Patent Description:
Creatinine is one of the metabolites of nitrogen compounds, present in all body fluids. It has no biological function and is removed from the bloodstream in kidneys (by glomerular filtration) and then excreted in urine from the body. Serum creatinine level is a marker of kidney filtration function and therefore a marker of a number of diseases.

In clinical laboratories, the accuracy and speed of a single creatinine determination in a clinically significant sample play an important role, and creatinine itself, beside glucose, is one of the most frequently determined metabolites. Creatinine level is routinely measured in serum and urine samples, mainly to assess kidney function, but also in diagnostics of diseases resulting in muscle loss, as an indication of urine dilution, and to predict the risk of acute renal failure.

There are many known methods of creatinine determination in biological samples. They cover the entire spectrum of instrumental techniques available today: from spectroscopic techniques (photometry, fluorimetry), through electrochemical techniques, including current and potentiometric methods, to separation techniques: chromatography and electromigration.

In analytical practice, the Jaffé method developed in <NUM> [<NPL>] is recommended for routine creatinine determination in clinically significant samples, despite its numerous disadvantages, including-Low selectivity for among others glucose and proteins. In the case of determinations in more advanced investigations, definitely more selective enzymatic methods are used although they are also more expensive.

The Jaffé method for the determination of creatinine in biological samples is based on the reaction between creatinine and picric acid (<NUM>,<NUM>,<NUM>-trinitrophenol) in an alkaline environment. The reaction product is an orange creatinine-picric acid complex/adduct, which can be determined spectrophotometrically in the wavelength range <NUM>-<NUM>. One of the postulated course of the reaction may be presented in the following reaction scheme:
<CHM>.

The Jaffé reaction was adapted to the colorimetric determination of creatinine in urine in <NUM> [<NPL>] and in deproteinised blood in <NUM> [<NPL>]. Typical reagent concentrations and analytical parameters for the Jaffé protocol are shown in Table <NUM>.

One of the most significant drawbacks and disadvantages of the Jaffé method is the fact that the course of the reaction is highly temperature dependent, and therefore the slope of the orange complex calibration curve is also temperature dependent. In the wavelength range of <NUM>-<NUM>, the absorbance of the creatinine-picric acid complex/adduct increases with increasing temperature, with the degree of increase in absorbance being different for different wavelengths at which it is measured. For this reason, an increase in temperature by <NUM> can cause an error in the measured creatinine concentration by up to <NUM>µmol·L-<NUM> if the measurement is made at <NUM>, while when the absorbance is measured at wavelengths longer than <NUM>, an error caused by temperature fluctuations is several times smaller. In addition, picric acid used in the Jaffé method, as a dry solid reagent, is an explosive substance and toxic to humans in case of contact with skin, inhalation and ingestion. Therefore, the preparation of the reagent must be carried out by trained personnel.

The main disadvantage of the Jaffé method, however, is its low selectivity. Creatinine is not the only compound that forms a red complex when reacted with picric acid, and a similar effect is also observed in the presence of acetone or glucose. The list of interferents of the Jaffé method includes more and more substances, including: proteins, bilirubin, glucose, ascorbic acid, acetoacetates and antibiotics belonging to the cephalosporin group, and the total number of known interferents already reaches over <NUM>. Some of them increase and other reduce the signal, while in comparison with more reliable methods, the Jaffé method increases the obtained results by about <NUM>µmol·L-<NUM>, at average [<NPL>].

Many attempts have been made to increase selectivity of the Jaffé method, but the only method routinely used in clinical analysis is a discriminant - sometimes called kinetic or two-point, measurement [<NPL>]. As the analytical signal, instead of the absorbance, the difference in absorbance measured at <NUM>th and <NUM>th second of the reaction at a wavelength of <NUM> is used. In this way, the influence of compounds, for example bilirubin, that react with picric acid with kinetics different to that of creatinine, is eliminated. Another advantage of using the two-point measurement is elimination of the influence of the sample color on the obtained result, because both blood serum and urine absorb radiation in the analytical range of the Jaffé method. The presented modification of the Jaffé method does not release it from all the known drawbacks. First of all, the influence of proteins on the measured creatinine concentration is not eliminated. Moreover, the double absorbance measurement creates a difficulty in the measurement methodology and causes the propagation of measurement uncertainty.

As an alternative to the attempts to modify the Jaffé method in order to improve its selectivity, it was proposed to replace picric acid with another nitro derivative of benzene, namely <NUM>,<NUM>-dinitrobenzoic acid (DNBA) [<NPL>; <NPL>; <NPL>]. The reaction is analogous to the reaction with picric acid according to the scheme:
<CHM>.

Due to the introduced change of the reagent, the method is more selective for creatinine than the classic Jaffé method, because compounds such as glucose, fructose, creatine or guanidine do not affect the measurement [<NPL>]. The kinetics of the creatinine reaction with <NUM>,<NUM>-dinitrobenzoic acid is largely dependent on the concentration of sodium hydroxide: at low concentrations the product forms slowly but is stable over time, while at higher concentrations the reaction kinetics are faster but the product decomposes. The results with the best reliability are obtained with a <NUM>,<NUM>-dinitrobenzoic acid solution in a concentration of <NUM> mmol·L-<NUM> and a sodium base solution of the same concentration, whereas the absorbance reading at <NUM> wavelength is made <NUM> minutes after mixing the reagents [<NPL>].

There are also reports on methods based on fluorimetric detection. The basis of the described fluorescent kidney function test is the creatinine reaction with an alkali metal salt of <NUM>,<NUM>-dinitrobenzoic acid [<NPL>; <CIT>)].

There is known a fluorimetric method of creatinine detection that employs a palladium (II) complex of naphthalimide derivative, which complex does not show fluorescence in a buffer of pH <NUM>. After addition of creatinine solution, the palladium ion is removed from the complex and is bound by the analyte molecule. The ligand itself (naphthalimide derivative), devoid of the palladium ion, fluoresces with an emission maximum at a wavelength of <NUM>, when excited at a wavelength of <NUM>. The dependence of the creatinine concentration on the fluorescence intensity can be approximated by the square function within the range of creatinine concentrations ranging from <NUM> to <NUM>µmol·L-<NUM> [<NPL>]. The developed protocol shows a correlation with the results obtained by the Jaffé method.

There is known a fluorimetric method of creatinine determination involving its reaction with a chalcone derivative [<NPL>]. This compound is characterised by a quantum yield of <NUM> at pH of <NUM> and, when excited by radiation of <NUM> wavelength, it has two emission maxima: one at a wavelength of <NUM> and the other at <NUM>, and the presence of creatinine shifts the emission maximum of the compound towards shorter waves. Other metabolites present in biological samples, such as urea, bilirubin, amino acids and proteins, and metal ions do not cause such a shift. The method is selective for creatinine, but is characterised by low precision (RSD <NUM>%) [<NPL>].

In literature there are reports on other systems for the fluorimetric determination of creatinine concentration. For example, there is known a method based on increasing the emission of rhodamine complex with gold(III) ions in the presence of creatinine [<NPL>], and a method employing an interaction of creatinine with quantum dots coated with thioglycolic acid [<NPL>].

An alternative type of creatinine determinations in body fluids are enzymatic methods. This group of analytical methods provides much greater selectivity in effect of the substrate specificity of the enzyme used. In such methods, the analyte is converted by one or more enzymatic reactions into a product that can be easily determined by spectroscopic or electrochemical techniques.

