Automated high-performance liquid chromatographic determination of 5-S-cysteinyl-3,4-dihydroxyphenylalanine in urine

An automated high-performance liquid chromatographic (HPLC) method has been developed for measurement of 5-S-cysteinyl-DOPA in urine (DOPA = 3,4-dihydroxyphenylalanine). The urinary sample was injected into an HPLC boronate column. With a mobile phase of 0.1 M phosphate buffer containing 0.2 mM disodium ethylenediaminetetraacetate (Na2EDTA) (pH 6.0) mixed with methanol (9:1), 5-S-cysteinyl-DOPA was adsorbed while most other compounds were washed away. By column switching, the column flow was reversed and 5-S-cysteinyl-DOPA was desorbed by a mobile phase of 0.1 M formic acid and 0.2 mM Na2EDTA at pH 3.0 and chromatographed on a reversed-phase column. The precision, as estimated from repeated analysis of an urinary sample and from duplicate analysis of a number of samples, ranged from 1.4 to 5.2% (coefficient of variation), and the analytical recovery was 93 +/- 4.1%. The method is suitable for use in the clinical laboratory.     

Determination of 9-[(2-phosphonylmethoxy)ethyl]adenine in rat urine by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic (HPLC) method for the determination of 9-[(2-phosphonylmethoxy)ethyl]adenine (PMEA) in urine is described. The procedure includes treatment of the urine sample with chloroacetaldehyde to form the fluorescent 1,N6-ethenoadenosine derivative, which was analyzed by reversed-phase HPLC with fluorometric detection. Validation of the method showed good sensitivity, precision and reproducibility. The method is useful for the study of urinary excretion of PMEA in the rat.     

尿中核苷排放模式测定的两种方法─-HPLC和CE法(英文)

癌症病人在尿中排放比常人高得多的叶啉和嘧啶,这些改性核耷是RNA的主要组成。由于其增加的排放与癌变的RNA周转有关,已被建议和研究作肿瘤标记物。文章给出测定尿中核苷的反相高效液相色谱法和毛细管电泳方法。两种方法所得数据一致。用此方法建立了正常人尿中核苷的排放水平,并测定了34个癌症病人尿中核苷浓度,观察到改性核着浓度明显的增高现象。结果袭明,两个方法均适于大量尿样的核苷与癌症关系的临床研究。     

尿中核苷排放模式测定的两种方法─-HPLC和CE法(英文)

 癌症病人在尿中排放比常人高得多的叶啉和嘧啶,这些改性核耷是RNA的主要组成。由于其增加的排放与癌变的RNA周转有关,已被建议和研究作肿瘤标记物。文章给出测定尿中核苷的反相高效液相色谱法和毛细管电泳方法。两种方法所得数据一致。用此方法建立了正常人尿中核苷的排放水平,并测定了34个癌症病人尿中核苷浓度,观察到改性核着浓度明显的增高现象。结果袭明,两个方法均适于大量尿样的核苷与癌症关系的临床研究。     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Quantitative Estimation of Urinary Metabolites in Ruminants by High-Performance Liquid Chromatography

Abstract

A reversed-phase high-performance liquid chromatographyc method for a simultaneous determination of cretinine (C); Uric Acid (A); Hipoxanthine (HX); Xanthine (X); Hippuric Acid (HA); Benzoic Acid (BA) and Phenylacetic Acid (PAA) in urine of ruminants is described.

Separation was achieved on a Nova-PaK column by-gradient elution. Quantitation and detection limits were determined Detection is effected by UV absorption at 254 nm with a total analysis time of less than 30 min. An aliquot of diluted urine is injected directly into the liquid chromatographic column.

The proposed HPLC method was verified for linearity, accuracy, precision and applicability.     

Simple high-performance liquid chromatographic method for the measurement of amiloride in body fluids

A simplified rapid high-performance liquid chromatographic (HPLC) procedure has been developed for the measurement of amiloride in plasma or urine. Solid-phase extraction columns provide quick, clean and simple sample preparation, allowing ten samples to be processed in less than 5 min. The HPLC system uses a standard reversed-phase (C18) column with detection by ultraviolet absorption at 365 nm. The assay has been used to define plasma amiloride concentration-time profiles and to quantitate urine amiloride recovery in healthy men after repeated administration at two doses.     

