CN115932111A

CN115932111A - Method for detecting dimethylglycine in serum and application thereof

Info

Publication number: CN115932111A
Application number: CN202211728016.5A
Authority: CN
Inventors: 谢宝刚; 刘晓丹; 张先超; 吴乐; 魏远旺; 徐金标
Original assignee: Jiaxing University
Current assignee: Jiaxing University
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-04-07

Abstract

The invention provides a method for detecting dimethylglycine in serum and application thereof, wherein the method comprises the following steps: (1) Mixing a sample to be tested with the internal standard solution, centrifuging to obtain a supernatant, and then performing column elution to obtain an eluted sample; (2) And mixing the eluted sample with a derivatization reagent and a reaction activator for derivatization reaction, carrying out HPLC (high performance liquid chromatography) detection on a product, and calculating the content of dimethylglycine in the sample to be detected according to a detection result. The detection method provided by the invention can efficiently and accurately detect the content of dimethylglycine in the serum, and provides a brand new method for detecting congenital heart disease.

Description

Method for detecting dimethylglycine in serum and application thereof

Technical Field

The invention belongs to the field of blood detection, particularly relates to a method for detecting dimethylglycine in serum and application thereof, and particularly relates to a brand-new and efficient method for detecting dimethylglycine in serum and application thereof.

Background

Congenital heart disease is the most common congenital malformation, and fetal ultrasound examination plays an important role in prenatal diagnosis of congenital heart disease, but clinical missed diagnosis often occurs due to the influence of various factors such as operator experience, fetal position and the like. The blood of the human body contains almost all metabolites of the human body, which can comprehensively reflect the physiological and biochemical states of the human body, and is widely used for disease research by researchers.

Choline is an important nutrient and precursor of betaine. Animal studies have shown that defects in choline metabolism are associated with fetal death or severe defects in neurogenesis. Dimethylglycine is a chemical complex that occurs in the pathway of choline metabolism to glycine, and in vivo the choline metabolite betaine transfers a methyl group under the action of betaine homocysteine methyltransferase to produce dimethylglycine. The human serum has complex components, the sensitivity for directly detecting the dimethylglycine is not high, and the interference of other impurities in the serum is easy to happen.

The pre-column derivatization method is a very important sample processing method in derivatization of a liquid chromatography-mass spectrometry combined technology, firstly, a sample is derivatized into a proper derivative in advance before chromatographic column separation, and then the derivative enters the chromatographic column for separation and then is detected, so that the sensitivity of analysis is improved. In recent years, chemical derivatization methods are increasingly applied to detection of various substances difficult to detect, and people are receiving more attention, however, at present, no literature reports a derivatization method of dimethylglycine in serum, so how to provide a method for quickly derivatizing and detecting dimethylglycine in serum becomes a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for detecting dimethylglycine in serum and application thereof, and particularly provides a brand-new and efficient method for detecting dimethylglycine in serum and application thereof. The detection method provided by the invention can efficiently and accurately detect the content of dimethylglycine in the serum, and provides a brand new method for detecting congenital heart disease.

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

in one aspect, the present invention provides a method for detecting dimethylglycine (N, N-dimethylglycine) in serum, comprising the steps of:

(1) Mixing a sample to be detected with an internal standard solution, centrifuging to obtain a supernatant, and then performing column elution to obtain an eluted sample;

(2) And mixing the eluted sample with a derivatization reagent solution and a reaction activator solution for derivatization reaction, carrying out HPLC (high performance liquid chromatography) detection on a product, and calculating the content of dimethylglycine in the sample to be detected according to a detection result.

According to the method, the dimethylglycine in the sample to be detected is extracted, then derivatization is carried out, the content is detected, the interference of other substances in the sample to be detected can be effectively eliminated, meanwhile, the dimethylglycine can be effectively detected, and the detection result is accurate.

Preferably, the internal standard of step (1) is dimethylglycine-D4 (NNDI-D4);

preferably, the concentration of the internal standard solution in the step (1) is 15-25 μ g/mL;

preferably, the volume ratio of the sample to be tested to the internal standard solution in the step (1) is 10 (2-3).

