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.     

Creatinine imprinted poly(hydroxyethyl methacrylate) based cryogel cartridges

Creatinine imprinted cryogel (MIP) cartridge was prepared with functional monomer N-methacryloyl-(L)-histidinemethylester (MAH) under frozen conditions. Creatinine adsorption studies and selectivity of MIP cryogel were evaluated in aqueous solution and artificial urine sample. Maximum adsorbed amount of creatinine was calculated as 6.83 mg/g polymer for MIP cryogel. Langmuir and Freundlich adsorption isotherm models were used to investigate the adsorption behaviour of creatinine. In the artificial urine sample; recovery amounts of creatinine were found 34.7–46.2%. Creatinine imprinted cryogel (MIP) cartridge recognized creatinine, 4.58 and 4.37 times greater competitive molecules. MIP cryogel catridge was repeatedly used many times for adsorption desorption cycles.     

A novel reversed-phase HPLC method for the determination of urinary creatinine by pre-column derivatization with ethyl chloroformate: comparative studies with the standard Jaffé and isotope-dilution mass spectrometric assays

Creatinine is an important biomarker for renal function diagnosis and normalizing variations in urinary drug/metabolites concentration. Quantification of creatinine in biological fluids such as urine and plasma is important for clinical diagnosis as well as in biomonitoring programs and urinary metabolomics/metabonomics research. Current methods for creatinine determination either are nonselective or involve the use of expensive mass spectrometers. In this paper, a novel reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of creatinine of high hydrophilicity by pre-column derivatization with ethyl chloroformate is presented. N-Ethyloxycarbonylation of creatinine significantly enhanced the hydrophobicity of creatinine, facilitating its chromatographic retention as well as quantification by HPLC. Factors governing the derivatization reaction were studied and optimized. The developed method was validated and applied for the determination of creatinine in rat urine samples. Comparative studies with isotope-dilution mass spectrometric method revealed that the two methods do not yield systematic differences in creatinine concentrations, indicating the HPLC method is suitable for the determination of creatinine in urine samples.

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.     

Ammonia abatement in an enzymatic flow system for the determination of creatinine in blood sera and urine

The abatement of ammonia in standard solutions, and in human blood and urine samples is achieved by adding suitable amounts of NADPH and α-ketoglutarate to the sample and passing it through a 2-m nylon tube with glutamate dehydrogenase immobilized on the inner wall. The procedure provides removal of 98% of the ammonia (1–5 × 10^?4 M) in the original sample in 50 s. The abatement of ammonia permits the use of an ammonia probe coupled with an immobilized degradative enzyme for the determination of creatinine. Creatinine was determined in clinical blood and urine samples by first removing the ammonia from the sample and then cleaving the creatinine to N-methylhydantoin and ammonia with immobilized creatininase. Only 200 μl of sample is needed and the entire process is conducted in a single flow stream.     

Continuous-flow enzymatic determination of creatinine with improved on-line removal of endogenous ammonia

A new continuous-flow automated enzymatic method suitable for the direct determination of creatinine in physiological samples is described. The proposed system utilizes an on-line gas predialysis unit in conjuction with a flow-through enzyme reactor coil and a potentiometric ammonia detector. The enzyme reactor contains immobilized creatinine iminohydrolase (EC 3.5.4.21) which converts creatinine to ammonia and N-methylhydantoin. Ammonia liberated from this reaction is detected downstream with the membrane electrode-based detector. The novel gas predialysis unit effectively removes >99.8% of endogenous ammonia (up to 1 mM) present in the sample. Thus, final peak potentials recorded by the electrode detector are directly proportional to the logarithm of creatinine concentrations present. The method is shown to be precise (<3%), selective, and capable of accurately determining creatinine in serum and urine samples containing abnormally high endogenous ammonia levels. Determinations of creatinine in serum samples (n = 30) using this new method correlate well with an existing Technicon AutoAnalyzer colorimetric method (r = 0.996).     

