A method for analyzing genotoxic impurities in sulbactam sodium raw material

Abstract

The invention discloses an analysis method for a genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in a sulbactam sodium raw material. The analysis method has strong specificity and high sensitivity, and can be used for monitoring the target product of the synthesis method of the invention, and can also be used alone for detecting the genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in the sulbactam sodium raw material, thereby ensuring the safety of the drug. The reaction raw materials of the synthesis method of the impurity are simple and easy to obtain, and the reaction is carried out in only one step, and the operation is simple.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and analysis, and specifically relates to an analysis method for impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in a sulbactam sodium raw material drug. Background Art

[0002]

[0003] Formula 1 (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid structure

[0004] (2S)-2-Amino-3-ethoxysulfinyl-3-methylbutyric acid belongs to the sulfinic ester compound, and its structure is shown in Formula 1. It is similar to the sulfonate compounds and may have potential genotoxicity. It was evaluated as positive by Derek and Sarah software and classified as Class 3. For drug safety considerations, it needs to be controlled as a genotoxic impurity.

[0005] (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid can be prepared by reacting (2S)-2-amino-3-sulfinyl-3-methylbutyric acid with an active ethoxy group, and the specific synthesis pathway is shown in Formula 2. (2S)-2-amino-3-sulfinyl-3-methylbutyric acid is a degradation impurity (EP impurity A) of sulbactam sodium listed in the European Pharmacopoeia, and the specific degradation pathway is shown in Formula 3.

[0006]

[0007] Formula 2 (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid synthesis route

[0008]

[0009] Formula 3: Production pathway of EP impurity A in sulbactam sodium

[0010] Since sulbactam sodium is a synergistic drug of β-lactam antibiotics, it is often used in combination with other antibiotics such as ampicillin, cefoperazone sodium, piperacillin, mezlocillin, etc. to increase antibacterial ability. The synthesis or refining process of sulbactam sodium often uses solvents containing active ethoxy groups such as ethanol and ethyl acetate. The specific reaction process is shown in Formula 4. Therefore, when using drugs containing sulbactam sodium, it is necessary to pay great attention to the control of potential genotoxic impurities (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid. As an antibiotic, the maximum daily dose of sulbactam sodium is 4g / day, and the treatment period is calculated as more than 1 to 12 months. The maximum daily intake of a single genotoxic impurity is 20μg / day. The LTL method is used to calculate the genotoxic impurity limit, and the single genotoxic impurity limit is 20 μg / 4g=5ppm (0.0005%), that is, the content of the genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in sulbactam sodium should not be higher than 5ppm. How to quickly and efficiently control the content of the genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in sulbactam sodium raw materials is extremely important for the safe use of drugs containing sulbactam sodium.

[0011]

[0012] Formula 4 Synthesis route of sulbactam sodium

[0013] There is no standard product of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid on the market, and the research can only be done by chemical synthesis and preparation of the impurity. No reports on the synthesis of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid were found through literature search. According to the basic principles of organic chemical synthesis, ethanol can be used to react with (2S)-2-amino-3-sulfinyl-3-methylbutyric acid (sulbactam sodium EP impurity A) to obtain the target product. It can be seen from its structure that the group that can be esterified by the substance includes a carboxyl group in addition to the sulfinic acid group. If the site-specific synthesis requires the protection of the carboxyl group, the reaction process is complicated, the operation steps are cumbersome, and the target product isomers are shown in Formula 5. Therefore, if there is a simple and convenient synthesis method and a method for analyzing the target product and its isomers that can be quickly distinguished, the complex site-specific synthesis of the sulfinic acid group can be omitted, and the cumbersome separation and purification of the target product is not required. If the analytical method has strong specificity and good sensitivity, it can also be used alone for the limit method determination of the impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid content in sulbactam sodium.

