Method for stabilizing creatinine-containing substance

Abstract

To provide a method for stabilizing a creatinine-containing substance in which, while a calibration substance of creatinine measurement in a sample, a standard substance of creatinine measurement in a sample or a reference substance of accuracy control of creatinine measurement in a sample, or the like that is a creatinine-containing substance to be used in creatinine measurement in a sample, needs to be in a liquid state during use, a concentration of creatinine varies with time in a creatinine-containing substance in the liquid state, and therefore the stability has been inadequate.SOLUTION: In a creatinine-containing substance of a liquid state and a refrigerated condition, in which a solvent is blood serum, blood plasma, or a protein-containing solution, creatine is made to co-exist with creatinine, and further, made to co-exist such that a molar ratio obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L) falls in the range of 2.0-2.8.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for stabilizing a substance containing creatinine, which can be a marker of kidney function. The present invention is useful in fields of life science such as clinical testing, clinical pathology, and medicine, and in fields of chemistry such as organic chemistry and analytical chemistry. [Background technology]

[0002] Creatinine in human serum is high in patients with diseases such as glomerulonephritis, renal failure, congestive heart failure, acromegaly, gigantism, etc., and is low in patients with diseases such as diabetes insipidus, muscular dystrophy, polymyositis, etc. Thus, creatinine in a sample is related to various diseases such as kidney disease and muscle disease, and its measurement is considered to be clinically important.

[0003] The method for measuring creatinine in a sample has traditionally been the alkaline picric acid method, which utilizes the Jaffe reaction in which creatinine and picric acid in an alkaline solution form a reddish-brown compound. However, this method has the drawback of being subject to many non-specific reactions and to the influence of drugs, which can result in errors in the measured values. Therefore, various enzymatic measurement methods have been developed to overcome these drawbacks.

[0004] In this enzymatic measurement method, for example, creatinine in a sample is first converted to creatine by creatinine amidohydrolase (creatinase). Next, the resulting creatine is converted to sarcosine by creatine amidinohydrolase (creatinase), and the sarcosine is further converted to hydrogen peroxide, formaldehyde, and glycine by sarcosine oxidase, and either the hydrogen peroxide or the formaldehyde is enzymatically measured. When hydrogen peroxide is measured, a measurement method in which it is mixed with a color-developing reagent using peroxidase to induce a color-developing system and the generated hydrogen peroxide is measured can be mentioned (see Non-Patent Document 1).

[0005] Conventionally, a known creatinine-containing substance is an aqueous creatinine solution in which the pH is made strongly acidic to stabilize the creatinine. In addition, it is known that the solvent for a creatinine-containing substance is serum, and the creatinine-containing substance is in a frozen state when manufactured, supplied, or sold, and when the creatinine-containing substance is used, the creatinine-containing substance is turned into a liquid state by placing the creatinine-containing substance at a low temperature or room temperature, but the usable period in the liquid state is only about one month.

[0006] In addition, a prior art document discloses "a composition for human consumption comprising creatine and a sufficient amount of creatinine to render the creatine substantially stable when provided in an aqueous solvent." This is a creatine composition that contains a sufficient amount of creatinine to render the creatine substantially stable. The invention described in this prior document is directed to stabilizing creatine, whereas the present invention is directed to stabilizing creatinine, and is therefore completely different (see Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2004-520060 [Non-patent literature]

[0008] [Non-Patent Document 1] Inspection and Technology, Vol. 15, No. 3, pp. 259-262, Yoshikazu Sasaki, 1987 Summary of the Invention [Problem to be solved by the invention]

[0009] Creatinine-containing substances used when measuring creatinine in a sample, such as calibration substances for measuring creatinine in a sample, standard substances for measuring creatinine in a sample, or control substances for quality control of measuring creatinine in a sample, need to be in a liquid state when used, but the concentration of creatinine in the creatinine-containing substance in the liquid state changes over time and its stability is insufficient.

