Method for separating and detecting components in butanedisulfonic acid ademetionine preparation

Abstract

The invention provides a method for separating and detecting components in butanedisulfonic acid ademetionine enteric-coated tablets, and belongs to the technical field of medicine quality analysis. According to the method, ammonium formate solution-water-acetonitrile and ammonium formate solution-acetonitrile-methyl tert-butyl ether are used as a mobile phase system, and the pH value of the ammonium formate solution is controlled to be 2.2-3.1, so that adenosine and adenine can be effectively separated; according to the method, the S-adenosyl-L-methionine glycine isomer 2, adenosine, adenine and S-adenosyl-L-homocysteine can be separated, the time consumption is short, the separation efficiency is high, the operation is simple, the separation cost is low, and the quality control of butanedisulfonic acid adenosyl methionine or related preparations can be improved. According to the method, the detection of the ademetionine and the related impurities is not influenced by auxiliary materials, the method is efficient and convenient, good in reproducibility, high in specificity, high in accuracy and high in sensitivity, and the quality control of the ademetionine sample can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of drug quality analysis, and in particular to a method for separating and detecting components in a succinate-adenosylmethionine preparation. Background Art

[0002] Adenosylmethionine disulfonate, the structural formula is shown in Formula 1, and the chemical name is (±)-5'-[( R* )-[( R* )-3-amino-3-carboxypropyl]methylsulfonyl]-5'-deoxyadenosine 1,4-butanedisulfonate, the molecular formula is C 15 H 23 N6O5S + ·C4H9O6S2 - 0.65C4H 10 O6S2, developed by Abbott Laboratories, is available in two dosage forms: freeze-dried powder injection and enteric-coated tablets. Its domestic market name is Sumeta, with a specification of 0.5g (calculated as adenosylmethionine). It is used to treat intrahepatic cholestasis before and due to cirrhosis, and is also used for intrahepatic cholestasis during pregnancy.

[0003]

[0004] Formula 1.

[0005] Adenosylmethionine butanedisulfonate preparations (such as enteric-coated tablets and lyophilized preparations) may contain process impurities and degradation impurities. Process impurities include decarboxylated adenosylmethionine and S-adenosyl-L-methionine glycine isomer 2. Degradation impurities include one or more of adenosine, adenine, dimethylthioadenosine, methylthioadenosine, and S-adenosyl-L-homocysteine. These impurities are all introduced into the finished product by the API or generated by API degradation. No new impurities or degradation impurities are generated during the formulation process.

[0006] Adenosine is both a process impurity and a degradation impurity. It can be produced or degraded during the fermentation process. Adenosine is a bioactive molecule that exerts its biological effects by binding to specific receptors. It participates in various physiological processes in the body, such as myocardial contraction, nerve conduction, and cell signal transduction. Therefore, it needs to be controlled. However, existing research does not have a method for detecting adenosine in adenosylmethionine butanedisulfate preparations. Summary of the Invention

[0007] In view of this, the present invention aims to provide a method for separating and detecting components in a succinate ademethionine preparation. The separation and detection method provided by the present invention can control impurities specified in existing quality standards and accurately detect adenosine in the succinate ademethionine preparation.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for separating components in a succinate-adenosylmethionine preparation, which is characterized by comprising the following steps: Dissolving and diluting a succinate ademethionine preparation to obtain a test solution; the succinate ademethionine preparation includes succinate ademethionine enteric-coated tablets and / or succinate ademethionine lyophilized agent; The test solution is subjected to high performance liquid chromatography separation to obtain component separation results; The components include adenosylmethionine and related impurities; the related impurities include one or more of decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthioadenosine, methylthioadenosine, adenosine, adenine and S-adenosyl-L-homocysteine; The mobile phase for the HPLC separation includes mobile phase A and mobile phase B; The mobile phase A is a mixed solution of a first ammonium formate solution, acetonitrile, and water, wherein the concentration of the first ammonium formate solution is 16-20 mmol / L and the pH value is 2.2-3.1; in the mobile phase A, the volume fraction of the first ammonium formate solution is 5-15%, the volume fraction of acetonitrile is 40-60%, and the volume fraction of water is 30-60%; The mobile phase B is a mixed solution of a second ammonium formate solution, acetonitrile and methyl tert-butyl ether, wherein the concentration of the second ammonium formate solution is 16-20 mmol / L and the pH value is 2.2-3.1; in the mobile phase B, the volume fraction of the second ammonium formate solution is 5-15%, the volume fraction of acetonitrile is 65-88%, and the volume fraction of methyl tert-butyl ether is 1-5%.

[0009] Preferably, when the succinate ademethionine preparation is succinate ademethionine enteric-coated tablets, the solvent used for dissolution is tetrahydrofuran and hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L; The mass ratio of the adenosylmethionine butanedisulfonate preparation to the volume of tetrahydrofuran is 1 g: 10-30 mL; The volume ratio of the succinate ademethionine preparation to the hydrochloric acid solution is preferably 2 g: 10-100 mL, more preferably 2 g: 20-80 mL, and even more preferably 2 g: 50-60 mL; When the succinate ademethionine preparation is a succinate ademethionine lyophilized agent, the solvent used for dissolution is water.

[0010] Preferably, the diluent used for the dilution is a mixture of hydrochloric acid solution and acetonitrile; the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L; and the volume fraction of the hydrochloric acid solution in the diluent is 10-50%.

[0011] Preferably, the concentration of the succinate-1,1-disulfonic acid ademethionine preparation in the test solution is 0.2-1.6 mg / mL.

[0012] Preferably, the reagent for adjusting the pH value of the first ammonium formate solution and the second ammonium formate solution is formic acid.

[0013] Preferably, the elution procedure of the high performance liquid chromatography separation is gradient elution, and the gradient elution procedure includes: 0-2 min, the volume fraction of the mobile phase A is 5-15%; From 2 to 17 minutes, the volume fraction of the mobile phase A increased from 5 to 15% to 50 to 60%; 17-27 min, the volume fraction of the mobile phase A is 50-60%; At 27-28 min, the volume fraction of the mobile phase A was reduced from 50-60% to 5-15%; 28-40 min, the volume fraction of the mobile phase A is 5-15%.

