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PubMed2003

Quantification of serum and urinary S-adenosylmethionine and S-adenosylhomocysteine by stable-isotope-dilution liquid chromatography-mass spectrometry.

BACKGROUND: We have developed an assay that uses stable-isotope-dilution liquid chromatography-mass spectrometry to assess S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in body fluids to investigate the relationship of these metabolites to hyperhomocysteinemia. METHODS: Commercially obtained SAM (D(3) methyl) and (13)C(5)-SAH uniformly labeled in the adenosyl moiety, which was synthesized using S-adenosylhomocysteine hydrolase, were added to samples followed by perchloric acid protein precipitation, C(18) chromatography, and analysis by liquid chromatography-mass spectrometry with quantification by comparison of the areas of internal standard and endogenous peaks. RESULTS: Estimates of intraassay imprecision (CV) were 5% and 17% for SAM and SAH, respectively (n = 10). SAM decreased and SAH increased in serum and plasma samples at both 4 degrees C and room temperature over 80 h. SAM and SAH were unstable in samples stored longer than 2 years at -20 degrees C. In 48 volunteers, the estimated reference intervals [from mean (2 SD) of log-transformed data] for serum SAM and SAH were 71-168 and 8-26 nmol/L, respectively. Fractional excretion of SAM was higher than that of SAH, and the urinary SAM:SAH ratio was much higher than the serum or erythrocyte SAM:SAH ratios. CONCLUSIONS: Stable-isotope-dilution liquid chromatography-mass spectrometry can be used to quantify SAM and SAH in biological fluids and tissues. Sample handling and storage must be stringently controlled for any epidemiologic or clinical use of such assays.

Adult↗

PubMed1988

[Triiodothyronine(T3) autoantibodies in a woman with nonthyroidal disorder: a study on preparation of serum IgG fraction employing protein A column chromatography].

A 48-year-old non-goitrous woman, who had undergone cardiac surgery for mitral stenosis under the extracorporeal circulation, showed high levels of serum T3 and free T3 in a recent follow-up study, employing antibody coated-bead RIA for T3 and -Amerlex M particle RIA for free T3. However, other thyroid function tests (T4, free T4, TSH and TBG) were normal. We suspected that thyroid hormone autoantibodies (THAA) in her serum interfered with T3 and free T3 analyses. The presence of THAA was demonstrated by the use of various procedures as follows. Firstly, the patient's serum was directly incubated with 125I-T3 or -T4 analog which did not bind to TBG, followed by B/F separation with polyethyleneglycol, counting the precipitates. Secondly, after the serum was treated with an acid-charcoal solution to remove circulating thyroid hormone, the measurement of THAA was made as stated above. Normal sera were used as controls. Both the non- and acid-charcoal-treated sera showed much higher percentages of 125I-T3 analog precipitation as compared with controls. In the case of 125I-T4 analog, there was no difference between them. In the third study, the presence of IgG antibodies that bound T3 but not T4 was investigated. The IgG fraction of the patient's serum was separated employing a Protein A-Sepharose CL-4B column chromatography. Then, the prepared IgG fraction was purified by a technique of gel filtration chromatography (Sephacryl S 200). Non-purified and purified-IgG fractions both revealed higher binding percentages of 125I-T3 analog than the control IgG fraction and non-IgG fraction of the patient. Furthermore, a good dose response was observed between the binding percentage of 125I-T3 analog and each dose of the patient's serum or IgG fraction. From these observations, it was clarified that this woman had anti-T3 IgG autoantibodies using a Protein A column chromatography with confirmation of gel filtration chromatography.

Autoantibodies↗

PubMed1985

Analysis of urinary aldosterone metabolites in the rabbit by gas chromatography-mass spectrometry.

After a large amount of aldosterone was injected into a male rabbit, urine was collected for 48 h. Separation of urinary aldosterone metabolites into monoglucosiduronate fraction and monosulphate fraction was carried out by a combination of countercurrent distribution and DEAE-Sephadex A-25 column chromatography. Each fraction was hydrolyzed with enzyme and free steroids released were separated by Sephadex LH-20 column chromatography. The free steroid was then identified by gas chromatography-mass spectrometry. In monoglucosiduronate fraction, 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were found. On the other hand, 3 alpha, 5 beta-tetrahydroaldosterone was the only aglycone detected in monosulphate fraction. These findings comfirmed results in the preceding paper, where the free steroid was characterized on the basis of the mobility of the steroid and its derivatives on paper chromatography.

