CN114965822A - Medicine analysis method for efficiently determining ganglioside GM1 and impurities thereof - Google Patents

Abstract

The invention discloses a medicine analysis method for efficiently determining ganglioside GM1 and impurities thereof, belonging to the technical field of medicine analysis. The method takes a chromatographic column with octadecylsilane chemically bonded silica as a filler as a stationary phase and takes a mixed solution of a buffer salt solution and an organic phase as a mobile phase, so that the related substances and the content of ganglioside GM1 can be effectively separated and determined.

Description

Medicine analysis method for efficiently determining ganglioside GM1 and impurities thereof

The application is a divisional application of patent application No. 201711030431.2 filed on 27/10/2017.

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a drug analysis method for efficiently determining ganglioside GM1 single components and related substances. The analysis method can effectively separate and measure the content of the ganglioside GM1 single component and related impurities, has simple and quick operation, and can be used as an important component for the quality control of the ganglioside GM 1.

Background

Gangliosides (gangliosides) are the most complex glycosphingolipids, widely distributed on the cell membranes of vertebrates, with the highest content in the central nervous system. It is composed of sialic acid-bearing oligosaccharide chains and ceramides, and gangliosides can be classified according to the number of sialic acid and oligosaccharide glycosyl groups and the differences of sialic acid attachment sites. Wherein the gangliosides with higher content are GM1, GDla, GDlb, GD3 and GTlb.

GM1 mainly comprises two components GM1A and GM1B, and GM1A and GM1B have the following structures. GM1 functions by "compensating" for Na +, K + -ATPase activity, plays an important role in Na +, K + -ATPase in maintaining membrane stability and excitability, and is important for normal cell function. The functions of GM1 can be broadly summarized as follows: 1. promoting normal development of nerve cells and brain tissue, and preventing and treating cerebral palsy; 2. repairing damaged nerve and brain tissue, and preventing and treating cerebral apoplexy; 3. enhancing memory function; 4. delaying the aging of nerve cells, and preventing and treating Parkinson's disease, senile dementia, etc. The function of GM1 is well-paid attention in the field of medicine, and a plurality of products are on the market at home at present.

The preparation of the ganglioside GM1 is derived from biological tissues and has complex components, and the types and the content of impurities in the product are directly related to the quality and the medication safety of the medicine, so how to accurately determine the related substances and the content of the single component of the ganglioside GM1 becomes a problem to be solved. National pharmacopoeia (2015) regarding pharmaceutical WS of ganglioside GM1 ₁ -XG-001-2015, WS for injection ₁ -XG-002 2015, injection WS ₁ The method for detecting related substances and content of ganglioside GM1 is specified in-XG-003-2015, and is characterized in that an amino column is used as a separation matrix, and a large amount of organic solvents are used as mobile phases for isocratic separation technology. The above methods have limited selectivity and weak separation ability, and have a risk of not effectively separating related substances with similar structures; the method has the advantages that the low ultraviolet wavelength of 205nm is used as the detection wavelength, and tetrahydrofuran is used as the mobile phase, so that the base line basic absorption value is increased, the base line noise is increased, the detection sensitivity of impurities is weakened, and the risk of underestimating the impurities and even incomplete impurity spectrum analysis exists; and the method has poor stability and reproducibility. In order to effectively analyze and detect the quality of the medicine and ensure the safety of the medicine, an analysis method for conveniently, efficiently and accurately determining the content of the ganglioside GM1 and related substances is still required to be developed.

Disclosure of Invention

The invention provides a high performance liquid chromatography method for measuring ganglioside GM1 and related substances, which adopts a chromatographic column using octadecylsilane chemically bonded silica as a filler and uses a buffer salt solution-organic phase as a mobile phase.

Wherein the buffered salt solution is selected from a solution of: phosphates, perchlorates, formates, acetates, preferably dihydrogen phosphates, more preferably potassium dihydrogen phosphates; the phosphate comprises orthophosphate, dibasic phosphate and monobasic phosphate; the salt is selected from sodium, potassium, rubidium, cesium, lithium, ammonium or amine, calcium salt; the organic phase is selected from one or a mixture of methanol, ethanol, acetonitrile, propanol and isopropanol, and is preferably acetonitrile.

Preferably, the pH value of the buffered salt solution is 6.0-9.0, preferably 6.5-7.5, more preferably 6.8, 7.0, 7.2.

