IP RP HPLC Research Articles

Separation and determination of some water-soluble vitamins in pharmaceutical preparation by IP RP-HPLC

A reversed phase(RP) HPLC with ion-pair reagent is used for simultaneous determinations of water-soluble vitamins (nicotineamide, nicotinic acid, folic acid, riboflavin (B2) and thiamin (B 1 )) in pharmaceutical preparations. The vitamins were analyzed using ZORBAX Cs (25cm x 4.6mm i.d) analytical column, 6-8µ111 particle size, by isocratic elution with a mobile phase consisted of 20%methanol, buffered with 0.05M KH2P04 (pH 3.5) , and 3x10-3M 1-octanesulfonic acid as an ion-pair reagent at a flow rate of (l.Oml/min.). The UV-Visible detection of the separated vitamins was made at 254nm for all water-soluble vitamins. The precision and accuracy of method were checked by calculating RSD% and relative error E%, which where found to be reasonable. A RSD% between (0.5-4.76) % and relative error -:\1etween (0.05-4.93)%.The limit of detection range was found to be (0.0244, \\ O.Q436µg/ml).

A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography

We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

Automated determination of early eluting oligonucleotide impurities using ion-pair reversed-phase liquid chromatography high resolution-mass spectrometry

A new method is presented for the automated determination of early eluting (E.E.) oligonucleotide impurities analyzed by IP-RP HPLC HRMS. E.E. are impurities shorter than the main drug component and are primarily formed by the mechanisms of coupling failure, and depurination. The method is based on the detection of the theoretically derived most abundant mass of an impurity in the experimental data. An exhaustive list of candidate impurities and their formulas is automatically generated using the parent sequence and the known mechanisms of impurity formation. The approach accounts for possible modifications in the individual oligonucleotide sequence moieties (e.g., linkage, sugar, and base, 3′, and 5′ ends). The detected ion signal is summed to provide four nested levels of impurity breakdown information. The approach allows for the rapid determination of relationships and trends of impurities in samples generated by different manufacturing processes and conditions. Representative examples are given to illustrate the capabilities and utility of the approach in synthesis and purification process optimization applications.

Genome-Wide Association Study to Identify Genes Related to Renal Mercury Concentrations in Mice.

Background:Following human mercury (Hg) exposure, the metal accumulates in considerable concentrations in kidney, liver, and brain. Although the toxicokinetics of Hg have been studied extensively, factors responsible for interindividual variation in humans are largely unknown. Differences in accumulation of renal Hg between inbred mouse strains suggest a genetic interstrain variation regulating retention or/and excretion of Hg. A.SW, DBA/2 and BALB/C mouse strains accumulate higher amounts of Hg than B10.S.Objectives:We aimed to find candidate genes associated with regulation of renal Hg concentrations.Methods:A.SW, B10.S and their F1 and F2 offspring were exposed for 6 weeks to 2.0 mg Hg/L drinking water. Genotyping with microsatellites was conducted on 84 F2 mice for genome-wide scanning with ion pair reverse-phase high-performance liquid chromatography (IP RP HPLC). Quantitative trait loci (QTL) were established. Denaturing HPLC was used to detect single nucleotide polymorphisms for haplotyping and fine mapping in 184 and 32 F2 mice, respectively. Candidate genes (Pprc1, Btrc and Nfkb2) verified by fine mapping and QTL were further investigated by real-time polymerase chain reaction. Genes enhanced by Pprc1 (Nrf1 and Nrf2) were included for gene expression analysis.Results:Renal Hg concentrations differed significantly between A.SW and B10.S mice and between males and females within each strain. QTL analysis showed a peak logarithm of odds ratio score 5.78 on chromosome 19 (p = 0.002). Haplotype and fine mapping associated the Hg accumulation with Pprc1, which encodes PGC-1-related coactivator (PRC), a coactivator for proteins involved in detoxification. Pprc1 and two genes coactivated by Pprc1 (Nrf1 and Nrf2) had significantly lower gene expression in the A.SW strain than in the B10.S strain.Conclusions:This study supports Pprc1 as a key regulator for renal Hg excretion.Citation:Alkaissi H, Ekstrand J, Jawad A, Nielsen JB, Havarinasab S, Soderkvist P, Hultman P. 2016. Genome-wide association study to identify genes related to renal mercury concentrations in mice. Environ Health Perspect 124:920–926; http://dx.doi.org/10.1289/ehp.1409284

11p15 DNA-methylation analysis in monozygotic twins with discordant intrauterine development due to severe twin-to-twin transfusion syndrome

