A reagent for determining the percentage of glycosylated hemoglobin and a detection method thereof

Abstract

The present invention discloses a reagent for determining the percentage of glycated hemoglobin and a detection method thereof. The reagent comprises an eluent A and an eluent B; the eluent A comprises HEPES and MES, and the concentrations of HEPES in the eluent A and MES in the eluent A are independently 0.05-20 g / L; the eluent B comprises HEPES and MES, and the concentrations of HEPES in the eluent B and MES in the eluent B are independently 0.05-40 g / L; the concentrations of HEPES in the eluent A and HEPES in the eluent B are different; and the concentrations of MES in the eluent A and MES in the eluent B are different. The reagent can effectively determine the percentage of glycated hemoglobin, and can directly determine the percentage of glycated hemoglobin without separately determining the total hemoglobin in the sample.

Description

Technical Field

[0001] The invention relates to a reagent for determining the percentage of glycosylated hemoglobin and a detection method thereof. Background Art

[0002] Diabetes is considered a serious public health problem due to its high prevalence and associated complications. Globally, 422 million adults have diabetes, and this number is expected to rise to 640 million by 2040. The increase in type 2 diabetes over the past 30 years has primarily mediated this epidemic, with considerable individual and global impacts. The risk of cardiovascular disease-related mortality in patients with diabetes is two to ten times that of patients with normoglycemia of the same age; and the risk of all-cause mortality, cardiovascular complications (such as coronary heart disease, heart failure, stroke, and peripheral arterial disease), and microvascular complications (including retinopathy, neuropathy, and nephropathy) are also significantly higher in patients with diabetes.

[0003] Glycated hemoglobin (GHb) is the product of the combination of hemoglobin in red blood cells and sugars (mainly glucose) in serum through non-enzymatic reactions. The non-enzymatic reaction to form glycated hemoglobin is characterized by persistence, slowness, and irreversibility. Therefore, the content of glycated hemoglobin is determined by the past rather than the immediate blood sugar concentration, and has nothing to do with factors such as whether the patient is fasting before the test, whether insulin is injected, and whether hypoglycemic drugs are taken. It is generally believed that the concentration of glycated hemoglobin can effectively reflect the average blood sugar level in the past 8-12 weeks. There are three types of hemoglobin in the blood of healthy adults: HbA, HbF, and HbA0. GHb is composed of HbA1a, HbA1b, HbA1c, etc., of which HbA1c accounts for about 70%, and has a relatively stable structure. It is often used as a monitoring indicator for diabetes control in clinical practice, and its concentration is expressed as a percentage of adult hemoglobin. HbA0 is the main component in the blood, accounting for 80-90%. HbF is the main interfering component.

[0004] Glycated hemoglobin levels can be used as a diagnostic test for diabetes. The Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020 Edition) formally included "glycated hemoglobin" in the diabetes diagnostic criteria for the first time, and glycated hemoglobin ≥ 6.5% can be used as a basis for diagnosing diabetes. However, a result below 6.5% does not rule out the possibility of diabetes, and the results of glucose determination should also be referred to. If glycated hemoglobin > 9.0%, it means that the patient has persistent hyperglycemia.

[0005] At present, the detection methods for HbA1c mainly include electrophoresis, immunoassay, affinity chromatography, chromatography, enzyme method, etc. Among them, electrophoresis requires specific instruments and equipment, which is expensive and not suitable for use in small and medium-sized hospitals; although the immunoassay is low in cost and the instrument is easy to operate, it has the disadvantages of low precision and single test results; affinity chromatography is simple and fast to operate, but the error is large, the test result is low in accuracy, and it measures the total amount of GHb; the enzyme method uses redox reaction to detect glycated hemoglobin, which requires the participation of multiple enzymes. Chromatography includes high-performance liquid cation exchange chromatography and high-performance liquid boric acid affinity chromatography. As the current gold standard method for HbA1c detection, high-performance liquid ion chromatography has the characteristics of high precision, simple operation, and good repeatability. It is the most frequently used in clinical practice and has high accuracy. However, when detecting the proportion of glycated hemoglobin components in blood samples, many HPLC detection reagents cannot effectively separate the components, thus affecting the HbA1c test results.