The only method of enzymatic creatinine determination, which apart from the Jaffé method, is routinely used in clinical laboratories, is the colorimetric method for the determination of hydrogen peroxide resulting from a cascade of enzymatic reactions:.

creatinine + H<NUM>O -EC <NUM>. <NUM> -→ creatine.

creatine + H<NUM>O -EC <NUM>. <NUM>-→ urea + sarcosine.

sarcosine + O<NUM> + H<NUM>O -EC <NUM>. <NUM>-→ glycine + H<NUM>O<NUM>.

The hydrogen peroxide is converted into the absorbing compound by another enzymatic reaction, catalysed by peroxidase (EC <NUM>. <NUM>) in the presence of an acceptor. The absorbance of the reaction product is measured at <NUM>. The linear range of the method is up to <NUM>µmol·L-<NUM> creatinine and the incubation time is <NUM> minutes. The method is characterised by high selectivity, however, in order to eliminate interference caused by ascorbic acid, the addition of ascorbate oxidase is necessary, while the addition of potassium ferrocyanide reduces the reduction of the signal caused by the presence of bilirubin [<NPL>].

Currently, a modified version of the above method is used in routine clinical analysis. <NUM>,<NUM>-Dichloro-<NUM>-hydroxybenzenesulfonic acid is replaced by a more effective hydrogen peroxide scavenger: <NUM>,<NUM>,<NUM>-triiodo-<NUM>-hydroxybenzoic acid. The product of the enzymatic reaction is determined colorimetrically at a wavelength of <NUM>. The method has a creatinine detection limit of <NUM>µmol·L-<NUM>, while its linear range is <NUM>µmol·L-<NUM>. The precision and repeatability, expressed as relative standard deviation, are less than <NUM>%. The selectivity of the colorimetric enzyme method is much betterthan that of the Jaffé method. No influence of bilirubin (up to <NUM>µmol·L-<NUM>), hemoglobin (up to <NUM>µmol·L-<NUM>), ascorbic acid (up to <NUM>µmol·L-<NUM>), creatine (up to <NUM> mmol·L-<NUM>) and most drugs for the results obtained has been noticed.

Isotope Dilution Mass Spectrometry (IDMS) is considered a completely selective (i.e. specific) method for determination of creatinine. The measurement procedure consists in enriching the sample with <NUM>C-labeled creatinine. The creatine is then separated from the sample using a weal< acid ion exchange resin column and the creatinine is converted to N-(<NUM>,<NUM>-dimethyl-<NUM>-pyrimidinyl)-N-methylglycine ethyl ester. The next step is the analysis by mass spectrometry of the intensity ratio of peaks related to the creatinine derivative being not enriched and the isotopically enriched creatinine derivative [<NPL>]. The method is characterised by a very high precision and selectivity and is currently considered the reference method for determining creatinine. Unfortunately, the measurement of creatinine concentration by this method is very laborious and takes more than <NUM> hours (including sample preparation), and that is why it is not used routinely.

Another available method for determining creatinine is capillary electrophoresis, employing differences in the rate of compounds migration in an electric field.

It is also known to use high-performance liquid chromatography to determine the concentration of creatinine in biological samples. Creatinine is separated from the other sample components on a Nucleosil <NUM>-<NUM> C18 packed column. A mixture of water and acetonitrile (<NUM>% v/v acetonitrile) with the addition of sodium salt of octane sulfonic acid is used as an eluent. The latter compound acts as a reagent for the formation of ion pairs, being recommended due to the fact that that creatinine is a cation under the conditions of the measurement. Spectrophotometric detection is made at a wavelength of <NUM> or <NUM>. In such a system, the creatinine retention time is <NUM> minutes. The limit of creatinine determination under optimised conditions is <NUM> pmol·L-<NUM>. This method has been proven effective in urine and blood serum samples. None of the other compounds present in such samples is eluted together with creatinine [<NPL>).

The publication No. <CIT> discloses a method for determining creatinine content or concentration in biological samples, wherein creatinine is enzymatically transformed to glycine, acetaldehyde and hydrogen peroxide and proportioning one of the reaction end products. It is possible to proportionate hydrogen peroxide enzymatically in the presence of compounds giving a chromogen compound effective to be colourimetrically analysed.

The <CIT> reveals methods for measuring analyte alone or in combination with total protein in biological samples using one or more colorimetric reagents alone or in combination with protein precipitation reagents. For determination of creatinine in a urine sample an aqueous solution of <NUM>,<NUM>-dinitrobenzoylchloride in phosphate buffer (pH <NUM>) or alternatively hydrogen peroxide and an oxidizable dye are used to obtain a fluorophore being further processed. A combination of both reagents, i.e., <NUM>,<NUM>-dinitrobenzoilchloride and hydrogen peroxide is not disclosed.

The reaction of p-dinitrobenzene with H<NUM>O<NUM> and NaOH in <NUM>%, <NUM>%, and <NUM>% aqueous dioxane carried out at <NUM>, involving formation of a reasonably stable intermediate which absorbs strongly in the visible region, has been an object of kinetic studies [<NPL>]. Although in the reaction mixture fluorophore is formed it is not suitable as a multi-compound fluorimetric reagent due to the fact that formation of fluorophore takes place immediately after mixing the p-dinitrobenzene with the remaining components and is not suitable for storage.

A fluorescent kidney function test based on reactivity of creatinine with alkaline <NUM>,<NUM>-dinitrobenzoate has been reported back in <NUM> [<NPL>]. A simple, sensitive and highly specific fluorimetric method for the creatinine determination has been reported. A fluorophore was produced in the reaction of creatinine with <NUM>,<NUM>-dinitrobenzoate under alkaline reaction conditions in aqueous and mixed solvent environment. The fluorophore had excitation and emission maxima near <NUM> and <NUM>, respectively. The calibration curve was linear in the range of creatinine concentration of <NUM>-<NUM>µmol·L-<NUM>, while the detection limit for creatinine was well below <NUM>µmοl·L-<NUM>.

In view of the absence in the prior art of an efficient, reliable and selective method of creatinine determination, that would involve additionally low analysis costs, it is the aim of the present invention to provide a new analytical method based to the maximum extent on known reagents, to be carried out using an optical detection and equipment commonly used in analytical laboratories, especially in clinical analytical laboratories.

This object is achieved in accordance with the invention, comprising a method for determining creatinine in clinically significant samples and a reagent to be used in the method.

A fluorimetric analytical method for the determination of creatinine in clinically significant biological samples, in which creatinine is reacted with <NUM>,<NUM>-dinitrobenzoic acid in a strongly alkaline aqueous-organic medium to form a fluorophore product, is characterised in that a clinically significant biological sample is mixed directly with a multi-component fluorimetric reagent containing <NUM>,<NUM>-dinitrobenzoate anions, an organic solvent, water, a base maintaining alkalinity of the reagent above pH = <NUM>, and an addition of hydrogen peroxide, whereas detection and/or quantification of the obtained product of creatinine reaction with <NUM>,<NUM>-dinitrobenzoate anions is carried out using the fluorimetric method, by taking a measurement of radiation having a wavelength of <NUM>-<NUM>, preferably <NUM>, emitted under the influence of an excitation radiation beam of a wavelength of <NUM>-<NUM>, preferably <NUM>, at a constant incubation time after the start of the reaction and determining creatinine concentration on the basis of separately prepared calibration curve that links creatinine concentration in the biological sample with the fluorimetric response characteristic for the given wavelength and incubation time.

