A method for detecting the content of avibactam sodium

Abstract

The invention discloses a method for detecting the content of avibactam sodium. The method adopts high performance liquid chromatography to analyze the avibactam sodium of the product, separates the avibactam sodium from impurities, effectively detects the content of the avibactam sodium, reduces the detection difficulty, has more accurate detection results, shortens the time consumption, and has higher detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular to a method for detecting the content of avibactam sodium. Background Art

[0002] Avibactam sodium belongs to the diazabicyclooctanone compound and is currently the most promising non-β-lactam inhibitor. It does not have significant antibacterial activity itself, but can inhibit type A (including ESBL and KPC) and type C β-lactamases. Therefore, when used in combination with various cephalosporins and carbapenem antibiotics, it has broad-spectrum antibacterial activity, especially against Escherichia coli and Klebsiella pneumoniae containing ultra-extended-spectrum β-lactamases, Escherichia coli containing excessive AmpC enzymes, and Escherichia coli containing both AmpC and ultra-extended-spectrum β-lactamases.

[0003] β-lactamase serine nucleophilically attacks the amide bond of avibactam, opens the ring to form a covalent bond, and obtains an enzyme-inhibitor complex, which is an enzyme-inhibiting form, and does not undergo hydrolysis. Then, cyclization forms a lactam ring to obtain avibactam. The rate of ring opening caused by nucleophilic attack is much greater than cyclization, causing β-lactamase to be basically in an inhibited state. In this process, the structure of avibactam itself can be restored by reverse reaction, so it has a long-lasting enzyme inhibitory effect. Avibactam is a new type of β-lactamase inhibitor with a non-lactam structural scaffold that irreversibly inhibits the lactamase of Mycobacterium tuberculosis.

[0004] At present, the material detection method of avibactam sodium is an important detection item in the drug detection process of avibactam sodium. Due to the structural characteristics of avibactam sodium, it is difficult to distinguish between the content and impurities during the detection of its content, which increases the difficulty of detection, makes the detection results inaccurate, and takes a long time, resulting in the inability to obtain evidence for the relevant detection content and low detection sensitivity. Therefore, a content detection method of avibactam sodium is urgently needed to solve the above problems. Summary of the invention

[0005] The invention provides a method for detecting the content of avibactam sodium, which is used to solve the technical problems that, in the process of detecting the content of avibactam sodium, the content and impurities are difficult to distinguish, the detection difficulty is increased, the detection result is inaccurate, the time is long, and the relevant detection content cannot be obtained and the detection sensitivity is low.

[0006] In view of this, the present invention provides a method for detecting the content of avibactam sodium, and adopts high performance liquid chromatography to detect the content of avibactam sodium in the product avibactam sodium.

[0007] Optionally, the HPLC conditions are:

[0008] Using porous graphite carbon as filler;

[0009] Mobile phase A: 0.1 mol / L potassium dihydrogen phosphate solution;

[0010] Mobile phase B: 0.2 mol / L potassium dihydrogen phosphate solution-acetonitrile, where the volume ratio of potassium dihydrogen phosphate solution to acetonitrile is 50:50;

[0011] Perform gradient elution;

[0012] The column temperature was 60°C;

[0013] The detection wavelength is 195nm;

[0014] Flow rate: 1.5 ml / min;

[0015] The injection volume was 5 μl.

[0016] Optionally, the gradient elution procedure is:

[0017] 0min, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%;

[0018] 15 min, the volume fraction of mobile phase A was 0%, and the volume fraction of mobile phase B was 100%;

[0019] 19 min, the volume fraction of mobile phase A was 0%, and the volume fraction of mobile phase B was 100%;

[0020] 19.1min, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%;

[0021] 24min, the volume fraction of mobile phase A was 95%, and the volume fraction of mobile phase B was 5%.

[0022] Optionally, the following detection steps are included:

[0023] S1: Preparation of solvent: water and acetonitrile were mixed in a volume ratio of 90:10 to prepare a solvent.

