Strong Anion-exchange Column Research Articles

Rapid purification method for fumonisin B1 using centrifugal partition chromatography

Fumonisin B1 (FB1) is a highly toxic mycotoxin produced by fungal strains belonging to the Fusarium genus, which can be found mainly in maize products, and is gaining interest in food safety. To produce large amounts of pure FB1, a novel purifying method was developed by using centrifugal partition chromatography, which is a prominent member of the liquid–liquid chromatographic techniques. Rice cultured with Fusarium verticillioides was extracted with a mixture of methanol/water and found to contain 0.87 mg of FB1 per gram. The crude extracts were purified on a strong anion-exchange column and then separated by using a biphasic solvent system consisting of methyl-tert-butyl-ether–acetonitrile–0.1% formic acid in water. The collected fractions were analysed by flow injection–mass spectrometry and high-performance liquid chromatography coupled with Corona-charged aerosol detector and identified by congruent retention time on high-performance liquid chromatography and mass spectrometric data. This method produced approximately 120 mg of FB1 with a purity of more than 98% from 200 g of the rice culture. The whole purification process is able to produce a large amount of pure FB1 for analytical applications or for toxicological studies.

Sensitive and simple determination of bromate in foods disinfected with hypochlorite reagents using high performance liquid chromatography with post-column derivatization

A novel analytical method for the quantification of bromate in fresh foods using high performance liquid chromatography (HPLC) with a post-column reaction has been developed. The fresh food sample solutions were pretreated with homogenization, centrifugal ultrafiltration and subsequent solid phase extraction using a strong anion-exchange resin. After separation on a strong anion-exchange chromatography column using a highly concentrated NaCl solution (0.3M) as the eluent, the bromate was quantified by detection using a post-column reaction with a non-carcinogenic reagent (tetramethylbenzidine). The developed HPLC technique made it possible to quantify bromate in salt-rich fresh foods. The recoveries from fresh foods spiked with bromate at low levels (2 or 10ng/g) satisfactorily ranged from 75.3 to 90.7%. The lowest quantification limit in fresh foods was estimated to be 0.6ng/g as bromic acid. The method should be helpful for the quantification of bromate in fresh foods disinfected with hypochlorite solutions.

Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.

Analysis of the Fusarium Mycotoxin Moniliformin in Cereal Samples Using 13C2-Moniliformin and High-Resolution Mass Spectrometry

Moniliformin is a mycotoxin produced by fungi of the Fusarium genus and occurs as a contaminant of different cereals worldwide. This study describes the first application of isotopically labeled (13)C(2)-moniliformin for the analysis of moniliformin in cereals. Moniliformin is a small and ionic molecule that forms only a single sensitive fragment ion in the collision cell of a tandem mass spectrometer. Therefore, the methods described in the literature for this kind of instrument observe only a single mass transition and show a relatively poor sensitivity. The use of high-resolution mass spectrometry was described to be a suitable alternative technique for the detection of this compound and was therefore applied in this study. The developed method is based on the use of strong anion exchange columns for cleanup prior to HPLC analysis and has a recovery rate of 75.3%, a limit of detection (LOD) of 0.7 μg/kg, and a limit of quantitation (LOQ) of 2.5 μg/kg. Twenty-three different cereal samples were analyzed for their moniliformin content. Twenty of them showed positive results with levels up to 126 ± 12.2 μg/kg.

Development and Validation of a Spectrophotometric Method for Quantification of Total Glucosinolates in Cruciferous Vegetables

Given their putative role in chemoprevention, validated methods are needed for quantification of total glucosinolates. Based on the colorimetric reaction of ferricyanide with 1-thioglucose, released by alkaline hydrolysis of glucosinolates, we developed a simple and sensitive method for spectrophotometric quantification of total glucosinolates in cruciferous vegetables. Lyophilized and ground vegetables are extracted with 80% boiling methanol. Extracted glucosinolates are isolated using a strong anion exchange column and then hydrolyzed with 2 N NaOH to release 1-thioglucose. Ferricyanide is added, and the decrease in absorbance is measured at 420 nm, with final values adjusted for background. Recovery of internal standard (sinigrin) was 107%. Intra- and interassay coefficients of variation were 5.4% and 15.8%, respectively. Dose response was linear with sinigrin and amount of plant material extracted (R(2) ≥ 0.99). Using sinigrin, the lower limit of quantification was 0.6 mg. This straightforward method may be an alternative to time-consuming and costly chromatographic methods.

