Ion-pair Reversed-phase High Performance Research Articles

Analysis of phosphorus herbicides by ion-pairing reversed-phase liquid chromatography coupled to inductively coupled plasma mass spectrometry with octapole reaction cell

A reversed phase ion-pairing high performance liquid chromatographic (RPIP-HPLC) method is developed for the separation of two phosphorus herbicides, Glufosinate and Glyphosate as well as Aminomethylphosphonic acid (AMPA), the major metabolite of Glyphosate. Tetrabutylammonium hydroxide is used as the ion-pairing reagent in conjunction with an ammonium acetate/acetic acid buffering system at pH 4.7. An inductively coupled plasma mass spectrometer (ICP-MS) is coupled to the chromatographic system to detect the herbicides at m/ z 31P. Historically, phosphorus has been recognized as one of the elements difficult to analyze in argon plasma. This is due to its relatively high ionization potential (10.5 eV) as well as the inherent presence of the polyatomic interferences 14N 16O 1H + and 15N 16O + overlapping its only isotope at m/ z = 31. An octapole reaction cell is utilized to minimize the isobaric polyatomic interferences and to obtain the highest signal-to-background ratio. Detection limits were found to be in the low ppt range (25–32 ng/l). The developed method is successfully applied to the analysis of water samples collected from the Ohio River and spiked with a standard compounds at a level of 20 μg/l.

Identification and characterization of selenium species in enriched green onion (Allium fistulosum) by HPLC-ICP-MS and ESI-ITMS

In this study speciation of selenium in selenium-enriched green onions (Allium fistulosum) was done with reversed-phase ion-pairing high performance liquid chromatography (RP-IP-HPLC) and size-exclusion chromatography (SEC) coupled on-line to ICP-MS for selenium specific detection. The plant extract obtained using sodium hydroxide (0.1 mol l−1) analyzed by SEC (CAPS 10 mmol l−1, pH 10.0) with ICP-MS detection showed the incorporation of selenium in both high (∼12 kDa) and low molecular weight (0.36–2 kDa) fractions. Presumably protein bound selenoamino acids were released using enzymes (Proteinase K and Protease XIV) and selenoamino acids found in cytosol in their free form were extracted using 0.1 M HCl. The extracts were analyzed for speciation studies by RP-IP-HPLC [0.1% (v/v) heptafluorobutyric acid, 5% (v/v) methanol, pH 2.5]. Matching the chromatographic retention times with commercially available standards provided evidence for the presence of Se-cystine, methylselenocysteine, Se-methionine, and inorganic selenium. Electrospray ionization ion trap mass spectrometry (ESI-ITMS) confirmed the presence of γ-glutamyl-Se-methylselenocysteine in green onions as has been reported in other onion types.

Molecular species analysis of phosphatidylcholine by reversed-phase ion-pair high-performance liquid chromatography.

The retention behavior of molecular species of phosphatidylcholine (PC) is studied by reversed-phase (RP) ion-pair high-performance liquid chromatography (HPLC). Mobile phases contain tetraalkyl ammonium phosphates (TAAPs) in methano-acetonitrile-water. The stationary phase is alkyl-bonded silica. Competitive interactions of TAAPs, analyte solutes, and an RP-HPLC column result in reduced retention of PC molecular species. PC molecular species are eluted at longer retention times with a larger size of TAAP in the mobile phase, and an increase in the TAAP concentration invariably causes a decrease in PC molecular species retention times. There is a linear correlation between the logarithmic retention factors (k) of PC molecular species and the total number of carbon atoms of TAAP, and the logarithm of k values of PC molecular species can be approximated as a linear function of the logarithm of the counter-ion concentration. There is found to be no distinct dependence between k values of PC molecular species and the mobile phase pH.

Simultaneous determination of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets by reversed-phase ion-pair high performance liquid chromatography.