[0006] In summary, the methods mentioned above either have high instrument costs and low accuracy, or measure the total amount of GHb, or the HPLC detection reagent cannot effectively separate the various components. Therefore, it is necessary to improve the existing reagents and methods for measuring the glycated hemoglobin ratio and provide a new reagent and its detection method to solve these problems. Summary of the invention

[0007] In order to overcome the shortcomings and deficiencies in the prior art, the purpose of the present invention is to provide a reagent for determining the percentage of glycated hemoglobin, which can effectively determine the percentage of glycated hemoglobin and can directly determine the percentage of glycated hemoglobin without the need to separately determine the total hemoglobin in the sample.

[0008] Another object of the present invention is to provide a method for determining the percentage of glycosylated hemoglobin, which utilizes high performance liquid ion chromatography to determine HbA1c in whole blood, and the determination result has high accuracy.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a reagent for determining the percentage of glycated hemoglobin, the reagent comprising an eluent A and an eluent B;

[0011] The eluent A comprises HEPES and MES, and the concentrations of HEPES in the eluent A and MES in the eluent A are independently 0.05 to 20 g / L;

[0012] The eluent B comprises HEPES and MES, and the concentrations of HEPES in the eluent B and MES in the eluent B are independently 0.05 to 40 g / L;

[0013] The concentrations of HEPES in the eluent A and the HEPES in the eluent B are different;

[0014] The concentrations of MES in the eluent A and the MES in the eluent B are different.

[0015] Preferably, the concentration of HEPES in the eluent A is 3-18 g / L, such as 4.3 g / L or 15 g / L, and the concentration of MES in the eluent A is 0.1-8 g / L, such as 0.2 g / L or 5 g / L.

[0016] Preferably, the concentration of HEPES in the eluent B is 2-20 g / L, such as 3.8 g / L or 18 g / L, and the concentration of MES in the eluent B is 0.1-10 g / L, such as 0.8 g / L or 8 g / L.

[0017] In the present invention, preferably, the pH value of the eluent A and the eluent B is 4.5 to 6, preferably 5 to 5.8, such as 5.2 to 5.5.

[0018] In the present invention, preferably, the eluent A and the eluent B further independently include one or more of a salt solution, a protonic acid and a preservative.

[0019] Wherein, the salt solution may be one or more of sodium chloride, sodium sulfate, and sodium nitrate.

[0020] Preferably, the salt solution in the eluent A is sodium chloride. The concentration of the salt solution in the eluent A is preferably 1-50 g / L.

[0021] More preferably, the concentration of sodium chloride in the eluent A is 10 g / L.

[0022] Preferably, the salt solution in the eluent B is sodium chloride. The preferred concentration of the salt solution in the eluent A is 1-100 g / L.

[0023] More preferably, the concentration of sodium chloride in the eluent B is 20 g / L.

[0024] The protonic acid may be one or more of trifluoroacetic acid, glycine, dilute nitric acid, and dilute hydrochloric acid, such as trifluoroacetic acid.

[0025] Preferably, the protonic acid in the eluent A is trifluoroacetic acid. The concentration of the protonic acid in the eluent A is preferably 0.01 to 20 g / L. More preferably, the concentration of trifluoroacetic acid in the eluent A is 1 g / L.

[0026] Preferably, the protonic acid in the eluent B is trifluoroacetic acid. The concentration of the protonic acid in the eluent A is preferably 0.01 to 20 g / L. More preferably, the concentration of trifluoroacetic acid in the eluent B is 1 g / L.

[0027] Wherein, the preservative may be one or more of sodium azide, Proclin 300, and thimerosal.

[0028] Preferably, the preservative in the eluent A is sodium azide. The concentration of the preservative in the eluent A is preferably less than 10 g / L.

[0029] More preferably, the concentration of sodium azide in the eluent A is 1 g / L.

[0030] Preferably, the preservative in the eluent B is sodium azide. The concentration of the preservative in the eluent B is preferably less than 10 g / L.

[0031] More preferably, the concentration of sodium azide in the eluent B is 1 g / L.

[0032] In the present invention, preferably, the reagent includes, in addition to the eluent A and the eluent B, a hemolytic agent.

[0033] Wherein, the hemolytic agent is two independent systems with the eluent A and the eluent B before elution, and can exist independently in the eluent system. The hemolytic agent can include a buffer.

[0034] The buffer may be one or more of a MES buffer, a HEPES buffer, and a phosphate buffer.

[0035] Preferably, the buffer in the hemolytic agent is MES buffer. The concentration of the buffer in the hemolytic agent is preferably 0.01-1 g / L.

