Detection method of bromoacetic acid impurity in cefathiamidine

Abstract

The present application relates to a method for detecting bromoacetic acid as an impurity in cefathiamidine. The detection method adopts high performance liquid chromatography-mass spectrometry, wherein the conditions of high performance liquid chromatography are: mobile phase A: water; mobile phase B: acetonitrile; wherein mobile phase A and mobile phase B are used for gradient elution. The method of the present application has a low detection limit of bromoacetic acid in cefathiamidine and high sensitivity, and can efficiently detect and control bromoacetic acid in cefathiamidine at the ng / ml level, which is of great significance to the quality control and pharmaceutical safety of cefathiamidine.

Description

Technical Field

[0001] The present invention relates to a method for detecting bromoacetic acid, and in particular to a method for detecting bromoacetic acid as an impurity in cefathiamidine by utilizing high performance liquid chromatography-mass spectrometry. Background Art

[0002] Impurities in drugs are likely to be toxic to the human body. Therefore, monitoring and controlling impurities in drugs during the drug production process and shelf life is an important part of improving drug quality. Among various impurities, genotoxic impurities refer to substances that can directly or indirectly damage cell DNA and produce mutagenic and carcinogenic effects. There are no genotoxic reactants in the synthesis of active pharmaceutical ingredients, but there are substances containing genotoxic structures in active pharmaceutical ingredients, which are called potential genotoxic impurities.

[0003] In the drug production process, the links that may produce genotoxic impurities include new drug synthesis, raw material purification, storage and transportation (contact with packaging materials), etc. Therefore, all genotoxic or carcinogenic chemicals involved should be specified in the new drug synthesis stage, such as reagents, intermediates, by-products, etc. Furthermore, genotoxic reactants and substances with genotoxic structures that do not appear in the active substances of the drug should be considered. Groups with toxic effects generally have the properties of electrophilic reagents. These groups undergo substitution reactions with nucleophilic centers in nucleic acids, proteins or other important components in the body under physiological conditions, causing irreversible damage to these components, which manifests as toxic, mutagenic or carcinogenic effects.

[0004] Cefathiamidine is the first generation cephalosporin developed in my country. It has strong antibacterial activity against Gram-positive bacteria and good antibacterial activity against a few Enterobacteriaceae. It has good antibacterial activity against viridans streptococci, hemolytic streptococci, non-hemolytic streptococci, diphtheria bacteria, Clostridium perfringens, tetani bacteria and anthrax bacteria.

[0005] When the existing industry prepares cefathiamidine, because the activity of bromine is strong, bromoacetyl bromide is used as an acyl donor, bromoacetyl bromide is reacted with 7-ACA (7-aminocephalosporanic acid) to generate an intermediate bromoacetyl 7-ACA, and then reacted with thiourea to generate cefathiamidine. This process makes the reaction of generating cefathiamidine easier to carry out, the reaction time is shorter, and the operation is simpler. When bromoacetyl bromide is used as a reactant, the bromoacetyl bromide that is not completely reacted cannot be stably present under the conditions of post-treatment (alkaline, aqueous solution), and it will be converted into bromoacetic acid, which will cause the residual risk of bromoacetic acid in cefathiamidine. According to Derec and Sarah software evaluation, bromoacetic acid is a genotoxic impurity, which needs to be monitored and controlled. Therefore, it is necessary to carry out qualitative and quantitative detection of the bromoacetic acid remaining in cefathiamidine. There is no report on the qualitative and quantitative detection of bromoacetic acid in cefathiamidine.

[0006] Establishment of bromoacetic acid limit: Cefathiamidine for injection, specification is 1.0g, clinical usage dosage is: 2g once, 2 to 4 times a day; the maximum daily dose is 8g. According to the ICH M7 guidelines and the conversion of medication intervals, assuming that the medication is used every day for 1 year, the impurities are controlled according to the limit of 1 to 12 months, and the limit calculation is as follows:

[0007]

[0008] Since the impurity limit of bromoacetic acid is relatively low, there is an urgent need for a method with high detection sensitivity and strong specificity that can perform qualitative and quantitative detection of bromoacetic acid as an impurity in cefathiamidine. Summary of the invention

[0009] In view of this, the main purpose of the present invention is to provide a method for qualitative and quantitative detection of bromoacetic acid as an impurity in cefathiamidine.

