Reversed-phase Ion-pair Chromatography Research Articles

Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids

The combined effect of the ion-pairing reagent concentration, C(ipr), and organic modifier content, phi, on the retention under phi-gradient conditions at different constant C(ipr) was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D phi-gradient retention data obtained under the same phi-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C(ipr) values in the eluent. The second approach is based on a retention model describing the combined effect of C(ipr) and phi on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C(ipr) values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C(ipr) values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by phi-variation.

Investigation of seleniummetabolites in Se-enriched kale, Brassica oleracea A, viaHPLC-ICPMS and nanoESI-ITMS

Kale (Brassica oleracea A.) can accumulate high concentrations of Se, as well as lutein and β-carotene, which have important human health benefits and possess strong antioxidant properties. In the present study, kale plants were cultivated in soil supplemented with sodium selenite or sodium selenate. The plants accumulated over 20 times higher concentration of selenium in the selenate-enriched soil compared with selenite-enriched soil. Selenium accumulation did not suppress the plant's uptake of the essential metals, Fe, Cu and Zn. Speciation of selenium in the plant was performed via size-exclusion chromatography and ion-pairing reversed phase chromatography using inductively coupled plasma mass spectrometry (ICPMS) and nano-electrospray ion trap mass spectrometry (nanoESI-ITMS) for detection. In ion-pairing reversed phase chromatography, 0.02% HFBA was found to facilitate separating the selenium metabolites without hampering ESI-ITMS ionization. MS2/MS3 spectra were successfully obtained for identification of Se-methylselenocysteine (MeSeCys). MeSeCys was the prominent selenium metabolite formed in kale, which has been suggested as a chemopreventive food supplement. The Se species in leaf are significantly different from those in stem. The leaf shows higher content of high molecular weight selenium than the stem, while the stem produced higher amounts of MeSeCys than the leaf, indicating the metabolism to MeSeCys competes with the production of high molecular weight selenium forms. The plant leaf, when supplemented with Se(IV), contained the highest amount of high molecular weight selenium (49%) as well as the lowest amount of MeSeCys (4%).

Ion-pair reversed-phase chromatography for speciation of organotin compounds

Objective: Ion-pair reversed-phase chromatography (IP-RP) has been investigated for speciation of dibutyltin (DBT), tributyltin (TBT), and triphenyltin (TPhT). The influence of several parameters (pH, alkyl chain length, concentration of the ion pair reagent) has been studied to determine the best chromatographic conditions. Methods: Separation of the three organotin compounds was achieved on a C8-bonded silica column using a mixture of methanol, water, and acetic acid (80:19:1) as eluent, containing 1 mmol/L decane sulfonic acid as ion pairing reagent. The eluates were detected on-line by hydride generation-quartz furnace atomic absorption spectrophotometry (HG-QFAAS). Results: The resolving power of this developed method for separation of the organotins species at above conditions was shown by the values of fundamental chromatographic parameters. The capacity factors (k’) for DBT, TBT and TPhT species were 0.27, 2.54, and 5.92 respectively. Resolution (Rs) values for DBT-TBT and TBT-TPhT separation were 2.92 and 2.42 respectively, while the selectivity for DBT-TBT and TBT-TPhT were 9.76 and 3.50 respectively. Conclusion: The developed IP-RP-HG-QFAAS chromatography technique can separate DBT, TBT, and TPhT with good performance which is shown by the chromatographic parameters produced.