[0036] More preferably, the concentration of MES buffer in the hemolytic agent is 0.5 g / L.

[0037] Preferably, the hemolytic agent may further include a protonic acid and / or a preservative.

[0038] The protonic acid may be one or more of trifluoroacetic acid, glycine, dilute nitric acid, and dilute hydrochloric acid.

[0039] Preferably, the protonic acid in the hemolytic agent is trifluoroacetic acid. The concentration of the protonic acid in the hemolytic agent is preferably 0.001 g / L to 5 g / L.

[0040] More preferably, the concentration of trifluoroacetic acid in the hemolytic agent is 0.1 g / L.

[0041] The preservative may be one or more of sodium azide, Proclin 300, and thimerosal.

[0042] Preferably, the preservative in the hemolytic agent is sodium azide. The concentration of sodium azide in the hemolytic agent is preferably less than 10 g / L.

[0043] More preferably, the concentration of sodium azide in the hemolytic agent is 1 g / L.

[0044] In a preferred embodiment of the present invention, the reagents include: eluent A includes 0.2 g / L MES, 4.3 g / L HEPES, 10 g / L sodium chloride, 1 g / L trifluoroacetic acid and 1 g / L sodium azide; eluent B includes 0.8 g / L MES, 3.8 g / L HEPES, 20 g / L sodium chloride, 1 g / L trifluoroacetic acid and 1 g / L sodium azide; the hemolytic agent includes 0.5 g / L MES, 0.1 g / L trifluoroacetic acid and 1 g / L sodium azide.

[0045] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0046] In a second aspect, the present invention provides a method for determining the percentage of glycated hemoglobin, which uses the reagent for determining the percentage of glycated hemoglobin as described above, and the method is the following method 1 or method 2:

[0047] Method 1: When the reagents include eluent A and eluent B, it comprises the following steps:

[0048] (1) eluting the hemolyzed sample with the eluent A and the eluent B in an analytical column to obtain HbA1c component and HbA0 component and then detecting them;

[0049] (2) Calculate the HbA1c test result.

[0050] Method 2: When the reagents include eluent A, eluent B and a hemolytic agent, it comprises the following steps:

[0051] (1) adding the hemolytic agent to the whole blood sample for hemolysis;

[0052] (2) eluting the hemolyzed sample in step (1) with the eluent A and the eluent B in an analytical column to obtain HbA1c component and HbA0 component, and then detecting them;

[0053] (3) Calculate the HbA1c test result.

[0054] The filler of the analytical column may be a cation exchange stationary phase.

[0055] The detection wavelength may be 415 nm.

[0056] The column temperature of the analytical column may be 37°C.

[0057] Taking the total volume of eluent A and eluent B as 100%, the elution gradient can be: 1s-6s, the volume of eluent A can be 70%, and the volume of eluent B can be 30%; 7s-25s, the volume of eluent A can be 50%, and the volume of eluent B can be 50%; 26s-50s, the volume of eluent A can be 0%, and the volume of eluent B can be 100%, or, the elution gradient can be: 1s-3s, the volume of eluent A can be 70%, and the volume of eluent B can be 30%; 3s-18s, the volume of eluent A can be 50%, and the volume of eluent B can be 50%; 18s-30s, the volume of eluent A can be 0%, and the volume of eluent B can be 100%.

[0058] The flow rates of the eluent A and the eluent B may be 1 mL / min to 2 mL / min, for example, 1.5 mL / min or 1.8 mL / min.

[0059] The injection volume can be 5 μL.

[0060] Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0061] The positive and progressive effects of the present invention are:

[0062] (1) The present invention provides a glycated hemoglobin detection reagent with a separation effect, which can effectively determine the percentage of glycated hemoglobin. The percentage of glycated hemoglobin can be directly determined without separately determining the total hemoglobin in the sample.

[0063] (2) Additives are added to the eluent A, eluent B and hemolytic agent, which can prevent the measured value of the sample from changing with the storage time of the sample. At the same time, the present invention can directly test the whole blood and mix and dilute it in the machine, reducing the complicated process of sample pretreatment. The hemolytic agent and the eluent belong to a similar system and will not have incompatible rejection, thereby achieving efficient and accurate detection of glycated hemoglobin.

