Method for detecting antioxidant in butanedisulfonic acid ademetionine for injection

Abstract

The invention relates to the technical field of medicine detection, and particularly discloses a method for detecting an antioxidant in butanedisulfonic acid ademetionine for injection. The method comprises the following steps: firstly, dissolving an ademetionine butanedisulfonate sample for injection in a solvent for injection for pretreatment, passing through a C18 solid-phase extraction column, and eluting with methanol, so that the antioxidant in the ademetionine butanedisulfonate sample for injection can be fully extracted, and the recovery rate is relatively high; then a C18 chromatographic column with a specific specification is adopted to carry out liquid chromatographic analysis on the test solution, and accurate detection of the antioxidant and the content of the antioxidant in the methionine butanedisulfonate for injection is realized at the same time; by limiting the conditions of the ultrahigh liquid chromatography, the peak response intensity of the test solution and the reference solution is improved, so that the detection precision is improved. The detection method can provide data support for the effective control of the quality of the ademetionine butanedisulfonate for injection, and ensures the safety of clinical medication.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting an antioxidant in adenosylmethionine butanedisulfonate for injection. Background Art

[0002] Adenosylmethionine butane disulfonate for injection, with the chemical name (±)-5'-[(R*)-[(R*)-3-amino-3-carboxypropyl]methylsulfonyl]-5'-deoxyadenosine 1,4-butane disulfonate, is suitable for intrahepatic cholestasis before and due to liver cirrhosis and intrahepatic cholestasis during pregnancy. The packaging components used for adenosylmethionine butane disulfonate for injection are generally halogenated (chlorinated) butyl rubber stoppers. According to the "Technical Guidelines for Compatibility Studies of Chemical Drugs and Elastomer Seals (Trial)", migration studies in packaging material compatibility studies are mandatory items. Compatibility studies are conducted to prove that there is no serious interaction between the packaging material and the drug, which will cause changes in the effectiveness and stability of the drug, or create safety risks.

[0003] Antioxidants are commonly used prescription additives in packaging materials. Antioxidants are added to halogenated (chlorinated) butyl rubber stoppers. Antioxidants 1010, 1076, 330 and 168 are commonly used antioxidants. There is a possibility of migration into the drug solution. Therefore, the content of antioxidants in adenosylmethionine butanedisulfonate for injection needs to be strictly controlled.

[0004] There is no report on the detection method of antioxidants in adenosylmethionine succinate for injection. In order to accurately detect the antioxidants in adenosylmethionine succinate for injection, multiple detections are required, and the operation process is cumbersome, time-consuming and labor-intensive, and the results are not ideal. Therefore, the art needs to provide a detection method that can accurately detect antioxidants in adenosylmethionine succinate for injection. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for detecting antioxidants in succinate ademethionine for injection. The ultrahigh performance liquid chromatography method can simultaneously meet the requirements of sensitivity and accurate quantitative detection of antioxidants 1010, antioxidants 1076, antioxidants 330 and antioxidants 168 in succinate ademethionine for injection, thereby effectively controlling the quality of antioxidants in succinate ademethionine for injection.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is: A method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection comprises the following steps: Preparation of the test solution: dissolve the injection adenosylmethionine butanedisulfonate sample in the injection solvent, add methanol aqueous solution to the obtained mixed system, pass through a C18 solid phase extraction column, wash with water, add methanol for elution, collect the eluate, and obtain the test solution; Preparation of reference solution: dissolving antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance in a mixed solution of methanol and tetrahydrofuran to obtain a reference solution; The reference solution and the test solution are detected by ultra high performance liquid chromatography, and the chromatographic conditions of the ultra high performance liquid chromatography include: A C18 chromatographic column with a size of 2.1 × 100 mm and a filler diameter of 1.7 μm was used; The mobile phase was acetonitrile, tetrahydrofuran and water in a volume ratio of (70~75):(15~20):(8~12).

