Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.     

The extraction of imperialine and imperialine-3 beta-glucoside from Fritillaria pallidiflora Schrenk and quantitative determination by HPLC-evaporative light scattering detection

The extraction procedure and quantitative determination by HPLC-evaporative light scattering detection (ELSD) of the main bioactive components, namely, imperialine (1) and imperialine-3 beta-glucoside (2), of bulbs of Fritillaria pallidiflora Schrenk have been investigated. The most efficient method for the simultaneous extraction of 1 and 2 involved pre-treatment of the bulb powder with ammonia, followed by reflux with dichloromethane:methanol at 90 degrees C for 4 h. Simultaneous determination of non-chromophoric 1 and 2 by HPLC-ELSD employed a Kromasil C18 column eluted with acetonitrile:water:diethylamine. The assay was accurate and reproducible with an overall variation lower than 4% and a sample recovery higher than 98%. The methods described have been successfully used to evaluate the quality of three batches of the crude traditional Chinese medicinal herb derived from the bulbs of F. pallidiflora.     

Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity

An unconventional use for the polarization optics, associated with a variety of commercially available fluorescence microplate readers, is reported. This novel application has allowed the discrimination of green fluorescent protein (GFP) fluorescence in genetically modified yeast cells from interfering autofluorescent species. The method exploits the unusually high fluorescence anisotropy of GFP compared to smaller fluorophores and autofluorescent species. The principle was successfully applied to resolve the induced GFP signal from that of autofluorescent test compounds, in an assay for genotoxic species. The use of fluorescence polarization enabled both proflavin and methapyrilene to be identified as genotoxic agents in the yeast assay. This would not have been possible using conventional fluorescence alone since these compounds were found to be intensely autofluorescent at the same wavelength as GFP and thus effectively mask the GFP signal.     

Determination of urinary 4-hydroxy-3-methoxyphenylethylene glycol in man by high performance liquid chromatography with electrochemical detection

A reverse phase high performance liquid chromatographic method for determining levels of 4-hydroxy-3-methoxyphenylethylene glycol (MHPG) in human urine that is virtually free of all interfering peaks has been developed. After addition of a homologous internal standard, enzymatic hydrolysis is performed. Samples are then placed onto columns containing AG1-X8, and the MHPG is collected in a phosphate buffer wash of the column. After ethyl acetate extraction and evaporation of the organic solvent, the dry residue is redissolved in mobile phase, and injected onto a reverse phase column. Results obtained with this assay were almost identical (101±5.6%, mean±SD, n=6) with those obtained using a gas chromatography - mass spectrometry (GCMS) assay.     

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min^-1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.     

HPLC-ELSD法测定发酵液中L-鸟氨酸的含量

采用HPLC-ELSD法,Prevail C18柱,体积分数0.06%HFBA溶液-乙腈(88∶12)为流动相,柱温30℃,流速为0.8 ml.min-1,ELSD漂移管温度110℃,气体流量2.8 L.min-1,建立了测定L-鸟氨酸的方法。鸟氨酸在0.1-0.6 g.L-1范围内,线性回归比非常显著(r2=0.9998),平均回收率98.94%。该法简便、快速、准确性高、重复性好,适合于L-鸟氨酸的定量分析。     

Isolation of N,N-dialkylated derivatives of valproylglycinamide from dog plasma by active charcoal adsorption and their quantification by high-performance liquid chromatography

A selective assay for quantification of N,N-dimethylvalproylglycinamide (DM-VGD) and N,N-diethylvalproylglycinamide (DE-VGD) in dog plasma utilizing reversed-phase high-performance liquid chromatography and UV detection has been developed. These compounds are derivatives of the potential anticonvulsant drug, valproylglycinamide, which is currently undergoing clinical trials. The method is based on extraction of dog plasma with activated charcoal, separation of the charcoal pallet and extracting it with methanol, evaporation of the solvent and injecting the reconstituted residue onto the column. The active charcoal adsorption method is reliable and reproducible, and it provides a chromatogram free of interfering endogenous plasma compounds. The assay was validated and provided a limit of quantification of 2.3 mmol/l for DE-VGD and 5.3 mmol/l for DM-VGD. Mean recovery of these compounds from plasma averages 75%. This analytical method is suitable for the quantitative determination of DM-VGD and DE-VGD in plasma and it has been applied to a pharmacokinetic study of these compounds in a dog.     

