Hemolytic agent, detection method and system for detecting glycosylated hemoglobin by high performance liquid chromatography

Abstract

The invention relates to the field of glycosylated hemoglobin detection, in particular to a hemolytic agent for detecting glycosylated hemoglobin through high performance liquid chromatography, a detection method and a system. The hemolytic agent comprises a polyoxyethylene ether nonionic surfactant, an organic alcohol preservative and a buffering agent, but does not comprise an ionic surfactant, under the synergistic effect of the polyoxyethylene ether nonionic surfactant and the organic alcohol preservative, the stability of a pre-diluted sample can be improved to 24 h or above, and the stability of the pre-diluted sample can be improved to 24 h or above. And accurate measurement of the pre-diluted sample after long-time storage is facilitated.

Description

Technical Field

[0001] This application relates to the field of glycated hemoglobin detection, specifically to a hemolysin, detection method, and system for high-performance liquid chromatography (HPLC) detection of glycated hemoglobin. Background Technology

[0002] Glycated hemoglobin (GHb) is a compound formed from hemoglobin in red blood cells and carbohydrates through non-enzymatic reactions. It is usually used as a reference standard for average plasma glucose concentration over a period of time. GHb testing can typically reflect a patient's blood glucose control over the past 8–12 weeks and is considered the "gold standard" for the diagnosis and treatment monitoring of diabetes. HbA1c is a type of GHb. Currently, commonly used HbA1c detection methods in clinical laboratories can be divided into two main categories: one based on the difference in charge between HbA1c and non-HbA1c, including cation exchange chromatography, electrophoresis, and isoelectric focusing; the other based on the difference in GHb group structure, including affinity chromatography, immunological methods, and enzymatic methods.

[0003] Ion-exchange high-performance liquid chromatography (HPLC) is currently considered the gold standard for HbA1c analysis. High-precision HPLC can separate HbA1c from total hemoglobin, yielding accurate values. HPLC detection of HbA1c typically requires pretreatment of blood samples with a hemolysin. This pretreatment serves two purposes: firstly, to dissolve red blood cell membranes and release hemoglobin; and secondly, to form stable derivatives with glycated hemoglobin. However, existing hemolysins have limited functionality. After mixing and dissolving the sample with the hemolysin, the resulting pre-diluted sample's hemoglobin mixture cannot remain stable for an extended period, thus affecting the detection results and failing to meet the requirements for detecting pre-diluted samples after long-term storage. Summary of the Invention

[0004] To address the aforementioned issues, this application provides a hemolysin, detection method, and system for the detection of glycated hemoglobin using high-performance liquid chromatography.

[0005] In a first aspect, this application provides a hemolysin for the detection of glycated hemoglobin by high performance liquid chromatography, wherein the hemolysin includes polyoxyethylene ether nonionic surfactants, organic alcohol preservatives and buffers, but does not include ionic surfactants.

[0006] It should be noted that the key to this application lies in the synergistic effect of polyoxyethylene ether nonionic surfactants and organic alcohol preservatives in the hemolysing agent, which can improve the stability of pre-diluted samples to more than 24 hours. The hemolysing agent formulation must not contain anionic surfactants and / or cationic surfactants, as ionic surfactants can react with the HPLC column or hemoglobin, affecting the glycated hemoglobin test.

[0007] According to some embodiments, the polyoxyethylene ether nonionic surfactant includes one or more of nonionic surfactants with polyoxyethylene polymer chain groups, such as alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ether, fatty alcohol polyoxyethylene ether, fatty amine polyoxyethylene ether, and polyoxyethylene oil ether. Preferably, it is one or more of polyoxyethylene (20) hexadecyl ether (Brij58), polyoxyethylene (10) oil ether (Brij O10), and polyepoxyethylene lauroyl ether (Brij 35).

[0008] According to some embodiments, the concentration of the polyoxyethylene ether nonionic surfactant in the hemolytic agent is 0.1-5 g / L, preferably 0.1-3 g / L.

[0009] According to some embodiments, the organic alcohol preservative includes one or more organic alcohols with one or more hydroxyl groups, such as propylene glycol, butanediol, benzyl alcohol, phenethyl alcohol, and phenoxyethanol.

[0010] According to some embodiments, the concentration of the organic alcohol preservative in the hemolytic agent is 0.1-5 g / L, preferably 1-3 g / L.