[0014]

[0015] Isomers generated by the synthesis reaction of formula 5 Summary of the invention

[0016] The invention provides a method for synthesizing and analyzing a genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in a sulbactam sodium raw material. The synthesis method is simple to operate, has only one-step reaction, and does not require purification or separation. The analysis method used has strong specificity and high sensitivity. When used in conjunction with the synthesis method, the isomers of the target product can be effectively distinguished. When used alone, the method can be used to determine the impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in sulbactam sodium. The method has the characteristics of strong specificity and high sensitivity.

[0017] One of the purposes of the present invention is to provide a method for synthesizing the genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in the raw material of sulbactam sodium, the synthetic method comprising the following steps:

[0018] (2S)-2-amino-3-sulfinyl-3-methylbutyric acid and anhydrous ethanol are refluxed at 50°C for 48 hours. The synthesized reaction solution does not need to be separated and purified, and only needs to be combined with the following analytical method to quickly distinguish and quantify the content of the target product. By searching the literature, no reports on the synthesis of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid have been found. This synthesis experiment proves that under not very intense reaction conditions, there is a risk of producing this potential genotoxic impurity in sulbactam sodium, which further proves the necessity of our invention of this method.

[0019] Wherein, the mass volume ratio of the (2S)-2-amino-3-sulfinyl-3-methylbutyric acid to anhydrous ethanol is 2 mg:1 mL.

[0020] Another object of the present invention is to provide a method for analyzing the genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in the raw material of sulbactam sodium, the method comprising the following steps:

[0021] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (CORTECS C18 3.0mm×75mm, 2.7μm or chromatographic column with equivalent performance); use water as mobile phase A and methanol as mobile phase B, and perform linear gradient elution according to the table below; flow rate is 0.4ml per minute; column temperature is 30℃; wavelength is 210nm, 230nm; injection volume is 10μl.

[0022]

[0023] Mass spectrometry condition 1: QTOF mass spectrometer; Dual ESI ion source; drying gas temperature of 350°C; drying gas flow rate of 12 L / min; capillary end voltage of 120 V; nebulization pressure of 35 psig; MS acquisition mode of separate acquisition of positive and negative ions; acquisition time of 0.1 to 14.9 min; acquired m / z range of 70 to 1700; analysis and processing method of 210 positive ion current graph extracted by integral processing.

[0024] Mass spectrometry condition 2: QTOF mass spectrometer; Dual ESI ion source; drying gas temperature of 350°C; drying gas flow rate of 12 L / min; capillary end voltage of 120 V; nebulization pressure of 35 psig; Targeted MS / MS acquisition mode is positive ion acquisition mode; acquisition time is 0.1 to 14.9 min; MS acquisition m / z range is 70 to 1700; MS / MS acquisition m / z range is 50 to 250; Targeted MS / MS acquisition list is as follows:

[0025]

[0026]

[0027] According to the present invention, the target product can be synthesized using the aforementioned synthesis method, and the reaction yield is 2.3% (the larger chiral isomer in the target product accounts for the percentage of the reaction raw material (2S)-2-amino-3-sulfinyl-3-methylbutyric acid), the reaction raw materials are simple and easy to obtain, the reaction steps are only one step, and there is no need for separation and purification of the product.

[0028] According to the present invention, by using the chromatographic conditions of the aforementioned analytical method in combination with mass spectrometry conditions 1, the target product in the reaction solution can be better separated from other impurities, with a minimum separation degree of 16.3, and the two chiral isomers of the target product can be effectively separated, with a separation degree of 1.8.

[0029] According to the present invention, the chromatographic conditions and mass spectrometry conditions 1 of the aforementioned analytical method are used in combination to detect the impurity in sulbactam sodium by limit method. The method has strong specificity and high sensitivity. When the concentration of the test solution is 15 mg / ml, the detection limit can reach 0.27 ppm, which is much smaller than the detection requirement of 5 ppm.