[0064] (3) The method for measuring glycated hemoglobin provided by the present invention can also achieve rapid and accurate detection of samples after storage. Therefore, for situations where the blood sample collection site and the test site are different, or a certain number of samples are collected for centralized testing, and the samples need to be stored before testing, the kit and the determination method provided by the present invention have very important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1This is a diagram showing the effect of the HPLC reagent in Example 1 on testing clinical samples.

[0066] Figure 2 This is a diagram showing the effect of the HPLC reagent in Example 2 on testing clinical samples.

[0067] Figure 3 This is a correlation trend diagram between the HPLC reagent in Example 4 and the test results of the Tosoh HPLC method.

[0068] Figure 4 This is a diagram showing the effect of the HPLC reagent in Example 6 on testing clinical samples.

[0069] Figure 5 This is a diagram showing the effect of the HPLC reagent in comparative example 1 on testing clinical samples. DETAILED DESCRIPTION

[0070] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0071] The present invention is further described below.

[0072] The present invention is a method for determining glycated hemoglobin in whole blood by high-performance liquid ion chromatography. First, when a blood sample is diluted with a hemolytic agent and passed through a high-performance liquid analysis column, components with different charges have different adsorption capacities with the analysis column; mixed eluents (eluent A and eluent B) of different proportions are used to separate the components in the sample. The eluted proteins pass through a detector in sequence, and the detector automatically records the change in its light absorption intensity at 415nm, and converts the light signal into an electrical signal to obtain a material signal spectrum. The collected spectrum data is analyzed and calculated to obtain the peak areas of HbA1c, HbA0, etc. The percentage of the HbA1c peak area to the total hemoglobin Hb peak area is the detection result of HbA1c in the human whole blood sample.

[0073] The raw materials used in the following examples are as follows:

[0074] HEPES buffer, MES buffer, sodium chloride, trifluoroacetic acid, sodium azide, purified water, quality control products and calibrators (all are lyophilized hemoglobin blood sample powder). The quality control products and calibrators are lyophilized hemoglobin blood sample powder (purchased from Bio-Rad). The lyophilized hemoglobin blood sample powder of the calibrator can be traced back through IFCC.

[0075] Example 1

[0076] A reagent for determining the percentage of glycosylated hemoglobin, wherein the eluent A comprises 0.2 g / L MES, 4.3 g / L HEPES, 10 g / L sodium chloride, 1 g / L sodium azide, and 1 g / L trifluoroacetic acid; the eluent B comprises 0.8 g / L MES, 3.8 g / L HEPES, 20 g / L sodium chloride, and 1 g / L trifluoroacetic acid; the hemolytic agent comprises 0.5 g / L MES, 0.1 g / L trifluoroacetic acid, and 1 g / L sodium azide; and the sample is a whole blood sample or a freeze-dried whole blood sample.

[0077] A method for determining glycated hemoglobin in whole blood comprises the following steps: adding a hemolytic agent to a sample for hemolysis, eluting the hemolyzed sample with an eluent in an analytical column to obtain HbA1c components and HbA0 components, and then detecting them, and calculating the detection result of HbA1c; the eluents are the eluent A and the eluent B.

[0078] The elution conditions are as follows: the filler of the analytical column is a cation exchange stationary phase, the filler particle size is 5 μm, the column length is 33 mm, and the column inner diameter is 4.0 mm; the detection wavelength is 415 nm; the column temperature is 37°C; the elution gradient is: 1s to 6s, 70% by volume of eluent A, 30% by volume of eluent B; 7s to 25s, 50% by volume of eluent A, 50% by volume of eluent B; 26s to 50s, 0% by volume of eluent A, 100% by volume of eluent B; the flow rate is 1.5 mL / min, and the injection volume is 5 μL.

[0079] The effect diagram of HPLC reagent testing clinical samples in Example 1 of the present invention is shown in Figure 1 , Figure 1 The percentage of substances corresponding to each characteristic peak is shown in the following table, where 24.2s is the special peak of HbA1c:

[0080] time Percentage content time Percentage content 3.5s 0.9％ 24.2s 7.5％ 7.3s 1.1％ 28.8s 3.2％ 16.8s 2.5％ 33.2s 84.8％

[0081] Example 2

[0082] The same as Example 1, except that the kit of Example 2 does not include protonic acid.

[0083] The specific preparation of the eluent A, eluent B and hemolytic agent in the kits of Example 1 and Example 2 is shown in Table 1.