Determination of Trace Level Oxytetracycline and Influences of Coexisting Pollutants on its Sorption by Three Kinds of Sludge

Strong anion exchange (SAX) columns and oasis HLB cartridges were combined to extract trace level oxytetracycline(OTC) and high performance liquid chromatography(HPLC) UV-detector were used to measure it in wastewater sample. The effect of mobile phase was investigated. Under the best analysis conditions, linear calibration equations were established for OTC. The linearity were in the range of 0.1-10g/mL, Correlation coefficient (R2) is about 0.9997. The recovery factors ranged from 82-92% with a relative standard deviation of 4.8-6.4%.Lab-scale experiments were conducted to investigate the sorption behavior of OTC by anaerobic sludge. Starch in the mixed liquor can decrease the adsorption capacity of OTC.

Inositol Hexakisphosphate Kinases Induce Cell Death in Huntington Disease

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.

Redesign of a Plugged β-Barrel Membrane Protein

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.

Determination of cyanuric acid in milk and milk powder by high performance liquid chromatography-tandem mass spectrometry

A method for the determination of cyanuric acid (CYA) in milk and milk powder was developed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS). The target analyte was extracted from samples by acetonitrile, and the proteins were precipitated at the same time. The supernatant was cleaned up and enriched with a strong anion exchange column, analysed by HPLC-MS/MS with an AX column, and quantified by internal standard method. Good linearity was obtained in the range of 50-2000 microg/L. The recoveries were all ranged from 97% to 121% at three levels, 200, 500 and 1000 microg/kg, and the relative standard deviations (RSDs) were all below 4.8%. Limit of quantification (LOQ) was 200 microg/kg. The method has high accuracy and precision, the operation is fast, easy and suitable for the determination of CYA in milk and milk powder.

Comprehensive Profiling of Phosphopeptides Based on Anion Exchange Followed by Flow-Through Enrichment with Titanium Dioxide (AFET)

For large-scale analysis of phosphorylation at proteome-wide scale, a variety of affinity-based strategies have been developed to enrich phosphopeptide. Because each method differed in their specificity of isolation, the global and unbiased enrichment of phosphopeptides remains a major technical challenge in phosphoproteomics. In the present work, we demostrate that the phosphopeptide enrichment method based on an online continuous pH gradient in a strong anion exchange column (SAX method) is highly complementary to the method based on titanium dioxide (TiO2) affinity enrichment. Moreover, we found that the flow-through fraction of either SAX or SCX is very phosphopeptide-rich, which necessitates further analysis by complementary method. Here, we developed a comprehensive phosphopeptides profiling strategy based on anion exchange followed by flow-through enrichment by TiO2 (AFET). In this strategy, SAX method was used as the first separation/enrichment step, which was online coupled with LC-MS/MS. The phosphopeptides in the SAX flow-through fraction were further enriched with TiO2. As a result, a more comprehensive, less biased phosphoproteome was aquired. Careful comparison of four different combination strategies reveal that the AFET method showed the advantages of more identified phosphopeptides, less mass spectrometry analysis time, as well as simple and automatic process step. It is well-suited for robust and reproducible phosphoproteomics, especially in the case of small amounts of sample.