Wherein, the concentration of the internal standard solution can be 15 μ g/mL, 16 μ g/mL, 17 μ g/mL, 18 μ g/mL, 19 μ g/mL, 20 μ g/mL, 21 μ g/mL, 22 μ g/mL, 23 μ g/mL, 24 μ g/mL or 25 μ g/mL, etc., and the volume ratio of the sample to be tested to the internal standard solution can be 10.

Preferably, the step (1) of column elution is performed by using a C18 column.

Preferably, the eluent for the elution in step (1) is an aqueous acetonitrile solution, wherein the volume fraction of acetonitrile in the aqueous acetonitrile solution is 3-7%, such as 3%, 4%, 5%, 6%, or 7%, but not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.

Preferably, the derivatizing agent in step (2) comprises any one of 4-bromo-N-methylbenzylamine, benzeneisothiocyanate or 1- [ (4-dimethylaminophenyl) carbonyl ] piperazine, preferably 4-bromo-N-methylbenzylamine.

The specific derivatization reagent can effectively perform derivatization on dimethylglycine, so that the detection accuracy is improved; while the effect of the derivatizing agent in the preferred range is further enhanced.

Preferably, the volume ratio of the sample after elution in the step (2) to the derivatization reagent solution and the reaction activator solution is 30 (3-4) to (0.8-1.2).

Preferably, the concentration of the derivatization reagent solution in the step (2) is 8-12mmol/L.

Preferably, the concentration of the reaction activator solution of step (2) is 0.4 to 0.6M.

Wherein, the volume ratio of the sample to the derivatizing reagent solution and the reaction activator solution after elution may be 30.

Preferably, the reaction activator of step (2) comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).

Preferably, the temperature of the derivatization reaction in the step (2) is 55-65 ℃ and the time is 25-35min. Wherein the temperature can be 55 deg.C, 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C, 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C or 65 deg.C, and the time can be 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min or 35min, but not limited to the above-mentioned values, and other values not listed in the above-mentioned value range can be applied.

Preferably, the HPLC detection in step (2) is performed by using a C18 reverse phase chromatographic column.

Preferably, the mobile phase of the HPLC detection in step (2) comprises mobile phase a and mobile phase B, wherein the mobile phase a comprises aqueous formic acid solution and the mobile phase B comprises acetonitrile.

Preferably, the mass fraction of the formic acid aqueous solution is 0.008-0.012%.

Preferably, the flow rate of the mobile phase detected by HPLC in step (2) is 0.25-0.35mL/min.

Preferably, the HPLC detection in step (2) has a column temperature of 43-47 ℃.

The mass fraction of the aqueous formic acid solution may be 0.008%, 0.009%, 0.01%, 0.011%, 0.012%, etc., the flow rate of the mobile phase may be 0.25mL/min, 0.26mL/min, 0.27mL/min, 0.28mL/min, 0.29mL/min, 0.3mL/min, 0.31mL/min, 0.32mL/min, 0.33mL/min, 0.34mL/min, 0.35mL/min, etc., the column temperature may be 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, etc., but is not limited to the above-mentioned values, and other values not listed in the above-mentioned range of values are also applicable.

Preferably, the elution procedure of the HPLC detection in step (2) is gradient elution, and the conditions of the gradient elution are as follows:

at 0-0.5min, the volume fraction of the mobile phase A is 90%, and the volume fraction of the mobile phase B is 10%;

at 0.5-3min, the volume fraction of the mobile phase A is changed from 90% to 10% and the volume fraction of the mobile phase B is changed from 10% to 90%;

at 3-5min, the volume fraction of the mobile phase A is 10%, and the volume fraction of the mobile phase B is 90%;

at 5-6min, the volume fraction of the mobile phase A is changed from 10% to 90% and the volume fraction of the mobile phase B is changed from 90% to 10%;

after 6min, the volume fraction of mobile phase a was 90% and the volume fraction of mobile phase B was 10%.

In another aspect, the present invention also provides a fetal congenital heart disease diagnostic kit comprising a dimethylglycine-d 4 solution, a 4-bromo-N-methylbenzylamine solution, and a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution.

Preferably, the concentration of the dimethylglycine-d 4 solution is 15-25 mug/mL, the concentration of the 4-bromo-N-methylbenzylamine solution is 8-12mmol/L, and the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 0.4-0.6M.

