CN108802211A - The liquid phase detection method of related substances in a kind of Cefquinome sulfate breast injection - Google Patents
The liquid phase detection method of related substances in a kind of Cefquinome sulfate breast injection Download PDFInfo
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Abstract
The present invention provides a kind of liquid phase detection methods of related substances in Cefquinome sulfate breast injection.The method of the present invention specificity is strong, and favorable reproducibility, this can make each impurity peaks separating effect more preferable, and testing result is more acurrate.
Description
Technical field
The present invention relates to detection technique fields, in particular to related in a kind of Cefquinome sulfate breast injection
The liquid phase detection method of substance.
Background technology
Cefquinome sulfate breast injection is that one kind is treated for mastitis for milk cows, is especially used for dry cow breast
The drug of room inflammation treatment, wherein active ingredient Cefquinome sulfate is a kind of aminothiazole oxidation of ketones imines cephalosporin, to being permitted
More Gram-positives and gramnegative bacterium have good spectrum antibacterial activity, absorb fast, peaking period, biology profit
Expenditure is higher, can reach higher tissue concentration in lung, breast tissue.
In existing Cefquinome sulfate breast injection in the liquid phase detection method in relation to substance, detached between each chromatographic peak
Though degree meet regulation it is relatively less than normal, cause the reproducibility of impurity peaks peak area poor, and using area normalization method calculating have
It closes content of material and makes easily to be influenced by the minor fluctuations of chromatographic condition in relation to material result, to be difficult to guarantee the standard of testing result
True property.
In view of this, special propose the present invention.
Invention content
The first object of the present invention is to provide a kind of liquid phase inspection of related substances in Cefquinome sulfate breast injection
Survey method.The method of the present invention specificity is strong, and favorable reproducibility, this can make each impurity peaks separating effect more preferable, and testing result is more accurate
Really.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
The liquid phase detection method of related substances in a kind of Cefquinome sulfate breast injection, including：
The related substances in Cefquinome sulfate breast injection are detected using high performance liquid chromatography；
Wherein, in the high performance liquid chromatography, gradient elution is carried out using following condition：
Wherein, mobile phase A is the mixed liquor of the buffer solution and acetonitrile of pH 3.6~4.0, and the ratio of buffer solution and acetonitrile is
85~95:5~15；
Mobile phase B is the mixed liquor of the buffer solution and acetonitrile of pH 3.6~4.0, the ratio of buffer solution and acetonitrile is 40~
60:40~60.
Preferably, in liquid phase detection method of the present invention, the high performance liquid chromatography is carried out using following condition
Gradient elution：
Preferably, in liquid phase detection method of the present invention, the high performance liquid chromatography is carried out using following condition
Gradient elution：
Preferably, in liquid phase detection method of the present invention, buffer solution and acetonitrile that the mobile phase A is pH 3.6
The ratio of mixed liquor, buffer solution and acetonitrile is 90:10；
And/or the mixed liquor for the buffer solution and acetonitrile that the Mobile phase B is pH 3.6, the ratio of buffer solution and acetonitrile are
50:50。
Preferably, in liquid phase detection method of the present invention, the high performance liquid chromatography uses following condition：
Flow velocity：0.8~1.2ml/min；
Column temperature：25~35 DEG C；
Detection wavelength：230~280nm；
And/or number of theoretical plate is calculated by Cefquinome sulfate and is not less than 2000.
Preferably, in liquid phase detection method of the present invention, the high performance liquid chromatography uses following condition：
Flow velocity：1.0ml/min；
Column temperature：30℃；
Detection wavelength：245nm.
Preferably, liquid phase detection method of the present invention includes：
It is prepared by test solution：It is added to Cefquinome sulfate breast injection sample full with Cefquinome sulfate reference substance
The extraction solution of sum, centrifuges after shaking up, and collects precipitation；It is handled 1-3 times under sediment the same terms, by last time processing gained
After drying precipitate, to flow phased soln, is diluted after ultrasound, shake up, filter, obtain test solution；
It is prepared by contrast solution 1：By test solution to shake up after flowing phase dilution, contrast solution 1 is obtained；
It is prepared by contrast solution 2：2,3- cyclohexyl pyrroles is shaken up with flowing to dilute after phased soln, obtains contrast solution 2；
Test solution, contrast solution 1 and contrast solution 2 are injected separately into high performance liquid chromatography, and record chromatography.
