A method for the isolation and detection of components in a preparation of ademetionine butanedisulfate

Abstract

The application provides a method for separating and detecting ingredients in a butanedisulfonic acid ademetionine enteric-coated tablet, and belongs to the technical field of drug quality analysis. The application uses ammonium formate solution-water-acetonitrile and ammonium formate solution-acetonitrile-methyl tert-butyl ether as a mobile phase system, and controls the pH value of the ammonium formate solution to be 2.2-3.1, so that adenosine and adenine can be effectively separated, and ademetionine, decarboxylated ademetionine, S-adenosyl-L-methionine glycine isomer 2, adenosine, adenine and S-adenosyl-L-homocysteine can be highly separated at one time, the method has the advantages of short time consumption, high separation efficiency, simple operation, low separation cost, and is favorable for improving the quality control of butanedisulfonic acid ademetionine or related preparations. In the application, the detection of ademetionine and related impurities is not affected by excipients, and has the advantages of high efficiency, convenience, good reproducibility, strong specificity, high accuracy and high sensitivity, and can realize the quality control of ademetionine samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical quality analysis, in particular to a method for separating and detecting components in ademetionine bistrisulfate preparation. BACKGROUND

[0002] Ademetionine bistrisulfate has a structural formula as shown in formula 1, and its chemical name is (±)-5'-[( R* )-[( R* )-3-amino-3-carboxypropyl]methylsulfonyl]-5'-deoxyadenosine 1,4-butanediol disulfate, and its molecular formula is C 15 H 23 N6O5S + ·C4H9O6S2 - ·0.65C4H 10 O6S2, which is developed by Abbott Laboratories, and its marketed dosage forms include lyophilized powder and enteric-coated tablets. Its domestic trade name is Simethy, and its specification is 0.5 g (calculated based on ademetionine). It is used for treating intrahepatic cholestasis caused by pre-cirrhosis and cirrhosis, and also for treating intrahepatic cholestasis during pregnancy.

[0003]

[0004] Formula 1.

[0005] There may be process impurities and degradation impurities in ademetionine bistrisulfate preparation (such as enteric-coated tablets and lyophilized agents). The process impurities include decarboxylated ademetionine and S-adenosyl-L-methionine glycine isomer 2, and the degradation impurities include one or more of adenosine, adenine, dimethylthioadenosine, methylthioadenosine and S-adenosyl-L-homocysteine. The above impurities are introduced into the finished product from the raw material or generated by degradation of the raw material, and no new impurities and no new degradation impurities are generated in the preparation process.

[0006] Among them, adenosine is both a process impurity and a degradation impurity, which can be produced or degraded in the biological fermentation process. Adenosine is a biologically active molecule, which exerts its biological effects by binding to specific receptors, and participates in various physiological processes in the body, such as myocardial contraction, nerve conduction and cell signal transduction, etc. Therefore, it needs to be controlled. However, there is no detection method for adenosine in ademetionine bistrisulfate preparation in the existing research. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a method for separating and detecting components in ademetionine bistrisulfate preparation. The separation and detection method provided by the present application can control the impurities specified in the existing quality standard, and can also accurately detect adenosine in ademetionine bistrisulfate preparation.

[0008] To achieve the above-mentioned purposes, the present application provides the following technical solutions.

[0009] The present application provides a separation method of components in a preparation of ademetionine bisulfate, characterized by comprising the following steps:

[0010] The preparation of ademetionine bisulfate is dissolved and diluted to obtain a test solution; the preparation of ademetionine bisulfate includes ademetionine bisulfate enteric-coated tablets and / or ademetionine bisulfate lyophilizate;

[0011] The test solution is subjected to high performance liquid chromatography separation to obtain component separation results;

[0012] The components include ademetionine and related impurities; the related impurities include one or more of decarboxylated ademetionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthetin, methylthetin, adenosine, adenine and S-adenosyl-L-homocysteine;

[0013] The mobile phase of the high performance liquid chromatography separation includes mobile phase A and mobile phase B;

[0014] 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 16-20 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%;

[0015] 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 16-20 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%.

[0016] Preferably, when the preparation of ademetionine bisulfate is ademetionine bisulfate enteric-coated tablets, the solvent used for dissolution is tetrahydrofuran and a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L;

[0017] The mass-to-volume ratio of the preparation of ademetionine bisulfate to tetrahydrofuran is 1 g: 10-30 mL;

[0018] The mass-to-volume ratio of the preparation of ademetionine bisulfate to the hydrochloric acid solution is preferably 2 g: 10-100 mL, more preferably 2 g: 20-80 mL, and further preferably 2 g: 50-60 mL;

[0019] When the ademetionine preparation is a lyophilized ademetionine preparation, the solvent used for the dissolving is water.

[0020] Preferably, the diluent used for the diluting is a mixture of hydrochloric acid solution and acetonitrile; the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L; the volume fraction of the hydrochloric acid solution in the diluent is 10-50%.

[0021] Preferably, the concentration of the ademetionine preparation in the test sample solution is 0.2-1.6 mg / mL.

[0022] Preferably, the reagent used for adjusting the pH values of the first ammonium formate solution and the second ammonium formate solution is formic acid.

[0023] Preferably, the elution procedure of the high performance liquid chromatography separation is gradient elution, and the procedure of the gradient elution comprises:

[0024] 0-2 min, the volume fraction of the mobile phase A is 5-15%;

[0025] 2-17 min, the volume fraction of the mobile phase A is increased from 5-15% to 50-60%;

[0026] 17-27 min, the volume fraction of the mobile phase A is 50-60%;

[0027] 27-28 min, the volume fraction of the mobile phase A is decreased from 50-60% to 5-15%;

[0028] 28-40 min, the volume fraction of the mobile phase A is 5-15%.

[0029] Preferably, the chromatographic column used for the high performance liquid chromatography separation comprises 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.