[0024] S2: Preparation of test solution

[0025] Take an appropriate amount of avibactam sodium, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.1 mg of avibactam sodium per 1 ml;

[0026] S3: Preparation of reference solution

[0027] Take an appropriate amount of avibactam sodium reference substance, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.1 mg of avibactam sodium per 1 ml;

[0028] S4: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the content of avibactam sodium in the test sample by the peak area according to the external standard method.

[0029] It can be seen from the above technical solutions that the embodiments of the present invention have the following advantages:

[0030] The present invention provides a method for detecting the content of avibactam sodium. High performance liquid chromatography is used to analyze the avibactam sodium of the present product, so that the avibactam sodium is separated from impurities, the content of the avibactam sodium is effectively detected, the detection difficulty is reduced, the detection result is more accurate, the time consumption is short, and the detection sensitivity is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0032] Figure 1 A comparison chart of the content of avibactam sodium of the present invention;

[0033] Figure 2 It is the system applicability spectrum of the related substances of the present invention;

[0034] Figure 3 is the applicability spectrum of the sodium ion system of the present invention;

[0035] Figure 4 is a reference spectrum of the residual solvent 1 of the present invention;

[0036] Figure 5 It is the reference spectrum of residual solvent 2 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the technical scheme of the present invention better understood by the personnel of the technical field, the technical scheme in the embodiment of the present invention is clearly and completely described below. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiment in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods.

[0038] Example 1

[0039] For easier understanding, see Figure 1The present invention provides a method for detecting the content of avibactam sodium, which uses high performance liquid chromatography to detect the content of avibactam sodium;

[0040] 1. Instruments and equipment: High performance liquid chromatograph (LC-2030), chromatographic column model is Thermo Scientific Hypercarb, specification is 100mm×4.6mm, 1 / 10,000 electronic balance (T-114), 1 / 100,000 electronic balance (AUW220D), pH meter (PB-10).

[0041] 2. Chromatographic conditions: porous graphite carbon was used as filler; mobile phase A: 0.1 mol / L potassium dihydrogen phosphate solution; mobile phase B: 0.2 mol / L potassium dihydrogen phosphate solution-acetonitrile, wherein the volume ratio of potassium dihydrogen phosphate solution to acetonitrile was 50:50; gradient elution was performed; column temperature was 60°C; detection wavelength was 195 nm; flow rate was 1.5 ml / min; injection volume was 5 μl.

[0042] The procedure of gradient elution is shown in Table 1 below:

[0043] Table 1: Gradient elution program

[0044] Time / (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 15 0 100 19 0 100 19.1 95 5 24 95 5

[0045] 3. Testing steps:

[0046] 101: Preparation of solvent: Mix water and acetonitrile in a volume ratio of 90:10 to prepare a solvent.

[0047] 102: Preparation of test solution

[0048] Take an appropriate amount of avibactam sodium (Formula I), accurately weigh it, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.1 mg of avibactam sodium per 1 ml;

[0049] 103: Preparation of reference solution

[0050] Take an appropriate amount of avibactam sodium reference substance, weigh it accurately, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.1 mg of avibactam sodium per 1 ml;

[0051] 104: According to the system suitability requirements, the number of theoretical plates calculated based on the avibactam sodium peak is greater than 5000, which meets the standard for avibactam sodium content.

[0052] 105: Determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the content of avibactam sodium in the test sample by the peak area according to the external standard method.

[0053] Example 2

[0054] For easier understanding, see Figures 2 to 5 , determination of related substances

[0055] 1. Determination of impurity B (Formula II), impurity C (Formula III), impurity D (Formula IV), impurity K (Formula V), AVB-1 (Formula VI), SMA (Formula VII), and AVB-2 (Formula VIII):

[0056] 1.1 Solution preparation

[0057] Test solution: Take an appropriate amount of avibactam sodium, weigh accurately, dissolve in water and dilute to make a solution containing approximately 5 mg per 1 ml.