Preferably, in liquid phase detection method of the present invention, the extraction being saturated with Cefquinome sulfate reference substance
Solution is the petroleum ether solution being saturated with Cefquinome sulfate reference substance；
Preferably, the boiling range of petroleum ether is 30~60 DEG C.
Preferably, in liquid phase detection method of the present invention, the preparation of contrast solution 1 includes：
1ml test solutions are placed in 100ml measuring bottles, then to flow phase dilution constant volume, are obtained after shaking up.
Preferably, liquid phase detection method of the present invention includes：If any impurity peaks in test solution chromatogram, contain 2,
3- cyclohexylmethylpyridines are not greater than 3 times of contrast solution main peak area, and other single contaminants are not greater than contrast solution main peak face
Product, total impurities are not greater than 4 times of contrast solution main peak area；
It ignores at peak in test solution chromatogram less than 0.5 times of contrast solution main peak area.
Compared with prior art, beneficial effects of the present invention are：
1. the present invention establish one can make related substance testing result reduce it is being influenced by chromatographic condition minor fluctuations,
The related substance detecting method of Cefquinome sulfate breast injection, the change in relation to substance computational methods can further enhance detection
As a result accuracy.
2. the present invention can make each impurity peaks separating effect more preferable, testing result is more acurrate, specificity is stronger.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
In view of in the presence of the liquid phase detection method of related substances in existing Cefquinome sulfate breast injection
The problems such as impurity separating degree is small, poor reproducibility, the present invention provides a kind of new detection method, the chromatography after present invention optimization
The separating effect of each ingredient in improvement of terms preparation, it is ensured that each impurity peak energy is preferably detached, to make testing result more
Accurately, specificity is stronger, and the change in relation to substance computational methods also further enhances the accuracy of testing result.
Specifically, detection method provided by the present invention, is that one kind being directed to the injection of dry breast phase Cefquinome sulfate breast
The liquid phase detection method of related substances, step can refer to as follows in agent：
It is prepared by test solution：It is added to Cefquinome sulfate breast injection (dry breast phase) sample with Cefquinome sulfate
The extraction solution of reference substance saturation, centrifuges after shaking up, and collects precipitation；It is handled 1-3 times under sediment the same terms, it will last time
After handling gained sediment drying, to flow phased soln, is diluted after ultrasound, shake up, filter, obtain test solution.
Preferably, the preparation of test solution includes the following steps：By dry breast phase Cefquinome sulfate breast injection sample
Product are placed in centrifuge tube, and saturation Cefquinome sulfate diethyl ether solution is then added (i.e. with Cefquinome sulfate standard items saturation
Diethyl ether solution, preparation method can refer to for：The Cefquinome sulfate of 9mg or so is taken, is added in 1000ml petroleum ethers, shakes up i.e.
Can), dry breast phase Cefquinome sulfate breast injection sample quality grams and saturation Cefquinome sulfate diethyl ether solution volume milli
The ratio for rising number is preferably 1~2:85~115, more preferably 1.5:100；
Then, it shakes up, centrifuges (rotating speed 4500r/min, 10~30min of time), discard supernatant liquid；
Sediment extracts again according to method as above, and repeats to extract 1~3 time；After last time extraction centrifugation
Gained sediment drying (is preferably dried up with protective gas such as nitrogen or argon gas)；
Then, appropriate mobile phase is added into the centrifuge tube equipped with the sediment after drying, is then centrifuged for mixture in pipe
System is transferred in measuring bottle (preferred, the nominal volume of dry breast phase Cefquinome sulfate breast injection sample quality grams and measuring bottle
The ratio of product ml is preferably 1~2:80~120, more preferably 1:100), 10~20min of ultrasound (preferably 15min),
It lets cool, scale is then diluted to mobile phase, filtered to get to test solution after shaking up.