[0011] According to some embodiments, the buffer includes one or more of a phosphate buffer pair, a sodium barbital-hydrochloric acid buffer pair, and a Tris-hydrochloric acid buffer pair. Preferably, the buffer is a phosphate buffer pair. More preferably, the buffer pair is selected from a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer pair.

[0012] According to some embodiments, the pH value of the hemolytic agent is 6 to 9, preferably 7.1 to 7.5.

[0013] It is understandable that the main function of the buffer is to maintain the pH stability of the hemolytic agent system. In addition to the buffers listed above, other types of buffers can be selected, as long as they can maintain the pH of the hemolytic agent system in the range of 6 to 9, preferably 7.1 to 7.5.

[0014] It should be noted that when the buffer is selected from phosphate buffer pairs, the hemolysin is more effective in improving the stability of pre-diluted samples.

[0015] According to some embodiments, the absolute value of the relative deviation between the second detection result obtained by high performance liquid chromatography (HPLC) of a blood sample treated with the hemolysin after waiting for a second time and the first detection result obtained by HPLC of a blood sample treated with the hemolysin after waiting for a first time is less than 3%, preferably less than 1%.

[0016] Wherein, the second time is greater than the first time, the first time is no more than 30 minutes, and the second time differs from the first time by at least 4 hours, preferably at least 8 hours, and more preferably at least 16 hours.

[0017] Secondly, this application provides the use of the above-mentioned hemolytic agent in the detection of glycated hemoglobin by high performance liquid chromatography.

[0018] Thirdly, this application provides a method for detecting glycated hemoglobin based on high performance liquid chromatography, including the step of using the above-mentioned hemolysin to perform high performance liquid chromatography detection of glycated hemoglobin on a blood sample.

[0019] According to some implementation methods, the method specifically includes the following steps:

[0020] A blood sample is mixed with the hemolysin to obtain a test sample; the test sample is injected into a separation system containing a chromatography column, and the test sample is eluted with elution buffers of different ionic strengths to separate glycated hemoglobin in the test sample; the signal of the separated glycated hemoglobin is collected; the collected signal is processed to obtain the detection result of glycated hemoglobin in the blood sample.

[0021] According to some implementation methods, the volume ratio of the blood sample to the hemolytic agent is 1:50 to 1:500.

[0022] According to some embodiments, the eluents with different ionic strengths include at least a first eluent and a second eluent, and the test sample is eluted sequentially with the first eluent and the second eluent; the first eluent is a low ionic strength eluent, and the second eluent is a high ionic strength eluent.

[0023] According to some embodiments, the low ionic strength eluent comprises citrate and / or succinate; the high ionic strength eluent comprises phosphate.

[0024] According to some embodiments, the elution is isocratic elution or gradient elution; the chromatography column is a cation exchange chromatography column, and the column packing material of the cation exchange chromatography column includes one or more of polymethacrylic acid resin, polystyrene acrylic resin, and polyglycerol methacrylate.

[0025] According to some embodiments, the step of mixing the blood sample with the hemolytic agent to obtain a test sample includes: mixing a portion of the blood sample with the hemolytic agent to obtain a first test sample, and mixing a portion of the blood sample with the hemolytic agent to obtain a second test sample;

[0026] The step of injecting the test sample into a separation system containing a chromatography column and eluting the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the test sample includes: injecting the first test sample into the separation system containing a chromatography column and eluting the first test sample with eluents of different ionic strengths to separate glycated hemoglobin from the first test sample; and injecting the second test sample into the separation system containing a chromatography column and eluting the second test sample with eluents of different ionic strengths to separate glycated hemoglobin from the second test sample; wherein the time when the first test sample is injected into the separation system and the time when the second test sample is injected into the separation system differs by at least 4 hours, preferably at least 8 hours, and more preferably at least 16 hours;

[0027] The acquisition of the signal of glycated hemoglobin includes: acquiring a first signal of glycated hemoglobin separated from the first test sample, and acquiring a second signal of glycated hemoglobin separated from the second test sample;

[0028] The step of processing the collected signal to obtain the detection result of glycated hemoglobin in the blood sample includes: processing the first signal to obtain a first detection result of glycated hemoglobin in the blood sample, and processing the second signal to obtain a second detection result of glycated hemoglobin in the blood sample; wherein the absolute value of the relative deviation between the first detection result and the second detection result is less than 3%, preferably less than 1%.

