柱前衍生高效液相色谱法测定氨基酸

本文阐述了以６－氨基酸喹啉基－Ｎ－羟基琥珀酰亚胺氨基甲酸酯为衍生剂，用紫外测检测柱前衍生反相高效液相色谱测定氨基酸的方法，并将实验结果和方法性能与经典的离子交换色谱法做了比较，该法不仅具有能与ＩＥＣ法相媲美的精密度，准确度，还不受样品基质，高盐乃至添加的大量其它电解质，维生素和微量元素的干扰，特别适于天然生物样品，食品及饲料的氨基酸分析。     

柱前衍生高效液相色谱测定小儿复方氨基酸注射液中氨基酸

建立了高效液相色谱(HPLC)测定小儿复方氨基酸注射液中各种氨基酸含量的方法.采用Kromasil C18(250×4.6 mm,5 μm)色谱柱,以2,4-二硝基氟苯为衍生试剂,梯度洗脱.流动相A为乙腈∶水(50∶50,V/V),流动相B为0.04 mol/L的磷酸二氢钾溶液(pH=6.8);流速为1.0 mL/min;检测波长为360 nm.所测氨基酸的线性范围为0.008～0.2213 mg/mL,平均回收率在97.85%～102.86%之间.     

Determination of Plasma Amino Acid Biomarkers by High Performance Liquid Chromatography for Diagnosis of Type 2 Diabetes Mellitus

The use of the fasting plasma glucose and 2-h plasma glucose tolerance tests leads to under-diagnosis of type 2 diabetes mellitus. To explore the contribution of metabolites for diagnosis for type 2 diabetes, plasma targeted metabolites (including free amino acids, lipids, and two hepatic aminotransferases) of 95 subjects were determined by high performance liquid chromatography and routine clinical methods. Receiver operating characteristic curve analysis was employed to evaluate potential diagnosis models. Based on the processing of orthogonal partial least squares-discriminant analysis, five amino acids (lysine, aspartate acid, threonine, methionine, and alanine) and two lipids (low density lipoprotein-cholesterol and high density lipoprotein-cholesterol) were discovered as potential biomarkers of type 2 diabetes. The area under the receiver operating characteristic curve built by the combination of these biomarkers and fasting plasma glucose was 0.994, which is significantly higher than that of the latter alone (0.907). These findings indicate that simultaneous measurement of specific metabolites with fasting plasma glucose from a single blood test for screening type 2 diabetes might be a more sensitive and specific strategy and provide additional insight regarding disease pathophysiologic mechanisms.     

Simultaneous Determination of Salicylic Acid and Salicyluric Acid in Plasma and Urine by High Pressure Liquid Chromatography

Abstract

A sensitive, rapid, and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of salicylic (SA) and salicyluric (SU) acids in plasma and urine. The compounds are extracted into ethyl ether at acid pH, evaporated, and reconstituted prior to instrumental separation. Overall recovery of both compounds is 90 ± 5%, and the sensitivity limits are 150 ng of SU and 300 ng SA per ml of biological fluid. The assay was used for the determination of both compounds in plasma and urine of man following oral doses of 40 mg/kg of sodium salicylate.     

Revised Method for Determination of Aspirin and Salicylic Acid in Human Plasma by High Pressure Liquid Chromatography

Abstract

The method for the analysis of aspirin and salicylic acid in human plasma has been updated to include advances in column technology, extraction procedures and absorbance detection. Aspirin and salicylic acid are extracted from acidified plasma into an organic solvent system containing internal standard. Following controlled evaporation under partial vacuum of the organic extract, the dried down-residue is reconstituted with mobile phase. Chromatography is ion suppression reverse phase on a 5 μm O.D.S. column with detection by absorbance at 237 nm and optional fluorescence. Concentration of aspirin as low as 0.20 μg/ml and salicyclic acid as low as 0.50 μg/ml can be quantitated.     

Determination of Glutamic Acid Decarboxylase Activity in Subregions of Rat Brain by High Pressure Liquid Chromatography

Abstract

A quantitative high pressure liquid chromato-graphic (HPLC) assay has been developed for the determination of glutamic acid decarboxylase (GAD) activity in subregions of rat brain. GAD activity was determined indirectly by measurement of gamma-aminobutyric acid (GABA). Fluorimetric detection was made possible by derivatization with ortho-phthalaldehyde and the limit of detection was 11 ng GABA.     

Use of Alkaline Degradation for the Analysis of Dihydrotriazines via High Pressure Liquid Chromatography

Abstract

An HPLC method is described for the determination of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3′,4′-dichlorobenzyloxy)-1,3,5-triazine hydrochloride (WR 38839). The procedure required the isomerization of the drug sample by alkaline treatment with sodium hydroxide, as the parent compound was retained by the column. The reaction product of the drug was analyzed by HPLC using a strong cation exchange resin as the stationary phase and glycine buffer, pH 10.4 as the mobile phase. The product was isolated and identified by TLC, UV, IR, mass spectroscopy and elemental analysis. The postulated mechanism indicates that this would be a general analytical method for dihydrotriazine compounds. This technique, developed for the assay of the dihydrotriazine in an aqueous system, was successfully applied to rat urine samples spiked with the drug.     