Preferably, the column is a reverse phase column selected from Kromasil, Apollo, Waters, Merk.

Preferably, the column has a size of 150mm x 4.6mm,3.5um, or other equivalent performance column.

Preferably, the column temperature of the process is 40 ℃; the detection wavelength is 200-210 nm, preferably 205 nm.

Preferably, the mobile phase flow rates of the process are: 0.5ml/min to 1.5ml/min, preferably 1.0 ml/min;

preferably, the pH value of the buffer salt solution can be adjusted by using phosphoric acid, potassium carbonate, sodium carbonate, triethylamine and a solution thereof; the concentration of the solution for adjusting the pH value is the conventional concentration in the field, and preferably 0.1-1.0 mol/L.

Preferably, the method employs gradient elution; specifically, the mobile phase comprises or consists of mobile phase A and mobile phase B; the mobile phase A is acetonitrile-dihydrogen phosphate solution mixed liquor, and the mobile phase B is acetonitrile; wherein the volume ratio of acetonitrile to dihydrogen phosphate solution in the mobile phase A is 30-80: 70-20, preferably 60-70: 40 to 30, more preferably 65 to 70: 35-30 parts of; in a preferred embodiment of the present invention, the volume ratio of acetonitrile to dihydrogen phosphate solution in the mobile phase a is 68: 32.

in the mobile phase of the invention, the concentration of the buffer salt solution is 0.001-0.50 mol/L, preferably 0.005-0.10 mol/L, 0.01-0.05 mol/L, more preferably 0.02mol/L or 0.01 mol/L; in a preferred technical scheme, the buffer salt solution is 0.01mol/L potassium dihydrogen phosphate solution, 0.01mol/L sodium dihydrogen phosphate or 0.01mol/L ammonium dihydrogen phosphate.

Preferably, the mobile phase of the invention is eluted with a gradient of:

note: 100-A means that all of the mobile phase B except the mobile phase A is the mobile phase B;

in one technical method of the invention, the invention employs an elution gradient as follows:

test samples, control solutions, system suitability solutions according to the national pharmacopoeia (2015) for the ganglioside GM1 bulk drug WS ₁ It can be prepared by the method specified in the standard XG-001-2015 or other conventional methods in the field. In one embodiment of the present invention, the test sample solution, the reference solution and the system suitability solution can be prepared according to the following methods:

(1) test solution: a proper amount of a test sample is taken and precisely weighed, and is dissolved and diluted by adding water or acetonitrile aqueous solution to prepare 0.5-10 mg/ml solution, preferably 1.0-5.0 mg/ml, more preferably 1.0mg/ml and 5.0mg/ml, as the test sample solution. More specifically, the test solution can be prepared by the following method: taking about 50mg of the product, precisely weighing, placing in a 10ml volumetric flask, adding water to dissolve and dilute to a scale, and shaking up to obtain a test solution with the concentration of 5.0 mg/ml.

(2) Control solution: taking a proper amount of ganglioside GM1 as a reference substance, precisely weighing, and dissolving and diluting with water or acetonitrile aqueous solution to prepare a solution of about 0.05-1.5 mg/ml (preferably 0.05mg/ml or 1mg/ml) as a reference substance solution. More specifically, the content control solution can be prepared by the following method: precisely weighing about 25mg of each of GM1A and GM1B as reference substances, placing in a 50ml volumetric flask, adding water to dissolve and dilute to scale, and shaking up; precisely measuring 5ml, placing in a 50ml measuring flask, adding water to dilute to scale, and shaking to obtain control solution. The reference substance solution can be prepared by the following method: precisely weighing about 25mg of monosialotetrahexosylganglioside A (namely the sodium salt form of GM 1) or monosialotetrahexosylganglioside B (namely the sodium salt form of GM 1B) as a control, placing the control in a 50ml volumetric flask, adding water to dissolve and dilute the control to a scale, and shaking up; precisely measuring 5ml of the solution, placing the solution in a 50ml volumetric flask, adding water to dilute the solution to the scale, and shaking up.

(3) System applicability solution: collecting gangliosides GM1A, GM1B, GD1a, GD ₃ The appropriate amount of each is precisely weighed, and dissolved and diluted with acetonitrile aqueous solution to prepare a mixed solution containing about 0.5-5 mg (preferably 1mg or 5mg) of ganglioside GM1 and 20-120 μ g (preferably 50 μ g) of each impurity in 1 ml. Optionally, the system suitability solution also contains an appropriate amount of sialic acid.

