CN114527190A

CN114527190A - Glycosylated hemoglobin detection kit and detection method thereof

Info

Publication number: CN114527190A
Application number: CN202210041604.5A
Authority: CN
Inventors: 孙朋卫; 逄晓云; 陈宏敏; 周晓光; 李运涛
Original assignee: Rongzhi Biotechnology Qingdao Co ltd
Current assignee: Rongzhi Biotechnology Qingdao Co ltd
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2022-05-24

Abstract

The invention discloses a glycosylated hemoglobin detection kit and a detection method thereof, wherein the kit comprises: solution A and solution B; the solution A consists of sinapic acid, acetonitrile, trifluoroacetic acid and water; the liquid B consists of trifluoroacetic acid and water. The glycosylated hemoglobin detection kit provided by the invention has the advantages of cheap and easily-obtained raw materials, simple process and suitability for large-scale production and application, and the glycosylated hemoglobin detection kit provided by the invention is used for detecting glycosylated hemoglobin, so that not only can a glycosylated hemoglobin variant be detected, but also detection errors caused by different glycosylated groups and special differences of the glycosylated hemoglobin variants can be effectively avoided, and the accuracy and precision of results are ensured.

Description

Glycosylated hemoglobin detection kit and detection method thereof

Technical Field

The invention relates to the technical field of glycosylated hemoglobin detection. More particularly, it relates to a glycosylated hemoglobin detection kit and a detection method thereof.

Background

Diabetes is an endocrine-metabolic disease, the incidence rate of the diabetes is second to cardiovascular diseases and tumors, and the incidence rate of the diabetes is on the rising trend in recent years, so that the diabetes is a worldwide public health problem seriously threatening human health. The traditional diabetes diagnosis mainly adopts methods such as fasting blood sugar, postprandial blood sugar or oral glucose tolerance test, but the blood sugar parameter measured by the method only represents the instant blood sugar level when blood is drawn, and the measurement result is not accurate.

Glycated hemoglobin (HbA1c) is a stable compound in which glucose in blood is covalently bound to the N-terminal valine residue of the beta chain of hemoglobin. The content of glycated hemoglobin depends mainly on the blood glucose concentration and the contact time between blood glucose and hemoglobin. The blood sugar enters the cells in a dispersion mode without the participation of insulin, the combination process of the blood sugar and the hemoglobin is slow and irreversible, the blood sugar and the hemoglobin exist all the time before the death of the red blood cells, the hemoglobin exists in each red blood cell, and the service life of the red blood cells is as long as 120 days. Therefore, compared with the conventional blood glucose measurement, the glycated hemoglobin measurement sample is not affected by diet, blood sampling time, and the like, and can reflect the average blood glucose level of 2 to 3 months in the past. However, glycated Hemoglobin includes specific variant Hemoglobin (Hemoglobin) in addition to conventional glycated Hemoglobin. The existing method for detecting the glycosylated hemoglobin mainly comprises the technologies of ion exchange chromatography, affinity chromatography, immunoassay, homogeneous enzyme method, capillary electrophoresis and the like. The above-mentioned techniques can be classified into two broad categories according to their measurement theories, and the first category is a method for measuring based on the difference between the charge of glycated hemoglobin and the charge of non-glycated hemoglobin, and includes electrophoresis and ion exchange high performance liquid chromatography. The capillary electrophoresis method can detect common variants theoretically and can prompt the existence of the variants, but the whole experiment is long in time consumption, the external interference factors to the capillary electrophoresis method are many, and the technical requirement on operators is high. Whether the ion exchange high performance liquid chromatography can indicate that the existence of the variant is influenced by the resolution of the instrument; the second type is a method of measurement using a difference in structure of glycated hemoglobin from non-glycated hemoglobin, and includes an enzymatic method, an affinity chromatography method, and an immunological method, none of which suggests the presence of specific variant hemoglobin.

Therefore, it is important to provide a glycated hemoglobin assay kit and a method for assaying glycated hemoglobin, which can assay specific variant hemoglobin, and which are easy to operate and highly accurate.

Disclosure of Invention

The first object of the present invention is to provide a glycated hemoglobin assay kit.

The second object of the present invention is to provide a method for assaying glycated hemoglobin assay kit which is compatible with the assay standards of the current assay methods.

In order to achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the present invention provides a glycated hemoglobin assay kit, the kit comprising: liquid A and liquid B; the solution A consists of sinapic acid, acetonitrile, trifluoroacetic acid and water; the liquid B consists of trifluoroacetic acid and water.