[0030] According to the present invention, the chromatographic conditions of the aforementioned analytical method and mass spectrometry conditions 2 are used in combination to confirm the structures of the isomers of the target product in the reaction solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0032] Figure 1 Positioning the chromatogram of each isomer of the target peak in the synthetic reaction solution in Example 1;

[0033] Figure 2 is the normalized peak area content of the target product in the synthetic reaction solution in Example 2;

[0034] Figure 3 The mass spectra of the target peak and its isomers in the synthetic reaction solution in Example 3;

[0035] Figure 4 The structure of the target product in the synthetic reaction solution in Example 3 is analyzed;

[0036] Figure 5 The structural analysis of the carboxylate isomers in the synthetic reaction solution of Example 3 is shown below;

[0037] Figure 6 This is the specificity and sensitivity mass spectrum in Example 4. DETAILED DESCRIPTION

[0038] The present invention can be further described by the following examples, however, the present invention is not limited to the following examples, and these examples do not limit the scope of the present invention in any way. Certain changes and adjustments made by those skilled in the art within the scope of the claims should also be considered to belong to the scope of the present invention.

[0039] Example 1

[0040] Instruments and conditions

[0041] Magnetic stirrer: Gongyi Yuhua ZNCL-BS intelligent magnetic stirrer.

[0042] Feed amount: (2S)-2-amino-3-sulfinyl-3-methylbutyric acid 10 mg, anhydrous ethanol 5 ml.

[0043] Reaction process: reflux reaction at 50°C for 48 hours.

[0044] The method is simple in synthesis, and only requires (2S)-2-amino-3-sulfinyl-3-methylbutyric acid to be dissolved in ethanol and then refluxed and heated for reaction, without the need for protection of specific reactive groups, and the reaction yield is 2.3%. In conjunction with Example 2 below, the reaction solution can be directly used as a reference substance, without the need for cumbersome separation, purification, and preparation work, and the operation is simple and easy.

[0045] Example 2

[0046] Instruments and conditions

[0047] HPLC-MS: Agilent 1290Infinity HPLC system + G6530B QTOF mass spectrometer Blank solvent: water

[0048] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (CORTECS C18 3.0mm×75mm, 2.7μm or chromatographic column with equivalent performance); use water as mobile phase A and methanol as mobile phase B, and perform linear gradient elution according to the table below; flow rate is 0.4ml per minute; column temperature is 30℃; detection wavelengths are 210nm and 230nm; injection volume is 10μl.

[0049]

[0050] Mass spectrometry conditions: QTOF mass spectrometer; Dual ESI ion source; drying gas temperature of 350°C; drying gas flow rate of 12 L / min; capillary end voltage of 120 V; nebulization pressure of 35 psig; MS acquisition mode of separate acquisition of positive and negative ions; acquisition time of 0.1 to 14.9 min; acquired m / z range of 70 to 1700; analysis and processing method of 210 positive ion current graph extracted by integral processing.

[0051] Solution preparation

[0052] Test solution 1: Transfer all the reaction solution in Example 1 to a 100 ml volumetric flask with water, add water to dissolve and dilute to the mark, and shake well.

[0053] Test solution 2: Measure 5 ml of test solution 1, place in a 50 ml volumetric flask, dilute to the mark with water, and shake well.

[0054] Reference solution: Take about 10 mg of sulbactam sodium EP impurity A reference, accurately weigh, place in a 100 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, measure 5 ml and place in a 50 ml volumetric flask, dilute to the scale with water, and shake well.

[0055] Accurately measure the test solution 1, test solution 2 and reference solution, and inject them into the HPLC-MS / MS instrument respectively, and record the chromatogram and mass spectrum.

[0056] Target impurity location: Extract the 210 positive ion chromatogram of the test solution 1 and determine the target peaks. The two peaks with a retention time of about 3 to 4 minutes are a pair of chiral isomers of the target product, and the peak with a retention time of about 6 minutes is the by-product carboxylate isomer.

