Cation-exchange High-performance Chromatography Research Articles

A Review of the Analytical Methods for the Determination of 4(5)-Methylimidazole in Food Matrices

4(5)-Methylimidazole (4(5)MEI) is a product of the Maillard reaction between sugars and amino acids, which occurs during the thermal processing of foods. This compound is also found in foods with caramel colorants additives. Due to its prevalence in foods and beverages and its potent carcinogenicity, 4(5)MEI has received federal and state regulatory agency attention. The aim of this review is to present the extraction procedures of 4(5)MEI from food matrices and the analytical methods for its determination. Liquid and gas chromatography coupled with mass spectrometry are the techniques most commonly employed to detect 4(5)MEI in food matrices. However, the analysis of 4(5)MEI is challenging due to the high polarity, water solubility, and the absence of chromophores. To overcome this, specialized sample pretreatment and extraction methods have been developed, such as solid-phase extraction and derivatization procedures, increasing the cost and the preparation time of samples. Other analytical methods for the determination of 4(5)MEI, include capillary electrophoresis, paper spray mass spectrometry, micellar electrokinetic chromatography, high-performance cation exchange chromatography, fluorescence-based immunochromatographic assay, and a fluorescent probe.

Comparative analytical profiling of bevacizumab biosimilars marketed in India: a national control laboratory study

In this study, analytical profiling of the bevacizumab (BVZ) biosimilars (N = 3) approved in India were evaluated for charge heterogeneity, isoelectric focusing, aggregation and in vitro potency analysis. The charge variants were characterized using high performance cation-exchange chromatography (CEX-HPLC), capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF). cIEF was also used for estimation of isoelectric point (pI value). In addition, aggregate analysis was done using size exclusion high performance chromatography (SEC-HPLC). The cell-based inhibition of proliferation assay using HUVEC cells, indirect ELISA and Western blot were performed for in vitro biological activity. In addition of cell-based cytotoxicity assay was also performed and found no cytotoxic effect on both HuT78 and WIL2S cells by bevacizumab biosimilars. The significant variations in acidic (p < 0.0001) and basic variants (p < 0.0001), pI value (p = 0.0035), aggregates (p = 0.0306) of biosimilars were found as compared to innovator product; however, cell-based potency analysis (p = 0.6047) and indirect ELISA (p = 0.1611) have shown no significant difference in the biological activity. The banding patterns of all biosimilars in western blot were found similar to the innovator product. The comparatively higher basic variants in the biosimilars were attributing to the high pI value of biosimilars to that of innovator product, although these variations were not affecting the biological activity of the biosimiars. This is a unique study, wherein the independent analysis by a National Control Laboratory (NCL) will not only help the National Regulatory Authority (NRA) to assess the quality and consistency in manufacturing of BVZ biosimilars marketed in India but also facilitate the uptake of BVZ biosimilars, and sustainable access to new medicines against the anti-angiogenic therapy.

Charge Variants of an Avastin Biosimilar Isolation, Characterization, In Vitro Properties and Pharmacokinetics in Rat.

The similarity between a proposed biosimilar product and the reference product can be affected by many factors. This study is designed to examine whether any subtle difference in the distribution of the charge variants of an Avastin biosimilar can affect its in vitro potency and in vivo PK. Here, the acidic, basic and main peak fractions of a biosimilar product were isolated using high-performance cation-exchange chromatography and were subjected to various studies to compare their in vitro properties and in vivo PK profile. A serial of analytical methods, including size exclusion chromatography (SEC), imaged capillary isoelectric focusing (icIEF) capillary zone electrophoresis (CZE) and cation-exchange chromatography (CEX-HPLC) were also used to characterize the isolated charge variants. The kinetics constant was measured using a Biacore X100 system. The study indicates the biosimilar product has a high similarity with avastin in physicochemical properties. The potency in vitro and PK profile in rat of charge variants and biosimilar product are consistent with avastin.

Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins

Sugar beet pectins (SBP) were precipitated from a purified extract in a 75% v/v ethanol solution at an initial extract pH (I-pH) ranging from 2.0 to 4.5. The effects of I-pH on pectin yield and pectin-cation interactions were studied. No simple correlation between pectin yield and I-pH was observed. The lowest pectin yield was obtained by precipitating the acidic pectin extract with ethanol at I-pH 4.5. Pectin yield increased to a maximum value as I-pH decreased from 4.5 to 3.0. These results indicated that decreased electrostatic repulsion between pectin chain segments enhanced pectin chain–chain interactions, thereby improving the precipitation effect. A decrease in pectin yield was observed as I-pH decreased below 3.0. The cation content of various samples was measured by high-performance cation-exchange chromatography in order to determine the content of SBP-bound cations during precipitation. Cation content and degree of cation binding were measured for pectins precipitated at I-pH 3.0, 2.5, and 2.0, and the results revealed that a decrease in pectin yield accompanied a decrease in cation–pectin interactions. These results suggest that the precipitation of SBP from an aqueous extract involves complex interactions between cations, solvent molecules, and pectin chain segments. The larger precipitation effect observed with divalent ions compared to monovalent ions may be due to enhanced inter-chain interactions between pectin molecules, probably via the formation of intermolecular bonds.

Simultaneous Analysis of 2- and 4-Methylimidazole in Caramel Color and Soft Drinks Using IC-PAD with Post-Column Addition of Hydroxide

High-performance cation exchange chromatography with pulsed integrated amperometric electrochemical detector (HPCEC-PAD) with post-column addition of hydroxide was first developed for simultaneous analysis of 4-methylimidazole (4-MeI) and 2-methylimidazole (2-MeI) in caramel color and soft drinks after solid-phase extraction (SPE). A CS12A cation exchange column was used to separate the targeted analytes with a perfect resolution (2.70). This method demonstrated low limit of quantification (0.05–10 mg/L) and excellent linearity with correlation of determination (R 2 > 0.999 for 2-MeI and 0.997 for 4-MeI). Low concentrations of 4-MeI were found in five soft drinks, whereas those in caramel color were generally high. Additionally, the suggested method showed higher resolution and sensitivity comparing with previous reported methods for 4-MeI and 2-MeI analyses.

A Case of Hemoglobin Beckman; An example of Hemoglobin Variants Interfering with HbA1c Measurement

Aim: It has been known for a long time that the presence of clinically silent hemoglobin (Hb) variants in blood samples could influence the measurement of glycohemoglobin (HbA1c) since these abnormal hemoglobins might interfere with some methodologies. In the present study we describe the first case of Hb Beckman in Turkey. Material and Methods: The variant was detected during the HbA1c measurement by cation exchange high performance chromatography (CE-HPLC) in a patient with diabetes mellitus. Since a HbA1c result could not be obtained by this method, the test was repeated by boronate affinity HPLC. Results: Further investigation performed in order to identify the variant revealed heterozygote Hb Beckman 135 GCT-GAT (Ala --> Asp) which explained the inappropriate HbA1c result by CE-HPLC. Conclusion: This new case of Hb Beckman is thought to be a modest contribution to the subject of abnormal hemoglobins that interfere the HbA1c measurement.

Analysis of Arsenic Speciation in Sea Fish and Shellfish by Using Anion/ Cation Exchange High Performance Chromatography Coupled with Inductively Coupled Plasma-Mass Spectrometry

Analysis of Arsenic Speciation in Sea Fish and Shellfish by Using Anion/ Cation Exchange High Performance Chromatography Coupled with Inductively Coupled Plasma-Mass Spectrometry

Supercritical fluid processing of proteins: lysozyme precipitation from aqueous solution.

Aqueous solutions of hen egg lysozyme (3% w/v) were dispersed and precipitated by a homogenous mixture of supercritical carbon dioxide-ethanol using the Solution Enhanced Dispersion by Supercritical fluid (SEDS) process. The effects of different working conditions, such as temperature, pressure and the flow rates of the solution and ethanol, on the particle-formation process were studied. The morphology, particle size and size distribution and biological activity of the protein were determined. The precipitates were examined with high-sensitivity differential scanning calorimetry (HSDSC) and high-performance cation-exchange chromatography. Particle size measurements showed the precipitates to be aggregates with primary particles of size 1-5 microm. The similarity of HSDSC data for unprocessed and processed samples indicated that the different physical forces that stabilise the native form of lysozyme are unchanged after SEDS processing. From FT-Raman spectroscopic studies secondary structural changes were observed in certain SEDS-produced lysozyme, with most processed samples displaying a slightly more disordered secondary structure than the unprocessed sample. However, SEDS samples produced at 200 bar and 40 degrees C exhibited negligible disturbance. Thus the SEDS process utilising aqueous solution was able to bring about size reduction of lysozyme with minimal loss of biological activity.

