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.     

A Uricase Method for the Peak Identification of Uric Acid Appeared in a Liquid Chromatogram Amperometrically Monitored

Abstract

A uricase method for the peak identification of uric acid appeared in a liquid chromatogram monitored by aid of an electrochemical detector has been developed. Uricase (EC 1.7.3.3, from Candida utilis) catalyzes the conversion of uric acid to allantoin. We have found that uric acid can be oxidized under the chromatographic conditions employed in this study, whereas allantoin cannot be oxidized. The complete disappearance of a uric acid peak in a chromatogram of a biological sample after the uricase treatment indicates that the uric acid peak does not contain any other electroactive components. We observed the complete disappearance of the uric acid peaks in the chromatograms of human serum and gastric body.     

HPLC Determination of 6-Thiouric Acid and 6-Mercaptopurine in Organ Transplant Patient Serum

Abstract

A sensitive high performance liquid chromatographic method for the simultaneous determination of 6-thiouric acid and 6-mercaptopurine in serum is described. Our intent was to develop a procedure that could be used for pharmacokinetic studies and therapeutic drug monitoring in organ transplant patients taking azathioprine. Serum samples were precipitated with acetonitrile containing 6-n-propyl-2-thiouracil as the internal standard. The chromatographic separation was performed with an octadecylsilane column and gradient solvent system consisting of acetonitrile and 0.01 M sodium dihydrogen phosphate, pH 6.1. An initial acetonitrile concentration of 1% was used to elute 6-thiouric acid but was increased to 16% to recover the 6-mercaptopurine and internal standard. The flow rate was increased from 1.3 ml/min to 1.5 ml/min during the analysis. The column effluent was monitored at 353 nm and 323 nm for detection of 6-thiouric acid and 6-mercaptopurine, respectively. Statistical analysis of standard curve data showed good intra- and inter-day accuracy, precision and reproducibility throughout a concentration range of 10–2500 ng for 6-thiouric acid and 10–500 ng for 6-mercaptopurine/ml of serum. The method has been applied to the quantification of 6-thiouric acid and 6-mercaptopurine in serum from two kidney allograft recipients.     

High Performance Liquid Chromatographic Analysis of Desipramine in Human Plasma Using a Microbore Column Technique

Abstract

A reversed-phase, isocratic HPLC method has been developed for the quantitation of desipramine in human plasma. the method involved the use of cloimpramine as an internal standard. the chromatographic separation was accomplished with a mobile phase comprising acetonitrile-aqueous solution (60:40. v/v) containing 10 mM disodium hydrogenphosphate and 80 mM sodium dodecyl sulfate adjusted to pH 2. the mobile phase was pumped at a flow rate of 0.5 ml/min. the column used was a microbore column (2 mm I.D. × 100 mm) packed with a C18 reversed-phase material (5μm ODS Hypersil). Plasma samples were extracted at basic pH with diethyl ether followed by back-extraction into 0.1 N sulfuric acid. Using UV detection at 250 nm, the lower limit of sensitivity was 10 ng/ml. the inter- and intra-assay coefficients of variation were found to be less than 10%. the assay procedure was applied to a long term oral dosing study in patients to monitor the plasma concentration of desipramine.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.     

Electrochemical Detection of Ascorbic and Uric Acids in the Eluate from Reversed-Phase High-Performance Liquid Chromatography with Newly Developed Rigid-Type Porous Polymer Packing (Polymetacrylate Gels)

Abstract

It has been found that the combination of ethylenediaminetetraacetate and metaphosphoric acid is useful for the stabilization of ascorbic acid (AA) and precipitation of proteins in body fluids and also that the complete separation of catecholamines, AA, and uric acid (UA) on a column packed with a newly developed rigid-type porous polymer packing (polymetacrtlate gels) can be achieved. On the basis of the above findings, we have developed a method for the determination of endogenous AA and UA in body fluids such as human serum, urine, and cerebrospinal fluid by reversed-phase high-performance liquid chromatography with electrochemical detection. Determination of AA and UA in each body fluid has been carried out by injecting 10 μl of each simply deproteinized body fluid into a chromatographic system.     