[0057] The content of sulbactam sodium EP impurity A in the test solution 2 at a wavelength of 230 nm was calculated by the peak area external standard method.

[0058] The percentage of the target product in the test solution 2 to the reaction raw material sulbactam sodium EP impurity A at a wavelength of 230 nm was calculated by the peak area normalization method. The content of the target product was calculated by combining the content of impurity A calculated by the peak area external standard method.

[0059] This analytical method can use the commercially available standard sulbactam sodium EP impurity A as an intermediate bridge to infer the content of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid, which currently has no standard, in the reaction solution, thereby creating conditions for its limit control. The mobile phase of this analytical method does not contain any buffer salt, and the target product has a large separation degree from other impurities, with a minimum separation degree of 16.3. If the analytical chromatographic column is replaced with a semi-preparative column, it can be directly used for the preparation and separation of the target product.

[0060] Example 3

[0061] Instruments and conditions

[0062] HPLC-MS: Agilent 1290Infinity HPLC system + G6530B Q-TOF mass spectrometer Blank solvent: water

[0063] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (CORTECS C18 3.0mm×75mm, 2.7μm or chromatographic column with equivalent performance); use water as mobile phase A and methanol as mobile phase B, and perform linear gradient elution according to the table below; flow rate is 0.4ml per minute; column temperature is 30℃; wavelength is 210nm; injection volume is 10μl.

[0064]

[0065] Mass spectrometry conditions: QTOF mass spectrometer; Dual ESI ion source; drying gas temperature of 350°C; drying gas flow rate of 12 L / min; capillary end voltage of 120 V; nebulization pressure of 35 psig; Targeted MS / MS acquisition mode is positive ion acquisition mode; acquisition time is 0.1-14.9 min; MS acquisition m / z range is 70-1700; MS / MS acquisition m / z range is 50-250; Targeted MS / MS acquisition list is as follows:

[0066]

[0067] The analytical method can perform secondary mass spectrometry analysis on the mass spectrometry peak of the suspected target product generated in the reaction solution, and ultimately determine the chromatographic peak position of the target product and its isomers, so that the target peak can be directly located by chromatographic conditions.

[0068] Example 4

[0069] Instruments and conditions

[0070] HPLC-MS: Agilent 1290 Infinity HPLC system + G6530B Q-TOF mass spectrometer Blank solvent: water

[0071] Chromatographic conditions: Use octadecylsilane bonded silica gel as filler (CORTECS C18 3.0mm×75mm, 2.7μm or chromatographic column with equivalent performance); use water as mobile phase A and methanol as mobile phase B, and perform linear gradient elution according to the table below; flow rate is 0.4ml per minute; column temperature is 30℃; wavelength is 210nm; injection volume is 10μl.

[0072]

[0073] Mass spectrometry conditions: QTOF mass spectrometer; Dual ESI ion source; drying gas temperature of 350°C; drying gas flow rate of 12 L / min; capillary end voltage of 120 V; nebulization pressure of 35 psig; MS acquisition mode of separate acquisition of positive and negative ions; acquisition time of 0.1 to 14.9 min; acquired m / z range of 70 to 1700; analysis and processing method of 210 positive ion current graph extracted by integral processing.

[0074] Solution preparation

[0075] Test solution: Take an appropriate amount of sulbactam sodium, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 15 mg per 1 ml.

[0076] Quantitative limit sensitivity solution: Take the test solution 2 in Example 2 as the initial concentration solution of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid, add water and dilute it step by step until the signal-to-noise ratio in the extracted 210 positive ion current graph is not less than 10.

[0077] Detection limit sensitivity solution: Take the quantification limit sensitivity solution and dilute it quantitatively until the signal-to-noise ratio in the extracted 210 positive ion current graph is not less than 3.

[0078] Spiked test solution 1: Take an appropriate amount of sulbactam sodium, weigh accurately, dissolve in the quantitation limit sensitivity solution and quantitatively dilute to make a solution containing approximately 15 mg of sulbactam sodium per 1 ml.