BackgroundPrenatal growth restriction and low birth weight have been linked to long-term alterations of health, presumably via adaptive modifications of the epigenome. Recent studies indicate a plasticity of the 11p15 epigenotype in response to environmental changes during early stages of human development.Study designWe analyzed methylation levels at different 11p15 loci in 20 growth-discordant monozygotic twin pairs. Intrauterine development was discordant due to severe twin-to-twin transfusion syndrome (TTTS), which was treated by fetoscopic laser coagulation of communicating vessels before 25 weeks of gestation. Methylation levels at age 4 were determined in blood and buccal cell-derived DNA by the single nucleotide primer extension reaction ion pair reverse-phase high performance liquid chromatography (SNuPE IP RP HPLC) assay. Methylation at LINE-1 repeats was analyzed as an estimate of global methylation.ResultsIn general, variance of locus-specific methylation levels appeared to be higher in buccal cell- as compared to blood cell-derived DNA samples. Paired analyses within the twin pairs revealed significant differences at only one CpG site (IGF2 dmr0 SN3 (blood), +1.9% in donors; P = 0.013). When plotting the twin pair-discordance in birth weight against the degree of discordance in site-specific methylation at age 4, only a few CpGs were found to interact (one CpG site each at IGF2dmr0 in blood/saliva DNA, one CpG at LINE-1 repeats in saliva DNA), with 26 to 36% of the intra-twin pair divergence at these sites explained by prenatal growth discordance. However, across the entire cohort of 40 children, site-specific methylation did not correlate with SD-scores for weight or length at birth. Insulin-like growth factor-II serum concentrations showed significant within-twin pair correlations at birth (R = 0.57) and at age 4 (R = 0.79), but did not differ between donors and recipients. They also did not correlate with the analyzed 11p15 methylation parameters.ConclusionIn a cohort of 20 growth-discordant monozygotic twin pairs, severe alteration in placental blood supply due to TTTS appears to leave only weak, if any, epigenetic marks at the analyzed CpG sites at 11p15.

Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides

Glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction of heparin currently used as an anticoagulant and antithrombotic drug, are arousing increasing interest in anticancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs heparins has not been studied yet in detail. In this work, ion pair reversed-phase high-performance liquid chromatography (IPRP–HPLC) coupled with electrospray ionization mass spectrometry (ESI–MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs heparins usually prepared from porcine mucosal heparin. The method was also found to be very effective in analyzing gs derivatives obtained from heparins of different animal and tissue origins. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs heparins contain mainly tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues was shown to be a marker of the gs-modified AT binding site within heparin chains.

Association of COMT genotypes with S-COMT promoter methylation in growth-discordant monozygotic twins and healthy adults

BackgroundCatechol-O-Methyltransferase (COMT) plays a key role in dopamine and estrogen metabolism. Recently, COMT haplotypes rather than the single polymorphism Val158Met have been reported to underlie differences in protein expression by modulating mRNA secondary structure. So far, studies investigating the epigenetic variability of the S-COMT (soluble COMT) promoter region mainly focused on phenotypical aspects, and results have been controversial.MethodsWe assessed S-COMT promoter methylation in saliva and blood derived DNA with regard to early pre- and postnatal growth as well as to genotype for polymorphisms rs6269, rs4633, and rs4680 (Val158Met) in 20 monozygotic twin pairs (mean age 4 years), who were discordant for intrauterine development due to severe feto-fetal-transfusion syndrome. Methylation levels of two previously reported partially methylated cytosines were determined by the quantitative SIRPH (SNuPE- IP RP HPLC) assay.ResultsOverall, we observed a high variability of S-COMT promoter methylation, which did not correlate with individual differences in the pre- or postnatal growth pattern. Within the twin pairs however we noted a distinct similarity that could be linked to underlying COMT genotypes. This association was subsequently confirmed in a cohort of 93 unrelated adult controls. Interestingly, 158Val-alleles were found at both ends of the epigenotypical range, which is in accordance with a recently proposed model of COMT haplotypes corresponding to a continuum of phenotypical variability.ConclusionThe strong heritable component of S-COMT promoter methylation found in our study needs to be considered in future approaches that focus on interactions between COMT epigenotype and phenotype.

Evaluation of cadmium species lability using ion-pair reversed phase HPLC coupled on-line with inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) coupled on-line with an ion-pair reversed phase HPLC (IP-RP HPLC) was developed for determining the lability of Cd species. The IP-RP HPLC–ICP-MS system measures chromatographic behaviors of Cd species in the presence of different model complexing agents (L) with stability constants (logKCdL) from 3.8 to 19.0. Cd species with logKCdL higher than 16, between 8 and 16, and smaller than 8 was then classified into inert, moderately labile, and labile species, respectively. The conditional stability constants and dissociation rate constants were also estimated from their corresponding chromatographic behavior. This method was applied to evaluating the lability-dependent biouptake of different Cd species in Phaeodactylum tricornutum, a typical unicellular marine diatom. IP-RP HPLC–ICP-MS is a useful and promising technique for determining the lability of noncovalent-bonded metal species (such as Cd species) in the environment and for forecasting their corresponding bioavailability especially when their speciation cannot be rigorously controlled and measured.

An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC.

We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader-Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite-deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods.

A Strategy for Examining Complex Mixtures of Deoxyoligonucleotides Using Ion-Pair–Reverse-Phase High-Performance Liquid Chromatography, Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry, and Informatics

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair–reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers.