[0010] In response to this, an object of the present invention is to provide a method for stabilizing a creatinine-containing substance. [Means for solving the problem]

[0011] The present inventors have conducted extensive investigations into methods for stabilizing a creatinine-containing substance in a liquid, refrigerated state using a solvent such as serum, plasma, or a protein-containing solution, and have found that the above-mentioned problems can be solved by allowing creatinine to coexist with creatine such that the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) is between 1.8 and 3.0, thereby completing the present invention.

[0012] That is, the present invention comprises the following inventions. (1) A method for stabilizing a creatinine-containing substance, comprising allowing creatine to coexist with creatinine in a refrigerated liquid creatinine-containing substance whose solvent is serum, plasma or a protein-containing solution, and allowing the creatine to coexist so that the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) is between 2.0 and 2.8. (2) The method for stabilizing a creatinine-containing substance according to (1) above, wherein the solvent is serum, plasma or a protein-containing solution and the creatinine-containing substance in a liquid and refrigerated state is a calibration material for measuring creatinine in a sample, a standard material for measuring creatinine in a sample, or a control material for quality control of measuring creatinine in a sample. Effect of the Invention

[0013] The method for stabilizing a creatinine-containing substance of the present invention is capable of stabilizing the creatinine-containing substance in a liquid and refrigerated state using a solvent such as serum, plasma or a protein-containing solution, so that the creatinine concentration of the creatinine-containing substance does not change for a long period of time.

[0014] For this reason, the calibration material for measuring creatinine in a sample, the standard material for measuring creatinine in a sample, and the control material for quality control of measuring creatinine in a sample, which are made of the stabilized creatinine-containing substance of the present invention, are stable for a long period of time and can contribute to obtaining accurate measurement values ​​of creatinine in a sample for a long period of time. As a result, the stabilized creatinine-containing substance of the present invention can contribute to the accurate diagnosis of diseases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] (1) Overview The method for stabilizing a creatinine-containing substance in the present invention is characterized in that, in a creatinine-containing substance in a liquid and refrigerated state in which the solvent is serum, plasma or a protein-containing solution, creatine is allowed to coexist with creatinine such that the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) is between 2.0 and 2.8. As a result, the method for stabilizing a creatinine-containing substance in the present invention exhibits the effects described in the above section [Effects of the Invention], such as that the creatinine concentration does not change for a long period of time even when the solvent is serum, plasma or a protein-containing solution and is in a liquid state and refrigerated, and the creatinine-containing substance is stable.

[0016] (2) Creatinine The creatinine used in the present invention may be any commercially available creatinine.

[0017] (3) Creatine The creatine used in the present invention may be any commercially available creatine.

[0018] (4) Coexistence ratio of creatinine and creatine In the present invention, the ratio of creatine to be coexisted with the creatinine-containing substance must be such that the molar ratio obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L) is between 2.0 and 2.8.

[0019] In the present invention, the lower limit of the molar ratio of creatine to be coexisted with the creatinine-containing substance, that is, the creatine concentration (mol / L) divided by the creatinine concentration (mol / L), is preferably 2.2, more preferably 2.4, from the viewpoint of its effectiveness.

[0020] In the present invention, the upper limit of the molar ratio of creatine to be coexisted with the creatinine-containing substance, that is, the creatine concentration (mol / L) divided by the creatinine concentration (mol / L), is preferably 2.6, more preferably 2.4, from the viewpoint of its effectiveness.

[0021] In the present invention, the molar ratio obtained by dividing the creatine concentration (mol / L), which is the ratio of creatine to be coexisted with the creatinine-containing substance, by the creatinine concentration (mol / L) is, when the lower limit of the molar ratio is 2.0, examples of the molar ratio include 2.0 to 2.4, 2.0 to 2.6, and 2.0 to 2.8.

[0022] In the present invention, the molar ratio of creatine coexisting in a creatinine-containing substance, which is the creatine concentration (mol / L) divided by the creatinine concentration (mol / L), may be 2.2 to 2.4, 2.2 to 2.6, or 2.2 to 2.8 when the lower limit of the molar ratio is 2.2.

[0023] In the present invention, the molar ratio of creatine coexisting in a creatinine-containing substance, that is, the creatine concentration (mol / L) divided by the creatinine concentration (mol / L), can be 2.4, 2.4 to 2.6, or 2.4 to 2.8 when the lower limit of the molar ratio is 2.4.