High Performance Liquid Chromatographic Determination of Triazolam in Human Serum

Abstract

A high performance liquid chromatographic method which utilizes UV-detection has been developed for the sensitive and specific determination of triazolam in human serum. Using 8-chloro-6-phenyl-l-ethoxymethyl-4H-s-triazolo[4, 3-a][1, 4]benzodiazepine as an internal standard, serum samples were buffered with 2 ml of 4M NaOH and extracted twice with 5 ml aliquots of toluene. The combined toluene extracts were evaporated to dryness and the residue dissolved in the chromatographic mobile phase. The samples were chromatography on a microparticulate reverse-phase column using a 0.06M acetic acid:acetonitrile (61:39) mobile phase. Known metabolites of triazolam did not interfere in the analysis. A linear relationship between peak height ratios and concentrations was observed, with the lower limit of detection being approximately 1 ng of triazolam. The utility of the method was demonstrated by administering therapeutic doses of the drug to human volunteers and monitoring serum triazolam concentrations as a function of time.     

HPLC Assay of 6-Methoxy-2-Naphthylacetic Acid,a Major Metabolite of Nabumetone,in Human Serum

Abstract

A modified high-performance liquid chromatographic method for the quantitative determination of 6-methoxy-2-naphthylacetic acid (6-MNA), a major metabolite of nabumetone, in human serum was developed and validated. The composition of the mobile phase in Daigneault & Ferslew's method was changed in this study to improve the separation of 6-MNA from serum components. The volume ratio of acetonitrile to 1.5% acetic acid solution was changed from 60: 40 to 25: 75 in this study. As a result of the modification, the separation of 6-MNA from 2-naphtol (internal standard) and serum components was greatly improved. At a flow rate of 3.0 ml/min of the mobile phase, retention times of 6-MNA and 2-naphtol were 13 and 9 min, respectively. The detection limit was 1 μg/ml. Intra- and interday variations of the assay were <10.0 and <7.28%, respectively. Intra- and interday relative errors were <7.82 and <6.76%, respectively. 6-MNA in human serum could be determined successfully after oral administration of nabumetone (1g). Thus, the modified UV-HPLC method was confirmed to be applicable to the pharmacokinetic study of 6-MNA after oral administration of nabumetone to human subjects.     

High Performance Liquid Chromatographic Separation of Tropatepine in Biological Fluids. Application to a Healthy Volunteer

Abstract

A high performance liquid chromatographic method for the determination of tropatepine in human plasma and urines is described here. After addition of an internal standard (2 chloro-11-(4-methyl piprazine 1-yl) dibenzo (b-f)(1–4) thiazepine) to the biological fluid and extraction at pH 12.0 in hexane, the analysis was performed on a reversed phase column (C18 microBondapak) with UV detection at 231 nm. The compound was eluted by a perchlorate buffer-acetonitrile mixture with a flow rate of 1.7 ml/min. The detection limit was about 25 ng/ml; reproducibility was around 7.5% for plasma concentrations below 50 ng/ml. Mass spectrometry by direct insertion probe had validated the chromatographic results. The method was successfully applied to plasma specimen collected from a healthy human volunteer following a single intravenous administration of 20 mg of tropatepine.     

Determination of Ciprofloxacin (Bay o 9867) in Biological Fluids by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for the analyses of ciprofloxacin (BAY o 9867) (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid hydrochloride) in human serum, plasma and urine samples is described. Diluted serum, plasma, and urine samples are injected onto a RP-18 column without prior extraction or clean-up procedure. Ciprofloxacin is separated from the ballast by an eluent consisting of an 0.025M H₃PO₄ solution adjusted to pH=3 with tetrabutylammonium hydroxide and acetonitrile.

Ciprofloxacin is detected fluorimetrically giving a detection limit of 8ng/ml in plasma and serum and of 50ng/ml in urine. A statistical evaluation of the assay showed acceptable accuracy and precision for 10 to 500ng of BAY o 9867 per ml in serum and plasma and for 50ng to 600ng of BAY o 9867 per ml of diluted urine specimens. This method was used to monitor the concentrations of BAY o 9867 in serum, plasma and urine of volunteers after oral administration of ciprofloxacin.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

Quantitative Determination of Uric Acid in Serum by Reversed-Phase Liquid Chromatography Using an Internal Standard

Abstract

A rapid and sensitive high-performance liquid chromatography procedure is reported for the analysis of uric acid in serum. The method employs a single treatment of serum with cyclohexane and an excess of ammonium sulfate to eliminate endogenous serum components which easily contaminate the chromatographic system. Reversed-phase chromatography is performed at room temperature using a binary-solvent gradient, monitoring the effluent at 290 nm. Quantitation is based on peak-height ratio of uric acid to internal standard (β-hydroxyethyltheophylline). A linear response is obtained to 160 mg/1. Within-day and between-day precisions for the mean of this range varied from 0.59 to 1.1% and from 0.71 to 1.2%, respectively. Numerous potential interfering substances tested do not interfere.     

A Rapid Liquid Chromatographic Method for Determination of Flecainide in Human Blood Plasma Using Ultraviolet Detection

Abstract

A liquid chromatographic method for the assay of the antiarrhythmic drug flecainide in plasma has been developed. The method is rapid, simple and with sufficient detection sensitivity to render it suitable for therapeutic drug monitoring. Flecainide and added internal standard, a non-fluorinated analogue, were extracted by a single ether extraction from alkalinized plasma followed by a back-extraction of the ether with dilute phosphoric acid. A portion of the acid extract was then applied directly to a 30 cm ODS column eluting isocratically with 30% acetonitrile in water containing 0.01M dibutylamine phosphate. Monitoring was by ultraviolet detection at 214 nm and the total run time was 8 min. This method is specific and can quantitate plasma levels to less than 30 ng/ml (free base) from 0.5 ml of plasma without interference from antiarrhythmic drugs commonly used in therapy.     

A Rapid High Pressure Liquid Chromatographic Assay of Benoxaprofen in Plasma

Abstract

A rapid high-performance liquid chromatographic assay for the determination of the anti-inflammatory drug benoxaprofen in human plasma, is described. Plasma samples of 1.0 ml, to which benoxaprofen, and warfarin as an internal standard, had been added, were extracted with ether under acidic conditions. The samples were analyzed on a MicroPak CN-10 column using 25% acetonitrile in water (pH 2.5 with phosphoric acid). Detection was made on a variable wavelength UV absorbance detector at 309 nm.

Samples containing 0.5–10 μg benoxaprofen gave a mean extraction recovery from control plasma of 90.6 ± 6.8% (n=18). Stability tests have shown that benoxaprofen in plasma is stable for at least two weeks after freezing.     

Extractionless HPLC Method for the Determination of Naproxen in Human Plasma and Urine

Abstract

A simple and rapid (extractionless) high-performance liquid chromatographic method with ultraviolet detection, at 278 nm, is described for the determination of naproxen in human plasma and urine. Niflumic acid is used as internal standard. The chromatographic system consists of a reversed-phase C18-Spherisorb column with acetonitrile/0.1 M sodium acetate (35:65 v/v, pH 6.14) as the mobile phase. The retention time is 3.0 min for naproxen and 3.8 min for niflumic acid. The total run time is 5 min and the typical assay time is 10 min. The method is sufficiently sensitive for biopharmaceutical studies, after the oral administration of a single sustained release dose.     