A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methylation analysis at specific CpG sites.

The precise mapping and quantification of DNA methylation as an epigenetic parameter during development and in diseased tissues is of great importance for functional genomics. Here we describe a rapid, quantitative method to assess methylation levels at specific CpG sites using PCR products of bisulfite-treated genomic DNA. Using single nucleotide primer extension (SNuPE) assays in combination with ion pair reverse phase high performance liquid chromatography (IP RP HPLC) separation techniques, methylated and unmethylated CpGs can be discriminated and quantified based on the different masses and hydrophobicities of the extended primer products. The assay is linear, highly reproducible and several sites can be measured simultaneously in one reaction. It can be semi-automated and eliminates the need for cloning and sequencing of individual bisulfite PCR products.

GENOTYPING OF A SHORT TANDEM REPEAT SYSTEM USING ION-PAIR REVERSE-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Ion pair-reverse phase high performance liquid chromatography (IPRP HPLC) is a method that can be used for the genotyping of short tandem repeats (STRs) in human nuclear DNA. The genotyping of STRs is useful in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. IPRP HPLC offers an attractive method for STR analysis because of the short analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHOl locus on chromosome 11p15.5. The IPRP HPLC conditions (column temperature, flow rate, and percent organic modifier per minute) were optimized for the separation of PCR products in the size range of 50–434 base pairs. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double stranded) with the use of internal markers. The analysis time for the HUMTHOl locus was less than 14 minutes, and the alleles could be peak captured for further examination such as sequencing.

RNA analysis by ion-pair reversed-phase high performance liquid chromatography.

Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) is presented as a new, superior method for the analysis of RNA. IP RP HPLC provides a fast and reliable alternative to classical methods of RNA analysis, including separation of different RNA species, quantification and purification. RNA is stable under the analysis conditions used; degradation of RNA during the analyses was not observed. The versatility of IP RP HPLC for RNA analysis is demonstrated. Components of an RNA ladder, ranging in size from 155 to 1770 nt, were resolved. RNA transcripts of up to 5219 nt were analyzed, their integrity determined and they were quantified and purified. Purification of mRNA from total RNA is described, separating mouse rRNA from poly(A)(+) mRNA. IP RP HPLC is also suitable for the separation and purification of DIG-labeled from unlabeled RNA. RNA purified by IP RP HPLC exhibits improved stability.

Analysis and purification of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Sizing of DNA fragments is a routine analysis traditionally performed on agarose or polyacrylamide gels. Electrophoretic analysis is labor-intensive with only limited potential for automation. Recovery of DNA fragments from gels is cumbersome. We present data on automated, size-based separation of DNA fragments by ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) — DNA chromatography — on the WAVE ® DNA Fragment Analysis System with the DNASep ® cartridge. This system is suitable for accurate and rapid sizing of double-stranded (ds) DNA fragments from 50 to ca. 2000 base pairs (bp). Fluorescently labeled DNA fragments are compatible with the technology. Length-dependent separation of dsDNA fragments is sequence independent and retention times are highly reproducible. The resolving capabilities of DNA chromatography are illustrated by the analysis of multiple DNA size markers. Resolved dsDNA fragments are easily collected and are suitable for downstream applications such as sequencing and cloning. DNA chromatography under denaturing conditions with fluorescently labeled DNA fragments offers a means for the separation and purification of individual strands of dsDNA. Analysis of DNA fragments on the WAVE System is highly automated and requires minimal manual intervention. DNA chromatography offers a reliable and automated alternative to gel electrophoresis for the analysis of DNA fragments.

DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography.

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.

Reverse-Phase High Performance Liquid Chromatography of Metal Chelates of 4-(2-Pyridylazo)resorcinol and 4-(2-Thiazolylazo)resorcinol. Simultaneous Determination of Low Concentrations of Co, Ni and Fe

The behaviour of stable and inert metal chelates of 4-(2-pyridylazo)resorcinol and 4-(2-thiazolylazo)resorcinol in RP HPLC and in its ion-pair modification (IP RP HPLC) on Separon SGX RPS silica gel was studied. Good results were obtained by the ion-pair variant in water methanol solutions at pH 7 in the presence of cetyltrimethylammonium bromide. The technique proved to be convenient for the preconcentration, separation and quantitation of low concentrations of Fe, Co, and Ni in waters.

HPLC Investigation of a Set of Local Anesthetic Aminoether Derivatives

Abstract A series of amino-alkylaryl-ether and thioether deriv—atives with local anesthetic activity was investigated in an IP-RP HPLC system. Good correlation was found between the retention (log k') and octanol-water partition coefficient (log P). The values of the correlation coefficients gradu—ally increased with the increasing concentration of the ion-pairing agent, sodium dodecyl sulfate (NaDS). This experience is interpreted by the similar interactions in the partition and chromatographic systems. The Alog k' and π values gener—ated from the log k' and log P values of the compounds hav—ing basic nucleus, showed also a good correlation.