[0084] Table 1 Preparation of eluent A, eluent B and hemolytic agent

[0085]

[0086]

[0087] The effect diagram of HPLC reagent testing clinical samples in Example 2 of the present invention is shown in Figure 2 , Figure 2 The percentages corresponding to the characteristic peaks are shown in the following table, where 23.2s is the special peak of HbA1c:

[0088] time Percentage content time Percentage content 3.3s 0.9％ 23.2s 7.8％ 6.7s 0.7％ 26.2s 3.4％ 9.9s 1.4％ 33.2s 85％ 15.4s 0.8％

[0089] Example 3

[0090] The sample is a quality control product, which is lyophilized powder of glycated hemoglobin blood sample.

[0091] The HbA1c in the quality control product was detected using the kits of Example 1 and Example 2. The detection instrument was a glycosylated hemoglobin meter, and the elution conditions were the same as those of Example 1.

[0092] Take Bio-rad's quality control products, low-value quality control product 1 and high-value quality control product 2, and re-dissolve the quality control products to obtain quality control product solutions. According to the volume ratio of 1:300, the hemolytic agents in Example 1 and Example 2 are added to the two groups of quality control product solutions with a volume of 20 μL for hemolysis; the quality control products after hemolysis are immediately eluted, separated and detected using an analytical column to obtain the HbA1c test results, and the HbA1c is tested on the 3rd and 7th days after being stored in a refrigerator at 2°C to 8°C. The test results are shown in Table 2.

[0093] Table 2 HbA1c test results in Example 1 and Example 2 (HbA1c%)

[0094]

[0095]

[0096] As can be seen from Table 2, as the storage time goes by, when the quality control product is detected using the kit of Example 1 and the determination method of this example, the result of HbA1c does not change substantially, while when the test is performed using the kit of Example 2, the result of HbA1c changes. Therefore, the trifluoroacetic acid added in Example 1 can effectively prevent the determination result of HbA1c from changing.

[0097] Example 4

[0098] The sample was EDTA anticoagulated venous whole blood, and the sample was tested immediately after collection. There were 49 samples in total, 20 μL of each sample was taken, and the G8 glycosylated hemoglobin test system automatically added the hemolytic agent of Example 1 at a ratio of 1:300 for hemolysis.

[0099] Take 12 samples from the above and conduct the following experiment:

[0100] A reagent for determining the percentage of glycated hemoglobin. The reagent of Example 1 is used to detect HbA1c in a sample under the determination method provided in Example 3. The clinical sample is taken back from the hospital, and the clinical sample is determined using the reagent and method of the present invention, and compared with the determination in the hospital. The test results are detailed in Table 3.

[0101] Table 3 HbA1c test results in Example 4 and hospital measurements (HbA1c%)

[0102]

[0103] As can be seen from Table 3, the HbA1c value detected by the kit of Example 1 is substantially consistent with the IFCC target value given by the IFCC standard laboratory. Therefore, the reagent provided in Example 1 and the detection method provided in Example 3 can accurately measure frozen samples after being stored for a period of time.

[0104] Take the remaining 37 samples mentioned above and conduct the following experiment:

[0105] The determination method adopts the HPLC analysis method of the G8 glycosylated hemoglobin test system of Tosoh Corporation of Japan, and the kit is the G8 glycosylated hemoglobin test kit of Tosoh Corporation of Japan.

[0106] The test results of Example 4 and the G8 glycosylated hemoglobin test system of Tosoh Corporation of Japan are shown in Table 4. Correlation analysis was performed on the test results of Example 4 and the G8 glycosylated hemoglobin test system of Tosoh Corporation of Japan, and a linear equation was obtained: y=0.9944x+0.0877, R 2 =0.9869, such as Figure 3 shown.

[0107] Table 4 Test results of HbA1c of Example 4 and G8 glycosylated hemoglobin test system of Tosoh Corporation of Japan

[0108]

[0109]

[0110] From Table 4 and Figure 3 It can be seen that the results obtained by measuring the whole blood sample stored at 2°C to 8°C for 7 days using the reagent of Example 1 and the measuring method of Example 3 are basically consistent with the test results of the G8 glycosylated hemoglobin test system of Tosoh Corporation of Japan, and the correlation is very good. Therefore, the reagent and measuring method of the present invention have high clinical application value.