[0030] The present application provides a method for detecting the components in an ademetionine preparation, comprising the following steps:

[0031] The components separated by the separation method are detected to obtain the detection results of the components in the ademetionine preparation.

[0032] The detection comprises ultraviolet detector detection or mass spectrometer detection.

[0033] Preferably, the detection wavelength of the ultraviolet detector is 210-290 nm.

[0034] The parameters of the mass spectrometer detection comprise:

[0035] Gas temperature: 300℃;

[0036] Gas flow rate: 7.0 L / min;

[0037] Spray pressure: 15 psi;

[0038] Sheath gas temperature: 250℃;

[0039] Sheath gas flow rate: 11.0 L / min;

[0040] Capillary voltage: 3500 V;

[0041] Nebulizer voltage: 500 V.

[0042] Preferably, the detection result comprises a qualitative detection result and / or a quantitative detection result.

[0043] The method for obtaining the quantitative detection result comprises an area normalization method, an internal standard method or an external standard method.

[0044] The application provides a method for separating components in a preparation of ademetionine bisulfate, comprising the following steps: dissolving and diluting the preparation of ademetionine bisulfate to obtain a test sample solution; and performing high performance liquid chromatography separation on the test sample solution to obtain a component separation result. The application uses ammonium formate solution-water-acetonitrile and ammonium formate solution-acetonitrile-methyl tert-butyl ether as a mobile phase system, and controls the pH value of the ammonium formate solution to be 2.2-3.1, so that adenosine and adenine can be effectively separated, and ademetionine, decarboxylated ademetionine, S-adenosyl-L-methionine glycine isomer 2, adenosine, adenine and S-adenosyl-L-homocysteine can be highly separated at one time, the method has short time consumption, high separation efficiency, simple operation and low separation cost, and is beneficial to improving the quality control of ademetionine bisulfate or related preparations.

[0045] In addition, the existing technology mobile phase adds ion pair reagents to separate impurities, which causes great damage to the chromatographic column and is not suitable for gradient elution. The mobile phase used in the application does not contain ion pair reagents, is suitable for gradient elution, and can efficiently separate impurities.

[0046] Further, the application uses a specific gradient elution program to optimize the separation effect and promote the effective separation of ademetionine and related impurities.

[0047] The application provides a detection method for components in a butanedisulfonic acid ademetionine preparation, and the obtained separation result is detected by using an ultraviolet detector or a mass spectrometer, so that qualitative and / or quantitative detection results of the components in the ademetionine sample can be obtained. The detection method provided by the application is not affected by excipients, is efficient and convenient, has good reproducibility, strong specificity, high accuracy and high sensitivity, and can realize quality control of the ademetionine sample. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The blank solvent HPLC chromatogram is shown in the following figure:

[0049] Figure 2 The blank excipient HPLC chromatogram is shown in the following figure:

[0050] Figure 3 The system suitability solution HPLC chromatogram is shown in the following figure:

[0051] Figure 4 The butanedisulfonic acid ademetionine enteric-coated tablet unbroken HPLC chromatogram is shown in the following figure:

[0052] Figure 5 The butanedisulfonic acid ademetionine enteric-coated tablet high-temperature broken HPLC chromatogram is shown in the following figure:

[0053] Figure 6 The butanedisulfonic acid ademetionine enteric-coated tablet light broken HPLC chromatogram is shown in the following figure:

[0054] Figure 7 The butanedisulfonic acid ademetionine enteric-coated tablet oxidation broken HPLC chromatogram is shown in the following figure:

[0055] Figure 8 The butanedisulfonic acid ademetionine enteric-coated tablet acid broken HPLC chromatogram is shown in the following figure:

[0056] Figure 9 The butanedisulfonic acid ademetionine enteric-coated tablet alkali broken HPLC chromatogram is shown in the following figure:

[0057] Figure 10 The limit of quantification HPLC chromatogram of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated ademetionine, S-adenosyl-L-methionine glycine isomer 2 and ademetionine is shown in the following figure:

[0058] Figure 11 The limit of detection HPLC chromatogram of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated ademetionine, S-adenosyl-L-methionine glycine isomer 2 and ademetionine is shown in the following figure:

[0059] Figure 12 The linear curve of dimethylthioadenosine is shown in the following figure:

[0060] Figure 13 linear curve for adenosylmethionine;

[0061] Figure 14 linear curve for methylthioadenosine;

[0062] Figure 15 linear curve for adenosine;

[0063] Figure 16 linear curve for decarboxylated adenosylmethionine;

[0064] Figure 17 linear curve for adenosylmethionine;

[0065] Figure 18 linear curve for S-adenosyl-L-methionine glycine isomer 2;

[0066] Figure 19 linear curve for S-adenosyl-L-homocysteine;

[0067] Figure 20 HPLC profile of Example 8;

[0068] Figure 21 HPLC profile of Comparative Example 1;

[0069] Figure 22 HPLC profile of Comparative Example 2;

[0070] Figure 23 HPLC profile of Comparative Example 3. DETAILED DESCRIPTION

[0071] The present application provides a method for separating components in a preparation of adenosylmethionine, comprising the following steps:

[0072] The preparation of adenosylmethionine is dissolved and diluted to obtain a test solution; the preparation of adenosylmethionine includes enteric-coated adenosylmethionine tablets and / or adenosylmethionine lyophilizate;

[0073] The test solution is subjected to high performance liquid chromatography separation to obtain component separation results.

[0074] In the present application, the components include adenosylmethionine and related impurities; the related impurities include one or several of decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthioadenosine, methylthioadenosine, adenosine, adenine and S-adenosyl-L-homocysteine. In the present application, 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:

[0075]

[0076] The present application dissolves and dilutes ademetionine butanedisulfonate preparation to obtain a test solution. In the present application, the ademetionine butanedisulfonate preparation includes ademetionine butanedisulfonate enteric-coated tablets and / or ademetionine butanedisulfonate lyophilizate, preferably a lyophilizate for injection.

