the BCA protein assay was selected. No significant differences were observed in total protein content measured by BCA assay, Kjeldahl and AA methods.ConclusionsHMA was reliable for the quantification of total fat and lactose content, but not for total protein one. The latter was measured by BCA assay, which yielded comparable results to Kjeldahl and AA methods.

Determination of cyanide in bamboo shoots by microdiffusion combined with ion chromatography-pulsed amperometric detection.

A practical method for the determination of cyanide in bamboo shoots has been developed using microdiffusion preparation integrated with ion chromatography–pulsed amperometric detection (IC-PAD). Cyanide was released from bamboo shoots after Conway cell microdiffusion, and then analysed by IC-PAD. In comparison with the previously reported methods, derivatization and ion-pairing agent addition were not required in this proposed microdiffusion combined with IC-PAD method. The microdiffusion parameters were optimized including hydrolysis systems, temperature, time, and so on. Under the optimum conditions, the linear range of the calibration curve for cyanide was 0.2–200.0 µg kg−1 with satisfactory correlation coefficients of 0.9996 and the limit of detection was 0.2 µg kg−1 (S/N = 3). The spiked recovery range was from 92.8 to 98.6%. The intra-day and inter-day relative standard deviations of cyanide were 2.7–14.9% and 3.0–18.3%, respectively. This method was proved to be convenient in operation with high sensitivity, precision and accuracy, and was successfully applied in the determination of cyanide in bamboo shoot samples.

In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry

Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS2 in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS2 permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

ABSTRACTFungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity.IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

Anticancer Activity of Sulfated Polysaccharides Isolated from the Antarctic Red Seaweed Iridaea cordata

This study aimed to isolate and characterize sulfated polysaccharides (SPs) from Iridaea cordata and evaluate their anticancer activity. SPs of the Antarctic red seaweed were obtained by CaCl₂ (SP1) and ethanol precipitations (SP2) following diluted acid extraction at room temperature. Yields of SP1 and SP2 were approximately 14% and 23%, respectively, of the dry weight of red seaweed. The average molecular mass of the SP1 and SP2 was estimated about 1.84 × 10³ and 1.42 × 10³ kDa, respectively, by size-fractionation High-Performance Liquid Chromatography (HPLC). From the High-Performance Anion-Exchange Chromatography-Pulsed Amperometric Detection (HPAEC-PAD) analysis, the main monosaccharide was galactose with glucose and fucose as minor components. The sulfate content of SP2 (40.4%) was slightly higher than that of SP1 (33.8%). The FT-IR spectra also showed characteristic band of carrageenan-like sulfated polysaccharides. Taken together the SPs are thought to be carrageenan-like sulfated galactan. The polysaccharides (SPs) from I. cordata exhibited weak antitumor activity against PC-3 (prostate cancer), HeLa (cervical cancer), and HT-29 (human colon adenocarcinoma). To our knowledge, this is the first data on biological activity of the Antarctic red seaweed I. cordata.

Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Liquid Chromatography-Pulsed Amperometric Detection with Column Switching: Collaborative Study, Final Action 2011.18.

AOAC First Action Method 2011.18, Myo-Inositol (Free and Bound as Phosphatidylinositol) in Infant and Pediatric Formulas and Adult Nutritionals, was collaboratively studied. With this method free myo-inositol and phosphatidylinositol bound myo-inositol are extracted using two different sample preparation procedures, separated by ion chromatography using a combination of Dionex Carbo Pac PA1 and MA1 columns with column switching, and detected with pulsed amperometry using a gold electrode. Free myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica SPE cartridges. Myo-inositol is then released from the glycerol backbone with concentrated acetic and hydrochloric acids at 120°C. During this collaborative study, nine laboratories from five different countries analyzed blind duplicates of nine infant and pediatric nutritional formulas for both free and phosphatidylinositol bound myo-inositol, and one additional laboratory only completed the free myo-inositol analyses. The method demonstrated acceptable repeatability and reproducibility and met the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs®) for free myo-inositol plus phosphatidylinositol bound myo-inositol for all the matrixes analyzed. SMPRs for repeatability were ≤5% RSD at myo-inositol concentrations of 2-68 mg/100 g ready-to-feed (RTF) liquid. SMPRs for reproducibility were ≤8% RSD in products with myo-inositol concentrations ranging from 2 to 68 mg/100 g RTF liquid. During this collaborative study, repeatability RSDs ranged from 0.51 to 3.22%, and RSDs ranged from 2.66 to 7.55% for free myo-inositol plus phosphatidylinositol bound myo-inositol.