[0024] In the present invention, the molar ratio obtained by dividing the creatine concentration (mol / L), which is the ratio of creatine to be coexisted with the creatinine-containing substance, by the creatinine concentration (mol / L) is, when the upper limit of the molar ratio is 2.8, examples of the molar ratio include 2.0 to 2.8, 2.2 to 2.8, and 2.4 to 2.8.

[0025] In the present invention, the molar ratio obtained by dividing the creatine concentration (mol / L), which is the ratio of creatine to be coexisted with the creatinine-containing substance, by the creatinine concentration (mol / L) is, when the upper limit of the molar ratio is 2.6, examples of the molar ratio include 2.0 to 2.6, 2.2 to 2.6, and 2.4 to 2.6.

[0026] In the present invention, the molar ratio of creatine coexisting in a creatinine-containing substance, that is, the creatine concentration (mol / L) divided by the creatinine concentration (mol / L), when the upper limit of the molar ratio is 2.4, can be 2.0 to 2.4, 2.2 to 2.4, or 2.4.

[0027] (5) Creatinine-containing substances in a liquid state The creatinine-containing substance in the method for stabilizing a creatinine-containing substance of the present invention is in a liquid state.

[0028] The solvent for the liquid creatinine-containing substance in the method for stabilizing a creatinine-containing substance of the present invention is serum, plasma or a protein-containing solution.

[0029] In the method for stabilizing a creatinine-containing substance of the present invention, the solvent for the liquid creatinine-containing substance is preferably serum or plasma, and more preferably serum.

[0030] Moreover, the protein-containing solution can include, for example, an albumin-containing aqueous solution. Examples of the albumin include human serum albumin (HSA) and bovine serum albumin (BSA).

[0031] Although there is no limitation on the protein concentration in the protein-containing solution, the protein concentration is preferably 0.1 to 20% (w / v).

[0032] (6) Creatinine-containing substances in a refrigerated state The creatinine-containing substance in the method for stabilizing a creatinine-containing substance of the present invention is in a refrigerated state. As for the refrigerated condition, the creatinine-containing substance in the stabilization method of the present invention is preferably kept at 2°C to 8°C, more preferably at 4°C to 6°C, and particularly preferably at 5°C.

[0033] (7) Calibration materials, reference materials, or quality control materials The creatinine-containing substance in the method for stabilizing a creatinine-containing substance of the present invention may be a calibration substance for measuring creatinine in a sample, a standard substance for measuring creatinine in a sample, or a control substance for quality control of measuring creatinine in a sample.

[0034] (8) Multiple calibrators, multiple standards, or multiple control materials for quality control The creatinine-containing substance in the stabilization method of the present invention may be a multi-calibrator, multi-standard, or multi-control substance for quality control, which contains one or more calibrators, standards, or control substances for quality control in addition to creatinine, creatine, and a solvent.

[0035] (9) Other substances contained The creatinine-containing substance in the method for stabilizing a creatinine-containing substance of the present invention may contain, in addition to creatinine, creatine, and a solvent, one or more of various proteins; various salts; various sugars; various buffers; various preservatives such as sodium azide or antibiotics; or various surfactants such as nonionic surfactants, amphoteric surfactants, cationic surfactants, or anionic surfactants, as appropriate.

[0036] The concentration of these when added to the creatinine-containing substance in the stabilization method of the creatinine-containing substance of the present invention is not particularly limited, but is preferably 0.001 to 10% (w / v), and more preferably 0.01 to 5% (w / v). EXAMPLES

[0037] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0038] [Example 1] (Confirmation of the stability of creatinine in the stored creatinine-containing substance of the present invention)

[0039] 1. Reagents (1) Creatinine measurement reagent Commercially available creatinine measurement reagents (in vitro diagnostic drugs) "Cygnus Auto CRE RI" and "Cygnus Auto CRE R-II" (both manufactured by Sinotest Co., Ltd.) were used as creatinine measurement reagents.

