Exchange High Performance Liquid Chromatography Research Articles

Analysis of the related substances in phosphorothioate antisense oligonucleotide FT19

A reversed-phase high performance liquid chromatographic (RP-HPLC) method was developed for the simultaneous determination of the contents of adapalene and the preservatives (2-phenoxyethanol and methyl-4-hydroxybenzoate) in adapalene gels. The chromatographic analysis was carried out on a Tigerkin C18 column (150 mm x 4.6 mm, 5 microm). The mobile phase was 0.02 mol/L ammonium acetate buffer (pH 3.0) and tetrahydrofuran-acetonitrile with gradient elution, and the detection wavelength was set at 270 nm. The calibration curves were linear over the ranges of 10-100 mg/L (r = 0.9999), 4-40 mg/L (r = 0.9999) and 4-40 mg/L (r = 0. 9999) for 2-phenoxyethanol, methyl-4-hydroxybenzoate and adapalene, respectively. The average recoveries of the three substances were within 98.0%-98.6%. The method is simple, reliable and suitable for the simultaneous determination of adapalene, 2-phenoxyethanol and methyl-4-hydroxybenzoate in adapalene gels.

Functional characterization of pathogen-responsive protein AtDabb1 with an antifungal activity from Arabidopsis thaliana

A plant antifungal protein was purified from Arabidopsis thaliana leaves by using a typical procedure consisting of anion exchange chromatography and high-performance liquid chromatography. We determined the amino acid sequence of the purified protein using MALDI-TOF/MS analysis, and found that the sequence matched that of a hypothetical Arabidopsis protein in GenBank (accession number NP_175547). We designated the protein as AtDabb1. After the cDNA encoding the AtDabb1 gene was cloned from an Arabidopsis leaf cDNA library, the recombinant protein was expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the AtDabb1 gene was induced by pathogen-related signaling molecules including salicylic acid and jasmonic acid. These results suggest that AtDabb1 may contribute to the induced plant defense mechanism against diverse pathogenic fungi.

Haemoglobin Noah Mehmet Oeztuerk (α 2 δ 2143 (H21)His → Tyr: A novel δ-chain variant in the 2,3-DPG binding site

A new δ-chain variant, δ143 (H21) His → Tyr or Hb Noah Mehmet Oeztuerk, was discovered during the investigation of the cause of hemolytic anaemia in a 6-month-old infant of Turkish descent. It was detected by Cation exchange high-performance liquid chromatography (CE-HPLC) using PolyCAT A column. P 50 was 20.6 ± 0.60 mmHg and 29.3 ± 0.40 mmHg for the carrier and the wild-type, respectively. This suggests an increase in oxygen affinity. On routine CE-HPLC Hb A 2 was low (1.2%) and the variant was not detected. An extended family study revealed that the variant was not associated with the anaemia or with any other clinical abnormality.

Evidence That Heparin Saccharides Promote FGF2 Mitogenesis through Two Distinct Mechanisms

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

BackgroundThe diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO2-) and nitrate (NO3-) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes.MethodWhile exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO2- and NO3- from mosquito mid-guts and haemolymph.ResultsThis method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO2- and NO3- in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1–100 μM) and from the extracted standards (1–100 μM) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO2- and NO3- in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO2- and NO3- in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology.ConclusionHPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.

Quantitative and Qualitative Evaluation of Chondroitin Sulfate in Dietary Supplements

We propose to evaluate the amount and quality of chondroitin sulfate (CS) from several food supplement preparations in the form of tablets, caplets, or capsules containing CS in varying contents and formulations in the presence of various additives and ingredients, with no other pretreatment than centrifugation to remove insoluble material. To quantify CS, two different analytical approaches were applied after their validation: specific and sensitive agarose-gel electrophoresis and strong-anion exchange–high-performance liquid chromatography (SAX-HPLC) determination of the constituent disaccharides after treatment with specific chondroitin lyases. The CS content in finished products evaluated using the two specific validated methods were found to conform to the label specifications. It is worth mentioning that the quantitative determinations have been performed using a very high pure European Pharmacopeia CS reference standard having substantially the same properties as the food supplement CS samples. Furthermore, by means of the specific agarose-gel elctrophoresis approach, we can exclude the presence in the nutraceuticals of other sulfated polysaccharides produced by extraction from tissues, in particular heparin, heparan sulfate, and dermatan sulfate. The SAX-HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and the possible origin. No disulfated disaccharides typical of CS from cartilaginous fishes, making the charge density lower than 1.0, were found, thus confirming the bovine or porcine origin, the most common sources of this “terrestrial” polysaccharide. Finally, high-performance size-exclusion chromatography (HPSEC) analysis was applied to evaluate the CS molecular mass in food supplements also in the presence of additives and other ingredients. This analytical approach confirmed that the nutraceutical CS samples are of high molecular mass and are not degraded during the food supplement preparations. In conclusion, this multianalytical approach can be used in the direct quantitation and evaluation of the quality and the possible origin of CS contained in food supplement formulations.

