HPLC Approach Research Articles

Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).

A New HPLC Approach for the Determination of Hydrophilic and Hydrophobic Components: The Case of Pseudoephedrine Sulfate and Loratadine in Tablets

Effective isocratic separations of decongestants and antihistamines is a challenging analytical task due to wild differences in their lipohilicities (hydrophilic decongestants and hydrophobic antihistamines). In this paper a new approach for resolving such a problem is described taking pseudoephedrine sulfate and loratadine as an example. The chromatographic behavior of pseudoephedrine sulfate and loratadine on RP C18 and C8 columns were studied in presence and absence of sodium lauryl sulfate (SLS). The effect of combining two different types of stationary phases (cyano and C18 or C8) on the relative retention of the two compounds was investigated. In conclusion, it was found that the combination of a C18 column followed by a standard cyano column provides a stationary phase that separates both compounds effectively and within a reasonable time. This approach was compared to a literature method and demonstrated to have superior selectivity.

Assay of Sudan I contamination of foodstuff by atmospheric pressure chemical ionization tandem mass spectrometry and isotope dilution.

Food safety represents one of the main issues of national and international agencies appointed to health control. In April 2003, a French agency disclosed that powdered or smashed hot chili pepper imported from India and Pakistan was heavily contaminated with a carcinogenic azo dye known as Sudan I. This paper deals with a modern approach for assaying the content of this colorant in foodstuff down to a limit of a few tens of parts per billion. The isotope dilution method combined with APCI tandem mass spectrometry was used. The internal standard, 1-(d5-phenylazo)-2-naphthalenol, was obtained by simple chemistry, and its structure was determined by 1H NMR spectroscopy. The mass spectrometric method is more sensitive than the HPLC approach by a factor of 20.

Generation and comparison of EST-derived SSRs and SNPs in barley (Hordeum vulgare L.).

The progress of genome sequencing projects of model plants like barley, combined with the recent advances of high throughput assays, has provided a wealth of sequence information. This information is being employed to develop a high density transcript map of barley (Hordeum vulgare L.). To achieve this goal, the available EST database is being used as a resource for the development of novel microsatellite (SSR) and single nucleotide polymorphism (SNP) markers. So far, a total of 692 microsatellites representing different di-, tri- and tetra-nucleotide repeats were identified from a set of 19,000 EST sequences. Non-redundant SSRs have been used for mapping and so far 76 microsatellite loci were mapped. In addition to the 180 SNP primer pairs, which were designed to target specific ESTs, 72 were polymorphic among the seven genotypes examined here. Of these, 60 SNPs have been mapped applying a denaturing HPLC approach. To examine the potential of the EST-derived markers for pedigree studies, EST-derived SSRs (75 loci) and SNPs (72 loci) were used to fingerprint a set of seven genotypes. The results show that although both marker types yielded similar groupings, a larger data set of both SSRs and SNPs is necessary to obtain stable clusters in unrelated germplasm.

Studies of the thermal degradation of acetaminophen using a conventional HPLC approach and electrospray ionization-mass spectrometry.

The thermal degradation of acetaminophen is studied via conventional accelerated aging studies by initially thermally stressing the compound at temperatures between 160 degrees C and 190 degrees C and measuring the rate of decomposition by reversed-phase high-performance liquid chromatography. Rates of decomposition of the compound in the dry state and the activation energy for the process are determined and compared with previously published kinetic and thermodynamic data for the degradation of acetaminophen in solution. In addition, the thermal fragmentation of acetaminophen under electrospray ionization (ESI) conditions using an interface with a heated capillary inlet is studied and the apparent activation energy for this process also is characterized. A comparison of the data shows that acetaminophen is significantly more stable in the dry state than in solution. However, the gas-phase fragmentation of acetaminophen under ESI conditions occurs more readily than either dry- or solution-state degradation. Although the resulting electrospray fragmentation mimics the breakdown product that is formed when the compound undergoes either acid or base catalyzed hydrolysis in aqueous solutions, the mechanism that produces the fragment ion appears to involve a two-step process. Initially, the parent ion forms of the analyte are produced in the spray region of the interface followed by wall-catalyzed decomposition and re-ionization in the heated inlet capillary of the spectrometer.

Pharmacokinetics and cellular uptake of imatinib and its main metabolite CGP74588.