Study of normal and modified nucleosides in serum by RP-HPLC

Summary Modified nucleosides derived predominantly from transfer ribonucleic acid (tRNA) have been studied as possible tumor markers. In this paper a reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been applied to study 15 normal and modified nucleosides in serum. The nucleoside levels in normal human serum were established, and the concentrations of 15 nucleosides in serum from 19 cancer patients were determined. It was found that the concentrations of modified nucleosides were significantly higher in patient sera. Based on 15 nucleoside concentrations in serum, factor analysis could classify correctly 90% of cancer patients from the normal humans. Further work showed that urine was slightly better than serum when determining nucleosides as biological marker candidates. More work is ongoing to determine the clinical usefulness of modified nucleosides as tumor markers.     

Microanalytical high-performance liquid chromatographic assay for cefpirome in human milk and urine.

To permit the characterization of cefpirome disposition in lactating females, a previously published high-performance liquid chromatographic (HPLC) method for determining the drug in serum was adapted for use with milk and urine. This automated, microanalytical technique requires 50 microliters of biological matrix, which is subjected to an isopropanol extraction. Chromatography was accomplished using a microbore HPLC system, a reversed-phase C18 column and a mobile phase of 0.3% triethylamine in water (pH 5.1). Cefpirome and the internal standard (beta-hydroxypropyltheophylline) were monitored using UV detection at 240 nm and had retention times of 2.84 and 5.05 min, respectively. The method was linear up to 500 mg/l for both matrices and had a limit of detection of 0.6 mg/l. The interday variation (relative standard deviation) at concentrations of 5.0, 50.0 and 500.0 mg/l was consistently less than 5% in both urine and breast milk. The method was found to be free from interference by other commonly administered medications and readily adaptable for use in clinical investigations. The ease of sample preparation, small sample volume requirement, short chromatographic time, apparent lack of interferences, analytical sensitivity and high precision and accuracy make this method ideal for use in pharmacokinetic investigations involving the determination of cefpirome in human milk and urine.     

肠癌患者尿中核苷排放的高效液相法研究

 用反相高效液相法测定尿中核苷。通过苯基硼酸亲和法提取尿中核苷,在柱(4 6mmi d ×250mm,5μm)上以25mmol/L磷酸二氢钾溶液(pH4 55)和60%的甲醇水溶液作为流动相进行二元梯度淋洗,于22℃下进行反相分离,260nm处紫外检测。用该法测定了41例肠癌患者和52例正常人尿中15种核苷的含量(用核苷与肌酐的摩尔比表示,下同),结果表明肠癌患者中有12种核苷的含量比正常人显著性增高(P<0 001)。以15种核苷的含量作为参量,结合主成分分析区分正常人和肠癌患者,对癌症病人的识别率达76%(31/41)。     

Direct injection analysis of hexamethylene bisacetamide in biological fluids by an HPLC column switching technique with a micellar mobile phase

Summary This paper describes the direct injection analysis of the anti-cancer drug hexamethylene bisacetamide (HMBA) in biological fluids by an HPLC column switching technique. The first chromatographic column, which provides for sample extraction and cleanup, employs a micellar mobile phase with SDS as the modifier. The second column, coupled on-line to the first, utilizes reversed-phase conditions for analysis. UV detection is employed at 210 nm. 282 samples from 12 cancer patients were analysed and good pharmacokinetics curves obtained. The drug gives recoveries of 94.0–100.9% with a relative standard deviation of 1.88%. A sample analysis is completed within 15 minutes. This method should be satisfactory not only for the analysis of HMBA but also, probably for other drugs in biological fluids.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

High-Performance Liquid Chromatographic Determination of Clavulanic Acid in Human Serum and Urine Using a Pre-Column Reaction with 1, 2, 4-Triazole

Abstract

A reversed-phase high-performance liquid chromatographic (RP-HPLC) assay for the determination of clavulanic acid in serum and urine is described. The clavulanic acid was assayed by reacting the sample with 1, 2, 4-triazole reagent which rapidly produces a derivative that has its UV absorption maximum at 317 nm. The resulting product was separated in a RP-18 column (μ-Bondapak, 10 μm) and detected at 313 nm. The method was applied to assays of clavulanate in serum and urine, and is reproducible with a lower limit of quantitation of 0.1 μ/Ml.     