Furthermore, in the solution A, the concentration of sinapic acid is 10-20mg/mL, the volume concentration of acetonitrile is 30-60%, and the volume concentration of trifluoroacetic acid is 0.1-0.5%.

Further, the volume concentration of trifluoroacetic acid in the solution B is 0.1-0.5%.

Further, the glycosylated hemoglobin detection kit also comprises a calibrator and a quality control product.

Further, the calibrator and the quality control material both consist of glycated hemoglobin and proclin 300.

In a second aspect, the present invention provides a method for detecting glycated hemoglobin by using a glycated hemoglobin detection kit, comprising the steps of:

1) 995 mu L B liquid and 5 mu L of blood sample to be tested are mixed. Obtaining a primary treatment sample;

2) mixing the solution A and the solution B in equal volume to obtain a mixed solution;

3) uniformly mixing 45 mu L of the mixed solution obtained in the step 2) with 5 mu L of the primary treatment sample obtained in the step 1) to obtain a secondary treatment sample;

4) spotting the secondary treatment sample obtained in the step 3) on a mass spectrum target plate, drying, and then performing test by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry;

5) calculating according to a linear equation to obtain the target; the linear equation is shown in formula (1):

y ═ a/(a + B) + B formula (1),

in the formula (1), A is the peak area of the N-terminal valine glycosylation modified beta globin with the mass-to-charge ratio of 15868 m/z; b is the peak area of the hemoglobin beta globin with the mass-to-charge ratio of 16030 m/z; y is the glycated value of glycated hemoglobin of the blood sample to be detected; b is a linear analytical constant.

Further, the drying is carried out at 25 to 42 ℃.

Further, the time for performing the test using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was less than 4 hours.

In addition, unless otherwise specified, all starting materials for use in the present invention are commercially available, and any range recited herein includes any value between the endpoints and any subrange between the endpoints and any value between the endpoints or any subrange between the endpoints. The percentages are mass percentages unless otherwise specified, and the solutions are aqueous solutions unless otherwise specified.

The invention has the following beneficial effects:

the glycosylated hemoglobin detection kit provided by the invention has the advantages of cheap and easily available raw materials and simple process, and is suitable for large-scale production and application.

The glycosylated hemoglobin detection kit provided by the invention is suitable for mass calibration within the range of 1kDa to 10 kDa.

The kit can be used for detecting the glycosylated hemoglobin variant, can also effectively avoid detection errors caused by different glycosylated groups and special differences of the glycosylated hemoglobin variant, and ensures the accuracy and precision of results.

When the glycosylated hemoglobin detection kit provided by the invention is used for detecting glycosylated hemoglobin, only simple dilution treatment needs to be carried out on a sample to be detected, so that the operation process is simplified, the use is more convenient, and the test time is effectively shortened.

When the glycosylated hemoglobin detection kit provided by the invention is used for detecting a sample with a glycosylation rate of 6 +/-0.5%, the analysis sensitivity is more than or equal to 0.05; coefficient of variation CV is less than or equal to 3 percent; the relative range of 96-person repeated determination of a single sample is less than or equal to 10 percent; the linear range of the sample saccharification rate is between 3.42% and 19.32%, and the whole measurement range of clinical sample measurement is covered; and in the interval of 3.45% -19.5%, the linear correlation coefficient r is more than or equal to 0.990; the variation coefficient CV in the bottles of the calibrator and the quality control material is less than or equal to 10 percent, and the variation coefficient CV among the bottles is less than or equal to 10 percent; the relative deviation of the accuracy of the method from national standard substances is not more than +/-10%, the relative deviation from enterprise reference substances is not more than +/-10%, and the single measurement deviation of a methodological comparison sample is not more than +/-5%.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 shows the mass spectrum of a first measurement blank (bovine serum).

Figure 2 shows the mass spectrum of a second measurement blank (bovine serum).

FIG. 3 shows the mass spectrum of the blank sample (bovine serum) for the third measurement.

FIG. 4 shows the test results of the test of kit-1 on the first working day of Experimental example 2.

FIG. 5 shows the results of the test conducted on kit-1 on the second working day of Experimental example 2.

FIG. 6 shows the test results of the test of kit-2 on the first working day of Experimental example 2.

FIG. 7 shows the results of the test conducted on kit-2 on the second working day of Experimental example 2.

