CN108802212B - Liquid phase detection method for cefquinome sulfate breast injectant-related substances in lactation period - Google Patents
Liquid phase detection method for cefquinome sulfate breast injectant-related substances in lactation period Download PDFInfo
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- KYOHRXSGUROPGY-OFNLCGNNSA-N (6r,7r)-7-[[(2z)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-8-oxo-3-(5,6,7,8-tetrahydroquinolin-1-ium-1-ylmethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid;hydrogen sulfate Chemical compound OS(O)(=O)=O.N([C@@H]1C(N2C(=C(C[N+]=3C=4CCCCC=4C=CC=3)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 KYOHRXSGUROPGY-OFNLCGNNSA-N 0.000 title claims abstract description 76
- 238000001514 detection method Methods 0.000 title claims abstract description 66
- 230000006651 lactation Effects 0.000 title claims abstract description 40
- 239000007791 liquid phase Substances 0.000 title claims abstract description 33
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- 230000014759 maintenance of location Effects 0.000 claims abstract description 28
- SITIYXVTKYBLMR-UHFFFAOYSA-N 3-cyclohexylpyridine Chemical compound C1CCCCC1C1=CC=CN=C1 SITIYXVTKYBLMR-UHFFFAOYSA-N 0.000 claims abstract description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 88
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 81
- 239000012071 phase Substances 0.000 claims description 53
- 239000000243 solution Substances 0.000 claims description 51
- 239000000523 sample Substances 0.000 claims description 28
- 239000007853 buffer solution Substances 0.000 claims description 24
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 24
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- 239000002244 precipitate Substances 0.000 claims description 14
- 229920006395 saturated elastomer Polymers 0.000 claims description 14
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- 239000012488 sample solution Substances 0.000 claims description 13
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
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- 239000007788 liquid Substances 0.000 claims description 11
- 238000010606 normalization Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 239000000706 filtrate Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 44
- 238000000926 separation method Methods 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 8
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- 238000012360 testing method Methods 0.000 description 22
- YWKJNRNSJKEFMK-PQFQYKRASA-N (6r,7r)-7-[[(2z)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-8-oxo-3-(5,6,7,8-tetrahydroquinolin-1-ium-1-ylmethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound N([C@@H]1C(N2C(=C(C[N+]=3C=4CCCCC=4C=CC=3)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 YWKJNRNSJKEFMK-PQFQYKRASA-N 0.000 description 20
- 229950009592 cefquinome Drugs 0.000 description 20
- 239000007979 citrate buffer Substances 0.000 description 14
- 239000000463 material Substances 0.000 description 8
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 description 6
- 229960004261 cefotaxime Drugs 0.000 description 6
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- 230000000052 comparative effect Effects 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 5
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- 238000004811 liquid chromatography Methods 0.000 description 4
- 239000012088 reference solution Substances 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
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- 241000283690 Bos taurus Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 239000013642 negative control Substances 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 208000004396 mastitis Diseases 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
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- 210000002345 respiratory system Anatomy 0.000 description 1
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- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 1
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
- IXGNPUSUVRTQGW-UHFFFAOYSA-M sodium;perchlorate;hydrate Chemical compound O.[Na+].[O-]Cl(=O)(=O)=O IXGNPUSUVRTQGW-UHFFFAOYSA-M 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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Abstract
The invention provides a liquid phase detection method for cefquinome sulfate breast injectant related substances in the lactation period. In the method, a related substance detection method is established, wherein the appearance time of a cefquinome sulfate breast injectant (in lactation period) special related substance (2, 3-cyclohexylpyridine) can be more fit with the retention time determined by a main drug self-contrast method, and the method has better tolerance, reproducibility and specificity. Meanwhile, the detection method provided by the invention can ensure that the separation effect of each impurity peak is better and the detection result is more accurate.
Description
Technical Field
The invention relates to the field of drug detection, and particularly relates to a liquid phase detection method for cefquinome sulfate breast injectant-related substances in a lactation period.
