A method for detecting the residual amount of glycerol and chloroglycerol in glycerophosphocholine

Abstract

The present invention belongs to the field of pharmaceutical analysis technology, and specifically discloses a quantitative separation and analysis method for the residual amount of glycerol and chloroglycerol in glycerophosphocholine. The method uses a special chromatographic column with H-type ligand exchange resin as filler, the column temperature is 40°C to 60°C, the mobile phase is a phosphoric acid solution of 0.02M to 0.05M, the flow rate is 0.3 to 0.5ml / min, and the diluent is purified water; a universal differential refractive index detector RID is used for detection; and the residual content of glycerol and chloroglycerol in the glycerophosphocholine test sample is calculated using an external standard method. The present invention provides a high-performance liquid chromatography analysis method that is easy to operate, has high accuracy and precision, good specificity and reproducibility of the method, and is convenient for standardized operation, which can separate the active drug L-α-glycerophosphocholine from glycerol and chloroglycerol and can be accurately quantified, greatly reducing the detection cost, and is simple to operate, has high accuracy and good reproducibility, and is very conducive to industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of medical analysis, and in particular to a method for detecting the residual amounts of glycerol and chloroglycerol in glycerophosphocholine. Background Art

[0002] L-α-Glycerophosphoryl Choline (L-α-GPC) is a naturally occurring water-soluble phospholipid metabolite in the body and a choline source for the synthesis of acetylcholine and phosphatidylcholine. It has important nutritional and health functions and medical application value. L-α-GPC can improve the cognitive ability of the brain and even repair the partially damaged cognitive ability of patients with early dementia. It can protect liver tissue from the penetration of fatty acids produced by toxic carbon tetrachloride and high-lipid protein foods. It has the effects of resisting hyperlipidemia and protecting blood vessels. It can also promote the physical growth of adolescents and improve memory. Therefore, it is widely used in medicine, health products and functional foods.

[0003] The chemical structure of L-α-GPC is as follows:

[0004]

[0005] The earliest L-α-GPC was extracted and purified from bovine pancreas. British patent GB2058792 and US patent US2864848 reported the technical process of preparing L-α-GPC by hydrolyzing and extracting egg yolk lecithin. Japanese patent (JP61158990) also reported a method of preparing L-α-GPC by hydrolyzing and extracting soybean lecithin as raw material. The above-mentioned extraction of L-α-GPC from natural substances has the following problems: due to the different sources of raw materials, there are great differences, and the impurity components are unevenly distributed, making the subsequent separation and purification process cumbersome and complicated, resulting in very unstable product quality. In addition, the operation process is complicated, resulting in high industrial production costs.

[0006] L-α-GPC can be chemically synthesized to avoid the problems existing in the natural material extraction method, so it has attracted widespread attention. The process of chemical synthesis of GPC uses chloroglycerol (chemical name: 3-chloro-1,2-propanediol) as a raw material. Some chloroglycerol will remain in the finished GPC, and it will also degrade to produce by-product glycerol. When the residual amount of glycerol is too much, it will further dehydrate and condense into glycidol, which has been listed as a Class 2A carcinogen by the International Agency for Research on Cancer of the World Health Organization (WHO); chloroglycerol is also suspected to be a substance that can cause genetic defects or carcinogens, and the content of both must be strictly controlled. Glycerol and chloroglycerol are both related impurities in the finished GPC products, so there must be relevant methods to measure and control their residual amounts.

[0007] In traditional methods, glycerol and chloroglycerol are usually detected by GC, but the boiling points of glycerol and chloroglycerol are relatively high (over 200°C), which requires stringent GC conditions; plus the main substance GPC is a viscous liquid or waxy solid with high viscosity, direct injection is likely to cause injection port and liner contamination and residue, and the high boiling points of glycerol and chloroglycerol limit the possibility of headspace injection, resulting in difficulties in detecting glycerol and chloroglycerol residues by gas chromatography. Therefore, it is necessary to seek a suitable HPLC method to detect the residual amount of glycerol and chloroglycerol.