The concentration of the dimethylglycine-d 4 solution may be 15. Mu.g/mL, 16. Mu.g/mL, 17. Mu.g/mL, 18. Mu.g/mL, 19. Mu.g/mL, 20. Mu.g/mL, 21. Mu.g/mL, 22. Mu.g/mL, 23. Mu.g/mL, 24. Mu.g/mL, or 25. Mu.g/mL, the concentration of the 4-bromo-N-methylbenzylamine solution may be 8mmol/L, 9mmol/L, 10mmol/L, 11mmol/L, or 12mmol/L, the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution may be 0.4M, 0.45M, 0.5M, 0.55M, or 0.6M, but not limited to the above-mentioned values, and any other values not mentioned in the above-mentioned range are also applicable.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for detecting dimethylglycine in serum, which can effectively eliminate the interference of other substances in a sample to be detected by extracting the dimethylglycine in the sample to be detected, performing derivatization and detecting the content, and can effectively detect the dimethylglycine at the same time, so that the detection result is accurate.

Drawings

FIG. 1 is a graph showing the results of quantification of the CHDP group and the NCP group in example 1;

FIG. 2 is a graph showing the effect of the 4-BNMA solution on the test results in example 2;

FIG. 3 is a graph showing the effect of the EDC solution in example 3 on the detection results;

FIG. 4 is a graph showing the effect of the temperature of the derivatization reaction on the measurement results in example 4;

FIG. 5 is a graph showing the effect of the time of the derivatization reaction on the measurement results in example 4;

FIG. 6 is a graph showing the effect of volume fraction of an aqueous acetonitrile solution on the results of measurement in example 5;

FIG. 7 is a graph showing the effect of the mass fraction of the mobile phase aqueous formic acid solution on the measurement results in example 6.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

In the following examples, 86 confirmed pregnant women with fetal congenital heart disease (CHDP group) and 92 normal pregnant women (NCP group) from women's child care institute in Jiangxi province were given consent from the test samples.

Example 1

The embodiment provides a method for detecting dimethylglycine in serum, which comprises the following specific steps:

preparing solution

(1) N, N-dimethylglycine standard solution

Accurately weighing 5mg of N, N-dimethylglycine in a 100.0mL volumetric flask, adding distilled water to a constant volume to a scale, repeatedly turning the volumetric flask upside down, fully mixing the solution to prepare N, N-dimethylglycine mother liquor with a certain concentration, and storing at 4 ℃ for later use.

(2) N, N-dimethyl glycine isotope standard solution

Precisely weighing 1mg of the N, N-dimethylglycine isotope standard in a 10.0mL volumetric flask, adding distilled water to a constant volume to scale, repeatedly turning the volumetric flask upside down and fully mixing the solution to prepare a mother solution of the N, N-dimethylglycine isotope standard with a certain concentration, and storing at 4 ℃ for later use.

(3) 1M Carboxylic acid activator EDC solution

0.775g of EDC was precisely weighed and dissolved in 1mL of 90% acetonitrile solution, mixed by vigorous vortex and stored at 4 ℃ for further use.

(4) 100mmoL 4-BNMA solution

Precisely transferring 20 mu L of 4-BNMA solution into 10mL of methanol solution, violently and uniformly mixing by vortex, and storing at 4 ℃ for later use. (10 mmol of 4-BNMA solution diluted accordingly with the mother liquor)

Preparation of the Standard Curve

Adding 400 mu L of acetonitrile, 20 mu L of NNDI-D4 internal standard solution with 20 mu G of N, N-dimethylglycine standard solution with different concentrations into 100 mu L of N, N-dimethylglycine standard solution, violently whirling and mixing, centrifuging at 12000G for 5min, taking 470 mu L of supernatant (a C18 column is firstly activated by 2mL of methanol and 2mL of water), adding 0.3mL of 5% of acetonitrile for washing twice, collecting the three times of supernatant in a 2mL EP tube together, adding 50 mu L of 10mmol of 4-BNMA solution and 15 mu L of 0.5M EDC solution, violently whirling and mixing, placing in a constant temperature shaker for derivatization at 60 ℃ for 30min, drying in a rotary evaporator at 50 ℃, adding 120 mu L of 50% acetonitrile solution for redissolving, violently whirling and centrifuging, and taking 100 mu L of supernatant for detection of N, N-dimethylglycine in the standard.