A reversed-phase ion-pair high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets. HPLC separation of the vitamins was performed on a Hypersil C(18) column and detected by ultraviolet absorbance at 280 nm. The use of methanol-aqueous 0.5% acetic acid solution (18:82, v/v; containing 2.5 mM sodium hexanesulfonate, pH = 2.8) as the mobile phase at a flow-rate of 1.2 mL/min enables the baseline separation of the four analytes free from interferences with isocratic elution at 30 degrees C. The analysis time was 17 min per injection. The method was linear in the ranges of 5-90, 2.5-90, 5-95 and 25-450 micro g/mL for thiamine mononitrate, riboflavin, pyridoxine hydrochloride and nicotinamide, respectively. The average coefficients of variation of within- and between-day assays were 2.2 and 3.6% for thiamine mononitrate, 1.8 and 2.4% for riboflavin, 1.3 and 1.7% for pyridoxine hydrochloride and 1.0 and 1.5% for nicotinamide, respectively. The average recoveries of thiamine mononitrate, riboflavin, pyridoxine hydrochloride and nicotinamide were 97.0, 97.2, 98.9 and 100.4% for the tablets, respectively. The method has been successfully applied to the simultaneous determination of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets.

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.

Cerebrospinal fluid levels of thiamine in patients with Alzheimer's disease.

Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism, such as the alpha-ketoglutarate dehydrogenase complex (KGDHC), pyruvate-dehydrogenase complex (PDHC), and transketolase. Some investigators reported decreased thiamine-diphosphate levels and decreased activities of KGDHC, pyruvate-dehydrogenase complex and transketolase in the brain tissue of Alzheimer's disease (AD) patients. We measured cerebrospinal (CSF) levels of thiamine-diphosphate, thiamine-monophosphate, free thiamine, and total thiamine, using ion-pair reversed phase high performance liquid chromatography, in 33 patients with sporadic AD and 32 matched controls. The mean CSF levels of thiamine-derivatives did not differ significantly from those of controls, while the mean plasma levels of thiamine-diphosphate, free and total thiamine were significantly lower in the AD-patient group. CSF and plasma thiamine levels were not correlated with age, age at onset, duration of the disease, and scores of the MiniMental State Examination, with the exception of plasma thiamine-diphosphate with MiniMental State Examination (r = 0.41, p < 0.05) in the AD-patients group. CSF and plasma values did not predict dementia progression, assessed with the MiniMental State Examination scores. These results suggest that CSF thiamine levels are not related with the risk for and the progression of AD.

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.

Electrospray ionization mass spectrometry coupled to reversed-phase ion-pair high-performance liquid chromatography for quantitation of sodium borocaptate and application to pharmacokinetic analysis.

We have developed a quantitative assay using electrospray ionization mass spectrometry coupled to reversed-phase ion-pair liquid chromatography (LC/MS) for quantitation of sodium borocaptate (BSH) in human plasma. The assay was developed using a Micromass Q-TOF II mass spectrometer equipped with an orthogonal electrospray source. The mobile phase was a 1:1 solution of methanol and 5 mM aqueous tetrabutylammonium acetate flowing at 0.2 mL/min, and the chromatography was performed using a Machery-Nagel Nucleosil C18 column. Plasma samples from patients who had received an intravenous infusion of sodium borocaptate (Na2B12H11SH), frequently referred to as BSH, were prepared for analysis by precipitation with acetonitrile. Following this, the supernatants were collected, and 40 microL was injected onto the column for analysis. The LC/MS assay was linear over a BSH plasma concentration range of 20-0.5 microg/mL with acceptable variability for both intra- and interassay precision. The LC/MS assay was used to generate pilot pharmacokinetic data for the plasma disposition of BSH in humans. The disposition of BSH was found to be consistent with a two-compartment model with first-order elimination from the central compartment. The mean total body plasma clearance was 95.7 +/- 30.8 m/min and the harmonic mean terminal half-life was 3.6 h.

Influence of hydrolysis, purification, and calibration method on furosine determination using Ion-pair reversed-phase high-performance liquid chromatography.

The influence of HCI concentration (6M, 8M, and 10M) and the ratio of sample protein to acid (1 or 5 mg of protein per mL of acid) on furosine formation during sample hydrolysis is studied. The conditions that maximize furosine formation are 10M HCI in the ratio of 1 mg of protein to 1 mL of acid. Purification of the hydrolysate by solid-phase extraction is also considered by examining the effect of hydrolysate volume and volume of 3M HCI used to elute the furosine. Furosine quantitation is carried out using the standard additions and external standard methods. The results indicate that there is no interference by the sample matrix and that external calibration is adequate.