[0007] Compared with the prior art, the detection method of antioxidants in adenosylmethionine for injection provided by the present invention is to first dissolve the adenosylmethionine for injection sample in an injection solvent for pretreatment, pass through a C18 solid phase extraction column, and elute with methanol, which can fully extract antioxidants 1010, antioxidants 1076, antioxidants 330 and antioxidants 168 in the adenosylmethionine for injection sample, with a high recovery rate; then use a C18 chromatographic column of a specific specification to perform liquid chromatography analysis on the test solution under specific conditions, and realize the accurate detection of antioxidants 1010, antioxidants 1076, antioxidants 330 and antioxidants 168 and their contents in adenosylmethionine for injection at the same time. The present invention improves the peak response intensity of the test solution and the reference solution by limiting the conditions of ultra-high liquid chromatography, thereby improving the detection accuracy. This detection method has strong specificity, low detection limit and quantification limit, good linear relationship, high recovery rate, good repeatability, strong stability, good durability, simple and quick operation, low detection cost, and accurate and reliable test results. It can provide data support for the effective control of the quality of adenosylmethionine butanedisulfonate for injection and ensure the safety of clinical drug use.

[0008] Preferably, the mass volume ratio of the adenosylmethionine butanedisulfonate sample for injection, the solvent for injection and the methanol aqueous solution is 1 g: (8-12) mL: (270-320) mL.

[0009] Preferably, the injection solvent contains L-lysine, and the concentration of the L-lysine is 80 mg / mL to 90 mg / mL.

[0010] Preferably, the injection solvent has a pH of 10.0-10.6.

[0011] For example, sodium hydroxide can be used to adjust the pH of the injection solvent.

[0012] The present invention is conducive to fully extracting antioxidant 1010, antioxidant 1076, antioxidant 330 and antioxidant 168 from the sample of succinate adenosylmethionine for injection by limiting the above conditions. Among them, the addition of L-lysine is conducive to fully dissolving the sample of succinate adenosylmethionine for injection, and can also improve the performance of the drug and enhance the efficacy.

[0013] Preferably, the volume ratio of methanol to water in the methanol aqueous solution is (5-8):1.

[0014] Preferably, the C18 solid phase extraction column is a NanoPu SelectCore C18 solid phase extraction column, the filler mass is 200 mg, the column volume is 6 mL, and the particle size of the spherical silica gel is 50 μm.

[0015] Preferably, before passing through the C18 solid phase extraction column, the C18 solid phase extraction column needs to be activated: The C18 solid phase extraction column was activated with 2.8 mL / 200 mg filler to 3.2 mL / 200 mg filler methanol and 2.8 mL / 200 mg filler to 3.2 mL / 200 mg filler water in sequence.

[0016] Preferably, the amount of water used in the water washing is 9.8 mL / 200 mg filler to 10.2 mL / 200 mg filler.

[0017] Preferably, the amount of methanol used is 9.8 mL / 200 mg filler to 10.2 mL / 200 mg filler.

[0018] Preferably, the volume ratio of methanol to tetrahydrofuran in the mixed solution of methanol and tetrahydrofuran is (5.0~5.8):1.

[0019] Preferably, the concentration of the antioxidant 1010 reference substance in the reference solution is 40 μg / mL~80 μg / mL, the concentration of the antioxidant 1076 reference substance is 1.2 μg / mL~5 μg / mL, the concentration of the antioxidant 330 reference substance is 25 μg / mL~50 μg / mL, and the concentration of the antioxidant 168 reference substance is 10 μg / mL~20 μg / mL.

[0020] Preferably, the chromatographic conditions of the ultra high performance liquid chromatography method further include: The C18 chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column or a Waters ACCQ-TAGTMULTRA C18 chromatographic column; Isocratic elution was used with a flow rate of 0.35 mL / min to 0.45 mL / min; The injection volume is 4.5 μL~5.5 μL; Column temperature is 28°C~32°C; The elution time is 14-16 minutes; The detection wavelength is 270nm~290nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a liquid chromatogram of blank solution 1 in Example 1 of the present invention; Figure 2 is a liquid chromatogram of blank solution 2 in Example 1 of the present invention; Figure 3 is a liquid chromatogram of the reference substance solution in Example 1 of the present invention; Figure 4 It is a liquid chromatogram of the test solution in Example 1 of the present invention; Figure 5 The liquid chromatogram of the spiked test solution in Example 1 of the present invention; Figure 6 It is a liquid chromatogram of the spiked test solution in Comparative Example 4 of the present invention; Figure 7 It is a liquid chromatogram of the spiked test solution in Comparative Example 5 of the present invention; Figure 8 This is a liquid chromatogram of the spiked test solution in Comparative Example 6 of the present invention; Fig. 9 It is a liquid chromatogram of the spiked test solution in Comparative Example 8 of the present invention; Fig.10 This is a liquid chromatogram of the spiked test solution in Comparative Example 9 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Example 1 This embodiment provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, comprising the following steps: (1) Solution preparation

[0024] Blank solution 1: methanol.

