Urinary Uric Acid Determination by Reversed-Phase High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

Determination of urinary uric acid has been attempted by reversed-phase high-performance liquid chromatography with electrochemical detection. We have found that the electrochemical detection method is suitable for monitoring eluate from reversed-phase column and also that the minimum detectable quantity of uric acid using en electrochemical detector is about 10 pg. Complete separation of uric acid was achieved in about 8 min under the present chromatographic conditions.     

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

The high-performance liquid chromatographic separation of enantiomers of N-protected non-protein amino acid esters was investigated by using a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiracel OD). The effect of the N-protecting groups and the ester groups on chiral discrimination was examined. The benzyloxycarbonyl (Z), 4-methoxybenzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl derivatives gave good enantiomeric separations, while the formyl and t-butoxycarbonyl groups marred them. Almost all the alkyl esters examined and the benzyl ester gave enantiomeric separations better than or of the same order as the methyl ester. The N-Z-protected methyl esters of a number of non-protein -amino acids were well resolved using hexane–2-propanol as a mobile phase. The resolution of -amino acid derivatives was inferior to that of the isomeric -amino acid derivatives.     

Determination of Catecholamines by Capillary Electrophoresis and Reversed-Phase High-Performance Liquid Chromatography

A method is proposed for the electrophoretic determination of adrenaline, noradrenaline, and dopamine with mass-spectrometric or UV detection. A procedure is proposed for the sample preparation of biological fluids with the use of solid-phase extraction on alumina. A comparative assessment of the determination of catecholamines by capillary electrophoresis and reversed-phase high-performance liquid chromatography was performed.     

Quantitative Determination of Tabersonine and Methoxytabersonine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A method for the quantitative determination of tabersonine and methoxytabersonine by reversed-phase high-performance liquid chromatography has been developed based on employing amine modifiers of the mobile phase as silanol group masking agents. Thus peak tailing is reduced and peak shapes are improved. The effect of pH of the eluent and the type of the organic modifier upon the overall separation has been examined. A mobile phase optimization procedure has been carried out for selecting the most favorable eluent mixture. The accuracy and precision achieved by the quantitative analysis are satisfactory.     

Cellular Fatty Acid Composition of Vibrio parahaemolyticus by Reversed-Phase High-Performance Liquid Chromatography

Abstract

High-performance liquid chromatography was used to separate and identify cellular fatty acids isolated from Vibrio parahaemolyticus, a gram-negative estuarine microorganism associated with seafood-borne enteritis in man. Fatty acids were isolated from statically grown bacterial cultures, saponified, and derivatized with an ultraviolet tag. Aliquots of derivatized fatty acids were injected onto a reversed-phase column with water:acetonitrile gradient as the mobile phase and ultraviolet detection at 254 nm. The predominant fatty acids found for the V. parahaemolyticus strains studied were C12, C14, C16:1, C16, C18:1, and C18. In addition, previously unreported fatty acids C13, C17, C19, and C21 were identified. Comparison of HPLC with GLC fatty acid separations showed good agreement with the exception that HPLC was able to resolve previously unidentified fatty acid constituents.     

Determination of Pharmaceuticals by Dynamic Cell Membrane Chromatography Coupled with High-Performance Liquid Chromatography

As a biological affinity chromatographic method, cell membrane chromatography (CMC) using a silica stationary phase covered with specific cell membrane has been used in screening active components. The innovation of this work is that the bioactive cell membrane and the chromatographic packing are mixed and absorbed for the first time to form the pre-column. The pre-column was placed in front of a C₁₈ column to create dynamic CMC online high-performance liquid chromatography (HPLC) system. The retention behavior and dynamic changes of pharmaceuticals were studied for this system. The results indicate that the retention time of the drug was increased and the symmetry factor reached the analytical level after the addition of the dynamic cell membrane pre-column. Therefore, the dynamic CMC coupled with HPLC system may be a potentially rapid and efficient drug analysis approach for the interaction of drug molecule and receptor on red blood cell membranes.     

反相液相色谱法分离2-(4-羟基苯氧基)丙酸酯对映体

合成了三-(4-甲基苯甲酸)纤维素酯(MCTB)手性固定相，用反相高效液相色谱法在该手性固定相上对2-(4-羟基苯氧基)丙酸酯对映体进行拆分．实验结果表明，在三-(4-甲基苯甲酸)纤维素酯手性固定相上，以甲醇与水的体积分数为75：25做流动相能较好的拆分2-(4-羟基苯氧基)丙酸甲酯、乙酯和丁酯对映体，其分离因子分别为1．38、1．49、0．98；同时还发现2-(4-羟基苯氧基)丙酸酯中酯基团的大小对其对映体的分离也有明显的影响，其中以乙酯的拆分效果最佳。     

Rapid Simultaneous Determination For Catecholamines By Reversed-Phase High-Performance Liquid Chromatography

Abstract

An improved reversed-phase high performance liquid chromatographic technique for the rapid simultaneous analysis of patecnolamines is presented.