Selective binding of nucleotide probes by eosinophilic cationic protein during in situ hybridisation.

During in situ hybridisation on frozen and paraffin-embedded sections of bowel for IkappaB alpha, oligodeoxyribonucleotide probes were found to bind more avidly to eosinophils than target mRNA. This binding could not be obviated using strategies previously employed to block either binding of long DNA probes (200-mers) to eosinophils in bone marrow smears, or of riboprobes to eosinophils in sections of bowel, without removing specific hybridisation of probes. That this binding could arise through interaction of anionic oligodeoxyribonucleotides with eosinophil cationic protein, which has an unusually high pI, and is abundant in cytoplasmic granules of eosinophils, was demonstrated in vitro using real-time biomolecular interaction analysis with a BiacoreX instrument. Finally, a relationship between probe hydrophobicity, measured by reverse phase ion-pair high performance liquid chromatography, and in situ binding of individual probes to eosinophils was demonstrated. Effective tissue penetration by hydrophobic probes and subsequent strong probe-eosinophilic cationic protein interactions therefore may confound the interpretation of in situ hybridisation performed with oligonucleotide probes in eosinophil-containing tissues, such as bowel and nasal polyps.

Simplex optimization of ion-pair reversed-phase high performance liquid chromatographic analysis of some heavy metals

The sequential simplex algorithm was used to optimize the ion-pair reversed phase high performance liquid chromatographic (IP-RPHPLC) analysis of 4-(2-pyridylazo)resorcinol (PAR) chelates of Co(II), Ni(II) and Cr(III). A chromatographic response function (CRF) which included the number of peaks, the resolution between adjacent peaks, a specified analysis time, and the individual retention times relative to a minimum retention time, was calculated to evaluate the quality of the individual chromatograms. The optimum conditions for the three experimental parameters were 28.6% acetonitrile in the mobile phase, 3.0 mmol l −1 concentration of acetate buffer at pH 6.0, and 5.2 mmol l −1 tetrabutylammonium bromide (TBABr). Optimum conditions were achieved in only 19 experiments. The order of elution was Co(II)PAR, Cr(III)PAR and Ni(II)PAR, respectively, with an analysis time of 15 min.

Uncertainty estimation on the quantification of major milk proteins by liquid chromatography.

A detailed uncertainty budget is evaluated for the quantification of major milk proteins separated by reversed phase ion-pair high performance liquid chromatography (RP-IP HPLC). Recommendations from the International Organisation for Standardisation, Guide to the Expression of Uncertainty and the EURACHEM/CITAC Guide "Quantifying Uncertainty in Analytical Measurement" were followed resulting in the expression of combined uncertainties as an expanded uncertainty. The authors have identified the lack of a detailed description on all possible sources of uncertainty on chromatographic milk protein separations. Up to now, uncertainty has only been expressed as a random chromatographic variability, being exclusively measured by regression equations. On the contrary, the establishment of a full uncertainty budget permits a complete identification of all sources of uncertainty associated with the measurement. Thus, a better comprehension of the method is obtained. Having identified all sources of uncertainty, to the best of our knowledge, they are minimised (whenever possible) and combined. The importance of this identification is crucial, in particular, concerning previously neglected sources, in this example the combination of the interpolated injected mass of protein and its purity, having a dominant influence on the total combined uncertainty. The quality of the measurement (measured by its associated uncertainty statement, which should be based on a full uncertainty budget evaluation) is demonstrated. Moreover, due to the high precision of the proposed method, the total protein content, in addition to the group protein composition (i.e. total casein and whey protein content) is calculated with high reliability, which is an extremely useful factor regarding the establishment of milk provenance/adulteration. Combined relative standard uncertainties ranging from 3.3 up to 12.5% for individual protein concentration were found, whereas for total protein content, an overall combined relative standard uncertainty of 2.7% (liquid) and 3.3% (powdered samples) was achieved.