[0058] Reference solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with water to make a solution containing approximately 5 μg per 1 ml.

[0059] System suitability solution: Take appropriate amounts of impurity B, impurity C, impurity D, impurity K, SMA, AVB-1, AVB-2 and avibactam sodium reference substance, dissolve in water and dilute to prepare a solution containing approximately 5 mg of avibactam sodium and 5 μg of each impurity per 1 ml.

[0060] 1.2 Chromatographic conditions

[0061] Porous graphite carbon was used as filler, mobile phase A was 0.1 mol / L potassium dihydrogen phosphate solution, wherein phosphoric acid was used to adjust the pH value to 2.5; mobile phase B was 0.1 mol / L potassium dihydrogen phosphate solution-acetonitrile, wherein phosphoric acid was used to adjust the pH value to 2.5, and the volume ratio of potassium dihydrogen phosphate solution to acetonitrile was 50:50; detection wavelength was 195 nm; flow rate was 1.5 ml / min; injection volume was 10 μl; column temperature was 60°C; elution mode was gradient elution;

[0062] The elution procedure is shown in Table 2 below:

[0063] Table 2: Gradient elution program

[0064] Time / (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 75 25 30 0 100 35 0 100 35.01 100 0 40 100 0

[0065] 1.3 System suitability requirements

[0066] In the system suitability solution chromatogram, Figure 2 As shown, the order of peaks is impurity B, impurity K, impurity C / D, avibactam sodium, AVB-1, SMA and AVB-2, and the separation between each chromatographic peak meets the requirements.

[0067] 1.4 Determination

[0068] Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, detect under the above chromatographic conditions, and record the chromatogram.

[0069] 1.5 Limits

[0070] If there are impurity peaks in the chromatogram of the test solution, the corrected peak area of ​​impurity B (multiplied by the correction factor of 2.81) shall not be greater than the main peak area of ​​the control solution (0.1%), the corrected peak area of ​​impurity K (multiplied by the correction factor of 0.78) shall not be greater than the main peak area of ​​the control solution (0.1%), the corrected peak area of ​​impurity C / D (multiplied by the correction factor of 1.20) shall not be greater than the main peak area of ​​the control solution (0.1%), and the corrected peak area of ​​AVB-1 (multiplied by the correction factor of 0.50) shall not be greater than The main peak area of ​​the control solution (0.1%), the peak area after SMA correction (multiplied by the correction factor 0.63) shall not be greater than the main peak area of ​​the control solution (0.1%), the peak area after AVB-2 correction (multiplied by the correction factor 0.31) shall not be greater than the main peak area of ​​the control solution (0.1%), and the peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the control solution (0.1%). The sum of the peak areas of all impurities calculated based on the corrected peak area shall not be greater than 10 times the main peak area of ​​the control solution (1.0%).

[0071] 2. Determination of sodium ions

[0072] 2.1 Solution preparation

[0073] Preparation of test solution

[0074] Take the avibactam sodium of this product, weigh it accurately, add water to dissolve it and quantitatively dilute it to make a solution containing about 0.5 mg of avibactam sodium per 1 ml.

[0075] Preparation of reference solution

[0076] Take an appropriate amount of sodium chloride reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing approximately 40 μg of sodium ions per 1 ml.

[0077] According to the system suitability requirements, the system suitability solution chromatogram is as follows Figure 3 As shown, the peak elution order is avibactam sodium and sodium ion, and the separation degree between the avibactam sodium peak and the sodium ion peak meets the requirements.

[0078] 2.2 Chromatographic conditions are as follows:

[0079] Use a cation exchange column, model Dionex IonPac TM CS12A 4*250mm; the detector is a conductivity detector; the detection mode is suppressed conductivity detection; the column temperature is 30℃; 0.025mol / L methanesulfonic acid solution is used as the eluent; the flow rate is 1.0ml per minute; the injection volume is 10μl.