The catecholamines are preconcentrated by employing a single preconcentration step, solid-liquid extraction, with active alumina.

A C₁₈ reversed-phase column is used and the catecholamines are detected at 280 nm. the retention time is 3.7 min for norepinophrine, 4.5 min for epinephrine and 6.2 min for dopamine using an eluting system of 0.165 M AcOH 1 pc in MeOH.

The within-run precision CVs are in the order of 7 pc whereas the day-to-day precision CVs are in the order of 10 pc for concentrations near the upper normal limits. the application of the technique to a series of hypertensive subjects indicated, after comparison with three patients with than sixfold of upper normal limits in addition to fourfold or more increase in DA excretion seems to be indicative for the presence of malignant pheochfomocytoma. to assess the efficiency of such a discriminating criterion a larger number of cases of pheochromocytoma and essential hypertension have to be studied. In conclusion, we propose a rapid, precise and low-cost HPLC technique for the determination of urinary catecholamines requiring only one pump and a UV-detector. the technique may prove useful in hospitals as a tool in discriminating between essential hypertention and pheochromocytoma.     

反相高效液相色谱技术鉴定小黑麦品种

通过反相高效液相色谱(RP-HPLC)分析小黑麦种子醇溶蛋白,对供试的18个不同品种的小黑麦进行了品种鉴定和倍性水平鉴定,同时也对育种系谱相同的品种进行了系谱证实。     

Reversed-Phase Ion-Pair High-Performance Liquid Chromatography of Phosphatidylinositols

Abstract

Reversed-phase ion-pal, high-performance liquid chromatographic (HPLC) separations of molecular species of phosphatidylinositols (PI) were studied. Mobile phases of acetonitrile-methanol-water containing various tetraalkylammonium phosphates (TAAP) were used for optimization. Stationary phases of macroporous polystyrene divinylbenzene (MPD), octylsilica, and octadecylsilica exhibited arying degrees of retentivity toward PI solutes. Without exception capacity factors (k') of PI on either alkylsilica or MPD increased with the alkyl chain length and the concentration of the quaternary ammonium counter ions evaluated. the results can be interpred in terms of an ion-pair retention mechanism. Logarithmic k' values were linearly related to the total number of carbons in TAAP. Perbenzoylation of PI yielded derivatives readily resolvable without the use of any mobile phase additive. Incorporation of TAAP to a mobile phase facilitated component separations of early-eluting perbenzoates. In HPLC with     

Urinary Uric Acid Determination by Reversed-Phase High Pressure Liquid Chromatography

Abstract

Reversed-phase high pressure liquid chromatography with UV detection was proven to be a powerful method for the separation and quantitation of urinary uric acid. We have compared three different treatments for urine samples previous chromatographic injection: alkaline methanol extraction, ethylacetate extraction and centrifugation. It was also studied storage conditions for urine samples.

Our findings show that the method has high specificity and reproducibility for urinary uric acid. Samples are stables and require only centrifugation previous injection to the chromatograph.     

Rapid Analysis for Codeine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A reversed phase high-performance liquid chromatographic (HPLC) method has been established for the separation and quantitative determination of the alkaloid codeine in pharmaceutical preparations and in body fluids. The minimum detectable concentrations for body fluids were 5ppb and 7ppb respectively for urine and whole blood with an analysis time of under 5 min. A RP-8 Spheri-5 guard column and a RP-8 Lichrosorb-10 column were used and codeine was detected at its absorption maximum wavelength of 212 nm using an eluting system of methanol: 0.5% w/v aqueous ammonium acetate (70:30) at a pH of about 7.0.     

Determination of Iodate in Iodized Salt by Reversed-Phase High-Performance Liquid Chromatography with UV Detection

A method for the determination of iodate was developed by reversed-phase high-performance liquid chromatography with UV detection. Iodate was converted to iodine, which was separated from the matrix using a reversed-phase Ultrasphere C₁₈ column (250 × 4.6 mm, 5 μm) with methanol-1 mmol L^?1 H₃PO₄ (20:80, v/v) as mobile phase at 1.00 mL min^?1 and UV detection at 224 nm. The calibration graph was linear from 0.05 μg mL^?1 to 5.00μg mL^?1 for iodine with a correlation coefficient of 0.9994 (n=7). The detection limit was 0.01 μg mL^?1. The method was successfully applied to the determination of iodate in iodized salt. The recovery was from 96% to 101% and the relative standard deviation was in the range of 1.5% to 2.9%.     