[0010] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0011] The present invention provides a method for detecting bromoacetic acid as an impurity in cefathiamidine, wherein the method adopts high performance liquid chromatography-mass spectrometry, wherein the conditions of high performance liquid chromatography are:

[0012] Mobile phase A: water;

[0013] Mobile phase B: acetonitrile;

[0014] Wherein, mobile phase A and mobile phase B are used for gradient elution, and the gradient and flow rate of the elution are:

[0015]

[0016]

[0017] Further, the gradient and flow rate of the elution are:

[0018] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.2 10 90 2.5 0.2 10 90 3.0 0.5 35 65 8.5 0.5 35 65 9.0 0.5 10 90 15 0.5 10 90 16 0.2 10 90 19 0.2 10 90

[0019] According to some embodiments, the conditions of the high performance liquid chromatography are:

[0020] Column: Shiseido PC HILIC, 100 mm × 2.0 mm, 3 μm;

[0021] Column temperature: 25-30°C;

[0022] Injection volume: 1.5~2.5μl;

[0023] The flow rate is 0.2~0.5ml / min.

[0024] Further, the conditions of the high performance liquid chromatography are:

[0025] Column: Shiseido PC HILIC, 100 mm × 2.0 mm, 3 μm;

[0026] Column temperature: 30°C;

[0027] Injection volume: 2 μl;

[0028] The flow rate is 0.2~0.5ml / min.

[0029] According to some embodiments, wherein the mass spectrometry conditions are:

[0030] Monitored ions: 138.9;

[0031] Collision induced dissociation voltage: 5~70V;

[0032] Atomizing gas pressure: 10~60psig;

[0033] Drying gas temperature: 200~350℃;

[0034] Drying gas flow rate: 6~13L / min.

[0035] Furthermore, the mass spectrometry conditions are:

[0036] Monitored ions: 138.9;

[0037] Collision induced dissociation voltage: 20~40V;

[0038] Atomizing gas pressure: 10~30psig;

[0039] Drying gas temperature: 320~350℃;

[0040] Drying gas flow rate: 6~13L / min.

[0041] Furthermore, the mass spectrometry conditions are:

[0042] Monitored ions: 138.9;

[0043] Collision-induced dissociation voltage: 25 V;

[0044] Atomizing gas pressure: 20psig;

[0045] Drying gas temperature: 350℃;

[0046] Drying gas flow rate: 13L / min.

[0047] The present invention has the following beneficial effects:

[0048] (1) It overcomes the interference of the solution at the bromoacetic acid peak position and quickly and efficiently detects the genotoxic impurity bromoacetic acid in cefathiamidine.

[0049] (2) improving the detection specificity of bromoacetic acid in cefathiamidine;

[0050] (3) The method of the present invention has a low detection limit for bromoacetic acid in cefathiamidine and high sensitivity, and can detect and control bromoacetic acid in cefathiamidine at the ppm level, which is of great significance in the quality control and pharmaceutical safety of cefathiamidine. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1A is a chromatogram of a blank solution under the conditions of Example 1 of the present invention, Figure 1B is a chromatogram of the reference substance solution under the conditions of Example 1 of the present invention, Figure 1C It is a chromatogram of the test solution under the conditions of Example 1 of the present invention.

[0052] Figure 2 This is a chromatogram of the 100% sample recovery solution according to Example 2 of the present invention under the conditions of Example 1.

[0053] Figure 3A Partial chromatogram of the detection limit level of the control solution of Example 1 of the present invention (retention time is 0-5min), Figure 3B It is a partial chromatogram at the quantitative limit level of the control solution of Example 1 of the present invention (retention time is 0-5min).

[0054] Figure 4A The chromatogram of the bromoacetic acid reference substance solution detected according to the conditions of Comparative Example 1, Figure 4B The chromatogram is obtained by testing the test solution according to the conditions of Comparative Example 1.

[0055] Figure 5A The chromatogram of the bromoacetic acid reference substance solution detected according to the conditions of Comparative Example 2, Figure 5B The chromatogram is obtained by testing the test solution according to the conditions of Comparative Example 2.

[0056] Fig. 6A The chromatogram of the blank solution detected according to the conditions of Comparative Example 3 is as follows: Figure 6B The chromatogram of the bromoacetic acid reference substance solution detected according to the conditions of Comparative Example 3, Figure 6C The chromatogram is obtained by testing the test solution according to the conditions of Comparative Example 3. DETAILED DESCRIPTION

[0057] The invention adopts liquid chromatography-mass spectrometry to carry out qualitative and quantitative detection of bromoacetic acid as an impurity in cefathiamidine.