5-Hydroxytryptophan: A method for monitoring human plasma content during clinical administration

A method is described for the assay of 5-hydroxytryptophan (5-HTP) in human blood plasma following administration of the compound. The procedure involves extracting the amino acid into butanol, returning the 5-HTP to an aqueous phase, and separating it from other interfering 5-hydroxyindoles on a column of Dowex 50W-X4, a strong cation-exchange resin. The concentration of 5-HTP in the buffer eluate is determined spectrofluorometrically in 3 N HCl; 25 ng/ml plasma can be detected. Using this method, it was possible to determine the time-course of plasma 5-HTP concentrations during the 24-h period after a single 300-mg dose. 5-HTP is being utilized in clinical trials for several psychiatric and neurological disorders.     

大花红天素分离、合成及其含量分析

研究大花红天素的分离、合成,并对大花红天素的含量进行分析测定。通过东京化成HP-20和硅胶柱色谱进行分离;以2-甲基-3-丁烯-2-醇和溴代四乙酰葡萄糖通过SN2反应合成大花红天素,合成收率为18％;通过HPLC-ELSD对大花红天素的含量进行分析,通过对8种样品的分析,测定大花红景天和红景天饮片中分别含大花红天素2．06％、0.83％,其他样品中未检出大花红天素。作者对大花红天素进行了首次合成并建立了相应的分析测定方法。     

重组水蛭素的纯化和鉴定

本文报导了化学合成的水蛭素基因在酵母细胞中得到表达,井能分泌水蛭素到胞外。将该菌株培养物的上清液经硫酸铵沉淀和Sephadex G-50过滤后,用DEAE-SephadexA-25进行阴离子交换层析,进而用HPLC反相层析,得到表达产物重组水蛭素。经SDS-PAGE,氨基酸序列分析,抗凝血酶活力分析及血浆滴定实验等方法鉴定,证明该基因表达产物与天然水蛭素HV_2相同。     

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.     

Determination of bevantolol enantiomers in human plasma by coupled achiral-chiral high performance-liquid chromatography

A coupled achiral-chiral high performance liquid chromatographic method was developed and fully validated for the determination of bevantolol enantiomers, (-)-(S)-bevantolol and (+)-(R)-bevantolol, in human plasma. Plasma samples were prepared by solid phase extraction with Sep-Pak Plus C18 cartridges followed by HPLC. Bevantolol enantiomers and (+)-(R)-Propranolol as internal standard (IS) were preseparated from interfering components in plasma on a Phenomenex silica column and bevantolol enantiomers and IS were resolved and determined on a Chiralcel OJ-H chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The Precolumn was used to concentrate bevantolol in the eluent from the achiral column before back flushing onto chiral phase. A detailed validation of the method was performed accordingly to FDA guidelines. For each enantiomer the assay was linear between 20 and 1600 ng/ml. The quantification limits of both bevantolol enantiomers were 20 ng/ml. The intraday variation was between 1.07 and 12.64% in relation to the measured concentration and the interday variation was 0.91 and 11.79%. The method has been applied to the determination of (-)-(S)- and (+)-(R)-bevantolol in plasma from healthy volunteers dosed with racemic bevantolol hydrochloride.     

酵母全细胞苯丙氨酸鲜氨酶（PAL）活性的紫外分光光度测定法

本文报告以L-苯丙氨酸 (L-phe) 为底物,酵母全细胞作酶源,酶促生成产物反式-肉桂酸 (t-Ca)测定苯丙氨解氨酸 (PAP,EC_(4.3.1.5) 活性的紫外分光光度法。测定程序包括标准物质t-Ca的加样试验,绝对回收率试验,线性回归分析的整套定量分析研宄步骤,建立了一套经过修改的Kalghatgi和Subba Rao(1975) PAL 活性测定法。此法具有良好的准确度和精密度,已经用于评价具有PAL活性的酵母菌株在液体培养物中细胞生长和PAL活性形成的时间过程研究。     

Identification and quantification of C21 steroidal saponins from Radix Cynanchi Atrati by high-performance liquid chromatography with evaporative light scattering detection and electrospray mass spectrometric detection

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-ESI/MS) and evaporative light scattering detection (HPLC-ELSD), respectively, has been performed for the simultaneous identification and quantification of six C(21) steroid saponins, including cynanversicoside A, B, D, G, glaucoside C and glaucogenin C-3-O-beta-d-thevetopyranoside in Radix Cynanchi Atrati. The extraction of the C(21) steroidal saponins was performed using a B-811 Buchi Universal Extraction System in Warm Solvent Mode, and the analyte was concentrated by column chromatography before HPLC analysis. The chromatographic separation was performed on an Agilent Zorbax Extend C(18) analytical column efficiently using gradient elution with acetonitrile and water. The method was validated with acceptable linearities (r > 0.9991) and recoveries (98.2-101.3%). The limits of detection of the C(21) steroid saponins were from 0.2 microg for glaucogenin C-3-O-beta-d-thevetopyranoside to 0.5 microg for cynanversicoside B. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. The method was successfully used to analyse 20 batches of Radix Cynanchi Atrati. The content of C(21) steroid saponins in the plant material varied significantly from habitat to habitat, confirming the necessity to control the quality of Radix Cynanchi Atrati during its preparation and application in the clinic.     