[0025] Blank solution 2: a mixed solution of methanol and tetrahydrofuran in a volume ratio of 7:3.

[0026] Reference substance stock solution: Take appropriate amounts of antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance, accurately weigh them, quantitatively dilute and dissolve them with blank solution 2, and prepare mixed solutions containing 5000μg, 150μg, 3000μg and 1250μg of each solution per 1mL to obtain reference substance stock solution. That is, in the reference substance stock solution, the concentrations of antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance are 5000μg / mL, 150μg / mL, 3000μg / mL and 1250μg / mL, respectively.

[0027] Reference substance solution: Accurately pipette 1 mL of reference substance stock solution into a 10 mL volumetric flask, dilute to the mark with blank solution 2, shake well to obtain the diluted stock solution, accurately pipette 1 mL of diluted stock solution into a 10 mL volumetric flask, dilute to the mark with methanol, shake well to obtain the reference substance solution.

[0028] Test solution: Take 0.1g of adenosylmethionine butanedisulfonate for injection and add it into 1mL of injection solvent (pH=10.3) containing 85.6mg L-lysine to dissolve it, then add methanol aqueous solution (mixed by 4mL water and 25mL methanol), mix well, pass through the activated Nanopore SelectCore C18 solid phase extraction column (referred to as Nanopore C18 SPE, activated with 3mL methanol and 3mL water in sequence), wash the solid phase extraction column with 10mL water, add 10mL methanol for elution, collect the eluate and put it into a 10mL volumetric flask, dilute to the scale with methanol, shake well to obtain the test solution.

[0029] Spiked test solution: accurately transfer 1 mL of reference stock solution into a 10 mL volumetric flask, dilute to the mark with blank solution 2, shake well to obtain the diluted stock solution; take 0.1 g of adenosylmethionine for injection and add it to 1 mL of injection solvent (pH = 10.3) containing 85.6 mg of L-lysine to dissolve, then add methanol aqueous solution (mixed by 4 mL of water and 25 mL of methanol) and 1 mL of diluted stock solution, mix well, pass through activated NanoSpec C18 SPE (activated with 3 mL of methanol and 3 mL of water in sequence), wash the solid phase extraction column with 10 mL of water, add 10 mL of methanol for elution, collect the eluate and place it in a 10 mL volumetric flask, dilute to the mark with methanol, shake well to obtain the spiked test solution.

[0030] (2) Use ultra high performance liquid chromatography to detect the blank solutions 1-2, reference solution, test solution and spiked test solution. The specific chromatographic conditions of ultra high performance liquid chromatography include: A Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) was used; The mobile phase was acetonitrile, tetrahydrofuran, and water in a volume ratio of 73:17:10; The flow rate was 0.4 mL / min, isocratic elution, and the elution time was 15 min; The injection volume was 5 μL; The detection wavelength is 280nm; The column temperature was 30°C; The liquid chromatogram results of blank solution 1~2, reference solution, test solution and spiked test solution are as follows Figures 1 to 5 As shown, the separation degree of each impurity in the spiked test solution was tested, and the test results are shown in Table 1. Figures 1 to 5 As can be seen from Table 1, in the method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection provided in this embodiment, blank solvents 1-2 do not interfere with the detection of each component, the separation between the components meets the requirements, and the method has good specificity.

[0031] Table 1 Retention time and separation of each impurity in the spiked test solution

[0032] Methodological validation Durability test The reference solution was prepared according to the method of Example 1, and only the flow rate and column temperature were changed to control the single factor variable. The detection was performed according to the detection method of Example 1, and the detection results are shown in Tables 2 and 3. It can be seen from Tables 2 and 3 that the separation degree of each impurity meets the requirements when the conditions are changed, indicating that the detection method provided by the present invention has good durability.

[0033] Table 2 Separation of impurities in reference solution at different flow rates

[0034] Table 3 Separation of impurities in reference solution at different column temperatures

[0035] Limit of detection and limit of quantitation test Accurately weigh appropriate amounts of antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance, prepare reference substance solutions according to the method in Example 1, and detect according to the chromatographic conditions in Example 1, record the chromatogram, and the peak height is 10 times the baseline noise as the quantitative limit, and the peak height is 3 times the baseline noise as the detection limit. The test results of the quantitative limit and the detection limit are shown in Table 4, and the repeatability test results of the quantitative limit are shown in Table 5. It can be seen from Tables 4 to 5 that the detection limit and the quantitative limit of the detection method provided by the present invention are low, and have the characteristics of high sensitivity; the relative standard deviation (RSD) of the peak area of ​​each antioxidant repeated 6 times is 7.91% at most, indicating that the detection method provided by the present invention has good repeatability of the quantitative limit.