Determination of EDTA in Dairy Wastewater and Adjacent Surface Water

An HPLC-UV analytical method was developed to determine ethylenediaminetetraacetic acid (EDTA) in dairy wastewater and surface water. The optimizing separation was achieved by reversed-phase ion-pair liquid chromatography on a C18 column using methanol as mobile phase solvent, tetrabutylammonium bromide as the ion-pair reagent in pH 3.3 formate buffer solution at a flow rate of 0.9 mL min -1 with a UV detector at 265 nm. No interference of Ca, Mg or NO3 - was detected. Method performance was evaluated in terms of linearity, repeatability and reproducibility. The method detection limit was 5 µg L -1 . The contents of EDTA in dairy effluents were 72 ~ 261 µg L -1 at a large dairy site. A change of EDTA concentration was observed downstream of the dairy effluent discharge, but this was well under the predicted no effect concentration for aquatic ecosystem. Keywords—Dairy wastewater, EDTA, HPLC, surface water. I. INTRODUCTION LEANING of equipment for production plants is a key process in the dairy industry to ensure safe products with high quality. It has been demonstrated (1) that using synergic effects of single components in chemical mixtures, for instance adding an effective complexing agent, is more likely to achieve an improved cleansing result with lower concentration compared to aqueous solutions merely containing the basic component of sodium hydroxide. The main function of complexing agents in a cleaning solution is to prevent precipitation of calcium, magnesium and heavy metal salts, which can cause deposits to appear on both the cleaning equipment and the plant to be cleaned (2). At present, ethylenediaminetetraacetic acid (EDTA) is the cheapest and most suitable complexing compound for many technical purposes, and is used in large quantities (3). In the New Zealand dairy industry, EDTA has been used as a cleaning additive to improve the cleansing efficiency during the clean-in-place (CIP) procedure. Nearly all applications eventually result in the release of EDTA to the aquatic environment (2), (3). In the late 1980s, the environmental impacts of EDTA were scrutinized in Europe, as EDTA occurred at a higher concentration in surface waters than any other identified anthropogenic organic compounds that may

Insights into the mechanism of separation of heparin and heparan sulfate disaccharides by reverse-phase ion-pair chromatography

Reverse-phase ion-pair high performance liquid chromatography (RPIP-HPLC) and ultra-performance liquid chromatography (RPIP-UPLC) are increasingly popular chromatographic techniques for the separation of organic compounds. However, the fine details of the RPIP separation mechanism are still being debated. Many factors including type and concentration of the ion-pairing reagent, mobile phase pH, organic modifier, ionic strength, and stationary phase all play a role in the overall efficiency and optimization of ion-pairing separations. This study investigates the role that competition between ion-pairing reagents with different steric bulk and hydrophobicity plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides. In addition to providing insights into the mechanism by which RPIP-HPLC can resolve closely related disaccharides, the use of competition between ion-pairing agents could lead to new methods for the separation of larger heparin and HS oligosaccharides. This approach should also be applicable to the analysis of other compound classes, and could lead to a general approach for the chromatographic resolution of mixtures of charged analytes having similar structures.

Fluorometric determination of pantothenic acid in human urine by isocratic reversed-phase ion-pair high-performance liquid chromatography with post-column derivatization

We describe here a method for the determination of pantothenic acid, vitamin B5, in human urine by isocratic reversed-phase ion-pair HPLC with post-column derivatization. Pantothenic acid in urine was separated using a Tosoh ODS-80Ts (4.6 i.d.×250mm) column with phosphate–sodium hydroxide buffer (pH 7.0) containing dodecyltrimethylammonium chloride. Following the isolation of pantothenic acid it was decomposed to pantoic acid and β-alanine by alkali treatment. The product β-alanine was post-derivatized to the fluorescent 1-alkylthio-2-alkylisoindole with orthophthaldialdehyde in the presence of 3-mercaptopropionic acid. In the proposed method, a urine sample can be directly injected into a HPLC system without any pre-clean up treatment. The limit of detection was 3pmol (ca. 650pg) per 20μL of urine at a signal-to-noise ratio of 5:1 and the limit of quantification was 5pmol (ca. 1000pg) per 20μL of urine, which was sufficiently sensitive for the determination of pantothenic acid in human urine. The total time required for the analysis was ca. 25min. The proposed method can be used to assess the pantothenic acid content of human urine as an alternative to the standard microbioassay for pantothenic acid.

Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA

Small interfering RNA (siRNA)-induced gene silencing shows great promise in genomic research and therapeutic applications. siRNA duplexes are typically assembled from complementary synthetic oligonucleotides. High-purity single-stranded species are required for in vivo applications. Methods for separation, characterization, and purification of short RNA strands have been developed based on reversed-phase ion-pair liquid chromatography. The purification strategies were developed for both single-stranded and duplex RNA species. The method of duplex purification uses on-column annealing of complementary RNA strands, followed by separation of the target duplex from truncated duplexes and single-stranded RNA forms. The proposed method significantly reduces the purification time of synthetic siRNA.