[0014] Preferably, the chromatographic column used for the HPLC separation includes an amino column, the column temperature is 25-35°C, the flow rate of the mobile phase is 0.2-1.0 mL / min, the separation wavelength is 210-290 nm, and the injection volume is 5-100 μL.

[0015] The present invention provides a method for detecting components in a succinate-adenosylmethionine preparation, comprising the following steps: The component separation results obtained by the above separation method are tested to obtain the test results of the components in the succinate ademethionine preparation; The detection includes ultraviolet detector detection or mass spectrometer detection.

[0016] Preferably, the detection wavelength of the ultraviolet detector is 210~290nm; The parameters detected by the mass spectrometer include: Gas temperature: 300℃; Gas rate: 7.0L / min; Spray pressure: 15psi; Sheath gas temperature: 250°C; Sheath gas rate: 11.0 L / min; Capillary voltage: 3500V; Nozzle voltage: 500V.

[0017] Preferably, the test results include qualitative test results and / or quantitative test results; The method for obtaining the quantitative detection result includes area normalization method, internal standard method or external standard method.

[0018] The present invention provides a method for separating components in a succinate ademethionine preparation, comprising the following steps: dissolving and diluting the succinate ademethionine preparation to obtain a test solution; and subjecting the test solution to high-performance liquid chromatography separation to obtain component separation results. The present invention uses ammonium formate solution-water-acetonitrile and ammonium formate solution-acetonitrile-methyl tert-butyl ether as mobile phase systems, and controls the pH value of the ammonium formate solution to 2.2-3.1. It can effectively separate adenosine and adenine, and can also highly separate succinate ademethionine, decarboxylated succinate ademethionine, S-adenosyl-L-methionine glycine isomer 2, adenosine, adenine, and S-adenosyl-L-homocysteine ​​at one time. The method is time-efficient, has high separation efficiency, is simple to operate, and has low separation cost, thereby facilitating improved quality control of succinate ademethionine or related preparations.

[0019] In addition, the existing mobile phase often contains ion-pairing agents to separate impurities, which causes significant damage to the chromatographic column and is not suitable for gradient elution. The mobile phase used in the present invention does not contain ion-pairing agents, is suitable for gradient elution, and can efficiently separate impurities.

[0020] Furthermore, the present invention adopts a specific gradient elution procedure to optimize the separation effect and promote the effective separation of ademetionine and related impurities.

[0021] The present invention provides a method for detecting components in a succinate-adenosylmethionine preparation. The method utilizes an ultraviolet detector or a mass spectrometer to detect the separation results, thereby obtaining qualitative and / or quantitative detection results for the components in the succinate-adenosylmethionine sample. The detection method provided by the present invention is unaffected by excipients, enabling the detection of succinate-adenosylmethionine and related impurities. The method is efficient, convenient, reproducible, highly specific, accurate, and sensitive, enabling quality control of succinate-adenosylmethionine samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the blank solvent HPLC spectrum; Figure 2 HPLC spectrum of blank excipient; Figure 3 HPLC spectrum of the system suitability solution; Figure 4 This is the HPLC spectrum of the enteric-coated tablets of ademethionine butanedisulfate without damage; Figure 5 This is the HPLC spectrum of enteric-coated ademethionine butanedisulfate tablets after high-temperature destruction; Figure 6 This is the HPLC spectrum of enteric-coated ademethionine butanedisulfate tablets after 24 hours of light destruction; Figure 7 This is the HPLC spectrum of succinate enteric-coated tablets after oxidation damage; Figure 8 This is the HPLC spectrum of enteric-coated tablets of adenosylmethionine disulfate after acid destruction; Figure 9 This is the HPLC spectrum of enteric-coated ademethionine butanedisulfate tablets after alkali destruction; Figure 10 The HPLC spectra of the limit of quantification of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and adenosylmethionine are shown; Figure 11 The detection limit HPLC profiles of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, and adenosylmethionine; Figure 12 is the linear curve of dimethylthioadenosine; Figure 13 is the linear curve of adenine; Figure 14 is the linear curve of methylthioadenosine; Figure 15 is the linear curve of adenosine; Figure 16 is the linear curve of decarboxylation of adenosylmethionine; Figure 17 is the linear curve of ademethionine; Figure 18 is the linear curve of S-adenosyl-L-methionine glycine isomer 2; Figure 19 is the linear curve of S-adenosyl-L-homocysteine; Figure 20 is the HPLC spectrum of Example 8; Figure 21 HPLC spectrum of Comparative Example 1; Figure 22 HPLC spectrum of Comparative Example 2; Figure 23 The HPLC spectrum of Comparative Example 3 is shown. DETAILED DESCRIPTION

[0023] The present invention provides a method for separating components in a succinate-adenosylmethionine preparation, comprising the following steps: Dissolving and diluting a succinate ademethionine preparation to obtain a test solution; the succinate ademethionine preparation includes succinate ademethionine enteric-coated tablets and / or succinate ademethionine lyophilized agent; The test solution is subjected to high performance liquid chromatography separation to obtain component separation results.

[0024] In the present invention, the components include adenosylmethionine and related impurities; the related impurities include one or more of decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthioadenosine, methylthioadenosine, adenosine, adenine, and S-adenosyl-L-homocysteine. In the present invention, the structural formulas of adenosine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthioadenosine, methylthioadenosine, adenine, and S-adenosyl-L-homocysteine ​​are as follows:

[0025] The present invention dissolves and dilutes a succinate ademethionine preparation to obtain a test solution. In the present invention, the succinate ademethionine preparation includes succinate ademethionine enteric-coated tablets and / or succinate ademethionine lyophilized agent, and the lyophilized agent is preferably a lyophilized agent for injection.