It is prepared by contrast solution 1：By test solution to shake up after flowing phase dilution, contrast solution 1 is obtained；
Preferably, the preparation of contrast solution 1 includes：1ml test solutions are placed in 100ml measuring bottles, then with flowing
Phase dilution constant volume obtains contrast solution 1 after shaking up
It is prepared by contrast solution 2：2,3- cyclohexyl pyrroles is shaken up with flowing to dilute after phased soln, obtains contrast solution 2；
2,3- cyclohexyl pyrroles is a kind of known impurity in dry breast phase Cefquinome sulfate breast injection
Preferably, the preparation of contrast solution 2 includes the following steps：Appropriate 2,3- cyclohexyl pyrroles is taken, is then mixed with flowing
It solves and dilutes so that the concentration of 2,3- cyclohexyl pyrroles reaches 10~30 (preferably 20) μ g/ml in solution.
Test solution, contrast solution 1 and contrast solution 2 are injected separately into high performance liquid chromatography, it is preferred that sample introduction
Amount is 20 μ L, and records chromatography.
If any impurity peaks in test solution chromatogram, the cyclohexylmethylpyridine containing 2,3- is not greater than contrast solution main peak area
3 times (3.0%), other single contaminants are not greater than contrast solution main peak area (1.0%), and it is molten that total impurities are not greater than control
4 times (4.0%) of liquid main peak area.Ignore not at peak in test solution chromatogram less than 0.5 times of contrast solution main peak area
Meter.
Different from external standard and the normalized detection method of area used by conventional liquid phase detection, employed in the present invention
Be Self-control method, and this be also the present invention for related substances detection method in traditional Cefquinome sulfate breast injection
One of improvement.In the way of own control, the accounting content repeatability of single impurity can be made more preferable, while also significantly
Improve the repeatability of total miscellaneous detection.
Chromatographic condition of the present invention is as follows：
High performance liquid chromatograph model：Agilent 1260.
Column model：Kromasil 100-5C18 (4.6*150mm, 5 μm)
Filler：Octadecylsilane key and silica gel.
Mobile phase：In the present invention, used is the mode of gradient elution, and gradient elution is then the another pass of the present invention
Where key point.
Mobile phase of the present invention includes mobile phase A and Mobile phase B；
Wherein, mobile phase A：The ratio of the buffer solution of pH 3.6~4.0 and the mixed liquor of acetonitrile, buffer solution and acetonitrile is 85
~95:5~15 (v/v)；
Preferably, mobile phase A is the mixed liquor of the buffer solution and acetonitrile of pH 3.6, and the ratio of buffer solution and acetonitrile is 90:
10(v/v)；
The preparation method of buffer solution is preferably as follows in mobile phase A：Citric acid 1.42g, disodium hydrogen phosphate 2.31g are taken, water is added
It dissolves and dilutes 1000ml (can reach pH3.6).
Mobile phase B：The ratio of the buffer solution of pH 3.6~4.0 and the mixed liquor of acetonitrile, buffer solution and acetonitrile is 40~60:
40~60 (v/v)；
Preferably, mobile phase A is the mixed liquor of the buffer solution and acetonitrile of pH 3.6, and the ratio of buffer solution and acetonitrile is 50:
50(v/v)；
The preparation method of buffer solution is preferably as follows in mobile phase A：Citric acid 1.42g, disodium hydrogen phosphate 2.31g are taken, water is added
It dissolves and dilutes 1000ml (can reach pH3.6).
Flow velocity：0.8~1.2ml/min；Preferably 1.0ml/min.
Column temperature：25~35 DEG C；Preferably：30℃.
Detection wavelength：230~280nm；Preferably 245nm.
Number of theoretical plate is calculated by Cefquinome sulfate peak is not less than 2000.
Meanwhile compared to traditional liquid chromatography detecting method for, the method for the present invention is without carrying out system suitability examination
It tests, and this is but also the method for the present invention is operationally also more convenient.