Background
The cefquinome sulfate is a special 4 th generation cephalosporin antibiotic for animals, has wide antibacterial spectrum and strong antibacterial activity, and can be used for clinically separating various G+Bacteria, G-MIC of bacteria50、MIC90The values are all small. The cefquinome sulfate is suitable for injection administration, has the advantages of quick absorption, short time to reach a peak value and higher bioavailability, can reach higher tissue concentration in tissues such as lung, mammary gland and the like, and is widely used for clinical treatment of porcine and bovine respiratory system infection and cow mastitis.
In the existing liquid phase detection method of related substances of cefquinome sulfate breast injectant (lactation period), the retention time of a special related substance (2, 3-cyclohexylpyridine) is determined by self-contrast with a main drug (cefquinome) peak, but in actual detection, the retention time of the separated 2, 3-cyclohexylpyridine peak is slightly different from the theoretical retention time in the method, and the retention time of the peak is easily influenced by different liquid chromatographs, chromatographic columns and mobile phase pre-preparation conditions of different brands and models, sometimes the separation effect of each impurity peak is also influenced, and the separation degree is relatively small although meeting the specification.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a liquid phase detection method for cefquinome sulfate breast injectant related substances in the lactation period.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a liquid phase detection method for substances related to cefquinome sulfate breast injectant in lactation period comprises the following steps:
detecting related substances of the cefquinome sulfate breast injectant in the lactation period by adopting a high performance liquid chromatography, and performing gradient elution according to the following conditions:
the mobile phase A is a mixed solution of citrate buffer solution and acetonitrile, wherein the ratio of the citrate buffer solution to the acetonitrile is as follows: 80-90: 10-20;
the mobile phase B is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the ratio of the citric acid buffer solution to the acetonitrile is as follows: 50-70: 30-50.
Preferably, the liquid phase detection method of the present invention performs gradient elution according to the following conditions:
the mobile phase A is a mixed solution of a citrate buffer solution and acetonitrile, wherein the pH value of the citrate buffer solution is 3.6-4.0, and the ratio of the citrate buffer solution to the acetonitrile is as follows: 85-90: 10-15;
the mobile phase B is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the pH value of the citric acid buffer solution is 3.6-4.0, and the proportion of the citric acid buffer solution to the acetonitrile is as follows: 60-70: 30-40.
Preferably, the liquid phase detection method of the present invention performs gradient elution according to the following conditions:
the mobile phase A is a mixed solution of citrate buffer solution and acetonitrile, wherein the ratio of the citrate buffer solution to the acetonitrile is as follows: 85: 15;
the mobile phase B is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the ratio of the citric acid buffer solution to the acetonitrile is as follows: 60:40.
Preferably, in the liquid phase detection method of the present invention, the high performance liquid chromatography conditions are as follows: flow rate: 0.8-1.2 ml/s; column temperature: 25-35 ℃; detection wavelength: 230 to 280 nm.
Preferably, in the liquid phase detection method of the present invention, the high performance liquid chromatography conditions are as follows: flow rate: 1.0 ml/s; column temperature: 30 ℃; detection wavelength: 254 nm; the number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate.
Preferably, in the liquid phase detection method according to the present invention, the liquid phase detection method includes: injecting the test solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main peak is 2 times;
the retention time of the 2, 3-cyclohexylpyridine peak relative to the cefquinome sulfate peak is 0.15-0.25.
Preferably, in the liquid phase detection method of the present invention, if there is an impurity peak in the chromatogram of the sample solution, the content of 2, 3-cyclohexylpyridine should not exceed 2.0%, the content of other single impurities should not exceed 1.0%, and the sum of the impurities should not exceed 4.0%, as calculated by peak area normalization.
Preferably, in the liquid phase detection method of the present invention, the preparation of the sample solution includes the following steps: adding an extraction solution saturated by a cefquinome sulfate reference substance into a breast injection of cefquinome sulfate in the lactation period, shaking uniformly, and centrifuging, wherein the obtained precipitate is extracted for 1-3 times under the same condition;
and drying the precipitate obtained in the last time, and dissolving the precipitate with a mobile phase to obtain a test solution.
Preferably, in the liquid phase detection method of the present invention, the extraction solution saturated with the cefquinome sulfate reference substance is a hexane solution saturated with the cefquinome sulfate reference substance.
