共查询到20条相似文献,搜索用时 8 毫秒
1.
A high-performance liquid chromatographic system for the separation of nonradiolabeled phosphoamino acids and orthophosphate by ion-pair reverse-phase chromatography has been developed. By the use of low-ionic-strength phthalate buffers at pH 6.3, the phosphoamino acids can be visualized by virtue of this uv-active eluant. The technique is sensitive to 200 pmol of phosphoamino acid and has been shown to be directly applicable to the analysis of isolated phosphoproteins. 相似文献
2.
Amino acids were derivatized with orthophthalaldehyde and separated by high-performance liquid chromatography on a polymer-based reverse-phase column (Hamilton PRP-1) at pH 7.2 using isocratic elution with 14.3 mM sodium phosphate, 1.1% tetrahydrofuran, 6.6% acetonitrile. Phosphorylated amino acids were eluted with baseline resolution in the following order: 1-phosphohistidine, phosphoserine, 3-phosphohistidine, phosphotyrosine, phosphothreonine, and phosphoarginine. Each of the phosphoamino acids was separated from its parent amino acid but aspartate and glutamate eluted in the same region as the phosphoamino acids. The sensitivity is in the picomole range and the separation time, injection to injection, is 15 min. The linearity for phosphothreonine extends at least from 30 pmol to 30 nmol. Quantitation by radioactivity is good for each of the phosphoamino acids except in the case of [1-32P]phosphohistidine, which coelutes with inorganic phosphate. 相似文献
3.
Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins. 相似文献
4.
5.
6.
A high-performance liquid chromatography system has been developed which resolves O-phosphoserine, O-phosphothreonine, and O4-phosphotyrosine. Separation is performed on an anion-exchange resin eluted with phosphate buffer of low pH. Detection of the amino acid derivatives is accomplished using O-phthalaldehyde in an in-stream fluorometric system. This highly sensitive method has been applied to the detection of phosphohydroxyamino acids in bovine myelin basic protein phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase and in whole bovine myelin phosphorylated by endogenous kinases. 相似文献
7.
A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs. 相似文献
8.
Imre Molnár Csaba Horváth 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1977,143(4):391-400
Over a hundred acidic urinary constituents were separated within 30 min by using 5-μm octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work. 相似文献
9.
All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH2-terminal analysis of lysozyme is described. 相似文献
10.
Mannes AJ Brown DC Keller J Cordes L Eckenhoff RG Caudle RM Iadarola MJ Meng QC 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,823(2):184-188
A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters. 相似文献
11.
Mannes AJ Cimino Brown D Perkowski SZ Keller J Caudle RM Iadarola MJ Meng QC 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,780(2):475-479
A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C(18) column and acetonitrile in 0.02 M NaH(2)PO(4) as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects. 相似文献
12.
Branched-chain ketoacids were isolated from plasma or serum samples by acidification, passage through a cationic exchange resin, ether extraction, and extraction of the ether layer with phosphate buffer. The recovery of 2-[1-14C]ketoisocaproate taken through these procedures averaged 95 +/- 3%. Branched-chain ketoacids were measured by high-performance liquid chromatography using a single mobile phase (sodium phosphate:acetonitrile). In normal human subjects, mean +/- SD fasting levels of 2-ketoisocaproate, 2-keto-3-methylvalerate, and 2-ketoisovalerate were 29 +/- 8, 18 +/- 4, and 12 +/- 3 microM, respectively. In normal rats, slightly different results were found: 24 +/- 10, 19 +/- 7 and 17 +/- 6 microM, respectively. In both species, levels of each ketoacid expressed as fractions of total branched-chain ketoacids were much less variable. 相似文献
13.
Previous studies suggest that plasma acetate may be an important fuel in man, accounting for approximately 10% of energy expenditure. Available methods for the determination of plasma acetate kinetics are difficult and time consuming. We describe here a procedure for the determination of plasma acetate concentration and specific activity using automated high-performance liquid chromatography that is precise and sensitive and accommodates large numbers of samples. The procedure involves extraction from plasma with diethyl ether, derivatization with bromoacetophenone, and separation on a C-18 reversed-phase column. The specific activities of D-beta-hydroxybutyrate and lactate can also be determined. Acetate turnover was measured in four dogs and was similar to that previously reported in sheep and humans. Transport of [14C]acetate into red blood cells was negligible. 相似文献
14.
15.
16.
James E. Evans Howard Tieckelmann Edwin W. Naylor Robert Guthrie 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1979,163(1):29-36
A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 103 concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed. 相似文献
17.
Ramsahoye BH 《Methods (San Diego, Calif.)》2002,26(2):156-161
Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA. 相似文献
18.
A method for the rapid and sensitive fluorometric analysis of porphyrin carboxylic acids by reverse-phase high-performance liquid chromatography is described. Separation of free porphyrin carboxylic acids was carried out with a microparticulate octadecylsilane column with elution by a gradient of methanol in phosphate buffer containing tetrabutylammonium hydroxide. Separation and quantitation of di-, tri-, tetra-, penta-, hexa-, hepta-, and octacar-boxylic porphyrins was achieved within 25 min at picomolar concentrations. The method is also capable of separating the type I and type III isomers of tetracarboxylic through hexacarboxylic porphyrins. By using a stopped flow technique, one can record fluorescence excitation and emission spectra of porphyrin carboxylic acids. This method is directly applicable to biological fluids such as urine, plasma, red cell lysates, or medium or extracts from cell culture. 相似文献
19.
20.
Atsuko Metori Akiri Ogamo Yasuhito Nakagawa 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1993,622(2):147-151
A high-performance liquid chromatographic (HPLC) procedure for the separation of hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecanoic acids (HODEs) after derivatization of the hydroxy group with 1-anthroylnitrile is described. Anthroyl esters of HETEs were separated from those of HODEs by reversed-phase HPLC. The positional isomers of the HETEs and HODEs were well separated by normal-phase HPLC. The fluorimetric HPLC method has a high sensitivity and naturally occurring HETEs can be quantitatively analyzed at the picomolar level. The amount of 5-HETE in A23187-stimulated polymorphonuclear leukocytes (PMNLs) was determined by the present method. PMNLs produced approximately 150 ng of 5-HETE per 107 cells at 5 min stimulation. The amount of 5-HETE determined by fluorimetric detection was consistent with that determined by ultraviolet detection (235 nm). 相似文献