[0040] (2) Creatinine and creatine measurement reagents A measurement reagent ("creatinine and creatine measurement reagent") for measuring the total amount of creatinine and creatine was prepared by mixing "Cygnus Auto CRE RI" (Cynotest) and "Cygnus Auto CRE R-II" (Cynotest), which are commercially available creatinine measurement reagents (in vitro diagnostic drugs), in a 3:1 ratio. (By mixing the two-reagent system "Cygnus Auto CRE RI" and "Cygnus Auto CRE R-II" to make a single reagent system, the elimination of creatinine, which is normally performed in the first reaction, is not carried out, and the total amount of creatinine and creatine is measured.)

[0041] 2. Sample (1) Pure water sample Pure water was prepared as a sample for reagent blank measurement.

[0042] (2) Creatinine standard solution A commercially available "CRE standard solution (5.0 mg / dL)" (Shinotest Co., Ltd.) was prepared as the creatinine standard solution.

[0043] (3) A creatinine-containing substance with a molar ratio of 0.2 (creatine concentration (mol / L) divided by creatinine concentration (mol / L)) Serum with a creatine concentration of 0.0844 mmol / L was selected, and creatinine was added to this serum so that the creatinine concentration became 0.422 mmol / L, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 0.2.

[0044] (4) A creatinine-containing substance with a molar ratio of 4.0, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). Creatine was added to the "creatinine-containing substance having a molar ratio of 0.2, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above, to give a concentration of 1.688 mmol / L, thereby preparing a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 4.0.

[0045] (5) A creatinine-containing substance with a molar ratio of 1.0, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 15.6 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 4.4 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 1.0.

[0046] (6) A creatinine-containing substance with a molar ratio of 1.6 (creatine concentration (mol / L) divided by creatinine concentration (mol / L)) 12.5 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 7.5 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 1.6.

[0047] (7) A creatinine-containing substance with a molar ratio of 1.8, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 11.5 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 8.5 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 1.8.

[0048] (8) A creatinine-containing substance with a molar ratio of 2.0, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 10.4 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 9.6 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 2.0.

[0049] (9) A creatinine-containing substance with a molar ratio of 2.2, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 9.4 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 10.6 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 2.2.

[0050] (10) A creatinine-containing substance with a molar ratio of 2.4, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 8.4 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 11.6 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 2.4.

[0051] (11) A creatinine-containing substance with a molar ratio of 2.6, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 7.3 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 12.7 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 2.6.

[0052] (12) A creatinine-containing substance with a molar ratio of 2.8, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 6.3 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 13.7 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 2.8.

[0053] (13) A creatinine-containing substance with a molar ratio of 3.0, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 5.2 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 14.8 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 3.0.

[0054] (14) A creatinine-containing substance with a molar ratio of 3.4, calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L). 3.2 mL of the "creatinine-containing substance having a molar ratio of 0.2, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (3) above was mixed with 16.8 mL of the "creatinine-containing substance having a molar ratio of 4.0, obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L)" prepared in (4) above, to prepare a creatinine-containing substance consisting of serum with a creatine (mol / L) / creatinine (mol / L) ratio of 3.4.

[0055] 3. Storage of creatinine-containing substances The 12 kinds of creatinine-containing substances prepared in the above 2 (3) to (14) were each divided into small portions, placed in bottles, and sealed with stoppers. Then, each of the bottles containing the 12 types of creatinine-containing substances prepared in (3) to (14) of Section 2 was stored at -80°C and at 2°C to 8°C, respectively. At the start of storage and after 6 months from the start of storage, the unopened bottles were opened and the creatinine concentration of each creatinine-containing substance and the total concentration of creatinine and creatine were measured.

[0056] 4. Measurement of creatinine concentration in creatinine-containing substances The creatinine concentration of the creatinine-containing substance was measured at each temperature and for each storage period in the above item 3.

[0057] Specifically, the measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Technologies Corporation), and 150 μL of "Cygnus Auto CRE RI" in 1(1) above was added and mixed to 6 μL of each of the creatinine-containing substances in 3 above, and the reaction was allowed to proceed at 37°C. Five minutes after the addition and mixing of the "Cygnus Auto CRE RI", 50 μL of "Cygnus Auto CRE R-II" in 1(1) above was added and mixed, and the reaction was allowed to proceed at 37°C.