Preferably, in the liquid phase detection method of the present invention, the preparation of the sample solution includes the following steps:
adding hexane solution saturated with cefquinome sulfate reference substance into breast injection of lactation period, shaking, centrifuging, extracting the obtained precipitate for 1 time under the same condition, drying, dissolving with mobile phase, ultrasonic treating, filtering, and injecting the filtrate into liquid chromatograph;
more preferably, the amount of sample is 20. mu.L.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention establishes a related substance detection method which can ensure that the peak emergence time of a special related substance (2, 3-cyclohexyl pyridine) of a cefquinome sulfate breast injectant (in the lactation period) can be more matched with the retention time determined by a main drug self-contrast method, and the method has better tolerance, reproducibility and specificity.
2. The detection method provided by the invention can ensure that the separation effect of each impurity peak is better and the detection result is more accurate.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention particularly provides a novel liquid phase detection method for related substances of a cefquinome sulfate breast injectant in the lactation period, aiming at the defects that the separation effect of each component in the preparation is poor, the impurity peak cannot be effectively separated and the like in the detection of related substance content of the existing cefquinome sulfate injectant in the lactation period, particularly in the detection of impurities such as 2, 3-cyclohexylpyridine and the like.
Specifically, the detection method provided by the invention comprises the following steps:
preparing a test solution: taking a cefquinome sulfate injection sample in the lactation period, adding the sample into a centrifuge tube, and preferably, the ratio of the mass milligrams of the cefquinome sulfate injection sample in the lactation period (calculated by cefquinome sulfate) to the calibrated volume milliliters of the centrifuge tube is (1-2): (4-6); more preferably, the ratio of the mass milligrams of the cefquinome sulfate injection sample (calculated by cefquinome sulfate) in the lactation period to the calibrated volume milliliters of the centrifuge tube is 1: 5;
then, an n-hexane solution saturated with cefquinome sulfate standard is added to the centrifuge tube (i.e., cefquinome sulfate is added to the n-hexane until cefquinome sulfate is saturated);
wherein, preferably, the ratio of the mass milligrams of the cefquinome sulfate injection sample (calculated by cefquinome sulfate) in the lactation period to the volume milliliters of the n-hexane solution saturated by the cefquinome sulfate standard is (10-20): (20-30); more preferably, the ratio of the mass milligrams of the cefquinome sulfate injection sample (calculated as cefquinome sulfate) in the lactation period to the volume milliliters of the n-hexane solution saturated with the cefquinome sulfate standard is 10: 25;
then, uniformly mixing, centrifuging (preferably, the rotating speed of the centrifugation is 4500r/min, and the time of the centrifugation is 10-30 min), and removing supernatant;
then, re-extracting the centrifuged precipitate for 1-3 times (preferably 1 time) by using an n-hexane solution saturated with a cefquinome sulfate standard according to the method;
discarding the supernatant in the centrifuge tube after the last extraction, volatilizing the precipitate to be dry at room temperature, and then adding a mobile phase into the centrifuge tube;
preferably, the ratio of the mass milligrams of the cefquinome sulfate injection sample (calculated by cefquinome sulfate) in the lactation period to the volume milliliters of the mobile phase is (1-2): (1-2); more preferably, the ratio of the mass milligrams of the cefquinome sulfate injectant sample (calculated as cefquinome sulfate) in the lactation period to the volume milliliters of the mobile phase is 1: 1;
and then, placing the centrifugal tube in a cold water bath for ultrasonic treatment (preferably ultrasonic treatment for 10min) to dissolve the cefquinome sulfate in the centrifugal tube, and filtering to obtain the test solution.
Then, 20. mu.L of the sample solution was subjected to liquid chromatography.
The liquid chromatography condition is one of the key points of the invention, and the adjustment and optimization of the traditional liquid chromatography condition also enable the detection method of the invention to ensure that the peak-appearing time of a special related substance (2, 3-cyclohexyl pyridine) in a cefquinome sulfate breast injectant (in lactation period) sample can be more suitable for the retention time determined by using a main drug self-contrast method.
