Weak Anion Exchange Chromatography Research Articles

Development and validation of a LC‐MS/MS assay to quantify intracellular tenofovir‐diphosphate (TFV‐DP) and emtricitabine‐triphosphate (FTC‐TP)

IntroductionCombination antiretroviral therapy has been associated with dramatic reductions in morbidity and mortality in HIV‐infected patients. The key to successful HIV treatment is strict adherence to the prescribed regimen. The combination of tenofovir, emtricitabine and efavirenz co‐formulated in a single tablet (Atripla) provides a ‘one tablet once a day’ regimen, making adherence easier for patients. Tenofovir and emtricitabine are converted intracellularly to active phosphate anabolites. Therefore, knowledge of intracellular pharmacokinetics should aid understanding of the virological response and toxicity of these agents. This intracellular methodology has been developed and applied to clinical trial samples.MethodPeripheral blood mononuclear cells (PBMC) were isolated from whole blood by gradient centrifugation using ficoll. PBMC were counted and lysed (70% MeOH) giving a final cell density of 2×106 cells/ml. Lysate was centrifuged, supernatant (intracellular matrix), was spiked with drug anabolites at various concentrations to produce standard curves and quality controls (QC). Samples were extracted by protein precipitation (acetonitrile), evaporated to dryness, internal standard (IS) added, and reconstituted in 5 mM ammonuim formate. Weak anion exchange chromatography with an optimised step‐wise gradient, interfaced with a triple quadrupole mass spectrometer operated in SRM with positive ionisation mode was used for quantification.ResultsAnalytes eluted within 12 minutes run‐time with adequate separation. Calibration curves were validated over the following range TFV‐DP = 0.35–10.91 ng/mL, FTC‐TP = 0.38–103.17 ng/mL (r2 values > 0.99; linear 1/x). The lower limit of quantification was < 20%, signal to noise was > 5% and carryover < 0.1%. The precision (relative standard deviation,% RSD) of the assays as determined from analysis of quality control samples was; TFV‐DP = 6.3–11% and FTC‐TP = 6–18.6%, and accuracy was TFV‐DP = 97.5–100.8%, FTC‐TP = 98–100.3%.ConclusionsA direct, highly sensitive intracellular anabolite assay has been developed and validated which elutes analytes and IS rapidly and has been applied to patient samples from a pivotal trial the results of which are crucial in our understanding of antiretroviral drug ‘forgiveness’.

Simultaneous Analysis of Steviol and Steviol Glycosides by Liquid Chromatography with Ultraviolet Detection on a Mixed-Mode Column: Application to Stevia Plant Material and Stevia-Containing Dietary Supplements

Simultaneous separation of steviol and steviol glycosides is challenging because of differences in their polarity and chemical structure. In this study, simultaneous analysis of steviol and steviol glycosides was achieved by LC with UV detection using a mixed-mode RP weak anion exchange chromatography column. Steviol and seven steviol glycosides were analyzed on an Acclaim Mixed-Mode Wax-1 (Dionex) column with a linear gradient of deionized water adjusted to pH 3.00 with phosphoric acid and acetonitrile. The extraction was performed by sonicating dry plant material at 40 degreesC in acetonitrile-water (30 + 70, v/v). LOQ values (mg/g dry weight of plant material) were rebaudioside B, 0.50; steviol, 0.70, dulcoside A, 1.0; steviolbioside, 1.2; stevioside and rebaudioside C, 2.0; rebaudioside D, 3.3; and rebaudioside A, 5.0. The method demonstrated suitable performance for all analytes tested with respect to accuracy (mean recoveries 95-99%), intraday and interday precision for retention times (0.070-0.28% and 0.33-1.0% RSD, respectively), and linearity. The method was used to authenticate steviol glycosides in several samples of Stevia plant material as well as to quantitate steviol glycosides in dietary supplements containing Stevia.