Effect of a carboxyl group on the chemiluminescent reaction of tris(2,2′-bipyridine)ruthenium(III) with aliphatic amines

The effect of a carboxyl group beside nitrogen of aliphatic amines on the tris(2,2′-bipyridine)ruthenium(III), Ru(bpy) 3 3+, chemiluminescent reaction was examined. It has been shown that a carboxylate anion promotes the chemiluminescent reaction at a lower pH and then the aliphatic amines with this substituent can be sensitively detected compared with corresponding aliphatic amines without this substituent. Based on this finding, preliminary studies on simultaneous determination of 4-hydroxyproline, N-methylglycine, N-methylalanine, proline, and pipecolic acid in human serum have been performed using isocratic reversed-phase ion-pair high-performance liquid chromatography (HPLC) with electrogenerated Ru(bpy) 3 3+ chemiluminescent detection. The detection limits (signal-to-noise ratio of 3) with the proposed method were 3.0, 12, 2.7, 4.6, and 10 nM for 4-hydroxyproline, N-methylglycine, N-methylalanine, proline, and pipecolic acid, respectively.

Triterpene Glycosides from Antarctic Sea Cucumbers. 1. Structure of Liouvillosides A1, A2, A3, B1, and B2 from the Sea Cucumber Staurocucumis liouvillei: New Procedure for Separation of Highly Polar Glycoside Fractions and Taxonomic Revision

Five new triterpene glycosides, liouvillosides A1 (1), A2 (2), A3 (3), B1 (4), and B2 (5), have been isolated from the Antarctic sea cucumber Staurocucumis liouviellei along with the known liouvilloside A(6), isolated earlier from the same species, and hemoiedemosides A (7) and B (8), isolated earlier from the Patagonian sea cucumber Hemioedema spectabilis. The isolation was carried out using a new chromatographic procedure including application of ion-pair reversed-phase chromatography followed by chiral chromatography on a cyclodextrin ChiraDex column. The structures of the new glycosides were elucidated using extensive NMR spectroscopy (1H and 13C NMR spectrometry, DEPT, 1H-(1)H COSY, HMBC, HMQC, and NOESY), ESI-FTMS, and CID MS/MS, and chemical transformations. Glycosides 1-3 are disulfated tetraosides and glycosides 4 and 5 are trisulfated tetraosides. Glycosides 2 and 3 contain 3-O-methylquinovose, found for the first time as a natural monosaccharide in sea cucumber glycosides. On the basis of analyses of glycoside structures a taxonomic revision is proposed.

Heterogeneity of depolymerized heparin SEC fractions: to pool or not to pool?

In the structural analysis of heparin and heparan sulfate, it is customary to combine or pool like-sized fractions obtained by size-exclusion chromatography (SEC) of enzymatically derived heparin oligosaccharides. In this study, we examine the heterogeneity of preparative-scale SEC fractions obtained from enzymatic digests of porcine intestinal mucosa heparin. Each fraction was profiled by capillary electrophoresis with UV detection (CE−UV) using a 60 mM formic acid running buffer at pH 3.43. Differences in the composition and relative concentration of components of the SEC fractions were observed for disaccharides and larger oligosaccharides. The heterogeneity of the fractions becomes more pronounced when heparin is digested using a heparin lyase cocktail. The heterogeneity of preparative SEC fractions was further investigated by reversed-phase ion-pairing ultraperformance liquid chromatography coupled with mass spectrometry (RPIP−UPLC−MS) using the ion-pairing reagent, tributylamine (Bu 3N). Our results suggest that preliminary profiling of preparative SEC fractions prior to pooling may simplify efforts to identify and/or isolate rare structures.

Simultaneous characterization of selenium and arsenic analytes via ion-pairing reversed phase chromatography with inductively coupled plasma and electrospray ionization ion trap mass spectrometry for detection: Applications to river water, plant extract and urine matrices

With an increased awareness and concern for varying toxicities of the different chemical forms of environmental contaminants such as selenium and arsenic, effective methodologies for speciation are paramount. In general, chromatographic methodologies have been developed using a particular detection system and a unique matrix for single element speciation. In this study, a routine method to speciate selenium and arsenic in a variety of “real world” matrices with elemental and molecular mass spectrometric detection has been successfully accomplished. Specifically, four selenium species, selenite, selenate, selenomethionine and selenocystine, and four arsenic species, arsenite, arsenate, monomethlyarsonate and dimethylarsinate, were simultaneously separated using ion-pairing reversed phase chromatography coupled with inductively coupled plasma and electrospray ionization ion trap mass spectrometry. Using tetrabutylammonium hydroxide as the ion-pairing reagent on a C 18 column, the separation and re-equilibration time was attained within 18 min. To illustrate the wide range of possible applications, the method was then successfully applied for the detection of selenium and arsenic species found naturally and spiked in river water, plant extract and urine matrices.