Fractionation of phosphopeptides on strong anion‐exchange capillary trap column for large‐scale phosphoproteome analysis of microgram samples

It is one of the key issues to develop powerful fractionating method to increase the identification of the low-abundance phosphopeptides. In this study, a semi-online 2-D LC separation strategy based on three-step fractionation of the enriched peptides on strong anion-exchange trap column was developed. It was demonstrated that the sensitivity and phosphoproteome coverage obtained by this fractionating method with strong anion-exchange trap column is much higher than those by the conventional methods based on C18 trap column. In addition, when the same amount of sample was loaded, the number of identified phosphopeptides had increased 108%. Combination of this three-step fractionation method with RPLC-MS/MS analysis by 300 min RP-gradient separation was applied to phosphoproteome analysis of human liver proteins, and 853 unique phosphopeptides was positively identified from 500 microg tryptic digest of human liver proteins. After three cycles' consecutive analyses, 1554 unique phosphopeptides and 1566 phosphorylated sites were totally identified from 735 phosphorylated proteins at a false discovery rate of <1% in about 54 h of analysis time.

Heparin identification test and purity test for OSCS in heparin sodium and heparin calcium by weak anion-exchange high-performance liquid chromatography

Heparin sodium and heparin calcium, which are widely used as anti-coagulants, are known to potentially contain the natural impurity dermatan sulfate (DS). Recently serious adverse events occurred in patients receiving heparin sodium in the US, and a contaminant oversulfated chondroitin sulfate (OSCS) was found to be a cause of the events. To ensure the quality and safety of pharmaceutical heparins, there is need of a physicochemical identification test that can discriminate heparin from the heparin-related substances as well as a sensitive purity test for OSCS. Recently, HPLC with a strong-anion exchange column was proposed as the methods for identifying heparin and determination of OSCS in heparin sodium. Although this method is convenient and easy to perform, the only column suitable for this purpose is the Dionex IonPac AS11-HC column. In this study, we developed alternative identification test and test for OSCS in both heparin sodium and heparin calcium using a weak anion-exchange column. The identification test allowed for separation of heparin from the impurity DS and contaminant OSCS in a shorter time. The purity test provided enough sensitivity, specificity, linearity, recovery and repeatability for OSCS. We believe that our methods will be useful for quality control of pharmaceutical heparins.

Coupling Strong Anion-Exchange Monolithic Capillary with MALDI-TOF MS for Sensitive Detection of Phosphopeptides in Protein Digest

Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the analysis of protein phosphorylation by detection of phosphopeptides in phosphoprotein digest. Enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC) followed with MALDI analysis is the common approach. However, the pH for loading and elution of phosphopeptides is incompatible with protein digestion as well as the preparation of the MALDI matrix solution. Therefore, some pretreatment steps, such as pH adjustment and desalting, are required, which make the approach tedious and insensitive. In this study, a strong anion-exchange (SAX) capillary monolith was prepared to enrich phosphopeptides from protein digest for MALDI-TOF MS analysis. It was found that phosphopeptides could be specifically retained on the SAX column at high pH around 8 and could be eluted by 5% formic acid. Thus, the protein digests without any pretreatment could be loaded onto the SAX column under basic pH condition; after removing nonphosphopeptides by washing, the bound phosphopeptides could be eluted directly onto a MALDI target and analyzed by MALDI-TOF MS. This approach significantly simplified the analytical procedures and reduced the sample loss. Because of the excellent MALDI MS compatible procedure and the microscale SAX column, a detection limit as low as 50 amol for the analysis of phosphopeptides from beta-casein digest was achieved. To circumvent the inconvenience of the sample loading, a new simple sample introducing method based on capillary action was proposed, which further reduced the detection limit to 10 amol.

β-Diketones: Peak shape challenges and the use of mixed-mode high-performance liquid chromatography methodology to obtain fast, high resolution chromatography

Historically, indirect methods have been used for the HPLC analysis of β-diketone compounds because of the very poor peak shapes and resolution obtained on conventional HPLC stationary phases. In this paper we demonstrate that it is possible to obtain good peak shapes for underivatised β-diketone compounds, in a simulated reaction mixture, using only conventional mobile phases with mixed-mode stationary phase HPLC columns. Optimum conditions were obtained using the mixed-mode reversed-phase strong anion exchange column Primesep B, supplied by SIELC Technologies, with a 0.1% aq. TFA/MeOH gradient method and a column temperature of 55 °C.