Despite the remarkable clinical response rates to imatinib in the treatment of bcr-abl leukemic patients, pharmacokinetic data on this relatively novel substance are needed to improve our understanding of the emergence of resistance, the interindividual variations of clinical response and the clinical and biologic relevance of its main metabolite N-desmethyl-imatinib. We present here pharmacokinetic data obtained with a newly designed HPLC approach in 97 patients with chronic myeloid leukemia or acute lymphatic leukemia (ALL) under treatment with imatinib that allowed us to calculate the AUC (39.5 microg.h/ml for an oral dose of 400 mg daily), the t(1/2) (18.2 h) and the peak concentration (1.92 micro/ml for an oral dose of 400 mg daily) of imatinib in plasma. In a subgroup of patients, the same parameters were analyzed for N-desmethyl-imatinib. We also provide data on the imatinib concentration in the cerebrospinal fluid (CSF) of ALL patients and demonstrate that oral administration of imatinib resulted only in a marginal flux across the blood-brain barrier. Finally, in an in vitro setting, we determined cellular concentrations of imatinib in HL-60 cells and showed an over-proportional uptake both in RPMI medium and in human plasma. Using an arithmetical approach combining all parameters obtained in imatinib-treated patients, we finally provide a conclusive approximation of basic pharmacokinetic data for both imatinib and its main metabolite N-desmethyl-imatinib.

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression.

We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis. The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests. The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components. Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30. However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

Comparison of different methods to determine polar compounds in frying oils

The validity of measurements of polar compounds in used frying oils was investigated. Compared were the reference method, the food oil sensor (FOS), and a newly developed HPLC method. It was shown that the HPLC values correlate better with the reference method than the FOS measurements. The standard deviation of the results obtained by the reference method was three times higher than those of the FOS and the HPLC methods. Because of the better precision and the higher speed, the HPLC approach is considered to be an attractive alternative to the reference method.

The HPLC Resolution of Nucleic‐Acid Bases and An a Log Hypoxanthine on R,S‐2‐Hydroxypropyl Derivatized β‐Cyclodextrin Bonded Chiral Stationary Phase Using a Water‐Based Mobile Phase

AbstractA HPLC approach using R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin packed column as the stationary phase was developed to resolve five nucleic‐acid bases and an a log hypoxanthine in the reversed‐phase mode. These bases are not only similar in structure but also very close in basicity. However, the resolution can be completed in less than ten minutes and is considered to be better carried out on the R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin phase than that obtained on the native β‐cyclodextrin phase under the same chromatographic conditions. The mechanism involved in the resolution is believed to be inclusion complexation between the analyte and the cavity of cyclodextrin in the reversed‐phase mode. The retention time was found relevant to the size of the analyte. The number of groups on analyte that is available to form hydrogen bonding with hydroxyl groups on CDs also affects the retention scale. Factors of introducing organic acid and base or organic modifier such as methanol to the water‐based mobile phase or increasing their percent ages in the mobile phase decreases the retention time without de grading the resolution significantly.

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides

Heparan sulphates are highly sulphated linear polysaccharides involved in many cellular functions. Their biological properties stem from their ability to interact with a wide range of proteins. An increasing number of studies, using heparan sulphate-derived oligosaccharides, suggest that specific structural features within the polysaccharide are responsible for ligand recognition and regulation. In the present study, we show that strong anion-exchange HPLC alone, a commonly used technique for purification of heparan sulphate-derived oligosaccharides, may not permit the isolation of highly pure heparan sulphate oligosaccharide species. This was determined by PAGE analysis of hexa-, octa- and decasaccharide samples deemed to be pure by strong anion-exchange HPLC. In addition, subtle differences in the positioning of sulphate groups within heparan sulphate hexasaccharides were impossible to detect by strong anion-exchange HPLC. PAGE analysis on the other hand afforded excellent resolution of these structural isomers. The precise positioning of specific sulphate groups has been implicated in determining the specificity of heparan sulphate interactions and biological activities; hence, the purification of oligosaccharide species that differ in this way becomes an important issue. In this study, we have used strong anion-exchange HPLC and PAGE techniques to allow production of the homogeneous heparan sulphate oligosaccharide species that will be required for the detailed study of structure/activity relationships.

Combined strong anion-exchange HPLC and PAGE approach for the purification of heparan sulphate oligosaccharides.