[0111] Example 5

[0112] Eluent A includes 5 g / L MES, 15 g / L HEPES, 10 g / L sodium chloride, 1 g / L sodium azide, and 1 g / L trifluoroacetic acid; eluent B includes 8 g / L MES, 18 g / L HEPES, 20 g / L sodium chloride, 1 g / L sodium azide, and 1 g / L trifluoroacetic acid; the hemolytic agent includes 0.5 g / L MES, 0.1 g / L trifluoroacetic acid, and 1 g / L sodium azide; the sample is a whole blood sample or a freeze-dried whole blood sample.

[0113] The elution conditions are as follows: the filler of the analytical column is a cation exchange stationary phase, the filler particle size is 5 μm, the column length is 33 mm, and the column inner diameter is 4.0 mm; the detection wavelength is 415 nm; the column temperature is 37°C; the elution gradient is: 1s to 3s, 70% by volume of eluent A, 30% by volume of eluent B; 3s to 18s, 50% by volume of eluent A, 50% by volume of eluent B; 18s to 30s, 0% by volume of eluent A, 100% by volume of eluent B; the flow rate is 1.8 mL / min, and the injection volume is 5 μL.

[0114] The effect diagram of HPLC reagent testing clinical samples in Example 6 of the present invention is shown in Figure 4 , Figure 4 The percentages corresponding to the characteristic peaks are shown in the following table, where 12.6s is the special peak of HbA1c:

[0115] time Percentage content time Percentage content 2.6s 2％ 15.1s 1.7％ 4.9s 0.5％ 18.2s 6.6％ 7.7s 1％ 33.2s 83.4％ 12.6s 4.8％

[0116] Comparative Example 1

[0117] The eluent A includes 7 g / L citric acid, 30 g / L sodium citrate, and 1 g / L sodium azide; the eluent B includes 28 g / L sodium citrate and 1 g / L sodium azide; the hemolytic agent includes 2 g / L sodium citrate and 1 g / L sodium azide; the sample is a whole blood sample or a freeze-dried whole blood sample.

[0118] The elution conditions are as follows: the filler of the analytical column is a cation exchange stationary phase, the filler particle size is 5 μm, the column length is 33 mm, and the column inner diameter is 4.0 mm; the detection wavelength is 415 nm; the column temperature is 37°C; the elution gradient is: 1s to 5s, 80% by volume of eluent A, 20% by volume of eluent B; 6s to 15s, 50% by volume of eluent A, 50% by volume of eluent B; 16s to 30s, 0% by volume of eluent A, 100% by volume of eluent B; the flow rate is 1.8 mL / min, and the injection volume is 5 μL.

[0119] The effect diagram of HPLC reagent testing clinical samples in Comparative Example 1 of the present invention is shown in Figure 5 , Figure 5 The percentages corresponding to the characteristic peaks are shown in the following table, where 15.2s is the special peak of HbA1c:

[0120] time Percentage content time Percentage content 2.8s 2.2％ 20s 85.1％ 7.1s 0.9％ 27.8s 3.4％ 15.2s 8.4％

[0121] Effect Example

[0122] Taking the hospital test value of HbA1C sample as a reference, the test values ​​of the reagents prepared in Example 1, Example 2, Example 5 and Comparative Example 1 were compared with the hospital test values, as shown in Table 5:

[0123] Table 5

[0124] sample Hospital test value Actual detection value deviation Example 1 7.7％ 7.5％ 2.59％ Example 2 7.7％ 7.8％ 1.29％ Example 5 4.9％ 4.8％ 2.04％ Comparative Example 1 7.7％ 8.4％ 9.09％

[0125] from Figure 1 , Figure 2 , Figure 4 and Figure 5 It can be concluded from the effect diagram that the HPLC reagents in Example 1, Example 2, Example 5 and Comparative Example 1 can basically achieve the separation of the characteristic peak of HbA1C from the characteristic peaks of other substances when testing clinical samples.

[0126] Those skilled in the art will appreciate that a deviation of less than 3% between the actual test value and the hospital test value is acceptable.

[0127] It can be seen from Table 5 that, compared with Comparative Example 1, Example 1 and Example 5, the HbA1C value detected by the reagent in Example 2 has the smallest deviation from the detection value of the hospital, and the result has the highest accuracy.