[0058] The absorbance difference was calculated by subtracting the absorbance (main wavelength: 600 nm, secondary wavelength: 750 nm) 4 minutes and 30 seconds after the addition and mixing of the "Cygnus Auto CRE RI" from the absorbance (main wavelength: 600 nm, secondary wavelength: 750 nm) 10 minutes after the addition and mixing of the "Cygnus Auto CRE RI".

[0059] The creatinine standard solution of 2(2) above was then measured in the same manner as the creatinine-containing substance, and the absorbance difference was calculated.

[0060] The pure water of 2(1) was then measured in the same manner as the creatinine-containing substance to determine the reagent blank value, which was then subtracted from the absorbance difference value obtained when the creatinine-containing substance was measured to determine the absorbance difference value. Furthermore, the reagent blank value was subtracted from the absorbance difference value obtained when the creatinine standard solution was measured to determine the absorbance difference value.

[0061] Then, based on the absorbance difference value of the creatinine standard solution, the creatinine concentration value of the creatinine-containing substance was calculated from the absorbance difference value of the creatinine-containing substance.

[0062] 5. Measurement of the total concentration of creatinine and creatine in creatinine-containing substances The total concentrations of creatinine and creatine in the creatinine-containing substances were measured at each temperature and for each storage period in the above item 3.

[0063] Specifically, the measurements were performed using a Hitachi 7180 automatic analyzer (Hitachi High-Technologies Corporation), and 200 μL of the "creatinine and creatine measurement reagent" in 1(2) above was added to and mixed with 6 μL of each of the creatinine-containing substances in 3 above, and the reaction was carried out at 37°C. The absorbance (main wavelength: 600 nm, secondary wavelength: 750 nm) was determined 10 minutes after the addition and mixing of the "creatinine and creatine measurement reagent".

[0064] The creatinine standard solution of 2(2) above was then measured in the same manner as the creatinine-containing substance, and the absorbance difference was calculated.

[0065] The pure water of 2(1) was then measured in the same manner as the creatinine-containing substance to determine the reagent blank value, which was then subtracted from the absorbance difference value obtained when the creatinine-containing substance was measured to determine the absorbance difference value. Furthermore, the reagent blank value was subtracted from the absorbance difference value obtained when the creatinine standard solution was measured to determine the absorbance difference value.

[0066] Then, based on the absorbance difference value of the creatinine standard solution, the total amount of creatinine and creatine in the creatinine-containing substance was calculated from the absorbance difference value of the creatinine-containing substance.

[0067] 6. Calculation of creatine concentration in creatinine-containing substances The creatinine concentration of the creatinine-containing substance at each temperature and storage period in 3 above, determined in 4 above, was subtracted from the total concentration of creatinine and creatine of the creatinine-containing substance at each temperature and storage period in 3 above, determined in 5 above, to calculate the creatine concentration of the creatinine-containing substance at each temperature and storage period in 3 above.

[0068] 7. Calculate the molar ratio by dividing the creatinine concentration (mol / L) of the creatinine-containing material by the creatinine concentration (mol / L) at the start of storage. (1) At the start of storage, the molar ratios were calculated by dividing the creatinine concentration (mol / L) determined in 6 above by the creatinine concentration (mol / L) determined in 4 above for a total of 12 creatinine-containing substances prepared in 2(3) to 2(14) above.

[0069] (2) As a result, for the creatinine-containing substance in 2(3) above, whose molar ratio obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L) was 0.2, the molar ratio (mol / L) obtained by dividing the calculated creatine concentration (mol / L) by the creatinine concentration (mol / L) was 0.2.

[0070] (2) In addition, for the creatinine-containing substance in 2(4) above, in which the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) was 4.0, the molar ratio of the calculated creatine concentration (mol / L) divided by the creatinine concentration (mol / L) was 4.0.

[0071] (3) Furthermore, for the creatinine-containing substance in 2(5) above, in which the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) was 1.0, the molar ratio of the calculated creatine concentration (mol / L) divided by the creatinine concentration (mol / L) was 1.0.