In one embodiment, the system suitability solution may be prepared by: taking about 2mg of GD1a reference substance and about 1mg of GD3 reference substance, putting the GD1a reference substance and the GD3 reference substance into the same container, adding 20ml of water for dissolving, shaking up, and taking the solution as a system applicability solution stock solution; about 5mg of each of GM1A and GM1B was added to 2ml of stock solution of the system compatible solution, and the mixture was dissolved and shaken up to obtain a system compatible solution).

The volume fraction of acetonitrile in the aqueous acetonitrile solution is 0% to 40%, preferably 0% to 20%, and more preferably 10%.

In a preferred embodiment of the present invention, the high performance liquid chromatography method for measuring ganglioside GM1 and related substances in the invention adopts the following chromatographic conditions and steps:

chromatographic conditions are as follows:

the type of the high performance liquid chromatograph has no special requirement; the following chromatographs can be selected from Waters e2695-2489 or Agilent 1260-VWD, Waters ACQUITY ARC-2489;

a chromatographic column: c18(Waters, 4.6X 150mm, 3.5 μm);

mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (30:70), and pH was adjusted to 7.0 with triethylamine.

Mobile phase B of acetonitrile

The gradient elution procedure was used:

flow rate: 1.0ml/min, detection wavelength 205nm, sample injection volume: 10 μ l, column temperature: at 40 ℃.

The invention provides a high performance liquid chromatography method for measuring sialic acid, which uses amino bonded silica gel as a filling agent; phosphoric acid solution (1 → 100) -acetonitrile-tetrahydrofuran (28: 62: 10) as the mobile phase; the detection wavelength was 205nm, the column temperature was 35 ℃ and the flow rate was 1.0ml per minute.

The high performance liquid chromatography method for measuring sialic acid comprises the following steps: precisely measuring 20 μ l of each of the system applicability solution, the reference solution and the sample solution, respectively injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram. Calculated as peak area by external standard method.

More specifically, the second aspect of the present invention provides a high performance liquid chromatography method for determining sialic acid, further comprising the steps of: taking a proper amount of sample, adding water to dissolve and dilute the sample to prepare a solution containing about 5mg of the sample in each 1ml of the sample solution; a proper amount of sialic acid control substance is dissolved in water and diluted to obtain a solution containing about 0.025mg in 1ml, which is used as a control substance solution. Appropriate amount of sialic acid control substance and GD3 control substance were dissolved in water and diluted to obtain 1ml solution containing sialic acid 0.025mg and GD30.05mg, which was used as system-compatible solution.

In the present invention, GM1, GM1A, GM1B refer not only to their free acid forms but also to their sodium salt forms or other salt forms. The samples or controls of GM1, GM1A, GM1B described herein refer to their sodium salt forms. The peak identified as "tys" in the figures of the present invention is the sialic acid peak.

The invention has the technical effects that:

the chromatographic column, the specific mobile phase and the elution gradient which adopt octadecylsilane chemically bonded silica as the filler optimize chromatographic separation materials and mobile phase conditions, have strong separation capability and excellent selectivity, ensure that the retention difference of impurities of each component on the chromatographic column is more obvious, effectively separate two components of the monosialotetrahexosylganglioside sodium, effectively separate dozens of related impurities, obtain excellent peak shape and separation selectivity, distinguish the ganglioside of different fatty chains and fucosylated ganglioside, and provide an effective tool for impurity research; and the method also has high resolution on ganglioside with sialic acid at different sugar residue connecting positions and other isomers, solves the problem of separation and determination of ganglioside and related substances thereof, and thus provides accurate basis for quality control of ganglioside product GM 1.

The method for determining sialic acid provided by the invention is modified from the self-comparison calculation of the main component GM1 to a sialic acid comparison product external standard method, the comparison peak shape is better, the accuracy of the sialic acid calculation result is improved, and the method is favorable for product quality control.