[0080] 2.3 Determination: Accurately measure the test solution and the reference solution, inject them into the ion chromatograph respectively, and record the chromatograms.

[0081] 2.4 Limit detection: Calculated by peak area using the external standard method, the sodium ion content is 7.5% to 8.5%.

[0082] Conclusion: It complies with the experimental requirements.

[0083] 3. Determination of residual solvent 1

[0084] 3.1 Solution preparation

[0085] Preparation of test solution

[0086] An appropriate amount of avibactam sodium is accurately weighed, dissolved in N,N-dimethylformamide and quantitatively diluted to prepare a solution containing approximately 200 mg of avibactam sodium per 1 ml.

[0087] Preparation of reference solution

[0088] Weigh appropriate amounts of methanol, ethanol, isopropanol, dichloromethane, tert-butanol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, benzene, methyl isobutyl ketone, acetic acid, toluene, benzyl alcohol, and isooctanoic acid, accurately weigh, and dilute with N,N-dimethylformamide to make a solution containing 1.0 mg each of ethanol, isopropanol, methyl tert-butyl ether, ethyl acetate, and acetic acid, 120 μg of dichloromethane, 0.7 mg of tert-butanol, 144 μg of tetrahydrofuran, 0.6 mg of methanol, 0.90 mg of methyl isobutyl ketone, 4 μg of benzene, 178 μg of toluene, 86.2 μg of benzyl alcohol, and 210 μg of isooctanoic acid per 1 ml.

[0089] 3.2 Chromatographic conditions are as follows:

[0090] The capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid is Agilent DB-624, with a specification of 60m×0.32mm; the starting temperature is 40°C, maintained for 10 minutes, and then increased to 125°C at a rate of 10°C per minute and maintained for 5 minutes; then increased to 180°C at a rate of 5°C per minute and maintained for 30 minutes; the injection port temperature is 200°C, the detector temperature is 250°C; the split ratio is 10:1; the flow rate is 2ml per minute; the carrier gas is nitrogen; the injection volume is 1μl.

[0091] 3.3 According to the system suitability requirements, the chromatogram of the reference solution is as follows Figure 4 As shown in the figure, the peak order is methanol, ethanol, isopropanol, dichloromethane, tert-butyl alcohol, methyl tert-butyl ether, ethyl acetate, tetrahydrofuran, benzene, methyl isobutyl ketone, acetic acid, toluene, benzyl alcohol, isooctanoic acid. The separation degree between each component peak meets the requirements.

[0092] 3.4 Determination: Take the test solution and the reference solution and inject them directly respectively, and record the chromatograms.

[0093] 3.5 Limit detection: Calculated by peak area using the external standard method, methanol is less than 0.3%, ethanol, isopropanol, methyl tert-butyl ether, ethyl acetate and acetic acid are less than 0.5%, dichloromethane is less than 0.06%, tert-butyl alcohol is less than 0.35%, tetrahydrofuran is less than 0.072%, benzene is less than 0.0002%, methyl isobutyl ketone is less than 0.45%, toluene is less than 0.089%, benzyl alcohol is less than 0.0431%, and isooctanoic acid is less than 0.105%.

[0094] 4. Determination of residual solvent 2

[0095] 4.1 Solution preparation

[0096] Preparation of test solution

[0097] Take an appropriate amount of avibactam sodium, weigh it accurately, add N,N-dimethylformamide to dissolve it and quantitatively dilute it to make a solution containing about 100 mg of avibactam sodium per 1 ml.

[0098] Preparation of reference solution

[0099] Take appropriate amounts of triethylamine, diethylamine, diisopropylethylamine, chlorobenzene, and dimethyl sulfoxide, weigh them accurately, and quantitatively dilute them with N,N-dimethylformamide to prepare a mixed solution containing approximately 500 μg of triethylamine, 55 μg of diethylamine, 74.2 μg of diisopropylethylamine, 36 μg of chlorobenzene, and 10 μg of dimethyl sulfoxide per 1 ml.