[0077] In the present application, when the ademetionine butanedisulfonate preparation is ademetionine butanedisulfonate enteric-coated tablets, the solvent used for the dissolution is preferably tetrahydrofuran and a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.05-0.2 mol / L. In the present application, the mass of the ademetionine butanedisulfonate preparation to the volume of tetrahydrofuran is preferably 1 g: 10-30 mL, more preferably 1 g: 20 mL; and the mass of the ademetionine butanedisulfonate preparation to the volume of the hydrochloric acid solution is preferably 1 g: 10-100 mL, more preferably 1 mg: 2-5 mL.

[0078] In the present application, when the ademetionine butanedisulfonate preparation is ademetionine butanedisulfonate lyophilizate, the solvent used for the dissolution is preferably water. The present application does not have special requirements for the amount of water, which can only dissolve the ademetionine butanedisulfonate lyophilizate.

[0079] In the present application, the solvent used for the dissolution is preferably a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.05-0.2 mol / L, more preferably 0.1 mol / L. In the present application, the mass of the ademetionine butanedisulfonate preparation to the volume of the hydrochloric acid solution is preferably 1 mg: 1-10 mL, more preferably 1 mg: 2-5 mL.

[0080] In the present application, the diluent used for the dilution is preferably a mixture of a hydrochloric acid solution and acetonitrile; the concentration of the hydrochloric acid solution is preferably 0.05-0.2 mol / L, more preferably 0.1 mol / L; and the volume fraction of the hydrochloric acid solution in the diluent is preferably 10-50%, specifically 10%, 20%, 30%, 40%, or 50%.

[0081] In the present application, the method for the dilution preferably includes mixing the dissolved solution of the ademetionine butanedisulfonate preparation with the diluent to obtain a diluted solution, i.e., a test solution. In the present application, the concentration of the ademetionine butanedisulfonate preparation in the test solution is preferably 0.2-1.6 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.

[0082] After obtaining the test sample solution, the test sample solution is subjected to high performance liquid chromatography separation to obtain component separation results. In the present application, the mobile phase of the high performance liquid chromatography separation comprises mobile phase A and mobile phase B. In the present application, 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 16-20 mmol / L, more preferably 18 mmol / L, and the pH value is 2.2-3.1, and specifically can be 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1; in the present application, the reagent for adjusting the first ammonium formate solution is preferably formic acid.

[0083] In the present application, the preparation method of the mobile phase A preferably comprises the following steps:

[0084] The pH value of the ammonium formate aqueous solution is adjusted to 2.2-3.1 using formic acid, water and acetonitrile are added to obtain the mobile phase A. In the present application, the concentration of the ammonium formate aqueous solution is preferably 16-20 mmol / L, more preferably 18 mmol / L.

[0085] In the present application, the volume fraction of the first ammonium formate solution in the mobile phase A 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 application, the volume fraction of acetonitrile in the mobile phase A is 40-60%, preferably 45-55%, and specifically can be 40%, 45%, 50%, 55% or 60%. In the present application, the volume fraction of water in the mobile phase A is 30-60%, preferably 40-50%, and specifically can be 30%, 40%, 50% or 60%.

[0086] In the present application, 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 16-20 mmol / L, more preferably 18 mmol / L, and the pH value is 2.2-3.1, and specifically can be 2.6, 2.7, 2.8, 2.9, 3.0 or 3.1; in the present application, the reagent for adjusting the second ammonium formate solution is preferably formic acid.

[0087] In the present application, the preparation method of the mobile phase B preferably comprises the following steps:

[0088] The pH value of the ammonium formate aqueous solution is adjusted to 2.2-3.1 using formic acid, acetonitrile and methyl tert-butyl ether are added to obtain the mobile phase B. In the present application, the concentration of the ammonium formate aqueous solution is preferably 16-20 mmol / L, more preferably 18 mmol / L.

[0089] In the present application, 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 application, 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 application, 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%.

[0090] In the present application, the elution procedure of the high performance liquid chromatography separation is gradient elution, and the procedure of the gradient elution preferably comprises:

[0091] 0-2 min, the volume fraction of the mobile phase A is preferably 5-15%, more preferably 10%, and specifically can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%;

[0092] 2-17 min, the volume fraction of the mobile phase A is increased from 5-15% to 50-60%, and in specific embodiments of the present application, 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%;

[0093] 17-27 min, the volume fraction of the mobile phase A is preferably 50-60%, more preferably 55%, and specifically can be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%;

[0094] 27-28 min, the volume fraction of the mobile phase A is decreased from 50-60% to 5-15%, and in specific embodiments of the present application, the volume fraction of the mobile phase A can be decreased 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%;

[0095] 28-40 min, the volume fraction of the mobile phase A is 5-15%, more preferably 10%, and specifically can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%.

[0096] In the present application, the chromatographic column used in the high performance liquid chromatography separation includes an amino column, and as a specific embodiment of the present application, the chromatographic column can be apHera NH2 polymer. In the present application, the column length of the chromatographic column 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 application, the size of the chromatographic column is 250 mm x 4.6 mm, and the particle size is 5 μm.

[0097] In the present application, the column temperature of the high performance liquid chromatography separation is preferably 25-35℃, and can be specifically 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃ or 35℃.

[0098] In the present application, the flow rate of the mobile phase of the high performance liquid chromatography separation is preferably 0.2-1.0 mL / min, and can be specifically 0.4 mL / min, 0.5 mL / min or 0.6 mL / min.

[0099] In the present application, the separation wavelength of the high performance liquid chromatography separation is preferably 210-290 nm.

[0100] In the present application, the injection amount of the high performance liquid chromatography separation is preferably 5-100 μL, and more preferably 20-80 μL, and can be 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.

[0101] In the present application, the temperature of the injection is preferably 2-12℃, and more preferably 5℃.