[0029] According to some embodiments, the step of injecting the test sample into a separation system containing a chromatography column and eluting the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the test sample includes: injecting a first portion of the test sample into the separation system containing a chromatography column, eluting the first portion of the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the first portion of the test sample, and injecting a second portion of the test sample into the separation system containing a chromatography column, eluting the second portion of the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the second portion of the test sample; wherein the time when the first portion of the test sample is injected into the separation system and the time when the second portion of the test sample is injected into the separation system differs by at least 4 hours, preferably at least 8 hours, and more preferably at least 16 hours;

[0030] The acquisition of the signal of glycated hemoglobin includes: acquiring a first signal of glycated hemoglobin separated from the test sample of the first part, and acquiring a second signal of glycated hemoglobin separated from the test sample of the second part.

[0031] The step of processing the collected signal to obtain the detection result of glycated hemoglobin in the blood sample includes: processing the first signal to obtain a first detection result of glycated hemoglobin in the blood sample, and processing the second signal to obtain a second detection result of glycated hemoglobin in the blood sample; wherein the absolute value of the relative deviation between the first detection result and the second detection result is less than 3%, preferably less than 1%.

[0032] Fourthly, this application provides a sample analysis system for detecting glycated hemoglobin based on high performance liquid chromatography (HPLC), including a glycated hemoglobin analysis device and a reagent kit, wherein the reagent kit includes the hemolysin described above, for use in the glycated hemoglobin analysis device to perform HPLC glycated hemoglobin detection on blood samples.

[0033] According to some embodiments, the glycated hemoglobin analysis device includes a first mixing chamber, a sample introduction system, a separation system containing a chromatography column, a detector, and a control system, wherein...

[0034] The first mixing chamber is used to mix the blood sample and the hemolysin to obtain the test sample, and to deliver the test sample to the sample introduction system;

[0035] The sample introduction system is used to filter the sample to be tested and deliver it to the separation system containing the chromatography column, and is also used to deliver the eluent to the separation system containing the chromatography column.

[0036] The separation system containing the chromatography column is used to pass the filtered test sample through the chromatography column to separate glycated hemoglobin in the test sample.

[0037] The detector is used to detect the isolated glycated hemoglobin signal;

[0038] The processor is used to process the signal to obtain the glycated hemoglobin test result of the blood sample.

[0039] According to some embodiments, the sample analysis system further includes a second mixing chamber for mixing to form an eluent.

[0040] Compared with the prior art, the advantage of this application is that the hemolysin pre-dilution function obtained by this application is more stable. After the hemolysin is mixed and dissolved with the sample, the resulting hemoglobin mixture can remain stable within 24 hours, which is beneficial for accurate measurement after long-term storage of the pre-diluted sample. Attached Figure Description

[0041] Figures 1A to 1C The chromatogram for HbA1c detection based on HPLC in Example 1 is shown below. Figure 1A This is a chromatogram of a whole blood sample. Figure 1B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 1C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0042] Figures 2A to 2C This is the chromatogram for Example 2, based on the HPLC method for the detection of HbA1c, wherein... Figure 2A This is a chromatogram of a whole blood sample. Figure 2B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 2C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0043] Figures 3A to 3C The chromatogram for Example 3, based on HPLC detection of HbA1c, is shown below. Figure 3A This is a chromatogram of a whole blood sample. Figure 3B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 3C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0044] Figures 4A to 4C The chromatogram for Example 4, based on HPLC detection of HbA1c, is shown below. Figure 4A This is a chromatogram of a whole blood sample. Figure 4B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 4C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0045] Figures 5A to 5C The chromatogram for Example 5, which is based on HPLC for the detection of HbA1c, is shown below. Figure 5A This is a chromatogram of a whole blood sample. Figure 5B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 5C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0046] Figures 6A to 6C The chromatogram for Example 6, based on HPLC detection of HbA1c, is shown below. Figure 6A This is a chromatogram of a whole blood sample. Figure 6BThis is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 6C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0047] Figures 7A to 7C The chromatogram for Example 7, which is based on HPLC for the detection of HbA1c, is shown below. Figure 7A This is a chromatogram of a whole blood sample. Figure 7B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 7C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0048] Figures 8A to 8C The chromatogram for Example 8, which is based on HPLC for the detection of HbA1c, is shown below. Figure 8A This is a chromatogram of a whole blood sample. Figure 8B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 8C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0049] Figures 9A to 9C The chromatogram for Example 9, based on HPLC detection of HbA1c, is shown below. Figure 9A This is a chromatogram of a whole blood sample. Figure 9B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 9C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0050] Figures 10A to 10C The chromatogram for HbA1c detection based on HPLC in Example 10 is shown below. Figure 10A This is a chromatogram of a whole blood sample. Figure 10B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 10C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0051] Figures 11A to 11C The chromatogram for HbA1c detection based on HPLC in Example 11 is shown below. Figure 11A This is a chromatogram of a whole blood sample. Figure 11B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 11C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0052] Figures 12A to 12C The chromatogram for HbA1c detection based on HPLC in Example 12 is shown below. Figure 12A This is a chromatogram of a whole blood sample. Figure 12B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 12C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0053] Figures 13A to 13C The chromatogram for Comparative Example 1, based on HPLC detection of HbA1c, is shown below. Figure 13A This is a chromatogram of a whole blood sample. Figure 13B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 13C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0054] Figures 14A to 14C The chromatogram for Comparative Example 2, based on HPLC detection of HbA1c, is shown below. Figure 14A This is a chromatogram of a whole blood sample. Figure 14B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 14C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0055] Figures 15A to 15C The chromatogram for Comparative Example 3, based on HPLC detection of HbA1c, is shown below. Figure 15A This is a chromatogram of a whole blood sample. Figure 15B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 15C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0056] Figures 16A to 16C The chromatogram for Comparative Example 4, based on HPLC detection of HbA1c, is shown below. Figure 16A This is a chromatogram of a whole blood sample. Figure 16B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 16C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0057] Figures 17A to 17C The chromatogram for Comparative Example 5, based on HPLC detection of HbA1c, is shown below. Figure 17A This is a chromatogram of a whole blood sample. Figure 17B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 17C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed.