Drawings

FIG. 1 is a high performance liquid chromatography profile determined in comparative example 1;

FIG. 2 is a high performance liquid chromatography chromatogram obtained by the measurement of example 1;

FIG. 3 is a high performance liquid chromatography chromatogram obtained by the measurement of example 2;

FIG. 4 is a high performance liquid chromatography profile obtained in example 3;

FIG. 5 System suitability solution profile;

FIG. 6 HPLC of the sialic acid control solution obtained from example 4;

FIG. 7 according to the national drug Standard WS ₁ Typical HPLC profile for the principal component control specified in XG-001-2015.

Detailed Description

The invention is further illustrated by the following specific examples. It should be understood that: the examples of the present invention are provided for illustration only and not for limitation of the present invention. The technical scheme obtained by simply improving the invention or equivalently replacing the conventional means or components on the basis of the technical scheme of the invention belongs to the protection scope of the invention. The impurity reference substance used in the present invention can be purchased or synthesized. In the examples, the term "C18" refers to a reverse phase chromatography column using octadecylsilane chemically bonded silica as a packing material

Comparative example 1

Taking a proper amount of ganglioside GM1 and related substances, dissolving a sample with water to prepare a sample solution containing about 1.0mg/ml of ganglioside GM 1. Press WS ₁ HPLC analysis was performed by the method prescribed in national drug Standard No. XG-001-2015, and a chromatogram was recorded, the HPLC analysis spectrum of which is shown in FIG. 1.

Example 1 determination of ganglioside GM1

Apparatus and conditions

High performance liquid chromatograph: waters e2695-2489

A chromatographic column: c18(Waters, 4.6X 150mm, 3.5 μm)

Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (30:70), pH 7.1 was adjusted with triethylamine.

Mobile phase B of acetonitrile

The gradient elution procedure was used:

flow rate: 1.0ml/min, detection wavelength 205nm, sample injection volume: 10 μ l, column temperature: at 40 ℃.

The experimental steps are as follows:

taking a proper amount of ganglioside GM1 and related substances, dissolving a sample with an aqueous solution to prepare a sample solution containing about 1.0mg/ml of ganglioside GM 1. Performing high performance liquid chromatography analysis according to the above test conditions, and recording chromatogram, wherein HPLC chromatogram is shown in FIG. 2.

The map result shows that the method has stronger separation capability and more excellent selectivity. Not only two single components of GM1 are effectively separated, but also tens of impurities associated therewith.

Example 2 determination of ganglioside GM1

Apparatus and conditions

High performance liquid chromatograph: agilent 1260-VWD

A chromatographic column: c18(Waters, 4.6X 150mm, 3.5 μm)

Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile (32:68), pH 7.1 was adjusted with triethylamine.

Mobile phase B of acetonitrile

The gradient elution procedure was used:

flow rate: 1.2ml/min, detection wavelength 205nm, sample injection volume: 10 μ l, column temperature: at 40 ℃.

The experimental steps are as follows:

taking a proper amount of ganglioside GM1 and related substances, dissolving a sample with an aqueous solution to prepare a sample solution containing about 5mg/ml of ganglioside GM 1. Performing high performance liquid chromatography analysis according to the gradient conditions, recording chromatograms, effectively separating two components of ganglioside GM1 and related substances, and making HPLC chromatogram basically consistent with that of figure 1.

Example 3 determination of ganglioside GM1

Apparatus and conditions

High performance liquid chromatograph: waters e2695-2489

A chromatographic column: c18(Symmetry RP18, 4.6X 150mm, 3.5 μm)

Mobile phase A: acetonitrile (35:65) as a 0.012mol/L potassium dihydrogen phosphate solution, and the pH was adjusted to 7.5 with triethylamine.

Mobile phase B acetonitrile

The gradient elution procedure was used:

flow rate: 1.2ml/min, detection wavelength 205nm, sample injection volume: 10 μ l, column temperature: at 40 ℃.

The experimental steps are as follows:

taking a proper amount of ganglioside GM1 and related substances, dissolving a sample with an aqueous solution to prepare a sample solution containing about 1.2mg/ml of ganglioside GM 1. Performing high performance liquid chromatography analysis according to the gradient conditions, and recording chromatogram, the result is shown in figure 4. Both components of ganglioside GM1 were effectively separated from their related substances.