Embodiment 1
Chromatographic condition and system suitability：High performance liquid chromatograph model：Agilent 1260；Column model：
Kromasil 100-5C18 (4.6*150mm, 5 μm)；Using octadecylsilane chemically bonded silica as filler；With pH3.6 buffer solutions
(taking citric acid 1.42g, disodium hydrogen phosphate 2.31g, be dissolved in water and dilute 1000ml)-acetonitrile (90:10) it is mobile phase A,
PH3.6 buffer solutions-acetonitrile (50:50) it is Mobile phase B, according to the form below progress linear gradient elution；Flow velocity is 1.0ml/min；Column temperature
It is 30 DEG C；Detection wavelength is 254nm.Number of theoretical plate is calculated by Cefquinome sulfate peak is not less than 2000.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 15 | 50 | 50 |
| 20 | 50 | 50 |
| 21 | 100 | 0 |
| 23 | 100 | 0 |
Detection method：Dry breast phase Cefquinome injection sample about 1.5g is taken, it is accurately weighed, it sets in centrifuge tube, is added and uses
The petroleum ether (30 DEG C~60 DEG C of boiling range) of Cefquinome sulfate reference substance saturation (takes Cefquinome sulfate about 9mg to add petroleum ether
1000ml) 20ml shakes up, and 4500 revs/min centrifuge 10 minutes, discard supernatant liquid；Primary with method operation, taking precipitate sets room temperature
It under state, is dried up with nitrogen, adds mobile phase appropriate, ultrasound shakes and is transferred in 100ml measuring bottles, ultrasound 15 minutes, after letting cool
It is diluted to scale with mobile phase, is shaken up, is filtered, as test solution；
Precision measures test solution 1ml, sets in 100ml measuring bottles with flowing phase dilution constant volume, shakes up, as a contrast solution
1；
It separately takes 2,3- cyclohexylmethylpyridines appropriate, adds flowing phased soln and dilution to be made in every 1ml and contain 20 μ g solution, as right
According to solution 2.
Test solution and contrast solution 1,2 each 20 μ l of contrast solution are respectively taken, liquid chromatograph is injected separately into, records color
Spectrum.If any impurity peaks in test solution chromatogram, the cyclohexylmethylpyridine containing 2,3- is not greater than 3 times of contrast solution main peak area
(3.0%), other single contaminants are not greater than contrast solution main peak area (1.0%), and total impurities are not greater than contrast solution master
4 times (4.0%) of peak area.It ignores at peak in test solution chromatogram less than 0.5 times of contrast solution main peak area.
Related substance-measuring method is investigated：
1. the determination of Detection wavelength
It weighs in Cefquinome reference substance 25mg to 250ml measuring bottles, adds flowing phased soln and constant volume, shake up, as wavelength
Test solution.It using ultraviolet spectrophotometry, is scanned within the scope of 190nm~600nm, is as a result shown in head at wavelength 254nm
Spore quinoline oxime has absorption maximum.
2. the determination of impurity computational methods
Take same sample repeat into 3 needles investigate impurity accounting content (peak area %), respectively compare area normalization method with
Self-control method, preferably more suitably impurity computational methods, the results are shown in table below：
By experimental result as above it is found that when with area normalization method, even same sample is repeated into 3 needles, it is also possible to
There is 2,3- cyclohexylmethylpyridines and other maximum single miscellaneous accounting content (peak area %) RSD situations bigger than normal, total miscellaneous accounting content
(peak area %) RSD is very big, and the result of Self-control method is obviously good compared with area normalization method, it can be seen that, with own control
Method can make the accounting content repeatability of single impurity more preferable, while also substantially improve the repeatability of total miscellaneous detection.It is therefore preferable that
Self-control method is the impurity computational methods of the present invention.
3. specificity is tested
The preparation of blank auxiliary solution：Cefquinome sulfate breast injection (dry breast phase) is prepared in formulation and technology ratio
Blank auxiliary solution.
Precision draws test solution, reference substance solution, blank auxiliary solution, each 20 μ l of mobile phase, is established by the present invention
Related substance-measuring method chromatographic condition sample introduction.As a result, there is chromatographic peak on a corresponding position with reference substance in test sample,
And baseline is steady in test sample chromatographic peak, each impurity peaks separating degree is good；Blank auxiliary solution, mobile phase are on each impurity position
No chromatographic peak occurs, and shows that mobile phase and negative control are noiseless to each impurity retention time.
4. the stability of sample solution
Test solution is taken, respectively at 0,2,4,6,10 hour, using identical chromatographic conditions, 20 μ l of sample introduction, according to measuring
Chromatographic peak area, test sample group RSD be 0.63% < 2.0%, prompt reference substance and test solution it is good in 10h internal stabilities
It is good.