A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers

BackgroundCell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time.ResultsTwo developmental stages (active growth and after growth arrest) were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function.ConclusionThis work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

Rapid quantification of global DNA methylation by isocratic cation exchange high-performance liquid chromatography

The DNA of many eukaryotes is methylated at specific cytosine residues in connection with gene regulation. Here we report a method for the quantification of global cytosine methylation based on enzymatic hydrolysis of DNA, dephosphorylation, and subsequent high-performance cation exchange chromatography. Nucleosides are separated in less than 3min under isocratic conditions on a benzenesulfonic acid-modified silica phase and detected by UV absorption. As little as 1μg of DNA is sufficient to measure 5-methyldeoxycytosine levels with a typical relative standard deviation of less than 3%. As a proof of concept, the method was applied for analysis of DNA from several Arabidopsis thaliana mutants affected in DNA methylation and from Medicago sativa seedlings treated with the environmental pollutant chromium(VI).

Analysis of Amadori Compounds by High-Performance Cation Exchange Chromatography Coupled to Tandem Mass Spectrometry

High-performance cation exchange chromatography coupled to tandem mass spectrometry or electrochemical detection was found to be an efficient tool for analyzing Amadori compounds derived from hexose and pentose sugars. The method allows rapid separation and identification of Amadori compounds, while benefiting from the well-known advantages of mass spectrometry, such as specificity and sensitivity. Glucose- and xylose-derived Amadori compounds of several amino acids, such as glycine, alanine, valine, leucine/isoleucine, methionine, proline, phenylalanine, and glutamic acid, were separated or discriminated using this new method. The method is suitable for the analysis of both model reaction mixtures and food products. Fructosylglutamate was found to be the major Amadori compound in dried tomatoes (approximately 1.5 g/100 g) and fructosylproline in dried apricots (approximately 0.2 g/100 g). Reaction of xylose and glycine at 90 degrees C (pH 6) for 2 h showed rapid formation of xylulosylglycine (approximately 12 mol %, 15 min) followed by slow decrease over time. Analysis of pentose-derived Amadori compounds is shown for the first time, which represents a major breakthrough in studying occurrence, formation, and decomposition of these labile Maillard intermediates.

Separation of Dissolved Aluminum in Neutral/Slightly Basic River Water by Cation-Exchange Chromatography with Fluorometric Detection of the Lumogallion–Aluminum Complex

Abstract The separation of dissolved aluminum in neutral/slightly basic river (Seta River) water was carried out using high-performance cation-exchange chromatography with a gradient elution of calcium ion in a dilute nitric acid solution (pH 4) and fluorometric detection of the lumogallion–aluminum complex. Although three peaks may appear, which correspond to species having a charge of less than +1, +2 and +3, only one major peak at the “3+” position appeared. From the sample pH and this result of separation, the main species of dissolved aluminum was concluded to be Al(OH)4−. In order to evaluate whether the elution was complete or not, the peak areas obtained with and without the cation-exchange column were compared. For an accurate evaluation of the elution peak, the residual solution of the solvent extraction with the 8-quinolinol/chloroform system was used. The elution of dissolved aluminum from the cation-exchange column was found to be incomplete (or not quantitative); i.e., the percentage of eluted aluminum was 79 and 70% for two Seta River water samples in October and January, respectively. The chemical form of the aluminum species which was adsorbed in the cation-exchange column is discussed.

Use of disposable gold working electrodes for cation chromatography–integrated pulsed amperometric detection of sulfur-containing amino acids

We have prepared disposable thin-film gold working electrodes on polymeric substrates. Our microfabrication process allows for inexpensive and reproducible mass production of such electrodes. We utilize this new type of electrode in flow-through electrochemical cells to replace the conventional non-disposable gold working electrodes for integrated pulsed amperometric detection (IPAD) of compounds separated by high-performance cation-exchange chromatography. Using two S-containing amino acids (homocysteine and cysteine) as test compounds, we have modified a previously reported waveform for optimum performance with disposable gold electrodes. With the help of the same two test substances we have characterized the analytical performance of disposable gold electrodes under the new conditions. Compared to non-disposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration and reproducibility. When used with a new IPAD waveform, the disposable electrodes functioned reproducibly for 3 days. At the end of the specified usage period of 3 days, the disposable electrodes are simply replaced. Reconditioning by polishing is thus no longer required.