Specifically, in the liquid phase detection method of the present invention, the chromatographic conditions are as follows:
the type of the high performance liquid chromatograph: agilent 1260;
the type of the chromatographic column: kromasil 100-5C18(4.6 x 150mm, 5 μm);
filling agent: octadecylsilane bond and silica gel;
flow rate: 1.0 ml/min;
column temperature: 30 ℃;
detection wavelength: 254 nm;
the number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate peak;
meanwhile, in the liquid phase detection method, a system adaptability experiment is not required. This also makes the method of the present invention more efficient.
Further, different from the isocratic elution method adopted in the existing detection, the method adopts a gradient elution method in the liquid chromatography detection process, and the gradient elution condition and the mobile phase are the other key points of the method.
Specifically, in the present invention, the mobile phase comprises two components, namely a mobile phase a and a mobile phase B:
wherein, the mobile phase A is preferably a mixed solution of citric acid buffer solution and acetonitrile with pH4.0, and the proportion of the two is preferably 85:15 (v/v);
in mobile phase a, a citrate buffer solution with ph4.0 can be prepared according to the following method: mixing 0.1mol/L citric acid solution and 0.1mol/L sodium citrate solution according to a ratio of 13.1:6.9(v/v) to obtain the citrate buffer solution with the pH value of 4.0.
Wherein, the mobile phase B is preferably a mixed solution of citric acid buffer solution and acetonitrile with pH4.0, and the ratio of the two is preferably 60:40 (v/v);
in mobile phase B, a citrate buffer solution with ph4.0 can be prepared according to the following method: mixing 0.1mol/L citric acid solution and 0.1mol/L sodium citrate solution according to a ratio of 13.1:6.9(v/v) to obtain the citrate buffer solution with the pH value of 4.0.
Preferably, the conditions for gradient elution are as follows:
| time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 15 | 70 | 30 |
| 20 | 70 | 30 |
| 21 | 100 | 5 |
| 23 | 100 | 5 |
Then, the chromatogram was recorded until the retention time of the 2 times that of the main peak was 2, and the retention time of the 2, 3-cyclohexylpyridine peak relative to the cefquinome peak was about 0.20;
meanwhile, if an impurity peak exists in a chromatogram of a sample solution, the content of 2, 3-cyclohexyl pyridine cannot exceed 2.0 percent, other single impurities cannot exceed 1.0 percent, and the sum of all the impurities cannot exceed 4.0 percent according to the calculation of a peak area normalization method.
In the detection method, the peak-off time of the 2, 3-cyclohexyl pyridine is more consistent with the retention time determined by the main drug self-contrast method through optimizing each chromatographic condition, and the detection method is more stable and better in tolerance and reproducibility, improves the separation effect of each component in the preparation, and ensures that each impurity peak can be better separated, so that the detection result is more accurate and the specificity is stronger.
Example 1
The chromatographic conditions and the system applicability test comprise the following types of high performance liquid chromatographs: agilent 1260; the type of the chromatographic column: kromasil 100-5C18(4.6 x 150mm, 5 μm); octadecylsilane chemically bonded silica is used as a filling agent; performing linear gradient elution with citrate buffer solution (ph4.0) (0.1 mol/L citric acid solution and 0.1mol/L sodium citrate solution are mixed according to the ratio of (V/V)13.1: 6.9) and acetonitrile (85:15) as mobile phase A and citrate buffer solution (ph4.0) -acetonitrile (60:40) as mobile phase B according to the following table; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength was 254 nm. The number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate peak. Linear gradient elution was performed according to the method shown in the following table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 15 | 70 | 30 |
| 20 | 70 | 30 |
| 21 | 100 | 0 |
| 23 | 100 | 0 |
The determination method comprises the following steps: taking a proper amount of the evenly mixed cefquinome sulfate breast injectant sample (about equivalent to 10mg of cefquinome) in the lactation period, putting the sample into a 50ml centrifuge tube, adding 25ml of n-hexane saturated with cefquinome sulfate, evenly mixing, centrifuging for 10 minutes (4500 r/min), discarding supernatant, repeatedly extracting for 1 time according to the method, and volatilizing the precipitate at room temperature until the precipitate is dry. Precisely adding 10ml of mobile phase, carrying out ultrasonic treatment in a cold water bath for 10 minutes to dissolve cefquinome sulfate, filtering, precisely measuring 20 mu l of a test sample continuous filtrate, injecting the filtrate into a liquid chromatograph, recording the chromatogram until the retention time of the chromatogram is 2 times of the retention time of a main peak, wherein the retention time of a 2, 3-cyclohexylpyridine peak relative to the cefquinome peak is about 0.20.
