Separation of Monosulfated Bile Acids by High-Performance Liquid Chromatography

Abstract

Separation of 3-, 7- and 12-monosulfates of cholate, chenodeoxycholate, deoxycholate, lithocholate and ursodeoxycholate and their glyco- and tauro-conjugates by high-performance liquid chromatography on a reversed phase column has been carried out. Effects of pH and salt concentration of a mobile phase on the k' value of sulfated bile acids were investigated with the μBondapak C₁₈ and ODS SC-02 columns. The 3-sulfated bile acids were efficiently separated on ODS SC-02 using three aqueous ammonium carbonate/acetonitrile systems. The chromatographic behaviors of 7-and 12-sulfated bile acids are also briefly discussed.     

Separation and Determination of Bile Acids in Human Bile by High-Performance Liquid Chromatography

Abstract

A method for simultaneous determination of major bile acids in human bile is described. The unconjugated, glycine- and taurine-conjugated bile acids are extracted with Sep-pak C¹⁸ and separated into groups by ion-exchange chromatography on a lipophilic gel. Subsequently, resolution of each group into ursodeoxycholate, cholate, chenodeoxycholate, deoxycholate and lithocholate is attained into two stages by high-performance liquid chromatography on a Radial-PAK A column. First, 0.3% ammonium phosphate (pH 7.7)/acetonitrile (19:8, v/v) is used for separation of the latter three as a mobile phase. Ursodeoxycholate and cholate are efficiently separated in 0.3% ammonium phosphate (pH 7.7)/acetonitrile (23:8, v/v). The present method is applicable to quantitation of bile acids in human bile with satisfactory accuracy and precision.     

Evaluation of Mobile and Stationary Phases in Reversed-Phase High-Performance Liquid Chromatography of Conjugated Bile Acids

Abstract

The reversed-phase high-performance liquid chromatographic separation of the ten major conjugated bile acids of man using isocratic conditions is described. Each component of the mobile and stationary phases was examined for its ability to influence the separation selectivity. Manipulation of pH, buffer species, organic modifier and different types of packings showed that optimal resolution was obtained with a mobile phase of methanol-0.02M sodium acetate (60:30) adjusted to pH 4.2 with phosphoric acid, on a Supelcosil LC-18-DB column. Advantages of the optimized phase system are the complete baseline separation of compounds within a short period of time, improved peak symmetry and a high rate of reproducibility. This new chromatographic method, coupled with UV detection at 205 nm, is suitable for the simultaneous determination of bile acid conjugates in routine clinical analysis.     

乳化液膜清除未结合胆红素

高未结合胆红素症 (Ｈｙｐｅｒｂｉｌｉｒｕｂｉｎｅｍｉａ)是新生儿常见疾病 .胆红素在血液中的浓度过高 ,会伤害婴儿的大脑 (核黄疸 ) [1].肝昏迷患者的症状与胆红素在血液中含量过高也有直接关系 ,迅速有效地将其清除可使患者症状缓解[2 ],迄今为止 ,清除胆红素的研究主要集中在以吸附剂吸附为基础的血液灌流方法 ,所用的吸附剂主要有活性炭和大孔吸附树脂 ,活性炭的吸附选择性和血液相容性都比较差 ;大孔吸附树脂的吸附选择性和血液相容性等方面有待进一步提高[3],特别是在吸附未结合胆红素时 ,清除率低、清除速度慢 ,难以满足临床方面…     

Separation of Ramipril Optical Isomers by High-Performance Liquid Chromatography

Abstract

High-performance liquid chromatographic methods for determining the optical purities of ramipril, a novel angiotensin converting enzyme inhibitor, and its synthetic intermediates have been developed. Analytical procedures were established for the resolution of optical isomers of two moieties, precursor I and precursor II, used for the preparation of ramipril. No occurrence of racemization during condensation reaction of the two precursors could be unambiguously confirmed by the proposed methods. On the basis of analytical data ramipril bulk material proved to be optically pure.     

反相高效液相色谱法测定鼠胆汁中游离与结合型胆汁酸

建立了一种柱前衍生反相高效液相色谱荧光检测大鼠胆汁中游离与结合型胆汁酸的方法.以4-溴甲基-7-甲氧基香豆素为衍生剂,月桂酸为內标,Waters Nova C18色谱柱分离,采用甲醇/乙腈和水梯度洗脱,荧光激发波长(λex)330 nm,发射波长(λem)400 nm,內标标准曲线法定量.各胆汁酸在5-2 400μmol/L范围内线性良好,r为0.998 9-0.999 7.最低检出限为1.2-2.5μmol/L.精密度日内在1.05%-3.67%之间,日间在3.16%-6.83%之间.平均回收率为87.8%-106.7%.该方法灵敏、重复性好,分析时间短,线性范围宽,适合于科研工作中对鼠胆汁中各胆汁酸进行定量检测.     