[0026] In the present invention, when the ademethionine butane disulfonate preparation is an enteric-coated ademethionine butane disulfonate tablet, the solvent used for dissolution is tetrahydrofuran and hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.05 to 0.2 mol / L. In the present invention, the mass ratio of the ademethionine butane disulfonate preparation to the volume of tetrahydrofuran is preferably 1 g:10 to 30 mL, more preferably 1 g:20 mL; in the present invention, the mass ratio of the ademethionine butane disulfonate preparation to the volume of the hydrochloric acid solution is preferably 1 g:10 to 100 mL, more preferably 1 mg:2 to 5 mL.

[0027] In the present invention, when the succinate ademethionine preparation is a succinate ademethionine lyophilized agent, the solvent used for dissolution is preferably water. The present invention has no special requirements for the amount of water used, as long as it can dissolve the succinate ademethionine lyophilized agent.

[0028] In the present invention, the solvent used for dissolution is preferably a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.05 to 0.2 mol / L, more preferably 0.1 mol / L. In the present invention, the mass ratio of the succinate ademethionine preparation to the hydrochloric acid solution is preferably 1 mg: 1 to 10 mL, more preferably 1 mg: 2 to 5 mL.

[0029] In the present invention, the diluent used for dilution is preferably a mixture of hydrochloric acid solution and acetonitrile; the concentration of the hydrochloric acid solution is preferably 0.05~0.2mol / L, more preferably 0.1mol / L; the volume fraction of the hydrochloric acid solution in the diluent is preferably 10~50%, specifically 10%, 20%, 30%, 40% or 50%.

[0030] In the present invention, the dilution method preferably comprises: mixing a solution of the succinate ademethionine preparation with a diluent to obtain a diluent, i.e., a test solution. In the present invention, the concentration of the succinate ademethionine preparation in the test solution is preferably 0.2 to 1.6 mg / mL, specifically 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, or 1.6 mg / mL.

[0031] After obtaining the test solution, the present invention performs high performance liquid chromatography separation on the test solution to obtain a component separation result. In the present invention, the mobile phase for high performance liquid chromatography separation includes mobile phase A and mobile phase B. In the present invention, the mobile phase A is a mixed solution of a first ammonium formate solution, acetonitrile and water, the concentration of the first ammonium formate solution is 16~20mmol / L, more preferably 18mmol / L, and the pH value is 2.2~3.1, specifically 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1; in the present invention, the reagent for adjusting the first ammonium formate solution is preferably formic acid.

[0032] In the present invention, the preparation method of the mobile phase A preferably comprises the following steps: The pH value of the ammonium formate aqueous solution is adjusted to 2.2-3.1 using formic acid, and water and acetonitrile are added to obtain mobile phase A. In the present invention, the concentration of the ammonium formate aqueous solution is preferably 16-20 mmol / L, more preferably 18 mmol / L.

[0033] In the present invention, in the mobile phase A, the volume fraction of the first ammonium formate solution is 5-15%, preferably 8-12%, specifically 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%. In the present invention, in the mobile phase A, the volume fraction of acetonitrile is 40-60%, preferably 45-55%, specifically 40%, 45%, 50%, 55% or 60%. In the present invention, in the mobile phase A, the volume fraction of water is 30-60%, preferably 40-50%, specifically 30%, 40%, 50% or 60%.

[0034] In the present invention, the mobile phase B is a mixed solution of a second ammonium formate solution, acetonitrile and methyl tert-butyl ether, the concentration of the second ammonium formate solution is 16~20mmol / L, more preferably 18mmol / L, and the pH value is 2.2~3.1, specifically 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1; in the present invention, the reagent for adjusting the second ammonium formate solution is preferably formic acid.

[0035] In the present invention, the preparation method of the mobile phase B preferably comprises the following steps: The pH value of the ammonium formate aqueous solution is adjusted to 2.2-3.1 using formic acid, and acetonitrile and methyl tert-butyl ether are added to obtain mobile phase B. In the present invention, the concentration of the ammonium formate aqueous solution is preferably 16-20 mmol / L, more preferably 18 mmol / L.

[0036] In the present invention, the volume fraction of the second ammonium formate solution in the mobile phase B is 5-15%, preferably 8-12%, and specifically can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%. In the present invention, the volume fraction of acetonitrile in the mobile phase B is 65-88%, more preferably 70-80%, and specifically can be 65%, 68%, 70%, 75%, 80%, 85% or 88%. In the present invention, the volume fraction of methyl tert-butyl ether in the mobile phase B is 1-5%, preferably 2-4%, and specifically can be 1%, 2%, 3%, 4% or 5%.

[0037] In the present invention, the elution procedure of the high performance liquid chromatography separation is gradient elution, and the gradient elution procedure preferably includes: 0-2 min, the volume fraction of the mobile phase A is preferably 5-15%, more preferably 10%, specifically 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%; 2-17 min, the volume fraction of the mobile phase A is increased from 5-15% to 50-60%. In a specific embodiment of the present invention, the volume fraction of the mobile phase A can be increased from any one of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15% to any one of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% and 60%; 17-27 min, the volume fraction of the mobile phase A is preferably 50-60%, more preferably 55%, specifically 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%; 27-28 min, the volume fraction of the mobile phase A is reduced from 50-60% to 5-15%. In a specific embodiment of the present invention, the volume fraction of the mobile phase A can be reduced from any one of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% and 60% to any one of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15%; 28~40min, the volume fraction of mobile phase A is 5~15%, more preferably 10%, specifically 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.

[0038] In the present invention, the chromatographic column used for the HPLC separation includes an amino column. As a specific embodiment of the present invention, the chromatographic column can be aphera NH2polymer. In the present invention, the column length is preferably 50-250 mm, the inner diameter is preferably 2.1-4.6 mm, and the particle size is preferably 3.5-5 μm. As a specific embodiment of the present invention, the column size is 250 mm × 4.6 mm, and the particle size is 5 μm.

[0039] In the present invention, the column temperature of the HPLC separation is preferably 25-35°C, specifically 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C.

[0040] In the present invention, the flow rate of the mobile phase for HPLC separation is preferably 0.2-1.0 mL / min, specifically 0.4 mL / min, 0.5 mL / min or 0.6 mL / min.

[0041] In the present invention, the separation wavelength of the HPLC separation is preferably 210-290 nm.