Heparan sulphates are highly sulphated linear polysaccharides involved in many cellular functions. Their biological properties stem from their ability to interact with a wide range of proteins. An increasing number of studies, using heparan sulphate-derived oligosaccharides, suggest that specific structural features within the polysaccharide are responsible for ligand recognition and regulation. In the present study, we show that strong anion-exchange HPLC alone, a commonly used technique for purification of heparan sulphate-derived oligosaccharides, may not permit the isolation of highly pure heparan sulphate oligosaccharide species. This was determined by PAGE analysis of hexa-, octa- and decasaccharide samples deemed to be pure by strong anion-exchange HPLC. In addition, subtle differences in the positioning of sulphate groups within heparan sulphate hexasaccharides were impossible to detect by strong anion-exchange HPLC. PAGE analysis on the other hand afforded excellent resolution of these structural isomers. The precise positioning of specific sulphate groups has been implicated in determining the specificity of heparan sulphate interactions and biological activities; hence, the purification of oligosaccharide species that differ in this way becomes an important issue. In this study, we have used strong anion-exchange HPLC and PAGE techniques to allow production of the homogeneous heparan sulphate oligosaccharide species that will be required for the detailed study of structure/activity relationships.

Use of directly coupled ion-exchange liquid chromatography–mass spectrometry and liquid chromatography–nuclear magnetic resonance spectroscopy as a strategy for polar metabolite identification

Ion-exchange LC–MS and LC–NMR have been successfully used to identify a novel N-acetyl metabolite of a highly polar drug candidate [2-(ethanimidoylamino)ethyl]sulfonyl alanine (GW273629) under development as a therapeutic agent. This has been achieved using a simple HPLC method without the need for complicated and time consuming pre- or post-column derivatisation. Ion-exchange chromatography using simple ionic strength buffer and organic solvent mobile phases, as applied here, should be suitable for the analysis of other charged polar species. Optimisation of the system described could result in the development of a rational generic HPLC approach specifically designed for the characterisation of polar drug molecules and their metabolites.

Ion pair-reversed phase HPLC approach facilitates subcloning of PCR products and screening of recombinant colonies.

The isolation of a single DNA molecule for cloning is technically difficult, and the subsequent screening of colonies for recombinant DNA clones is time consuming. Ion pair-reversed phase HPLC (IP-RP-HPLC) analysis of nucleic acids improves the resolution and isolation of PCR products to be cloned. We demonstrate that PCR products analyzed and collected using the IP-RP-HPLC approach (WAVE DNA Fragment Analysis System) can be cloned directly into a plasmid vector. In addition, we demonstrate that when IP-RP-HPLC analysis is extended to the colony screening process, the time required for these procedures is reduced.

Ion chromatographic analysis of hydronium ion using ODS modified with dodecylsulfate as the stationary phase.

The hydronium ion (H3O+), the most essential species to every field of chemistry, was successfully separated from other cations. This major goal was achieved by a HPLC approach in which a reversed-phase ODS which had been initially modified with dodecylsulfate was used as the stationary phase and an aqueous KNO3 solution was used as the mobile phase. Of all tested monovalent cations (analyte ions), the hydronium ion (H3O+) showed the highest affinity towards the stationary phase, followed by NH4+, Na+, and then Li+. The hydronium ion, after the separation, was directly detected using conductivity and the detection signal (conductivity) was in linear proportion (linear range 7.8 x 10(-4)-0.45 M; injection volume, 100 microL) to the concentration of the acid species of the tested sample. This proposed HPLC method was applicable to separate/detect H3O+ from both strong and weak acidic samples.

Stability of reconstituted solutions of ceftazidime for injections: an HPLC and CE approach

The stability of aqueous reconstituted ceftazidime injection vials containing ceftazidime pentahydrate blended with anhydrous sodium carbonate was investigated in different storage conditions (4°C and 10°C for 7 days in a refrigerator, 20 and 30°C for 24 h) with validated HPLC and (micellar) CE methods. Stability indicating data were obtained for ceftazidime and two degradation products: pyridine and the Δ 2-ceftazidime isomer. Other degradation products were also identified (the complementarity of the two used experimental procedures was useful in such exercise) and characterized by their UV spectra and retention times. Stability data (7 days at 4°C in a refrigerator and 18 h at room temperature) resulted in agreements with the manufacturers prescription and point out the need of a strict temperature control of the refrigerator’s compartment used to store the reconstituted solution.