According to the invention, as a component containing <NUM>,<NUM>-dinitrobenzoate anions, a solution of <NUM>,<NUM>-dinitrobenzoic acid, a <NUM>,<NUM>-dinitrobenzoate salt, preferably sodium <NUM>,<NUM>-dinitrobenzoate or <NUM>,<NUM>-dinirobenzoic acid ester, preferably methyl <NUM>,<NUM>-dinitrobenzoate is used, the solution comprising an organic solvent, a mixture of organic solvents, water, or a mixture thereof. A solvent which is highly miscible with water, preferably a monohydric alcohol or a polyhydric alcohol is used as the organic solvent, wherein as the monohydric alcohol, methanol, ethanol, propanol or butanol is used, and <NUM>,<NUM>-butanediol, <NUM>,<NUM>-propanediol or ethylene glycol is used as the polyhydric alcohol, <NUM>,<NUM>-butanediol being most preferred. A solution having a concentration greater than <NUM> mol·L-<NUM> is used as the base-containing component, which solution comprises water, an organic solvent or a mixture of organic solvents, or a mixture thereof, wherein as a base, a metal hydroxide, ammonium hydroxide or an organic base that does not significantly interfere with the fluorescence of the reaction product of creatinine with a <NUM>,<NUM>-dinitrobenzoate anion, preferably an alkali metal hydroxide (LiOH, NaOH, KOH, RbOH, CsOH), ammonium hydroxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrabutylammonium hydroxide, or mixtures thereof, most preferably LiOH, NaOH or KOH, is used. As a component containing hydrogen peroxide, an aqueous or aqueous-organic hydrogen peroxide solution is used.

According to the invention, the fluorimetric reagent is made of components including <NUM>,<NUM>-dinitrobenzoic acid, <NUM>,<NUM>-butanediol, water, NaOH and hydrogen peroxide, the components being mixed in any order and combination prior to adding the biological sample of clinical significance, wherein, stable component solutions are primarily prepared, preferably a solution containing <NUM>,<NUM>-dinitrobenzoic acid, <NUM>,<NUM>-butanediol, water and hydrogen peroxide, and a component solution containing water and NaOH, which component solutions are mixed immediately before adding the biological sample of clinical significance.

According to the invention, for samples containing creatinine in a concentration lower than or equal to <NUM>µmol·L-<NUM>, the reagent is used in which:.

According to the invention, the volume ratio of the fluorimetric reagent to the clinically significant biological sample is from <NUM>:<NUM> to <NUM>:<NUM>, preferably <NUM>:<NUM>. The measurement of the fluorescence intensity is performed in the range of the observed linear dependence of the fluorescence intensity on the creatinine concentration, i.e. after the time of <NUM>-<NUM>, preferably <NUM>. The creatinine determination range is within the range of <NUM>-<NUM>µmol·L-<NUM> for the incubation time of <NUM>-<NUM>, preferably <NUM>-<NUM>µmol·L-<NUM> for the incubation time of <NUM>.

The method according to the invention is selective for creatinine in the presence of sugars, proteins and bilirubin. A clinically significant biological sample is a blood serum at a <NUM>-<NUM>-fold dilution, preferably at <NUM>-fold dilution; a urine at a <NUM>-<NUM>-fold dilution, preferably at <NUM>-fold dilution; or a dialysate at a <NUM>-<NUM>-fold dilution, preferably at <NUM>-fold dilution.

A multi-component fluorimetric reagent, being alkaline and containing <NUM>,<NUM>-dinitrobenzoate anions in an aqueous-organic environment, characterised in that it additionally contains hydrogen peroxide and <NUM>,<NUM>-butanediol, is suitable for use in the analytical fluorimetric method for determination of creatinine in clinically significant biological samples, and after direct mixing with a clinically significant biological sample containing creatinine, selectively produces a fluorophore in the reaction between creatinine and a <NUM>,<NUM>-dinitrobenzoate anion, which fluorophore under the influence of an excitation radiation beam of a wavelength of <NUM>-<NUM>, preferably <NUM>, emits radiation of a wavelength of <NUM>-<NUM>, preferably <NUM>, wherein the reagent contains <NUM>,<NUM>-dinitrobenzoate ions at a concentration of from <NUM> mmol·L-<NUM> up to saturation, preferably <NUM> mmol·L-<NUM>; the base at a concentration of <NUM>-<NUM> mol-L<NUM>, preferably <NUM> mol·L-<NUM>; hydrogen peroxide at a concentration of <NUM>-<NUM> mmol·L-<NUM>, preferably <NUM> mmol·L-<NUM>; and the mutual ratio of the solvents ranges from completely organic to fully aqueous, and the optimal ratio of organic solvent to water in the aqueous-organic mixture is from <NUM>:<NUM>, which fluorimetric reagent is stable at room temperature for over <NUM> hours after its preparation and can be used for the selective determination of creatinine at concentrations ranging from <NUM>-<NUM>µmol·L-<NUM> in the presence of glucose, proteins and bilirubin, for an incubation time of <NUM>-<NUM>, preferably <NUM>.

When using the solution according to the invention, it is possible to accurately and precisely determine the concentration of creatinine in biological samples. The results obtained with the method according to the invention are consistent with the results obtained in the Clinical Laboratory, which routinely uses the enzymatic method, which requires the use of as many as four enzymes and a number of other reagents. Enzyme preparations are relatively expensive and must be stored in refrigerators at low temperatures, and in solutions are often quite unstable. In addition, a decisive difficulty of polyenzymatic methods is to find the optimal reaction conditions, especially the pH of the solution and the temperature at which all enzymes work satisfactorily. Overcoming these drawbacks by using the simple, one-point method according to the invention is very attractive, that being its most important advantage over the prior art methods for creatinine determination.

It is also an advantage of the present invention that the measurement in the disclosed method is a single point one and the reagents are non-toxic, which allows for the development of mobile measurement systems. Using the method according to the invention, it is possible to create systems ready to use at any time and place, in accordance with the concept of the point of care testing, i.e. carrying out analytical tests directly at the place of patient care, for example at the bedside in the hospital, in ambulances or in the offices of primary contact (GP) doctors, by personnel not being trained in clinical analytics.

The possibility of abandoning the two-point measurement in the practice of analytical laboratories is a great help in routine determinations, and most importantly, it reduces the measurement uncertainty. In the case of the two-point measurement, the analytical signal is a difference of two values, both of which are subject to measurement uncertainty. In the simplest terms, the error of the difference between the two values will be the square root of the sum of the squared errors of the two measurements, therefore the obtained error value is greater than that of the single-point measurement.

The invention will now be described in detail by way of example with reference to the accompanying drawing. The values in the graph correspond to the final concentrations in the reagent.

Despite the long history of determinations of creatinine in clinical biological samples, it is still a significant problem to find a reliable, cheap and convenient analytical method for this purpose, which would be characterised by greater reliability than the routine Jaffé method, as well as lower price and lower complexity than the routinely used enzymatic methods. This is evidenced, for example, by numerous reviews and research papers published in recent years on the subject, that may be related to the ever-increasing number of people suffering from chronic kidney disease (<NUM>% increase in the incidence in <NUM>-<NUM>) [<NPL>].

