High-performance Anion-exchange Chromatography Method Research Articles

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.

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

A Validated Method for Detection and Quantitation of 2’Fucosyllactose in Infant Formula Matrices

Analytical methods to assess the content of free carbohydrates in solution range from simple tests of reductive power to combinations of chromatography and mass spectrometry. Soluble carbohydrates such as lactose, maltose, fructooligosaccharides, and galactooligosaccharides are commonly found in infant formulas either as sources of energy or soluble fibers. On the other hand, a rich repertoire of lactose-based carbohydrates occurs naturally in human milk. The advent of novel biosynthesis technologies resulted in the availability of human milk oligosaccharide structures that are being used as ingredients in infant formulas. Notably, 2’Fucosyllactose has been tested in preclinical and clinical studies to determine its safety and to explore its potential health benefits in the context of pediatric nutrition. Several chromatographic methods for the analysis of human milk oligosaccharides have been published and, the main challenge associated with 2’Fucosyllactose quantitation has been to improve its resolution from lactose, which is present at concentrations around 70 g/l in both, infant formula and human milk. We developed a high-performance anion exchange chromatography method to detect and quantify 2’ Fucosyllactose in the presence of lactose by expanding the elution time between these saccharides. We validated the analytical procedure which behaved linearly (average R=0.99951) at concentrations as low as 1.75 µg/ml (limit of quantitation) with an average limit of detection of 0.43 µg/ml.

Monosaccharide spectrum of arabinoxylane fraction of grain winter rye different genotypes

The analysis of the content and monosaccharide composition of the rye meal carbohydrate fraction of different varieties was carried out using high-performance anion-exchange chromatography method. The dominant component was glucose, in approximately equal proportions arabinose, xylose and galactose were detected. Our research has established partial degradation of arabinoxylans and starch at the beginning of water extraction at a temperature of 30оC that as a result of the work of exo-enzymes rye grain. Water extracts of meal contain from 52 to 74% carbohydrates in oligo - and polymeric form. Based on the content of xylose monosaccharide, rye Ogonek, Podarok and Marusenka are characterized as low pentosan, and Tatarskaya 1, Radon and Pamyati Kunakbaev as highly pentosan (9-11 and 14-16 mg of xylose/ 1 ml of extract, respectively).

The Determination of Sugars in Dairy Products: Development of a New Standard Method for the International Dairy Federation and the Internal Organization for Standardization.

A method using high-performance anion-exchange chromatography (HPAEC) with a pulsed amperometric detector (PAD) for the determination of mono- and disaccharides is described. The method was accepted by the International Dairy Federation and the Internal Organization for Standardization as a new work item for the determination of sugars in dairy matrixes, and the Milk and Milk Products technical committee of ISO/TC 34/SC 5 accepted the topic "Milk and milk products - Determination of the sugar contents - High-performance anion-exchange chromatographic method (HPAEC-PAD)" as a new work item. The proposed method consists of an aqueous ethanol extraction of the sugars in the dairy sample, followed by clarification with Carrez I and II reagents. The clarified filtrate is diluted and then directly introduced in the HPAEC-PAD system for quantification of the sugars. A single-laboratory validation of the proposed method has been scheduled for spring 2017.

The free and total myo-inositol contents of early lactation and seasonal bovine milk

A high performance anion-exchange chromatographic method employing pulsed amperometric detection was applied to the determination of endogenous free and total myo-inositol in bovine milk, for which there is limited information. The contents and trend variability of myo-inositol in milk from extensively pasture-fed cows during early lactation and across a production season were therefore evaluated. Free and total myo-inositol in seasonal milk were within the ranges of 2.3–4.5 mg 100 g−1 and 5.3–8.7 mg 100 g−1, respectively. This novel information will both improve understanding of the expression of innate myo-inositol in bovine milk, and provide manufacturers with information that can enhance formulation capability related to the production of cow's milk-based products

High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the sensitive determination of hyaluronan oligosaccharides.

High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.

Determination of the xylan backbone distribution of arabinoxylan-oligosaccharides

Arabinoxylan-oligosaccharide samples (AXOS) present themselves as mixtures of different molecular entities with xylan backbones of different length and with different levels of arabinose substitution. Their prebiotic properties depend on their degree of polymerisation (DP) and degree of arabinose substitution (DAS). Therefore, structural characterisation of AXOS samples is important. Gas chromatography (GC) is most frequently used for quantification of AXOS levels and for determination of the average DP (avDP) and average DAS (avDAS), yet it does not provide information on the molecular mass distribution of the xylan backbones of the different AXOS entities present in the mixture. This manuscript evaluates a method based on high performance anion exchange chromatography (HPAEC) involving quantification of xylo-oligosaccharides (XOS) after either acidic or enzymic removal of arabinose substituents for its ability to determine such distribution. Results show that despite the fact that a small fraction of the arabinoses could not be removed, representative DP distributions of xylan backbones in complex AXOS samples were obtained. The similarity of the avDP determined with GC or determined with the new HPAEC method using enzymic removal of arabinose substituents confirmed this. It can be concluded that the HPAEC method involving enzymic removal of arabinoses provides useful insight in the DP distribution of the xylan backbones in complex AXOS samples.

Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases

We present an evaluation of HPLC-based analytical tools for the simultaneous analysis of native and oxidized cello-oligosaccharides, which are products of enzymatic cellulose degradation. Whereas cello-oligosaccharides arise from cellulose depolymerization by glycoside hydrolases, oxidized cello-oligosaccharides are produced by cellobiose dehydrogenase and the recently identified copper dependent lytic polysaccharide monooxygenases (LPMOs) currently classified as CBM33 and GH61. The latter enzymes are wide-spread and expected to play crucial roles in further development of efficient enzyme technology for biomass conversion. Three HPLC approaches with well documented performance in the field of oligosaccharide analysis have been investigated: high-performance anion-exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and porous graphitized carbon liquid chromatography (PGC-LC). HPAEC with pulsed amperometric detection (PAD) was superior for analysis of oxidized oligosaccharides, combining the best separation with superior sensitivity for oligosaccharide species with a degree of polymerization (DP) ranging from 1 to 10. Furthermore, the HPAEC method can be optimized for operation in a high-throughput manner (run time 10min). Both PGC-LC and HILIC allow reasonable run times (41 and 25min, respectively), with acceptable separation, but suffer from poor sensitivity compared to HPAEC-PAD. On the other hand, PGC-LC and HILIC benefit from being fully compatible with online mass spectrometry. Using an LC–MS setup, these methods will deliver much better sensitivity than what can be obtained with conventional detectors such as ultraviolet-, charged aerosol-, or evaporative light scattering and may reach sensitivities similar to or even better than what is obtained in HPAEC-PAD. Pure oxidized cello-oligosaccharide standards, ranging from DP2 to DP5, were obtained by semi-preparative PGC and characterized by MS and NMR analysis.

Complete Monosaccharide Analysis by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

Monosaccharide analysis is a critical way to profile the composition of complex carbohydrates. Methods to analyze neutral and amino sugars have been established for a long time, but methods for acidic sugars are rare. The acidic sugars, including uronic acids and sialic acids, are also important components in some complex carbohydrates. In this report, a high-performance anion-exchange chromatography method with pulsed amperometric detection was initially developed to analyze acidic sugars including different uronic acids and sialic acids. Subsequently, a method to profile complete monosaccharides, including most neutral, amino, and acidic sugars, was developed. This method has a limit of quantitation of ~12.5 × 10(-3) nmol for each sugar as well as good linearity over a wide range. This is a convenient procedure because it avoids additional derivatization of monosaccharides and has a broad application to a wide range of complex carbohydrates. The monosaccharide compositions of a variety of complex carbohydrates such as different glycosaminoglycans, alginate, fucoidan, and glycans were profiled by this comprehensive method. In addition, the hydrolysis patterns of these complex carbohydrates are discussed.

Simultaneous determination of two Amadori compounds in Korean red ginseng ( Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC–PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng ( Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15–102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.

Determination of phytic acid and inositol pentakisphosphates in foods by high-performance ion chromatography.

A high-performance anion exchange chromatographic method was adapted for the quantitative determination of phytic acid and inositol pentakisphosphate isomers (excluding enantiomers) in foods. Because of the cost and limited availability of inositol phosphate standards, a phytic acid sodium salt standard was used for the calculation of an average relative response factor for the quantification of inositol pentakisphosphate isomers, and the purity of phytic acid sodium salt standard was also accurately established. The detection limits (S/N = 3) for phytic acid and inositol pentakisphosphates were in the range of 1.5-3.4 microM (0.1-0.2 microg/100 microL). This method has been successfully applied to the determination of phytic acid and inositol pentakisphosphates in a variety of beans and nuts after extraction with 0.5 M HCl and cleanup with solid phase extraction cartridges. The results demonstrated that there was a strong correlation between either the phytic acid content or the total content of phytic acid together with inositol pentakisphosphates and the total dietary fiber content in the group of all raw dry beans and in the group of raw dry black beans but not in the group of raw dry red kidney beans, which was probably due to the insufficient number of the raw dry red kidney bean samples.