Supercritical fluid processing of proteins: I: Lysozyme precipitation from organic solution

The solution enhanced dispersion by supercritical fluid (SEDS) process was used to evaluate the effect of the processing variables on the biological and physicochemical characteristics of lysozyme protein particles produced from an organic solution of dimethylsulfoxide (DMSO) using an experimental design procedure. The processing variables were temperature, pressure, solution concentration and the flow-rates of supercritical carbon dioxide and a protein solution. Solutions of hen egg lysozyme (0.5–1%, w/v) in DMSO were dispersed using supercritical carbon dioxide as the antisolvent, and particles precipitated in a particle formation vessel. The morphology, particle size and size distribution and biological activity of the protein were determined. The precipitates were also examined with high sensitivity differential scanning calorimetry (HSDSC) and high-performance cation-exchange chromatography. The amount of residual DMSO was determined using headspace gas chromatography. Particle size measurements showed the precipitates to be agglomerates with primary particles of size 1–5 μm, containing <20 ppm of residual solvent. The activity of the precipitates varied between 44 and 100% depending on the experimental conditions. The similarity of HSDSC data for unprocessed and processed samples indicated that the SEDS process does not cause major denaturation of lysozyme when prepared from DMSO solutions. By optimising of working conditions, the SEDS process can produce micron-sized particles of lysozyme with minimal loss of biological activity.

High-Performance Cation-Exchange Chromatography and Pulsed Amperometric Detection for the Separation, Detection, and Quantitation of N-Alkylated Imino Sugars in Biological Samples

The use of imino sugars for the potential treatment of lysosomal glycolipid storage diseases and hepatitis virus infections requires accurate, quantitative measurement of these compounds in biological samples. We demonstrate here the versatility of cation-exchange chromatography and pulsed amperometric detection of a range of compounds that differ in both isometric structure and N-alkyl chain length. Although column retention appears dependent upon residual charge on the imine function, successful isocratic separation can be achieved by secondary hydrophobic interactions. A series of N-alkylated deoxynojirimycin compounds containing C1–10 alkyl chains are readily separated and detected by pulsed amperometry after cation suppression. Using experimentally derived response factors for imino sugars and measurement of peak areas we have developed a reliable method for quantitatively determining concentrations in solution. A rapid protocol for the removal of protein and contaminants in biological samples is described. This has allowed the successful measurement of imino sugars in animal tissues and will be useful for understanding the factors involved in compound bioavailability and in the design of novel therapeutics.

Waveform optimization for integrated square-wave detection of biogenic amines following their liquid chromatographic separation

Results are described from research designed to optimize the potential–time ( E– t) waveform applied in integrated square-wave detection (ISWD). More specifically, goals of this study included the minimization of background signal with maximization of the signal-to-background ratio ( S/ B) for application of ISWD to polyamines separated by high performance cation-exchange liquid chromatography (LC). This effort included optimization of the separation procedure because the background signal was determined to be a sensitive function of the composition of the chromatographic mobile phase. Initial estimates of potential parameters were obtained from an off-line voltammetric study of 1,3-diaminopropane at a gold rotated disk electrode (RDE). Final waveform optimization was based on data obtained during on-line application of the ISWD waveform for 1,3-diaminopropane injected at various times during execution of the mobile phase gradient. The maximum potential in the waveform ( E MAX) was chosen to achieve formation of surface oxide (AuO) with concomitant oxidation of the amine without evolution of O 2. The minimum potential in the waveform ( E MIN) was chosen to achieve reduction of the surface oxide generated at E MAX with minimal reduction of dissolved O 2.

Dissolution of well and poorly crystallized kaolinites; Al speciation and effects of surface characteristics