[0102] The present application provides a detection method for components in a butanedisulfonic acid ademetionine preparation, which comprises the following steps:

[0103] The detection result of the components in the butanedisulfonic acid ademetionine preparation is obtained by detecting the separation result of the components obtained by the above separation method.

[0104] In the present application, the detection includes ultraviolet detector detection or mass spectrometer detection.

[0105] In the present application, the detection wavelength of the ultraviolet detector is preferably 210-290 nm, and can be specifically 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm or 290 nm.

[0106] In the present application, the parameters detected by the mass spectrometer preferably include:

[0107] Gas temperature: 300℃;

[0108] Gas rate: 7.0 L / min;

[0109] Spray pressure: 15 psi;

[0110] Sheath gas temperature: 250℃;

[0111] Sheath gas rate: 11.0 L / min;

[0112] Capillary voltage: 3500 V;

[0113] Nozzle voltage: 500 V.

[0114] In the present application, the detection result preferably includes a qualitative detection result and / or a quantitative detection result; the method for obtaining the quantitative detection result preferably includes area normalization method, internal standard method or external standard method. The present application is not particularly limited to the area normalization method, internal standard method or external standard method, and the area normalization method, internal standard method or external standard method well known to those skilled in the art can be used for quantification.

[0115] The separation and detection method of the components in the ademetionine butanedisulfonate preparation provided in the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0116] In the examples of the present application, the manufacturer of the ademetionine butanedisulfonate enteric-coated tablets is ABBVIE S.R.L., and the specification is 0.5 g / tablet, 10 tablets / box in terms of ademetionine; the manufacturer of the ademetionine butanedisulfonate for injection is ABBVIE S.R.L., and the specification is 0.5 g / branch, 10 branches / box in terms of ademetionine.

[0117] In the embodiment of the present application, the conditions (optimum conditions) of HPLC chromatographic separation and detection are as follows: the high performance liquid chromatograph is Agilent 1260; the chromatographic column is apHera NH2 polymer, 250 mm x 4.6 mm, 5 μm; the column temperature is 30 DEG C; the mobile phase: the mobile phase A is ammonium formate solution-acetonitrile-water, the volume ratio is 10:50:40; the mobile phase B is ammonium formate solution-acetonitrile-methyl tert-butyl ether, the volume ratio is 10:88:2; wherein the preparation method of ammonium formate solution in the mobile phase A and the mobile phase B is as follows: taking ammonium formate 1.18 g, dissolving in 1000 mL of water, and adjusting the pH value to 2.8+ / -0.2 with formic acid.

[0118] The ultraviolet detector detects, and the detection wavelength is 260 nm; the flow rate of the mobile phase is 0.5 mL / min; the injection amount is 5 μL, and the injection temperature is 5 DEG C; the gradient elution mode is adopted, and the gradient elution procedure is shown in Table 1.

[0119] Table 1 Gradient elution procedure

[0120]

[0121] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0122] Example 1

[0123] Methodology validation-system suitability

[0124] 1. Preparation method of solution

[0125] (1) Blank solution / diluent: 0.1 mol / L hydrochloric acid aqueous solution (take 9 mL of hydrochloric acid into 1000 mL of water, mix well, and then obtain): acetonitrile (20:80).

[0126] (2) Ado-methionine control stock solution: accurately weigh 37.40 mg of adenosylmethionine disulfonate control sample, place in a 50 mL volumetric flask, dissolve in 10 mL of water, and then dilute to the mark with the diluent, and shake well, and then obtain;

[0127] (3) Impurity stock solution:

[0128] Dimethylthioadenosine stock solution: accurately weigh 27.13 mg of dimethylthioadenosine, place in a 50 mL volumetric flask, dissolve and dilute to the mark with the diluent, shake well, accurately take 5 mL, place in a 50 mL volumetric flask, dissolve and dilute to the mark with the diluent, and shake well, and then obtain the dimethylthioadenosine stock solution;

[0129] Methylthioadenosine stock solution: accurately weigh 19.66 mg of methylthioadenosine, place in a 50 mL volumetric flask, dissolve and dilute to the mark with the diluent, and shake well.

[0130] Adenosine stock solution: Take adenosine 20.02 mg, accurately weigh, place in a 50 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well, and then accurately take 5 mL into a 50 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well, and use as adenosine stock solution;

[0131] Adenosine stock solution: Take adenosine 20.02 mg, accurately weigh, place in a 50 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well, and then accurately take 5 mL into a 50 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well, and use as adenosine stock solution;

[0132] Decarboxylated adenosylmethionine stock solution: Take decarboxylated adenosylmethionine 26.04 mg, accurately weigh, place in a 100 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well;

[0133] S-Adenosyl-L-methionine glycine isomer 2 stock solution: Take S-adenosyl-L-methionine glycine isomer 2 25.46 mg, accurately weigh, place in a 200 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well;

[0134] S-Adenosyl-L-methionine glycine isomer 2 stock solution: Take S-adenosyl-L-methionine glycine isomer 2 25.46 mg, accurately weigh, place in a 200 mL volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well;

[0135] (4) Component positioning solution: adenosylmethionine reference substance 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 each diluted 50 times with solvent.

[0136] (5) System suitability solution: accurately weigh 38.43 mg of adenosylmethionine reference substance, place in a 50 mL volumetric flask, dissolve with 10 mL of water, and then accurately take 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 adenosylmethionine 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 in the volumetric flask, dissolve and dilute to the mark with appropriate diluent, shake well;

[0137] (6) Blank adjuvant solution: take blank adjuvant 233.66 mg (about equivalent to adenosylmethionine 0.5 g), put into 50 mL volumetric flask, add 10 mL tetrahydrofuran, shake for 40 minutes, then add 25 mL 0.1 mol / L hydrochloric acid aqueous solution, shake for 40 minutes, then dilute to the mark with 0.1 mol / L hydrochloric acid aqueous solution, shake well, centrifuge at 3000 rpm per minute for 5 min, accurately take 2 mL supernatant into 50 mL volumetric flask, dilute to the mark with diluent, shake well, filter to obtain.

