High-performance Liquid Chromatography Patterns Research Articles

HPLC fingerprint and total antioxidant capacity of the extracts from the aerial part of Paeonia lactiflora Pall

ABSTRACT High-performance liquid chromatography (HPLC) fingerprinting is a reliable quality control method for traditional Chinese medicine (TCM). As a research method combining fingerprint and pharmacodynamics of TCM, the TCM spectral effect relationship is widely used in the quality control of TCM and its preparations, which can better reflect the intrinsic quality of TCM and is of great significance for the research on the material basis of TCM efficacy. In this study, an HPLC fingerprint of the extracts from the aerial part of Paeonia lactiflora Pall (APPE) was first established. Then, the HPLC fingerprint of APPE was evaluated by stoichiometry analysis methods such as similarity analysis, hierarchical clustering analysis, and principal component analysis. At the same time, the spectral effect relationship between the HPLC pattern and the total antioxidant capacity of APPE was analyzed by Partial Least Squares Regression (PLSR). The results showed that the similarity correlation coefficient of the 12 batches was ≥0.994, and the total antioxidant capacity was stable. In addition, PLSR analysis showed that 13 chemical components were closely related to the antioxidant activity of APPE.

Understanding the Working Mechanism of the Novel HKUST-1@BPS Composite Materials as Stationary Phases for Liquid Chromatography.

Composite materials have been used based on coordination polymers or microporous metal-organic frameworks (MOFs) combined with mesoporous matrices for adsorption-related techniques, which enable outflanking some adverse phenomena manifested during pristine components operation and enhance the performance and selectivity of the resulting materials. In this work, for the first time, the novel HKUST-1@BPS composites synthesized by the microwave-assisted (MW) technique starting from microporous HKUST-1 (Cu3(btc)2) MOF and biporous silica matrix (BPS) with bimodal mesopore size distribution were comparatively studied as materials for liquid-phase adsorption techniques utilizing the high-performance liquid chromatography (HPLC) method and benzene as a model adsorbate. It was established that the studied HKUST-1@BPS composites can function as stationary phases for HPLC, unlike the pristine HKUST-1 and bare BPS materials, due to the synergetic effect of both components based on the preliminary enhanced adsorbate mass transfer throughout the silica mesopores and, subsequently, its penetrating into HKUST-1 micropores. The suggested mechanism involves the initial deactivation of open metal Cu2+ sites in the HKUST-1 framework structure by isopropanol molecules upon adding this polar component into the mobile phase in the region of the isopropanol concentration of 0.0 to 0.2 vol.%. Thereafter, at the medium range of varying the isopropanol concentration in the eluent of 0.2 to 0.3 vol.%, there is an expansion of the previously inaccessible adsorption centers in the HKUST-1@BPS composites. Subsequently, while further increasing the isopropanol volume fraction in the eluent in the region of 0.3 to 5.0 vol.%, the observed behavior of the studied chromatographic systems is similar to the quasi-normal-phase HPLC pattern. According to the obtained thermodynamic data, benzene adsorption into HKUST-1 micropores from solutions with a vol.% of isopropanol in the range of 0.4 to 5.0 follows the unique entropy-driven mechanism previously described for the MIL-53(Al) framework. It was found that HKUST-1 loading in the composites and their preparation conditions have pronounced effects on their physicochemical properties and adsorption performance, including the adsorption mechanism.

Hematological and Molecular Findings in the First Case of Hb J-Norfolk [HBA2: c.173G>A (or HBA1] in an Indian Patient

We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.