FIG. 8 shows the results of the sensitivity analysis test of the glycated hemoglobin measurement kit of test example 4.

FIG. 9 shows the results of the linear correlation analysis test of the glycated hemoglobin measurement kit of test example 5.

FIG. 10 shows the results of the accuracy analysis test of the glycated hemoglobin assay kit of test example 6.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are included merely to further illustrate the features and advantages of the invention and are not intended to limit the invention to the claims.

The invention provides a glycosylated hemoglobin detection kit, which comprises: liquid A and liquid B; the solution A consists of sinapic acid, acetonitrile, trifluoroacetic acid and water; the liquid B consists of trifluoroacetic acid and water.

In the detection of the glycated hemoglobin test kit provided by the present invention, the reagents (solution a and solution B) in the kit are mixed with the blood sample to form a co-crystal, and under the irradiation of laser light, the reagents can absorb the laser energy and then transfer the energy to the surrounding blood sample molecules, so that the glycated hemoglobin in the sample is depolymerized into α -globin and β -globin monomers. And finally, specifically detecting mass spectrum peaks of beta globin (m/z is 158683) and N-terminal valine glycosylation modified beta globin (m/z is 160303) in the hemoglobin, and performing relative quantification by utilizing the peak area ratio to determine the glycosylation value of the hemoglobin in the human whole blood sample.

When the components in the solutions a and B are within the above ranges, the laser irradiation can absorb more laser energy, thereby depolymerizing glycated hemoglobin in the sample more rapidly, i.e., the detection speed can be further increased.

The quality control material can control the quality of the determination accuracy of the kit, specifically, the saccharification value of the quality control material can be detected simultaneously when the kit is used, and if the determination deviation is more than 10%, the kit in the batch is considered to have quality problems, and the determination result is unreliable.

Wherein proclin300 is a biological preservative, so that the deterioration of the glycosylated hemoglobin can be avoided, and the detection result cannot be influenced.

The invention provides a method for detecting glycosylated hemoglobin by a glycosylated hemoglobin detection kit, which comprises the following steps:

y ═ a/(a + B) + B formula (1),

The detection kit of the invention can be matched with the detection method to better detect the glycosylated hemoglobin. The specific amount of each reagent in the preparation method is only one preferred embodiment of the invention, and the protection scope of the invention should not be limited by the specific amount, i.e. in principle, normal detection can be performed as long as the ratio of each reagent is within the range.

According to an embodiment of the present invention, the fitting of the linear equation may be performed analytically by taking any two calibrators of known glycation values. Preferably, the diluted calibrator is subpackaged and stored at-20 ℃, repeated freezing and thawing should be avoided as much as possible, and repeated freezing and thawing does not exceed 3 times at most.

Further, the blood sample to be tested is obtained by diluting fresh or frozen reconstituted whole blood by 200 times.

Wherein, the kit is suitable for fresh or frozen reconstituted blood samples.

Among them, if the testing time is too long, the ionization efficiency of each component of the kit is influenced, and the result of poor measuring stability is caused, and the result of higher measured value may be caused by long-time storage of the glycosylated hemoglobin at normal temperature.

Normally, the reference interval of the glycated hemoglobin is 4% -6.5% (20mmol/mol-42mmol/mol), and if the glycated hemoglobin of the sample is detected to be more than 15% according to the method provided by the invention, the sample should be retested, and if the retested result is not changed, the hemoglobin variant may exist in the sample.

Unless otherwise specified, the preparation processes of the present invention are conventional processes, and the starting materials used are commercially available or can be prepared according to the prior art as disclosed, unless otherwise specified, and the percentages are by mass.

In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Before the test, preparation work is carried out, which comprises the following steps: the blank sample (bovine serum) was analyzed 3 times by matrix assisted laser desorption ionization time of flight mass spectrometry, and the results are shown in FIGS. 1-3.

As can be seen from FIGS. 1-3, no significant mass spectrum peak was detected for the blank reagent, indicating that the blank reagent did not affect the detection.