Studies on Steroids CCXXXXIV. Separation and Characterization of C-25 Epimers of Unconjugated and Conjugated Trihydroxycholestanoic Acids in Urine from a Patient with Zell Weger Syndrome by High-Performance Liquid Chromatography

Abstract

The separation and characterization of C-25 epimers of unconjugated and glycine- and taurine-conjugated 3α, 7α, 12α - trihydroxy-5β-cholestanoic acid (THCA) in biological fluids by high-performance liquid chromatography (HPLC) are described. The 5β-cholestanoic acid fraction was obtained from a urine specimen from a patient with Zellweger syndrome by passing it through a Sep-pak C₁₈ cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile. The derivatives were separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on alipophilicgel, piperidinohydroxypropyl Sephadex LH-20. Sub-sequent resolution of each fractionin to (25s)- and (25R)-THCA was attained by HPLC on a Cosmosil 5C column. The C-25 epimers of unconjugated and conjugated Tk%A were unequivocally identified on the b asis of the irbehaviors in HPLC using mobile phases of different pHs. The ratios of the unconjugated, glycine- and taurine-conjugated (25RbTHCA to the corresponding (25S)-epimers were 16:1, 5:4 and 3:2, respectively .     

反相高效液相色谱对DABTH—氨基酸的分离与鉴定

应用反相高效液相色谱(RP-HPLC)技术,采用等度洗脱。对20种标准DABTH-氨基酸进行了分离与鉴定,灵敏度达到pmol水平;并以溶菌酶为模式蛋白,经手工DABITC/PITC双偶联方法对其N端部分序列进行Edman降解,其降解产物DABTH-氨基酸用RP-HPLC技术进行了鉴定,蛋白质微量序列分析的灵敏度达到nmol水平.     

高效液相色谱法制备人胆汁中结合胆红素

针对单结合胆红素（ｍｏｎｏｃｏｎｊｕｇａｔｅｄｂｉｌｉｒｕｂｉｎ，ＭＣＢ）理化性质极不稳定而造成对其分析、制备的困难性，在Ｓｐｉｖａｋ和Ｔｒｏｔｍａｎ方法的基础上建立了一个简单可行的人胆汁结合胆红素（ｃｏｎｊｕｇａｔｅｄｂｉｌｉｒｕｂｉｎ，ＣＢ）高效液相色谱（ＨＰＬＣ）二次洗脱的制备方法，并对方法的可靠性作了论证，得到了ＭＣＢ较为满意的量与纯度，为进一步探讨ＭＣＢ的理化性质及其与胆石形成的关系提供了条件。     

Separation of Multicomponent Mixture of Naphthalene Derivatives by Reversed-Phase High-Performance Liquid Chromatography

Separation of a mixture of naphthalene derivatives by reversed-phase high-performance liquid chromatography with UV detection was studied. Chromatographic characteristics were calculated for nine compounds, including naphthalene.     

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

Abstract

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu²⁺) in the hydro-organic eluent.     

The Separation of Bilirubin,Photobilirubin and their Major Isomers by Ion-Pair High Pressure Liquid Chromatography

Abstract

The separation of the three major isomers and the main photoproducts of bilirubin by Ion-Pair High Pressure Liquid Chromatography is described. Chromatographic procedures found in the literature proved difficult to reproduce, had unacceptably long elution times (over 2 hours), required complex gradients or two or more columns. As these reports provided no data to show why specific chromatographic conditions were chosen an optimization study was carried out. The separation is shown to be dependent on pH, organic content and buffer concentration of the mobile phase, ion-pairing agent concentration and injection solvent. A separation, carried out in less than thirty minutes on octadecyl silica using isocratic conditions with detection at 455 nm is demonstrated. This technique will be used in the elucidation of the mechanism involved in phototherapy treatment of neonatal jaundice.     

Determination of Isomeric Nitriles with High-Performance Liquid Chromatography after Separation by Derivatization

A method is proposed for the separation of nitriles from other functional groups by derivatization. The resultingO-carboxamide amidoxime derivative of the nitrile is then identified by the use of HPLC retention times. The method has been successfully applied to isomeric monocyanopyridine and cyanotoluenes at a 1 mg/ml limit of detection.     