EXAMPLE 4 measurement of sialic acid content

Chromatographic conditions are as follows: amino bonded silica gel is used as a filling agent; phosphoric acid solution (1 → 100) -acetonitrile-tetrahydrofuran (28: 62: 10) as the mobile phase; the detection wavelength is 205nm, the column temperature is 35 ℃, and the flow rate is 1.0ml per minute;

precisely measuring sialic acid reference solution and sample solution 20 μ l each, injecting into liquid chromatograph, measuring according to the above chromatographic conditions, and recording chromatogram. Calculating by peak area according to an external standard method; the HPLC profile of the obtained sialic acid control solution is shown in FIG. 6;

according to the national drug Standard WS ₁ A typical profile of the principal component control specified in-XG-001-2015 is shown in FIG. 7.

And (3) testing the applicability of the system:

under the above-identified chromatographic conditions, the system suitability solution is used to analyze whether the chromatographic conditions meet the requirements, and as can be seen from fig. 5, under the conditions, the separation degree between each related substance and the main peak meets the requirements, and the peak purity meets the requirements.

Specificity property

(1) Acid degradation:

related substance test solution: taking about 50mg of a sample, placing the sample in a 10ml volumetric flask, adding 2ml of water to dissolve the sample, adding 3ml of hydrochloric acid solution with the concentration of 1mol/L, degrading for 18h, adding 1mol/L of sodium hydroxide solution for neutralization, adding water to dilute to the scale, and shaking up.

Content test solution: precisely measuring 2ml of test solution under related substance items, placing the test solution in a 10ml volumetric flask, adding water to dilute the test solution to a scale, and shaking up.

Alkali degradation:

related substance test solution: taking about 50mg of a sample, placing the sample in a 10ml volumetric flask, adding 2ml of water to dissolve the sample, adding 3ml of 1mol/L hydrochloric acid solution, degrading for 18 hours, adding 1mol/L hydrochloric acid solution to neutralize, adding water to dilute to a scale, and shaking up.

Content test solution: precisely measuring 2ml of test solution under related substance items, placing the test solution in a 10ml volumetric flask, adding water to dilute the test solution to a scale, and shaking up.

(2) Oxidative degradation:

related substance test solution: taking about 50mg of a sample, placing the sample in a 10ml volumetric flask, adding 2ml of water to dissolve the sample, adding 3ml of 30% hydrogen peroxide, degrading for 20 hours, adding water to dilute to a scale, and shaking up.

Content test solution: precisely measuring 2ml of test solution under related substance items, placing the test solution in a 10ml volumetric flask, adding water to dilute the test solution to a scale, and shaking up.

(3) And (3) high-temperature degradation:

a) related substance test solution: taking about 50mg of a sample, placing the sample in a 105 ℃ oven for 10 hours, cooling to room temperature, adding water to dissolve and dilute to a scale, and shaking up.

Content test solution: precisely measuring 2ml of test solution under related substance items, placing the test solution in a 10ml volumetric flask, adding water to dilute the test solution to a scale, and shaking up.

b) Related substance test solution: taking about 50mg of the sample, adding water to dissolve and dilute to the scale, and shaking up. Taking a proper amount of the test solution, placing the test solution in a 105 ℃ oven for 10 hours, and cooling the test solution to room temperature to obtain the related substance test solution.

Content test solution: precisely measuring 2ml of test solution under related substance items, placing the test solution in a 10ml volumetric flask, adding water to dilute the test solution to a scale, and shaking up.

(4) Hydrolysis:

related substance test solution: taking about 50mg of a sample, putting the sample into a 10ml volumetric flask, adding 2ml of water for dissolving, adding 3ml of water, standing at room temperature for 20 hours, adding water for diluting to the scale, and shaking up.

Content test solution: precisely measuring 2ml of test solution under related substance items, placing the test solution in a 10ml volumetric flask, adding water to dilute the test solution to a scale, and shaking up.

(5) Factor of influence degradation test:

and (3) high-temperature degradation:

related substance test solution: taking about 50mg of the sample which is respectively placed for 5 days and 10 days at the high temperature of 40 ℃ and 60 ℃, putting the sample into a 10ml volumetric flask, adding water to dissolve and dilute the sample to the scale, and shaking up.

Content test solution: taking about 50mg of the sample which is respectively placed for 5 days and 10 days at the high temperature of 40 ℃ and 60 ℃, placing the sample in a 50ml volumetric flask, adding water to dissolve and dilute the sample to the scale, and shaking up.