[0058] Figures 18A to 18C The chromatogram for Comparative Example 6, based on HPLC detection of HbA1c, is shown below. Figure 18A This is a chromatogram of a whole blood sample. Figure 18B This is a chromatogram of a pre-diluted sample immediately before it is processed. Figure 18C This is a chromatogram of a pre-diluted sample stored for 24 hours before being processed. Detailed Implementation

[0059] The technical solution of this application will be clearly and completely described below with reference to the implementation methods and embodiments. Obviously, the specific implementation methods described are only a part of the implementation methods, not all of them. Based on these implementation methods, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0060] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0061] In this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a method or product that comprises a list of elements includes not only the elements expressly stated, but also other elements not expressly listed, or elements inherent to carrying out the method or product.

[0062] Unless otherwise stated, the singular forms “a / kind” and “the / said” as used herein include the plural form of the noun they refer to.

[0063] Examples 1-9

[0064] In some embodiments of this application, the polyoxyethylene ether nonionic surfactants used include one or more of polyoxyethylene (20) hexadecyl ether (Brij 58), polyoxyethylene (10) oil ether (Brij O10), and polyoxyethylene lauroyl ether (Brij 35); the organic alcohol preservatives used include one or more of phenoxyethanol, phenethyl alcohol, and propylene glycol; and the buffer pair used is a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer pair.

[0065] Prepare the hemolytic agent according to the formula in Table 1:

[0066] Table 1 Hemolytic agent formulations for each embodiment

[0067]

[0068]

[0069] Comparative Examples 1-6

[0070] Comparative Examples 1-3 provided in this application are formulations containing only polyoxyethylene surfactants, and Comparative Examples 4-6 are formulations containing only organic alcohol preservatives.

[0071] Prepare the hemolytic agent according to the formula in Table 2:

[0072] Table 2 shows the hemolytic agent formulations for each comparative example.

[0073]

[0074]

[0075] Test Example 1

[0076] Following the instructions for the Mindray H50 glycated hemoglobin analyzer, the following samples were tested for HbA1c in closed mode:

[0077] Group 1: The same whole blood sample was artificially pre-diluted outside the machine using the hemolysing agents described in Examples 1-12 and Comparative Examples 1-6 (the volume ratio of whole blood sample to hemolysing agent was 1:100), and the pre-diluted sample was immediately tested on the machine.

[0078] Group 2: After placing the samples from Group 1 for 24 hours, test them on the machine.

[0079] Group 3: Using the whole blood mode of the instrument, the same whole blood sample was automatically pre-diluted in the machine with the hemolysing agents described in Examples 1-12 and Comparative Examples 1-6 (the volume ratio of whole blood sample to hemolysing agent was 1:100). The pre-diluted sample was immediately tested on the machine.

[0080] The obtained chromatograms are shown in Figures 1-18, and the obtained HbA1c content (the ratio of HbA1c to total hemoglobin, %) is shown in Table 3.

[0081] Table 3 shows the HbA1c content and accuracy obtained from Test Example 1.