Structural investigation of water-soluble polysaccharides extracted from the fruit bodies of Coprinus comatus

Water-soluble polysaccharide material, extracted from the stipes of the fruit bodies of Coprinus comatus by hot water, was fractionated by sequential weak anion-exchange and size-exclusion chromatography. The relevant fractions were subjected to structural analysis, including (d/l) monosaccharide/methylation analysis and 1D/2D NMR spectroscopy. Besides the disaccharide α,α-trehalose [α-d-Glcp-(1↔1)-α-d-Glcp], high-molecular-mass α-d-glucans (the most abundant component) consisting of [→4)-α-d-Glcp-(1→]n backbones with ∼10% branching at C-6 by terminal α-d-Glcp-(1→6)- or α-d-Glcp-(1→6)-α-d-Glcp-(1→6)- units, lower-molecular-mass linear β-d-glucans consisting of [→6)-β-d-Glcp-(1→]m sequences, and a lower-molecular-mass pentasaccharide-repeating α-l-fuco-α-d-galactan, {→6)-α-d-Galp-(1→6)-[α-l-Fucp-(1→2)-]α-d-Galp-(1→6)-α-d-Galp-(1→6)-α-d-Galp-(1→}p, were found to be present.

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.

Improving Depth in Phosphoproteomics by Using a Strong Cation Exchange-Weak Anion Exchange-Reversed Phase Multidimensional Separation Approach

Several enrichment and separation strategies are available that allow nearly pure phosphopeptide pools to be created. These phosphopeptide pools are too complex to be completely unraveled by RP-LC-MS analysis alone. Here, we implement weak anion exchange (WAX) chromatography as an additional, complementary dimension to strong cation exchange (SCX) and reversed phase (RP). Initially, we used SCX to fractionate a human lysate digest to generate a fraction highly enriched for phosphopeptides. Analysis of this single fraction by RP-LC-MS with a 140 min gradient method allowed the identification of 4045 unique phosphopeptides (false discovery rate (FDR) < 1%; Mascot score > 20) using an Orbitrap Velos. Triplicate analysis (420 min total gradient time) of the same sample increased the total to just over 5000 unique phosphopeptides. When we separated the same sample by WAX and analyzed 14 WAX fractions by 30 min gradient RP-LC-MS (420 min total gradient time) we were able to identify 7251 unique phosphopeptides, an approximate increase of 40%, while maintaining the same total gradient time. We performed a more comprehensive, albeit also more time-consuming, analysis of the same 14 WAX fractions by the use of 140 min gradient LC-MS analyses, which resulted in the detection of over 11 000 unique phosphopeptides. Our results clearly demonstrate that additional separation dimensions are still necessary for in-depth phosphoproteomics and that WAX is a suitable dimension to be combined and sandwiched between SCX and RP chromatography.

Anti-tumor activity of components isolated from purple sweet potato polysaccharides

To isolate and purify components from polysaccharides of purple sweet potato (PPSP) and to test their anti-tumor activity. DEAE-Cellulose and CM-Cellulose exchange chromatography were applied to separate components of PPSP. The anti-tumor activities of each component were measured by MTT assay on Hela and HepG(2) cells and their monosaccharide composition were analyzed by TLC chromatography, followed by infrared spectroscopy studies. Through weak anion exchange chromatography and gradient elution by sodium chloride solution, four components were separated and named as PPSP, PPSPII, PPSPIII and PPSPIV, respectively. MTT tests showed that PPSP II and PPSPIII inhibited Hela and HepG2 tumor cells in a certain extent. The structural analysis revealed that PPSPI was mainly composed of glucose and galactose, PPSP II was composed of glucose and had a typical absorption peak of β-D-glucose chitosan pyranose, PPSP III was a glycoprotein showing a protein absorption peak. Four components were separated from PPSP successfully, among which PPSP II and PPSP III shows anti-tumor activities on Hela and HepG(2) cells in vitro.

One proteoglycan from the fruiting bodies of Chroogomphis rutilus (Schaeff.: Fr.) O.K. Miller: purification and structural features

A water-soluble proteoglycan (CRP-W2) was purified from the fruiting bodies of Chroogomphis rutilus by boiled water extraction, ethanol precipitation, weak anion exchange and size exclusion chromatography. Its molecular weight (Mw) was estimated to be about 1.3×104Da using high-performance size-exclusion chromatography (HPSEC). As a precondition to understand the bioactivity, the structural features of CRP-W2 was analyzed using chemical methods, IR spectroscopy and NMR spectroscopy. The results indicated that CRP-W2 had a backbone consisting of 1,6-linked and 1,2,6-linked-α-d-Gal, which was terminated with 1-linked-α-d-Man and 1-linked-α-d-Glc residues at the O-2 position of 1,2,6-linked-α-d-Gal in the molar ratio of 2:2:1:1.