高效液色谱法测定蛇胆和人工蛇胆中的结合胆汁酸

1　引言 　　人工蛇胆是天然蛇胆的人工模拟物，其中含有牛磺胆酸(taurocholic axid,TCA), 牛磺去氧胆酸(taurodeoxycholic acid,TDCA)，胆绿素，粘蛋白等成分。本品是正在开发的一类新药，为了制定质量标准，需要建立检测方法。以往天然蛇胆的质量检测方法主要采用薄层扫描法和先将TCA和TDCA水解成游离胆酸，再做成各种衍生物测定，操作繁琐费时，且精确性及重复性不如高效液相色谱法(HPLC)。本文采用HPLC法，直接测定人工蛇胆中TCA和TDCA，选择二苯胺为内标物，操作简单，方法准确，并用于测定天然蛇胆干燥品（乌梢蛇）的含量同时与人工蛇胆进行了比较。     

Tocainide Determination by High-Performance Liquid Chromatography

Abstract

Tocainide has been assayed after serum deproteinization with acetonitrile by HPLC. The pH was critical in separating the drug from other interferences in the serum. The method is simple and fast.     

Separation of Chlorinated Phenols by Isocratic High-Performance Liquid Chromatography on Reverse Phase Column

Abstract

A method is described for isocratic highperformance liquid chromatographic separation of chlorinated phenols using methanol : phosphate buffer - pH 7.20 (50:50) solvent system. 13 out of 15 congeners of chlorophenols and phenol have been separated in 32 minutes at a flow rate of 0.8 ml per minute. The system is found to be useful for the separation of chlorophenols extracted from mouse liver fed with hexachlorocyclohexane.     

Enantiomeric Separation of N-Protected Non-Protein Amino Acid Esters by Chiral High-Performance Liquid Chromatography

Abstract

We have found that high-performance liquid chromatographic analysis of enantiomeric N-protected amino acid esters on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiralcel OD) can be utilized as one of the procedures for determining the optical purities of non-protein amino acids. The methyl esters of the N-benzyloxycarbonyl (Z) derivatives of a number of non-protein amino acids showed excellent to good enantiomeric separations using hexane - 2-propanol as a mobile phase. There was a regularity in the elution order of enantiomers: the L-isomer had a shorter retention time than the D-isomer. We have also investigated the effect of the N-protecting groups and the ester groups on the enantiomeric separation. The Z, 4-methoxybenzyloxycarbonyl (Z(OMe)), and 9-fluorenylmethoxycarbonyl (Fmoc) derivatives gave exceptionally good resolutions. By contrast, the formyl and t-butoxycarbonyl (Boc) groups impaired the enantiomeric separation. Almost all the alkyl esters examined and the benzyl ester gave resolutions better than or of the same order as the methyl ester. The resolution of β-amino acids was worse than that of the corresponding α-amino acids.     

Separation of Thiamin,Thiamin Antagonists and Their Phosphate Esters by High-Performance Liquid Chromatography

Abstract

Methods are presented for the separation of the phosphate esters of thiamin, oxythiamin or pyrithiamin and the separation of thiamin and oxythiamin by high-performance liquid chromatography. Detection by means of a UV monitor and fluorometer with a post-column oxidation system is described which allows for selective detection of thiochrome, pyrichrome and their respective phosphate esters. Anion exchange and ion-pair reverse phase chromatography are the chromatographic modes of separation.     

Separation and Determination of 2,5-Dimethoxy-4-Methylamphetamine Enantiomers in Plasma by High-Performance Liquid Chromatography

Abstract

A method for quantitation of 2,5-dimethoxy-4-methylamphetamine (DOM) enantiomers in plasma by high-performance liquid chromatography (HPLC) is described, d- and l-DOM were readily converted to the amides by condensation with a newly developed chiral reagent, succinimidyl ester of l-α-methoxy-α-methyl-1-naphthaleneacetic acid. The yielded diastereomers were separated on the μPorasil column with cyclohexane/ethyl acetate (3:1) exhibiting satisfactory k' and R values. The clean-up procedure by use of Sep-pak C₁₈ and carboxymethyl Sephadex LH-20 (CM-LH-20) proved to be effective for determination of the drug in biological fluids by HPLC. The plasma levels of d- and l-DOM after administration of the racemate to the rabbit were determined by the method thus established.     

A Rapid Isocratic Reversed-Phase Separation and Determination of Some Barbiturates by High-Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic method for the rapid separation and quantitative determination of five barbiturates in pharmaceutical preparations and body fluids has been developed. A C₁₈ reversed phase column was used and the barbiturates were detected at 235nm.

A number of eluting systems were examined, the most suitable of them being, Ethanol:Propanol:Methanol:Water (26:5:29:40) at pH = 8.8.