[0042] In the present invention, the injection volume of the HPLC separation is preferably 5 to 100 μL, more preferably 20 to 80 μL, specifically 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, 85 μL, 90 μL, 95 μL or 100 μL.

[0043] In the present invention, the injection temperature is preferably 2-12°C, more preferably 5°C.

[0044] The present invention provides a method for detecting components in a succinate-adenosylmethionine preparation, comprising the following steps: The component separation results obtained by the above separation method are tested to obtain the test results of the components in the succinate ademethionine preparation.

[0045] In the present invention, the detection includes ultraviolet detector detection or mass spectrometer detection.

[0046] In the present invention, the detection wavelength of the ultraviolet detector is preferably 210~290nm, specifically 210nm, 215nm, 220nm, 225nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm or 290nm.

[0047] In the present invention, the parameters detected by the mass spectrometer preferably include: Gas temperature: 300℃; Gas rate: 7.0L / min; Spray pressure: 15psi; Sheath gas temperature: 250°C; Sheath gas rate: 11.0 L / min; Capillary voltage: 3500V; Nozzle voltage: 500V.

[0048] In the present invention, the test results preferably include qualitative test results and / or quantitative test results; and the method for obtaining the quantitative test results preferably includes area normalization, internal standard method, or external standard method. The present invention is not particularly limited to the area normalization, internal standard method, or external standard method, and quantification can be performed using the area normalization, internal standard method, or external standard method well known to those skilled in the art.

[0049] The following examples will describe in detail the separation and detection methods of the components in the succinate ademethionine preparation provided by the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0050] In the embodiments of the present invention, the manufacturer of enteric-coated ademethionine butanedisulfonate tablets is ABBVIE SRL, and the specification is 0.5 g / tablet, 10 tablets / box, calculated as ademethionine; the manufacturer of lyophilized ademethionine butanedisulfonate for injection is ABBVIES.RL, and the specification is 0.5 g / tube, 10 tubes / box, calculated as ademethionine.

[0051] In an embodiment of the present invention, the conditions for HPLC chromatographic separation and detection (optimal conditions) are as follows: the high performance liquid chromatograph is Agilent 1260; the chromatographic column is aphera NH2polymer, 250 mm×4.6 mm, 5 μm; the column temperature is 30°C; the mobile phase is: mobile phase A is ammonium formate solution-acetonitrile-water, with a volume ratio of 10:50:40; the mobile phase B is ammonium formate solution-acetonitrile-methyl tert-butyl ether, with a volume ratio of 10:88:2; the preparation method of the ammonium formate solution in mobile phase A and mobile phase B is: take 1.18 g of ammonium formate, add 1000 mL of water to dissolve it, and add formic acid to adjust the pH value to 2.8±0.2.

[0052] UV detector detection, detection wavelength is 260nm; mobile phase flow rate is 0.5mL / min; injection volume is 5μL, injection temperature is 5℃; gradient elution mode is adopted, and the gradient elution program is shown in Table 1.

[0053] Table 1 Gradient elution program

[0054] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0055] Example 1 Methodology Validation-System Suitability 1. Solution preparation method (1) Blank solution / diluent: 0.1 mol / L hydrochloric acid aqueous solution (9 mL of hydrochloric acid is added to 1000 mL of water and mixed): acetonitrile (20:80).

[0056] (2) Adenosylmethionine reference stock solution: Accurately weigh 37.40 mg of adenosylmethionine butanedisulfonate reference solution into a 50 mL volumetric flask, add 10 mL of water to dissolve, and then dilute to the mark with diluent, shake well, and the solution is ready. (3) Impurity stock solution: Dimethylthioadenosine stock solution: accurately weigh 27.13 mg of dimethylthioadenosine into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, then accurately measure 5 mL into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and use this as the dimethylthioadenosine stock solution; Methylthioadenosine stock solution: Take 19.66 mg of methylthioadenosine, accurately weigh it, place it in a 50 mL volumetric flask, add appropriate amount of diluent to dissolve it and dilute it to the scale, shake well; Adenosine stock solution: accurately weigh 20.02 mg of adenosine and place it in a 50 mL volumetric flask. Add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, then accurately measure 5 mL and place it in a 50 mL volumetric flask. Add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and this is the adenosine stock solution. Adenine stock solution: accurately weigh 24.49 mg of adenine into a 25 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, then accurately measure 5 mL into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and use this as the adenine stock solution. Decarboxylated adenomycin stock solution: Take 26.04 mg of decarboxylated adenomycin, accurately weigh it, place it in a 100 mL volumetric flask, add appropriate amount of diluent to dissolve it and dilute it to the scale, shake well; S-adenosyl-L-methionine glycine isomer 2 stock solution: Take 25.46 mg of S-adenosyl-L-methionine glycine isomer, accurately weigh it, place it in a 200 mL volumetric flask, add appropriate amount of diluent to dissolve it and dilute it to the scale, shake well; S-adenosyl-L-homocysteine ​​stock solution: accurately weigh 24.43 mg of S-adenosyl-L-homocysteine ​​into a 25 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, then accurately measure 5 mL into a 50 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, shake well, and prepare the S-adenosyl-L-homocysteine ​​stock solution; (4) Component location solution: Adenosylmethionine reference stock solution, dimethylthioadenosine stock solution, methylthioadenosine stock solution, adenine stock solution, adenosine stock solution, decarboxylated adenosylmethionine stock solution, S-adenosyl-L-methionine glycine isomer 2 stock solution, and S-adenosyl-L-homocysteine ​​stock solution were diluted 50 times with solvent respectively.