High-humidity degradation:

related substance test solution: taking about 50mg of the sample which is respectively placed for 5 days and 10 days under the conditions of saturated sodium chloride solution and saturated potassium nitrate solution, placing the sample in a 10ml volumetric flask, adding water to dissolve and dilute the sample to the scale, and shaking up.

Content test solution: taking about 50mg of the sample which is respectively placed for 5 days and 10 days under the conditions of saturated sodium chloride solution and saturated potassium nitrate solution, placing the sample in a 50ml volumetric flask, adding water to dissolve and dilute the sample to the scale, and shaking up.

Degradation by illumination:

related substance test solution: taking about 50mg of the sample which is respectively placed for 5 days and 10 days under the condition of the illumination of 4500lx +/-500 lx, placing the sample in a 10ml volumetric flask, adding water to dissolve and dilute the sample to the scale, and shaking up.

Content test solution: taking about 50mg of the sample which is respectively placed for 5 days and 10 days under the condition of the illumination intensity of 4500lx +/-500 lx, placing the sample in a 50ml volumetric flask, adding water to dissolve and dilute the sample to the scale, and shaking up.

After degradation is finished, the sample preparation method and chromatographic condition are detected according to the invention, and chromatogram is recorded, and the result shows that the product can be effectively separated from main components at each degradation impurity peak.

(6) Linearity

Related substances are as follows: linear solutions containing the GM1A control at LOQ concentrations, 5. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml, 250. mu.g/ml, 300. mu.g/ml were prepared, respectively. Linear solutions containing the GM1B control at LOQ concentrations, 5. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml, 250. mu.g/ml, 300. mu.g/ml were prepared, respectively. According to the chromatographic condition detection, the result shows that the measured concentration of the ganglioside GM1A is in the range of 1.5348 mu g/ml-294.975 mu g/ml, y is 3342.8419x-1434.5325, the correlation coefficient r is 1.0000, the correlation coefficient is more than 0.99, and the linearity meets the requirement. The measured concentration of ganglioside GM1B is in the range of 1.8428 mug/ml-284.9352 mug/ml, y is 3230.8702x-2308.7666, correlation coefficient r is 1.0000, correlation coefficient is greater than 0.99, and linearity meets the requirement.

The content is as follows: linear solutions containing approximately 0.4mg/ml, 0.45mg/ml, 0.5mg/ml, 0.55mg/ml, 0.6mg/ml of GM1A control were prepared, respectively. Linear solutions containing approximately 0.4mg/ml, 0.45mg/ml, 0.5mg/ml, 0.55mg/ml, 0.6mg/ml of GM1B control were prepared, respectively. According to the chromatographic condition detection, the result shows that the measured concentration of the ganglioside GM1A is in the range of 0.39691 mg/ml-0.55936 mug/ml, y is 2932303.2462x +20988.1709, the correlation coefficient r is 0.9999, the correlation coefficient is more than 0.999, and the linearity meets the requirement. The measured concentration of ganglioside GM1B is in the range of 0.38824 mg/ml-0.53449 microgram/ml, y is 2991291.3924x +41939.0354, correlation coefficient r is 0.9998, correlation coefficient is greater than 0.999, and linearity meets the requirement.

(7) Stability of solution

The ganglioside GM1 related substances and content test solution are injected at 0, 7, 14 and 21 hours respectively, the stability of the sample solution is examined, the results are respectively shown in tables 1 and 2, and the solution is stable within 24 hours.

TABLE 1 results for the materials

Table 2 content results

(8) Durability

Since the chromatographic conditions of the product are gradient elution and the types, the column temperature, the flow rate, the pH value and the like of a chromatographic column are specified, the conditions are correspondingly finely adjusted, the durability of the method is investigated, and the result shows that the method has good durability on the column temperature, the flow rate, the pH value and the like, and the separation degree of the ganglioside GM1 and adjacent impurities thereof can be effectively separated under the conditions of different column temperatures, flow rates, pH values and the like.