The research on the present invention was inspired by the desire to provide a method of high reliability (selective against other components of biological samples), which would be easy, fast and cheap to be prepare. As a result, the new method would be competitive to the Jaffé method, which is highly susceptible to interferences, and to the enzymatic methods, the disadvantageous due to the need of using expensive preparations with limited durability and to the need of using long incubation times.

Research has focused on the development of a fluorimetric method for determining creatinine in serum samples, although the analysis of creatinine concentration in urine and dialysate is also very important, mainly because creatinine is used as an indicator of urine dilution and is proportional to the patient's muscle mass. In addition, urine and dialysate have a much poorer matrix than blood serum, they should not contain proteins and bilirubin, the level of creatinine in the urine is <NUM> orders of magnitude higher than in the blood serum and the level of creatinine in the dialysate is similar to the level of creatinine in the blood serum.

However, the differences in the characteristics of the above-mentioned biological samples do not affect the applicability of the creatinine determination method according to the invention, which may be successfully applied to all those types of samples, just taking into account the necessary dilutions to ensure optimal creatinine content in the analysed samples.

Literature reports indicated that it is possible to determine creatinine fluorimetrically on the basis of the creatinine reaction with <NUM>,<NUM>-dinitrobenzoic acid [<NPL>], however, so far no one has reproduced the results. In the course of the research related to the present invention, it was proven that the previously described method does not work even under the proposed conditions and that it is not possible to obtain a fluorimetric signal even when larger amounts of the analyte are used (<FIG>).

In the course of the current research, however, a significant analytical potential of the fluorimetric method of creatinine determination based on its reaction with ions <NUM>,<NUM>-dinitrobenzoate ions has been confirmed (the ions being introduced into the reaction system as a reagent comprising <NUM>,<NUM>-dinitrobenzoic acid, a salt or an ester thereof).

The observation that allowed for further experiments was the finding that the presence of hydrogen peroxide in the reaction mixture leads to the formation of a fluorescent product in the creatinine reaction with <NUM>,<NUM>-dinitrobenzoic acid dissolved in <NUM>,<NUM>-butanediol in a strongly alkaline medium. The emission spectrum of the fluorophore after excitation by radiation with a wavelength of <NUM> (maximum of the absorption spectrum) is shown in <FIG>. The maximum emission of the fluorophore occurs at a wavelength of <NUM>.

The key to the effectiveness of the creatinine determination method was the addition of hydrogen peroxide to the reaction environment, conditioning and enabling the occurrence of the fluorescence phenomenon in the presence of the product of creatinine reaction with <NUM>,<NUM>-dinitrobenzoate ions. The use of H<NUM>O<NUM> in the fluorescence determination of creatinine has not been previously known in the literature.

Although the mechanism of hydrogen peroxide action in the creatinine reaction with <NUM>,<NUM>-dinitrobenzoate ions in an alkaline environment is still unknown, a reproducible and reliable effect is achieved. Currently, it has been excluded that hydrogen peroxide oxidises the solvent used to prepare the acid solution, because while it is possible to oxidise alcohols by peroxide, the reaction requires the presence of a catalyst in the form of sodium tungstate and tetraalkyl-ammonium hydrogen sulfate, a significantly increased concentration of hydrogen peroxide and the elevated temperature [<NPL>]. Moreover, the fluorescent reaction product is formed not only when hydrogen peroxide is previously added to the component solution containing <NUM>,<NUM>-dinitrobenzoic acid and an organic solvent (e.g. <NUM>,<NUM>-butanediol), but also when it is mixed with the component solution containing the base (e.g. LiOH, NaOH, KOH), and also when it is added separately just before the reaction. It was also confirmed that the replacement of the H<NUM>O<NUM> addition with the enrichment of the reaction reagent with the solvent oxidation product (e.g. succinic acid, which is the <NUM>,<NUM>-butanediol oxidation product) does not allow for fluorescence in the tested system.

Without wishing to limit the scope of possible explanations, it is necessary to mention the observation made, which may contribute to explaining the mechanism of the taking place reaction. For all tested NaOH concentrations, after approx. <NUM>-<NUM> seconds of incubation, the phase of intense signal increase begins, followed by a decrease in the fluorescence intensity to a value close to the signal corresponding to the blank sample, and after approx. <NUM> the fluorescence disappears (<FIG>). This suggests that the fluorescent compound may be an intermediate product of the creatinine reaction with <NUM>,<NUM>-dinitrobenzoate anions, which intermediate product is transformed after some time into a non-fluorescent final product.

In the first phase, the conditions of the reaction between creatinine and <NUM>,<NUM>-dinitrobenzoic ions, leading to the formation of the previously described product (complex/adduct) absorbing and/or emitting electromagnetic radiation, were investigated and optimised. Then, the calibration characteristics of the developed method and its analytical parameters, such as the limits of detection and quantification, linear range and precision, were determined. The selectivity of the method according to the invention was also tested and compared with the selectivity of the routinely used Jaffé method. The working examples demonstrate the use of the developed protocols for the determination of creatinine in serum, urine and dialysate samples.

In the course of the work, the main problem turned out to be a significant reduction of the analytical signal caused by the presence of interferents, especially proteins, in biological samples. In such cases, the most common remedy is the addition of denaturing agents to denature proteins, or the addition of a constant amount of protein to the calibrators. Unfortunately, in the case of the method according to the invention, no effective denaturing agent was found, which does not exclude the possibility of developing such an agent in future. In turn, the possible addition of protein to the calibration solutions would significantly deteriorate the sensitivity of the reaction, because the presence of even small amounts of albumin (<NUM>·L-<NUM>) causes a decrease in the fluorescence of the reaction according to the invention by as much as <NUM>%. This effect can be minimised by significant (e.g. <NUM>-fold) dilution of biological samples, which is, however, inconvenient and practically excludes the possibility of carrying out measurements in accordance with the concept of point of care testing, i.e. conducting analytical tests directly at the place of patient care - at the bedside in hospitals, in ambulances or in GP practices by staff not being trained in clinical analytics.

The optimisation of the method according to the invention consisted in determining the optimal composition of a fluorimetric reagent, containing all the necessary components for the creatinine reaction with the <NUM>,<NUM>-dinitrobenzoate anion.

During the optimisation experiments, the following were determined:.

During optimisation experiments, a fluorimetric reagent was produced using mainly <NUM>,<NUM>-dinitrobenzoic acid, <NUM>,<NUM>-butanediol, water, hydrogen peroxide and sodium hydroxide, due to their easy availability and low cost. Typically, a component solution containing <NUM>,<NUM>-dinitrobenzoic acid, <NUM>,<NUM>-butanediol, water and hydrogen peroxide at appropriate concentrations was formed and mixed with the component solution containing NaOH just prior to the experiment.