Determination of neomycin in plasma and urine by high-performance liquid chromatography. Application to a preliminary pharmacokinetic study.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in plasma and urine. The plasma was deproteinated with trichloroacetic acid and centrifuged. The supernatant was mixed with ion-pair concentrate and centrifuged again. The resultant supernatant was analyzed by HPLC. Urine was centrifuged to remove debris, if any, mixed with ion-pair concentrate and analyzed directly by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall average recovery of neomycin was 97 and 113% from plasma spiked at 0.25-1.0 micrograms/ml, using standard curves prepared in plasma extract and in water, respectively, and 94% for urine spiked at 1-10 micrograms/ml using a standard curve prepared in water. The method was used to detect neomycin in plasma and urine obtained from animals injected intramuscularly with neomycin. Various pharmacokinetic parameters of neomycin were also determined from its profile of plasma concentration versus time.     

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

HPLC method for the analysis of cyproterone acetate in tablets

A reversed-phase high-performance liquid chromatographic (HPLC) method is described for the analysis of cyproterone acetate in tablets. This steroid is extracted and quantitated using a C18 column. This procedure is shown to be rapid, simple, and valid in terms of recovery, linearity, and precision. Application of this method to analyze dilute samples obtained from dissolution testing is demonstrated.     

High-performance liquid chromatographic determination of alpha-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the determination of alpha-keto acids in human serum and urine is described. In an acidic solution, twelve species of alpha-keto acids examined were converted by reaction with 1,2-diamino-4,5-methylenedioxy-benzene into highly fluorescent derivatives. The derivatives were separated isocratically by reversed-phase HPLC on a TSK gel ODS-80TM column and detected fluorimetrically. Eight alpha-keto acids in human serum and eleven alpha-keto acids in human urine can be determined simultaneously. The detection limits (signal-to-noise ratio = 5) are 6-44 fmol in an injection volume of 5 microliters. The intra-assay relative standard deviations for both serum and urine sample analyses are usually ca. 5%.     

Liquid chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection

A liquid chromatographic method using a solid-phase extraction procedure for the quantification of sotalol in plasma and urine is described. Sotalol is eluted from an extraction column with ethyl acetate-acetonitrile (1:2) and, after separation by reversed-phase high-performance liquid chromatography on a mu Bondapak C18 column, is quantified by fluorescence detection at excitation and emission wavelengths of 240 and 310 nm, respectively. The method has been demonstrated to be linear over the concentration ranges 10-6000 ng/ml in plasma and 0.5-100 micrograms/ml in urine. Mean inter-assay accuracy of the method for plasma ranged from 93 to 100% and for urine from 102 to 114%; precision ranged from 0.5 to 1.6% for plasma over a concentration range of 200-4000 ng/ml and for urine from 0.7 to 2.0% at concentrations of 2-50 micrograms/ml. Mass spectrometry confirmed the presence of sotalol in isolated chromatographic fractions of plasma and urine extracts from subjects given sotalol orally.     

Analysis of normal and modified nucleosides in urine by capillary electrophoresisa)

Summary Modified nucleosides excreted in urine have been studied as potential diagnostic markers for cancer and AIDS, and as indicators for the whole-body turnover of RNA. Until now, reversed-phase (RP) HPLC and, to some extent, immunoassays are the preferred analytical methods for urinary nucleosides. A new capillary electrophoretic method for the analysis of normal and modified nucleosides in urine has been developed and optimized in our laboratory. The separation of nucleosides extracted from normal human urine on phenyl boronic acid affinity chromatography columns was performed in uncoated 565 mm (500 mm to detection window) × 50 μm i.d. capillary tubing using a 300 mM SDS—25 mM borate—50 mM phosphate buffer (pH 6.7), a 45-s load, a voltage of 7.5 kV (41 μA) and UV detection at 260 and 210 nm. The average recovery of the nucleosides was 91 %. The calibration curves were linear over all physiological and pathophysiological concentration ranges and the limits of detection were at micromolar levels. Reproducibility of migration times were better than 1 % (coefficient of variation,CV), and the reproducibilities of the determined concentrations were better than 5 % for standards and 6–15 % for extracted urine. The developed method was used to quantify 15 normal and modified nucleosides in 25 normal urines to establish reference ranges. The analysis time was less than 45 min. Dedicated to Professor E. Bayer on the occasion of his 70th birthday. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.