Serum sample pretreatment

Unfreezing a serum sample at 20 ℃, adding 400 mu L of acetonitrile, 20 mu L of 20 mu g/mL NNDI-D4 internal standard solution into 100 mu L of serum, violently whirling and mixing, centrifuging at 12000g for 5min, taking 470 mu L of supernatant (a C18 column is firstly activated by 2mL of methanol and 2mL of water), adding 0.3mL of 5% acetonitrile aqueous solution for washing, collecting three times of supernatant in a 2mL EP tube (450 mu L of liquid), adding 50 mu L of 10 mmol/L4-BNMA solution and 15 mu L of 0.5M EDC solution, violently whirling and mixing, placing in a constant temperature shaker for derivatization at 60 ℃ for 30min, drying at 50 ℃ in a rotary evaporator after derivatization, adding 120 mu L of 50% acetonitrile aqueous solution for redissolution, whirling and centrifuging, and taking 100 mu L of supernatant for detecting N, N-dimethylglycine in the serum.

Detection method and detection conditions

Chromatographic conditions are as follows: a single quadrupole rod LC-MS is adopted as a technical platform. And (3) chromatographic column: ACOQUITY

BEH C18 column (1.7 μm, 2.1X 50 mm). The composition of the mobile phase was: 0.01% aqueous formic acid (a) -acetonitrile (B); the flow rate is 0.3mL/min; the sample injection volume is 5.0 mu L; the separation time is 10.0min; the column temperature was set at 45 ℃.

Mobile phase gradient elution procedure

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Mass spectrum conditions: the electrospray ion source adopts positive ion detection, the voltage of an electrospray capillary is set to be 3.0kV, nitrogen is used as a drying gas for solvent evaporation, the evaporation temperature is 172 ℃, and the temperature of an ion transmission pipe is 300 ℃.

Mass spectrometric quantitation of parameters for NNDI and NNDI-D4

Linear range

And performing linear fitting by taking the N, N-dimethylglycine standard substances with different concentrations as abscissa and the peak area ratio of the N, N-dimethylglycine standard substances to the NNDI-D4 (internal standard substance) as ordinate to obtain a linear equation.

The regression equation and linear range based on N, N-dimethylglycine in the HPLC detection method are shown in Table 1.

TABLE 1N regression equation and Linear Range for N-dimethylglycine

Precision degree

Randomly and uniformly mixing the serum samples of 3 pregnant women, taking out 300 mu L of the serum samples from the mixed serum, dividing into 3 parts, performing derivatization treatment according to the sample treatment method to serve as quality control samples during detection, repeatedly injecting the sample into each quality control sample for 3 times after 1 st, 3 th and 5 th days respectively, comparing with the detection result of the first day, obtaining the Relative Standard Deviation (RSD) analyzed by the derivatization method, and calculating the intra-day precision and the inter-day precision of detecting the N, N-dimethylglycine.

The detection result of the serum quality control sample shows that the detection result RSD of the method for detecting dimethylglycine in the same day is between 3.2 and 8.7 percent, the detection result RSD of the method in the second day is between 1.8 and 13.2 percent, and the detection result RSD of the method in the third and fifth days is less than 15 percent, which indicates that the precision of the detection method is good.

Sample recovery rate

Serum samples of 5 pregnant women were randomly taken and mixed, 50. Mu.L of serum was taken, 50. Mu.L of 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL of N, N-dimethylglycine standard solution and 50. Mu.L of a blank solvent (distilled water) were added, samples of different concentrations were prepared, 3 samples of each concentration were processed in parallel, and HPLC assay was performed after the same processing as the above sample processing method.

Accuracy refers to the degree to which the results determined by this method are close to the true or reference value, and is generally expressed in terms of recovery, which is generally considered reasonable from 90% to 110%. The sample recovery for NNDI detection by this method is shown in Table 2.

TABLE 2 sample recovery of NNDI

Matrix effect

mu.L of NNDI-D4 standard solution was taken and 50. Mu.L of distilled water or 50. Mu.L of serum (three samples per group were processed in parallel) was added thereto and subjected to HPLC determination after the same treatment as described above for sample processing.