Aldosterone↗

PubMed1988

Cathepsin D from human leukocytes. Purification by affinity chromatography and properties of the enzyme.

Cathepsin D of human leukocytes was isolated and characterized. Purified leukocytes were lysed under nitrogen pressure and the proteinase activity precipitated by centrifugation at 48,000 x g. The precipitate was extracted by various buffers. The yield of cathepsin D was almost pH-independent but could be increased by Triton X-100. Employing gel chromatography the activity was found at a molecular mass close to 42,000 Da. Purification of the enzyme was performed by a two-step procedure using pepstatin-Sepharose chromatography and ion exchange chromatography. Three multiple forms of the enzyme were separated by ion exchange chromatography. The isoelectric points of the three forms of the enzyme were close to pH 5.0. The enzyme showed the typical characteristics of the acid proteinase cathepsin D. Enzyme activity was influenced by heavy metals such as Hg2 and Fe3 as well as by typical inhibitors for carboxyl-proteinases such as diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(4-nitrophenoxy)propane and 4-bromo-phenacylbromide. An immunological comparison with cathepsin D from human liver by immunodiffusion and immunoelectrophoresis indicates identity of the two enzymes.

Cathepsin D↗

PubMed1982

The measurement of glycosylated hemoglobin in man and animals by aminophenylboronic acid affinity chromatography.

Glycosylated hemoglobin (GHb) was estimated in normal and diabetic human, rat, and dog hemolysate by m-aminophenylboronic acid (PBA) affinity chromatography and the results compared with the values determined using two ion-exchange (ION-E) methods and a colorimetric thiobarbituric acid (TBA) method. There was a good correlation between the values estimated by PBA and both ION-E chromatography methods for the human samples (r = 0.83, P less than 0.0002, r = 0.86, P less than 0.002). In diabetic rat and dog hemolysates, PBA chromatography demonstrated higher glycosylated hemoglobin than in normal hemolysate. In both species, there was an excellent correlation between the PBA-estimated GHb and plasma glucose levels (rat r = 0.79, P less than 0.001; dog r = 0.67, P less than 0.003). The ION-E and TBA methods were not as effective in separating diabetic from normal samples and correlated less well with plasma glucose levels. PBA chromatography relies on the interaction of m-amino phenylboronate with the hydroxyl groups of the glucose residues attached to hemoglobin. It is not affected by intra- or interspecies variations in the hemoglobin moiety and should be adaptable for measurement of GHb in a number of laboratory animals and in patients with hemoglobinopathy. It is not affected significantly by temperature and may offer advantages over the ION-E method in the routine determination of human glycosylated hemoglobin.

Animals↗

PubMed1985

A widely applicable analytical system for biological stains: reverse-phase thin layer chromatography.

The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. Rf values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.

Chemical Phenomena↗

PubMed1999

Quantitative analysis of pharmacokinetic study samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).

The basis of all pharmacokinetic evaluations are powerful assays to quantify drugs and/or metabolites in biological matrices using modern sensitive instrumental analytical techniques, such as capillary gas chromatography and high-performance liquid chromatography (HPLC). Being both specific and universal, mass spectrometry (MS) is an ideal chromatographic detector. Due to recent exciting achievements in the interfacing of liquid chromatography (LC) and MS, LC-MS, like the successfully preceding hyphenated technique gas chromatography-mass spectrometry (GC-MS), has now become a valuable technique in the analyst's toolbox. The key features of LC-MS are explained and four examples demonstrating its potential for highly specific and sensitive routine drug assays with the option of high sample throughput in pharmacokinetic investigations are presented.

Animals↗

PubMed2000

Temperature-modulated high-performance liquid chromatography for detecting variation in human genome.

