Pair Reversed-phase High-performance Liquid Research Articles

Simultaneous Determination of Protoberberine Alkaloids Derived from Coptidis Rhizoma and Phellodendri Cortex in Pharmaceuticals by On-line Solid-Phase Extraction (SPE)—High Performance Liquid Chromatography with Ultraviolet Detection (HPLC-UV)

An automated on-line solid-phase extraction (SPE) high-performance liquid chromatography (HPLC) method was developed for the determination of six protoberberine alkaloids in compound Chinese medicines containing Coptidis Rhizoma and Phellodendri Cortex. The sample solution extracted by acidic-methanol was loaded into the constructed system directly to perform enrichment and the purification of the analytes was performed automatically. A cation-exchanged sorbent based on turbulent chromatography was employed for on-line SPE to remove macro and neutral matrix interferences from samples. The target alkaloids were retained on the SPE column and eluted onto the trapping C18 column by a mobile phase of methanol/500 mM ammonium acetate (85:15) at a flow rate of 0.3 mL/min. Ion pair reverse phase high-performance liquid chromatography was employed to complete the determination of six analytes. The results showed the linear correlation coefficients for all analytes were above 0.999. The peak area of relative standard deviation in reproducibility measurements were from 0.55% to 2.39%. Recovery measurements spiked at two levels were from 92.71% to 104.28%. The reported method was suitable for the determination of six active alkaloids in multicomponent Chinese medicines containing Coptidis rhizoma and Phellodendri cortex.

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

All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover, error hydrolysis, and site selectivity

Sialyltransferases are important enzymes of glycobiology and the related biotechnologies. The development of sialyltransferases calls for access to quick, inexpensive, and robust analytical tools. We have established an assay for simultaneous characterization of sialyltransferase activity, error hydrolysis, and site selectivity. The described assay does not require expensive substrates, is very sensitive (limit of detection=0.3μU), and is easy to perform. It is based on sialylation of nitrophenyl galactosides; the products thereof are separated and quantified by ion pair reversed phase high-performance liquid chromatography with ultraviolet detection.

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.

CYP2D6 genotyping in breast cancer patients by liquid chromatography-electrospray ionization mass spectrometry

The application of cytochrome P450 2D6 (CYP2D6) genotyping to allow a personalized treatment approach for breast cancer patients undergoing endocrine therapy has been repeatedly discussed. However, the actual clinical relevance of the CYP2D6 genotype in the endocrine treatment of breast cancer still remains to be elucidated. A major prerequisite for the successful and valid evaluation of the CYP2D6 genotype with regard to its pharmacokinetic and clinical relevance is the availability of a comprehensive, accurate and cost-effective CYP2D6 genotyping strategy. Herein we present a CYP2D6 genotyping assay employing polymerase chain reaction (PCR)-ion pair reversed-phase high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (ICEMS). The genotyping strategy involves the simultaneous amplification of nine variable regions within the CYP2D6 gene by a two-step PCR protocol and the direct analysis of the generated PCR amplicons by ICEMS. The nucleotide composition profiles generated by ICEMS enable the differentiation of 37 of the 80 reported CYP2D6 alleles. The assay was applied to type the CYP2D6 gene in 199 Austrian individuals including 106 breast cancer patients undergoing tamoxifen treatment. The developed method turned out to be a highly applicable, robust and cost-effective approach, enabling an economical CYP2D6 testing for large patient cohorts.

Determination of arsenic species in human urine using high performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS)

A speciation technique for arsenic (As) has been applied using ion pair reverse phase-high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (RP-HPLC-ICP-MS). Six As-species (arsenite, arsenate, dimethylarsinic acid, dimethylarsinous acid, monomethylarsonic acid and monomethylarsonous acid) have been separated with isocratic elution within less than 6 minutes. A cation exchange column was used for separation of AsB, AsC, Tetra (Me4As+) and TMAsO. The As chemical form and oxidation state is highly important in respect to toxicity; therefore total As-determination is insufficient for a complete toxicological evaluation and risk assessment. Arsenic-speciation has been studied on urine samples of children from an As-affected area in the Iron Quadrangle, Brazil. DMAsV and MMAsV were the major urinary metabolites in these samples. The mean value for total As-concentration of all samples (n = 15) was 26.3 ng As mL−1 with a range of 16.1 to 55.2 ng As mL−1. TMAsO and AsC (arsenocholine) were not detected in this study. In most samples, monomethylarsonous acid [MMAsIII] was detected up to 2.0 ng As mL−1. DMAsIII was not detected at any time, most probably due to volatilisation and some oxidation to DMAsV. The chromatographic recoveries, calculated from ([sum(species) × 100]/total As in urine samples), ranged from 77.4 to 94.9%. This work also contributes to the pertinent discussion regarding the reliability of MMAsIII/DMAsIII speciation in urine.