[0058] In the following specific examples, Agilent 1260-Agilent 6120 was used, wherein the liquid chromatograph was Agilent 1260 and the mass spectrometer was Agilent 6120.

[0059] The cefathiamidine sample was first separated by a liquid chromatograph, and the liquid flow from the retention time of 0 to 3.0 min entered the mass spectrometer for detection, and the liquid flow from 3.1 min to 19 min did not enter the mass spectrometer for detection. During the test, a blank solution was first injected to determine that the blank solvent and the system had no interference with the detection of bromoacetic acid, and a bromoacetic acid reference solution was injected to determine that the instrument response met the detection requirements, and then the cefathiamidine sample was injected for detection.

[0060] The following examples are used to further illustrate the present invention but are not intended to limit the scope of the present invention.

[0061] Example 1

[0062] First, prepare the following solution:

[0063] a) Prepare reference solution:

[0064] Dissolve bromoacetic acid in water to prepare a bromoacetic acid solution with a concentration of about 250 ng / ml as a reference solution;

[0065] b) Prepare the test solution:

[0066] Cefathiamidine was dissolved in water to prepare a cefathiamidine solution with a concentration of about 100 mg / ml as a test solution;

[0067] c) Blank solution: water

[0068] Then, the test solution, reference solution and blank solution were tested according to the following liquid chromatography-mass spectrometry conditions, and the spectra were recorded:

[0069] Liquid chromatography-mass spectrometry conditions:

[0070] Instrument model Agilent 1260-Agilent 6120

[0071] Mass spectrometry conditions:

[0072] Scan mode: Selected ion monitoring (SIM)

[0073] Polarity: Negative ion mode

[0074] Ion source: electrospray ion source

[0075] Monitored ions: 138.9

[0076] Mass spectrometry parameters: capillary voltage 4000V

[0077] Atomizing gas pressure: 20psig

[0078] Drying gas temperature: 350℃

[0079] Drying gas flow rate: 13L / min

[0080] Collision-induced dissociation: 25V

[0081] Liquid chromatography conditions:

[0082] Column: Shiseido PC HILIC, 100 mm × 2.0 mm, 3 μm

[0083] Mobile phase A: water Mobile phase B: acetonitrile

[0084] Column temperature: 30°C; injection volume: 2 μl;

[0085] Running time: 19 min (mass spectrometry acquisition time 0 to 3.0 min).

[0086] Gradient elution program:

[0087]

[0088]

[0089] By the attached Figures 1A to 1C It can be seen that bromoacetic acid and cefathiamidine can be completely separated, and cefathiamidine can be completely and quickly eluted without being affected by other impurities in the test solution. And when the bromoacetic acid content is >250ng / ml, this method can detect it quickly and efficiently.

[0090] Example 2

[0091] Preparation of injection recovery solution

[0092] Cefathiamidine and bromoacetic acid were dissolved in water to prepare a solution containing about 100 mg / ml of cefathiamidine and 250 ng / ml of bromoacetic acid as a 100% sample recovery solution; in the same way, solutions with bromoacetic acid contents of 50% and 150% were prepared as 50% and 150% sample recovery solutions, wherein the concentrations of bromoacetic acid were 125 ng / ml and 375 ng / ml, respectively.

[0093] The 100% added sample recovery solution was tested according to the liquid chromatography-mass spectrometry conditions in Example 1, and the spectrum was recorded. The results are as follows: Figure 2 shown.

[0094] From the above, it can be seen that bromoacetic acid is completely separated from the main peak of the test sample, the test sample does not interfere with the detection of the target impurity peak, and the method has good specificity.

[0095] The recoveries of 50% recovery solution, 100% recovery solution and 150% recovery solution were 104% to 110%, with an RSD of 3%. The accuracy of this method was good.

[0096] Example 3

[0097] Determine the limit of detection and limit of quantification

[0098] The reference solution (250 ng / ml) in Example 1 was accurately measured and diluted step by step until the solution with a peak height of 10 times the baseline noise was the quantitative limit solution, and the solution with a peak height of 3 times the baseline noise was the detection limit solution.

[0099] The quantitative limit solution and the detection limit solution were tested according to the liquid chromatography-mass spectrometry conditions of Example 1, and the spectra were recorded. Figure 3A and 3B As shown, the test results are as follows:

[0100]

[0101] From the table above, the signal-to-noise ratios of the detection limit and the quantification limit meet the requirements. The quantification limit of this method is about 12.95ng / ml (0.13ppm after conversion), which is less than the impurity limit requirement. Therefore, the sensitivity of this method meets the detection requirements.