[0057] (5) System suitability solution: Accurately weigh 38.43 mg of adenosyl methionine disulfonate reference substance and place it in a 50 mL volumetric flask. After dissolving with 10 mL of water, accurately measure 1 mL of dimethylthioadenosine stock solution, 1 mL of methylthioadenosine stock solution, 1 mL of adenine stock solution, 1 mL of adenosine stock solution, 1 mL of decarboxylated adenosyl methionine stock solution, 1 mL of S-adenosyl-L-methionine glycine isomer 2 stock solution, and 1 mL of S-adenosyl-L-homocysteine ​​stock solution. Place them in a volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the mark, and shake well. (6) Blank excipient solution: Weigh 233.66 mg of blank excipient (equivalent to 0.5 g of adenosylmethionine) into a 50 mL volumetric flask, add 10 mL of tetrahydrofuran and shake for 40 minutes, then add 25 mL of 0.1 mol / L hydrochloric acid aqueous solution and shake for 40 minutes, then dilute to the mark with 0.1 mol / L hydrochloric acid aqueous solution, shake well, centrifuge at 3000 rpm for 5 minutes, accurately measure 2 mL of supernatant into a 50 mL volumetric flask, dilute to the mark with diluent, shake well, and filter.

[0058] 2 HPLC separation The blank solvent (diluent), blank excipient solution and system suitability solution were injected and separated by HPLC under the optimal HPLC detection conditions. Figure 1 is the blank solvent HPLC spectrum, Figure 2 is the HPLC spectrum of blank excipient, Figure 3 is the HPLC spectrum of the system suitability solution, Figures 1 to 3 It can be seen that the excipients in the enteric-coated ademethionine butanedisulfonate tablets do not interfere with the impurity detection, and each impurity can be completely separated from the main peak of ademethionine.

[0059] The results of HPLC testing of system suitability solutions under different column temperatures, mobile phase A pH values, and mobile phase flow rates are shown in Tables 2 and 3. In Tables 2 and 3, columns 1# and 2# represent different batches of the same column model.

[0060] Table 2 Results of the durability test under varying chromatographic conditions

[0061] Table 3 Results of the durability test under varying chromatographic conditions

[0062] Example 2 Methodology Validation-Stability Undamaged solution: Take 49.12 mg of fine powder of this product (adenylmionine butanedisulfonate enteric-coated tablets), accurately weigh it, place it in a 50 mL volumetric flask, add 10 mL of water and shake for 30 minutes to dissolve it, then dilute it to the scale with diluent, shake well, and filter it.

[0063] Acid destruction solution: Take 49.91 mg of fine powder of this product, accurately weigh it, place it in a 50 mL volumetric flask, add 1 mL of 0.1 mol / L hydrochloric acid solution, let it stand for 24 hours, add 1 mL of 0.1 mol / L sodium hydroxide solution to neutralize it, add 10 mL of water and shake for 30 minutes to dissolve it, then dilute it to the scale with diluent, shake well, and filter it.

[0064] Alkali destruction solution: Take 47.53 mg of fine powder of this product, accurately weigh it, place it in a 50 mL volumetric flask, add 1 mL of 0.1 mol / L sodium hydroxide solution, let it stand for 2.5 hours, add 1 mL of 0.1 mol / L hydrochloric acid solution to neutralize it, add 10 mL of water and shake for 30 minutes to dissolve it, then dilute it to the scale with diluent, shake it well, and filter it.

[0065] Oxidative destruction solution: Take 47.36 mg of fine powder of this product, accurately weigh it, place it in a 50 mL volumetric flask, add 1 mL of 10% hydrogen peroxide, leave it for 24 hours, add 10 mL of water and shake for 30 minutes to dissolve it, then dilute it to the scale line with diluent, shake well, and filter it.

[0066] High-temperature solid destruction solution: Take 50.43 mg of sample fine powder that has been placed at high temperature of 60℃ for 5 hours, accurately weigh it, put it into a 50mL volumetric flask, add 10mL of water and shake for 30 minutes to dissolve it, then dilute it to the scale line with diluent, shake well, and filter it.

[0067] Light irradiation solid destruction solution: Take 48.38 mg of sample fine powder that has been exposed to light (4500±500lx) for 24 hours, accurately weigh it, place it in a 50mL volumetric flask, add 10mL of water and shake for 30 minutes to dissolve it, then dilute it to the scale line with diluent, shake well, and filter it.

[0068] The undamaged solution and each damaged solution were sampled and separated by HPLC. The results are shown in Figures 4 to 9 .

[0069] Figure 4 The HPLC spectrum of the undamaged enteric-coated ademethionine tablets is Figure 4 It can be seen that the degradation impurities methylthioadenosine, adenine, S-adenosyl-L-homocysteine ​​(abbreviated as homocysteine) and process impurity S-adenosyl-L-methionine glycine isomer 2 (abbreviated as isomer 2) can be detected in the undamaged solution.

[0070] Figure 5 This is the HPLC spectrum of ademetionine enteric-coated tablets after high temperature destruction. Figure 5 It can be seen that under high temperature conditions of 60℃, ademethionine enteric-coated tablets are unstable and degrade into dimethylthioadenosine.

[0071] Figure 6 This is the HPLC spectrum of ademetionine enteric-coated tablets after 24 hours of light destruction. Figure 6 It can be seen that ademethionine enteric-coated tablets are relatively stable under 24h of light irradiation and no new impurities are degraded.

[0072] Figure 7 This is the HPLC spectrum of ademetionine enteric-coated tablets after oxidation damage. Figure 7 It can be seen that ademethionine enteric-coated tablets can be degraded into unknown impurity 1 (retention time 14.867 min) and unknown impurity 2 (retention time 15.019 min) under oxidative conditions, and are unstable under oxidative conditions.

[0073] Figure 8 This is the HPLC spectrum of enteric-coated ademethionine tablets after acid destruction. Figure 8 It can be seen that ademethionine enteric-coated tablets are relatively stable under acidic conditions and no new impurities are degraded.

[0074] Figure 9 The HPLC spectrum of alkali destruction of enteric-coated ademethionine tablets is Figure 9 It can be seen that ademethionine enteric-coated tablets are unstable under alkaline conditions and degrade into methylthioadenosine and adenosine.