柱前衍生–高效液相色谱法测定白刺果中氨基酸含量

建立一种快速测定不同产地的白刺果中氨基酸含量的HPLC方法。采用柱前邻苯二甲醛(OPA)和氯甲酸芴甲酯(FMOC)联合在线衍生、二元梯度洗脱(流动相A:40 mmol/L NaH2PO4·H2O,pH 7.8;流动相B:乙腈–甲醇–水的体积比为4.5∶4.5∶1)、反相C18短柱分离(色谱柱:Zorbax Eclipse AAA C18柱,75 mm×4.6 mm,3.5μm)、二极管阵列检测器(检测波长:338 nm;参考波长:390 nm)和荧光检测器(激发波长:340 nm;发射波长:450nm)联合检测,内标法定量。各氨基酸含量在4.5~900μmol/L范围内线性关系良好,相关系数为0.991 2~0.999 8,除了蛋氨酸(部分氧化降解)加标回收率为78.1%外,其它各氨基酸的加标回收率为93.1%~105.1%,相对标准偏差为3.21%~6.23%(n=5)。对产自青海、新疆和内蒙古等3个地区的白刺果中氨基酸含量进行了测定,氨基酸总量分别为11.23,10.47,8.84 g/(100 g),并对各种不同类型氨基酸占氨基酸总量的比例进行了分析。该法适合于白刺果氨基酸含量的测定。     

The Determination of Folic Acid (Pteroylmonoglutamic Acid) in Fortified Products by Reversed Phase High Pressure Liquid Chromatography

Abstract

A reversed phase HPLC method was developed for the separation and determination of pteroylglutamic acid (PGA) in fortified foods. Extraction was carried out by heating with phosphate-citrate buffer, pH 8.0 containing ascorbate, and incubation with papain at 40°C for 4 hrs. The extracts were purified and concentrated on a short DEAE column which was rinsed with phosphate buffer, pH 7.0, of increasing molarity. PGA was eluted with 0.1M phosphate buffer, pH 7.0, containing 0.5M NaCl. The eluants were chromatographed on a Spherisorb ODS 10 μm column (250 × 4.6 mm) using a 30 min linear gradient of 2% to 30% acetonitrile in 0.1M acetate buffer, pH 4.0, at 1 ml/min and an absorbance detector at 280 nm. The coefficients of variation on analysis of 8 replicate samples of a milk and soy protein based infant formulas were 5.9% (at 4.6 ng/50 μl inject) and 6.8% (at 1.8 ng/50 μl inject) respectively.     

Application of High Pressure Liquid Chromatography to the Determination of Diastereoisomer in Methylphenidate Hydrochloride

Abstract

A procedure for the quantitative analysis of the diastereoisomer in methylphenidate hydrochloride is described. The method is based on a high pressure liquid chromatographic separation of the isomers on a Sil-X column with a mobile phase containing ethanol, cyclohexane, chloroform and ammonium hydroxide. The method is simple, precise and accurate and has a limit of detection of 0.1% of the diastereoisomer in the drug substance.     

Determination of Aspirin,Salicylic Acid,Salicyluric Acid,and Gentisic Acid in Human Plasma and Urine by High Pressure Liquid Chromatography

Abstract

A method has been developed to simultaneously determine aspirin, salicylic acid, salicyluric acid, and gentisic acid concentrations in human plasma and urine. An extraction and dry-down step prepare the sample for resolution by reverse-phase high pressure liquid chromatography. The column effluent is monitored by both ultraviolet absorbance and fluorescence to optimize sensitivity and specificity for the compounds of interest.     

高效液相色谱法测定生物催化法生产中的己二酸

己二酸是一种重要有机化合物,具有强极性和离子性的特点,由于在210 nm以上有弱的紫外吸收,故用气相色谱(GC)或高效液相色谱(HPLC)直接分离测定均有一定困难。为此,本文从研究乙酸缓冲溶液中己二酸-铜络合物的紫外吸收光谱出发,建立了高效液相色谱紫外检测己二酸的方法。通过实验确定了最优色谱条件。本法测定己二酸的线性相关系数为0.998,回收率为97.5%,检出限为0.32 mg/mL。此方法可用于监测生物催化法生产的己二酸,优化生产条件。     

The Use of High Pressure Liquid Chromatography for the Identification and Preparation of Pigments Concerned in Photosynthesis

Abstract

The chlorophylls and carotenoids present in preparations from chloroplasts of marine algae can be extracted and separated by high pressure liquid chromatography (H.P.L.C.). The reverse-phase columns; Partisil 10 ODS (Whatman), μ Bondapak C18 and μ Bondapak CN (Waters Associates) gave good separation of the different pigments. Addition of the ion-pairing agent tetrabutylamnonium phosphate to the methanol/water solvents gave improved separations with complex mixtures and identification was facilitated by examination of the column eluant at 440nm where a ll the pigments absorbed and at 650nm where only chlorophylls absorbed. The method allowed the isolation of individual pigments for further study.     