[0008] Since glycerophosphocholine (GPC) and its impurities glycerol and chloroglycerol do not have ultraviolet absorption, the HPLC method to control its impurity limit has very high requirements for chromatographic conditions, and good sensitivity must be achieved to effectively control the impurities. Moreover, the polarity of these three is very large, and they are almost not retained in general reversed-phase chromatography, making it difficult to separate the target impurities. Therefore, it is urgent to establish a dedicated, sensitive and stable high-performance liquid chromatography method to monitor the residual amount of glycerol and chloroglycerol in GPC. Summary of the invention

[0009] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a high performance liquid chromatography analysis method which is simple to operate, has high accuracy and precision, good method specificity and reproducibility, and is convenient for standardized operation, so that the active drug L-α-glycerophosphocholine can be separated from glycerol and chloroglycerol and can be accurately quantified.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A method for detecting the residual amounts of glycerol and chloroglycerol in glycerophosphocholine mainly comprises the following steps:

[0012] 1) Prepare glycerol, chloroglycerol reference solution and glycerophosphocholine test solution;

[0013] 2) respectively injecting the prepared glycerol, chloroglycerol reference solution and glycerophosphocholine test solution into a high performance liquid chromatograph, separating and eluting according to the chromatographic conditions of the present invention, and recording a chromatogram, wherein the chromatographic conditions are: selecting a special chromatographic column with H-type ligand exchange resin as filler, a column temperature of 40° C. to 60° C., a mobile phase of 0.02M to 0.05M phosphoric acid solution, a flow rate of 0.3 to 0.5 ml / min, and a diluent of purified water;

[0014] 3) Calculate the residual amount of glycerol and chloroglycerol in the glycerophosphocholine test sample using the external standard method.

[0015] That is, a standard curve is determined using the concentration and peak area of ​​the reference solution, and the value of the correction factor is obtained according to the calculation formula of the correction factor; the peak areas of glycerol and chloroglycerol in the glycerophosphocholine test solution are obtained in the same manner as above, and the residual amounts of glycerol and chloroglycerol in the glycerophosphocholine test solution are calculated using the standard curve and the correction factor.

[0016] In the present invention, a universal differential refractive index detector RID is used for detection, and the detector temperature is 25°C to 40°C.

[0017] In the present invention, the chromatographic column adopts a Hi-Plex H chromatographic column with a specification of 300 mm*6.5 mm and 8 μm.

[0018] In the present invention, the running time is 20 minutes to 70 minutes.

[0019] In the present invention, the preferred flow rate is 0.5 ml / min; the column temperature is 50° C., the mobile phase is 0.04 M phosphoric acid solution, and the running time is 70 min.

[0020] The injection volume during the detection was 90 ml.

[0021] In the present invention, the reference solution and the glycerophosphocholine test solution are prepared as follows:

[0022] 1) Take the glycerol reference substance and dissolve it with a diluent to form a reference substance solution a with a concentration of 1.0 mg / ml. Prepare two parallel portions, marked as a1 and a2 respectively;

[0023] 2) Take the chloroglycerol reference substance and dissolve it with a diluent to form a reference substance solution b with a concentration of 1.0 mg / ml. Prepare two parallel portions and mark them as b1 and b2 respectively;

[0024] 3) Take 1.0 ml of the reference solution a1 and b1 respectively, and dilute them with diluent to form a mixed reference solution c1 containing 0.05 mg / ml of each reference substance; then take 1.0 ml of the reference solution a2 and b2 respectively, and dilute them with diluent to form a mixed reference solution c2 containing 0.05 mg / ml of each reference substance.

[0025] 4) Take the glycerophosphocholine sample to be tested and dilute it with a diluent to a glycerophosphocholine test solution A with a concentration of 10.0 mg / ml; prepare 2 portions in parallel and mark them as A1 and A2 respectively;

[0026] The present invention also includes an HPLC system adaptability test: 1 ml of each of solutions a1, b1 and A1 is taken and placed in a 100 ml volumetric flask, water is added to dilute to the scale, and shaken to obtain a mixed sample solution d.

[0027] Respectively take 90 μl of the above solutions d, c1, c2 and glycerophosphocholine test solutions A1 and A2, inject them into the liquid chromatograph, record the chromatogram, and calculate the residual amount of glycerol and chloroglycerol in the sample by the peak area according to the external standard method.

[0028] In the present invention, the diluent is purified water.

[0029] In summary, the advantages and beneficial effects of the present invention are as follows:

[0030] 1. The present invention can separate glycerol, chloroglycerol and glycerophosphocholine well, with strong specificity and good reproducibility.