Solution stability testing result：
5. linear relationship
Precision weighs Cefquinome reference substance 18.75mg and is placed in 100ml measuring bottles, and mobile phase is added and is allowed to after being completely dissolved
Constant volume shakes up, as storing solution.The storing solution 1,2,4,8,16,24ml are measured again respectively in 100ml measuring bottles, uses mobile phase
It is diluted to scale, is shaken up, with 0.45 μm of membrane filtration, obtains 1.875 μ g/ml, 3.75 μ g/ml, 7.5 μ g/ml, 15 μ g/ml, 30
μ g/ml, 45 μ g/ml Series Measurement strength solutions.By the chromatographic condition that the present invention establishes, 20 μ l of sample introduction, determination sample chromatographic peak
Area.
Linear test result：
| Concentration (μ g/ml) | 1.875 | 3.75 | 7.5 | 15 | 30 | 45 |
| Peak area | 20.6 | 46.6 | 94.7 | 193.4 | 383.8 | 572.8 |
With peak area and a concentration of coordinate, standard curve is established, obtaining equation is：Y=12.792X-1.2179 (R2=
0.9999), prompting related material concentration, there are good linear passes with peak area within the scope of 1.875 μ of μ g/ml~45 g/ml
System.
6. precision test
By the test solution of 0h under " sample solution stability " item, (i.e. a concentration of 0.75mg/ml's containing Cefquinome is molten
Liquid), it under identical chromatographic conditions, repeats sample introduction 6 times, every time 20 μ l, measures 2,3- cyclohexylmethylpyridine peak areas respectively, investigate instrument
Device precision, and the relative standard deviation RSD obtained by 6 groups of data is calculated, result is 0.61% < 2.0%, it can be seen that, this hair
The related substance-measuring method precision of bright foundation is good.Precision test result is as follows：
7. repetitive test
Selection is with 6 parts of lot number Cefquinome sulfate breast injection (dry breast phase), after accurately weighed, is grasped according to test sample item
Solution is prepared, using identical chromatographic conditions, 20 μ l of sample introduction acquire the chromatogram of 6 parts of samples, and the relative standard for calculating solution is inclined
Poor RSD, result are 1.61% < 2.0%.It can be seen that the related substance-measuring method repeatability that the present invention establishes is good.It repeats
Property experimental result is specific as follows：
8. reappearance test
Selection is with 6 parts of lot number Cefquinome sulfate breast injection (dry breast phase), by another analyst in different experiments
Room, different working days, using different liquid chromatographs, different chromatographic columns is operated with " repetitive test ", is measured related
Content of material accounting.Reproducible test results is as follows：
The result shows that：In two analyst's testing results, 2,3- cyclohexylmethylpyridines, other maximum single miscellaneous, total miscellaneous equal < of RSD
2.0%, difference is small.It can be seen that the related substance-measuring method reproducibility that the present invention establishes is good.
9. accuracy test
It is accurately weighed by 80%, 100%, 120% difference of Cefquinome sulfate breast injection (dry breast phase) labelled amount
Known each 3 parts of Cefquinome sulfate breast injection (dry breast phase) sample in relation to amount of substance, is respectively placed in 9 centrifuge tubes,
Test sample and contrast solution are prepared according to experimental implementation, related substance is measured by the related substance-measuring method that the present invention establishes
Amount calculates the rate of recovery.Accuracy test result is as follows：
From result as above：The rate of recovery of the present invention and average recovery rate 92%~105%, RSD all 2% with
It is interior, it can be seen that, the related substance-measuring method accuracy that the present invention establishes is good.
10. serviceability test
It takes with a collection of Cefquinome sulfate breast injection (dry breast phase) in right amount, investigates change in flow ± 0.2ml/ respectively
The variation of instrument chromatographic behavior, sees when acetonitrile ratio changes ± 2%, different chromatographic columns in min, ± 5 DEG C of column temperature variation, mobile phase
Baseline separation situation is examined, the average value and RSD values of the data obtained, serviceability test result are as follows under the conditions of calculating respectively：
Test result shows the related substance-measuring side of Cefquinome sulfate breast injection (dry breast phase) Cefquinome sulfate
In method serviceability test, flow velocity, column temperature, chromatographic column, flowing phase composition, each impurity peaks reach baseline separation, it is each under the conditions of institute
2,3- cyclohexylmethylpyridines, the equal < 2% of other maximum single miscellaneous, total miscellaneous RSD values in relation to substance are surveyed, prompts the present invention to establish related
Substance-measuring method good tolerance.
