Purification of 125I-labeled succinyl cyclic nucleotide tyrosine methyl esters by high-performance liquid chromatography

Carrier free 125I-labeled succinyl cyclic adenosine monophosphate (ScAMP) and succinyl cyclic guanosine monophosphate (ScGMP) tyrosine methyl esters (TME) were purified by reversed phase high-performance liquid chromatography (HPLC) or descending paper chromatography. Using an isocratic buffer for HPLC, mono-ScAMP-125I-TME and mono-ScGMP-125I-TME were eluted from a C18 column at 8.9 and 6.9 min, respectively. Both of the mono-iodinated radioligands were completely separated from their noniodinated precursors and other iodinated products. The radioligands purified by HPLC or paper chromatography were used for the radioimmunoassay (RIA) of cAMP and cGMP. Cyclic AMP or cGMP inhibited binding of the HPLC purified radioligands at three- to fivefold lower concentrations than the paper chromatography purified radioligands. The sensitivity of the RIA decreased with time if paper chromatography purified radioligands were used, but remained stable for 4 months if the HPLC purified compounds were used, even with storage at 4 degrees C. We attribute these results to better purification of radioligands by the HPLC than by the paper chromatography. Using optimal conditions the HPLC method takes only 10 min and results in a high yield (greater than 95%) of added 125I into the monoiodinated products.     

Purification and analysis of a 5kDa component of enamel matrix derivative

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.     

Comparison of reversed-phase and ion-pair chromatography for the determination of strychnine in animal tissues

Ion-pair and reversed-phase high-performance liquid chromatography (HPLC) were evaluated for quantification of strychnine in mountain beaver tissues. Retention time shifts hindered strychnine quantification with both HPLC systems. Co-extracted free fatty acids released during storage formed ion-pairs with strychnine, resulting in increased retention by reversed-phase HPLC. Competition with co-extracted basic compounds is likely responsible for the decreased retention of strychnine by ion-pair HPLC. Following an acid-base clean-up, optimal results were obtained with reversed-phase HPLC. Ion-pair chromatography was then used for qualitative confirmation of strychnine residues.     

Steroid metabolism in teleost gonads: purification and identification of metabolites by high-performance liquid chromatography.

A simple, efficient, and comprehensive technique for the purification, identification, and quantitation of the common steroid metabolites synthesized by the gonads of teleosts involving five systems of high-performance liquid chromatography (HPLC) was developed. Steroid standards were identified in HPLC by UV absorption at 254 nm or 280 nm, by differential refractive index, or by using radioactive standards. Metabolites that do not absorb UV light and are not resolved in the isocratic HPLC systems were identified in thin-layer chromatography following purification by HPLC. By using this technique, most of the steroid metabolites, including some polar metabolites, synthesized by the gonadal tissues of the teleosts can be purified within three steps of chromatography. The HPLC systems reported here are also useful in identifying the chromium trioxide oxidized products of metabolites, such as triols and tetrols, which considerably narrows down the number of probable metabolites.     

银耳多糖单糖组成分析的三种色谱方法比较

为选择一种准确快捷的方法测定银耳多糖的单糖组成,对薄层色谱法(TLC)、气相色谱法(GC)、高效液相色谱法(HPLC)三种色谱方法进行比较。结果表明,前两种方法的测定结果均不理想,而HPLC法,操作简便,灵敏度高,分离效果好,信息完整。测定结果为由葡萄糖、甘露糖、葡萄糖醛酸、木糖、岩藻糖组成,其摩尔比为0.24∶1.00∶0.06∶0.29∶0.25。HPLC法对酸性杂多糖组成糖分析是一种比较理想的选择。     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

Determination of aldosterone secretion rate utilizing mixed mode and high performance liquid chromatography

A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates.     

Purification of Antibiotics from Physarum Gyrosum by High Pressure Liquid Chromatography

A family of five antibiotic substances was isolated from the slime mold Physarum gyrosurn by high pressure liquid chromatography (HPLC). For this purpose, mold was cultured for two weeks in a liquid medium. Soluble products were harvested by rotary evaporation of medium and extraction with 1-butanol. Paper chromatography in ethyl acetate :pyridine:water (2:2:1 v/v) was used for preliminary fractionation. Active components were separated by HPLC with a reverse-phase column packed with Bondapack C₁₈/Porasil B (Waters Associates) and were eluted with a linear gradient of methanol:water increasing from 70 to 100% methanol over 90 minutes. Puri-fication was completed by rechromatographing individual fractions. Purity of the active components was verified by HPLC and thin layer chromatography. Activity assays against Bacillus cereus showed these materials to be bacteriostatic rather than bacteriocidal.     