[0031] 2. Since the polarity of glycerol, chloroglycerol and GPC is relatively large, conventional reversed-phase high performance liquid chromatography is not suitable for separating the three. In the existing HPLC method, silica chromatographic column and water are used as mobile phase, which easily causes column bleeding of the chromatographic column, not only resulting in large column loss and high detection cost, but also greatly limiting reproducibility. The present invention uses a highly selective ligand exchange column, a dilute acid solution as the mobile phase, and adopts ordinary high performance liquid chromatography, which is simple and easy to use, with high accuracy and good reproducibility.

[0032] 3. The present invention uses isocratic elution and RID detector, which can effectively separate the GPC main peak, glycerol, chloroglycerol and other related substances. It is efficient, simple and stable, and is very conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a typical HPLC spectrum for the determination of glycerol and chloroglycerol in GPC in the HPLC system suitability test;

[0034] Figure 2 The HPLC spectrum of glycerol and chloroglycerol in the glycerophosphocholine test solution in the embodiment is used;

[0035] Figure 3 It is the linear relationship diagram between the glycerol peak area and concentration in the linear range test;

[0036] Figure 4 This is the linear relationship diagram between the peak area and concentration of chloroglycerol in the linear range test;

[0037] Figure 5 To measure the spectrum of related substances in GPC test samples using Silica chromatographic columns; DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0039] Example

[0040] A method for detecting the residual amount of glycerol and chloroglycerol in glycerophosphocholine, the specific operation method is as follows:

[0041] 1. Solution preparation

[0042] 1) Take 50 mg of glycerol reference substance, place it in a 50 ml volumetric flask, add diluent to dilute to the mark, and obtain glycerol reference substance solution a; prepare 2 portions in parallel, and mark them as a1 and a2 respectively;

[0043] 2) Take the chloroglycerol reference substance, place it in a 50 ml volumetric flask, add diluent to dilute to the mark, and obtain chloroglycerol reference substance solution b; prepare 2 portions in parallel, and mark them as b1 and b2 respectively;

[0044] 3) Take 1.0 ml of each of the reference substance solutions a1 and b1, place them in the same volumetric flask with a capacity of 20 ml, and add diluent to dilute them into a mixed reference substance solution c1 containing 0.05 mg / ml of each reference substance; then take 1.0 ml of each of the reference substance solutions a2 and b2, place them in another volumetric flask with a capacity of 20 ml, and add diluent to dilute them into a mixed reference substance solution c2 containing 0.05 mg / ml of each reference substance;

[0045] 4) Take the glycerophosphocholine sample to be tested and dilute it with a diluent to a glycerophosphocholine test solution A with a concentration of 10.0 mg / ml; prepare 2 portions in parallel and mark them as A1 and A2 respectively;

[0046] 5) Take 1 ml each of solutions a1, b1, and A1, place them in a 100 ml volumetric flask, add water to dilute to the scale, shake well, and obtain a mixed sample solution d.

[0047] 6) Take 90 μl of the above solutions d, c1, c2 and glycerophosphocholine test solutions A1 and A2 respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the residual amount of glycerol and chloroglycerol in the sample by the peak area according to the external standard method.

[0048] The source of the glycerophosphocholine sample to be tested is the GPC sample refined by the Bailing Company Research Institute.

[0049] 2. Select chromatographic conditions

[0050] The chromatographic conditions used in this method are shown in Table 1.

[0051] Table 1 Chromatographic system conditions

[0052] Chromatographic column model and specifications Hi-Plex H, 300mm*6.5mm, 8μm Detector Differential refractometer, detector temperature 35°C Column temperature (℃) 50 Injection volume (μl) 90 Thinner Purified water Mobile phase 0.04M phosphoric acid solution Flow rate (ml / min) 0.5 Running time (min) 70

[0053] 3.HPLC system suitability test

[0054] (1) Take 1 μl of each of the reference solution a1, b1, and the glycerophosphocholine test solution A1, inject them into the HPLC, and record the chromatogram. Figure 1 As shown, the retention times of the three substances were determined.

[0055] (2) Take 90 μl of the mixed sample solution d and inject it into a high performance liquid chromatograph to record the chromatogram. The separation of the three substances should meet the requirements (separation greater than 1.5), and the theoretical plate number should be no less than 2000 based on glycerol.

[0056] 4. Determination of correction factors for glycerol and chloroglycerol

[0057] Take mixed control solutions c1 and c2 as correction factor determination solutions, take 90 μl of each, inject twice each, and take the average of the four correction factors.