A comparison of pulsed amperometric detection and spectrophotometric detection of carbohydrates in cider brandy by liquid chromatography

A high-performance anion-exchange chromatographic method that uses an alkaline mobile phase (20 mM NaOH) on an anion exchange column (RCX-30 Hamilton) at 30 °C coupled with pulsed amperometric detection was applied to the analysis of the glucose, galactose, xylose, arabinose, ribose, fucose, rhamnose and fructose in cider brandies. Data were compared with those obtained from aldoses- and uronic acids- p-aminobenzoic ethyl ester derivatives analysis by HPLC on a C 8 column at 45 °C with spectrophotometric detection. Both methods were validated and their advantages and drawbacks taken into account.

Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications

A high-performance anion-exchange chromatographic method was developed for the separation of phytic acid and other inositol phosphates ( myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates) with gradient elution and ultraviolet absorbance detection after post-column derivatization. With the acidic eluents, the combination of anion-exchange and ion suppression retention mechanisms led to the separation of 35 inositol phosphates (excluding enantiomers) into 27 peaks for the first time, and the retention behaviors of all myo-inositol bis- to hexakisphosphate isomers were studied. The whole separation procedure was completed within 65 min. Based on the investigations of nonenzymatic hydrolysis of phytic acid under different conditions by using this method, an in-house reference standard solution was produced, which can be used for method development. In addition, by applying this method to in vitro kinetic studies, at least one new enzymatic hydrolysis pathway of phytic acid was found, and one rule of enzymatic dephosphorylation of inositol phosphates (position effect) was proposed and another one (neighboring effect) was confirmed. The principle of the proposed identification approach for several inositol phosphate isomers based on hydrolysis products study will be applicable to other natural products analysis, for which standards are very expensive or not available.

Determination of Nucleotides and Sugar Nucleotides Involved in Protein Glycosylation by High-Performance Anion-Exchange Chromatography: Sugar Nucleotide Contents in Cultured Insect Cells and Mammalian Cells

We have developed a simple and highly sensitive HPLC method for determination of cellular levels of sugar nucleotides and related nucleotides in cultured cells. Separation of 9 sugar nucleotides (CMP-Neu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) was examined by reversed-phase HPLC and high-performance anion-exchange chromatography (HPAEC). Although the reversed-phase HPLC, using an ion-pairing reagent, gave a good separation of the 12 nucleotides, it did not separate sufficiently the sugar nucleotides for quantification. On the other hand, the HPAEC method gave an excellent and reproducible separation of all nucleotides and sugar nucleotides with high sensitivity and reproducibility. We applied the HPAEC method to determine the intracellular sugar nucleotide levels of cultured Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five, BTN-TN-5B1-4) insect cells, and compared them with those in Chinese hamster ovary (CHO-K1) cells. Sf9 and High Five cells showed concentrations of UDP-GlcNAc, UDP-Gal, UDP-Glc, GDP-Fuc, and GDP-Man equal to or higher than those in CHO cells. CMP-Neu5Ac was detected in CHO cells, but it was not detected in Sf9 and High Five cells. In conclusion, the newly developed HPAEC method could provide valuable information necessary for generating sialylated complex-type N-glycans in insect or other cells, either native or genetically manipulated.

Method development for the quantitative determination of lactulose in heat-treated milks by HPAEC with pulsed amperometric detection.

A robust, rapid, and sensitive high-performance anion-exchange chromatographic method for the separation and quantitative determination of lactulose in heated milks, along with other common milk carbohydrates, has been developed. Complete separation of galactose, glucose, N-acetylgalactosamine, lactose, lactulose, and epilactose was isocratically accomplished in about 22 min by an anion-exchange column eluted with 10 mM NaOH spiked with 2 mM Ba(OAc)2. The within-day repeatability was lower than 2.1% for 10 repetitive injections. Under optimized conditions, there was no need either of post-column addition of strong bases to the eluent for enhancing detection sensitivity or, even more important, for column regeneration between chromatographic runs. Upon 100-fold sample dilution, the amperometric response of lactulose in milk samples was found to be linear up to 100 microM (r = 0.99935) with a limit of detection equal to 1.2 microM (S/N = 3). The lactulose content in ultrahigh-temperature (UHT) and sterilized milks was evaluated by a calibration graph using 2-deoxyglucose as the internal standard, making the proposed method very useful in discriminating among heat-treated milks. Whereas the mean value of lactulose in skimmed, partially skimmed, and whole UHT milks ranged from 10 to 90 mg/100 mL, lactulose content in bottle-sterilized whole milk (two samples) was higher than 140 mg/100 mL. The presence of epilactose, which is another isomer of lactose, was also ascertained in sterilized milk.