Approaches to a markedly increased sensitivity of the radioimmunoassay for thyrotropin-releasing hormone by derivatization

Antisera to thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) have previously been produced in rabbits by immunization with a conjugate having TRH linked to a carrier protein by means of dinitrophenylene (Dnp) moiety. Studies on the specificity of the antisera obtained suggested that the sensitivity of the radioimmunoassay for TRH may be increased substantially by prior conversion of the hormone in to dinitrophenylene derivatives. To test this possibility, several TRH-Dno derivatives were prepared by reaction of TRH with equimolar amounts of 1,5-difluoro-2,4-dinitrobenzene yielding Nim-(5-fluoro-2,4-dinitrophenyl)TRH. This intermediate was reacted with ammonia, histamine, tyramine or N alpha-acetyl-lysine methyl ester (N alpha Ac-Lys-OMe) to yield the respective unsubstituted and N-substituted Nim-(5-amino-2,4-dinitrophenyl)TRH derivatives: TRH-Dnp-NH2, TRH-Dnp-histamine, TRH-Dnp-tyramine and TRH-Dnp-N alpha Ac-Lys-OMe. Nim-(2,4-Dinitrophenyl)TRH was prepared similarly by reaction of TRH with 1-fluoro-2,4-dinitrobenzene. The products were isolated by means of high-performance liquid chromatography (HPLC) and were found to be pure by HPLC and thin-layer chromatography using several solvent systems. TRH-Dnp-histamine and TRH-Dnp-tyramine were labelled with 125I using the chloramine-T method. The labelled products were purified to homogeneity by ion-exchange chromatography on SP-Sephadex and adsorption chromatography on Sephadex LH-20, respectively, and were found by HPLC to be pure.     

Analysis of Poly-beta-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-beta-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.     

Analysis of oligosaccharides by on-line high-performance liquid chromatography and ion-spray mass spectrometry.

Oligosaccharides were analyzed by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). First, oligosaccharides labeled with 2-aminopyridine were studied to see if they could be analyzed by MS under the conditions used for separation by HPLC. Pyridylamino (PA)-oligosaccharides could be analyzed under these conditions, although the mass spectra were affected. Then, liquid chromatography-mass spectrometry was used to analyze a PA-oligosaccharide mixture derived from human immunoglobulin G. The PA-oligosaccharides were separated on a reversed-phase column and mass-analyzed directly. The observed molecular weights were close to or identical to those expected from the structures, which were estimated from the elution position on HPLC. This method is rapid and simple, as the mass spectrometer can give the accurate molecular weight of each PA-oligosaccharide in one chromatography run, even if the HPLC separation is incomplete. This method can be used to extend the so-called two-dimensional mapping of PA-oligosaccharides. The structure can be studied in greater detail by tandem MS.     

Analysis methods of ginsenosides

Ginsenosides are considered the main active principles of the famous Chinese traditional medicine "ginseng". For more than 30 years many researchers developed methods for the identification and quantification of ginsenosides in ginseng plant material, extracts and products. Separation of ginsenosides has been achieved using thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). Among these techniques HPLC is by far the most employed. Ultraviolet (UV), evaporative light scattering (ELSD), fluorescence and, recently, mass spectrometry (MS) were coupled with HPLC for the detection of ginsenosides. The most recent methods are here discussed together with a critical evaluation of the published results. Furthermore new techniques such as near infrared spectroscopy (NIRS) and enzyme immunosassay (EIA) recently used for the determination of ginsenosides will be discussed.     

The presence of free D-alanine, D-proline and D-serine in mice.

The presence of free D-alanine, D-proline and D-serine was demonstrated in mammalian tissues, using a mutant mouse strain lacking D-amino acid oxidase. In the experiment, free amino acids from the kidney and serum were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) to diastereomers, separated by two-dimensional thin-layer chromatography (TLC), and analysed by reversed-phase high-performance liquid chromatography (HPLC) for the resolution of D- and L-isomers. D/L ratios of alanine, proline and serine were obtained based on the peak areas of HPLC.     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon alpha2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this way native conditions during SEC and structural changes of the protein during RP-HPLC were demonstrated. These results were confirmed by stand-alone fluorescence and CD measurements. With respect to HPLC tandem detection, the fluorescence detector compared favourably to the UV and CD detector regarding linearity, sensitivity and selectivity. SEC combined with intrinsic fluorescence scanning detection permits conformational analysis of small amounts of aggregates in the presence of excess native monomeric protein. In conclusion, HPLC with on-line UV and intrinsic fluorescence detection provides a promising concept for analysing the amount and conformational properties of a biopharmaceutical and its impurities.     