[0058] Correction factor calculation formula: F = Cr / Ar

[0059] In the formula, F is the correction factor, Cr is the reference concentration, and Ar is the reference peak area.

[0060] The average correction factor of glycerol was Fa = 7.9584 × 10-7, RSD = 0.33%

[0061] The average correction factor of chloroglycerol is Fb = 8.0634 × 10-7, RSD = 0.51%

[0062] 5. Glycerophosphocholine test sample determination

[0063] Take 90 μl of each of the glycerophosphocholine test solutions A1 and A2, inject each solution twice, and calculate the residual amounts of glycerol and chloroglycerol in the glycerophosphocholine test solution by the external standard method using the peak area:

[0064] Residual glycerol concentration (Ca) = Fa*Aa

[0065] Where Ca is the residual glycerol concentration, Fa is the correction factor of glycerol, and Aa is the peak area of ​​glycerol in the test sample.

[0066] Chloroglycerol residual concentration (Cb) = Fb*Ab

[0067] Where, Cb is the residual concentration of chloroglycerol, Fb is the correction factor of chloroglycerol, and Ab is the peak area of ​​chloroglycerol in the sample.

[0068] 6. Sample measurement results

[0069] according to Figure 2 The residual glycerol and chloroglycerol were calculated from the chromatogram.

[0070] Calculation of residual glycerol: Substitute the glycerol chromatographic peak area in the test result of the test sample into the formula and calculate according to the external standard method. The residual concentration of glycerol in the test sample is 0.08%, that is, 1g of glycerophosphocholine sample contains glycerol impurities = 1g × 0.08% = 0.0008g = 0.8mg, that is, the residual glycerol is 0.8mg / g, or 800ppm, which meets the standard requirement of "not more than 0.5%".

[0071] Calculation of residual chloroglycerol: Substitute the chromatographic peak area of ​​chloroglycerol in the test sample into the formula, and calculate according to the external standard method. The residual concentration of chloroglycerol in the test sample is "not detected", and the detection limit of chloroglycerol is 0.05%, that is, the chloroglycerol contained in the sample is less than 0.05%, which meets the standard requirement of "not more than 0.5%".

[0072] Methodological investigation

[0073] In order to further verify the feasibility of this method, the following experiments were carried out:

[0074] 1. Specificity test

[0075] According to the product process route, appropriate amounts of glycerol and chloroglycerol control samples (analytical grade) that may be impurities in glycerophosphocholine products were taken, and the diluents were added to dissolve and fixed to a concentration of 0.5 mg / ml for HPLC positioning. The results showed that in this detection method, the above substances can be well separated and the peak reproducibility is good. The test has good specificity for determining the residual glycerol and chloroglycerol in GPC.

[0076] 2. Limit of detection and limit of quantification

[0077] Stock solution: weigh 50 mg of glycerol and chloroglycerol reference substance and dilute to 50 ml to make the stock solution.

[0078] Quantitative limit solution: accurately pipette 1 ml of the stock solution into a 100 ml volumetric flask, add diluent to make up to the mark, and shake well; then pipette 2.0 ml of the solution into a 20 ml volumetric flask, add diluent to make up to the mark, and shake well.

[0079] Detection limit solution: Accurately pipette 3 ml of the quantitation limit solution into a 10 ml volumetric flask, add diluent to the mark, shake well, and the solution is ready.

[0080] Detection limit determination results: When the signal-to-noise ratio S / N of the test solution concentration is ≈ 3, it is determined as the detection limit; when S / N ≈ 10, it is determined as the quantification limit. The test results are shown in Tables 2 and 3.

[0081] Table 2 Results of glycerol quantitative limit determination

[0082] Serial number 1 2 3 4 5 6 Peak area 2509 2774 2603 3206 3364 2470 Retention time 16.746 16.716 16.732 16.740 16.742 16.741 S / N 11.51 15.99 13.52 13.57 15.90 12.07

[0083] Among them, the concentration of glycerol solution is 1.66μg / ml.

[0084] Conclusion: When the concentration of glycerol solution is 1.66 μg / ml, it is equivalent to 3% of the limit concentration, S / N≈10, which is the quantitative limit of glycerol under this method.

[0085] Table 3 Results of determination of quantification limit of chloroglycerol

[0086] Serial number 1 2 3 4 5 6 Peak area 1534 1516 1701 1539 1380 1615 Retention time 15.487 15.493 15.485 15.475 15.509 15.482 S / N 16.6 18.34 27.55 25.6 19.86 22.34

[0087] Among them, the concentration of chloroglycerol solution is 1.55μg / ml.