Human Lipocalin-1 Association with 3H-Testosterone and 3H-Estradiol

Purpose: Topical androgens and estrogens have been studied for use in treating ocular conditions such as dry eye. The aim of this study was to identify proteins from normal human tears that associated with exogenously added sex steroid hormones. One of the major proteins in ocular tears is lipocalin-1. It binds a variety of lipids and other hydrophobic molecules and is proposed to function as a carrier protein or a lipid scavenger.Methods: Normal human tears were incubated with 3H-testosterone or 3H-estradiol. Labeled tear proteins were separated on a Q Sepharose Fast Flow (QFF) Hi Trap strong anion exchange column with a step gradient of NaCl. 3H-testosterone or 3H-estradiol was measured in aliquots of eluted fractions using scintillation counts, and the remainder of each sample was gel electrophoresed and silver stained. In separate experiments, 3H-steroid-labeled tear proteins were electrophoresed in 15% polyacrylamide gels and excised from the gels. Tritium content of the proteins was measured in a scintillation counter. Immunoblots with antibodies to lipocalin-1 verified the migration of lipocalin-1 in the gels.Results: 3H-steroid labeled tear proteins were found in the 0.15 M NaCl fractions of QFF strong anion exchange columns. 18 kD lipocalin-1 (among other tear proteins) eluted in the 0.15 M NaCl fraction. Excision of labeled tear proteins from 15% polyacrylamide gels indicated that radioactive label was associated with an 18 kD protein. Immunoblots verified that lipocalin-1 migrated as an 18 kD protein.Conclusions: The sex steroid hormones testosterone and estradiol associated with 18 kD lipocalin-1 in human tears.

Two-chaperone assisted soluble expression and purification of the bacteriophage T4 long tail fibre protein gp37

Bacteriophage T4 recognises its host cells through its long tail fibre protein gene product (gp) 37. Gp37 is a protein containing 1026 amino acids per monomer, forming a fibrous parallel homotrimer at the distal end of the long tail fibres. The other distal half-fibre protein, gp36, is much smaller, forming a trimer of 221 amino acids per monomer. Functional and structural studies of gp37 have been hampered by the inability to produce suitable amounts of it. We produced soluble gp37 by co-expression with two bacteriophage T4-encoded chaperones in a two-vector system; co-expression with each chaperone separately did not lead to good amounts of correctly folded, trimeric protein. An expression vector for the bacteriophage T4 fibrous protein chaperone gp57 was co-transformed into bacteria with a compatible bi-cistronic expression vector containing bacteriophage T4 genes 37 and 38. A six-histidine tag is encoded amino-terminal to the gp37 gene. Recombinant trimeric gp37, containing the histidine tag and residues 12–1026 of gp37, was purified from lysed bacteria by subsequent nickel-affinity, size exclusion and strong anion exchange column chromatography. Yields of approximately 4 mg of purified protein per litre of bacterial culture were achieved. Electron microscopy confirmed the protein to form fibres around 63 nm long, presumably gp36 makes up the remaining 11 nm in the intact distal half-fibre. Purified, correctly folded, gp37 will be useful for receptor-binding studies, high-resolution structural studies and for specific binding and detection of bacteria.