Changes in the High-Performance Liquid Chromatography Pattern of Hemoglobin Lepore After Treatment With Hydroxyurea

Hemoglobin (Hb) Lepore is a β+-thalassemic hemoglobin variant composed of 2 α-globin and 2 δβ-globin hybrid chains. Three common Hb Lepore variants have been identified based on the primary sequence of the fusion proteins. In most cases, diagnosis of Hb S/Hb Lepore compound heterozygosity is determined using a combination of high-performance liquid chromatography (HPLC) testing and acid/alkaline gel electrophoresis. Herein, we report an unusual case of Hb S/Hb Lepore, in which hydroxyurea therapy resulted in a change to the typical HPLC pattern, which confounded diagnosis. Our results emphasize the importance of having a detailed clinical history for proper diagnosis. In particular, the case reported herein demonstrates how diagnosis of a compound heterozygous Hb S/Hb Lepore genotype can be misinterpreted during routine Hb evaluation via HPLC if the patient has been treated with hydroxyurea. * HPLC : high-performance liquid chromatography; Hb : hemoglobin; PCR : polymerase chain reaction

The Use of High Performance Liquid Chromatography to Speciate and Characterize the Epidemiology of Mycobacteria

We evaluated high-performance liquid chromatography (HPLC) for species identification of mycobacteria from various clinical specimens in an urban hospital in South Korea between January 2005 and December 2009. In the study period 24,774 cultures were completed, yielding the 3215 clinical isolates cultivated for mycobacteria and positive cultures that had mycolic acid investigated by HPLC. For species identification, we compared HPLC patterns of clinical isolates with 33 standard Mycobacterium species. There were 3 different HPLC groups with single, double, and triple-cluster patterns representing 9, 20, and 4 mycobacterial species, respectively. Species identification rates of HPLC for Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) were found to be 100% and 95.6%, respectively. Among mycobacterial isolates, 12.1% were NTM-positive. There were 20 different NTM species with frequencies of 0.3%~15.5%. The HPLC method was highly sensitive identifying NTM isolated from clinical specimens.

홍삼의 에탄올 추출물의 감마선 조사를 통한 항암효과 증대

Red ginseng is one of the most popular traditional medicines in Korea. In this study, we developed a new technique in which ethanol extract of ed inseng (HRG) was exposed to the gamma radiation ranging from 1~5 kGy. The irradiated HRG (IHRG) were analyzed by high performance liquid chromatography (HPLC) to determine any compositional changes of ginsenosides due to irradiation. No appreciable difference was observed in the HPLC pattern of ginsenosides of HRG. Using MTT assay, the cytotoxicity effect was significantly increased by IHRG compared to HRG. The concentration was for IHRG-1 (1 kGy), and for IHRG-5 (5 kGy). The evidences of apoptosis, such as nuclei cleavage and Annexin V staining, were observed in the human prostate cancer PC-3 cells treated with the IHRG. Additionally, the level of reactive oxygen species (ROS) was apparently elevated by IHRG. We also studied the inhibitory effect of IHRG on the growth rate of tumor xenografts in BALB/c male mice. The tumor growth rates were inhibited by 56.9 and 76.1% in mice treated with 10 mg/kg of IHRG-1 and IHRG-5, respectively, compared with control group (21.1%). These results suggest that some biologically active and soluble components in HRG can be more effectively enhancement of anti-tumor effects using irradiation.

Drought Tolerance in Cotton: Involvement of Non‐enzymatic ROS‐Scavenging Compounds

AbstractCompounds with reactive oxygen species (ROS)‐scavenging ability were studied. High‐performance liquid chromatography (HPLC) pattern of polyphenols, contents of proline and carotenoids, and antiradical (AR) capacity were determined. The malonyldialdehyde (MDA) level was also assessed. Tolerant and sensitive cotton genotypes were compared, grown in the Aegean region of Turkey at normal (field capacity) and limited (1/3 field capacity) water supply. Chlorogenic acid isomers and flavonoids were identified in HPLC pattern of polyphenols. At normal water supply, the tolerant genotype was distinguished by a higher content of all polyphenol types, higher proline, carotenoids and AR capacity and lower MDA level compared with the sensitive genotype. In plants subjected to water deficit, a decline of all polyphenol compounds, carotenoids and AR capacity was observed. However, this response was less pronounced in the tolerant than in the sensitive genotype, i.e. despite the stress conditions imposed, the tolerant plants maintained a more effective defence system. The data are corroborated by the weaker structural membrane damage in the drought‐exposed tolerant vs. sensitive genotype, according to the MDA test. Hence, diverse chemical types are involved in the non‐enzymatic ROS‐scavenging system of cotton plants and can be related to the drought tolerance of this important crop.