[0088] Conclusion: When the concentration of chloroglycerol test solution is 1.55 μg / ml, it is equivalent to 3% of the limit concentration, S / N≈10, which is the quantitative limit of chloroglycerol under this method.

[0089] 3. Linear range test

[0090] Linear stock solution: Weigh 55.63 mg of glycerol and 51.54 mg of chloroglycerol reference respectively, add diluent to dissolve and dilute to 50 ml, shake well, and the solution is ready.

[0091] Prepare a series of solutions at different concentration levels for analysis. The linear relationship between the peak area of ​​glycerol or chloroglycerol and the concentration in the linear range test is as follows: Figure 3 and Figure 4 The linear test results are shown in Table 4.

[0092] Table 4 Configuration of linear solutions at each concentration level

[0093]

[0094] The results show that: under this method, the peak area and concentration of glycerol have a good linear relationship in the concentration range of 1.70-221.9μg / ml, and the correlation coefficient is r=0.99999; this method is used to detect glycerol impurities in the range of 3% to 444% of the residual requirement limit, which can well control the residual amount of glycerol. The peak area and concentration of chloroglycerol have a good linear relationship in the concentration range of 1.50-206.2μg / ml, and the correlation coefficient is r=0.9999; this method is used to detect chloroglycerol impurities in the range of 3% to 412% of the residual requirement limit, which can well control the residual amount of chloroglycerol.

[0095] 4. Recovery (accuracy) test

[0096] Standard solution: Take 50 mg each of glycerol and chloroglycerol reference substances, accurately weigh, add diluent to dissolve and make up to 50 ml, then accurately pipette 1.0 ml and dilute to 20 ml to make a standard solution containing 0.05 mg of glycerol and chloroglycerol per 1 ml. Make two parallel aliquots, inject 2 times in each aliquot, and calculate the correction factor.

[0097] Recovery rate stock solution: Accurately weigh 215.57 mg of glycerol reference substance and 251.62 mg of chloroglycerol into a 50 ml volumetric flask, then accurately pipette 5.0 ml to dilute to 100 ml, shake well, and obtain.

[0098] Recovery test solution: Take 250 mg of GPC test sample, accurately weigh it, put it into a 25 ml volumetric flask, add diluent to dissolve it, and transfer the recovery stock solution into each volumetric flask as follows, dilute to the mark with diluent, and shake well; each concentration level is repeated 3 times.

[0099] (1) 80% control concentration: 4.0ml → 50ml

[0100] (2) 100% control concentration: 5.0 ml → 50 ml

[0101] (3) 120% control concentration: 6.0ml → 50ml

[0102] Take 90 μl of the standard solution and recovery test solution respectively, inject them into the liquid chromatograph, record the chromatogram, and determine the recovery rates by the external standard method. The results are shown in the following table.

[0103] Table 5 Recovery results

[0104]

[0105] Conclusion: At three concentrations, the recovery rate of glycerophosphocholine was between 100.3% and 102.2%, and the RSD of the recovery rate at each concentration was no more than 2.0%; the total average recovery rate of the three concentration levels was 101.2%, and the RSD was 0.58%, which met the requirements of the validation scheme (recovery rate 91.0% to 106.0%, RSD ≤ 2.0%), confirming that the method has good accuracy.

[0106] 5. Precision test

[0107] Take the test solution under the content determination item, inject it continuously for 6 times, and record the peak area of ​​the main component of glycerophosphocholine. The precision results are shown in the following table.

[0108] Table 6 Precision results

[0109] Serial number 1 2 3 4 5 6 average RSD Peak area 355831 355988 355042 354803 355285 351775 354787 0.44％ Retention time 16.373 16.375 16.375 16.379 16.382 16.384 16.378 0.03％ Theoretical plates 12059 12051 12080 12120 12128 12189 / / Tailing Factor 1.406 1.408 1.400 1.41 1.404 1.400 / /

[0110] Summary: As can be seen from the table, in the repeated injections of 6 injections of the test solution, the RSD of the GPC peak area was 0.44% (standard ≤2.0%); the RSD of the retention time was 0.03% (standard ≤1.0%); the theoretical plate number was greater than 3000, and the tailing factor was <1.6; it can be seen that the precision of the method is good.