Negligible Expression of Arsenic in Some Commercially Available Brands of Portland Cement and Mineral Trioxide Aggregate

Introduction This study was designed aiming to determine and compare the amount of arsenic in some brands of mineral trioxide aggregate (MTA) and Portland cement. Methods In the present study, arsenic species (As[III], As[V], and dimethylarsinic acid) were separated by high-performance liquid chromatography (HPLC) using a strong anion exchange column and converted into arsines by online HG. The instrumental coupling, HPLC-HG-AFS, was applied to 0.2 g of each cement that was prior digested in a solution of HCl, HNO 3, and HBF 4. Data were expressed as a part per million, and the preliminary analysis of the raw pooled data revealed a bell-shaped distribution. Statistical analysis was performed using one-way analysis of variance for multiple comparisons. Results In all chromatograms obtained, only type III arsenic could be detected. The minimum amount of arsenic was detected in samples of white MTA ProRoot (3.3 × 10-4) and the maximum in the samples MTA Bio Angelus (Angelus, Londrina, PR, Brazil) (8.6 × 10-4). In the Gray MTA (Angelus), gray ProRoot MTA (Tulsa/Dentsply, Tulsa, OK) and CP Juntalider (Brasilatex Ltda, Diadema, SP, Brazil) did not detect any trace of arsenic. The values of arsenic found in CP Irajazinho (Votorantim Cimentos, Rio Branco, SP, Brazil) and white MTA Angelus were intermediaries to minimum and maximum values. The nonparametric test Kruskal-Wallis showed statistically similar results among all cements tested (p > 0.5). Conclusions Overall, the present study showed that all cements showed insignificant amounts of type III arsenic as well as no trace of arsenic DMA and type V could be detected.

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP–sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE–sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP–sugar pyrophosphorylase and further converted to UDP–glucuronic acid. This UDP–sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min –1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.

Simultaneous determination of inorganic and organic antimony species by using anion exchange phases for HPLC–ICP-MS and their application to plant extracts of Pteris vittata

Antimony is a common contaminant at abandoned sites for non-ferrous ore mining and processing. Because of the possible risk of antimony by transfer to plants growing on contaminated sites, it is of importance to analyze antimony and its species in such biota. A method based on high performance liquid chromatographic separation and inductively coupled plasma mass spectrometric detection (HPLC–ICP-MS) was developed to determine inorganic antimony species such as Sb(III) and Sb(V) as well as possible antimony-organic metabolisation products of the antimony transferred into plant material within one chromatographic run. The separation is performed using anion chromatography on a strong anion exchange column (IonPac AS15/AG 15). Based on isocratic optimizations for the separation of Sb(III) and Sb(V) as well as Sb(V) and trimenthylated Sb(V) (TMSb(V)), a chromatographic method with an eluent gradient was developed. The suggested analytical method was applied to aqueous extracts of Chinese break fern Pteris vittata samples. The transfer of antimony from spiked soil composites into the fern, which is known as a hyperaccumulator for arsenic, was investigated under greenhouse conditions. Remarkable amounts of antimony were transferred into roots and leaves of P. vittata growing on spiked soil composites. Generally, P. vittata accumulates not only arsenic (as shown in a multiplicity of studies in the last decade), but also antimony to a lower extent. The main contaminant in the extracts was Sb(V), but also elevated concentrations of Sb(III) and TMSb(V) (all in μg L −1 range). An unidentified Sb compound in the plant extracts was detected, which slightly differ in elution time from TMSb(V).

Analysis of heparin sodium by SAX/HPLC for contaminants and impurities

A chromatographic method was developed for the detection and quantification of the contaminant oversulfated chondroitin sulfate (OSCS) and the impurity dermatan sulfate in heparin active pharmaceutical ingredient (API). The HPLC analysis of heparin is carried out using a polymer-based strong anion exchange (SAX) column with gradient elution from 0.125M sodium chloride to 2.5M sodium chloride buffered mobile phase. The limit of detection (LOD) and limit of quantitation (LOQ) for the contaminant OSCS in heparin were determined to be 0.03% and 0.1%, respectively. The LOD and LOQ for dermatan sulfate, an impurity in heparin sulfate, were determined to be 0.1% and 0.8%, respectively. This manuscript is not a policy document and is not intended to replace either of the methods (capillary electrophoresis and NMR) currently required by the FDA.