[0138] 2 HPLC separation

[0139] The optimal HPLC detection conditions were used to sample the blank solvent (diluent), blank adjuvant solution and system suitability solution, and HPLC separation was performed. Figure 1 The HPLC chromatogram of the blank solvent is, Figure 2 The HPLC chromatogram of the blank adjuvant is, Figure 3 The HPLC chromatogram of the system suitability solution is, Figures 1-3 It can be seen that the adjuvant in the adenosylmethionine enteric-coated tablet does not interfere with the impurity detection, and each impurity can be completely separated from the adenosylmethionine main peak.

[0140] Under different column temperature, pH value of mobile phase A and mobile phase flow rate conditions, the HPLC detection results of system suitability solution sampling are shown in Tables 2 and 3. In Tables 2 and 3, 1#, 2# chromatographic column represents different batches of the same type of chromatographic column.

[0141] Table 2 Determination results of chromatographic condition change resistance experiment

[0142]

[0143] Table 3 Determination results of chromatographic condition change resistance experiment

[0144]

[0145] Example 2

[0146] Methodology verification-stability

[0147] Undamaged solution: take the fine powder of the product (ademetionine butanedisulfonate enteric-coated tablet) 49.12 mg, accurately weigh, put into 50 mL volumetric flask, add 10 mL water, shake for 30 minutes to dissolve, then dilute to the mark with diluent, shake well, filter to obtain.

[0148] Acidic degradation solution: Take 49.91 mg of the fine powder of the product, accurately weigh, and place in a 50 mL volumetric flask. Add 1 mL of 0.1 mol / L hydrochloric acid solution, and stand for 24 hours. Add 1 mL of 0.1 mol / L sodium hydroxide solution to neutralize, add 10 mL of water, shake for 30 minutes to dissolve, then dilute to the calibration line with diluent, shake well, and filter to obtain.

[0149] Alkaline degradation solution: Take 47.53 mg of the fine powder of the product, accurately weigh, and place in a 50 mL volumetric flask. Add 1 mL of 0.1 mol / L sodium hydroxide solution, and stand for 2.5 hours. Add 1 mL of 0.1 mol / L hydrochloric acid solution to neutralize, add 10 mL of water, shake for 30 minutes to dissolve, then dilute to the calibration line with diluent, shake well, and filter to obtain.

[0150] Oxidative degradation solution: Take 47.36 mg of the fine powder of the product, accurately weigh, and place in a 50 mL volumetric flask. Add 1 mL of 10% hydrogen peroxide solution, and stand for 24 hours. Add 10 mL of water, shake for 30 minutes to dissolve, then dilute to the calibration line with diluent, shake well, and filter to obtain.

[0151] High-temperature solid degradation solution: Take 50.43 mg of the fine powder of the sample that has been placed at a high temperature of 60°C for 5 hours, accurately weigh, and place in a 50 mL volumetric flask. Add 10 mL of water, shake for 30 minutes to dissolve, then dilute to the calibration line with diluent, shake well, and filter to obtain.

[0152] Illumination solid degradation solution: Take 48.38 mg of the fine powder of the sample that has been placed under illumination (4500±500 lx) for 24 hours, accurately weigh, and place in a 50 mL volumetric flask. Add 10 mL of water, shake for 30 minutes to dissolve, then dilute to the calibration line with diluent, shake well, and filter to obtain.

[0153] The undegraded solution and each degradation solution were injected into HPLC for separation, and the results are shown in Table 1. Figures 4-9 .

[0154] Figure 4 The HPLC chromatogram of the undegraded adenosylmethionine enteric-coated tablets is shown in Figure 1. Figure 4 As can be seen from Figure 1, the degradation impurities methylthioadenosine, adenine, S-adenosyl-L-homocysteine (abbreviated as homocysteine), and the process impurity S-adenosyl-L-methionine glycine isomer 2 (abbreviated as isomer 2) can be detected in the undegraded solution.

[0155] Figure 5 The HPLC chromatogram of the adenosylmethionine enteric-coated tablets after high-temperature degradation is shown in Figure 2. Figure 5 As can be seen from Figure 2, the adenosylmethionine enteric-coated tablets are unstable under the condition of a high temperature of 60°C, and degrade to dimethylthioadenosine.

[0156] Figure 6 The HPLC chromatogram of the adenosylmethionine enteric-coated tablets after illumination degradation for 24 hours is shown in Figure 3.Figure 6 It can be seen that the adenosylmethionine enteric-coated tablets are relatively stable under light for 24 h, and no new impurities are degraded.

[0157] Figure 7 The HPLC chromatogram of the adenosylmethionine enteric-coated tablets after oxidation is shown in Figure 2. Figure 7 It can be seen that the adenosylmethionine enteric-coated tablets can degrade unknown impurity 1 (retention time of 14.867 min) and unknown impurity 2 (retention time of 15.019 min) under oxidation conditions, and are unstable under oxidation conditions.

[0158] Figure 8 The HPLC chromatogram of the adenosylmethionine enteric-coated tablets after acid destruction is shown in Figure 3. Figure 8 It can be seen that the adenosylmethionine enteric-coated tablets are relatively stable under acidic conditions, and no new impurities are degraded.

[0159] Figure 9 The HPLC chromatogram of the adenosylmethionine enteric-coated tablets after alkaline destruction is shown in Figure 4. Figure 9 It can be seen that the adenosylmethionine enteric-coated tablets are unstable under alkaline conditions, and degrade methylthioadenosine and adenosine.