SIMULTANEOUS LIQUID CHROMATOGRAPHIC DETERMINATION OF INDINAVIR, SAQUINAVIR, AND RITONAVIR IN HUMAN PLASMA WITH COMBINED ULTRAVIOLET ABSORBANCE AND ELECTROCHEMICAL DETECTION

A method is described for the simultaneous determination of protease inhibitors Indinavir, Saquinavir, and Ritonavir in human plasma using liquid chromatography with ultraviolet and electrochemical detection. Sample pre-treatment consisted of solid-phase extraction prior to ion-pair reversed phase high performance liquid chromatography with ultraviolet detection at 240 nm (Saquinavir and Ritonavir) and electrochemical detection at + 750 mV (Indinavir and Saquinavir). The method was validated with respect to specificity, linearity, limits of detection and quantification, precision, and accuracy. The proposed LC assay was utilized to analyze plasma samples from patients treated with HIV-protease inhibitors.

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.

Analysis of sulfonated compounds by reversed-phase ion-pair chromatography—Mass spectrometry with on-line removal of non-volatile tetrabutyl ammonium ion-pairing agents

An ion-pair reversed-phase high performance liquid chromatography method with tetrabutyl ammonium salts as ion-pairing agents and mass spectrometric detection has been developed for sulfonated compounds with special focus on structural identification. A cation exchange suppressor cartridge placed between the UV detector and the ion source of the mass spectrometer completely removed the non-volatile ion-pairing agent resulting in excellent conditions for both electrospray ioniziation and atmospheric pressure chemical ioniziation. Peak broadening caused by the dead volume of the suppressor is negligible. The application range of this method is demonstrated by the structural identification of individual compounds within a complex mixture of model substances consisting of sulfonated aromatics, textile dyes and detergents with as many as four sulfonic acid groups, all largely differing from each other in their structural properties. The influence of the ion-pairing agent's counter ion on retention behaviour is also discussed.

Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here. This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.

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.

Amino acid profile of milk-based infant formulas

The protein content and amino acid profile of three milk-based infant formulas, two of which were powdered (adapted and follow-on) and the third liquid, were determined to check their compliance with the EU directive and to evaluate whether or not they fulfil an infant's nutritional needs. To obtain the amino acid profile proteins were subjected to acid hydrolysis, prior to which the sulfur-containing amino acids were oxidized with performic acid. The amino acids were derivatized with phenylisothiocyanate (PITC) and then determined by ion-pair reverse phase high performance liquid chromatography (HPLC) In the case of tryptophan a basic hydrolysis was applied and there was no need of derivatization. The protein contents of the analysed formulas were in the ranges established by the EU directive for these products and the amino acid contents were in the ranges reported by other authors for these types of formulas. In all cases the tryptophan content determined the value of the chemical score, which was always lower than 80% of the reference protein but in the ranges reported by other authors. The analysed adapted infant formula provides amino acids in amounts higher than the established nutritional requirements.

A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection

A new method has been developed that is capable of providing a complete profile of the most common monothiols and disulfides present in plasma or tissue extracts. The method utilizes reversed phase ion-pairing high performance liquid chromatography coupled with coulometric electrochemical detection to simultaneously quantify free oxidized and reduced aminothiols or total aminothiols after chemical reduction. The method is extremely sensitive, with limits of detection in the 5 fmol/mL range for monothiols and 50 fmol/mL for dithiols. The interassay and intraassay coefficients of variation for total and free aminothiols ranged between 1.2 and 5.8%. The mean recoveries for total and plasma aminothiols ranged between 97.1 and 102.8%. The aminothiols are quantified directly, without derivatization, and include methionine, homocysteine, homocystine, cystathionine, cysteine, cystine, cysteinylglycine, and oxidized and reduced glutathione. Because a complete aminothiol profile of metabolites in both the remethylation (anabolic) and transulfuration (catabolic) pathways of homocysteine metabolism can be determined simultaneously, this new method should be useful in determining the metabolic etiology of homocysteinemia and in designing appropriate nutritional intervention strategies. Basic research applications of this method should lead to an increased understanding of the metabolic pathology of aminothiol imbalance.