Test example 1: fitted linear equation

Fitting a linear equation comprising the steps of:

1) the glycation values of the calibrators at two different glycation values are taken as abscissa (where glycation value of calibrator 1 is: 6.11% uncertainty 0.03%, glycation number of calibrator 2: 8.97% uncertainty 0.03%);

2) mixing 995 mu L B liquid with 5 mu L of the calibrator in the step 1) respectively to obtain a primary treatment sample;

3) mixing the solution A and the solution B in equal volume to obtain a mixed solution;

4) uniformly mixing 45 mu L of the mixed solution obtained in the step 3) with 5 mu L of the primary treatment sample obtained in the step 2) to obtain a secondary treatment sample;

5) spotting the secondary treatment sample obtained in the step 4) on a mass spectrum target plate, drying, and then performing test by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry; and then reading and calculating the peak area of the glycosylated hemoglobin/(the peak area of the non-glycosylated hemoglobin + the peak area of the glycosylated hemoglobin) corresponding to the different calibrators, and fitting by taking the value as a vertical coordinate to obtain a linear fitting equation Y which is A/(A + B) + B. Wherein, B is the peak area of hemoglobin beta globin (m/z: 15868); a is the peak area of the N-terminal valine glycosylation modified beta globin (m/z: 16030).

Test example 2: repeatability test

The test kit of the test example repeatedly utilizes quality control products in each kit, and a person in the art can select a calibrator or other blood samples according to needs.

Preparation work: two types of glycated hemoglobin assay kits (kit-1 and kit-2) each include: liquid A, liquid B, a calibrator and a quality control material; the solution A comprises sinapic acid with concentration of 10-20mg/mL (M: V), acetonitrile with concentration of 10-20% (V: V), trifluoroacetic acid with concentration of 0.1-0.5% (V: V) and proper amount of water; the solution B comprises trifluoroacetic acid with concentration of 0.1-0.5% (V: V) and appropriate amount of water, and the quality control product and the calibrator comprise glycosylated hemoglobin and proclin300, wherein the glycosylated value of the glycosylated hemoglobin in the calibrator is 5.93 +/-0.03%. The difference is that the glycation value of the glycated hemoglobin in the quality control product in the kit-1 is 6.11% + -10%, and the glycation value of the glycated hemoglobin in the quality control product in the kit-2 is 8.97% + -10%.

(II) testing: the test was performed on each of the kits-1 and-2, 10 replicates per working day for a total of two working days (results are shown in FIGS. 4 to 7). The specific testing steps comprise:

1) before use, the solution A and the solution B of the kit are balanced to room temperature (25 ℃) for 5-10 min;

2) adding 45 mu L of the mixed solution of the solution A and the solution B in equal proportion into a 0.6mL EP tube (mixing uniformly in advance), adding 5 mu L of quality control product into the tube, and mixing uniformly by vortex to obtain the quality control product to be detected;

3) taking 2.5 mu L of a target plate for the quality control product to be detected, pointing at 3 target plate hole sites, and naturally drying at room temperature or drying and crystallizing at 38-42 ℃ by a heating plate to obtain the target plate to be detected;

4) and (4) loading the target plate to be detected obtained in the step 3) on a MALDI-TOF MS machine for analysis and test, wherein the test is completed within 4h, and then calculating the glycation value of the hemoglobin in the quality control product according to the linear equation obtained in the test example 1.

(III) analysis: calculating the average value of the measured glycation values of hemoglobin according to the formula (1)

And standard deviation(s), and the daily and diurnal Coefficient of Variation (CV) of the quality control product was calculated according to the formula (2), and the results are shown in Table 1.

In the formula: x … … hemoglobin glycation number;

n … … number of measurements.

Table 1:

and (4) conclusion: as can be seen from Table 1, the daytime variation coefficient CV of different quality control products measured by using the kit provided by the invention is less than or equal to 3 percent, so that the kit provided by the invention has better stability.

Test example 3: inter-batch difference testing

The test kit of the test example uses the quality control product in each kit, and the skilled person can select the calibrator or other blood samples according to the needs.

Preparation work: three different batches of kits (denoted as batches 1-3) were prepared, the composition of which was identical to that of kit-2 of test example 2.

(II) testing: and testing the hemoglobin glycosylation value of the quality control product in the three different batches of kits, and respectively repeating the measurement for 3 times. The specific testing steps comprise:

3) taking 2.5 mu L of target plate of the quality control point to be detected, carrying out point condensation on 3 target plate hole sites, and naturally drying at room temperature or drying and crystallizing at 38-42 ℃ by a heating plate to obtain the target plate to be detected;

4) and (3) loading the target plate to be tested obtained in the step 3) on a MALDI-TOF MS machine for analysis and test, wherein the test is completed within 4h, and then calculating the glycosylation value of the hemoglobin in the quality control product according to the linear equation of the experimental example 1.