Recently, temperature modulated heteroduplex analysis (TMHA) based upon ion-pair reversed-phase high-performance liquid chromatography has been developed as a tool for polymorphisms and novel mutation detection in human genome. This new approach, with increased accuracy and throughput over traditional methods. This new approach is also known as denaturing high performance liquid chromatography(DHPLC). However, the terms of both TMHA and DHPLC should be considered carefully. Firstly, it is DNA molecular that is denatured in high performance liquid chromatography but not the method or the instrument. Secondly, the temperature in the liquid chromatograph is modulated according to Tm (melting temperature) of the DNA fragments. As it is known, Tm is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands, and under this temperature, half of the DNA molecules is still not denaturing. Thirdly, the targets for detection are both the homoduplexes and the mismatched heteroduplexes, which are double-stranded nucleic acid molecules and are annealing. The term of DHPLC may lead to confusion in annealing and denaturing. In addition, since TMHA can be carried out using gel electrophoresis and HPLC, there is a need to distinguish HPLC from gel electrophoresis. Therefore, we suggest a new term for this technology, which is TmHPLC (temperature-modulated high- performance liquid chromatography). This term not only provides a clear definition for this new method, but also includes both TMHA and HPLC. More importantly, TmHPLC implies that the temperature in HPLC is modulated according to Tm of the DNA fragments. We also review advances in detecting variation through TmHPLC. This new approach leads to significant reduction of sequencing efforts for searching novel mutations and polymorphisms, and because of its high sample throughput, to faster data generation. This implies that TmHPLC is a sensitive, accurate, and cost effective approach to screening sequence variation in human genome.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

The role of planar chromatography in medicinal plant research.

This paper summarizes the role of planar chromatography (PC) in medicinal and aromatic plant (MAP) research and development, and demonstrates the importance of the technique, after extraction, in the analysis of MAP (identification and quantitative determination of the separated compound/s), in the purification and isolation process, and in different types of screening procedure. Special attention is paid to analytical, micropreparative and preparative forced-flow techniques, for example overpressured-layer chromatography (OPLC) and rotation planar chromatography (RPC). The special features of analytical, micropreparative, and preparative layer chromatography (PLC), OPLC, and RPC are compared in tables. Purification and isolation procedures using forced-flow techniques are shown in flowcharts. Some applications, relating to different classes of substance, are presented to demonstrate the versatility of various planar chromatographic techniques.

Chromatography↗

PubMed2002

[Plate theory model under non-ideal chromatography].

The mobile phase in chromatography is regarded as to be composed of many continual plates, whose height is the same as that of one plate in the stationary phase. The solute in initial concentration of mobile phase is seen to be concentrated in the first plate. The solute is exchanged dynamically between the mobile phase and stationary phase. When the mobile phase passes the distance of one plate in the stationary phase, the solute cannot be immediately released from the stationary phase to mobile phase, because of the dynamic factors of chromatographic system. The solute distribution between mobile and stationary phases is controlled by two factors, i.e. the equilibrium coefficient P, which represents chromatographic thermodynamic properties, and release probability factor alpha, which represents chromatographic dynamic properties. This process is considered as non-ideal chromatography. In the view of statistics, authors studied the influences integrated of the release property factor and equilibrium coefficient factor upon chromatographic distribution, and simulated the chromatographic distribution curve by means of computer. As a result, a new distribution model has been established, the distribution equation under the non-ideal chromatography was deduced and, the integrative action of the release probability and the equilibrium coefficient was revealed. The release probability factor can be regarded as an index in chromatography and the peak width can be deduced and hence the resolution can be calculated through this index.

Chromatography↗

PubMed2000

[Purification of follicle-stimulating hormone by radial-flow chromatography].

By comparing the purification results of radial-flow chromatography with conventional chromatography, a new purification process of follicle-stimulating hormone (FSH) from crude sample of human menopausal gonadotrophin has been developed. The results showed that the specific activity, activity recovery, and purification factor of FSH purified by radial-flow chromatography were higher than those by conventional chromatography in large scale. The specific activity of FSH purified was 180 IU/mg protein, activity recovery 56%, purification factor 150. The purified product meets the quality requirement of the Standard of Chinese Pharmacopoeia.

Chromatography, Ion Exchange↗

PubMed2002

[Studies on the retention mechanism of N-methylaniline bonded silica for high performance liquid chromatography].

