Patent WO-2021086209-A1 (WO)
Title: Fluorimetric analytical method for the determination of creatinine in clinically significant biological samples and a fluorimetric reagent for use in this method
Inventor: LEWIŃSKA IZABELA
IPC: G01N33/70
Filed: 2020-10-30
Abstract: A fluorimetric analytical method for the determination of creatinine in clinically significant biological samples, in which creatinine is reacted with 3,5-dinitrobenzoic acid in a strongly alkaline aqueous-organic medium to form a fluorophore product is characterized in that a clinically significant biological sample is mixed directly with a multi-component fluorimetric reagent containing 3,5-dinitrobenzoate anions, an organic solvent, water, a base maintaining alkalinity of the reagent above pH = 12, and additionally hydrogen peroxide, whereas detection and/or quantification of the obtained product of creatinine reaction with 3,5-dinitrobenzoate anions is carried out using the fluorimetric method, by taking a measurement of radiation having a wavelength of 450-500 nm, preferably 480 nm, emitted under the influence of an excitation beam of radiation of a wavelength of 380-420 nm, preferably 405 nm, 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. A multi-component fluorimetric reagent for use in the analytical fluorimetric method for determination of creatinine in clinically significant biological samples, being alkaline and containing 3,5-dinitrobenzoate anions in an aqueous-organic environment is characterized in that it additionally contains hydrogen peroxide, and after direct mixing with a clinically significant biological sample containing creatinine, selectively produces a fluorophore in the reaction between creatinine and a 3,5-dinitrobenzoate anion, which fluorophore under the influence of an excitation beam of radiation of 380-420 nm wavelength, preferably of 405 nm wavelength, emits radiation of 450-500 nm wavelength, preferably of 480 nm wavelength. The fluorimetric method according to the invention allows 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. It is a single point method that requires relatively short incubation times (200-630 s), which allows to avoid the inconvenience of enzymatic methods. The creatinine determination range is 0.5-1000 μmol-L -1 , for incubation times of 20-3600 s, preferably 3-750 μmol-L -1  for 300 s. The method according to the invention allows the measurement of creatinine in blood serum samples, urine or dialysate after adequate dilution. The speed of measurements and simplicity of measurements, as well as the use of non-toxic components of the fluorimetric reagent, make it possible to create measurement systems ready for use at any time and place, in accordance with the point of care testing concept. The unit measurement price is competitive with other routinely used methods of creatinine determination in biological samples.