Analytical speciation of chromium in in-vitro cultures of chromate-resistant filamentous fungi

In this work, different analytical speciation schemes have been used to study the reduction of Cr(VI) by a chromate-resistant strain of filamentous fungi Ed8 (Aspergillus sp), indigenous to contaminated industrial wastes. As demonstrated previously, this strain has the capability to reduce chromate present in the growth medium without its accumulation in the biomass, yet the reduced chromium end-products have not been characterized. Liquid growth medium, initially containing 50 mg L(-1) Cr(VI), was analyzed for Cr(III)/Cr(VI) and for total Cr at different time intervals (0-24 h) after inoculation with fungi. Three hyphenated procedures, based on the Cr(III)-EDTA formation and species separation by anion-exchange or ion-pairing reversed-phase chromatography with ICP-MS or DAD detection were used. The results obtained for Cr(VI) in each case were consistent, demonstrating efficient reduction of chromate during 24 h of Ed8 growth. However, pre-column complexation with EDTA did not ensure complete recovery of the reduced forms of chromium in the above procedures. An alternative speciation scheme, based on extraction of Cr(VI)-benzyltributylammonium bromide (BTAB) ion pairs into chloroform and subsequent determination of residual chromium by ICP-MS has provided evidence on the effective conversion of chromate into reduced chromium species in the growth medium. The results indicate the feasibility of using Ed8 strain for chromate bioremediation purposes. Analytically it can be concluded that speciation of chromium in biological systems should not be limited to its two most common oxidation states, because the actual reduced chromium species are not converted quantitatively to Cr(III)-EDTA.

The Effect of Glutathione Modulation on the Concentration of Homocysteine in Plasma of Rats

Elevated plasma homocysteine concentration in humans is associated with increased risk of arteriosclerosis and ischaemic heart disease. We studied whether the plasma homocysteine concentration could be changed by administration of drugs that modulate the concentration of glutathione in both plasma and tissue. Male wistar rats received reduced glutathione (0.5 mmol/kg), N-acetylcysteine (0.5 mmol/kg), L-buthionine-[S,R]-sulfoximine (2 mmol/kg) or Ringer acetate intravenously. Twenty min. later an arterial blood sample was drawn for the measurement of homocysteine and other thiols in the plasma. The thiols were quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The total homocysteine concentration in plasma of fasted rats was 6.1±0.5 μM. Intravenous administration of reduced glutathione or N-acetylcysteine reduced the homocysteine concentration in plasma significantly by 51% to 3.0±0.3 μM and 63% to 2.2±0.2 μM, respectively (P<0.05). In contrast, L-buthionine-[S,R]-sulfoximine increased the concentration of homocysteine by 41% to 8.6±0.6 μM (P<0.05). The glutathione concentration in plasma was 19.5±1.9 μM in controls and was unchanged by N-acetylcysteine administration. Reduced glutathione increased plasma glutathione to 379.7±22.9 μM (P<0.05), whereas L-buthionine-[S R]-sulfoximine lowered the plasma glutathione concentration to 5.3±0.4 μM. Homocysteine was negatively correlated to the glutathione (r=−0.399, P<0.01) and the cysteine (r=−0.52, P<0.01) concentrations in plasma. Our conclusion is that modulation of the glutathione levels influences the concentration of homocysteine in plasma of rats.

Measurement of Acetylcholine in Rat Brain Microdialysates by LC - Isotope Dilution Tandem MS.

An LC-MS/MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,1,2,2-d(4)) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 μl/min flow rate. The instrument's response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d(4) in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1,1,2,2-d(4) added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.

Simultaneous Tracking of Sulfur Species in the Oxidation of Thiourea by Hydrogen Peroxide

We employ reversed-phase ion-pair high-performance liquid chromatography to quantitatively characterize the oxidation kinetics of thiourea oxidation by hydrogen peroxide. The HPLC technique makes it possible to monitor the concentrations of a variety of sulfur-containing species with different oxidation states and to elucidate the relative phase relations among them. The experimental results are in good agreement with simulations from an 8-step reaction mechanism.