Claims

1. A reagent for determining the percentage of glycosylated hemoglobin, characterized in that: The reagents include eluent A and eluent B; Wherein, the eluent A comprises HEPES and MES, the concentration of HEPES in the eluent A is 3 to 18 g / L; the concentration of MES in the eluent A is 0.1 to 8 g / L; Wherein, the eluent B comprises HEPES and MES, the concentration of HEPES in the eluent B is 2-20 g / L; the concentration of MES in the eluent B is 0.1-10 g / L; Wherein, the concentration of HEPES in the eluent A is different from that in the eluent B; the concentration of MES in the eluent A is different from that in the eluent B; The eluent A and the eluent B further independently include a protonic acid, and the protonic acid is trifluoroacetic acid; The pH values ​​of the eluent A and the eluent B are 4.5 to 6; The eluent A and the eluent B further independently include a salt solution and / or a preservative; The salt solution is one or more of sodium chloride, sodium sulfate and sodium nitrate; The preservative is one or more of sodium azide, Proclin 300 and thimerosal.

2. The reagent for measuring the percentage of glycated hemoglobin according to claim 1, characterized in that: The concentration of HEPES in the eluent A is 4.3 g / L or 15 g / L; And / or, the concentration of MES in the eluent A is 0.2 g / L or 5 g / L; And / or, the concentration of HEPES in the eluent B is 3.8 g / L or 18 g / L; And / or, the concentration of MES in the eluent B is 0.8 g / L or 8 g / L; And / or, the pH value of the eluent A and the eluent B is 5 to 5.

8.

3. The reagent for measuring the percentage of glycated hemoglobin according to claim 2, characterized in that: The pH values ​​of the eluent A and the eluent B are 5.2-5.

5.

4. The reagent for measuring the percentage of glycated hemoglobin according to claim 1, characterized in that: The salt solution in the eluent A is sodium chloride; the concentration of the salt solution in the eluent A is 1 to 50 g / L; the salt solution in the eluent B is sodium chloride; the concentration of the salt solution in the eluent B is 1 to 100 g / L; The concentration of proton acid in the eluent A is 0.01 to 20 g / L; the concentration of proton acid in the eluent B is 0.01 to 20 g / L; The concentration of the preservative in the eluent A is less than 10 g / L; the concentration of the preservative in the eluent B is less than 10 g / L.

5. The reagent for measuring the percentage of glycated hemoglobin according to claim 4, characterized in that: The concentration of sodium chloride in the eluent A is 10 g / L; And / or, the concentration of sodium chloride in the eluent B is 20 g / L; And / or, the concentration of trifluoroacetic acid in the eluent A is 1 g / L; And / or, the concentration of trifluoroacetic acid in the eluent B is 1 g / L; And / or, the preservative in the eluent A is sodium azide; the concentration of sodium azide in the eluent A is 1 g / L; And / or, the preservative in the eluent B is sodium azide; the concentration of sodium azide in the eluent B is 1 g / L.

6. The reagent for measuring the percentage of glycated hemoglobin according to claim 1, characterized in that: The reagents include not only the eluent A and the eluent B, but also a hemolytic agent; the hemolytic agent includes a buffer solution; the hemolytic agent also includes a protonic acid and / or a preservative.

7. The reagent for measuring the percentage of glycated hemoglobin according to claim 6, characterized in that: In the hemolytic agent, the buffer is one or more of MES buffer, HEPES buffer and phosphate buffer.

8. The reagent for measuring the percentage of glycated hemoglobin according to claim 7, characterized in that: In the hemolytic agent, the concentration of the buffer is 0.01 to 1 g / L; And / or, in the hemolytic agent, the protonic acid is one or both of trifluoroacetic acid and glycine; in the hemolytic agent, the concentration of the protonic acid is 0.001 g / L to 5 g / L; And / or, in the hemolytic agent, the preservative is one or more of sodium azide, Proclin 300 and thimerosal; in the hemolytic agent, the concentration of the preservative is less than 10 g / L.

9. The reagent for measuring the percentage of glycated hemoglobin according to claim 8, characterized in that: In the hemolytic agent, the buffer is MES buffer; in the hemolytic agent, the concentration of the MES buffer is 0.5 g / L; And / or, in the hemolytic agent, the protonic acid is trifluoroacetic acid; in the hemolytic agent, the concentration of the trifluoroacetic acid is 0.1 g / L; And / or, in the hemolytic agent, the preservative is sodium azide; in the hemolytic agent, the concentration of sodium azide is 1 g / L.

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