11. detection limit
It takes Cefquinome reference substance to be made into the testing liquid of 7.5 μ g/ml with flowing phased soln, and dilutes step by step, adjust examination
Testing solution concentration makes the response of Cefquinome be about 3 times of noise level, show that the detection of Cefquinome is limited to 1.5 μ g/ml,
And provide that " ignoring at peak of 0.5 times less than contrast solution main peak area " (is ignored not at the peak for being less than 3.75 μ g/ml in method
Meter), therefore the related substance-measuring method detection limit that the present invention establishes can meet detection needs.
12. the related substance-measuring of Cefquinome sulfate in sample
The related substance-measuring method established by the present invention measures three batches of Cefquinome sulfate breast injections (dry breast respectively
Phase) in the related substance of Cefquinome sulfate, it is as a result as follows：
By result as above it is found that the related substance RSD of three batches of samples is respectively less than 2%, difference is small in batch.
Comparative example 1
Chromatographic condition and system suitability：Using octadecylsilane chemically bonded silica as filler；With 0.345% 1 water
Close sodium perchlorate solution-phosphoric acid-acetonitrile (1000:12:115) it is mobile phase (to adjust pH value to 3.6 with triethylamine), and flow velocity is
1.0ml/min；Column temperature is 30 DEG C；Detection wavelength is 270nm.Cefotaxime about 25mg is taken, adds mobile phase 100ml to make dissolving, separately
Cefquinome about 25mg is taken, is set in 25ml measuring bottles, above-mentioned cefotaxime solution 1ml is added in precision, and scale is diluted to mobile phase.
The separating degree of 20 μ l injection liquid chromatographs of precision measurement, record chromatogram, Cefquinome and cefotaxime should be greater than 1.0.
Assay method：Dry breast phase Cefquinome sulfate breast injection about 1.5g is taken, it is accurately weighed, it sets in centrifuge tube, adds
Entering the petroleum ether (30 DEG C~60 DEG C of boiling range) being saturated with Cefquinome sulfate reference substance (takes Cefquinome sulfate about 9mg to add oil
Ether 1000ml) 20ml, it shakes up, 4500 revs/min centrifuge 10 minutes, discard supernatant liquid；Primary with method operation, taking precipitate sets room
It under temperature state, is dried up with nitrogen, adds mobile phase appropriate, ultrasound shakes and is transferred in 100ml measuring bottles, and ultrasound 15 minutes is let cool
It is diluted to scale with mobile phase afterwards, is shaken up, is filtered；
It separately takes 2,3- cyclohexylmethylpyridines appropriate, adds flowing phased soln and dilution to be made in every 1ml and contain 20 μ g solution, as right
According to product solution.
Test solution and each 20 μ l of reference substance solution are taken, liquid chromatograph is injected separately into, records chromatogram.Test sample is molten
It if any impurity peaks in liquid chromatography figure, is calculated by peak area normalization method, 3.0% must not be crossed containing 2,3- cyclohexylmethylpyridines, it is other single
Impurity must not cross 1.0%, and total impurities must not cross 4.0%.
1. specificity is tested
(1) preparation of blank auxiliary solution：Cefquinome sulfate breast injection (dry breast is prepared in formulation and technology ratio
Phase) blank auxiliary solution.
Precision draws test solution, reference substance solution, blank auxiliary solution, each 20 μ l of mobile phase, is established by the present invention
Related substance-measuring method chromatographic condition sample introduction.As a result, there is chromatographic peak on a corresponding position with reference substance in test sample,
And baseline is steady in test sample chromatographic peak, each impurity peaks separating degree is good；Blank auxiliary solution, mobile phase are on each impurity position
No chromatographic peak occurs, and shows that mobile phase and negative control are noiseless to each impurity retention time.
But the peak-to-peak separating degree of each impurity of test sample, peak purity, the stability of retention time are general, poor compared with embodiment 1,
Data comparison is specific as follows：
Note：Peak purity ratio=peak purity up to standard reaches 98% impurity peaks number/total impurities peak number * 100%
By Experimental comparison results as above it is found that the related substance detecting method of embodiment 1 is compared with 1 specificity of comparative example, reproduction
Property is stronger.