Identification of green tea’s ( Camellia sinensis (L.)) quality level according to measurement of main catechins and caffeine contents by HPLC and support vector classification pattern recognition

High performance liquid chromatography (HPLC) was identified green tea’s quality level by measurement of catechins and caffeine content. Four grades of roast green teas were attempted in this work. Five main catechins ((−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epicatechin (EC), and (+)-catechin (C)) and caffeine contents were measured simultaneously by HPLC. As a new chemical pattern recognition, support vector classification (SVC) was applied to develop identification model. Some parameters including regularization parameter ( R) and kernel parameter ( K) were optimized by the cross-validation. The optimal SVC model was achieved with R = 20 and K = 2. Identification rates were 95% in the training set and 90% in the prediction set, respectively. Finally, compared with other pattern recognition approaches, SVC algorithm shows its excellent performance in identification results. Overall results show that it is feasible to identify green tea’s quality level according to measurement of main catechins and caffeine contents by HPLC and SVC pattern recognition.

Identification of Mycobacteria Species by HPLC and Species Distribution during Five Years at Ulsan University Hospital

Infections caused by mycobacteria have been significantly increasing. Due to the difficulty of making a decision about the pathogenicity of mycobacteria, species-level identification is very important for patients' diagnosis and treatment. The purpose of this study was to identify mycobacteria species using a high performance liquid chromatography (HPLC) method and to provide an initial database for the distribution of mycobacteria in Korea. Acid fast bacteria isolated from 3,107 clinical specimens were identified by mycolic acid analysis using HPLC. The HPLC patterns were compared with those of standard mycobacteria species. The HPLC patterns were divided into single, double, and triple cluster groups, each group comprising 9, 20, and 4 species, respectively. Mycobacteria and non-tuberculous mycobacteria (NTM) were identifies by HPLC at the rates of 99.5% and 95.6%, respectively. NTM was isolated in 12.4% of the mycobacteria positive specimens. This study also found that there were 20 different NTM species with the distribution of each species ranging from 0.3% to 15.9% of the total NTM. While the rate of NTM has been increasing in Korea, M. avium-intracellulare, M. fortuitum, and M. chelonae are relatively decreasing, and M. kansasii and M. gordonae are relatively increasing. HPLC method was highly discriminative for the identification of NTM in clinical specimens.

The first case of Mycobacterium sherrisii disseminated infection in a child with AIDS

The first case of Mycobacterium sherrisii disseminated infection in a child with AIDS

A New Approach to Plant Diversity Assessment Combining HPLC Data, Simplex Mixture Design and Discriminant Analysis

The quantitative assessment of plant diversity and its monitoring with time represent a key environmental issue for management and conservation of natural resources. Assessment of plant diversity could be based on chemical analyses of secondary metabolites (e.g. flavonoids, terpenoids), because of the substantial quantitative and qualitative between-individual variability in such compounds. At a geographical scale, the plant populations become widely dispersed, and their monitoring from numerous routine individual analyses could become restricting. To overcome such constraint, this study develops a multivariate calibration model giving the relative frequency of a particular taxon from a simple high-performance liquid chromatography (HPLC) analysis of a plant mixture. The model was built from a complete set of mixtures combining different taxons, according to an experimental design (Scheffe’s matrix). For each mixture, a reference HPLC pattern was simulated by averaging the individual HPLC profiles of the constitutive taxons. The calibration models, based on Bayesian discriminant analysis (BDA), gave statistical relationships between the contributions of each taxon in mixtures and reference HPLC patterns of these mixtures. Finally, these models were validated on new mixtures by using outside plants. This new biodiversity survey approach is illustrated on four chemical taxons (four chemotypes) of Astragalus caprinus (Fabaceae). The more differentiated the taxon, the better predicted its contributions (in mixtures) were by BDA calibration model. This new approach could be very useful for a global routine survey of plant diversity.