This study compared surface characteristics and dissolution behavior of well-crystallized (KGa-1b) and poorly crystallized (KGa-2) kaolinite standards. Atomic force microscopy (AFM) revealed that particles of KGa-1b generally have nicely hexagonal micromorphology and crystallographically controlled microtopographic features. Particles of KGa-2 are also hexagonal, but their micromorphology tends to be more rounded. Basal-plane surfaces tend to be more irregular with fewer clearly crystallographically controlled features. KGa-1b particles tend to be larger in diameter and thicker than KGa-2 particles. Micromorphologic measurements showed that both KGa-1b and KGa-2 have modal edge- to total surface-area ratios of approximately 0.1 (mean ~ 0.2), although these measurements did not include the potentially large contribution of basal-plane step edges (additional 20% or more). Dissolution experiments were conducted in oxalic acid and inorganic acids at pH 3, 22 °C, I = 0.01 M, under batch dissolution conditions. Dissolution rates (measured as Si release) in 1 m M oxalic acid were approximately twice as fast for KGa-2 as for KGa-1b (2.27 vs. 0.96 nmol/m 2 ·h). Rates for KGa2 and KGa-1b were similar in HNO3 (0.86 and 1.16 nmol/m 2 ·h, respectively). The comparable rates for these two sedimentary kaolinites and for a hydrothermal kaolinite studied by Wieland and Stumm (1992) suggests that the fundamental structure of kaolinite, rather than specific surface details, exerts the greatest influence on dissolution kinetics. High-performance cation exchange chromatography (HP-CEC) was used to determine the distributions of monomeric Al species over the course of kaolinite dissolution. For dissolution in 1 m M oxalate, Al-oxalates were observed almost exclusively in agreement with results of equilibrium speciation calculations. For dissolution in HNO 3, the peak representing uncomplexed Al species, Al f, was predominant but not exclusive, as predicted by calculations. A peak having a retention time characteristic of species with +2 charge may be evidence for an AlOSi(OH) 3 2+ species, and warrant further investigation.

Capillary Zone Electrophoresis for the Diagnosis of Congenital Hemoglobinopathies

The analysis of human hemoglobins (Hbs) is of medical importance in a number of congenital defects. The hemoglobinopathies are grouped into defective variants of Hb, such as Hb S and >600 other variants, and thalassemias characterized by abnormal expression of the genes for normal globin chains (1)(2)(3). Alkaline electrophoresis, performed historically on cellulose acetate and currently on agarose, combined with citrate agar electrophoresis at acidic pH is still widely used (4)(5)(6)(7), but high-performance cation-exchange chromatography (HPCEC) offers superior resolution, speed, and automation (8)(9)(10). Capillary electrophoresis uses numerous separation principles and shares with HPLC the advantages of high resolution and automation, with on-line detection and direct quantification (11)(12)(13)(14)(15). Capillary isoelectric focusing on coated capillaries can be used to study Hb (16)(17)(18)(19)(20)(21), but is slower than HPCEC (21). The first reported assays based on capillary zone electrophoresis (CZE) gave poor resolution of Hbs or were not quantitative (22)(23)(24). The aim of the present work was to evaluate a rapid CZE assay with dynamic coating of the fused-silica at alkaline pH to detect abnormal Hbs and to quantify Hbs for the diagnosis of thalassemias, and at acidic pH to confirm the identity of Hb variants. Reagents were obtained from Analis. The alkaline kit A2 contained a hemolyzer, an initiator consisting of a polycation, and an arginine buffer (pH 8.8) containing a polyanion. The acidic kit A1 has been described elsewhere for Hb A1c measurement (25). Adult and newborn samples were collected in EDTA-containing tubes at the maternity unit of the Hopital Saint-Antoine (AP-HP, Paris, France) and received in the laboratory for Hb analysis. Whole blood samples …

Degree of acetylation of heteropolysaccharides

The acetyl groups in polysaccharides and glycoproteins have been determined using 4 N HCl at 120 °C for acid hydrolysis. Acetic acid and hexosamine were determined by high-performance cation-exchange chromatography with UV detection and high-performance anion-exchange chromatography with pulsed amperomeric detection, respectively. The method compares well with other procedures and shows an additional advantage of being able to analyze for hexosamine in the same hydrolyzate, thus permitting the degree of acetylation of hexosamine-containing biopolymers to be determined directly without correction for additional components in the material of interest.

Determination of melamine derivatives, melame, meleme, ammeline and ammelide by high-performance cation-exchange chromatography

The thermal decomposition of melamine produces melame and meleme, while alkaline hydrolysis of melamine produces ammeline and ammelide. These four melamine derivatives were determined by high-performance cation-exchange chromatography using phosphate buffer as the eluent. The elution behavior during the isocratic and gradient elutions was examined and the determination was simultaneously achieved using photodiode array UV–Vis detection. An isocratic elution with 50 m M phosphate buffer (pH 2.5) seemed most suitable for the rapid and quantitative analysis. Three types of gradient elutions involving phosphate- and NaCl-concentrations, and pH also showed satisfactory separations for the melamine derivatives.