High-performance liquid chromatography and radioimmunoassay of rat plasma corticosterone

Problems inherent in corticosterone radioimmunoassay (RIA) led to consideration of alternative methods. A high-performance liquid chromatography (HPLC) procedure was evaluated that separated and quantitated dichloromethane-extracted corticosterone by reverse-phase chromatography. The results were correlated (r = 0.92) with an RIA procedure. The HPLC recovered nearly 100% of corticosterone added to rat plasma and had excellent reproducibility. In addition, chromatogram profiles of dichloromethane-soluble components obtained from rat plasma, derived from drug effect studies, could have value for characterizing response patterns. Without automated sample injection equipment, HPLC is more appropriately applied in monitoring RIA results than in processing large numbers of samples.     

Isolation of insulin-like growth Factors I and II from human plasma

Insulin-like growth factors I and II have been isolated from Cohn fraction IV-1 of human plasma using gel permeation chromatography, ion exchange chromatography, reversed phase chromatography, isoelectric focusing (IEF) and high performance liquid chromatography(HPLC). IGF I of specific activity 89 U/g, as measured by the isolated rat adipocyte assay, and IGF II, of specific activity 78 U/g, were obtained in yields of 16 micrograms and 34 micrograms respectively per 100g of Cohn fraction. Although this process yields IGF I which is contaminated with IGF II (due to the relatively large amount of the latter present in the original plasma), the IGF II preparations appear to be relatively free from IGF I. This separation was mainly achieved with IEF since the two factors elute close together on HPLC. Nevertheless, HPLC is important for their subsequent purification. The process is thus especially suitable for the preparation of IGF II and appears to give better yields than those obtained by earlier methods which used acid-ethanol extraction, gel permeation chromatography and polyacrylamide gel electrophoresis.     

Purification and Characterization of a Glycine-rich Protein from the Aleurone Layer of Soybean Seeds

Glycine-rich protein (GRP) was extracted with hot water from cell walls of the aleurone layer of soybean seeds and solubilized by pectinase treatment. GRP was purified by Sephadex G-100 gel chromatography, anion exchange HPLC, and reverse-phase HPLC. Two GRP fractions that had almost the same amino acid composition were found by gel chromatography. The high-molecular-mass G RP seems to be an associated form of the low-molecular-mass GRP (30-kDa). Thirty-kDa GRP was separated into a major GRP-I and a minor GRP-II by anion exchange HPLC. The major amino acids of GRP-I purified by reverse-phase HPLC were glycine (68%) and serine (12%). GRP-I contained a small proportion of sugar, approximately 9% (w/w), and mannose, arabinose, glucose, xylose, and galactose were included in the sugar moiety. The N-terminal amino acid sequence of GRP-I was a novel polyglycine structure including at least 20 glycine-repeated sequence. The GRP-I might be a novel type of extracellular matrix protein specific to the aleurone layer.     

Modification of the C-terminal octapeptide of cholecystokinin with a high-specific-activity iodinated imidoester: preparation, characterization, and binding to isolated pancreatic acinar cells

Proteoglycans were separated by high-performance liquid chromatography (HPLC), using two coupled Aquapore columns containing glycerylpropylsilane groups covalently linked to large-pore (50–100 nm) silica spheres. This two-column HPLC system was effective in separating cartilage proteoglycan aggregates and monomers, without altering their biochemical integrity. This system was also effective in resolving small amounts of isotopically labeled proteoglycans synthesized by cultured mammalian cells. The small sample size, short analysis time, and high reproducibility represent improvements in the study of proteoglycans over conventional soft-gel chromatography.     

Purification and biochemical characteristics of two distinct human interleukins 1 from the myelomonocytic THP-1 cell line

An effective induction protocol for the production of interleukin 1 (IL 1) by human myelomonocytic cell line (THP-1) cells was developed, and two biochemically distinct human IL 1 peptides were purified. Lipopolysaccharide, silica, and hydroxyurea by themselves did not induce IL 1 production, but these three stimulants in combination had a synergistic effect on the production of IL 1 by THP-1 cells. A 17-kilodalton (kDa) form of human IL 1 with a pI of 7.0 (IL 1-beta) was purified to homogeneity by sequential chromatography on DEAE-Sephacel, Sephacryl S-200, CM high-performance liquid chromatography (HPLC), and hydroxyapatite HPLC. The recovery of IL 1-beta activity was 45%, and the specific activity was 2.3 X 10(7) units/mg. Both IL 1-beta and a second 17-kDa IL 1 moiety with a pI of 5.0 (IL 1-alpha) were also extracted from stimulated THP-1 cells and purified to homogeneity by sequential chromatography on Sephacryl S-200, ion exchange HPLC, and hydroxyapatite HPLC. The recovery of IL 1-beta from cell extracts was 5.6%, and the specific activity was 3 X 10(7) units/mg. In contrast, only 0.85% of IL 1-alpha was recovered with a specific activity of 5.3 X 10(7) units/mg.(ABSTRACT TRUNCATED AT 250 WORDS)