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten: Studies on gluten, gliadin and glutenin

The effect of hydrostatic pressure (0.1–800 MPa) in combination with various temperatures (30–80 °C) on the chemical and physical properties of wheat gluten, gliadin and glutenin was studied. Chemical changes of proteins were determined by extraction, reversed-phase high-performance liquid chromatography (HPLC), sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy, thiol measurement and studies on disulphide bonds. Rheological changes were measured by extension tests and dynamic stress rheometry. Treatment of gluten with low pressure (200 MPa) and temperature (30 °C) increased the proportion of the ethanol-soluble fraction (ESF) and decreased gluten strength. The enhancement of both pressure and temperature provoked a strong reduction of the ESF and the thiol content of gluten. Within gliadin types, cysteine containing α- and γ-gliadins, but not cysteine-free ω-gliadins were sensitive to pressure and were transferred to the ethanol-insoluble fraction. Disulphide peptides isolated from treated gluten confirmed that cleavage and rearrangement of disulphide bonds were involved in pressure-induced reactions. Increased pressure and temperature induced a significant strengthening of gluten, and under extreme conditions (e.g. 800 MPa, 60 °C), gluten cohesivity was lost. Isolated gliadin and glutenin reacted differently: solubility, HPLC and SDS-PAGE patterns of gliadin having a very low thiol content were not influenced by pressure and heat treatment; only conformational changes were detected by CD spectroscopy. In contrast, the properties of isolated glutenin having a relatively high thiol content were strongly affected by high pressure and temperature, similar to the effects on total gluten.

DNA Fingerprinting of Cannabis sativa Using Inter-Simple Sequence Repeat (ISSR) Amplification

Chemical analysis of cannabinoid, and Inter-Simple Sequence Repeat (ISSR) fingerprinting of DNA were used to identify different samples of Cannabis sativa L. for forensic purposes. Three samples were classified into two types, tetrahydrocannabinol (THC) and cannabidiol (CBD) chemo-types, by high performance liquid chromatography (HPLC). The two samples of the CBD type were not distinguished by their HPLC patterns. ISSR fingerprinting identified polymorphic DNA patterns between these samples. ISSR fingerprinting clearly differentiated between cannabis samples that could not be achieved by HPLC analysis.

Seed globulins in the Old World Lupinus species: Comparative study by HPLC

Seed globulins isolated from 58 accessions representing 12 Old World Lupinus species were studied using two techniques of high-performance liquid chromatography (HPLC): reversed-phase (RP-HPLC) and ion-exchange (IE-HPLC). Differences in quantitative and qualitative composition of globulins between smooth-seeded and rough-seeded lupins appear to be significant. Each investigated species/subspecies is distinguished by its specific protein HPLC pattern of globulins. The number of globulin peaks recorded in particular species varied from four to eight. In total, 72 retention times of protein peaks were distinguished in the investigated taxa. Chromatographic data were subjected to statistical analysis using hierarchical UPGMA grouping of the examined taxa. Heterogeneous smooth-seeded lupins proved to be distantly related to rather homogeneous rough-seeded lupins except for the new species L. anatolicus. Within the rough-seeded lupins three subgroups are distinguished: (1) L. atlanticus, L. cosentinii and L. digitatus, (2) L. palaestinus and L. pilosus and (3) L. princei. The obtained data are discussed with reference to taxonomic relationships in the Old World lupins.