[0111] 6. Repeatability test

[0112] Take the same glycerophosphocholine test sample, accurately weigh 100 mg, add diluent to dissolve and make up to 10 ml, inject into liquid chromatograph, parallel 6 parts, inject each solution once, calculate the residual amount of glycerol and chloroglycerol in the sample by external standard method, the repeatability of the test results is shown in the table below.

[0113] Table 7 Glycerol repeatability test results

[0114]

[0115]

[0116] Conclusion: The RSD of the 6 determination results was 0.00% (the standard requirement is ≤2.0%), and the repeatability was good.

[0117] Table 8 Chloroglycerol repeatability test results

[0118]

[0119] Conclusion: Chloroglycerin was not detected in the 6 determination results, and the repeatability was good.

[0120] Comparative Example

[0121] This comparative example adopts the traditional method of detecting GPC impurities, using a Silica chromatographic column and water as the mobile phase. The detection results of the relevant substances in the test sample are as follows Figure 4 As shown, its peak table is shown in Table 9.

[0122] Table 9 Peak list of GPC related substances measured by Silica chromatographic column

[0123]

[0124] Figure 4 The results in Table 9 show that the separation of glycerol and adjacent impurity peaks is poor; chloroglycerol peaks with other impurities, and its residual amount cannot be accurately determined. In addition, the silica column uses water as the mobile phase, and the column loss is very large, which is not suitable for large-scale production to monitor product quality.

[0125] We tried to use gas chromatography to measure and found that in the same sample, the peak area of ​​glycerol injected continuously increased and could not reach a stable peak. We found that the reason was that both GPC and glycerol were very viscous and had caused residue in the pipeline.

[0126] In summary, the analytical method of the present invention can accurately, simply and reliably determine the residual amount of glycerol and chloroglycerol in GPC. Using H-type ligand exchange column and RID detection, the method is accurate, simple and easy to implement, and easy to promote; the method can be used for industrial quality control of GPC products, overcoming the shortcomings of traditional methods.

Claims

1. A method for detecting the residual amount of glycerol and chloroglycerol in glycerophosphocholine, characterized in that The main steps include: 1) Prepare glycerol, chloroglycerol reference solution and glycerophosphocholine test solution with a diluent, wherein the diluent is purified water; 2) Inject the glycerol, chloroglycerol reference solution and glycerophosphocholine test solution prepared in step 1 into the high performance liquid chromatograph respectively to obtain a chromatogram. The chromatographic conditions are as follows: a Hi-PlexH column with a specification of 300 mm*6.5 mm and 8 μm is selected, the column temperature is 40°C to 60°C, the mobile phase is a 0.02M to 0.05M phosphoric acid solution, the flow rate is 0.3 to 0.5 ml / min, and the running time is 20min to 70min; a universal differential refractive index detector RID is used for detection, and the detector temperature is 25°C to 40°C; 3) Calculate the residual amounts of glycerol and chloroglycerol in the glycerophosphocholine test sample using the external standard method.

2. The method for detecting the residual amounts of glycerol and chloroglycerol in glycerophosphocholine according to claim 1, characterized in that The flow rate was 0.5 ml / min; the column temperature was 50° C., the mobile phase was 0.04 M phosphoric acid solution, and the running time was 70 min.

3. The method for detecting the residual amounts of glycerol and chloroglycerol in glycerophosphocholine according to claim 1, characterized in that The preparation method of the reference solution and the glycerophosphocholine test solution in step 1 is as follows: 1) Take the glycerol reference substance and dissolve it with a diluent to make a reference solution a with a concentration of 1.0 mg / ml. Prepare two parallel portions and mark them as a1 and a2 respectively. 2) Take the chloroglycerol reference substance and dissolve it with a diluent to make a reference solution b with a concentration of 1.0 mg / ml. Prepare two parallel portions and mark them as b1 and b2 respectively; 3) Take 1.0 ml of each of the reference substance solutions a1 and b1, respectively, and dilute them with diluent to form a mixed reference substance solution c1 containing 0.05 mg / ml of each reference substance; then take 1.0 ml of each of the reference substance solutions a2 and b2, respectively, and dilute them with diluent to form a mixed reference substance solution c2 containing 0.05 mg / ml of each reference substance; 4) Take the glycerophosphocholine sample to be tested and dilute it with a diluent to a glycerophosphocholine test solution A with a concentration of 10.0 mg / ml; prepare 2 parallel portions and mark them as A1 and A2 respectively.

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