(III) analysis: calculating the average value of the measurement results for each kit (a)

1, 2, 3) and the total average of the results of three kit measurements

Then, the relative range (R) between batches is calculated according to the formulas (3) and (4). The results are shown inShown in table 2.

In the formula:

maximum value of (1);

minimum value of (1);

table 2:

and (4) conclusion: as can be seen from Table 1, the difference R between quality control product batches of the kits of different batches is less than or equal to 3 percent and is far higher than the requirement of the industry standard of less than or equal to 10 percent.

Test example 4: sensitivity analysis

The sensitivity of the test kit of the test example uses the calibrator in each kit, and those skilled in the art can select the quality control product or other blood samples with known glycated hemoglobin values as required.

Preparation work: the kit components used in this test example were the same as those of kit-2 of test example 2.

(II) testing: the above kit was tested 3 times repeatedly (results are shown in fig. 8) according to the following method, and the specific test steps included:

2) adding 45 mu L of the mixed solution of the solution A and the solution B in equal proportion into a 0.6mL EP tube (mixing uniformly in advance), adding 5 mu L of the calibrator into the tube, and performing vortex mixing to obtain the calibrator to be tested;

3) taking 2.5 mu L of a to-be-detected calibrator point target plate, pointing 3 target plate hole sites, and naturally drying at room temperature or drying and crystallizing at 38-42 ℃ by a heating plate to obtain the to-be-detected target plate;

4) and (3) loading the target plate to be detected obtained in the step 3) on a MALDI-TOF MS machine for analysis and test (completed within 4 h), and then calculating the glycosylation value of the hemoglobin in the calibrator according to a linear equation obtained in the experimental example 1.

(III) analysis: the analytical sensitivity was calculated according to the formula (5), and the results are shown in Table 3.

Analytical sensitivity peak area ratio determination mean/actual sample concentration 6% … … … … … … (5)

Table 3:

peak area 1	Peak area 2	Peak area 3	Mean value of	Saccharification rate of sample	Sensitivity of analysis
						6.5	6.3	6.5	6.4	5.93	0.065

And (4) conclusion: as can be seen from Table 3, the assay sensitivity of the kit measured in this test example was 0.065, which is higher than the requirement of the industry standard 0.05.

Test example 5: linear correlation analysis

Preparation work: 1) the kit components used in this test example were the same as those of kit-2 of test example 2.

2) Test blood samples R1-R5 were prepared at different levels of 5 known glycation values in a linear range (see Table 4 for specific glycation values).

And (II) testing: R1-R5 were tested 3 times, respectively, according to the following method (results are shown in FIG. 9). The specific testing steps comprise:

2) mixing 995 mu L B liquid with 5 mu L of blood sample to be detected to obtain a first-stage treatment sample;

4) uniformly mixing 45 mu L of the mixed solution with 5 mu L of the primary treatment sample to obtain a secondary treatment sample;

5) taking 3 target plate hole sites of 2.5 mu L secondary treatment sample points, and then naturally drying at room temperature or drying and crystallizing at 38-42 ℃ by a heating plate to obtain a target plate to be detected;

6) and (3) carrying out analysis and test (completed within 4 h) on the target plate to be tested obtained in the loading step 5) of the MALDI-TOF MS machine, and then calculating the glycation value of the hemoglobin in the blood sample to be tested according to a linear equation obtained in the test example 1.

(III) analysis: the expected concentration (xi) was used as an independent variable, the measurement result mean (yi) was used as a dependent variable, the correlation coefficient r was calculated according to formula (6), and the relative deviation was calculated according to formula (7), and the results are shown in table 4.

Wherein xi is the actual hemoglobin glycation value of the sample;

the average value of the actual hemoglobin glycation value of the sample is obtained;

yi is the measured hemoglobin glycosylation value of the sample;

the average value of the hemoglobin glycosylation values of the measured samples was obtained.

Relative deviation (%) ═ (assay mean-target value)/target value 100% … … … … … … (7)

Table 4:

and (4) conclusion: as can be seen from Table 4, the linear correlation coefficient r of the kit of the present invention is in the range of 3.44% to 19.57% linear²0.998, indicating a good linear correlation of the results determined using the kit of the invention with the true values.

Test example 6: accuracy analysis

The accuracy of the test kit of this experimental example utilized 2 national standards of known glycation values: national standard 1 and

national standard

2 and 2 enterprise reference products with known glycation values: c1 and C2, from which other blood samples with known glycated hemoglobin values can be selected as desired by those skilled in the art.