Quantitative detection method of triglycerides in serum lipoproteins and serum-free glycerol by high-performance liquid chromatography

We have developed a simple and reliable method for quantitative detection of triglycerides (TG) in serum lipoproteins and serum-free glycerol (FG) by high-performance liquid chromatography (HPLC). After separation of serum constituents using a new gel-permeation column (TSK gel Lipopropak XL, Tosoh) and a new eluent (TSK eluent LP-2, Tosoh), TG and FG were detected by on-line reaction using a modified reagent which contained glycerol kinase, glycerol-3-phosphate oxidase and lipoprotein lipase. HPLC patterns showed five peaks corresponding to chylomicrons, very-low-density, low-density, high-density lipoproteins and FG. Absolute concentrations of TG in each lipoprotein fraction and serum FG were calculated from the corresponding peak areas using standard FG as a calibrator. Due to its very high sensitivity of peak detection, this method has become desirable for the analyses of lipoproteins of very low concentrations such as in cell culture systems. This technique will contribute to a better understanding of lipoprotein TG and serum FG distribution in human and nonhuman subjects.

POSSIBLE HAZARDS OF ROUTINE BACILLUS CALMETTE-GUÉRIN IMMUNIZATION IN HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CHILDREN

POSSIBLE HAZARDS OF ROUTINE BACILLUS CALMETTE-GUÉRIN IMMUNIZATION IN HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CHILDREN

Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry.

Disease-associated proteins separated by two-dimensional electrophoresis (2-DE) are often in the femtomole range. Identification of 2-DE separated proteins by sequencing and amino acid analysis is limited to the lower picomole range. Identification down to the femtomole range can be achieved by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). We optimized the measurement by MALDI-MS for the analysis of proteolytic digests of 2-DE-separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive protein identification. In some cases, more information about the protein can be obtained by separating the peptides by micro high-performance liquid chromatography (HPLC) before employing MALDI-MS analysis. More peptides are found than in the mixtures, and comparison of HPLC patterns can reveal some differences to be post-translational modifications of proteins, even in the case of identical peptide mass fingerprints. Furthermore, carboxy-terminal sequencing by on-target carboxypeptidase P digestion can be used to confirm the obtained result without the need for more material. The search program FRAGFIT was modified and renamed FRAGMOD to include the modifications of methionine and tryptophan oxidation and alkylation of cysteine by acrylamide into the mass search. By applying this procedure, 15 proteins were identified, among them two different putative phosphorylated forms of two proteins, a putative N-terminal blocking group and four dilated cardiomyopathy-associated proteins. The resulting approach for the identification may be used for large-scale investigations of 2-DE-separated proteins.

Kappa marker typing with high-performance liquid chromatography: identification of kappa marker specific tryptic peptide from the kappa light chain of immunoglobulin G

Human serum immunoglobulin G was separated into its heavy and light chains by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred electrophoretically to a polyvinylidenedifluoride membrane. Peptide fragments liberated from the light chain by in situ digestion with trypsin were then analyzed by reversed-phase high-performance liquid chromatography (HPLC). On comparing the HPLC patterns of these fragments derived from three major kappa marker (Km) types, two distinct peaks specific for the Km types were detected. Sequencing of the two specific peak peptides confirmed that they were identical to a stretch comprising residues 191–207 of the immunoglobulin kappa light chain, which contains valine/leucine allotypic variation at position 191.

High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms.

A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory.

Identification of mycobacteria by high-performance liquid chromatography

Mycolic acids extracted from saponified mycobacterial cells were examined as p-bromophenacyl esters by high-performance liquid chromatography (HPLC). Standard HPLC patterns were developed for species of Mycobacterium by examination of strains from culture collections and other well-characterized isolates. Relative retention times of peaks and peak height comparisons were used to develop a differentiation scheme that was 98% accurate for the species examined. A rapid, cost-effective HPLC method which offers an alternative approach to the identification of mycobacteria is described.