[0072] (4) Furthermore, for the creatinine-containing substance in 2(6) above having a molar ratio of 1.6 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the molar ratio obtained by dividing the calculated creatine concentration (mol / L) by the creatinine concentration (mol / L) was also 1.6.

[0073] (5) Furthermore, for the creatinine-containing substance in 2(7) above having a molar ratio of 1.8 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the molar ratio obtained by dividing the calculated creatine concentration (mol / L) by the creatinine concentration (mol / L) was also 1.8.

[0074] (6) Furthermore, for the creatinine-containing substance in 2(8) above having a molar ratio of 2.0 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the molar ratio obtained by dividing the calculated creatine concentration (mol / L) by the creatinine concentration (mol / L) was also 2.0.

[0075] (7) Furthermore, for the creatinine-containing substance in 2(9) above having a molar ratio of 2.2 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the molar ratio calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L) was also 2.2.

[0076] (8) Furthermore, for the creatinine-containing substance in 2(10) above having a molar ratio of 2.4 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the molar ratio obtained by dividing the calculated creatine concentration (mol / L) by the creatinine concentration (mol / L) was also 2.4.

[0077] (9) In addition, for the creatinine-containing substance in 2(11) above having a molar ratio of 2.6 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the molar ratio calculated by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L) was also 2.6.

[0078] (10) In addition, for the creatinine-containing substance in 2(12) above having a molar ratio of 2.8 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the calculated molar ratio of 2.8 was obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L).

[0079] (11) In addition, for the creatinine-containing substance in 2(13) above having a molar ratio of 3.0 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the calculated molar ratio of the creatine concentration (mol / L) by the creatinine concentration (mol / L) was 3.0.

[0080] (12) In addition, for the creatinine-containing substance in 2(14) above having a molar ratio of 3.4 obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L), the calculated molar ratio of 3.4 was obtained by dividing the creatine concentration (mol / L) by the creatinine concentration (mol / L).

[0081] 8. Relative ratio of creatinine concentration in creatinine-containing materials after 6 months of storage The relative ratio (unit: percentage) calculated by dividing the creatinine concentration value of the creatinine-containing substance stored at 2 to 8°C for 6 months calculated in 4 above by the creatinine concentration value of the creatinine-containing substance stored at -80°C for 6 months calculated in 4 above is shown in Table 1.

[0082] [Table 1]

[0083] 9.Measurement results From Table 1, it can be seen that when the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) is between 1.8 and 3.0, the relative ratio of the creatinine concentration is close to 100%, and the creatinine concentration of the refrigerated creatinine-containing substance does not change for a long period of time, and the creatinine-containing substance can be stabilized. In particular, when the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) is between 2.0 and 2.8, the relative ratio of the creatinine concentration is close to 100%, and the creatinine concentration of the refrigerated creatinine-containing substance does not change for a long period of time, and the creatinine-containing substance can be stabilized.

[0084] This confirms that the creatinine concentration of the creatinine-containing substance of the present invention does not change for a long period of time, and the creatinine-containing substance can be stabilized. It has been confirmed that the calibration material for measuring creatinine in a sample, the standard material for measuring creatinine in a sample, and the control material for quality control of measuring creatinine in a sample, which are made of the stabilized creatinine-containing material of the present invention, are stable for a long period of time and can contribute to obtaining accurate measurement values ​​of creatinine in a sample for a long period of time. This confirms that the stabilized creatinine-containing substance of the present invention can contribute to the accurate diagnosis of diseases.

Claims

1. A method for stabilizing a creatinine-containing substance, comprising allowing creatine to coexist with creatinine in a refrigerated liquid state creatinine-containing substance in which the solvent is serum, plasma or a protein-containing solution, and allowing the coexistence such that the molar ratio of the creatine concentration (mol / L) divided by the creatinine concentration (mol / L) is between 2.0 and 2.

8.

2. The method for stabilizing a creatinine-containing substance according to claim 1, wherein the solvent is serum, plasma or a protein-containing solution, and the creatinine-containing substance in a liquid and refrigerated state is a calibration material for measuring creatinine in a sample, a standard material for measuring creatinine in a sample, or a control material for quality control of measuring creatinine in a sample.

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