High-Performance Anion-Exchange Chromatography Method for Analysis of Propylene Glycol Alginate in Beer

A method was developed for the quantification of propylene glycol alginate (PGA) in lager beer. It employed a two-step acid hydrolysis of PGA into guluronic and mannuronic acid as constituent acidic sugars (uronic acids), followed by high-performance anion-exchange chromatography (HPAEC). PGA was precipitated from beer with ethanol at 70% (v/v), subsequently hydrolyzed by methanol-HCl and trifluoroacetic acid, and finally analyzed by HPAEC. A good separation between both uronic acids was obtained, enabling PGA analysis in the relevant application range. Galacturonic acid was used as an internal standard. In spiked beers, PGA was recovered analytically for ≈60%. PGA concentrations >10 mg/L could be determined quantitatively, whereas below this level, only qualitative results were obtained. The detection level of the method is affected by the occurrence of glucuronic acid in beer.

Separation and determination of four artificial sweeteners and citric acid by high-performance anion-exchange chromatography

A high-performance anion-exchange chromatographic method for the simultaneous separation and determination of four artificial sweeteners (sodium saccharin, aspartame, sodium cyclamate and acesulfame-K) and citric acid in a single injection is proposed. The separation was performed by an anion-exchange gradient program and determination by ultraviolet absorbance detection for sodium saccharin, aspartame, acesulfame-K in combination with conductivity detection for sodium saccharin, sodium cyclamate, acesulfame-K as well as citric acid in series. Good linearities between the concentrations of all analytes and relevant peak area responses were achieved over the range 2–100 μg/ml. The detection limits (signal-to-noise ratio 3:1) were 0.019 μg/ml for sodium saccharin, 0.035 μg/ml for aspartame, 0.044 μg/ml for acesulfame-K by ultraviolet absorbance detection and 0.26 μg/ml for sodium saccharin, 0.16 μg/ml for sodium cyclamate, 0.23 μg/ml for acesulfame-K and 0.22 μg/ml for citric acid by conductivity detection. The method has been successfully applied to the determination of the four sweeteners and citric acid in drinks and powdered tabletop sweeteners, and the average recoveries for various samples ranged from 93 to 107%.

Determination of total nonstructural carbohydrates in tree species by high-performance anion-exchange chromatography with pulsed amperometric detection

Tracing shifts in carbohydrate pools has become an important tool for studying the integration of plant responses to natural and anthropogenic stresses on tree species. As a result, the need for a rapid and sensitive analytical technique to measure the components in the total nonstructural carbohydrate pool in various plant tissues has increased. In this paper we report carbohydrate data for five species that were obtained using a high-performance anion-exchange chromatography method that uses an alkaline, isocratic mobile phase (160 mM NaOH) with triple-pulsed amperometric detection. This approach has been applied to the analysis of sugar alcohols, monosaccharides, disaccharides, and other oligosaccharides in ponderosa pine (Pinusponderosa Dougl. ex Laws.), black cherry (Prunusserotina Ehrh.), and other species roots, branches, stems, and needles. The results show the range of tissue concentrations found and suggest the usefulness of the anion-exchange method for the routine quantification of the total nonstructural carbohydrate pool in trees with minimal sample preparation and cleanup. Base-line resolution of the soluble carbohydrates was accomplished in less than 13 min. The chromatographic analysis of starch as glucose was complete in less than 4 min. Electrochemical detection enabled selectivity of the carbohydrates and higher sensitivity over the conventional colorimetric assays or high-performance liquid chromatography with refractive index detection. The minimum levels of quantification were 4.5 ng for myo-inositol, 4.6 ng for sorbitol, 4.5 ng for glucose and fructose, 17.1 ng for sucrose, and 29.7 ng for raffinose.

Comparison of micro-enzymatic and high-performance liquid chromatographic methods for the assay of serum 1,5-anhydroglucitol

Serum 1,5-anhydro- d-glucitol (AG) has been proposed as a marker of glycaemic control in diabetic patients. Two methods have been developed which could be applied to routine clinical monitoring of serum AG. We have compared the assay characteristics of an adapted enzyme assay, based on the enzyme pyranose oxidase, with a high-performance anion-exchange chromatographic method using pulsed amperometric detection (HPAEC-PAD). Linearity and minimum detectable concentrations were practically identical (to at least 400 μmol/l and 4 μmol/l AG, respectively), though intra- and inter-assay precision was better with the HPAEC PAD system at a clinically relevant concentration of 40 μmol/l AG (9.8% vs. 11.8% and 9.2% vs. 13.0%, respectively). The recovery of added AG to serum samples was lower with the micro-enzyme assay than the HPAEC-PAD assay (74 ± 15% vs. 102 ± 8%). Despite good agreement by linear regression ( r = 0.974, P < 0.005), the assay methods did not demonstrate satisfactory agreement when using a difference plot (limits of agreement −16.l to 18.7 μmol/l). We conclude that although both assay methods are applicable to routine analyses, the HPAEC-PAD is more precise and is more specific for AG than the enzyme based assay.