Preparation of a reference material “Creatinine in Human Urine”

Two batches of a reference material “Creatinine in Human Urine” have been prepared with creatinine concentrations at the physiological level, and used in interlaboratory comparisons in which up to 26 laboratories participated employing up to 4 independent methods. The 95% confidence intervals obtained for the certified creatinine concentrations are better than the “acceptable ranges” of commercially control samples available for clinical laboratories, the certified values being traceable to mean values of the commercial control samples. Thus, a suitable reference material has been prepared for the quality assurance of environmental and occupational health studies in which the concentration of a pollutant or its metabolites in human urine has to be related to the creatinine concentration.     

Rapid Determination of Creatinine in Human Urine by Microchip Electrophoresis with LED Induced Fluorescence Detection

In clinical medicine, urine creatinine concentration is an important marker in the evaluation of renal function and muscular dysfunctions. Herein, we reported a novel method for rapid determination of creatinine in urine by microchip electrophoresis with light-emitting diode induced fluorescence detection. Creatinine was derivatized by fluorescein isothiocyanate, and then quantitatively detected by the developed microchip LED induced fluorescence detection system. The excitation and emission wavelengths were 490 and 523 nm, respectively. The urine samples were analyzed after centrifuge and filtration. A baseline separation was obtained in <30 s using 10 mM borate buffer (pH 9.0, containing 45 mM sodium dodecylsulfate), with separation voltage of 1.5 kV. Good linearity was obtained (r ² = 0.9978) in the concentration range of 10.0–2.00 × 10³ μM, and the limit of detection was 2.87 μM (S/N = 3). The recovery was 96.0–107 %, and the interday precision was <4.5 % (n = 6). To validate assay results, we compared the present method with the Jaffe’s colorimetric assay by measuring real urine samples. The method was reliable, sensitive, high-speed, low-cost and suitable for the routine analysis of creatinine in biofluids.

     

Clinical analysis of human urine by means of potentiometric Electronic tongue

The Electronic tongue (ET) composed of different kind of potentiometric chemical sensors has been applied for the detection of urinary system dysfunctions and creatinine levels. The creatinine contents evaluated by ET were compared with those obtained by automated Jaffe’s method and GC-MS, obtaining a satisfying agreement for both methods. Partial least square regression discriminate analysis (PLS-DA) and feed forward back-propagation neural network (FFBP NN) classified 51 urine specimens from healthy volunteers in four classes, according to the creatinine content, showing that both techniques can satisfactorily differentiate urines according to this parameter. The best accuracy result of 92.2% correct classification of unknown samples was achieved with FFBP NN. Moreover, the possibility of ET system to distinguish between urine samples of healthy patients, and those with malignant and non-malignant tumor diagnosis of bladder has been shown.     

Creatinine biosensor based on ammonium ion selective electrode and its application in flow-injection analysis

A new, highly sensitive, fast responding and stable potentiometric biosensor for creatinine determination is developed. The biosensor is based on an ammonium ion-selective electrode. Creatinine deiminase (EC 3.5.4.21) is chemically immobilized on the surface of the polymeric ion-sensitive membrane in the form of monomolecular layer using a simple, one-step carbodiimide covalent attachment method. The resulting enzyme electrodes are useful for measurement under flow injection analysis (FIA) conditions. The biosensors exhibit excellent operational and storage stability. The enzyme electrodes retain over 70% of initial sensitivity after ten weeks of work under FIA conditions. The storage stability at 4 °C is longer than half a year without loss of sensitivity. Under optimized conditions near 30 samples per hour can be analyzed and the determination range (0.02-20.0 mmol l⁻¹) fully covers creatinine concentrations important from clinical and biomedical point of view. The simple biosensor/FIA system has been successfully used for determination of creatinine in urine, serum and posthemodialysate samples.     