Identification of low-abundance proteins via fractionation of the urine proteome with weak anion exchange chromatography

BackgroundLow-abundance proteins are difficultly observed on the two-dimensional gel electrophoresis (2-DE) maps of urine proteome, because they are usually obscured by high-abundance proteins such as albumin and immunoglobulin. In this study, a novel fractionation method was developed for enriching low-abundance proteins by removing high-abundance proteins and progressive elution with salts of various concentrations.ResultsStepwise weak anion exchange (WAX) chromatography, which applied DEAE-Sephacel resin with non-fixed volume elution, was used to fractionate urine proteome prior to performing 2-DE. Urine proteome was separated into four fractions by progressively eluting the column with 0 M, 50 mM, 100 mM, and 1 M NaCl solutions. Most of the heavy and light immunoglobulin chains appeared in the eluent. After the high-abundance proteins were removed, various low-abundance proteins were enriched and could be easily identified. The potential of this method for obtaining diversified fractionations was demonstrated by eluting the column separately with Na2SO4 and MgCl2 solutions. The 2-DE maps of the fractions eluted with these different salt solutions of identical ionic strength revealed markedly different stain patterns.ConclusionThe present study demonstrated that this fractionation method could be applied for purposes of enriching low-abundance proteins and obtaining diversified fractionations of urine, and potentially other proteomes.

Physical fractionation of aquatic viral assemblages

Aquatic viruses are extraordinarily diverse and have major influences on plankton ecology and evolution, but the majority of these viruses remain uncharacterized. Most viruses have not been cultivated and cultivationindependent means to explore viral diversity have provided only a fragmented view of viral genomic information. In this study, ultracentrifugation and chromatographic methods were evaluated for their ability to fractionate aquatic viruses based on their differing physical properties with the aim of physically enriching subsets of aquatic viruses for further study. Centrifugation in continuous cesium chloride gradients, strong and weak anion‐exchange chromatography, and size‐exclusion chromatography with Sephacryl S‐1000 were found to separate aquatic viruses based on their differing buoyant densities, surface charges, and sizes, respectively. Sucrose density gradients, cation‐exchange chromatography, and size‐exclusion chromatography using controlled‐pore glass were found to have insufficient resolution to effectively separate viral assemblages. Using the successful methods listed above, we were able to separate complex assemblages of viruses into fractions that had distinguishable subsets of the viruses present in the original community as determined by pulsed‐field gel electrophoresis. Strong anion‐exchange chromatography was particularly effective at separating viruses in the samples and resulted in one to six dominant viral genome bands in most fractions. Our results suggest that individual populations of viruses may be physically enriched, and possibly isolated, from complex assemblages without cultivation. These fractionation methods are expected to improve our ability to characterize the genotypes and phenotypes of the uncultivated majority of viruses in aquatic environments.

Solid-support fluorescent derivatization of picomoles of protein at low concentration with FITC

A new method based on solid-support reaction is described to realize fluorescent derivatization of proteins at concentrations as low as 10−8M. A simple, low-cost homemade capillary C18 cartridge was fabricated as the solid-support reactor. Using bovine serum albumin (BSA) as a test protein, we demonstrated that the protein can be captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. The analysis by nano-HPLC with laser-induced fluorescence (LIF) detection was described. The effect of reaction conditions on the derivatization has been evaluated and discussed. The use of the solid-support reactor allows easy handling of as little as 8.5pmol of BSA. A fraction from weak anion-exchange chromatography (WAX) of human liver extract was used as an illustrative example of application to real samples.

Weak anion and cation exchange mixed-bed microcolumn for protein separation

To separate proteins with a wide distribution of pIs under the conditions compatible to online tryptic digestion (with preferable pH=8.0), weak anion and cation exchange chromatography (WAX/WCX) mixed-bed microcolumn has been developed. With a mixture of five proteins with pIs ranging from 4.2 to 11.4, the effect of WAX/WCX ratio on the separation performance was investigated, and an optimum packing ratio of 1:1 w/w was obtained. Moreover, the undesirable hydrophobic interaction between the proteins and the stationary phase was suppressed with 10% ACN v/v added in the mobile phases. Under the optimized conditions compatible to tryptic digestion, basic and acidic proteins were resolved simultaneously, with RSDs of relative retention time on six columns less than 6%, indicating the good resolution and packing reproducibility. Furthermore, one RPLC fraction of proteins extracted from rat middle brain and the whole protein mixture extracted from rat liver were analyzed, respectively. The results demonstrated better separation performance on WAX/WCX microcolumns than that on both weak anion exchange chromatography and weak cation exchange chromatography at pH ∼8. We anticipate that WAX/WCX microcolumns are promising for the integration of protein separation and tryptic digestion aiming at high-throughput proteome study.