Preparation example 1 preparation of monosialotetrahexosylgangliosides GM1A and GM1B

Taking 600mg of crude ganglioside, dissolving in 10mL of methanol-10 mM ammonium dihydrogen phosphate buffer saline solution (pH is 2.5), and sampling 10 mL; using a reverse phase chromatography column (column size 50X 250mm, particle size 10 μm, pore diameter)

Bonding filler mass 300g), flow rate 0.6 mm/s; the mobile phase was methanol-10 mM ammonium dihydrogen phosphate buffer salt (pH 2.5) with a volume ratio of methanol/buffered saline solution of 60/40; an ultraviolet detector for detecting the wavelength of 205nm, collecting sample eluent in stages, and respectively evaporating the solvent to obtain corresponding GM1A and GM 1B; the HPLC purity was determined to be 99.80% and 99.20%, respectively; wherein the 'reversed phase chromatographic column' column refers to a dynamic axial compression preparation column, and the filler (namely bonding filler) is octadecyl bonding silica gel.

Attached: national drug Standard WS ₁ GM1 content determination method specified in-XG-001-2015:

[ CONTENT DETERMINATION ] is determined according to high performance liquid chromatography (appendix VD of the second part of the 2010 edition of Chinese pharmacopoeia).

Chromatographic conditions and system applicability test: using amino silane bonded silica gel as filler, using phosphoric acid solution (1 → 100) -acetonitrile-tetrahydrofuran (30: 60: 10) as mobile phase, and detecting wavelength 205 nm. Taking appropriate amount of monosialotetrahexosylganglioside sodium reference substance, sialic acid reference substance, disialotetrahexosylganglioside (GD1a) reference substance and disialotetrahexosylganglioside (GD3) reference substance, adding water to dissolve and dilute to prepare mixed solution containing 1mg, 25 mu g, 100 mu and 50 mu g in each 1ml, and using the mixed solution as system applicability solution. And (4) injecting 20 mu l of the mixture into a liquid chromatograph, and recording the chromatogram. The number of theoretical plates is not less than 1000 according to the monosialotetrahexosylganglioside peak, and the separation degree between the monosialotetrahexosylganglioside peak and adjacent impurity peaks and among all known impurity peaks meets the requirement.

The determination method comprises the following steps: taking the product, precisely measuring, dissolving in water, quantitatively diluting to obtain solution containing about 1mg per 1ml, precisely measuring 20 μ l, injecting into liquid chromatograph, and recording chromatogram; taking monosialotetrahexosyl ganglioside sodium reference substance, and determining by the same method. Calculating by the cover area according to an external standard method to obtain the product.

Claims (10)

1. A high performance liquid chromatography method for measuring ganglioside GM1 and its related substances adopts chromatographic column using octadecylsilane chemically bonded silica as filler, and its mobile phase comprises mobile phase A and mobile phase B; the mobile phase A is mixed solution of acetonitrile-dihydrogen phosphate solution, and the pH value is adjusted to 6.5-7.5 by triethylamine; the mobile phase B is acetonitrile; the volume ratio of acetonitrile to dihydrogen phosphate solution in the mobile phase A is 65-70: 35-30 parts of; and elution was carried out with the following gradient

The phosphate solution is 0.01mol/L potassium dihydrogen phosphate solution and 0.01mol/L sodium dihydrogen phosphate.

2. The process according to claim 1, characterized in that the volume ratio of acetonitrile to dihydrogen phosphate solution in mobile phase a is 68: 32.

3. the process of claim 1, wherein the mobile phase a is adjusted to a pH of 6.8, 7.0, or 7.2 with triethylamine.

4. The method of claim 1, wherein the chromatography column has a size of 150mm x 4.6mm,3.5 μm.

5. The method of claim 1, wherein the column temperature is 40 ℃ and the detection wavelength is 205 nm.

6. The method of claim 1, wherein the related substances comprise impurities GD3, GD1a-1, GD1 a-2.

7. The method of claim 1, wherein the method has a mobile phase flow rate of 0.5 to 1.5 mL/min.

8. The method according to claim 1, wherein the flow rate of the mobile phase of the method is 1.0 mL/min.

9. The method according to claim 1, characterized in that the following elution gradient is used:

10. a high performance liquid chromatography method for measuring ganglioside GM1 and its related substances adopts the following chromatographic conditions:

a chromatographic column: c18, Waters, 4.6X 150mm, 3.5 μm;

mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution-acetonitrile with the volume ratio of 30:70, and triethylamine is used for adjusting the pH value to 7.0;

mobile phase B of acetonitrile

The gradient elution procedure was used:

flow rate: 1.0mL/min, detection wavelength 205nm, sample injection volume: 10 μ L, column temperature: at 40 ℃.

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