Optimization of Quantitative MGMT Promoter Methylation Analysis Using Pyrosequencing and Combined Bisulfite Restriction Analysis

Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension- and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pair-reverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens.

High-Performance Liquid Chromatographic/Mass Spectrometric Studies on the Susceptibility of Heparin Species to Cleavage by Heparanase

Heparanase is an endo-beta-D-glucuronidase that cleaves the heparan sulfate chains of heparan sulfate proteoglycans and is implicated in angiogenesis and metastasis. With the aim of establishing a simple and reliable method for studying the susceptibility of heparin/heparan sulfate oligosaccharides to be cleaved by heparanase, an on-line ion pair reversed-phase high-performance liquid chromatographic/electrospray ionization mass spectrometric method was set up. The method works in the micromolar range of concentration and does not require derivatization of the substrate or of the products. It is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal heparin disaccharide standard. Substrates were (1) the synthetic pentasaccharides GlcN (NS,6S) - GlcA - GlcN (NS,3S,6S) - IdoA (2S) - GlcN (NS,6S) - OMe (AGA*IA (M)) and GlcN (NS,6S) - GlcA - GlcN (NS,6S) - IdoA (2S) - GlcN (NS,6S) - OMe (AGAIA (M)), corresponding to the heparin/heparan sulfate active site for antithrombin, and to the same sequence devoid of the 3- O-sulfate group in the central glucosamine, respectively; and (2) two natural heparin octasaccharides containing the AGA*IA sequence in different locations along the chain. The two pentasaccharides exhibited a higher susceptibility to heparanase cleavage with respect to the octasaccharides. The commercial availability of AGA*IA (M) makes it an ideal substrate to determine the specific activity of heparanase preparations. The present method could also be used for rapid screening of potential heparanase inhibitors.

Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography

Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.

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.

Simultaneous Determination of Pyridoxine Hydrochloride and Doxylamine Succinate from Tablets by Ion Pair Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)

A new, simple, precise, and rapid ion pair reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed for the simultaneous determination of pyridoxine hydrochloride (PYR) and doxylamine succinate (DOX) in tablets. The stationary phase was a Microbondapak C18 column (10 μ, 300 mm × 3.9 mm i.d.). The mobile phase was water:methanol (60:40) containing 10 mM heptanesulfonic acid and 0.25% triethylamine and adjusted to pH 2.2 with orthophosphoric acid. Detection was carried out at 263 nm using an ultraviolet (UV) detector. The flow rate was 1.0 ml/min, and retention times were 3.65 min and 7.32 min for PYR and DOX, respectively. The linearity was obtained in the concentration range 0.5–500 μg/ml for PYR and DOX. Mean percentage recoveries were 100.20% and 101.20% for PYR and DOX, respectively.

Separation and quantitation of some metal ions by RP-HPLC using EDTA as complexing agent in mobile phase

Ion pair reversed-phase high performance liquid chromatography has been used to achieve the simultaneous separation of some transition metal ions including Ni(II), Co(II), Cu(II), Mn(II), Fe(III), Pb(II), Cr(III), and Hg(II) as complexes of ethylenediaminetetraacetic acid (EDTA) originated, in the column itself. Due to the negative charge of the metal complexes, the separation of metal ions from inorganic anions such as Cr2O72− and NO3− is possible by employing a positively charged counterion in the mobile phase. The influence of the nature and concentration of counterion, organic modifier content, ionic strength, EDTA concentration and pH of the mobile phase on the retention and selectivity has been investigated. Quantitation of metal ions was achieved with spectrophotometric detection. Detection limits ranged from 300 pg for Fe(III) to 35 ng for Cr(III) with a linearity of 2–3 orders of magnitude. Some applications of the technique to the separation of multielement mixtures are shown.