[0036] Table 4 Test results of limit of quantitation and limit of detection

[0037] Table 5 Results of repeatability test of limit of quantitation (peak area)

[0038] Linear relationship test Appropriate amounts of antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance were accurately weighed, and sample solutions of 7 concentration levels were prepared respectively. The above sample solutions were tested according to the chromatographic conditions in Example 1, and the peak area of ​​the chromatogram was determined. The component concentration in the sample solution was used as the abscissa and the peak area was used as the ordinate to draw a standard working curve and establish a linear equation. The final test results are shown in Tables 6 to 9. From the data in Tables 6 to 9, it can be seen that in the detection method provided by the present invention, the concentrations of antioxidant 1010, antioxidant 1076, antioxidant 330 and antioxidant 168 have a good linear relationship with the peak area measured by the chromatogram.

[0039] Table 6 Linear relationship test results of antioxidant 1010

[0040] Table 7 Linear relationship test results of antioxidant 330

[0041] Table 8 Linear relationship test results of antioxidant 1076

[0042] Table 9 Linear relationship test results of antioxidant 168

[0043] Recovery test Accurately weigh appropriate amounts of antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance, quantitatively dilute and dissolve with blank solution 2, and prepare solutions containing 5000μg, 150μg, 3000μg and 1250μg per 1mL as stock solutions. Take 0.8mL, 1.0mL and 1.2mL of the stock solutions respectively, place them in 10mL volumetric bottles, dilute to the mark with blank solution 2, shake well, and use them as stock solutions with low, medium and high recovery rates. Take 0.1g of injection adenosylmethionine succinate and add it to 1mL of injection solvent (pH=10.3) containing 85.6mg L-lysine to dissolve, then add methanol aqueous solution (mixed by 4mL water and 25mL methanol) and 1mL recovery rate stock solution, mix evenly, pass through activated nano-spectrum C18 SPE (activated by 3mL methanol and 3mL water in sequence), wash the solid phase extraction column with 10mL water, add 10mL methanol for elution, collect the eluate and place it in a 10mL volumetric flask, dilute to the scale with methanol, shake well, as the recovery rate test solution, each concentration is parallel 3. Detection was performed according to the detection method and chromatographic conditions in Example 1. The recovery rates of each antioxidant in the injection adenosylmethionine succinate were finally measured as shown in Tables 10 to 13. It can be seen from Tables 10 to 13 that the recoveries of the antioxidants at three different addition amounts were all between 96% and 104%, and the maximum RSD was 2.44%, indicating that the detection method provided by the present invention has good accuracy.

[0044] Table 10 Recovery test results of antioxidant 1010

[0045] Table 11 Recovery test results of antioxidant 330

[0046] Table 12 Recovery test results of antioxidant 1076

[0047] Table 13 Recovery test results of antioxidant 168

[0048] Repeatability test Six groups of succinate adenosylmethionine for injection samples from the same batch were taken, and test solutions were prepared according to the method in Example 1. The samples were injected according to the chromatographic conditions in Example 1, and the contents of various antioxidants in different groups of succinate adenosylmethionine for injection samples were detected respectively. The test results showed that antioxidant 1010, antioxidant 1076, antioxidant 330 and antioxidant 168 were not detected, with good repeatability.

[0049] Solution stability test The reference solution was prepared according to the method in Example 1, and after being placed at room temperature for 0 h, 1 h, 2 h, 4 h, 6 h, 9 h and 12 h, ultra high performance liquid chromatography was performed under the chromatographic conditions in Example 1, and the stability test results of the solution are shown in Table 14. As can be seen from Table 14, the reference solution was placed at room temperature for 12 h and the same method was used to perform the test, and the results obtained were basically consistent, and the RSD of the peak area of ​​each impurity was less than 1.66%, indicating that the reference solution prepared by the present invention has good stability.

[0050] Table 14 Stability of reference solution (peak area)

[0051] Example 2 This embodiment provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to embodiment 1, except that the chromatographic column is replaced with Waters ACCQ-TAG TM ULTRA C18 chromatographic column (2.1×100 mm, 1.7 μm). The other conditions are the same as those in Example 1 and will not be described in detail.