Compound Heterozygosity for Hemoglobin C and Hemoglobin Korle-Bu

We describe a case of compound heterozygosity for hemoglobin C (beta6 Glu-->Lys) and hemoglobin Korle-Bu (beta73 Asp-->Asn). To our knowledge, this is the third case report of this unusual hemoglobin genotype, and the first to integrate data from cation exchange high-performance liquid chromatography, hemoglobin electrophoresis, and beta-globin gene sequencing. The principal hematological finding in our case was microcytosis without significant anemia. The previous case reports of this hemoglobinopathy also describe microcytosis, with varying degrees of hemolytic anemia. Given the relative gene frequencies of hemoglobins C and Korle-Bu, the occurrence of this compound-heterozygous genotype should be anticipated in people of African ancestry.

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).

Two New Hemoglobin Variants: Hb Sinai-Greenspring [β34(B16)Val→Ile, GTC > ATC] and Hb Sinai-Bel Air [β53(D4)Ala→Asp, GCT > GAT

Neonatal screening for hemoglobinopathies occasionally results in the detection of novel hemoglobin (Hb) variants. Two heterozygous infants were found with different β chain mutations, neither of which produced obvious clinical or laboratory abnormalities on routine examinations. The variants were characterized by cation exchange high performance liquid chromatography (HPLC), reversed phase HPLC, and sequencing of amplified β-globin genes. Functional studies could not be performed at this time.

Hb Gerland [α55(E4)Val→Ala]: A Mutation Found on the α1-Globin Gene

The Hb Gerland [α55(E4)Val→Ala] mutation has been described in the α2-globin gene. We report here the same mutation in the paralogous α1-globin gene. This variant was found in a healthy 18-month-old boy of Chinese origin. Abnormal hemoglobin (Hb) fractions were visible on isoelectric focusing (IEF) and cation exchange high performance liquid chromatography (HPLC), with elution patterns differing from one system to another. Direct sequencing of the α-globin genes revealed a GTT>GCT (Val→Ala) transversion at codon 55 of the α1-globin gene. We propose to name the variant encoded by the α1-globin gene Hb Gerland [A1], and the variant that is encoded by the α2-globin gene Hb Gerland [A2].

Quantitative arsenic speciation in two species of earthworms from a former mine site

The relationship between the total arsenic concentration and the chemical speciation of arsenic in two species of earthworm (Lumbricus rubellus and Dendrodrilus rubidus) in relation to the host soil, was investigated for 13 sites of varying arsenic content, including a background level garden soil and a former mine site at the Devon Great Consols, UK. Earthworms were collected with the host soil (As soil concentration range 16-12, 466 mg kg(-1) dry weight) and measured for their total arsenic (concentration range 7-595 mg kg(-1) dry weight) using inductively coupled plasma mass spectrometry (ICP-MS). A methanol-water mixture was used to extract arsenic species from the earthworms prior to determination of the individual arsenic species by a combination of anion and cation exchange high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). A gradient elution anion exchange method is presented whereby nine arsenic species could be measured in one sample injection. Arsenic species were identified by comparison of retention times and sample spiking with known standards and a fully characterised seaweed extract. Arsenic was generally present in the earthworm as arsenate (As(V)) or arsenite (As(III)) and arsenobetaine (AB). Methylarsonate (MA), dimethylarsinate (DMA) and three arsenosugars (glycerol, phosphate, sulfate) were present as minor constituents. These results are discussed in relation to the mechanisms for coping with exposure to soil bound arsenic.

Assessment of total arsenic and arsenic species stability in alga samples and their aqueous extracts

In order to achieve reliable information on speciation analysis, it is necessary to assess previously the species stability in the sample to analyse. Furthermore, in those cases where the sample treatment for species extraction is time-consuming, an assessment of the species integrity in the extracts is of paramount importance. Thus, the present paper reports total arsenic and arsenic species stability in alga samples ( Sargassum fulvellum and Hizikia fusiformis), as well as in their aqueous extracts, which were stored in amber glass and polystyrene containers at different temperatures. Total arsenic determination was carried out by inductively coupled plasma atomic emission spectroscopy (ICP-AES), after sample acid digestion in a microwave oven, while arsenic speciation was conducted by anion exchange high performance liquid chromatography on-line coupled to ICP-AES, with and without sample introduction by hydride generation (HPLC-ICP-AES and HPLC-HG-ICP-AES), after aqueous microwave-assisted extraction. The results obtained for solid alga samples showed that total arsenic (for Hijiki alga) and arsenic species present (As(V) for Hijiki and NIES No. 9 Sargasso) are stable for at least 12 months when samples are stored in polystyrene containers at +20 °C. On the other hand, a different behaviour was observed in the stability of total arsenic and As(V) species in aqueous extracts for both samples, being the best storage conditions for Sargasso extracts a temperature of −18 °C and polystyrene containers, under which they are stable for at least 15 days, while Hijiki extracts must be stored in polystyrene containers at +4 °C in order to ensure the stability for 10 days.

Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices.