Development of a fast capillary electrophoresis method for determination of creatinine in urine samples

The aim of this work was to develop a fast method using capillary electrophoresis for the determination of creatinine in human urine samples. The pH and constituents of the background electrolyte were selected by inspection of effective mobility of creatinine and candidate urine interferents versus pH curves. The tendency of the analyte to undergo electromigration dispersion and the buffer capacity were evaluated by the Peakmaster software and considered in the optimization of the background electrolyte, composed by 10 mmol L(-1) tris(hydroxymethyl)aminomethane and 20 mmol L(-1) 2-hydroxyisobutyric acid (HIBA) at pH 3.93. Separation was conducted in a fused-silica capillary (32 cm total length and 8.5 cm effective length, 50 microm I.D.), with short-end injection configuration and direct UV detection at 215 nm. The migration time of creatinine was only 22s. A few figures of merit of the method are as follows: good linearity in the concentration interval of 5-70 mg L(-1) (R(2)>0.99), limit of detection of 0.5 mg L(-1), inter-day precision better than 2.7% (n=9) and recovery in the range 99.0-103.7% at three concentration levels (50, 100 and 150 mg L(-1)). Urine samples were prepared by deproteination with acetonitrile (1:3 sample:acetonitrile, v/v), centrifugation and dilution of a deproteinated aliquot with 12.5 mmol L(-1) HIBA (1:4, v/v). Creatinine concentrations between 489 and 1063 mg L(-1) were obtained in the urine of four healthy volunteers.     

Determination of polyamines in urine of normal human and cancer patients by capillary gas chromatography

A capillary gas chromatographic method is described for the determination of polyamines (putrescine, spermidine and spermine) in the urine of normal human and cancer patients. Morning urine after acid hydrolysis is cleaned up on a silica gel column and derivatized with trifluoroacetic-anhydride. Creatinine in human urine is used as internal standard. Recoveries of polyamines are 96.7% putrescine, 102.6% spermidine (Spd), and 98.7% spermine. SD of the method for Spd is 1.949 +/- 0.041 (micrograms/mg creatinine, mean +/- SD, n = 5). The results show that the mean level of polyamines in cancer patients urine is much higher than that in normal human urine. The mean of total polyamines in the normal human and the cancer patients is 2.01 and 44.74, respectively (g/mg creatinine).     

Stopped-flow determination of creatinine in human serum and food samples

Summary The results obtained by applying a modular stopped-flow system for the determination of creatinine by the alkaline picrate method are reported. The stopped-flow technique allows the simple and rapid determination of creatinine in human serum and food samples. The calibration graph is linear in the range 1–100 g ml^–1 of creatinine and the precision is close to 3%. The method features acceptable selectivity. The initial rate is measured in only 5 s, which allows the easy application of the method to routine analyses. No pretreatment of the serum samples is required. For food samples, total creatine and creatinine have been determined in dehydrated soup and meat extract samples.

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Stopped-flow-Bestimmung von Creatinin in Humanserum und Nahrungsmitteln

     

Alternative method for measurement of albumin/creatinine ratio using spectrophotometric sequential injection analysis

A simple, automatic and practical system for successive determination of albumin and creatinine has been developed by combining sequential injection analysis (SIA) and highly sensitive dye-binding assays. Albumin detection was based on the increase in the absorbance due to complex formation between albumin and eosin Y in acidic media. The absorbance of the complex was monitored at 547 nm. For the creatinine assay, the concentration of creatinine was measured by reaction with alkaline picrate to form a colored product which absorbs at 500 nm. The influences of experimental variables such as effects of pH, reagent concentration, standard/sample volume and interferences were investigated. Under optimal conditions, the automated method showed linearity up to 20 mg L⁻¹ for albumin and 100 mg L⁻¹ for creatinine. The 3σ detection limits were 0.6 and 3.5 mg L⁻¹ for albumin and creatinine, respectively, and the relative standard deviations (n = 10) were 2.49% for 20 mg L⁻¹ albumin, and 3.14% for 20 mg L⁻¹ creatinine. Application of the proposed method to the direct analysis of urinary samples yielded results which agreed with those obtained from the Bradford protein assay and a creatinine enzymatic assay according to a paired t-test. The results obtained should be a step towards developing a fully automated and reliable analytical system for clinical research, which requires direct determination of albumin and creatinine and/or its ratios.     