However, the established procedure does not exclude replacing of the above components with their chemical equivalents, which will play an identical function in the fluorescence process of the creatinine-DNBA reaction product. The task of the <NUM>,<NUM>-dinitrobenzoic acid compound is to form a complex/adduct in the course of its reaction with creatinine. The purpose of the organic solvent is to facilitate the dissolution of the <NUM>,<NUM>-dinitrobenzoic acid compound since, for example, <NUM>,<NUM>-dinitrobenzoic acid is poorly soluble in water. However, this does not exclude carrying out measurements in a pure-water solution, as long as it ensured to be alkaline, which enables the formation of <NUM>,<NUM>-dinitrobenzoate anions. The role of the base is to provide an alkaline environment that stabilises the <NUM>,<NUM>-dinitrobenzoate anions and enables the fluorescence process to take place, and in the case of measurements in an aqueous medium, also to facilitate the dissolution of the <NUM>,<NUM>-dinitrobenzoate compound. The role of hydrogen peroxide is to enable the fluorescence process to take place.

According to the invention, it is possible to use <NUM>,<NUM>-dinitrobenzoic acid, <NUM>,<NUM>-dinitrobenzoate salt (e.g. sodium salt) or <NUM>,<NUM>-dinirobenzoate ester (e.g. methyl <NUM>,<NUM>-dinitrobenzoate) as the source of <NUM>,<NUM>-dinitrobenzoate anions. Any organic solvent that dissolves the <NUM>,<NUM>-dinitrobenzoate compounds and is highly miscible with water may be used. For example, monohydric alcohol (methanol, ethanol, propanol or butanol), polyhydric alcohol (<NUM>,<NUM>-butanediol, <NUM>,<NUM>-propanediol or ethylene glycol) or mixtures thereof can be used. Any aqueous or aqueous-organic solution of a base in concentration above <NUM> mol·L-<NUM> can be used, as long as the base (organic or inorganic) does not significantly interfere with the fluorescence of the product of creatinine reaction with <NUM>,<NUM>-dinitrobenzoate, such as for example alkali metal hydroxide (LiOH, NaOH, KOH, RbOH, CsOH), ammonium hydroxide, organic bases (tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrabutylammonium hydroxide) or mixtures thereof may be used. It is possible to use an aqueous or a water-organic hydrogen peroxide solution.

According to the invention, the fluorimetric reagent is prepared from suitable components which are mixed in any order and combination prior to addition of a clinically significant biological sample. The fluorescent reagent is stable at room temperature for more than <NUM> hours.

Unless otherwise stated, in all fluorimetric measurements the operating voltage of the photomultiplier was <NUM> V, while the width of the slots for both the excitation beam and the emitted beam was <NUM>. A beam excitation with a wavelength of λex = <NUM> (maximum of the absorption spectrum of the creatinine-DNBA reaction product, <FIG>) was used, and the fluorescence intensity was recorded for the wavelength λem = <NUM> (the maximum emission spectrum of the creatinine-DNBA reaction product, <FIG>). In kinetic measurements, the signal was recorded every second. All optimisation runs are standardised. Scinco FluoroMate FS-<NUM> spectrofluorimeter was used.

In order to select the optimal solvent for <NUM>,<NUM>-dinitrobenzoate compounds, which are the main component of the fluorescent reagent, a series of measurements was performed. The criteria that must be met by an optimal solvent are: first of all miscibility with water, low volatility and low toxicity. Initially, monohydroxy alcohols (methanol, ethanol, propanol), polyhydroxy alcohols (ethylene glycol, <NUM>,<NUM>-propanediol, <NUM>,<NUM>-butanediol) and aqueous all<aline solutions were selected. A series of fluorimetric reagent solutions were prepared using pre-selected solvents in a ratio of <NUM>:<NUM> with water, containing DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>). After mixing the reagent with the standard solution of creatinine (<NUM>µmol·L-<NUM>) in a volume ratio of <NUM>:<NUM>, the variability of the fluorescence intensity (λem = <NUM>) was recorded after excitation by irradiation of the wavelength λex = <NUM>. Measurable fluorescence was recorded in each of the tested alcohols. Among the solvents used, <NUM>,<NUM>-butanediol allows for the fastest reaction between DNBA - and creatinine (maximum intensity approx. <NUM> counts, after an incubation time of approx. Methanol also seems to be a very promising solvent (<NUM> counts/<NUM>), but due to its volatility, there were large differences between the obtained results, and its toxicity could exclude it from applicability. Of all the solvents tested <NUM>,<NUM>-propanediol could be an alternative for <NUM>,<NUM>-butanediol (<NUM> counts/<NUM>) because it is non-toxic, readily available and inexpensive. The results are shown in <FIG>.

In order to select the optimal concentration of H<NUM>O<NUM> in the fluorimetric reagent, a series of reagent solutions containing DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>) <NUM>-butanediol and water in a volume ratio of <NUM>:<NUM>, and H<NUM>O<NUM> in various concentrations ranging from <NUM>-<NUM> mmol·L-<NUM> were prepared. After mixing the reagent with the creatinine standard solution (<NUM>µmol·L-<NUM>) in a <NUM>:<NUM> volume ratio, the dependence of the fluorescence intensity on time was investigated (λex = <NUM>, λem = <NUM>). The optimal kinetics (<NUM> counts/<NUM>) was observed for an H<NUM>O<NUM> concentration of <NUM> mmol·L-<NUM>. A slightly higher intensity of fluorescence was observed for the concentration of H<NUM>O<NUM> <NUM> mmol·L-<NUM>, but a longer incubation (<NUM> counts/<NUM>) was required, which would be burdensome with the routine use of the method. The results are shown in <FIG>.

In order to select the optimal volume ratio of <NUM>,<NUM>-butanediol to the component of the H<NUM>O<NUM> solution in water (initial concentration C°H<NUM>O<NUM> = <NUM> mmol·L-<NUM>), a series of reagent solutions containing DNBA (<NUM> mmol·L-<NUM>), NaOH ( <NUM> mol·L-<NUM>) were prepared. The range of changes in the concentration of H<NUM>O<NUM> in the fluorimetric reagent was: <NUM>-<NUM> mmol·L-<NUM>. The range of changes in the volume ratio <NUM>,<NUM>-butanediol to water ranged from <NUM>:<NUM> to <NUM>:<NUM>. After mixing the reagent with the standard creatinine solution (<NUM>µmol·L-<NUM>) in the volume ratio of <NUM>:<NUM>, the dependence of fluorescence intensity on time was investigated (λex = <NUM>, λem = <NUM>). The most favorable volume ratio of BTD to H<NUM>O<NUM> aqueous solution was found to be the ratio <NUM>:<NUM> (<NUM> counts/<NUM>). The results are shown in <FIG>.

In order to select the optimal concentration of DNBA in the fluorimetric reagent, a series of reagent solutions containing NaOH (<NUM> mol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>), <NUM>,<NUM>-butanediol and water in the volume ratio of <NUM>:<NUM>, and DNBA at various concentrations ranging from zero to saturation were prepared. After mixing the reagent with the creatinine standard solution (<NUM>µmol·L-<NUM>) in a volume ratio of <NUM>:<NUM>, the dependence of the fluorescence intensity on time was investigated (λex = <NUM>, λem = <NUM>). The optimal kinetics (<NUM> counts/<NUM>) was observed for DNBA concentration of <NUM> mmol·L-<NUM>. The results are shown in <FIG>.