[0160] Example 3

[0161] Method validation-quantitative limit and detection limit

[0162] Preparation of the quantitative limit solution:

[0163] The quantitative limit stock solution: 0.5 mL of the adenosylmethionine control stock solution prepared in Example 1, 5 mL of the dimethylthioadenosine stock solution, 0.5 mL of the methylthioadenosine stock solution, 2 mL of the adenine stock solution, 5 mL of the adenosine stock solution, 1 mL of the decarboxylated adenosylmethionine stock solution, 2 mL of the S-adenosyl-L-methionine glycine isomer 2 stock solution, and 2 mL of the S-adenosyl-L-homocysteine stock solution were accurately measured into the same 100 mL volumetric flask, dissolved and diluted to the mark with the diluent, and shaken well.

[0164] The quantitative limit solution: 5 mL of the quantitative limit stock solution was accurately measured into a 50 mL volumetric flask, diluted to the mark with the diluent, and shaken well. (Equivalent to 0.05% of the limit concentration)

[0165] The detection limit solution: 3 mL of the quantitative limit solution was accurately measured into a 10 mL volumetric flask, diluted to the mark with the diluent, and shaken well. (Equivalent to 0.015% of the limit concentration)

[0166] 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 the peak height to the noise (signal-to-noise ratio) was calculated, and the chromatogram was recorded. The results are shown in Tables 4-5 and Figures 10-11 , Figure 10Limit of quantitation HPLC chromatogram of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine, Figure 11 Limit of detection HPLC chromatogram of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine.

[0167] Table 4 Limit of quantitation determination results

[0168]

[0169]

[0170] From Table 4 and Figure 10 It can be seen that under the HPLC chromatographic conditions of the present application, the limit of quantitation of adenosylmethionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine meets the requirement that S / N is greater than 10, and the limit of quantitation of each component is about equivalent to 0.05wt% of the concentration of adenosylmethionine in the test sample solution.

[0171] Table 5 Limit of detection determination results

[0172]

[0173] From Table 5 and Figure 11 It can be seen that under the HPLC chromatographic conditions of the present application, the limit of detection of adenosylmethionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine meets the requirement that S / N is greater than 3, and the limit of detection of each component is about equivalent to 0.01wt% of the concentration of adenosylmethionine in the test sample solution.

[0174] Example 4

[0175] Methodology validation - linearity

[0176] Preparation of linear solutions

[0177] Mixed stock solution: precisely pipette 2 mL of the ademetionine reference substance stock solution, 20 mL of the dimethylthioadenosine stock solution, 20 mL of the methylthioadenosine stock solution, 20 mL of the adenine stock solution, 20 mL of the adenosine stock solution, 30 mL of the decarboxy ademetionine stock solution, 30 mL of the S-adenosyl-L-methionine glycine isomer 2 stock solution, and 20 mL of the S-adenosyl-L-homocysteine stock solution into the same 200 mL volumetric flask, dissolve and dilute to the calibration mark with a suitable diluent, and shake well; (limit 500%)

[0178] L1 (equivalent to the limit of quantification concentration): take the limit of quantification solution in Example 3.

[0179] L2 (equivalent to 30% of the limit concentration): precisely pipette 3 mL of the mixed stock solution into a 50 mL volumetric flask, dilute to the calibration mark with a diluent, and shake well.

[0180] L3 (equivalent to 50% of the limit concentration): precisely pipette 5 mL of the mixed stock solution into a 50 mL volumetric flask, dilute to the calibration mark with a diluent, and shake well.

[0181] L4 (equivalent to 100% of the limit concentration): precisely pipette 5 mL of the mixed stock solution into a 25 mL volumetric flask, dilute to the calibration mark with a diluent, and shake well.

[0182] L5 (equivalent to 200% of the limit concentration): precisely pipette 10 mL of the mixed stock solution into a 25 mL volumetric flask, dilute to the calibration mark with a diluent, and shake well.

[0183] Take each of the above linear solutions L1~L5 for injection 1 time, HPLC separation and detection, and the experimental results are shown in Figures 12-19 and Tables 6~13, wherein, Figures 12-19 The linear curves of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxy ademetionine, ademetionine, S-adenosyl-L-methionine glycine isomer 2, and S-adenosyl-L-homocysteine are shown in order.

[0184] According to the slope of the main component and each impurity linear equation, the correction factor f (f=k 主成分 / k 斜率 ) of each impurity is calculated, and the results are shown in Tables 6~13, wherein, k 主成分 is the slope of the linear regression equation of the main component concentration (X)-response value (Y), and k 杂质 is the slope of the linear regression equation of the impurity concentration (X)-response value (Y).

[0185] Table 6 Linear determination results of dimethylthioadenosine

[0186]

[0187] Table 7 Methioaspartate linear assay results

[0188]

[0189] Table 8 Adenine linear assay results

[0190]

[0191] Table 9 Adenosine linear assay results

[0192]

[0193] Table 10 Decarboxyaspartomethionine linear assay results

[0194]

[0195] Table 11 S-Adenosyl-L-methionine linear assay results

[0196]

[0197] Table 12 S-Adenosyl-L-methionine glycine isomer 2 linear assay results

[0198]

[0199] Table 13 S-Adenosyl-L-homocysteine linear assay results

[0200]

[0201] From Figures 12-19 As can be seen from Tables 6-13, under the HPLC chromatographic conditions of the present application, the concentrations of S-adenosyl-L-methionine and its impurities dimethylthioaspartate, methioaspartate, adenine, adenosine, decarboxyaspartomethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine have a good linear relationship with peak area.

[0202] Example 5

[0203] Method Validation - Accuracy

[0204] Mixed Impurity Reference Solution: Accurately weigh 5 mL of the mixed stock solution from Example 4 into a 25 mL volumetric flask, dilute to the mark with diluent, and mix. (Equivalent to 100% of the limit concentration)

[0205] Preparation of Accuracy Solutions:

[0206] A1 (equivalent to the quantitative limit concentration): take 40 mg of the test sample fine powder, place it in a 50 mL volumetric flask, add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution, then add 5 mL of the quantitative limit stock solution in Example 3, shake for 30 minutes to dissolve, dilute to the mark with acetonitrile, shake well, filter, and prepare 3 parallel samples.