Short communication: Quantification of carbohydrates in whey permeate products using high-performance anion-exchange chromatography with pulsed amperometric detection

A method was developed for the characterization and quantification of the disaccharide lactose and 3 major bovine milk oligosaccharides (BMO) in dairy streams. Based on high-performance anion-exchange chromatography-pulsed amperometric detection (HPAE-PAD), this method is advantageous because it requires minimal sample preparation and achieves good chromatographic separation of oligosaccharide isomers within 30min. The linear dynamic range and limit of detection were 0.1 to 10mg/L and 0.03 to 0.22mg/L, respectively. Mean recoveries of the BMO were excellent and ranged from 98.4 to 100.4%. Without complicated sample preparation procedures, this HPAE-PAD method measured BMO [3′-sialyllactose (3′SL), 6′-sialyllactose (6′SL), and 6′-sialyllactosamine (6′SLN)] and lactose using a single instrument, therefore increasing the accuracy of the measurement and applicability for the dairy industry. In colostrum whey permeate, 3′SL, 6′SL, and 6′SLN were 94, 29, and 46mg/L, respectively. This work is the first to demonstrate that some commercial products, currently marketed for supporting a healthy immune system, contain significant amounts of bioactive BMO and therefore, carry additional bioactivities.

Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Ion Chromatography-Pulsed Amperometric Detection and Column Switching: Collaborative Study, Final Action 2011.18

Determination of Myo-Inositol in Infant, Pediatric, and Adult Formulas by Ion Chromatography-Pulsed Amperometric Detection and Column Switching: Collaborative Study, Final Action 2011.18

HPAEC-PAD quantification of Haemophilus influenzae type b polysaccharide in upstream and downstream samples

Due to the rapidly increasing introduction of Haemophilus influenzae type b (Hib) and other conjugate vaccines worldwide during the last decade, reliable and robust analytical methods are needed for the quantitative monitoring of intermediate samples generated during fermentation (upstream processing, USP) and purification (downstream processing, DSP) of polysaccharide vaccine components. This study describes the quantitative characterization of in-process control (IPC) samples generated during the fermentation and purification of the capsular polysaccharide (CPS), polyribosyl-ribitol-phosphate (PRP), derived from Hib. Reliable quantitative methods are necessary for all stages of production; otherwise accurate process monitoring and validation is not possible. Prior to the availability of high performance anion exchange chromatography methods, this polysaccharide was predominantly quantified either with immunochemical methods, or with the colorimetric orcinol method, which shows interference from fermentation medium components and reagents used during purification. Next to an improved high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method, using a modified gradient elution, both the orcinol assay and high performance size exclusion chromatography (HPSEC) analyses were evaluated. For DSP samples, it was found that the correlation between the results obtained by HPAEC-PAD specific quantification of the PRP monomeric repeat unit released by alkaline hydrolysis, and those from the orcinol method was high (R2=0.8762), and that it was lower between HPAEC-PAD and HPSEC results. Additionally, HPSEC analysis of USP samples yielded surprisingly comparable results to those obtained by HPAEC-PAD. In the early part of the fermentation, medium components interfered with the different types of analysis, but quantitative HPSEC data could still be obtained, although lacking the specificity of the HPAEC-PAD method. Thus, the HPAEC-PAD method has the advantage of giving a specific response compared to the orcinol assay and HPSEC, and does not show interference from various components that can be present in intermediate and purified PRP samples.

Development of a liquid chromatographic method for the quantification of paromomycin. Application to in vitro release and ex vivo permeation studies

We have developed a reversed phase high performance liquid chromatography pulsed amperometric detection (RPHPLC-PAD) method for the determination of paromomycin. It is sensitive, repeatable, and selective without the pretreatment step.Trifluoroacetic acid–water was utilized as the eluent and detected by PAD under NaOH alkaline conditions. The paromomycin detection limit (S/N=3.3) was 2μgmL−1 and the quantification limit (S/N=10) was 6μgmL−1. Coefficients of linear regression were higher than 0.99 for concentrations between 6.25 and 200μgmL−1. The intra and inter-day precision (RSD) was less than 6.5%. The average recoveries were 97.53–102.01%. The proposed HPLC-PAD method presented advantageous performance characteristics and it can be considered suitable for the evaluation of paromomycin loaded nanogel formulation in ex vivo permeation and in vitro release studies.