[0102] Comparative Example 1

[0103] The reference solution and the test solution in Example 1 were tested according to the following liquid chromatography-mass spectrometry conditions, and the chromatograms were recorded.

[0104] Mass spectrometry conditions:

[0105] Scan mode: Selected ion monitoring (SIM)

[0106] Polarity: Negative ion mode

[0107] Ion source: electrospray ion source

[0108] Monitored ions: 138.9

[0109] Mass spectrometry parameters: capillary voltage 4000V

[0110] Atomizing gas pressure: 20Psig

[0111] Drying gas temperature: 350℃

[0112] Drying gas flow rate: 13L / min

[0113] Collision-induced dissociation: 25V

[0114] Liquid chromatography conditions:

[0115] The mobile phase A-mobile phase B (20:80) was used for isocratic elution.

[0116] Column: Shiseido PC HILIC, 100 mm × 2.0 mm, 3 μm

[0117] Mobile phase A: water Mobile phase B: acetonitrile

[0118] Flow rate: 0.2 ml / min; column temperature: 30 °C; injection volume: 3 μl;

[0119] The recorded chromatogram is attached. Figure 4A and 4B As shown in the figure, it can be seen that when eluted isocratically with mobile phase A-mobile phase B (20:80), bromoacetic acid and cefathiamidine cannot be completely separated.

[0120] Comparative Example 2

[0121] The reference solution and the test solution in Example 1 were tested according to the following liquid chromatography-mass spectrometry conditions, and the chromatograms were recorded.

[0122] Mass spectrometry parameters:

[0123] Scan mode: Selected ion monitoring (SIM)

[0124] Polarity: Negative ion mode

[0125] Ion source: electrospray ion source

[0126] Monitored ions: 138.9

[0127] Mass spectrometry parameters: capillary voltage 4000 V;

[0128] Atomizing gas pressure: 20Psig;

[0129] Drying gas temperature: 350℃;

[0130] Drying gas flow rate: 13L / min;

[0131] Collision-induced dissociation: 25V

[0132] Liquid chromatography conditions:

[0133] The column was eluted isocratically with mobile phase A-mobile phase B (10:90), injection volume was 2 μl, flow rate was 0.2 ml / min, and column temperature was 30°C.

[0134] The results are as follows Figure 5A and 5B As shown in the figure, the peak area of ​​cefathiamidine is too broad and the retention time is long.

[0135] Comparative Example 3

[0136] HPLC method detection

[0137] The blank solvent, quantitative limit solution and test solution were tested by liquid chromatography. The liquid chromatography conditions were as follows:

[0138] Chromatographic column: Shiseido MGII C18, 250 mm × 4.6 mm, 5 μm;

[0139] Mobile phase A: 0.05 mol / l potassium dihydrogen phosphate (pH 2.50): acetonitrile (97:3);

[0140] Mobile phase B: acetonitrile

[0141] Flow rate: 1.0ml / min, injection volume: 40μl; column temperature: 30℃; detection wavelength: 210nm. Perform gradient elution according to the following table:

[0142]

[0143] 1) Blank solvent (diluent): Mobile phase A; Chromatogram as attached Fig. 6A shown.

[0144] 2) Quantitative limit solution:

[0145] Weigh an appropriate amount of bromoacetic acid reference substance, add diluent to dissolve and dilute step by step to investigate the quantitative limit concentration, and obtain C LOQ = 0.2μg / ml quantitative limit solution. The chromatogram is as attached Figure 6B shown.

[0146] According to the ICH M7 guidelines and the conversion of the medication interval, the impurity limit of bromoacetic acid is 20μg / day / 8g / day = 2.5ppm. LOQ / C 进 ≤ limit * 50% to calculate the sample injection concentration (C 进 ) should be at least 160mg / ml.

[0147] 3) Test solution:

[0148] Weigh an appropriate amount of cefathiamidine raw material sample, add diluent to dissolve and dilute to make a solution containing about 160 mg of cefathiamidine per 1 ml. The chromatogram is shown in the attached figure. Figure 6C shown.

[0149] By the attached Figures 6A-6C It can be seen that the impurity peak area of ​​the high-concentration test solution is too large, and there is interference at the bromoacetic acid peak position (around 8 minutes). The specificity and sensitivity of the above-mentioned method for detecting bromoacetic acid do not meet the requirements.