The Analysis of Insulin-Related Peptides by Reversed-Phase High-Performance Liquid Chromatography

Abstract

This paper describes the use of high performance liquid chromatography (HPLC) for the rapid analysis and purification of insulin-related peptides prepared by solid-phase synthetic procedures. Examples include the bovine insulin C-peptide (34–45), the porcine insulin C-peptide (41–53) and the insulin B-chain fragment (22–27). Chromatographic elution systems containing reducing reagents like B-mercaptoethanol allow the direct analysis of insulin reduction products. Similar systems should allow the rapid analysis of disulphide bond pairing patterns in appropriate polypeptides and proteins either directly or following proteolytic digestion.

Reversed-phase high performance liquid chromatography (RP-HPLC) is a versatile and rapid technique useful for the analysis and purification of biological substances. In a series of recent publication^1–4 we have described methods for the analysis of underivatised amino acids, peptides and proteins on reversed-phase packings using ion-pairing or stationary phase modifying reagents as components of the mobile phase. These studies demonstrated that excellent resolution of closely related peptides can be achieved under a variety of elution conditions. The addition of low levels of phosphoric acid, inorganic or organic phosphates to a mobile phase (generally water-organic solvent mixtures), in particular, allows rapid and reproducible analysis of peptidic compounds with high sensitivity detection at wavelengths down to 190nm^5,6. It is the purpose of this report to show that these chromatographic conditions allow the facile analysis, and purification, of a variety of insulin-related peptides.     

Reversed-Phase High-Performance Liquid Chromatography of Unsubstituted Aminobenzoic Acids

Abstract

High-performance liquid chromatographic (HPLC) characteristics of three position isomers of aminobenzoic acids (potential metabolites of important anesthetic drugs), were delineated with respect to their interactions with various mobile phases and stationary phases. HPLC with five hydrocarbonaceous phase, β-cyclodextrin silica (CDS), macrophase MP-1 polymer (MP), macroporous polystyrene/divinylbenzene (MPD), octadecylsilica (ODS), and propylphenylsilica (PPS), yielded results explicable in terms of substituent effects derived from the bifunctional amino– and carboxy groups. For cases where mobile phases contained sulfonates or quaternary ammonium salts both having longer chain alkyls, retention of analytes on all but CDS appeared to proceed predominantly via an ion-pairing mechanism. The extent of the corresponding counter-ion effects decreased in the order: MPD > ODS > PPS > MP, while the analyte retention order paralleled thier pH₂ values. On the other hand, an inverse relationship between the magnitude of capacity factors (k′) and pK₁ values of the title compounds was observed in experiments that produced retention data incompatible with ion-pair interaction rationales. The unique HPLC results obtained with the CDS phase are compared with those obtained with other phases.     

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.     

Separation of Enantiomers of Non-Protein Amino Acids by High-Performance Liquid Chromatography on a Chiral Ligand-Exchange Column

The HPLC separation of enantiomers 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). Good enantiomeric separations were achieved with a variety of -amino acids carrying aliphatic or aromatic side chains, cyclic imino acids, and -amino acids, together with -methyl--amino acids, by optimizing the amount (0–20%, v/v) of 2-propanol as the organic component and the concentration (1–5 mM) of Cu²⁺ as the complexing metal ion in the aqueous-organic eluent.     

Propylamido-Modified C18 Reversed-Phase for High-Performance Liquid Chromatography

The synthesis and chromatographic properties of a propylamido-modified C18 stationary phase are described. The propylamido-modified C18 phase was prepared by bonding stearic acid to 3-aminopropyl-silica gel. The synthesis was simple and reproducible through amide formation by using N-hydroxysuccinimide and dicyclohexylcarbodiimide. This prepared stationary phase exhibited excellent abilities to separate polycyclic aromatic hydrocarbons (PAHs), polysiloxanes and phenols by HPLC. The chromatographic results show that both the C3 moiety and the amido-group in this phase contribute significantly to the HPLC separation process. The chromatographic behavior of the prepared phase and commercial C18 phases was also compared in analyses of PAHs and phenols. Although the commercial C18 phases have been extensively used, both the unique selectivity and the highly reproducible synthesis of our prepared phase make it an excellent complement to ordinary C18 phases.     

Determination of Triandrin and Salicin in Salix viminalis L. by Reversed-Phase High-Performance Liquid Chromatography

The optimal separation of phenolic components of the bark of basket willow Salix viminalis L. was attained using reversed-phase high-performance liquid chromatography with isocratic elution. Triandrin (1-O--D-glucopyranoside of p-coumaryl alcohol) and salicin (1-O--D-glucopyranoside of salicyl alcohol) were identified. Regularities in the retention of triandrin and salicin in the Separon SGX C₁₈–binary mobile phase (acetonitrile–water) system were considered. A procedure was developed for the quantitative determination of triandrin in raw basket-willow bark and its extract.