Investigation of related substance measurement methods:
1. determination of detection wavelength
Weighing 25mg to 250ml of cefquinome reference substance in a measuring flask, adding a mobile phase for dissolving, fixing the volume and shaking up to obtain the wavelength test solution. By utilizing an ultraviolet spectrophotometry, scanning is carried out within the range of 190 nm-600 nm, and the result shows that the cefquinome has the maximum absorption at the wavelength of 254 nm.
2. Determination of impurity calculation method
Taking the same batch of samples to examine retention time and the same sample, repeating the step of examining 3 needles to examine the content of the impurity in percentage (peak area%), respectively comparing an area normalization method with a self control method, and preferably selecting a more appropriate impurity calculation method, wherein the experimental result is as follows:
from the above experimental results, it can be seen that: when the area normalization method is used, the retention time RSD of 2, 3-cyclohexyl pyridine and other maximum single impurities among 3 samples of the same batch of samples is larger (more than 2 percent), and when the self-control method is adopted, the retention time RSD is less than 0.5 percent.
Therefore, the adoption of the self-contrast method can lead the peak emergence time of a single impurity to be more stable, and the tolerance and the reproducibility of the method are better; when the area normalization method is adopted, even if the same sample is repeatedly fed into 3 needles, the situation that the RSD of 2, 3-cyclohexyl pyridine and other maximum single impurity ratio content (peak area%) is larger can occur, the RSD of the total impurity ratio content (peak area%) is even up to 4.97%, the result of the self-comparison method is obviously better than that of the area normalization method, and the self-comparison method is proved to be capable of enabling the ratio content repeatability of a single impurity to be better and greatly improving the repeatability of the total impurity detection. Therefore, the self-control method is preferably the impurity calculation method of the present invention.
3. Specificity test
Preparing a blank auxiliary material solution: preparing blank auxiliary material solution of cefquinome sulfate breast injectant (in lactation period) according to the formula process proportion.
Precisely sucking 20 mul of each of the sample solution, the reference solution, the blank auxiliary material solution and the mobile phase, and injecting samples according to the chromatographic condition of the related substance determination method established by the invention. The result shows that chromatographic peaks appear on the corresponding positions of the sample and the reference substance, the base line in the chromatographic peaks of the sample is stable, and the separation degree of each impurity peak is good; the blank auxiliary material solution and the mobile phase have no chromatographic peak at each impurity position. It can be seen that the mobile phase and the negative control did not interfere with the retention time of each impurity.
4. Sample solution stability test
Sample injection of 20 mul of the test solution is carried out for 0, 2, 4, 6 and 10 hours respectively under the same chromatographic conditions, and the RSD of the test group is 0.88 percent to less than 2.0 percent according to the measured chromatographic peak area, so that the reference substance and the test solution have good stability within 10 hours.
The solution stability test results are as follows:
5. linear relation
Precisely weighing 20mg of cefquinome reference substance, placing the cefquinome reference substance into a 200ml measuring flask, adding a mobile phase to completely dissolve the cefquinome reference substance, fixing the volume, and shaking up to obtain a stock solution. Then, 1, 5, 10, 20, 40 and 60ml of the stock solutions were measured out and placed in a 100ml measuring flask, diluted to the scale with the mobile phase, shaken up and filtered through a 0.45 μm filter membrane to obtain solutions of 0.001mg/ml, 0.005mg/ml, 0.01mg/ml, 0.02mg/ml, 0.04mg/ml and 0.06mg/ml in the series of concentrations measured. According to the chromatographic conditions established by the invention, 20 mu l of sample is injected, and the chromatographic peak area of the sample is determined. The results of the linearity test are as follows:
| concentration (mg/ml) | 0.001 | 0.005 | 0.01 | 0.02 | 0.04 | 0.06 |
| Peak area | 6.7 | 36.3 | 73.8 | 151.4 | 295.4 | 442.7 |
Establishing a standard curve by taking the peak area and the concentration as coordinates to obtain an equation: Y7385.6X +0.3086 (R)20.9999). Therefore, the concentration of related substances in the cefquinome sulfate is in a good linear relation with the peak area within the range of 0.001 mg/ml-0.06 mg/ml.