Three N-methylaniline bonded stationary phases containing different bonding amounts were prepared by modifying silica gel with N-methylanilinetriethoxysilane. Influences of the bonding amounts on the retentions and selectivity of polycyclic aromatic hydrocarbons(PAHs), polar-, acid- and basic-substituted aromatic compounds were investigated in reversed- and normal-phase high performance liquid chromatography. In the reversed-phase liquid chromatography, the plots of logk' against the carbon atom numbers for alkylbenzenes are linear, and the retentions and relative retentions of the solutes(including alkylbenzenes, PAHs and nitrobenzenes) increased with the increase of the bonding amounts and water volume fractions in mobile phase. The results indicated that the hydrophobic interaction played a major role in the retention process. In the normal-phase liquid chromatography, the retentions of PAHs increased and the retentions of nitrobenzenes decreased with the increase of bonding amounts. With the increase of bonding amounts, the retentions of acidic compounds increased and the retentions of basic solutes decreased considerably. It is indicated that the number of imino groups increased and the activity of residual silanols diminished with the increase of bonding amounts. The retentions of solutes on the stationary phases result from acid-basic, dipole-dipole, pi-pi and charge transfer interactions in the normal phase chromatography.

Aniline Compounds↗

PubMed2001

[Simultaneous purification and renaturation of recombinant human interferon-alpha expreessed by E. coli by high-performance hydrophobic interaction chromatography].

One-step simultaneous purification and renaturation of interferon-alpha from the inclusion body of E. coli expreessed by high-performance hydrophobic interaction chromatography(HPHIC) are presented. The inclusion body was dissolved by 8 mol/L guanidine hydrochloride. The sample lysis was centrifuged and the solution was renaturated by three different methods, i.e. hydrophobic interaction chromatography, dilution and dialysis. The total activity yield renaturation by hydrophobic interaction chromatography was 2 times and 2.6 times as much as that by dilution and by dialysis. The purity of the target product was 95% according to high-performance size exclusion chromatography and the specific activity was 1.8 x 10(8) IU/mg. The method developed is simpler and more efficient than others.

Chromatography, High Pressure Liquid↗

PubMed1992

Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.

Biotechnology↗

PubMed2004

The role of liquid chromatography in proteomics.

Proteomics represents a significant challenge to separation scientists because of the diversity and complexity of proteins and peptides present in biological systems. Mass spectrometry as the central enabling technology in proteomics allows detection and identification of thousands of proteins and peptides in a single experiment. Liquid chromatography is recognized as an indispensable tool in proteomics research since it provides high-speed, high-resolution and high-sensitivity separation of macromolecules. In addition, the unique features of chromatography enable the detection of low-abundance species such as post-translationally modified proteins. Components such as phosphorylated proteins are often present in complex mixtures at vanishingly small concentrations. New chromatographic methods are needed to solve these analytical challenges, which are clearly formidable, but not insurmountable. This review covers recent advances in liquid chromatography, as it has impacted the area of proteomics. The future prospects for emerging chromatographic technologies such as monolithic capillary columns, high temperature chromatography and capillary electrochromatography are discussed.

Chromatography, Liquid↗

PubMed2004

[Analyses of gasoline range hydrocarbons by gas chromatography].

Analyses of gasoline range hydrocarbons by gas chromatography are reviewed with 66 references. Methods of high resolution capillary gas chromatography, multidimensional gas chromatography, and gas chromatography with atomic emission detection after bromination are summarized respectively. The advantages and limitations of each method are discussed and solutions for some existing problems are proposed.

Chromatography, Gas↗

PubMed2004

[Purification and relative molecular mass determination of tea glycoprotein by high performance liquid chromatography-electrospray ionisation mass spectrometry].

Tea glycoprotein (TGC) was extracted from coarse tea leaves produced in Jiangxi Province. The separation and purification were carried out by using gel chromatography and high performance liquid chromatography. Furthermore, relative molecular mass (M(r)) of TGC was determined by gel chromatography and high performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS), respectively. It is 51 500.

Chromatography, High Pressure Liquid↗

PubMed2004

[Studies on chromatography fingerprint of huangqi by HPLC].

OBJECTIVE: To study on chromatography fingerprint(CFP) of Huangqi herbs. METHOD: HPLC was used on a Hypersil ODS column with water-acetonitriles gradient elution. CFP was referenced to formononetin, calycosin and then the contents of astragaloside IV, formononetin and calycosin was determinted. RESULT: 26 common peaks were pointed out, their similarity of 12 samples were more than 90%. CONCLUSION: The handling of samples were simple. The study method on chromatography fingerprint of Huangqi were reliable. Good repetition and their similarity accorded with the demand on "the technology guide to experiment studies on chromatography fingerprint of injection of traditional Chinese medicine (TCM)", which was issued by the nation pharmacopoeial commission. The chromatography fingerprint and determination of index composition could be used to comprehensively control the quality of Huangqi.

Astragalus propinquus↗