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.

Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography

We present a simple and fast method for large-scale purification of RNA oligonucleotides suitable for biochemical and structural studies. RNAs are transcribed in vitro with T7 RNA polymerase using linearized plasmid DNA templates. After addition of EDTA, the crude transcription reaction is subjected directly to weak anion-exchange chromatography using DEAE-sepharose to separate the T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, and the plasmid DNA template from the desired RNA product. The novel method does neither require tedious phenol/chloroform extraction of the T7 RNA polymerase nor denaturation of the RNA, which is desirable especially for larger RNAs. In addition, isotopically labeled rNTPs can be easily recycled from the column flow-through and oligomeric RNA aggregates can be separated from the natively folded monomeric RNA product.

Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within −10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within −14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.

Characterization of Heparin Impurities with HPLC-NMR Using Weak Anion Exchange Chromatography

A weak anion exchange (WAX) chromatography method coupled to online UV and NMR detection was developed for the separation and structural identification of heparin impurities. This work describes the separation of dermatan sulfate, chondroitin sulfate A, heparin, and the semisynthetic fully sulfated chondroitin sulfate, FSCS, through a displacement-based mechanism. The WAX separation requires significantly less salt than comparable strong anion exchange chromatography, facilitating NMR detection using a standard commercially produced flow probe. Retention and elution of both heparin and FSCS were found to be highly dependent on pH/pD and salt concentration. The elution time of each analyte could be controlled simply by adjusting a delay prior to introduction of the appropriate salt concentration, with little impact of the extent of the delay on chromatographic peak shape. Both on-flow and stop-flow proton ((1)H) NMR spectra were acquired to monitor and identify eluting components following separation. The approach described for the online separation of heparin and its potential impurities adds to the arsenal of techniques available for the rapid identification of new natural and/or intentional contaminants that may be introduced into the heparin supply chain.

Refolding of reduced/denatured bovine pancreatic insulin with ion-exchange chromatography coupled with MALDI-TOF MS

The refolding of the reduced/denatured insulin from bovine pancreas as the model protein was investigated with weak anion exchange chromatography (WAX) coupled with MALDI-TOF MS. The results indicated that the disulfide bonds almost cannot be formed correctly with the common mobile phase by WAX. However, with the urea gradient elution and in the presence of GSSG/Cyst as the ratio 1:6 in the mobile phase employed, the disulfide exchange of reduced/denatured insulin can be accelerated resulting in forming the correct three disulfide bonds. The protein refolding efficiency of reduced/denatured insulin can be increased from 3% to 34%. The effects of urea gradient and the oxidant and reductant groups, such as GSSG/GSH, Cyst, and GSSG/Cyst, on the forming the disulfide bonds of reduced/denatured insulin were investigated in detail. The results were further tested by the separation of the WAX fraction of reduced/denatured insulin with RPLC and MALDI-TOF MS.

Mixed retention mechanism of proteins in weak anion-exchange chromatography

Using four commercial weak anion-exchange chromatography (WAX) columns and 11 kinds of different proteins, we experimentally examined the involvement of hydrophobic interaction chromatography (HIC) mechanism in protein retention on the WAX columns. The HIC mechanism was found to operate in all four WAX columns, and each of these columns had a better resolution in the HIC mode than in the corresponding WAX mode. Detailed analysis of the molecular interactions in a chromatographic system indicated that it is impossible to completely eliminate hydrophobic interactions from a WAX column. Based on these results, it may be possible to employ a single WAX column for protein separation by exploiting mixed modes (WAX and HIC) of retention. The stoichiometric displacement theory and two linear plots were used to show that mechanism of the mixed modes of retention in the system was a combination of two kinds of interactions, i.e., nonselective interactions in the HIC mode and selective interactions in the IEC mode. The obtained U-shaped elution curve of proteins could be distinguished into four different ranges of salt concentration, which also represent four retention regions.

Fingerprinting complex pectins by chromatographic separation combined with ELISA detection

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-α- l-arabinan and (1,4)-β- d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.

The HIV-1 Envelope Glycoprotein gp120 Features Four Heparan Sulfate Binding Domains, Including the Co-receptor Binding Site

It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg(419), Lys(421), and Lys(432), which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition.