Quantitation of Creatinine in Urine and Plasma Samples by Reversed Phase HPLC

Abstract

Creatinine determination in urine and plasma affords an index of the renal function. Reversed-phase high pressure liquid chromatography was used for the separation and quantitation of creatinine in normal and arsenic exposed human urine samples. Acetonitrile/water (1:1) was the mobile phase. The method was compared with the Jaffé alkaline picrate reaction. Results show that the HPLC procedure has high reproducibility and samples are stable at the storage conditions. Plasma samples required depro-teinization and extraction with CH₃CN prior to HPLC analysis, while urine samples required only centrifugation.     

Chromatographic Properties of Ethanol/Water Mobile Phases on Silica Based Monolithic C18

A simple and reliable method based on capillary electrophoresis with electrochemical detection (CE–ED) was applied to study the effect of aerobic exercises on creatinine and uric acid concertration in saliva and urine. The pH value, the running buffer concentration, the SDS concentration, separation voltage, injection time and the potential applied to the working electrode were investigated to find the optimum conditions. The detection limits (S/N = 3) for creatinine and uric acid were 3.6 μmol L⁻¹ and 0.86 μmol L⁻¹, respectively. This method was successfully used in the rapid analysis of creatinine and uric acid in saliva samples. After aerobic exercises, creatinine concentration decreased, and uric acid concentration increased in saliva. In urine, the concentrations of creatinine and uric acid both increased after exercise.

     

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.     

HPLC Analysis of Cefmetazole and Nocardicins A and E in Human Serum and Urine Using Solid-Phase Extraction

Abstract

A method for extraction and quantification of cefmetazole and nocardicins A and E in serum and urine samples is described in this paper. Sample pretreatment is carried out using solid-phase extraction cartridges, resulting in very high extraction recoveries of these β-lactam antibiotics. The procedure, which prepares biological fluids for reversed-phase high-performance liquid chromatographic analysis is convenient, rapid and reproducible. An water-methanol-acetic acid mobile phase was used with benzotriazole as an internal standard. The detection limit was 0.2 μg/ml at 280 nm.     

Revised HPLC Determination of Atenolol in Plasma and Urine

Abstract

An HPLC method for analysis of atenolol in human plasma and urine is presented. Based on alkaline extraction, acid backextraction and reverse phase ion-pair chromatography this method is quite specific for atenolol. For a 0.5 ml plasma sample the sensitivity ranges from 20 ng/ml in fasted healthy volunteers to 50 ng/ml in various groups of patients. A sensitivity in urine of 1.0 mcg/ml was sufficient for all samples studied. As presented this method has been used in several clinical pharmacokinetic studies involving hundreds of samples.     

Enzymatic Fluorimetric Determination of Ursodeoxycholic Acid in Urine Using Clostridium Absonum 7β-Hydroxysteroid Dehydrogenase

Abstract

A simple and rapid enzymatic fluorimetric method for the determination of ursodeoxycholic acid (UDCA) and its glycine (GUDCA) and taurine (TUDCA) conjugates in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C₁₈) are used for the solid-phase extraction of bile acids (BA) from urine samples. the method is based on the fluorimetric monitoring of NADPH formed via the reaction of 7β hydroxylated BA (7β-BA) with β-nicotinamide adenine dinucleotide phosphate (β-NADP⁺) catalysed by 7β hydroxysteroid dehydrogenase (7β-HSD). the 7β-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC # 27555) and purified by affinity chromatography.

The method has a limit of detection of 2 μmol/L (initial sample concentration), within-run precision varied from 8.3% to 5.3% and between-run precision varied from 12% to 1.8% for low and high concentrations respectively. the recovery of ursodeoxycholic acid added to urine samples was about 98% (range 88–110%). the method was successfully applied for UDCA determination in urine samples from patients subjected to UDCA therapy. Randomly collected urine samples from patients and controls were used and the results were expressed as ratio of [UDCA]/[creatinine] to correct for variation in urine flow.     

Comparison of a GC Method and Two HPLC Methods for the Determination of p-Hydroxyphenytoin in Urine

Abstract

Three chromatographic methods for determining p-hydroxy-phenytoin (p-HPT) in urine were compared: (1) GC with derivatisation of the samples, (2) HPLC after extraction with ethyl acetate and (3) HPLC using a column switching system for direct injection of samples. In all three methods the p-HPT glucuronides were first hydrolysed using concentrated mineral acid at boiling point. For method (1) the acidic hydrolysate was adjusted to pH 7–8.5. Benzenetetrahydrofuran was used for extraction of p-HPT. The extract was evaporated to dryness, taken up in trimethyl-aniliniumhydroxide and injected. For method (2) the acidic hydrolysate was buffered with tri-sodium phosphate. An aliquot of the buffered solution was extracted with ethyl acetate. The extract was evaporated to dryness, taken up in methanol and injected. For method (3) the hydrolysate was diluted with water/acetonitrile (9:1), centrifuged and directly injected onto the pre-column for the sample washing step.     

CE Determination of Creatinine and Uric Acid in Saliva and Urine During Exercise

A simple and reliable method based on capillary electrophoresis with electrochemical detection (CE–ED) was applied to study the effect of aerobic exercises on creatinine and uric acid concertration in saliva and urine. The pH value, the running buffer concentration, the SDS concentration, separation voltage, injection time and the potential applied to the working electrode were investigated to find the optimum conditions. The detection limits (S/N = 3) for creatinine and uric acid were 3.6 μmol L^?1 and 0.86 μmol L^?1, respectively. This method was successfully used in the rapid analysis of creatinine and uric acid in saliva samples. After aerobic exercises, creatinine concentration decreased, and uric acid concentration increased in saliva. In urine, the concentrations of creatinine and uric acid both increased after exercise.     

A Fiber Optic Biosensor for Uric Acid Based on Immobilized Enzymes

Abstract

A simplified enzyme based fiber optic uric acid biosensor is reported. It is constructed by coimmobilizing uricase and horseradish peroxidase [HRP] to bovine albumin via glutaraldehyde. A new fluorimetric substrate, thiamine, is used to indicate the sensing process. Under optimized conditions, the linear range of uric acid concentration is 0.5 to 5.0 ug/mL with a correlation coefficient of 0.997, and the detection limit is 0.15 ug/mL with excellent reversibility and stability. The sensor can be used at least two months at room temperature. It is possibly to determine uric acid directly in serum and urine samples with satisfactory results.     

Quantitation of Benzodiazepine Hydrolysis Products in Urine Using Solid-Phase Extraction and High Performance Liquid Chromatography

Abstract

Solid-phase extraction and high performance liquid chromatography (HPLC) were used to indirectly (via products of acid hydrolysis) confirm the presence of benzodiazepine metabolites in the urine of patients who had received overdoses of these compounds. The use of solid-phase extraction method for quantitation of benzodiazepine hydrolysis products in urine offers numerous advantages in comparison to extraction with chloroform. The chromatograms of urine extracts were free of interferences. The recoveries of the benzodiazepine hydrolysis products and the internal standard were greater than 96%, which makes this method highly reliable for quantitative analytical purposes.     

The Kinetics and Mechanism of the Hydrolysis of Creatinine in Urine

Abstract

Creatinine in urine concentrations are routinely measured at Aldermaston by an autoanalyser, using the Jaffe reaction, as an index of urinary excretion rates. These values are used in calculations to estimate the body content of radionuclides from their urinary excretion rates.

Unfortunately, creatinine in urine concentrations gradually decrease with sample age due to pseudo first order hydrolysis of creatinine to give creatine in the presence of ammonia. This reaction may be arrested or reversed by mineral acid.

After storage at ambient temperatures for several weeks the creatinine in urine concentration falls by around 20%, so it is good practice to analyse samples soon after provision.

The activation energy for the hydrolysis of creatinine in urine is around 60 KJ/mol over the range 20–70 °C. Hence, raising the temperature by 10 [ddot]C approximately doubles the reaction rate.     

The Determination Ofritalinic Acid in Urine by Gas Chromatography

Abstract

A gas chromatographic method for the analysis of ritalinic acid, the major metabolite of methylphenidate, in urine has been developed. Solid phase extraction is employed to afford high recovery of ritalinic acid. The method has a lower limit for reliable quantitation of 1 μ/ml. The reliability and sensitivity of this method make it suitable for human studies.     

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.     

An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH₂Cl₂ containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH₃CN containing IS.

Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH₂PO₄, pH 3.5 - CH₃OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Separation and Quantitation of Urinary Phthalates by HPLC

Abstract

A method was developed for the separation and quantitation of plasticizers and their metabolites from human urine using HPLC, Urine was diluted with an equal volume of water and extracted at pH 2.0 with diethyl ether, The extract was dried, the solvent vacuum stripped, and the residue dissolved in methanol for injection into the chromatograph. A C₁₈ reverse phase column containing 10 μ particles was used for the analysis. Ionic suppression, 0.5% acetic acid in water, at pH 3.0 was used to resolve the acidic components. A step gradient of acetonitri1e:water (containing acetic acid) was used to elute the polar metabolites as well as the non-polar plasticizers. Mass spectrometry was used t o identify the compounds in the HPLC fractions. From the HPLC fractions of the urine extract collected, phthalic acid, MEHP, DEHP and normal urinary constituents (e.g., hippuric and benzoic acid derivatives) were identified     

A Semi-Automated Colorimetric Method for the Analysis of Dihydroxyphenylacetic Acid (DOPAC) in Urine of Parkinsonian Patients Receiving L-Dopa

Abstract

An analytical system is presented which permits the separation and analysis of urinary dihydroxyphenylacetic acid (DOPAC). This method is suitable for analyzing the urine from both normal and Parkinsonian patients receiving L-Dopa.     

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.     

Non-enzymatic sensing of uric acid using a carbon nanotube ionic-liquid paste electrode modified with poly(β-cyclodextrin)

We describe a nonenzymatic electrochemical sensor for uric acid. It is based on a carbon nanotube ionic-liquid paste electrode modified with poly(β-cyclodextrin) that was prepared in-situ by electropolymerization. The functionalized multi-walled carbon nanotubes and the surface morphology of the modified electrodes were characterized by transmission electronic microscopy and scanning electron microscopy. The electrochemical response of uric acid was studied by cyclic voltammetry and linear sweep voltammetry. The effects of scan rate, pH value, electropolymerization cycles and accumulation time were also studied. Under optimized experimental conditions and at a working voltage of 500 mV vs. Ag/AgCl (3 M KCl), response to uric acid is linear in the 0.6 to 400 μΜ and in the 0.4 to 1 mΜ concentration ranges, and the detection limit is 0.3 μΜ (at an S/N of 3). The electrode was successfully applied to the detection of uric acid in (spiked) human urine samples.

SEM images of (a) carbon ionic liquid electrode (CILE) (b) MWNT-CILE (c) β-CD/CILE (d) β-CD/ MWNT-CILE. The surfaces of carbon ionic liquid electrode (CILE) (a) and MWNT-CILE (b) were homogenous and no separated carbon layers can be observed; After β- cyclodextrin (CD) was modified on CILE and MWNT-CILE, the surfaces of β-CD modified electrodes (c and d) exhibited loose and porous morphologies.

     

Determination of uric acid and creatinine in human urine using hydrophilic interaction chromatography

Uric acid is the end-product of purine metabolism and a major antioxidant in humans. The concentrations of uric acid in plasma and urine are associated with various diseases and routinely measured in clinical and biomedical laboratories using enzymatic conversion and colorimetric measurement. In this study a hydrophilic interaction chromatographic (HILIC) method was developed for simultaneous determination of uric acid and creatinine, a biomarker of urine dilution and renal function, in human urine. Urine samples were pretreated by dilution, protein precipitation, centrifugation and filtration. Uric acid and creatinine were separated from other components in urine samples and quantified using HILIC chromatography. A linear relationship between the ratio of the peak area of the standards to that of the internal standard and the concentration of the standards was obtained for both uric acid and creatinine with the square of correlation coefficients >0.999 for both analytes. The detection limits were 0.04 μg/mL for creatinine and 0.06 μg/mL for uric acid. The described HILIC method has proved to be simple, accurate, robust and reliable.     

High Pressure Liquid Chromatographic Determination of Mecillinam in Human Plasma and Urine

Abstract

A simple, specific, rapid and sensitive method for the analysis of mecillinam in plasma and urine using high pressure liquid chromatography is described. The assay is performed by direct injection of a plasma protein free supernatant or a dilution of urine. A μBondapak phenyl column with an eluting solvent of 16% CH₃CN-0.2% H₃PO₄ was used, with UV detection of the effluent at 220 nm. Desacetyl-cephalothin was used as the internal standard and quantitation was based on peak height ratio of mecillinam to that of the internal standard. The lowest concentration detectable without extraction was 0.25 μg/ml for plasma and 8.9 μg/ml for urine. No interference from plasma and urine was noted.