Chromatographic behavior of doxylamine succinate, phenylpropanolamine hydrochloride, chlorpheniramine maleate, dextromethorphan hydrobromide, paracetamol, and guaifenesin in ion pair reverse-phase high performance liquid chromatography

A study of the chromatographic behavior of some cough syrup ingredients has led to the optimum chromatographic separation of four ingredients (doxylamine succinate, phenylpropanolamine hydrochloride, chlorpheniramine maleate, and dextromethorphan hydrobromide). The paracetamol and guaifenesin were overlapping under all chromatographic conditions. The application of 1% chlorotrimethylsilane in methanol to the column (Partisil 5 CCS/C 8) was found to improve the column efficiency significantly. This separation can be applied for the analysis of cough syrup for these ingredients after a study of the interferences due to normal excipients. 2 2 Excipient: any inert substance used as a diluent or vehicle for a drug.

A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells

A simple and fast ion pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of ATP, ADP, AMP, GTP, GDP, IMP, NADP +, NADPH, NAD +, NADH, ADP-ribose, inosine, adenosine, hypoxanthine, and xanthine. This method allows us to have a complete picture of the most important nucleotides present in fresh human erythrocytes. Furthermore it is particularly useful in the study of the erythrocyte adenine nucleotide catabolism allowing the detection of degradation products such as IMP, inosine, adenosine, hypoxanthine, and xanthine. The separation of the compounds under investigation is achieved in less than 15 min using a reversed-phase 3-μm Supelcosil LC-18 column and adding tetrabutylammonium, as ion-pair agent, to the buffers. The short time of analysis, the high reproducibility of the system, and the accurate evaluation of the compounds of interest make this method particularly suitable for routine analysis. Finally it is possible to use this assay as an alternative method of measuring activities of enzymes which catalyze reactions involving some of these compounds, as in the case of Na +K + ATPase, AMP deaminase, and adenosine deaminase.

Determination of benzoylecgonine in urine from drug abusers using ion pair high performance liquid chromatography.

An ion pair reversed-phase high performance liquid chromatographic (HPLC) method for the determination of the cocaine metabolite benzoylecgonine in urine is presented. It includes sample purification with solid phase extraction (Sep-Pak C18) and ultraviolet (UV) detection at 233 nm. It is a relatively simple and sensitive method developed for confirmation of positive results from radioimmunoassay screening for benzoylecgonine in urine from drug abusers. Detection of benzoylecgonine concentrations down to 20 ng/mL is possible. Linear standard curves have been obtained from 30 ng/mL to 6 micrograms/mL.

Quantitative Analysis of 5-Fluorouracil in Human Serum by High-Performance Liquid Chromatography

Abstract A modified ion pair reverse-phase high-performance liquid chromatographic (HPLC) method for examining 5-fluorouracil levels in human serum was developed. The method employs the use of an internal standard and an organic extraction procedure. An isocratic mobile phase consisting of tetraethylammonium bromide, tetrabutylammonium hydrogen sulfate, methanol, sodium acetate, and phosphate buffer was used at a flow rate of 0.7 ml/min. 5-Fluorouracil was detected by UV absorption at a wavelength of 266 nm. The HPLC method described is a convenient and sensitive assay for examining 5-fluorouracil concentration in human serum.

Separation of angiotensins and assay of angiotensin-generating enzymes by high-performance liquid chromatography

A rapid and sensitive separation of [Ile 5 ]angiotensin II, [Val 5 ]angiotensin II, des[Asp 1 ],[Ile 5 ]angiotensin II, [Ile 5 ]angiotensin I, des[Asp 1 ],[Ile 5 ]angiotensin I, the dipeptide His-Leu, and NH 2 -terminal tetradecapeptide of equine angiotensinogen has been achieved employing perchlorate ion pair reverse-phase high-performance liquid chromatography. Complete resolution of the angiotensins is effected in 20 min under isocratic conditions. Peptide concentrations as low as 10 pmol in 100 μl may be directly quantitated by monitoring the uv absorbance of the column effluent at 200 nm. Applications of this method to the measurement of renin activity and converting-enzyme activity are presented.