[0075] Example 3 Methodology Validation - Limit of Quantitation and Limit of Detection Preparation of quantitation limit solution: Quantitation limit stock solution: Accurately measure 0.5 mL of the ademethionine reference substance stock solution prepared in Example 1, 5 mL of dimethylthioadenosine stock solution, 0.5 mL of methylthioadenosine stock solution, 2 mL of adenine stock solution, 5 mL of adenosine stock solution, 1 mL of decarboxylated ademethionine stock solution, 2 mL of S-adenosyl-L-methionine glycine isomer 2 stock solution, and 2 mL of S-adenosyl-L-homocysteine ​​stock solution, and place them in the same 100 mL volumetric flask. Add diluent to dissolve and dilute to the mark, and shake well.

[0076] Quantitation limit solution: Accurately measure 5 mL of quantitation limit stock solution, place in a 50 mL volumetric flask, add diluent to dilute to the mark, and shake well. (Equivalent to 0.05% of the limit concentration) Detection limit solution: Accurately measure 3 mL of the quantification limit solution, place it in a 10 mL volumetric flask, add diluent to dilute to the mark, and shake well. (Equivalent to 0.015% of the limit concentration) The above quantitative limit solution was continuously injected into HPLC for separation and detection 6 times, and the detection limit solution was continuously injected into HPLC for detection 3 times. The ratio of peak height to noise (signal-to-noise ratio) of each peak was calculated and the chromatogram was recorded. The results are shown in Tables 4 to 5 and Figure 10-11 , Figure 10 The HPLC spectrum of the limit of quantification of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine ​​isomer. Figure 11 The detection limit HPLC spectrum of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine.

[0077] Table 4 Quantitation limit determination results

[0078]

[0079] From Table 4 and Figure 10 It can be seen that under the HPLC chromatographic conditions of the present invention, the quantification limits of ademethionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated ademethionine, ademethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine ​​meet the requirement of S / N greater than 10, and the quantification limit of each component is approximately equivalent to 0.05 wt% of the ademethionine concentration in the test solution.

[0080] Table 5 Detection limit determination results

[0081] From Table 5 and Figure 11 It can be seen that under the HPLC chromatographic conditions of the present invention, the detection limits of ademethionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated ademethionine, ademethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine ​​meet the requirement of S / N greater than 3, and the detection limit of each component is approximately equivalent to 0.01 wt% of the ademethionine concentration in the test solution.

[0082] Example 4 Methodology Validation-Linear Relationship Preparation of linear solution Mixed stock solution: Accurately measure 2 mL of the ademethionine reference substance stock solution, 20 mL of dimethylthioadenosine stock solution, 20 mL of methylthioadenosine stock solution, 20 mL of adenine stock solution, 20 mL of adenosine stock solution, 30 mL of decarboxylated ademethionine stock solution, 30 mL of S-adenosyl-L-methionine glycine isomer 2 stock solution, and 20 mL of S-adenosyl-L-homocysteine ​​stock solution in Example 1, place them in the same 200 mL volumetric flask, add an appropriate amount of diluent to dissolve and dilute to the scale, and shake well; (limit 500%) L1 (equivalent to the quantitative limit concentration): Take the quantitative limit solution in Example 3.

[0083] L2 (equivalent to 30% of the limit concentration): Accurately measure 3 mL of the mixed stock solution, place it in a 50 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0084] L3 (equivalent to 50% of the limit concentration): Accurately measure 5 mL of the mixed stock solution, place it in a 50 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0085] L4 (equivalent to 100% of the limit concentration): Accurately measure 5 mL of the mixed stock solution, place it in a 25 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0086] L5 (equivalent to 200% of the limit concentration): Accurately measure 10 mL of the mixed stock solution, place it in a 25 mL volumetric flask, add diluent to dilute to the scale, and shake well.

[0087] Take the above linear solutions L1~L5 and inject them once, separate and detect by HPLC. The experimental results are shown in Figures 12 to 19 and Tables 6 to 13, among which, Figures 12 to 19 The linear curves are dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine, respectively.

[0088] According to the slope of the linear equation between the main component and each impurity, the correction factor f (f=k 主成分 / k 斜率 ), the results are shown in Table 6~Table 13, where k 主成分 The slope of the linear regression equation of the principal component concentration (X)-response value (Y), k 杂质 It is the slope of the linear regression equation of impurity concentration (X)-response value (Y).

[0089] Table 6 Linear determination results of dimethylthioadenosine

[0090] Table 7 Linear determination results of methylthioadenosine

[0091] Table 8 Adenine linearity determination results

[0092] Table 9 Adenosine linearity determination results

[0093] Table 10 Decarboxylation of adenosylmethionine linear determination results

[0094] Table 11 Adenosylmethionine linear determination results

[0095] Table 12 Linear determination results of S-adenosyl-L-methionine glycine isomer 2

[0096] Table 13 S-adenosyl-L-homocysteine ​​linear determination results

[0097] Depend on Figures 12 to 19 As shown in Tables 6 to 13, under the HPLC chromatographic conditions of the present invention, the concentrations of adenosylmethionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, and S-adenosyl-L-homocysteine ​​have a good linear relationship with the peak area.

[0098] Example 5 Methodological Validation-Accuracy Mixed impurity reference solution: Accurately measure 5 mL of the mixed stock solution in Example 4, place it in a 25 mL volumetric flask, add diluent to the mark, and shake well. (Equivalent to 100% of the limit concentration) Preparation of accuracy solution: A1 (equivalent to the quantification limit concentration): Take 40 mg of the test sample fine powder and place it in a 50 mL volumetric flask. Add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution and 5 mL of the quantification limit stock solution in Example 3. Shake for 30 minutes to dissolve. Dilute to the mark with acetonitrile, shake well, filter, and prepare three parallel portions.

[0099] A2 (equivalent to 50% of the limiting concentration): Take 40 mg of the test sample powder and place it in a 50 mL volumetric flask. Add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution and 5 mL of the mixed stock solution in Example 4. Shake for 30 minutes to dissolve. Dilute to the mark with acetonitrile, shake well, filter, and prepare three parallel portions.

[0100] A3 (equivalent to 100% of the limiting concentration): Take 40 mg of the test sample powder and place it in a 50 mL volumetric flask. Add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution and 10 mL of the mixed stock solution prepared in Example 4. Shake for 30 minutes to dissolve. Dilute to the mark with acetonitrile, shake well, filter, and prepare three replicates.