Measurement of Cefixime in Serum and Cerebrospinal Fluid by High-Performance Liquid Chromatography

Abstract

Cefixime is a new cephalosporin antibiotic for oral administration. A high-performance liquid chromatographic (HPLC) method was developed to measure cefixime in small volumes of serum and cerebrospinal fluid (CSF) to conduct a pharmacokinetics study in pediatric patients. The assay involved precipitation of serum proteins with 6% trichloroacetic acid, using 7-hydroxycoumarin as an internal standard. Chromatographic separation was accomplished using ultrasphere C8 column and mobile phase containing 15% acetonitrile in a buffer at a detection wave length of 280 nm. The retention time of cefixime and 7-hydroxycoumarin was about 5 and 9 minutes, respectively. The method was suitable for quantitation of cefixime at a concentration ranging from 0.05 to 10 μg/ml. The coefficient of variation was less than 3%. The technique was used successfully to measure cefixime in serum and CSF obtained from an infant receiving cefixime.     

Simple High-Performance Liquid Chromatographic Method for the Analysis of Quinine in Human Plasma without Extraction

Abstract

A simple, rapid and sensitive assay, capable of quantitating quinine (Q) in human plasma samples is reported. The assay uses a reversed-phase C18 HPLC column packed with 5 μ ODS Hypersil. The chromatographic separation was accomplished with an isocratic mobile phase comprising acetonitrile-aqueous phosphate buffer pH 2 (50:50, v/v) containing 25 mM sodium dodecyl sulfate and 3 mM tetrabutylammonium bromide at a flow rate of 0.5 ml/min. The eluant was monitored by a fluorescence detector (excitation wavelength at 350 nm and emission wavelength at 450 nm). The assay was based on a simple plasma protein precipitation technique. To 200 μ of plasma sample, 400 μ of internal standard (cinchocaine 30 μ/ml in methanol) was added. After brief vortexing and centrifugation, the clear supernatant was injected onto the HPLC column. The inter- and intra-assay coefficients of variation were found to be less than 10%. The lowest limit of detection for Q in plasma was 18 ng/ml.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

High Pressure Liquid Chromatographic Assay of Cefamandole in Serum Following Intravenous and Intraperitoneal Administration

Abstract

Following cesarean section 102 women were treated with cefamandole by either perioperative intravenous administration or intraperitoneal irrigation. High-pressure liquid chromatographic (HPLC) methods for the quantitation of the low serum levels of cefamandole following intraperitoneal lavage were developed. The antibiotic was assayed in the serum using a standard microbiological assay and two types of reverse phase column technology for HPLC. The two HPLC systems were almost identical in performance. Both HPLC methods were at least 10-fold more sensitive than the microbiological assay. The correlation between the three methods was 0.9739. The half-life of cefamandole was 37 min, which was not significantly different from the half-life of the drug in serum of non-pregnant women. The peak serum levels were 47.6 ± 36.8 μg/ml and 1.98 ± 1.5 μg/ml for the intravenous and intraperitoneal methods of administration, respectively.     

Sensitive High-Performance Liquid Chromatographic Determination of Mebeverine in Plasma Using Fluorescence Detection

Abstract

A sensitive high-performance liquid chromatographic (HPLC) method for mebeverine (MB) determination in plasma is described. Sample preparation involves extraction of MB and Ibuprofen (internal standard) from 0.5 ml plasma. The analysis is carried out on reversed-phase chromatographic system using U-Bondapack C₁₈ column with a mobile phase consisting of water: acetonitrile:acetic acid (59:40:1) mixture. The effluent was monitored using a fluoremetric detection at excitation and emission wave lengths 270 and 362 nm, respectively. The method gave accurate, precise and reproducible results with high sensitivity. The within-day coefficients of variation ranged from 2.5 to 6.1% and between-days from 7.5 to 13.5% at four different concentrations. Injection-volumes containing as small amount of MB as 0.5 ng in plasma was detected. This method was applied to a bioavailability study with a single 10 mg/kg oral dose in two rabbits.