6. Precision test
The sample solution of 0h in the item of the stability of the sample solution (namely the solution containing cefquinome with the concentration of 1.0 mg/ml) is repeatedly injected for 6 times under the same chromatographic condition, 20 mul is injected each time, the peak area of 2, 3-cyclohexylpyridine is respectively measured, the precision of the instrument is inspected, the relative standard deviation RSD obtained by 6 groups of data is calculated, and the result is 0.55 percent (< 2.0 percent).
The precision test result is as follows:
7. repeatability test
Selecting 6 parts of cefquinome sulfate breast injectant (in lactation period) of the same batch number, accurately weighing, preparing a solution according to the operation of a test item, adopting the same chromatographic conditions, injecting 20 mu l of sample, collecting the chromatogram of 6 parts of samples, and calculating the relative standard deviation RSD of the solution, wherein the result is 1.07% (< 2.0%). Therefore, the related substance determination method established by the invention has good repeatability.
The results of the repeatability tests are as follows:
8. reproducibility test
6 parts of cefquinome sulfate breast injectant (in lactation period) in the same batch is selected, and another analyst uses different liquid chromatographs and different chromatographic columns in different laboratories and different working days to measure the content ratio of related substances in the same operation of a 'repeatability test'.
The results of the reproducibility test were as follows:
the results show that: in the detection results of the two analysts, the RSD of the 2, 3-cyclohexyl pyridine, other maximum single impurities and total impurities is less than 2.0 percent, and the difference values are small, so that the method for determining the related substances established by the invention is good in reproducibility.
9. Accuracy test
Accurately weighing 3 parts of cefquinome sulfate breast injectant (lactation period) samples with known related substances according to 80%, 100% and 120% of the marked amount of the cefquinome sulfate breast injectant (lactation period), respectively placing the cefquinome sulfate breast injectant (lactation period) samples into 9 100ml centrifuge tubes, preparing a test sample and a reference solution according to experimental operation, measuring the related substances according to the related substance measuring method established by the invention, and calculating the recovery rate.
The accuracy test results are as follows:
from the above experimental results, it can be seen that: the recovery rate and the average recovery rate are both 92-105%, and the RSD is within 2%. Therefore, the related substance measuring method established by the invention has good accuracy.
10. Durability test
Taking a proper amount of cefquinome sulfate breast injectant (in lactation period) of the same batch, respectively inspecting the change of flow rate +/-0.2 ml/min, the change of column temperature +/-5 ℃, the change of acetonitrile proportion +/-2% in a mobile phase, the change of pH of citrate buffer solution +/-0.2 (namely 0.1mol/L citric acid solution: 0.1mol/L sodium citrate solution (V/V) ═ 14.0:6.0 or 12.3:7.7) in the mobile phase) and the change of instrument chromatographic behavior in different chromatographic columns, observing baseline separation conditions, and calculating the average value and RSD value of the obtained data under each condition, wherein the test results are as follows:
test results show that in the durability test of the method for measuring cefquinome sulfate related substances of the cefquinome sulfate breast injectant (in lactation period), the flow rate, the column temperature, the chromatographic column, the mobile phase composition and the PH slightly change, all impurity peaks reach baseline separation, and the RSD values of 2, 3-cyclohexylpyridine, other maximum single impurity related substances and total impurity related substances measured under all conditions are less than 2%. Therefore, the related substance measuring method established by the invention has good durability.
11. Detection limit
Dissolving a cefquinome reference substance into a test solution of 0.01mg/ml by using a mobile phase, diluting the test solution step by step, adjusting the concentration of the test solution to enable the response value of the cefquinome to be about 3 times of the noise level, and obtaining that the detection limit of the cefquinome is 3 mug/ml, wherein the method provides that the peak which is less than 0.5 times of the main peak area of the reference solution is ignored (namely the peak which is less than 5 mug/ml is ignored). Therefore, the detection limit of the related substance determination method established by the invention can meet the detection requirement.