[0101] A4 (equivalent to 200% of the limiting concentration): Take 40 mg of the test sample powder and place it in a 50 mL volumetric flask. Add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution and 20 mL of the mixed stock solution prepared in Example 4. Shake for 30 minutes to dissolve. Dilute to the mark with acetonitrile, shake well, filter, and prepare three replicates.

[0102] Each accuracy solution was injected, separated and detected by HPLC, and the recovery rate was calculated according to the external standard method. The results are shown in Table 14.

[0103] Table 14 Spike recovery results

[0104] As shown in Table 14, under the HPLC chromatographic conditions of the present invention, the accuracy of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine ​​was good.

[0105] Example 6 Methodology Validation-Precision Spiked test solution: Take 10 tablets of the test sample, place them in a 500mL measuring flask, add 100mL of tetrahydrofuran and shake for 40 minutes, then add 250mL of 0.1mol / L hydrochloric acid aqueous solution and shake for 40 minutes to completely disintegrate the tablets, then dilute to the mark with 0.1mol / L hydrochloric acid aqueous solution, shake well, centrifuge at 3000 rpm for 5min, accurately measure 2mL of the supernatant to a 50mL measuring flask, add 5mL of the mixed stock solution in Example 4, dilute to the mark with diluent, shake well, and filter to obtain the result. Prepare 6 portions in parallel (based on adenosylmethionine, concentration: 0.4mg / mL) and inject each solution into HPLC for separation and detection. Calculate the recovery by the external standard method. The results are shown in Table 15.

[0106] Table 15 Precision results (wt%)

[0107] As shown in Table 15, the precision of the spiked test solution for the detection of related substances in enteric-coated ademethionine butanedisulfate tablets was good.

[0108] Example 7 Methodology Validation-Solution Stability Reference substance mixed solution: the reference substance mixed solution prepared in Example 4.

[0109] Spiked test solution: the spiked test solution prepared in Example 6.

[0110] The reference mixed solution and the spiked test solution were placed at 5°C for 0 h, 10 h, 20 h, and 30 h, respectively, and then injected and separated and detected by HPLC. The results are shown in Tables 16 and 17.

[0111] Table 16 Stability of reference substance mixed solution

[0112] Table 17 Stability of spiked test solution

[0113] As shown in Tables 16 and 17, under the HPLC chromatographic conditions provided by the present invention, the reference solution and the test solution of adenosylmethionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine ​​are stable at 5°C for 30 hours.

[0114] Example 8 Preparation of test sample: Take 4 bottles of lyophilized adenosine butanedisulfonate for injection, transfer all the contents to a 250mL volumetric flask with diluent, dilute to the scale with water, shake well, and use as the test sample stock solution. Accurately measure 5mL and place it in a 100mL volumetric flask, dilute to the scale with diluent, shake well to obtain the test sample solution (concentration is 0.4mg / mL), and accurately measure 5μL and inject into the liquid chromatograph.

[0115] HPLC chromatographic separation and detection conditions: high performance liquid chromatograph is Agilent 1260; chromatographic column is apheraNH2polymer, 250mm×4.6mm, 5μm; column temperature is 30℃; mobile phase: mobile phase A is ammonium formate solution-acetonitrile-water, with a volume ratio of 10:50:40; mobile phase B is ammonium formate solution-acetonitrile-methyl tert-butyl ether, with a volume ratio of 10:88:2; the preparation method of ammonium formate solution in mobile phase A and mobile phase B is as follows: take 1.18g of ammonium formate, add 1000mL of water to dissolve it, and add formic acid to adjust the pH value to 2.8±0.2.

[0116] UV detector detection, detection wavelength is 260nm; mobile phase flow rate is 0.5mL / min; injection volume is 5μL, injection temperature is 5℃; gradient elution mode is adopted, and the gradient elution program is shown in Table 18. The obtained chromatogram is shown in Figure 20 shown.

[0117] Table 18 Gradient elution program

[0118] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0119] The results show that the detection method provided by the present invention can successfully separate the impurities of adenosylmethionine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, adenosine, adenine and S-adenosyl-L-homocysteine ​​in adenosylmethionine butanedisulfonate for injection.

[0120] Comparative Example 1 HPLC chromatographic separation and detection conditions: high performance liquid chromatograph is Agilent 1260; chromatographic column is apheraNH2polymer, 250mm×4.6mm, 5μm; column temperature is 30℃; mobile phase: mobile phase A is ammonium formate solution-acetonitrile-water, with a volume ratio of 10:50:40; mobile phase B is ammonium formate solution-acetonitrile, with a volume ratio of 10:90; the preparation method of ammonium formate solution in mobile phase A and mobile phase B is as follows: take 1.18g of ammonium formate, add 1000mL of water to dissolve it, and add formic acid to adjust the pH value to 3.2.

[0121] UV detector detection, detection wavelength is 260nm; mobile phase flow rate is 0.5mL / min; injection volume is 5μL, injection temperature is 5℃; gradient elution mode is adopted, and the gradient elution program is shown in Table 19. The obtained chromatogram is shown in Figure 21 shown.

[0122] Table 19 Gradient elution program

[0123] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0124] The results showed that the analytical method used in Comparative Example 1 could not separate adenine and adenosine, and the peak shape of decarboxylated adenosylmethionine was poor, showing a split peak.

[0125] Comparative Example 2 HPLC chromatographic separation and detection conditions: high performance liquid chromatograph is Agilent 1260; chromatographic column is apheraNH2polymer, 250mm×4.6mm, 5μm; column temperature is 30℃; mobile phase: mobile phase A is ammonium formate solution-acetonitrile-water, with a volume ratio of 10:50:40; mobile phase B is ammonium formate solution-acetonitrile-methyl tert-butyl ether, with a volume ratio of 10:88:2; the preparation method of ammonium formate solution in mobile phase A and mobile phase B is as follows: take 1.18g of ammonium formate, add 1000mL of water to dissolve it, and add formic acid to adjust the pH value to 3.2.