The matrix effect refers to the influence of other components in a sample except a target analyte on a measured value of an analyte, and the value is generally considered to be free from the matrix effect when the value is 85% -115%. The matrix effect of the method is 98.23% when the method is used for detection, which indicates that the method is reliable.

Detection limit

The detection Limit (LOD) of this method was calculated to be 10ng/mL and the quantitation Limit (LOQ) was calculated to be 20ng/mL.

Quantitative detection result of N, N-dimethylglycine in serum of pregnant woman with fetal congenital heart disease

The quantitative detection of N, N-dimethylglycine in CHDP (N = 86) and NCP (N = 92) serum samples proves that the concentration of N, N-dimethylglycine in the serum of the CHDP group is significantly higher than that in the NCP group, and further ROC analysis is carried out according to the content parameters of N, N-dimethylglycine in the serum of the CHDP and the NCP groups, so that the N, N-dimethylglycine has a good effect on distinguishing the CHDP from the NCP (AUC =0.863, a 95% confidence interval is 0.879-0.947), the specificity is 85.80%, and the sensitivity is 73.80%.

The results of the CHDP and NCP groups are shown in FIG. 1.

Example 2

This example investigated the effect of 4-BNMA solution on the assay results in the pretreatment of serum samples, wherein the concentration of 4-BNMA solution was replaced by 100, 10, 5 and 1mmol/L based on example 1 (unlike the sample of example 1), and the results are shown in FIG. 2.

As a result, it was found that the peak areas after 10 and 100 mmol/L4-BNMA-derivatized NNDI were not very different, and the peak areas of the remaining groups were lower, while the peak shapes after 100 mmol/L4-BNMA-derivatized NNDI were worse, and finally 10 mmol/L4-BNMA was selected as the best group.

Example 3

This example investigated the effect of EDC solution on assay results in a pre-treatment of serum samples, wherein based on example 1 (unlike the example 1 sample, in which the serum1 and serum2 samples differ), EDC solution was replaced with 15. Mu.L/25. Mu.L of 0.25M/0.5M/0.8M/1M EDC solution, respectively, and the results are shown in FIG. 3.

By examining the addition of 15. Mu.L/25. Mu.L of 0.25M/0.5M/0.8M/1M EDC as the carboxylic acid activator, it was found that the addition of 15. Mu.L/25. Mu.L of 0.5M EDC provides good derivatization conditions for 4-BNMA, which provides high sensitivity to N, N-dimethylglycine in serum and good peak shape.

Example 4

In this example, the influence of the temperature and time of the derivatization reaction on the detection result in the pretreatment of the serum sample was studied, and based on example 1 (different from the sample in example 1), the derivatization time was fixed for 30min, and the derivatization was performed by changing the reaction temperatures to 25 ℃, 37 ℃, 50 ℃, 60 ℃ and 75 ℃, respectively; then, the reaction temperature was fixed at 60 ℃ and the reaction times were varied at 30min, 60min, 90min and 120min, respectively, and the results are shown in FIGS. 4 to 5.

The relative peak areas of N, N-dimethylglycine detected by derivatization at 25 ℃, 37 ℃, 50 ℃, 60 ℃ and 75 ℃ for 30min are compared, and the experimental result shows that the sensitivity of the N, N-dimethylglycine is higher and the peak shape is good when the derivatization is carried out at 60 ℃ for 30 min. Then, the reaction time is optimized at 60 ℃, and the derivatization time is considered to be 30min, 60min, 90min and 120min, so that the derivatization time is 30min and has a higher response value at 60 ℃.

Example 5

This example investigated the effect of column elution on the assay results of volume fraction of acetonitrile in the pretreatment of serum samples, and based on example 1 (unlike the sample of example 1), the aqueous acetonitrile solution passed through the C18 column was replaced with 0.3mL of an aqueous acetonitrile solution having a volume fraction of 5%/10%/20%/30%/40%/50%, respectively, and the results are shown in FIG. 6.

As a result, it was found that most of N, N-dimethylglycine in serum was eluted with 5% acetonitrile.