[0082]

[0083]

[0084] In the hemolysin provided in this application, polyoxyethylene surfactants and organic alcohol preservatives exhibit a synergistic effect, enabling pre-diluted samples to maintain good stability within 24 hours. As shown in Figures 1-12, the chromatograms of samples pre-diluted immediately after use with the hemolysin described in Examples 1-12, samples stored for 24 hours after pre-diluted use, and whole blood samples showed no significant changes. Table 3 shows that the relative deviation of HbA1c content in samples pre-diluted immediately after use with the hemolysin described in Examples 1-12, samples stored for 24 hours after pre-diluted use, and whole blood samples did not exceed 3%. These data indicate that the hemolysin provided in this application can effectively improve the stability of pre-diluted blood samples, and that the amount of polyoxyethylene surfactants and organic alcohol preservatives has almost no effect on the sample test results within a certain range. As shown in Figures 13-18 and Table 3, hemolysins containing only polyoxyethylene surfactants or only organic alcohol preservatives cannot meet the requirement of maintaining the stability of pre-diluted samples within 24 hours.

Claims

1. A hemolysin for the detection of glycated hemoglobin by high performance liquid chromatography, characterized in that: The hemolytic agents include polyoxyethylene ether nonionic surfactants, organic alcohol preservatives, and buffers, but do not include ionic surfactants.

2. The hemolytic agent according to claim 1, characterized in that: The polyoxyethylene ether nonionic surfactants include one or more of alkylphenol polyoxyethylene ethers, fatty acid polyoxyethylene ethers, fatty alcohol polyoxyethylene ethers, and fatty amine polyoxyethylene ethers.

3. The hemolytic agent according to claim 1, characterized in that: The concentration of the polyoxyethylene ether nonionic surfactant in the hemolytic agent is 0.1–5 g / L, preferably 0.1–3 g / L.

4. The hemolytic agent according to claim 1, characterized in that: The organic alcohol preservatives include one or more of propylene glycol, butanediol, benzyl alcohol, phenethyl alcohol, and phenoxyethanol.

5. The hemolytic agent according to claim 1, characterized in that: The concentration of the organic alcohol preservative in the hemolytic agent is 0.1–5 g / L, preferably 1–3 g / L.

6. The hemolytic agent according to claim 1, characterized in that: The buffer includes one or more of the following: phosphate buffer pairs, sodium barbital-hydrochloric acid buffer pairs, and Tris-hydrochloric acid buffer pairs.

7. The hemolytic agent according to claim 1, characterized in that: The hemolytic agent has a pH value of 6 to 9, preferably 7.1 to 7.

5.

8. The hemolytic agent according to claim 1, characterized in that: The absolute value of the relative deviation between the second test result obtained by high performance liquid chromatography (HPLC) of a blood sample treated with the hemolysin after a second waiting period and the first test result obtained by HPLC of a blood sample treated with the hemolysin after a first waiting period is less than 3%, preferably less than 1%. Wherein, the second time is greater than the first time, the first time is no more than 30 minutes, and the second time differs from the first time by at least 4 hours, preferably at least 8 hours, and more preferably at least 16 hours.

9. The use of a hemolytic agent as described in any one of claims 1 to 8 in the detection of glycated hemoglobin by high performance liquid chromatography.

10. A method for detecting glycated hemoglobin based on high performance liquid chromatography, characterized in that: The method includes the step of using the hemolytic agent described in any one of claims 1 to 8 to perform high-performance liquid chromatography (HPLC) detection of glycated hemoglobin in a blood sample.

11. The method according to claim 10, characterized in that: The step of using the hemolytic agent according to any one of claims 1 to 8 to perform high-performance liquid chromatography (HPLC) detection of glycated hemoglobin in a blood sample specifically includes the following steps: A blood sample is mixed with the hemolytic agent described in any one of claims 1 to 8 to obtain a test sample; The test sample is injected into a separation system containing a chromatography column, and the test sample is eluted with eluents of different ionic strengths to separate glycated hemoglobin from the test sample. The signal from the isolated glycated hemoglobin was collected; The collected signals are processed to obtain the detection results of glycated hemoglobin in the blood sample.

12. The method according to claim 11, characterized in that: The volume ratio of the blood sample to the hemolytic agent is 1:50 to 1:

500.

13. The method according to claim 11, characterized in that: The eluents with different ionic strengths include at least a first eluent and a second eluent, and the test sample is eluted sequentially with the first eluent and the second eluent; the first eluent is a low ionic strength eluent, and the second eluent is a high ionic strength eluent.