[0126] UV detector detection, detection wavelength is 260nm; mobile phase flow rate is 0.5mL / min; injection volume is 5μL, injection temperature is 5℃; gradient elution mode is adopted, and the gradient elution program is shown in Table 20. The obtained chromatogram is shown in Figure 22 shown.

[0127] Table 20 Gradient elution program

[0128] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0129] The results showed that the analytical method used in Comparative Example 2 could not separate adenine and adenosine, and the peak shape of decarboxylated adenosylmethionine was poor, showing a split peak.

[0130] Comparative Example 3 HPLC chromatographic separation and detection conditions: high performance liquid chromatograph is Agilent 1260; chromatographic column is apheraNH2polymer, 250mm×4.6mm, 5μm; column temperature is 30℃; mobile phase: mobile phase A is ammonium formate solution-acetonitrile-water, with a volume ratio of 10:50:40; mobile phase B is ammonium formate solution-acetonitrile, with a volume ratio of 10:90; the preparation method of ammonium formate solution in mobile phase A and mobile phase B is as follows: take 1.18g of ammonium formate, add 1000mL of water to dissolve it, and add formic acid to adjust the pH value to 2.8±0.2.

[0131] The detection wavelength was 260 nm using a UV detector; the mobile phase flow rate was 0.5 mL / min; the injection volume was 5 μL, and the injection temperature was 5°C; a gradient elution method was used, and the gradient elution program is shown in Table 21. The chromatogram obtained is shown in Figure 23 shown.

[0132] Table 21 Gradient elution program

[0133] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0134] The results showed that the analytical method used in Comparative Example 3 could not separate adenine and adenosine, but the peak shape of decarboxylated adenosylmethionine was improved to form a symmetrical peak.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for separating components in a succinate-1,2-disulfonic acid ademethionine preparation, characterized in that: The following steps are involved: Dissolving and diluting a succinate ademethionine preparation to obtain a test solution; the succinate ademethionine preparation includes succinate ademethionine enteric-coated tablets and / or succinate ademethionine lyophilized agent; The test solution is subjected to high performance liquid chromatography separation to obtain component separation results; The components include adenosylmethionine and related impurities; the related impurities include one or more of decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthioadenosine, methylthioadenosine, adenosine, adenine and S-adenosyl-L-homocysteine; The mobile phase for the HPLC separation includes mobile phase A and mobile phase B; The mobile phase A is a mixed solution of a first ammonium formate solution, acetonitrile, and water, wherein the concentration of the first ammonium formate solution is 16-20 mmol / L and the pH value is 2.2-3.1; in the mobile phase A, the volume fraction of the first ammonium formate solution is 5-15%, the volume fraction of acetonitrile is 40-60%, and the volume fraction of water is 30-60%; The mobile phase B is a mixed solution of a second ammonium formate solution, acetonitrile and methyl tert-butyl ether, wherein the concentration of the second ammonium formate solution is 16-20 mmol / L and the pH value is 2.2-3.1; in the mobile phase B, the volume fraction of the second ammonium formate solution is 5-15%, the volume fraction of acetonitrile is 65-88%, and the volume fraction of methyl tert-butyl ether is 1-5%.

2. The separation method according to claim 1, wherein When the succinate ademethionine preparation is a succinate ademethionine enteric-coated tablet, the solvent used for dissolution is tetrahydrofuran and hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L; The mass ratio of the adenosylmethionine butanedisulfonate preparation to the volume of tetrahydrofuran is 1 g: 10-30 mL; The volume ratio of the succinate-adenosylmethionine preparation to the hydrochloric acid solution is 2 g: 10-100 mL; When the succinate ademethionine preparation is a succinate ademethionine lyophilized agent, the solvent used for dissolution is water.

3. The separation method according to claim 1 or 2, characterized in that The diluent used for the dilution is a mixture of hydrochloric acid solution and acetonitrile; the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L; and the volume fraction of the hydrochloric acid solution in the diluent is 10-50%.

4. The separation method according to claim 1, wherein The concentration of the succinate-1,1,2-disulfonic acid ademethionine preparation in the test solution is 0.2-1.6 mg / mL.

5. The separation method according to claim 1, characterized in that The reagent for adjusting the pH value of the first ammonium formate solution and the second ammonium formate solution is formic acid.

6. The separation method according to claim 1, characterized in that The elution procedure of the high performance liquid chromatography separation is gradient elution, and the procedure of the gradient elution includes: 0-2 min, the volume fraction of the mobile phase A is 5-15%; From 2 to 17 minutes, the volume fraction of the mobile phase A increased from 5 to 15% to 50 to 60%; 17-27 min, the volume fraction of the mobile phase A is 50-60%; At 27-28 min, the volume fraction of the mobile phase A was reduced from 50-60% to 5-15%; 28-40 min, the volume fraction of the mobile phase A is 5-15%.

7. The separation method according to claim 1 or 6, characterized in that The chromatographic columns used for HPLC separation include amino columns, the column temperature is 25-35°C, the flow rate of the mobile phase is 0.2-1.0 mL / min, the separation wavelength is 210-290 nm, and the injection volume is 5-100 μL.

8. A method for detecting components in a succinate-1,2-disulfonic acid ademethionine preparation, characterized in that: The following steps are involved: Detecting the component separation results obtained by the separation method according to any one of claims 1 to 7 to obtain detection results of the components in the succinate ademethionine preparation; The detection includes ultraviolet detector detection or mass spectrometer detection.

9. The detection method according to claim 8, characterized in that The detection wavelength of the ultraviolet detector is 210~290nm; The parameters detected by the mass spectrometer include: Gas temperature: 300℃; Gas rate: 7.0L / min; Spray pressure: 15psi; Sheath gas temperature: 250°C; Sheath gas rate: 11.0 L / min; Capillary voltage: 3500V; Nozzle voltage: 500V.

10. The detection method according to claim 8, characterized in that The test results include qualitative test results and / or quantitative test results; The method for obtaining the quantitative detection result includes area normalization method, internal standard method or external standard method.

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