Example 6

This example investigated the effect of the mass fraction of aqueous formic acid in the mobile phase during HPLC on the results of the assay, and based on example 1 (unlike the sample of example 1), the mass fraction of aqueous formic acid in the chromatographic conditions was replaced with 0.01%/0.05%/0.1%, respectively, and the results are shown in FIG. 7.

As a result, it was found that the sensitivity of detecting dimethylglycine using 0.01% formic acid water-acetonitrile as a mobile phase was high and the peak shape was good.

Example 7

This example provides a method for detecting dimethylglycine in serum, which is the same as example 1 except that 4-BNMA is replaced with an equal amount of 1- [ (4-dimethylaminophenyl) carbonyl ] piperazine.

According to calculation, the detection Limit (LOD) of the method is 50ng/mL, the quantification Limit (LOQ) is 100ng/mL, and the target chromatographic peak and the detection sensitivity are not as good as those of 4-BNMA.

From the results of examples 1 and 7, it can be found that the present invention can further improve the detection limit, the quantification limit and the sensitivity of the detection method by selecting a specific derivatization reagent.

The applicant states that the present invention is illustrated by the above examples to the method for detecting dimethylglycine in serum and the application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

Claims

1. A method for detecting dimethylglycine in serum is characterized by comprising the following steps:

(1) Mixing a sample to be tested with the internal standard solution, centrifuging to obtain a supernatant, and then performing column elution to obtain an eluted sample;

(2) And mixing the eluted sample with a derivatization reagent solution and a reaction activator solution for derivatization reaction, carrying out HPLC (high performance liquid chromatography) detection on a product, and calculating the content of the dimethylglycine in the sample to be detected according to a detection result.

2. The detection method according to claim 1, wherein the internal standard of step (1) is dimethylglycine-d 4;

preferably, the volume ratio of the sample to be detected to the internal standard solution in the step (1) is 10 (2-3).

3. The detection method according to claim 1 or 2, wherein the step (1) of column elution is performed using a C18 column;

preferably, the eluent eluted in the step (1) is acetonitrile aqueous solution, and the volume fraction of acetonitrile in the acetonitrile aqueous solution is 3-7%.

4. The detection method according to any one of claims 1 to 3, wherein the derivatizing reagent of step (2) comprises any one of 4-bromo-N-methylbenzylamine, benzeneisothiocyanate or 1- [ (4-dimethylaminophenyl) carbonyl ] piperazine, preferably 4-bromo-N-methylbenzylamine;

preferably, the volume ratio of the sample after elution in the step (2) to the derivatization reagent solution and the reaction activator solution is 30 (3-4) to (0.8-1.2);

preferably, the concentration of the derivatization reagent solution in the step (2) is 8-12mmol/L;

preferably, the concentration of the reaction activator solution of step (2) is 0.4-0.6M;

preferably, the reaction activator of step (2) comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

5. The detection method according to any one of claims 1 to 4, wherein the temperature of the derivatization reaction in step (2) is 55 to 65 ℃ and the time is 25 to 35min.

6. The detection method according to any one of claims 1 to 5, wherein the HPLC detection in step (2) is performed using a C18 reverse phase chromatography column.

7. The detection method according to any one of claims 1 to 6, wherein the mobile phase of the HPLC detection of step (2) comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an aqueous formic acid solution, and the mobile phase B comprises acetonitrile;

preferably, the mass fraction of the formic acid aqueous solution is 0.008-0.012%;

preferably, the flow rate of the mobile phase detected by HPLC in the step (2) is 0.25-0.35mL/min;

8. The detection method according to any one of claims 1 to 7, wherein the elution procedure of the HPLC detection of step (2) is gradient elution under the following conditions:

when the time is 0.5-3min, the volume fraction of the mobile phase A is changed from 90% to 10% and the volume fraction of the mobile phase B is changed from 10% to 90%;

9. The kit for diagnosing the fetal congenital heart disease is characterized by comprising a dimethylglycine-d 4 solution, a 4-bromo-N-methylbenzylamine solution and a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution.

10. The diagnostic kit for fetal congenital heart disease according to claim 9, wherein said dimethylglycine-d 4 solution has a concentration of 15-25 μ g/mL, said 4-bromo-N-methylbenzylamine solution has a concentration of 8-12mmol/L, and said 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution has a concentration of 0.4-0.6M.