Changes of Serum Glycans During Sepsis and Acute Pancreatitis

Acute inflammatory response is a complex process associated with the production of both pro- and anti-inflammatory mediators. Although it is generally considered to be a single homeostatic mechanism, there are differences associated with the nature and the site of inflammation. We examined the changes of N-linked glycans released from the serum of a patient with sepsis and a patient with acute pancreatitis during the first eight days of the disease. Sera were taken from patients at the time of reporting to hospital and then three more times. The blood from a healthy individual was drawn on one occasion only. Glycans were released using N-glycosidase F and were subjected to normal phase and weak anion exchange high-performance liquid chromatography, exoglycosidase digestions, and mass spectrometry. The levels of identified structures have been followed through the course of disease and compared to the control levels. Changes in serum glycans were found to occur very early in acute inflammation. The most prominent differences include the increase in ratio of outer arm to core fucose, increase in the amount of tetrasialylated structures, changes in the levels of mannose structures, and in the degree of branching. The relative proportions of different glycans changed daily and some differences were also observed between sepsis and pancreatitis, probably reflecting that in these two conditions, the acute phase response is triggered by a different stimulus that is associated with different patterns of production of cytokines.

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205 nm. The novel method described here uses a running buffer of aqueous 200 mM NaH 2PO 4 (pH 7.05) containing 100 mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.

Characterization of Fusarium graminearum inhibitory lipopeptide from Bacillus subtilis IB

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.

Validation of a high-performance liquid chromatographic assay for the quantification of Reovirus particles type 3

An anion exchange high-performance liquid chromatography (HPLC) method for the quantification of human Reovirus type 3 particles was validated according to the performance criteria of precision, specificity, linearity of calibration and working range, limits of detection and quantification, accuracy and recovery. Samples taken at various stages of Reovirus purification were used for the validation of the method. The method was specific for Reovirus which eluted around 9.8 min without interference from any other component in the sample. Reovirus can be detected between 0.32E+12 and 2.10E12 VP/mL by the proposed method that has the correlation coefficient of linearity equal to 0.9974 and the slope of linearity equal to 5.74E−07 area units/(VP mL).

Use of stable isotopically enriched proteins and directly coupled high-performance liquid chromatography inductively coupled plasma mass spectrometry for quantitatively monitoring the transfer of metals between proteins

Studies have shown that metallothionein (MT) may play an important role in modulating the activity of certain Zn-regulated enzymes under various oxidoreductive conditions by either donating or removing Zn. To better determine the role of MT in interprotein metal transfer, we describe a procedure that uses stable isotopically enriched 67Zn 7 metallothionein 2 ( 67Zn 7–MT-2) to quantitatively determine the stoichiometry of transfer of Zn from the protein to a recipient apo-metalloenzyme, apo-carbonic anhydrase (apo-CA) by directly coupled ion exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry. Quantitatively, the transfer of 67Zn was consistent with the enzymatic activation of the apo-enzyme as judged by its esterase activity and ability to cleave p-nitrophenyl acetate. Maximum enzyme activation occurred at an MT-2:apo-CA molar ratio of 1, implying the release of a single atom of Zn from MT-2. Preincubation of 67Zn 7–MT-2 with an excess of oxidized glutathione (GSSG) increased metal donation fourfold, whereas reduced glutathione (GSH) inhibited donation by approximately 50%. By using multiple recipient and donor proteins having different stable isotopic signatures, the technique has the potential for quantitatively studying the kinetic and thermodynamic aspects of Zn transfer between numerous competing ligands in vitro, an important first step toward understanding the regulatory role of this metal in protein functioning and cellular metabolism in vivo.

Evaluation of Transformation Ability of Caesium Chloride Density Gradient Centrifugation and Ion Exchange High Performance Liquid Chromatography Purified Plasmids

Evaluation of Transformation Ability of Caesium Chloride Density Gradient Centrifugation and Ion Exchange High Performance Liquid Chromatography Purified Plasmids

Direct Determination of Ofloxacin Enantiomers in Human Urine by Ligand Exchange Chromatography

A sensitive and simple method for the determination of ofloxacin enantiomers in human urine was developed by ligand exchange high performance liquid chromatography. Chiral separation was performed on a C18 column, where the mobile phase consisted of a methanol‐water solution (containing1.2 mmol L−1 L‐phenylalanine and 1.0 mmol L−1 copper sulphate) (15∶85, v/v) and its flow rate was set at 1.0 mL min−1. After centrifugation, the human urine was injected into a C18 column directly. Baseline separation of ofloxacin enantiomers in human urine were obtained with a resolution of 3.24 in less than 25 min, and no interference by the protein or endogenous compounds were observed. The effects of different separation conditions were investigated and the concentration of ligand and pH of the mobile phase play a critical role in the enantioseparation. The standard curves showed excellent linearity over the concentration range from 0.8 to 400 µg mL−1 for ofloxacin enantiomers. The linear correlation equations are: Y=171.11X+ 504.13 (r=0.9995) and Y=169.01X+631.59 (r=0.9994) for (S)‐ofloxacin and (R)‐ofloxacin, respectively. The average recovery of ofloxacin enantiomers from human urine samples was more than 96%. The procedure developed was successfully applied to investigate the stereoselectivity and pharmacokinetics of ofloxacin enantiomers in the human body.