12. Determination of cefquinome sulfate-related substances in sample
The method for determining related substances established by the invention respectively determines related substances of cefquinome sulfate in three batches of cefquinome sulfate breast injectants (in lactation period), and the results are as follows:
as can be seen, the RSD of the related substances of the three batches of samples is less than 2%, and the intra-batch difference is small.
Comparative example 1
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking sodium perchlorate buffer solution (taking 3.45g of sodium perchlorate monohydrate, adding 1000ml of water for dissolution, adding 12ml of phosphoric acid and 115ml of acetonitrile, and adjusting the pH to 3.6 +/-0.1 by triethylamine) as a mobile phase, wherein the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength was 270 nm.
Taking about 25mg of cefotaxime, adding 100ml of mobile phase to dissolve the cefotaxime, taking about 25mg of cefotaxime and 1ml of cefotaxime solution, diluting the cefotaxime solution to 25ml by using the mobile phase, and shaking up the solution to be used as a system applicability test solution. Injecting 20 μ l into a liquid chromatograph, and recording the chromatogram, wherein the separation degree between the cefquinome peak and the cefotaxime peak is more than 1.0.
The determination method comprises the following steps:
taking a proper amount of the evenly mixed cefquinome sulfate breast injectant sample (about equivalent to 10mg of cefquinome) in the lactation period, putting the sample into a 50ml centrifuge tube, adding 25ml of n-hexane saturated with cefquinome sulfate, evenly mixing, centrifuging for 10 minutes (4500 r/min), discarding supernatant, repeatedly extracting for 1 time according to the method, and volatilizing the precipitate at room temperature until the precipitate is dry. Precisely adding 10ml of mobile phase, placing in a cold water bath for ultrasonic treatment for 10 minutes to dissolve the cefquinome sulfate, and filtering. And precisely measuring 20 mu l of subsequent filtrate, injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the main peak is 2 times that of the chromatogram, wherein the retention time of the 2, 3-cyclohexylpyridine peak relative to the cefquinome peak is about 0.3. If an impurity peak exists in the chromatogram of the test solution, the content of 2, 3-cyclohexyl pyridine cannot exceed 2.0 percent, other single impurities cannot exceed 1.0 percent, and the sum of all the impurities cannot exceed 4.0 percent according to the calculation of a peak area normalization method.
1. Specificity test
(1) Preparing a blank auxiliary material solution: preparing blank auxiliary material solution of cefquinome sulfate breast injectant (in lactation period) according to the formula process proportion.
Precisely sucking 20 mul of each of the sample solution, the reference solution, the blank auxiliary material solution and the mobile phase, and injecting samples according to the chromatographic condition of the related substance determination method established by the invention. As a result, chromatographic peaks appear on the corresponding positions of the sample and the reference, the base line in the chromatographic peaks of the sample is stable, and the separation degree of each impurity peak is good; the blank auxiliary material solution and the mobile phase have no chromatographic peak at each impurity position, which indicates that the mobile phase and the negative control have no interference on the retention time of each impurity.
(2) Comparing example 1 and comparative example 1, chromatograms were recorded, and the degrees of separation of the respective impurity peaks, and the ratio of the retention time of 2, 3-cyclohexylpyridine to cefquinome were observed.
The detection results of the cefquinome sulfate breast injectant (in lactation period) in the example 1 and the comparative example 1 are as follows:
the results show that: the chromatographic condition of the embodiment 1 can better ensure better separation degree, and the retention time ratio of the 2, 3-cyclohexylpyridine to the cefquinome is more stable, so that the peak-out time of the 2, 3-cyclohexylpyridine can be more fit with the retention time determined by the main drug self-contrast method.
From the above experimental results, although the cefquinome peak in example 1 and comparative example 1 can not be interfered by the mobile phase and the blank preparation control, and both meet the basic requirement of specificity, the detection method of the related substances in example 1 has stronger specificity and reproducibility than that in comparative example 1, and the peak emergence time of 2, 3-cyclohexylpyridine can better fit the retention time determined by the main drug self-control method.
2. The determination results of cefquinome related substances in three samples:
as can be seen from the above experimental control results, the RSD of the related substances of the three samples is less than 2%, but the reproducibility result of example 1 is better than that of comparative example 1.
The results show that the specificity and the reproducibility of the detection method for the liquid phase related substances of the optimized cefquinome sulfate breast injectant (in the lactation period) are improved.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. A liquid phase detection method of cefquinome sulfate breast injectant 2, 3-cyclohexylpyridine in the lactation period is characterized by comprising the following steps:
detecting the cefquinome sulfate breast injectant 2, 3-cyclohexylpyridine in the lactation period by adopting a high performance liquid chromatography, and performing gradient elution according to the following conditions:
the mobile phase A is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the ratio of the citric acid buffer solution to the acetonitrile is as follows: 80-90: 10-20;
the mobile phase B is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the ratio of the citric acid buffer solution to the acetonitrile is as follows: 50-70: 30-50;
the high performance liquid chromatography conditions were as follows:
flow rate: 0.8-1.2 ml/s;
column temperature: 25-35 ℃;
detection wavelength: 230 to 280 nm.
2. The liquid phase detection method according to claim 1, wherein the liquid phase detection method is characterized by performing gradient elution under the following conditions:
the mobile phase A is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the pH value of the citric acid buffer solution is 3.6-4.0, and the proportion of the citric acid buffer solution to the acetonitrile is as follows: 85-90: 10-15;
the mobile phase B is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the pH value of the citric acid buffer solution is 3.6-4.0, and the proportion of the citric acid buffer solution to the acetonitrile is as follows: 60-70: 30-40.
3. The liquid phase detection method according to claim 2, wherein the liquid phase detection method performs gradient elution under the following conditions:
the mobile phase A is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the ratio of the citric acid buffer solution to the acetonitrile is as follows: 85: 15;
the mobile phase B is a mixed solution of a citric acid buffer solution and acetonitrile, wherein the ratio of the citric acid buffer solution to the acetonitrile is as follows: 60:40.
4. The liquid phase detection method according to claim 1, wherein the high performance liquid chromatography conditions are as follows:
flow rate: 1.0 ml/s;
column temperature: 30 ℃;
detection wavelength: 254 nm;
the number of theoretical plates is not less than 2000 calculated according to cefquinome sulfate.
5. The liquid phase detection method according to claim 1, characterized by comprising:
injecting the test solution into a liquid chromatograph, and recording the chromatogram until the retention time of the main peak is 2 times;
the retention time of the 2, 3-cyclohexylpyridine peak relative to the cefquinome sulfate peak is 0.15-0.25.
6. The liquid phase detection method according to claim 5, wherein the chromatogram of the sample solution contains impurity peaks, and the content of 2, 3-cyclohexylpyridine is not more than 2.0%, the content of other single impurities is not more than 1.0%, and the sum of the respective impurities is not more than 4.0%, as calculated by a peak area normalization method.
7. The liquid-phase detection method according to claim 5, wherein the preparation of the sample solution comprises the steps of: adding an extraction solution saturated by a cefquinome sulfate reference substance into a breast injection of cefquinome sulfate in the lactation period, shaking uniformly, and centrifuging, wherein the obtained precipitate is extracted for 1-3 times under the same condition;
and drying the precipitate obtained in the last time, and dissolving the precipitate with a mobile phase to obtain a test solution.
8. The liquid phase detection method of claim 7, wherein the extraction solution saturated with the cefquinome sulfate control is a hexane solution saturated with the cefquinome sulfate control.
9. The liquid-phase detection method according to claim 8, wherein the preparation of the sample solution comprises the steps of:
adding saturated hexane solution of cefquinome sulfate as reference substance into breast injection of cefquinome sulfate in lactation period, shaking, centrifuging, extracting the obtained precipitate for 1 time under the same condition, drying, dissolving with mobile phase, ultrasonic treating, filtering, and injecting the filtrate into liquid chromatograph.
10. The liquid phase detection method according to claim 9, wherein the amount of sample is 20 μ L.
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