14. The method according to claim 13, characterized in that: The low ionic strength eluent comprises citrate and / or succinate; the high ionic strength eluent comprises phosphate.

15. The method according to claim 11, characterized in that: The elution is either isocratic elution or gradient elution; The chromatography column is a cation exchange chromatography column, and the column packing material of the cation exchange chromatography column includes one or more of polymethacrylic acid resin, polystyrene acrylic resin, and polyglycerol methacrylate.

16. The method according to claim 11, characterized in that: The step of mixing a blood sample with the hemolytic agent according to any one of claims 1 to 8 to obtain a test sample includes: mixing a portion of the blood sample with the hemolytic agent to obtain a first test sample, and mixing a portion of the blood sample with the hemolytic agent to obtain a second test sample; The step of injecting the test sample into a separation system containing a chromatography column and eluting the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the test sample includes: injecting the first test sample into the separation system containing a chromatography column and eluting the first test sample with eluents of different ionic strengths to separate glycated hemoglobin from the first test sample; and injecting the second test sample into the separation system containing a chromatography column and eluting the second test sample with eluents of different ionic strengths to separate glycated hemoglobin from the second test sample; wherein the time when the first test sample is injected into the separation system and the time when the second test sample is injected into the separation system differs by at least 4 hours, preferably at least 8 hours, and more preferably at least 16 hours; The acquisition of the signal of glycated hemoglobin includes: acquiring a first signal of glycated hemoglobin separated from the first test sample, and acquiring a second signal of glycated hemoglobin separated from the second test sample; The step of processing the collected signal to obtain the detection result of glycated hemoglobin in the blood sample includes: processing the first signal to obtain a first detection result of glycated hemoglobin in the blood sample, and processing the second signal to obtain a second detection result of glycated hemoglobin in the blood sample; wherein the absolute value of the relative deviation between the first detection result and the second detection result is less than 3%, preferably less than 1%.

17. The method according to claim 11, characterized in that: The step of injecting the test sample into a separation system containing a chromatography column and eluting the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the test sample includes: injecting a first portion of the test sample into the separation system containing a chromatography column, eluting the first portion of the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the first portion of the test sample, and injecting a second portion of the test sample into the separation system containing a chromatography column, eluting the second portion of the test sample with eluents of different ionic strengths to separate glycated hemoglobin from the second portion of the test sample; wherein the time when the first portion of the test sample is injected into the separation system and the time when the second portion of the test sample is injected into the separation system differs by at least 4 hours, preferably at least 8 hours, and more preferably at least 16 hours; The acquisition of the signal of glycated hemoglobin includes: acquiring a first signal of glycated hemoglobin separated from the test sample of the first part, and acquiring a second signal of glycated hemoglobin separated from the test sample of the second part. The step of processing the collected signal to obtain the detection result of glycated hemoglobin in the blood sample includes: processing the first signal to obtain a first detection result of glycated hemoglobin in the blood sample, and processing the second signal to obtain a second detection result of glycated hemoglobin in the blood sample; wherein the absolute value of the relative deviation between the first detection result and the second detection result is less than 3%, preferably less than 1%.

18. A sample analysis system for detecting glycated hemoglobin based on high performance liquid chromatography, characterized in that: The invention includes a glycated hemoglobin analysis device and a kit, wherein the kit includes a hemolysin as described in any one of claims 1 to 8, for use in the glycated hemoglobin analysis device for high-performance liquid chromatography detection of glycated hemoglobin in blood samples.

19. The sample analysis system according to claim 18, characterized in that: The glycated hemoglobin analysis device includes a first mixing chamber, a sample introduction system, a separation system containing a chromatography column, a detector, and a processor. The first mixing chamber is used to mix a blood sample with the hemolytic agent according to any one of claims 1 to 8 to obtain a test sample, and to deliver the test sample to the sample introduction system; The sample introduction system is used to filter the sample to be tested and deliver it to the separation system containing the chromatography column, and is also used to deliver the eluent to the separation system containing the chromatography column. The separation system containing the chromatography column is used to pass the filtered test sample through the chromatography column to separate glycated hemoglobin in the test sample. The detector is used to detect the isolated glycated hemoglobin signal; The processor is used to process the signal to obtain the glycated hemoglobin test result of the blood sample.

20. The sample analysis system according to claim 19, characterized in that: The sample analysis system also includes a second mixing chamber for mixing to form the eluent.

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