On Line Detection of Plasma Borne Vasoconstrictor by the Use of High Pressure Liquid Chromatography

Abstract

An on line detection system for a plasma borne vasoconstrictor was developed using a rat heart bioassay and a reversed-phase high pressure liquid chromatograph (HPLC). Partially purified plasma borne vasoconstrictor, which is yet to be characterized, was fractionated by HPLC, and the output from the unit was introduced into the rat heart bioassay system directly. The on line HPLC-rat heart bioassay system detected the active fraction from approximately 20 substances in the partially purified plasma. This system enabled rapid and reproducible identification of the active vasoconstrictor in plasma.     

Use of Cycloalkylcarbonyl Derivatives for the Determination of Amino Acid Methyl Esters by Gas Chromatography–Mass Spectrometry

Cycloalkylcarbonyl (cycloalkyl = cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl) derivatives were proposed for the determination of amino acid methyl esters by gas chromatography–mass spectrometry. These compounds are readily formed with the quantitative yield on heating hydrochlorides of amino acid methyl esters with cycloalkylcarbonyl chlorides. Although these derivatives exhibit lower chromatographic mobility than acetyl or perfluoroacyl derivatives, they form symmetric peaks. Mechanisms of the fragmentation of the new class of compounds under electron ionization conditions were studied in detail using high-resolution mass spectra and analysis of metastable ions. It was demonstrated that cyclopentylcarbonyl and cyclohexylcarbonyl derivatives can decompose with the cleavage of carbocycles.     

猪毛水解母液中氨基酸的反相高效液相色谱分析

A new method for separation and quantification of amino acids by pre-column derivatization withphenylisothiocyanate(PITC ) and reversed-phase HPLC is described. Seventeen amino acids were separatedin 30 min at room temperature.The method has been successfully applied to the determination of amino acidcomposition in the solution which was prepared by hydrolyzing pig hair and extration of cystine.     

Use of Ethoxy-Homologs as Internal Standards for Determination of Urinary Vanillylmandelic Acid and Normetanephrine in Man by High Performance Liquid Chromatography

Abstract

Synthesis of the ethoxy-homologs of vanillylmandelic acid (3-ethoxy-4-hydroxymandelic acid, EMA) and normetanephrine (3-ethoxy-4-hydroxyphenylethanolamine, EHPEA) and their use in minor modifications of existing assays on human urine are described. For analysis of vanillylmandelic acid, the homologous internal standard is added to an aliquot of urine, organic acids are then extracted into ethyl acetate and back extracted into 10% K₂CO₃, and finally extracted a second time into ethyl acetate. After evaporation of the ethyl acetate, the residue is redissolved in the mobile phase to be used in the chromatography, and injected onto a reverse phase column. Comparison of results with a gas chromatography-mass spectrometry (GCMS) assay gave almost identical results (96% ± 2%, mean ± SE, n=10). Analysis of normetanephrine is performed by addition of internal standard to an aliquot of urine before heat hydrolysis of amine conjugates. The amines are adsorbed onto columns of Bio Rex 70, and then eluted with a solution of 1 M NH₄OH, which is then evaporated to dryness. After residue is redissolved in borate buffer, the amines are extracted into toluene:isoamyl alcohol and back extracted into 0.1 M acetic acid before injection onto a reverse phase column. Correlation with a GCMS assay gave similar results (102% ± 4%, mean ± SE, n=9).     

Method for Determination of Codeine and Morphine in Rat Whole Blood by High Pressure Liquid Chromatography

Abstract

A HPLC method to simultaneously determine codeine and morphine in rat whole blood has been developed and evaluated. This method is based on a selective extraction and reversed-phase chromatography which results in chromatograms with 220 to 3500 theoretical plates for morphine and codeine. Detection is by electrochemical oxidation at +1.2V vs Ag/AgCl. In this method, the procedure of blood centrifugation for plasma preparation is eliminated. Therefore, the blood volume required is decreased and the sensitivity of analysis is considerably increased. Concentrations of codeine and morphine are low as 2ng/ml can be quantitated in as little as 100 mcl of rat whole blood.     

反相高效液相色谱测定敦煌壁画胶结材料中氨基酸

用邻苯二甲醛（OPA）柱前衍生，反相高效液相色谱法测定了敦煌壁画中15种氨基酸成份，采用Watersodsc18柱分离，PH=6.5的醋酸钠缓冲溶液和甲醇二元梯度洗脱，荧光检测，24min内15种氨基酸标准样品达到分离，待测样品中氨基酸也有很好的分离效果。