[0150] Example 4

[0151] Mass spectrometry parameter optimization process

[0152] First, set up the chromatographic conditions as follows:

[0153] Column: Shiseido PC-HILIC 2.0×100mm, 3μm

[0154] Mobile phase A: water Mobile phase B: acetonitrile Mobile phase A-mobile phase B (10:90) Injection volume: 2μl

[0155] Column temperature: 30℃ Flow rate: 0.2ml / min

[0156] Reference solution: 250ng / ml (bromoacetic acid)

[0157] In the following, the collision-induced dissociation voltage, nebulizer gas pressure, drying gas temperature and drying gas flow rate will be screened.

[0158] a. Screening collision-induced dissociation voltage: range 5~70V

[0159] Target impurity: Bromoacetic acid

[0160] Mass spectrometry conditions:

[0161] Scan mode: Selected ion monitoring (SIM)

[0162] Polarity: Negative ion mode

[0163] Ion source: electrospray ion source

[0164] Monitored ions: 138.9

[0165] Mass spectrometry parameters: capillary voltage: 4000 V;

[0166] Atomizing gas pressure: 40Psig;

[0167] Drying gas temperature: 350℃;

[0168] Drying gas flow rate: 13L / min;

[0169] Table 1 Corresponding results of different collision-induced dissociation voltages

[0170]

[0171] b. Screening atomizing gas pressure: range 10~60psig

[0172] Target impurity: Bromoacetic acid

[0173] Mass spectrometry conditions:

[0174] Scan mode: Selected ion monitoring (SIM)

[0175] Polarity: Negative ion mode

[0176] Ion source: electrospray ion source

[0177] Monitored ions: 138.9

[0178] Mass spectrometry parameters: Capillary voltage: 4000V

[0179] Drying gas temperature: 350℃

[0180] Drying gas flow rate: 13L / min

[0181] Collision-induced dissociation: 25V

[0182] Table 2 Corresponding results of different atomizing gas pressures and voltages

[0183]

[0184] c. Screen the drying gas temperature, 200 ~ 350 ℃.

[0185] Target impurity: Bromoacetic acid

[0186] Mass spectrometry conditions:

[0187] Scan mode: Selected ion monitoring (SIM)

[0188] Polarity: Negative ion mode

[0189] Ion source: electrospray ion source

[0190] Mass spectrometry parameters: Monitoring ion: 138.9

[0191] Capillary voltage: 4000V

[0192] Atomizing gas pressure: 20psig

[0193] Drying gas flow rate: 13L / min

[0194] Collision-induced dissociation: 25V

[0195] Table 3 Corresponding results of different drying gas temperatures

[0196]

[0197] d. Screening drying air flow rate: 6~13L / min

[0198] Mass spectrometry conditions:

[0199] Target impurity: Bromoacetic acid

[0200] Scan mode: Selected ion monitoring (SIM)

[0201] Polarity: Negative ion mode

[0202] Ion source: electrospray ion source

[0203] Monitored ions: 138.9

[0204] Mass spectrometry parameters: Capillary voltage: 4000V

[0205] Atomizing gas pressure: 20Psig

[0206] Drying gas temperature: 350℃

[0207] Collision-induced dissociation: 25V

[0208] Table 4 Corresponding results of different drying gas flow rates

[0209]

[0210] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. A method for detecting bromoacetic acid as an impurity in cefathiamidine, wherein: The method adopts high performance liquid chromatography-mass spectrometry, wherein the conditions of high performance liquid chromatography are: Column: Shiseido PC HILIC, 100 mm × 2.0 mm, 3 μm; Mobile phase A: water; Mobile phase B: acetonitrile; Column temperature: 30°C; Injection volume: 1.5~2.5μl; Flow rate: 0.2-0.5 ml / min; Wherein, mobile phase A and mobile phase B are used for gradient elution, wherein the gradient and flow rate of the elution are: The mass spectrometry conditions are: Monitored ions: 138.9; Collision induced dissociation voltage: 5~70V; Atomizing gas pressure: 10~60psig; Drying gas temperature: 200~350℃; Drying gas flow rate: 6~13L / min.

2. The method according to claim 1, wherein the injection volume of the HPLC is 2 μl.

3. The method according to claim 1 or 2, wherein: The mass spectrometry conditions are: Collision induced dissociation voltage: 20~40V; Atomizing gas pressure: 10~30psig; Drying gas temperature: 320~350℃.

4. The method according to claim 1 or 2, wherein: The mass spectrometry conditions are: Collision-induced dissociation voltage: 25 V; Atomizing gas pressure: 20psig; Drying gas temperature: 350℃; Drying gas flow rate: 13L / min.

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