Simultaneous determination of creatinine and uric acid in human plasma and urine by micellar electrokinetic chromatography

High performance capillary electrophoresis using a buffer solution containing micelles of ionic surfactant (e.g. sodium dodecyl sulfate), called micellar electrokinetic chromatography, has been applied to the separation and simultaneous determination of creatinine and uric acid in human plasma and urine. The sample was introduced into the capillary by siphoning an appropriate volume of untreated plasma or urine spiked with an internal standard (antipyrine). Creatinine, uric acid, and antipyrine were separated mutually, and from other endogeneous components within 18 min. The calibration plots showed good linearity (correlation coefficient > 0.999) over the concentration range needed for clinical analysis. Standard addition tests indicated that the recoveries of creatinine and uric acid from urine samples ranged, respectively, from 97 % to 106 % and 97.4 % to 108 % with a coefficient of variation (C.V.) of 3.3 % (n = 5), and that those from plasma samples ranged, respectively, from 100 % to 112 % and 101 % to 107 % with a C.V. of 4.7 % (n = 5). The results were in agreement with those obtained by conventional methods.     

Spectrophotometric simultaneous determination of creatinine and creatine by flow injection with reagent injection

The reactions of creatinine with picric acid and of creatine with 1-naphthol and biacetyl, both in an alkaline medium, have been used to develop a flow injection method for the simultaneous determination of creatinine and creatine, respectively. The sample containing both analytes is continuously merged with a picrate stream and mixed through the reactors; the coloured stream passes through a flow cell in a spectrophotometer set at 520 nm, recording a continuous signal proportional to the creatinine concentration. The mixture of the reagents 1-naphthol and biacetyl is inserted into the stream by use of the injection valve, which results in a peak (superimposed on the continuous signal), proportional to the creatine concentration. Linear calibration graphs for both analytes were obtained up to 30 mg l^–1 with relative standard deviations <2%, and a sampling rate of 42 measurements h^–1. The method was applied to the determination of creatinine and creatine in broth cube samples.     

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from Cl was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from Cl after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/1 in water). Diluted serum and urine could be injected onto C1, and Cl was backflushed after the transfer of uric acid from Cl to C5.

Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 μmol/1, respectively) was 98.9±0.56% and 100.9±1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 μmol/l, respectively) was 99.4±0.72% and 98.7±1.45%, respectively (mean±R.S.D., n=6).     

Study of the stoichiometry and the kinetics of the Jaffé reaction with a picrate ion-selective electrode

Potentiometric studies with the picrate ion-selective electrode indicate that the species formed as the product of reaction between alkaline picrate and creatinine in the Jaffé reaction is a 1:1 complex. Kinetic studies indicate that the forward reaction is first order with respect to picrate, creatinine, and hydroxide concentration. The second-order rate constant k was found to be in the range 9.1–10.4 M⁻² sec⁻¹ at 27 °C and μ = 1.00, with creatinine or picrate in excess, k increases with increasing μ and temperature. An activation energy of 10.1 kcal/mol was calculated for the Jaffé reaction, with creatinine in excess.     

高效毛细管区带电泳测定人尿液中尿酸、肌酐和伪尿核苷的含量

报道了采用高效毛细管区带电泳技术直接将人尿液注入毛细管进行尿液中肌酐、尿酸及伪尿核苷含量测定的新方法。试验表明,以磷酸盐（ｐＨ６．１）作缓冲液,对人体尿液中肌酐、尿酸及伪尿核苷进行直接分析具有较高的灵敏度和较好的重复性。     

Gas chromatographic determination of 2-ethylhexanoic acid in urine as its pentafluorobenzyl ester

A gas chromatographic (GC) method was developed for the determination of 2-ethylhexanoic acid (2-EHA), initially in the urine of animals, but subsequently in samples of urine from sawmill workers in order to evaluate their exposure to 2-EHA which is used as a wood preservative. The 2-EHA was derivatised to the pentafluorobenzyl ester, which was then analysed by means of a cross-linked methyl silicone GC column with electron capture detection. Gas chromatography-mass spectrometry was used to confirm the identity of the GC peaks. The analytical range of the method was 0.03-2.70 mmol of 2-EHA per mol of creatinine in urine and the limit of detection was 0.01 mmol per mol of creatinine. The recovery of 2-EHA was 81-90% with a coefficient of variation of 9.8%. The amount of 2-EHA excreted in urine was corrected for the excretion of creatinine. The concentration of 2-EHA in the urine of the workers studied varied from 0.01 to 5.40 mmol per mol of creatinine; the median was 0.1 mmol per mol of creatinine.