[0207] A2 (equivalent to 50% of the limit concentration): take 40 mg of the test sample fine powder, place it in a 50 mL volumetric flask, add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution, then add 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 3 parallel samples.

[0208] A3 (equivalent to 100% of the limit concentration): take 40 mg of the test sample fine powder, place it in a 50 mL volumetric flask, add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution, then add 10 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 3 parallel samples.

[0209] A4 (equivalent to 200% of the limit concentration): take 40 mg of the test sample fine powder, place it in a 50 mL volumetric flask, add 10 mL of 0.1 mol / L hydrochloric acid aqueous solution, then add 20 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 3 parallel samples.

[0210] Inject each accuracy solution, separate and detect by HPLC, calculate the recovery rate by external standard method, and the results are shown in Table 14.

[0211] Table 14: Results of standard addition recovery rate

[0212]

[0213] As can be seen from Table 14, under the HPLC chromatographic conditions of the present application, the accuracy of dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated adenosylmethionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine is good.

[0214] Example 6

[0215] Methodology verification-precision

[0216] Spiked sample solution: 10 tablets of the test sample were taken and placed in a 500 mL volumetric flask. 100 mL of tetrahydrofuran was added and shaken for 40 minutes. Then 250 mL of 0.1 mol / L hydrochloric acid aqueous solution was added and shaken for 40 minutes to completely disintegrate the tablets. The solution was diluted to the mark with 0.1 mol / L hydrochloric acid aqueous solution, shaken well, and centrifuged at 3000 rpm per minute for 5 minutes. 2 mL of the supernatant was accurately taken into a 50 mL volumetric flask, 5 mL of the mixed stock solution in Example 4 was added, diluted to the mark with the diluent, shaken well, and filtered to obtain the solution. Six solutions (0.4 mg / mL of ademetionine) were prepared in parallel, and each solution was injected into HPLC for separation and detection. The recovery rate was calculated by the external standard method, and the results are shown in Table 15.

[0217] Table 15 Precision results (wt%)

[0218]

[0219] As can be seen from Table 15, the spiked sample solution of the ademetionine butanedisulfonate enteric-coated tablet related substance detection method has good precision.

[0220] Example 7

[0221] Method validation-solution stability

[0222] Reference mixture solution: the reference mixture solution prepared in Example 4.

[0223] Spiked sample solution: the spiked sample solution prepared in Example 6.

[0224] The reference mixture solution and the spiked sample solution were respectively placed at 5°C for 0h, 10h, 20h, 30h, injected, and HPLC separated and detected. The results are shown in Tables 16-17.

[0225] Table 16 Stability of reference mixture solution

[0226]

[0227] Table 17 Stability of spiked sample solution

[0228]

[0229] As can be seen from Tables 16-17, under the HPLC chromatographic conditions provided by the present application, the reference solution and the test sample solution of ademetionine and its impurities dimethylthioadenosine, methylthioadenosine, adenine, adenosine, decarboxylated ademetionine, ademetionine, S-adenosyl-L-methionine glycine isomer 2 and S-adenosyl-L-homocysteine are stable at 5°C within 30 hours.

[0230] Example 8

[0231] Preparation of the test sample: 4 vials of the methionine adenosyltransferase freeze-dried injection were taken, and the contents were transferred into a 250 mL volumetric flask with diluent, diluted with water to the calibration mark, shaken well, and used as the test sample stock solution. 5 mL of the test sample stock solution was accurately measured into a 100 mL volumetric flask, diluted with diluent to the calibration mark, shaken well, and obtained the test sample solution (0.4 mg / mL). 5 μL of the test sample solution was injected into the liquid chromatograph.

[0232] The HPLC chromatographic separation and detection conditions were as follows: the high performance liquid chromatograph was Agilent 1260; the chromatographic column was apHera NH2 polymer, 250 mm x 4.6 mm, 5 μm; the column temperature was 30 DEG C; the mobile phase was as follows: the mobile phase A was ammonium formate solution-acetonitrile-water, the volume ratio was 10:50:40; the mobile phase B was ammonium formate solution-acetonitrile-methyl tert-butyl ether, the volume ratio was 10:88:2; the preparation method of the ammonium formate solution in the mobile phase A and the mobile phase B was as follows: 1.18 g of ammonium formate was dissolved in 1000 mL of water, and formic acid was added to adjust the pH value to 2.8±0.2.

[0233] The ultraviolet detector was used for detection, the detection wavelength was 260 nm, the flow rate of the mobile phase was 0.5 mL / min, the injection amount was 5 μL, the injection temperature was 5 DEG C, the gradient elution mode was used, and the gradient elution program was shown in Table 18. Figure 20

[0234] Table 18 Gradient elution program

[0235]

[0236] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0237] The results show that the detection method can successfully separate the impurities of methionine adenosyltransferase, decarboxy methionine adenosyltransferase, S-adenosyl-L-methionine glycine isomer 2, adenosine, adenine and S-adenosyl-L-homocysteine in the methionine adenosyltransferase injection.

[0238] Comparative Example 1

[0239] The HPLC chromatographic separation and detection conditions were as follows: the high performance liquid chromatograph was Agilent 1260; the chromatographic column was apHera NH2 polymer, 250 mm x 4.6 mm, 5 μm; the column temperature was 30 DEG C; the mobile phase was as follows: the mobile phase A was ammonium formate solution-acetonitrile-water, the volume ratio was 10:50:40; the mobile phase B was ammonium formate solution-acetonitrile, the volume ratio was 10:90; the preparation method of the ammonium formate solution in the mobile phase A and the mobile phase B was as follows: 1.18 g of ammonium formate was dissolved in 1000 mL of water, and formic acid was added to adjust the pH value to 3.2. ​

[0240] Detection was performed using a UV detector at a wavelength of 260 nm; the mobile phase flow rate was 0.5 mL / min; the injection volume was 5 μL; and the injection temperature was 5 °C. Gradient elution was used, and the gradient elution program is shown in Table 19. The resulting chromatogram is shown below. Figure 21 As shown.