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography

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.     

Fast analysis of pravastatin in production media

High throughput methods (high performance liquid chromatography and capillary electrophoresis) were developed to determine pravastatin in production media. The analyses were performed on particle column, monolithic column and silica capillary filled with borate buffer pH 9.3 containing 20 mM SDS. All three methods successfully separate pravastatin from interfering compounds (matrix, mevastatin and 6-epi pravastatin) and runtimes are shorter than 1 min. Solvent consumptions for methods using small particle column, monolith column and MECK were 132, 510 and 1.5 mL h(-1). The most sensitive was the method using particle column (LOD was about 10(-5) mg mL(-1)), followed by the system using monolith column (LOD was 2 x 10(-4) mg mL(-1)) and the MECK method (LOD was about 0.02 mg mL(-1)).     

An assay for ribonucleotide reductase based on ion-exchange chromatography of the reaction product

A rapid and convenient assay for ribonucleotide reductase has been developed in which the reaction product, deoxycytidine diphosphate (dCDP), is isolated without further conversion. The enzymatic reaction is terminated by the addition of ethanol and the sample is chromatographed on a single, small, and disposable column of polyethylenimine cellulose. A two-step elution is conducted with buffers containing 25% ethanol. First, contaminants and byproducts such as cytidine and its monophosphate are removed at low ionic strength while the diphosphates are retained. Then dCDP is selectively eluted as a sharp peak with a strong borate buffer. Under these conditions, the excess substrate, cytidine diphosphate, remains on the column, presumably as the borate complex. The assay is linear with time for 15 min at 25 degrees C and linear with the amount of enzyme even at very low concentrations. With slight modifications, the assay seems applicable to the use of UDP or ADP as substrates. The method is not suitable for samples which contain nucleotide kinase or other interfering enzymes which convert a significant amount of dCDP into byproducts. However, another chromatographic system based on similar principles has been found which could be used to measure any dCTP produced in this way.     

虹鳟LECT2的酵母表达、纯化及生物活性分析

白细胞衍生趋化因子2 (leukocyte cell-derived chemotaxin 2,LECT2)是一个参与多种生理和病理过程的分泌型细胞因子.该文采用毕赤酵母表达体系分泌表达虹鳟LECT2,用阳离子交换柱结合分子筛层析方法分离纯化目的蛋白,并获得纯度为96％,得率为120 mg/L的重组虹鳟(Oncorhynchus mykiss)LECT2酵母培养物.生物学活性验证表明该重组蛋白能趋化虹鳟头肾来源的巨噬细胞,增强其呼吸爆发和杀菌能力,并改变其细胞因子等基因的表达.综上,该实验建立了一种快速有效的虹鳟LECT2活性重组蛋白的制备方法,为后续相关蛋白的功能研究奠定了基础.     

Assay for phosphatidylserine decarboxylase utilizing DEAE-cellulose column chromatography

A simple assay for phosphatidylserine decarboxylase is described. Following incubation of a mitochondrial fraction from Saccharomyces cerevisiae with purified, exogenous phosphatidyl[3H]serine, the lipid extract is applied to a small DEAE-cellulose column equilibrated in CHCI3-CH3OH (1:1). The unreacted substrate, phosphatidyl[3H]serine, is quantitatively bound by the ion-exchange column while the product, phosphatidyl[3H]ethanolamine, is eluted by sequential washing with CHCI3-CH3OH (1:1) and CH3OH. The organic solvents are evaporated, and the amount of radiolabeled phosphatidyl[3H]ethanolamine formed by enzymatic decarboxylation is determined by liquid scintillation spectrometry. The reliability of this assay was established by showing that several enzymatic properties of the yeast enzyme, defined by the new assay, were essentially identical to the properties characterized by a more tedious paper chromatographic assay described previously. Virtually identical rates of enzymatic decarboxylation of phosphatidyl[3H]serine were also obtained for mitochondrial fractions from pig brain and rat liver when the activities were compared by the column and paper chromatographic methods.