Ultraperformance Ion-Pair Liquid Chromatography Coupled to Electrospray Time-of-Flight Mass Spectrometry for Compositional Profiling and Quantification of Heparin and Heparan Sulfate

Heparin and heparan sulfate (HS) are important pharmaceutical targets because they bind a large number of proteins, including growth factors and cytokines, mediating many biological processes. Because of their biological significance and complexity, there is a need for development of rapid and sensitive analytical techniques for the characterization and compositional analysis of heparin and HS at the disaccharide level, as well as for the structure elucidation of larger glycosaminoglycan (GAG) sequences important for protein binding. In this work, we present a rapid method for analysis of disaccharide composition using reversed-phase ion-pairing ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry ((RPIP)-UPLC-MS). Heparin disaccharide standards were eluted in less than 5 min. The method was used to determine the constituents of GAGs from unfractionated heparin/HS from various bovine and porcine tissues, and the results were compared with literature values.

Changes in heparan sulfate are associated with delayed wound repair, altered cell migration, adhesion and contractility in the galactosyltransferase I (ß4GalT-7) deficient form of Ehlers–Danlos syndrome

Reduced activity of beta4-galactosyltransferase 7 (beta4GalT-7), an enzyme involved in synthesizing the glycosaminoglycan linkage region of proteoglycans, is associated with the progeroid form of Ehlers-Danlos syndrome (EDS). In the invertebrates Drosophila melanogaster and Caenorhabditis elegans, mutations in beta4GalT-7 affect biosynthesis of heparan sulfate (HS), a modulator of several biological processes relevant to wound repair. We have analyzed structural alterations of HS and their functional consequences in human beta4GalT-7 Arg270Cys mutant EDS and control fibroblasts. HS disaccharide analysis by reversed phase ion-pairing chromatography revealed a reduced sulfation degree of HS paralleled by altered immunostaining patterns for the phage-display anti-HS antibodies HS4E4 and RB4EA12 in beta4GalT-7 mutant fibroblasts. Real-time PCR-analysis of 44 genes involved in glycosaminoglycan biosynthesis indicated that the structural alterations in HS were not caused by differential regulation at the transcriptional level. Scratch wound closure was delayed in beta4GalT-7-deficient cells, which could be mimicked by enzymatic removal of HS in control cells. siRNA-mediated knockdown of beta4GalT-7 expression induced morphological changes in control fibroblasts which suggested altered cell-matrix interactions. Adhesion of beta4GalT-7 deficient cells to fibronectin was increased while actin stress fiber formation was impaired relative to control cells. Also collagen gel contraction was delayed in the beta4GalT-7 mutants which showed a reduced formation of pseudopodia and filopodia, less efficient penetration of the collagen gels and a diminished formation of collagen suprastructures. Our study suggests an HS-dependent basic mechanism behind the altered wound repair phenotype of beta4GalT-7-deficient EDS patients.

Avant-propos

RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 μg of RNA from only 108 Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.

Stereoselective Synthesis of Sugar Nucleotides Using Neighboring Group Participation

A straightforward, efficient method for the chemical synthesis of sugar nucleotides derived from D-mannose and L-fucose precursors is described. This synthetic strategy involves the coupling of acylated glycosyl bromides with nucleoside 5'-diphosphates, which enables the exploitation of neighboring group participation to exclusively prepare diastereomerically pure sugar nucleotides of desired 1,2-trans anomeric configuration. This is the first stereoselective direct coupling approach to sugar nucleotide synthesis. Following deprotection using triethylamine and purification via C18 reversed-phase ion-pair chromatography, UDP- and GDP-alpha-D-mannose as well as UDP- and GDP-beta-L-fucose were obtained in good yield in only four synthetic steps from D-mannose and L-fucose.

Simultaneous determination of silicon and phosphorus in soil and plants by reversed-phase ion-pair chromatography

A reversed-phase ion-pair high-performance liquid chromatographic method, using tetrabutylammonium bromide (TBABr) as ion-pair reagent, has been developed for the simultaneous analysis of silicon (Si) and phosphorus (P) as heteropoly acids in soil and plant samples. The effect of the concentrations of ion-pair reagent, acetate buffer and organic modifier as well as the pH of buffer on separation was made clear. The reaction conditions and stability of heteropoly acids were investigated. Furthermore, the phenomenon occurred in the optimized process was also further researched. The separation was performed on a reversed-phase C 18 column within 11 min with 40:60 (v/v) 0.1 M acetate buffer (pH 3.9)–acetonitrile (ACN) containing 0.8 mM TBABr as a mobile phase. The linear ranges of the peak area calibration curves for Si and P were 0.08–50 mg/L and 0.40–50 mg/L, respectively. The detection limits calculated at S/N = 3 were 0.0057 mg/L and 0.0280 mg/L for Si and P, respectively. The method was successfully applied to the analysis of soluble and total contents of Si and P in soil and plant samples.