In order to select the optimal concentration of NaOH in the fluorimetric reagent ensuring maximum sensitivity of the method, a series of reagent solutions containing DNBA (<NUM> mmol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>), <NUM>,<NUM>-butanediol and water in the volume ratio of <NUM>:<NUM>, and NaOH at various concentrations ranging from <NUM>-<NUM> mol·L-<NUM> were prepared. After mixing the reagent with the standard creatinine solution (<NUM>µmol·L-<NUM>) in the volume ratio of <NUM>:<NUM>, the dependence of the fluorescence intensity on the time was investigated (λex = <NUM>, λem = <NUM>). The change in NaOH concentration within the range of <NUM>-<NUM> mol·L-<NUM> does not significantly affect the kinetic course of the reaction. <NUM> mol·L-<NUM> was selected as the optimal concentration as it provides a slightly faster reaction between creatinine and DNBA (<NUM> counts/<NUM>). Interestingly, after an hour, the fluorescence intensity for all tested concentrations is equal to the fluorescence intensity recorded at the beginning of the chemical reaction, which means that the fluorescent product is likely to completely degrade during this time. The results are shown in <FIG>.

In result of the optimisation experiments, the following concentrations of the components in the fluorimetric reagent were selected: DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>), <NUM>,<NUM>-butanediol and water in a volume ratio of <NUM>:<NUM>.

After optimisation of the fluorimetric reagent composition, the calibration characteristics and validation parameters of the method according to the invention were determined. For this purpose, a reagent with the optimal composition indicated above was prepared, as well as a series of creatinine standard solutions with concentrations ranging from <NUM>-<NUM>µmοl·L-<NUM> After mixing the reagent with the creatinine standard solution in a volume ratio of <NUM>:<NUM>, the fluorescence intensity of the creatinine-DNBA reaction product was measured after three different incubation times: <NUM>, <NUM>, <NUM> (λex = <NUM>, λem = <NUM>). The calibration dependencies are shown in <FIG>, and the basic validation parameters are given in Table <NUM>.

The developed creatinine protocol shows linearity within the range <NUM>-<NUM>µmol·L-<NUM>. The sensitivity of the method increases with the incubation time in the first phase (average <NUM>-<NUM>), and then decreases due to the varying fluorescence intensity over time (<FIG>), with an intense increase in fluorescence intensity following a <NUM> second incubation. This means that the optimal time for carrying out the measurement is on average in the range of up to about <NUM>, but it is possible to conduct research in the entire range of fluorescence time (<NUM>-<NUM>). The method according to the invention can be used to test biological samples because the physiological range of creatinine in these fluids is within or is greater than the linear range of the method, which allows for possible dilution of the fluids (blood serum <NUM>-<NUM>µmol·L-<NUM>, urine <NUM>-<NUM> mmol·L-<NUM>, dialysate <NUM>-<NUM>µmol·L-<NUM>).

In order to check protein interference in the method of creatinine determination according to the invention, the dependence of the fluorescence intensity on the time (λex = <NUM>, λem = <NUM>) was recorded for the creatinine standard solution of the concentration of <NUM>µmol·L-<NUM> and for the same standard containing the addition of albumin (<NUM>-<NUM>·L-<NUM>). The results of this experiment are shown in <FIG>. It was observed that the presence of albumin starts to influence the fluorimetric signal after a certain time and not from the beginning of the reaction. Initially, i.e. up to <NUM> seconds of the reaction time, the fluorescence intensity is independent of the albumin concentration in the sample. This result suggests that it is possible to determine creatinine in the blood serum and without diluting the sample. It is worth noting that lower concentrations of albumin (<NUM>-<NUM>·L-<NUM>) begin to reduce the signal only after about <NUM> seconds of the reaction between creatinine and DNBA. These are the concentrations of proteins lower than in the blood serum (approx. <NUM>·L-<NUM>), but in the case of dilution it is possible to extend the measurement time, which is advantageous due to the method sensitivity increasing with time (<NUM>-<NUM>).

In order to checl< whether the degree of albumin interference depends on the creatinine concentration, a series of creatinine standard solutions with the addition of albumin at a concentration of <NUM>·L-<NUM> were prepared. The dependences of the fluorescence intensity (λex = <NUM>, λem = <NUM>) were recorded for the incubation time of <NUM>. The obtained results are shown in <FIG>. It was observed that the protein at the physiological concentration is not a significant interferent of the method according to the invention at creatinine concentrations below <NUM>µmol L-<NUM>, because the calibration curve recorded with and without protein overlaps in this range. This means that irrespective of the creatinine concentration, it is possible to discriminate between the influence of proteins on the determination by measuring the fluorescence intensity after a maximum of <NUM> seconds for undiluted serum and for diluted serum after at least <NUM> seconds.

The optimisation of the reaction conditions began with the selection of the optimal concentration of H<NUM>O<NUM> in a fluorimetric reagent in which DNBA was dissolved in an aqueous NaOH solution. A series of reagent solutions containing DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>) and H<NUM>O<NUM> in various concentrations ranging from <NUM>-<NUM> mmol·L-<NUM> were prepared. After mixing the reagent with the standard creatinine solution (<NUM>µmol·L-<NUM>) in the volume ratio of <NUM>:<NUM>, the dependence of the fluorescence intensity on the time was investigated (λex = <NUM>, λem = <NUM>). Concentration of <NUM> mmol·L-<NUM> was selected as the optimal concentration of H<NUM>O<NUM>, analogous to the experiment with <NUM>,<NUM>-butanediol. The results are shown in <FIG>.

In order to optimise the concentration of DNBA in the fluorimetric reagent, a series of reagent solutions containing H<NUM>O<NUM> (<NUM> mmol·L-<NUM>) and DNBA dissolved in NaOH solution in the DNBA/NaOH molar ratio equal to <NUM>:<NUM> was prepared, with the DNBA concentration fluctuating in the range of <NUM>-<NUM> mmol·L-<NUM>. After mixing the reagent with the creatinine standard solution (<NUM>µmol·L-<NUM>) in a volume ratio of <NUM>:<NUM>, the dependence of the fluorescence intensity on time was investigated (λex = <NUM>, λem = <NUM>). The highest fluorescence intensity was recorded for a DNBA concentration of <NUM> mmol·L-<NUM>. The results are shown in <FIG>.

The optimal concentration of NaOH in the fluorimetric reagent was determined after the preparation of a series of reagent solutions containing DNBA (<NUM> mmol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>) and NaOH in various concentrations ranging from <NUM>-<NUM> mol·L-<NUM>. After mixing the reagent with the standard creatinine solution (<NUM>µmol·L-<NUM>) in the volume ratio of <NUM>:<NUM>, the dynamics of the analytical signal increase (λex = <NUM>, λem = <NUM>) was tested. It was observed that the use of NaOH in a concentration in the range of <NUM>-<NUM> mol·L-<NUM> gives similar results, but the concentration of <NUM> mol·L-<NUM> gives the greatest sensitivity. The results are shown in <FIG>.

In effect of the optimisation experiments, the following concentrations of the components in the fluorimetric reagent not containing organic solvents were selected: DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>).

Calibration characteristics of the method were determined in accordance with the developed protocol. For this purpose, creatinine standard solutions with the concentration range of <NUM>-<NUM>µmol·L-<NUM> were prepared and the fluorescence intensity was recorded after three reaction times: <NUM>, <NUM>, <NUM>. The calibration curves for the fluorimetric creatinine determination method are shown in <FIG> (λex = <NUM>, λem = <NUM>). The analytical parameters are presented in Table <NUM>.