[0241] Table 19 Gradient elution program

[0242]

[0243] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0244] 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.

[0245] Comparative Example 2

[0246] HPLC chromatographic separation and detection conditions: The high-performance liquid chromatograph was an Agilent 1260; the chromatographic column was an apoHera NH2 polymer, 250 mm × 4.6 mm, 5 μm; the column temperature was 30 ℃; the mobile phases were: mobile phase A was ammonium formate solution-acetonitrile-water, with a volume ratio of 10:50:40; mobile phase B was 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 phases A and B was as follows: 1.18 g of ammonium formate was dissolved in 1000 mL of water, and the pH was adjusted to 3.2 with formic acid.

[0247] Detection was performed using a UV detector at a wavelength of 260 nm; the mobile phase flow rate was 0.5 mL / min; the injection volume was 5 μL; and the injection temperature was 5 °C. Gradient elution was used, and the gradient elution program is shown in Table 20. The resulting chromatogram is shown below. Figure 22 As shown.

[0248] Table 20 Gradient elution program

[0249]

[0250] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0251] 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.

[0252] Comparative Example 3

[0253] HPLC chromatographic separation and detection conditions: high performance liquid chromatograph is Agilent 1260; chromatographic column is apHera NH2 polymer, 250 mm x 4.6 mm, 5 μm; column temperature is 30 DEG C; mobile phase: mobile phase A is ammonium formate solution-acetonitrile-water, volume ratio is 10:50:40; mobile phase B is ammonium formate solution-acetonitrile, volume ratio is 10:90; wherein the preparation method of ammonium formate solution in mobile phase A, mobile phase B is: taking ammonium formate 1.18 g, dissolving in 1000 mL water, adding formic acid to adjust pH to 2.8±0.2.

[0254] UV detector detects, detection wavelength is 260 nm; mobile phase flow rate is 0.5 mL / min; injection volume is 5 μL, injection temperature is 5 DEG C; gradient elution mode is used, and the gradient elution program is shown in Table 21. The obtained chromatogram is shown in Figure 23

[0255] Table 21 Gradient elution program

[0256]

[0257] Diluent: 0.1 mol / L hydrochloric acid solution: acetonitrile = 20:80 (volume ratio).

[0258] The results show that: the analysis method used in Comparative Example 3 cannot separate adenine and adenosine, but the peak type of decarboxylated adenosylmethionine is improved, which is a symmetrical peak.

[0259] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A method for detecting components in a preparation of ademetionine butyldi sulfate, characterized by, The method comprises the following steps: dissolving and diluting a preparation of ademetionine butanedisulfate to obtain a test solution; the preparation of ademetionine butanedisulfate comprises ademetionine butanedisulfate enteric-coated tablets and / or ademetionine butanedisulfate lyophilizate; subjecting the test solution to high performance liquid chromatography separation to obtain component separation results; the components comprise ademetionine and related impurities; the related impurities are decarboxylated ademetionine, S-adenosyl-L-methionine glycine isomer 2, dimethylthetin, methylthetin, adenine and S-adenosyl-L-homocysteine; the mobile phase of the high performance liquid chromatography separation comprises mobile phase A and mobile phase B; 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 16-20 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; 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%; the elution procedure of the high performance liquid chromatography separation is gradient elution, and the procedure of the gradient elution comprises: 0-2 min, the volume fraction of the mobile phase A is 5-15%; 2-17 min, the volume fraction of the mobile phase A is increased from 5-15% to 50-60%; 17-27 min, the volume fraction of the mobile phase A is 50-60%; 27-28 min, the volume fraction of the mobile phase A is decreased from 50-60% to 5-15%; 28-40 min, the volume fraction of the mobile phase A is 5-15%; the chromatographic column used in the high performance liquid chromatography separation is an amino column; detecting the obtained component separation results to obtain detection results of the components in the preparation of ademetionine butanedisulfate; the detection is mass spectrometer detection.

2. The detection method according to claim 1, characterized in that, the detection results comprise qualitative detection results and / or quantitative detection results; the method for obtaining the quantitative detection results comprises area normalization method, internal standard method or external standard method.

3. The method of claim 1, wherein when the preparation of ademetionine butanedisulfate is ademetionine butanedisulfate enteric-coated tablets, the solvent used for dissolution is tetrahydrofuran and a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 0.05-0.2 mol / L; the mass of the preparation of ademetionine butanedisulfate to the volume of tetrahydrofuran is 1 g:10-30 mL; the mass of the preparation of ademetionine butanedisulfate to the volume of the hydrochloric acid solution is 2 g:10-100 mL; when the preparation of ademetionine butanedisulfate is ademetionine butanedisulfate lyophilizate, the solvent used for dissolution is water.

4. The method of claim 1, wherein, The diluent used in 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%.

5. The method of claim 1, wherein The concentration of the ademetionine butanedisulfonate preparation in the test solution is 0.2-1.6 mg / mL.

6. The method of claim 1, wherein The reagent for adjusting the pH values of the first ammonium formate solution and the second ammonium formate solution is formic acid.

7. The method of claim 1, wherein, The column temperature for high performance liquid chromatography separation is 25-35 DEG C, the flow rate of the mobile phase is 0.2-1.0 mL / min, and the injection volume is 5-100 mu L.

8. The method of claim 1, wherein, The parameters detected by the mass spectrometer include: Gas temperature: 300 DEG C; Gas rate: 7.0 L / min; Spray pressure: 15 psi; Sleeve flow gas temperature: 250 DEG C; Sleeve flow gas rate: 11.0 L / min; Capillary voltage: 3500 V; Nozzle voltage: 500 V.

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