[0079] Spiked test solution 2: Take an appropriate amount of sulbactam sodium, accurately weigh it, dissolve it in the detection limit sensitivity solution and quantitatively dilute it to make a solution containing about 15 mg of sulbactam sodium per 1 ml.

[0080] Accurately measure the test solution, each sensitivity solution and each spiked test solution, inject them into the HPLC-MS / MS instrument respectively, and record the mass spectrum.

[0081] Extract 210 positive ion chromatogram, the minimum separation of the three target peaks is 1.8, which is the separation between the two chiral isomers of the target product, and the separation between the carboxylate isomers can reach 16.3. According to the content of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in the test solution 2 calculated in Example 2, the concentration of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in the quantitative limit sensitivity solution is 4.1×10 -6 mg / ml, based on the test sample of 15 mg / ml, the limit detection can be 0.27ppm, which is much less than the limit detection requirement of 5ppm. This shows that the method has strong specificity and good sensitivity, and can be used for the limit method detection of the impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in sulbactam sodium raw materials.

Claims

1. A method for analyzing genotoxic impurities in sulbactam sodium bulk drug, characterized in that: The genotoxic impurity is (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid. The analysis method adopts high performance liquid chromatography and mass spectrometry. The high performance liquid chromatography uses water as mobile phase A and methanol as mobile phase B, and performs linear gradient elution according to the following table; the flow rate is 0.4 ml per minute; the column temperature is 30°C; the high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler, the wavelength is 210nm, 230nm, and the injection volume is 10μL; the mass spectrometry conditions are QTOF mass spectrometry detector, Dual ESI ion source, drying gas temperature is 350°C, drying gas flow rate is 12L / min, capillary end voltage is 120V, nebulization pressure is 35psig, MS acquisition mode is positive and negative ion separate acquisition, acquisition time is 0.1-14.9min, the acquired m / z range is 70-1700, and the analysis and processing method is the integral processing of the extracted 210 positive ion flow chart; 2. The analysis method according to claim 1, characterized in that The mass spectrometry conditions are QTOF mass spectrometer, Dual ESI ion source, drying gas temperature of 350°C, drying gas flow of 12 L / min, capillary end voltage of 120 V, nebulization pressure of 35 psig, Targeted MS / MS acquisition mode of positive ion acquisition mode, acquisition time of 0.1 to 14.9 min, MS acquisition m / z range of 70 to 1700, MS / MS acquisition m / z range of 50 to 250, and the Targeted MS / MS acquisition list is as follows:

3. The analysis method according to claim 1, characterized in that The method can separate the target product from other impurities in the reaction solution with a minimum separation degree of 16.3, and can effectively separate two chiral isomers of the target product with a separation degree of 1.

8.

4. The analysis method according to claim 1, characterized in that The method can be used for the limit method detection of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid in sulbactam sodium. When the concentration of the test solution is 15 mg / ml, the detection limit can reach 0.27 ppm.

5. The analysis method according to claim 2, characterized in that The method can be used for confirming the structures of each isomer of the target product in the synthesis reaction solution of (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid.

6. The analysis method according to claim 1, characterized in that The genotoxic impurity (2S)-2-amino-3-ethoxysulfinyl-3-methylbutyric acid is prepared by the following method: (2S)-2-amino-3-sulfinyl-3-methylbutyric acid and anhydrous ethanol were used as reaction materials, and the temperature was controlled at 50° C. for reflux reaction for 48 hours.

7. The analysis method according to claim 6, characterized in that The mass volume ratio of (2S)-2-amino-3-sulfinyl-3-methylbutyric acid and ethanol is 2 mg:1 mL.

8. The analysis method according to claim 6, characterized in that After the reflux reaction is completed, the reaction solution does not need to be separated and purified, and the content of the target product can be quickly distinguished and quantified by the analytical method.

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