It was observed that elimination of the organic solvent from the reaction medium causes a great loss of sensitivity of the method according to the invention and an increase in the limits of quantification and detection of creatinine. Wiling to determine serum creatinine without the use of an organic solvent, even a fivefold dilution may be at risk of obtaining a solution with a creatinine concentration lower than its limit of quantification. A possible solution to this problem may be to increase the width of the slots for the excitation and the emitted beams, but such an approach may reduce the selectivity of the method.

In order to investigate the influence of proteins on the kinetics of fluorescence (λex = <NUM>, λem = <NUM>), a creatinine standard solution (<NUM>µmol·L-<NUM>) was prepared with different albumin content (<NUM>-<NUM>·L-<NUM>) and the dependence of fluorescence intensity on time was recorded after mixing it with the reagent of optimal composition presented above. The results are shown in <FIG>. It was observed that the influence of proteins is significant after a very short incubation time (approx. <NUM>), which makes difficult the reliable determination of serum creatinine carried out according to the method of the invention using an aqueous reagent.

To investigate whether the observed relationship is present for other creatinine concentrations, a calibration curve was constructed without protein and in the presence of <NUM>·L-<NUM> albumin in the standard. The time when the presence of proteins starts to lower the signal is short, and is approx. <NUM> seconds. For time in which the sensitivity of the method would be the highest due to the potentially high intensity of fluorescence (approx. <NUM>), interferences from proteins prevent a reliable determination of creatinine. The results are shown in <FIG>.

Optimisation of the volume ratio of the reagent to a sample is needed due to the two opposing trends: increasing the sample volume increases the amount of analyte and lowering the limit of detection, and in turn reducing the sample volume, the degree of interference of other components of the sample decreases. Control measurements were carried out by mixing the water-organic reagent with the sample in volume ratios of <NUM>:<NUM>, <NUM>:<NUM> and <NUM>:<NUM>, and the concentrations of components in the obtained mixture were constant and amounted to <NUM> mmol·L-<NUM> DNBA, <NUM> mol·L-<NUM> NaOH, <NUM> mmol·L-<NUM> H<NUM>O<NUM>, and the volume ratio of <NUM>,<NUM>-butanediol and water was <NUM>:<NUM>. A series of creatinine standard solutions (<NUM>µmol·L-<NUM>) containing a number of potential interferents (glucose, uric acid, bilirubin, albumin, phosphates, urea) were prepared. After mixing the reagent with standard solutions in appropriate volume ratios, the dependence of fluorescence intensity on time was examined (λex = <NUM>, λem = <NUM>) and the comparative results are presented in <FIG>. In the presence of interferents, the highest fluorescence intensity was observed for the systems in ratio of <NUM>:<NUM>, and the <NUM>:<NUM> and <NUM>:<NUM> systems showed lower fluorescence intensity.

The interference of creatinine determination due to the presence of proteins (e.g. albumin) decreased with the increase in the ratio of the fluorescent reagent volume to the sample volume, because the amount of interferents in the tested mixture also decreased. For the <NUM>:<NUM> samples, the relative error due to the presence of proteins was as high as <NUM>% (<FIG>), and for the <NUM>:<NUM> samples it dropped to as little as <NUM>% (<FIG>). In turn, the detection limit increased with an increase in the VREAGENT/VSAMPLE ratio. Significant dilution of the sample at a ratio of <NUM>:<NUM> during the measurement increased the limit of detection for creatinine to a value of about <NUM>µmol·L-<NUM>, which still allowed the assay to be performed, but indicated that further increasing the volume ratio would practically exclude the usefulness of such a protocol. On the other hand, the use of the VREAGENT/VSAMPLE = <NUM>:<NUM> protocol allowed to reduce the limit of creatinine detection significantly below <NUM>µmol·L-<NUM>, (Table <NUM>), but caused a significant (<NUM>%) extinction of the fluorescence of the product of creatinine reaction with anions of <NUM>,<NUM>-dinitrobenzoic acid caused by the interfering proteins present in significant concentrations in the sample.

Analytical measurements of the mixture of fluorimetric reagent and a sample in a volume ratio of <NUM>:<NUM> were considered to be the optimal protocol. When using such a protocol, the method of the invention has low detection and quantification limits, as well as high selectivity, because none of the potential interferents significantly influenced the recorded fluorimetric signal - the error caused by the presence of most interferents is not greater than ±<NUM>% for incubation times below <NUM>.

The selectivity of the inventive fluorimetric creatinine determination method was compared to the Jaffé single point and two-point methods and to the photometric method based on o the creatinine reaction with DNBA by examining creatinine standard solutions containing admixtures of significant interferents present in biological samples (<FIG>). As can be seen, the method according to the invention has the greatest selectivity for creatinine. Importantly, the error caused by the presence of bilirubin in the sample has been almost completely eliminated. In case of the Jaffé method, to get similar result it is necessary to make a two-point measurement, which is not preferred in analytical methods. Moreover, compared to other methods, the error caused by the presence of proteins is significantly reduced. In this form, the fluorimetric method for determining creatinine can be successfully applied to the determination of creatinine in blood serum, but also in urine and dialysate, where the problem of protein interference generally does not occur.

In order to verify the suitability of the method according to the invention for clinical trials, measurements were carried out on synthetic sera with normal (Cormay Serum HN) and pathological (Cormay Serum HP) content of creatinine and other components, as well as in their mixtures in dilutions ranging from <NUM> to <NUM> (Table <NUM>).

The creatinine content of the samples was determined by the method of the invention using a fluorimetric reagent of an optimised composition: DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>), VBTD/VH<NUM>O = <NUM>:<NUM>, VREAGENT/VSAMPLE = <NUM>:<NUM>. The cuvette was closed with a stopper and its contents was mixed. The results are shown in <FIG> and Table <NUM>.

It has been observed that dilution of the serum sample prior to measurement is necessary to obtain reliable results. Creatinine concentrations determined in the undiluted serum sample significantly deviate from the nominal concentrations. The measurement time should depend on the degree of sample dilution - the more diluted the sample, the longer the incubation time should be used, for example: <NUM> for a 2x dilution, <NUM> for 5x, <NUM> sec for 10x. This trend is consistent with the results described above (<FIG>, <FIG>). For high sample dilution, the influence of proteins on the measurement is eliminated, but a long incubation time is advantageous due to the increased sensitivity of the method. On the other hand, for smaller protein dilutions, proteins reduce the analytical signal and therefore it is necessary to shorten the incubation time.

An optimised protocol was used for the analysis of biological samples. Serum samples were selected for testing because they usually have a greater number of interferents in comparison with urine and dialysate samples which are usually easier to analyse. Samples were obtained from patients of the Independent Public Central Clinical Hospital. During the measurement carried out according to the method of invention, each one of the samples was diluted five times, and the reaction time was chosen to be <NUM> seconds. Detailed protocol for creatinine determination is provided in the working example. Comparative results were obtained using the enzymatic method at the Central Clinical Laboratory of the Medical University of Warsaw [<NPL>)], and using the Jaffé protocols for one-point and two-point measurement modes.