[0052] The recovery test was performed according to the detection method and chromatographic conditions in this embodiment. The spiked recovery of each antioxidant in adenosylmethionine succinate for injection was finally measured as follows: 97.70% for antioxidant 1010, 96.18% for antioxidant 330, 100.95% for antioxidant 1076, and 92.50% for antioxidant 168. It can be seen that the recovery of each antioxidant at 3 different addition amounts is between 96% and 101%, indicating that the detection method provided by the present invention has good accuracy.

[0053] Comparative Example 1 This comparative example provides a method for detecting antioxidants in adenosylmethionine succinate for injection, which is similar to Example 1, except that the methanol aqueous solution is replaced with an acetonitrile aqueous solution (mixed with 4 mL of water and 25 mL of acetonitrile). The remaining conditions are the same as those in Example 1 and will not be repeated.

[0054] The recovery test was carried out according to the detection method and chromatographic conditions in this comparative example, and the methanol aqueous solution in the recovery stock solution was replaced with an acetonitrile aqueous solution (mixed with 4 mL of water and 25 mL of acetonitrile). The final recovery of the spiked antioxidants in adenosylmethionine succinate for injection was measured as shown in Table 15.

[0055] Comparative Example 2 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that the methanol aqueous solution is replaced with an ethanol aqueous solution (mixed with 4 mL of water and 25 mL of ethanol). The remaining conditions are the same as those in Example 1 and will not be repeated.

[0056] The recovery test was carried out according to the detection method and chromatographic conditions in this comparative example, and the methanol aqueous solution in the recovery stock solution was replaced with an ethanol aqueous solution (mixed by 4 mL of water and 25 mL of ethanol). The final recovery of the spiked antioxidants in adenosylmethionine succinate for injection was measured as shown in Table 15.

[0057] Table 15 Spiked recovery test results in different solvents

[0058] Comparative Example 3 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that 10 mL of methanol (elution solvent) is replaced with a mixed solution of 7 mL of methanol and 3 mL of dichloromethane. The remaining conditions are the same as those in Example 1 and will not be repeated.

[0059] The recovery test was carried out according to the detection method and chromatographic conditions in this comparative example, and 10 mL of methanol (elution solvent) in the recovery stock solution was replaced with a mixed solution of 7 mL of methanol and 3 mL of dichloromethane. The results showed that: when running for a long time, the column efficiency and peak shape deteriorated.

[0060] Comparative Example 4 This comparative example provides a method for detecting antioxidants in adenosylmethionine succinate for injection, which is similar to Example 1, except that the mobile phase is replaced with water and acetonitrile in a volume ratio of 10:90, and the elution time is 20 minutes. The other conditions are the same as those in Example 1 and will not be repeated.

[0061] The liquid chromatogram results of the spiked test solution are as follows Figure 6 As shown, antioxidant 330, antioxidant 1076 and antioxidant 168 did not show any peaks.

[0062] Comparative Example 5 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that the mobile phase is replaced with a 0.02 mol / L ammonium formate aqueous solution (containing 0.3% formic acid) and acetonitrile in a volume ratio of 10:90, and the elution time is 20 min. The remaining conditions are the same as those in Example 1 and will not be repeated.

[0063] The liquid chromatogram results of the spiked test solution are as follows Figure 7As shown, antioxidant 330, antioxidant 1076 and antioxidant 168 did not show any peaks.

[0064] Comparative Example 6 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that the mobile phase is replaced with water, acetonitrile and tetrahydrofuran in a volume ratio of 10:80:10, and the elution time is 20 minutes. The other conditions are the same as those in Example 1 and will not be repeated.

[0065] The liquid chromatogram results of the spiked test solution are as follows Figure 8 As shown, the peak is late (causing antioxidant 168 to not peak), the test time is long and the efficiency is low.

[0066] Comparative Example 7 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that the mobile phase is replaced with water, methanol and tetrahydrofuran in a volume ratio of 10:73:17. The remaining conditions are the same as those in Example 1 and will not be repeated.

[0067] The quantitative limit detection was performed according to the chromatographic conditions in this comparative example, and the test results were as follows: antioxidant 1010 was 0.52 μg / mL, antioxidant 330 was 0.33 μg / mL, antioxidant 1076 was 1.02 μg / mL, and antioxidant 168 was 1.45 μg / mL.

[0068] Comparative Example 8 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that the Nanopore C18 SPE is replaced with Nanopore HLB SPE (Nanopore SelectCore HLB solid phase extraction column). The remaining conditions are the same as those in Example 1 and will not be repeated.