(II) testing: the four samples to be tested were tested in the following manner, and each sample was tested 3 times (results are shown in FIG. 10). The specific testing steps comprise:

2) adding 45 mu L of the mixed solution of the solution A and the solution B in equal proportion into a 0.6mL EP tube (mixing uniformly in advance), adding 5 mu L of a sample to be detected into the tube, and mixing uniformly in a vortex manner to obtain a standard sample to be detected;

3) taking 2.5 mu L of standard sample to be detected, spotting the standard sample on a target plate, spotting 3 target plate holes, and naturally drying at room temperature or drying and crystallizing at 38-42 ℃ by a heating plate to obtain the target plate to be detected;

4) and (3) loading the target plate to be detected obtained in the step 3) on a MALDI-TOF MS machine for analysis and test (completed within 4 h), and then calculating the glycation value of the hemoglobin in the sample according to a linear equation obtained in the test example 1.

(III) analysis: the mean values of the measurement results of the different samples to be measured were calculated, and the relative deviations of the target values of the samples were calculated according to the formula (7) of test example 5, and the results are shown in table 5.

Table 5:

sample(s)	1	2	3	Mean value of	Standard value	Relative deviation (%)
							National standard 1	6.99	6.78	6.87	6.88	6.86	0.3
National standard 2	9.50	9.48	9.41	9.46	9.34	1
							C1	5.81	5.68	5.80	5.76	5.93	3
C2	10.66	10.46	10.38	10.50	10.48	0.2

And (4) conclusion: as can be seen from Table 5, the measurement deviation of the glycosylation value and the actual value of the hemoglobin tested by using the kit of the invention is less than or equal to 3 percent and is far higher than the requirement of the industry standard of less than or equal to 10 percent.

Test example 7

Taking 10 different blood samples to be detected C1-C10; then, the analysis and test of C1-C10 were carried out by the method of the present invention, which is labeled as Quan TOF (kit: kit-2 of test example 2), electrophoresis and HPLC, respectively, and the results are shown in tables 6-9:

table 6:

table 7:

table 8:

table 9:

the above results show that: compared with the existing traditional measuring methods (HPLC and electrophoresis methods) in the market, the kit and the detection method thereof have equivalent measuring results.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

Claims

1. A glycated hemoglobin assay kit, comprising: liquid A and liquid B; the solution A consists of sinapic acid, acetonitrile, trifluoroacetic acid and water; the liquid B consists of trifluoroacetic acid and water.

2. The glycated hemoglobin assay kit as set forth in claim 1, wherein the solution A contains sinapic acid at a concentration of 10-20mg/mL, acetonitrile at a volume concentration of 30-60%, and trifluoroacetic acid at a volume concentration of 0.1-0.5%.

3. The glycated hemoglobin assay kit as set forth in claim 1, wherein the trifluoroacetic acid is present in the solution B at a concentration of 0.1-0.5% by volume.

4. The glycated hemoglobin assay kit of claim 1, wherein the glycated hemoglobin assay kit further comprises a calibrator and a quality control.

5. The glycated hemoglobin assay kit as set forth in claim 4, wherein the calibrator and the quality controller each comprise glycated hemoglobin and proclin 300.

6. A method of assaying based on the glycated hemoglobin assay kit according to any one of claims 1 to 5, comprising the steps of:

1) mixing 995 mu L B liquid with 5 mu L of blood sample to be detected to obtain a first-stage treatment sample;

4) spotting the secondary treatment sample obtained in the step 3) on a mass spectrum target plate, drying, and then performing test by using matrix-assisted laser desorption ionization time-of-flight mass spectrum;

5) calculating according to a linear equation to obtain a glycated hemoglobin value of the blood sample to be detected; the linear equation is shown in formula (1):

y ═ a/(a + B) + B formula (1),

in the formula (1), A is the peak area of the N-terminal valine glycosylation modified beta globin with the mass-to-charge ratio of 15868 m/z; b is the peak area of the hemoglobin beta globin with the mass-to-charge ratio of 16030 m/z; y is the glycated hemoglobin value of the blood sample to be measured, and b is a linear analysis constant.

7. The method of claim 6, wherein the test blood sample is prepared by 200-fold dilution of fresh or frozen reconstituted whole blood.

8. The method according to claim 6, wherein the drying is performed at 25-42 ℃.

9. The method of claim 6, wherein the matrix-assisted laser desorption ionization time-of-flight mass spectrometry is performed for less than 4 hours.