A correlation was observed between the results obtained by the method according to the invention and the results obtained at the Central Clinical Laboratory using the enzymatic method. The comparison of the results with the determined <NUM>% confidence interval is shown in <FIG>. The linear regression equation of the obtained points is y = (<NUM> ± <NUM>) x + (<NUM> ± <NUM>) with the Prearson's coefficient r being <NUM>. This result indicates a correlation between the two data sets. Moreover, the agreement between the results obtained by the fluorimetric method and the enzymatic method is shown in the Bland-Altman plot (<FIG>), which is a graphic illustration of the agreement between the two measurement methods [<NPL>]. All the points of the plot are within the agreement interval, i.e. inside the region between the standard deviation lines, which means that the enzymatic method and the method of the invention can be used interchangeably for the determination of creatinine in biological samples.

Moreover, in order to verify whether the obtained results differ significantly from the results obtained by the enzymatic method, two-tailed Student's t-test was performed for the confidence interval of <NUM>% and <NUM> degrees of freedom. The determined value of the texp parameter is <NUM>, while the critical (tabular) value of the tcrit parameter for this type of test is <NUM>. This means that the creatinine concentrations determined by the method of the invention do not differ significantly from the creatinine concentrations determined by the enzymatic method, which is considered the most reliable routine method for creatinine detection.

During the analysis of the results obtained by the Jaffé method, two samples were rejected due to the very large standard deviation of the measurement series. The Student's t-test was used again to check the statistical differences in the results, but after the rejection of two samples, the number of degrees of freedom decreased to <NUM>. Comparing the results obtained with the method according to the invention and the two-point Jaffé method, the texp parameter was <NUM>, which is less than the critical tcrit value for this test of <NUM>. It is also worth mentioning that when comparing the results obtained in the Clinical Laboratory by the enzymatic method with the one-point Jaffe method for a measurement time of <NUM> seconds, the determined value of the texp parameter is <NUM>, i.e. it is higher than the critical value. This means that the concentrations determined by the one-point method of the invention are in better agreement with the results obtained with the enzymatic method in the Clinical Laboratory than the results obtained by the one-point Jaffé method.

The method for determining creatinine according to the invention shows exceptional selectivity, accuracy and precision when compared to the known fluorimetric methods. The results obtained with the method of the invention are comparable with standard measurement methods of other types, but in contrast, the method according to the invention is the only one that uses commercially available reagents. This is beneficial because the dangers and risks of their use, their impact on analysts' health, and recommended waste disposal methods are well known.

The comparison of the cost of performing <NUM> analyses (the cost of the reagents alone in terms of the total volume of the reagents and the sample, amounting to <NUM>), shows a similar price range of the optimised method according to the invention (approx. <NUM> PLN) and the Jaffé method (approx. <NUM> PLN). It should be noted, however, that <NUM>% of the determination costs in the method according to the invention relates to the purchase cost of <NUM>,<NUM>-butanediol, and replacing it with another solvent (e.g. propylene glycol) can significantly reduce the total cost of the reagents below <NUM> PLN, i.e. below the cost level of the Jaffé method. For comparison, a ready-made commercial enzyme kit allowing to perform <NUM> determinations costs almost € <NUM> (about <NUM>,<NUM> PLN) [https://www. com/creatinine-assay-kit-ab65340. html, <NUM>/<NUM>/<NUM>]. The cost difference between the method according to the invention and the enzyme method is significant.

The fluorimetric method according to the invention allows for a reliable and precise determination of creatinine concentration in clinically significant biological samples. The results obtained by the method according to the invention are consistent with the results obtained in the Clinical Laboratory using a routine enzymatic method. It is a one-point method, requiring relatively short incubation times (<NUM>-<NUM>), which eliminates the inconvenience of enzymatic methods. The determination range of creatinine is in the range of <NUM>-<NUM>µmol·L-<NUM>, for incubation times of <NUM>-<NUM>, preferably <NUM>-<NUM>µmol·L-<NUM> for <NUM>. The method according to the invention allows the measurement of creatinine content in blood serum samples, urine or dialysate after appropriate dilution, and the dialysate often does not need to be diluted.

The speed and simplicity of measurements, as well as the use of non-toxic components of the fluorimetric reagent, make it possible to create measuring systems ready for use at any time and place, in accordance with the concept of point of care testing. The unit measurement price is competitive with other routinely used methods of creatinine determination in biological samples.

The invention is described below in the working examples illustrating the implementation of the method according to the invention using the reagent according to the invention, by means of their use for the determination of creatinine in clinically significant biological samples.

Example <NUM>. Fluorimetric determination of creatinine in blood serum: Two component solutions were prepared. Solution <NUM>: aqueous-organic solution of <NUM>,<NUM>-dinitrobenzoic acid (<NUM> mmol·L-<NUM>) and hydrogen peroxide (<NUM> mmol·L-<NUM>) in a mixture of <NUM>,<NUM>-butanediol and water in a <NUM>:<NUM> volume ratio. Solution <NUM>: NaOH aqueous solution (<NUM> mol·L-<NUM>). Into the fluorimetric cuvette there were successively introduced: <NUM> of Solution <NUM>, and then <NUM> of Solution <NUM>, thus obtaining the fluorimetric reagent of the following composition: DNBA (<NUM> mmol·L-<NUM>), NaOH (<NUM> mol·L-<NUM>), H<NUM>O<NUM> (<NUM> mmol·L-<NUM>), <NUM>,<NUM>-butanediol and water in a <NUM>:<NUM> volume ratio. The reaction was initiated by introducing a <NUM> of a sample of <NUM>-fold diluted blood serum into a fluorimetric cuvette, resulting in a mixture of <NUM>:<NUM> volume ratio of the reagent and sample. The content of the cuvette was mixed for <NUM>, and then the registration of changes in the fluorescence intensity of the creatinine-DNBA reaction product was started. The value recorded after <NUM> of incubation (λex = <NUM>, λem = <NUM>) was compared with a previously prepared calibration curve to determine the creatinine concentration in the sample, and the reading was compared with the reference data. The obtained result was within the <NUM>% confidence interval of the reference method (Hitachi Roche Cobas <NUM>) - enzyme protocol.

Example <NUM>. Fluorimetric determination of creatinine in urine. The procedure was as in Example <NUM>. The reaction was initiated by loading into a fluorimetric cuvette a <NUM> sample of <NUM>-fold diluted urine to obtain a <NUM>:<NUM> volume ratio of reagent and sample. The obtained result was within the <NUM>% confidence interval of the reference method (Hitachi Roche Cobas <NUM>) - Jaffé protocol.

Claim 1:
A fluorimetric analytical method for the determination of creatinine in clinically significant biological samples, in which creatinine is reacted with <NUM>,<NUM>-dinitrobenzoic acid in a strongly all<aline aqueous-organic medium to form a fluorophore product, characterised in that a clinically significant biological sample is mixed directly with a multi-component fluorimetric reagent containing <NUM>,<NUM>-dinitrobenzoate anions, an organic solvent, water, a base maintaining alkalinity of the reagent above pH = <NUM>, and an addition of hydrogen peroxide, whereas detection and/or quantification of the obtained product of creatinine reaction with <NUM>,<NUM>-dinitrobenzoate anions is carried out using the fluorimetric method, by taking a measurement of radiation having a wavelength of <NUM>-<NUM>, preferably <NUM>, emitted under the influence of an excitation radiation beam of a wavelength of <NUM>-<NUM>, preferably <NUM>, at a constant incubation time after the start of the reaction and determining creatinine concentration on the basis of separately prepared calibration curve that links creatinine concentration in the biological sample with the fluorimetric response characteristic for the given wavelength and incubation time.