[0069] The liquid chromatogram results of the spiked test solution are as follows Fig. 9 As shown, antioxidant 1076 was not detected.

[0070] The recovery test was carried out according to the detection method and chromatographic conditions in this comparative example, and the Napu C18 SPE was replaced by the Napu HLB SPE accordingly. The results were as follows: Antioxidant 1010 was 77.33%, Antioxidant 330 was 76.25%, Antioxidant 1076 was 0%, and Antioxidant 168 was 72.07%.

[0071] Comparative Example 9 This comparative example provides a method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, which is similar to Example 1, except that the Waters ACQUITY UPLC BEH C18 column is replaced with Waters XSelectHSS T3 (2.1×100 mm, 2.5 μm). The remaining conditions are the same as those in Example 1 and will not be described in detail.

[0072] The liquid chromatogram results of the spiked test solution are as follows Fig.10 The peak area results are shown in Table 16.

[0073] Table 16 Test results of spiked test solution under different chromatographic columns (peak area)

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection, characterized in that: The following steps are involved: Preparation of the test solution: dissolve the injection adenosylmethionine butanedisulfonate sample in the injection solvent, add methanol aqueous solution to the obtained mixed system, pass through a C18 solid phase extraction column, wash with water, add methanol for elution, collect the eluate, and obtain the test solution; Preparation of reference solution: dissolving antioxidant 1010 reference substance, antioxidant 1076 reference substance, antioxidant 330 reference substance and antioxidant 168 reference substance in a mixed solution of methanol and tetrahydrofuran to obtain a reference solution; The reference solution and the test solution are detected by ultra high performance liquid chromatography, and the chromatographic conditions of the ultra high performance liquid chromatography include: A C18 chromatographic column with a size of 2.1 × 100 mm and a filler diameter of 1.7 μm was used; The mobile phase was acetonitrile, tetrahydrofuran and water in a volume ratio of (70~75):(15~20):(8~12).

2. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection as claimed in claim 1, characterized in that: The mass volume ratio of the injection adenosylmethionine butanedisulfonate sample, the injection solvent and the methanol aqueous solution is 1 g: (8-12) mL: (270-320) mL; The injection solvent contains L-lysine, and the concentration of the L-lysine is 80 mg / mL to 90 mg / mL; The pH of the injection solvent is 10.0-10.6; The volume ratio of methanol to water in the methanol aqueous solution is (5-8):

1.

3. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection as claimed in claim 1, characterized in that: The C18 solid phase extraction column is a NanoPu SelectCore C18 solid phase extraction column, the filler mass is 200 mg, the column volume is 6 mL, and the particle size of the spherical silica gel is 50 μm.

4. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection according to claim 1 or 3, characterized in that: The amount of water used in the water washing is 9.8 mL / 200 mg filler to 10.2 mL / 200 mg filler; The amount of methanol used is 9.8 mL / 200 mg filler to 10.2 mL / 200 mg filler.

5. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection as claimed in claim 1, characterized in that: The volume ratio of methanol to tetrahydrofuran in the mixed solution of methanol and tetrahydrofuran is (5.0-5.8):

1.

6. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection as claimed in claim 1, characterized in that: The concentration of the antioxidant 1010 reference substance in the reference substance solution is 40 μg / mL to 80 μg / mL, the concentration of the antioxidant 1076 reference substance is 1.2 μg / mL to 5 μg / mL, the concentration of the antioxidant 330 reference substance is 25 μg / mL to 50 μg / mL, and the concentration of the antioxidant 168 reference substance is 10 μg / mL to 20 μg / mL.

7. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection as claimed in claim 1, characterized in that: The chromatographic conditions of the ultra high performance liquid chromatography method also include: the C18 chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column or a Waters ACCQ-TAGTM ULTRA C18 chromatographic column.

8. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection as claimed in claim 1, characterized in that: The chromatographic conditions of the ultra high performance liquid chromatography also include: isocratic elution with a flow rate of 0.35 mL / min to 0.45 mL / min.

9. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection according to claim 1 or 8, characterized in that: The chromatographic conditions of the ultra high performance liquid chromatography also include: an injection volume of 4.5 μL to 5.5 μL; and / or The elution time is 14min~16min.

10. The method for detecting antioxidants in adenosylmethionine butanedisulfonate for injection according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography method also include: a column temperature of 28° C. to 32° C.; and / or The detection wavelength is 270nm~290nm.

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