[0100] 4.2 Chromatographic conditions are as follows:

[0101] The chromatographic column was a capillary column with polyethylene glycol as the stationary liquid, with a starting temperature of 50°C, which was increased to 100°C at a rate of 8°C per minute, maintained for 10 minutes, and then increased to 180°C at a rate of 15°C per minute, maintained for 5 minutes; the split ratio was 10:1; the flow rate was 2 ml per minute; the injection port temperature was 200°C; the detector was a FID detector; the detector temperature was 250°C; the carrier gas was nitrogen; and the injection volume was 1 μl.

[0102] 4.3 According to the system suitability requirements, the chromatogram of the reference solution is as follows Figure 5 As shown in the figure, the peak order is triethylamine, diethylamine, diisopropylethylamine, chlorobenzene and dimethyl sulfoxide. The separation degree between each component peak meets the requirements.

[0103] 4.4 Determination: Take the test solution and the reference solution and inject them directly respectively, and record the chromatograms.

[0104] 4.5 Limit detection: calculated by peak area using the external standard method, triethylamine is less than 0.5%, diethylamine is less than 0.055%, diisopropylethylamine is less than 0.0742%, chlorobenzene is less than 0.036%, and dimethyl sulfoxide is less than 0.01%.

[0105] In summary, the present invention provides a method for detecting the content of avibactam sodium, using high performance liquid chromatography to detect avibactam sodium, related substances, impurity J, isomers, tetrabutylammonium, sodium ions, residual solvent 1 and residual solvent 2 in the product avibactam sodium, through an elution procedure, avibactam sodium and other impurities in the product avibactam sodium are analyzed to separate peak values, the content of avibactam sodium is accurately detected, and the detection sensitivity is improved.

[0106] In addition, the structural formula of the product avibactam sodium and related substances of the present invention is as follows:

[0107]

[0108]

[0109] As described above, the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of avibactam sodium and its related substances, characterized in that: The content of avibactam sodium is detected by high performance liquid chromatography; the structural formula of avibactam sodium is Formula I; The HPLC conditions are: Porous graphite carbon was used as filler, mobile phase A was 0.1 mol / L potassium dihydrogen phosphate solution, wherein phosphoric acid was used to adjust the pH value to 2.5; mobile phase B was 0.1 mol / L potassium dihydrogen phosphate solution-acetonitrile, wherein phosphoric acid was used to adjust the pH value to 2.5, and the volume ratio of potassium dihydrogen phosphate solution to acetonitrile was 50:50; detection wavelength was 195 nm; flow rate was 1.5 ml / min; injection volume was 10 μl; column temperature was 60°C; elution mode was gradient elution; The elution procedure is shown in the following table: The related substances include impurity B with the structural formula of formula II, impurity C with the structural formula of formula III, impurity D with the structural formula of formula IV, impurity K with the structural formula of formula V, AVB-1 with the structural formula of formula VI, SMA with the structural formula of formula VII, AVB-2 with the structural formula of formula VIII, and the chemical structural formulas are as follows:

2. A method for detecting the content of avibactam sodium and its related substances according to claim 1, characterized in that: The following detection steps are included: S1: Preparation of solvent Water and acetonitrile were mixed in a volume ratio of 90:10 to prepare a solvent; S2: Preparation of test solution Take an appropriate amount of avibactam sodium, weigh accurately, dissolve in water and dilute to make a solution containing about 5 mg per 1 ml; S3: Preparation of reference solution Accurately measure an appropriate amount of the test solution and dilute it quantitatively with water to make a solution containing about 5 μg per 1 ml; S4: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the content of avibactam sodium in the test sample by the peak area according to the external standard method.

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