Rapid estimation of octanol-water partition coefficients of pesticides by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) was evaluated as a new technique for the rapid estimation of octanol-water partition coefficient (logKow). Retention measurements for more than 40 reference pesticides with varied structural characteristics and hydrophobicity were carried out in two MEKC systems, based on sodium dodecyl sulfate (SDS) and sodium cholate (SC), respectively. To enable an accurate determination of capacity factors in the SC-MEKC system, cypermethrin (a synthetic pyrethroid insecticide) was utilized instead of Sudan III as the SC micelle tracer, since a few highly hydrophobic pesticides were found to elute after Sudan III. The linear correlation between logarithmic capacity factor (logk') and logKow in the two systems was examined. It was found that, under the typical buffer condition (10 mM sodium phosphate with 60 mM surfactant, pH 7.0), the SDS-MEKC system provided a somewhat wider dynamic range for hydrophobicity (logKow from -1.0 to 4.5). However, the correlation of logk' with logKow was not very high when all the reference pesticides were included in one single calibration set. For the SC-MEKC system, the dynamic range for logKow was in the range of 1.0-5.5, and a good linear correlation existed between logk' and logKow, even when all reference pesticides were incorporated into a single calibration group. By comparing the regression line of the reference pesticides with that of a group of simple aromatic derivatives, it was discovered that molecular size and functionality posed a less significant effect on the measurement of logKow in the SC-MEKC system than in the SDS-MEKC system. Thus, SC-MEKC shall be the system of choice for the estimation of logKow. The typical error on logKow determination using the current MEKC technique was within 0.5 units, suggesting that MEKC can be a valuable complement to reversed phase high performance liquid chromatography (RP-HPLC) for the indirect determination of logKow. Besides maintaining all the advantages of the HPLC approach, the MEKC technique showed some unique benefits, such as better inter-column reproducibility, higher throughput, and less handling of toxic pesticides and solvents.

Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection

This paper describes the development and validation of a new off-line approach to quantitate both covalent and noncovalent, in-solution aggregates present in protein formulations and compares the new assay to established HPLC methods. This off-line analysis is well suited for use in QC release testing, formulation development and stability indicating applications. An inexpensive, continuous source HPLC fluorometer has been adapted with the addition of second order filters for use as a sensitive right-angle scatterometer which can determine the molecular weight of protein aggregates in solution. When used as an HPLC detector, right-angle light scattering is a sensitive method which can determine the molecular weight of peaks separable by HPLC, thus discriminating between monomers of different conformations and aggregates. The weight-averaged molecular weight of aggregate peaks can be calculated with system calibration, yielding the average number of monomers per aggregate. If the protein concentration is high enough for an adequate signal, the off-line technique of right-angle light scattering of protein formulations has advantages of convenience and speed over the HPLC approach. Samples are placed in standard fluorometer cuvettes and toluene is used as a calibrator. Data are presented which show the off-line (static) method to be extremely rapid, rugged and precise. The accuracy of this approach is demonstrated through cross-validation to traditional GPC analysis of protein aggregate distributions. This non-invasive light scattering approach is particularly useful when non-covalent protein aggregation is reversible and readily altered by chromatographic separations typically used for characterizing aggregates.

Positive and negative regulation of type II TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.

The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively.

A novel enzymic determination of maltobionate

A novel enzymic determination of maltobionate with three enzymes (a new enzyme, maltobionate α- d-glucohydrolase [EC 3.2.1.-], gluconate kinase [EC 2.7.1.12], and 6-phosphogluconic dehydrogenase [EC 1.1.1.44]) is described. It fulfilled the need for an accurate, specific, and rapid assay of maltobionate, as shown by the procedure, the linearity of calibration curve, and the analytical precision, and was simple as compared to an HPLC approach. There was a good correlation ( r = 0.998) between the results obtained by the enzymic and HPLC methods. As an application, the concentrations of maltobionate in the culture broth of Pseudomonas taetrolens IFO 3460 were determined by the proposed method, affording the maltobionate production from maltose in the course of cultivation of this strain.

Quantitative determination of a dipeptide in personal wash liquid by capillary electrophoresis

The dipeptide alanylglutamine is quantified in a commercial personal cleaning fluid using micellar electrokinetic capillary chromatography. Quantitation is achieved using an internal standard approach with either normalised peak height or area measurements. Correlation coefficients for the calibration graphs were typically 0.999 with data generated over a two-month period and using different capillaries. The data was in good agreement with that of an HPLC approach which utilised pre-column derivatisation with dabsyl chloride.