(2) comparative example 1 and comparative example 1 record chromatogram, observe separating degree, the peak purity ratio up to standard of each impurity peaks
Example, and take the stability of same batch of sample investigation impurity peaks retention time.
Testing result of the Cefquinome sulfate breast injection (dry breast phase) under embodiment 1, comparative example 1 is as follows：
Note：Peak purity ratio=peak purity up to standard reaches 98% impurity peaks number/total impurities peak number * 100%
It is compared from experimental result as above：Embodiment 1 more can guarantee that more preferably separating effect, each impurity component can be by more
It detaches well, and the stability of impurity peaks retention time is more preferable, to can guarantee that detection method specificity, reproducibility are stronger, inspection
It is more acurrate to survey result.
In conclusion embodiment 1 and comparative example 1 can be such that Cefquinome peak is not done by mobile phase, blank formulation control
It disturbs, meets specificity basic demand.However, each peak separating degree effect of 1 energy test sample of embodiment, peak purity are more preferable, and impurity
Peak retention time is more stable, it was demonstrated that the embodiment 1 after preferably is more preferably compared with comparative example 1.
2. the related substance-measuring result of Cefquinome in three batches of samples：
By experimental result as above it is found that the related substance RSD of three batches of samples is respectively less than 2%, but 1 reproducibility result of embodiment
It is more preferable compared with comparative example 1.
In summary, we can see that the related substance of Cefquinome sulfate breast injection (dry breast phase) liquid phase after present invention optimization
Improved in terms of detection method specificity, reproducibility.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. the liquid phase detection method of related substances in a kind of Cefquinome sulfate breast injection, which is characterized in that including：
The related substances in Cefquinome sulfate breast injection are detected using high performance liquid chromatography；
Wherein, in the high performance liquid chromatography, gradient elution is carried out using following condition：
Wherein, mobile phase A is the mixed liquor of the buffer solution and acetonitrile of pH 3.6~4.0, the ratio of buffer solution and acetonitrile is 85~
95:5~15；
Mobile phase B is the mixed liquor of the buffer solution and acetonitrile of pH 3.6~4.0, and the ratio of buffer solution and acetonitrile is 40~60:40
~60.
2. liquid phase detection method according to claim 1, which is characterized in that in the high performance liquid chromatography, using such as
Lower condition carries out gradient elution：
3. liquid phase detection method according to claim 2, which is characterized in that in the high performance liquid chromatography, using such as
Lower condition carries out gradient elution：
4. liquid phase detection method according to claim 1, which is characterized in that the mobile phase A is the buffer solution of pH 3.6
With the mixed liquor of acetonitrile, the ratio of buffer solution and acetonitrile is 90:10；
And/or the mixed liquor for the buffer solution and acetonitrile that the Mobile phase B is pH 3.6, the ratio of buffer solution and acetonitrile is 50:
50。
5. liquid phase detection method according to claim 1, which is characterized in that the high performance liquid chromatography uses following item
Part：
Flow velocity：0.8~1.2ml/min；
Column temperature：25~35 DEG C；
Detection wavelength：230~280nm；
And/or number of theoretical plate is calculated by Cefquinome sulfate and is not less than 2000.
6. liquid phase detection method according to claim 5, which is characterized in that the high performance liquid chromatography uses following item
Part：
Flow velocity：1.0ml/min；
Column temperature：30℃；
Detection wavelength：245nm.
7. liquid phase detection method according to claim 1, which is characterized in that including：
It is prepared by test solution：It is saturated to the addition of Cefquinome sulfate breast injection sample with Cefquinome sulfate reference substance
Solution is extracted, is centrifuged after shaking up, precipitation is collected；It is handled 1-3 times under sediment the same terms, last time is handled into gained precipitation
After object drying, to flow phased soln, is diluted after ultrasound, shake up, filter, obtain test solution；
It is prepared by contrast solution 1：By test solution to shake up after flowing phase dilution, contrast solution 1 is obtained；
It is prepared by contrast solution 2：2,3- cyclohexyl pyrroles is shaken up with flowing to dilute after phased soln, obtains contrast solution 2；
Test solution, contrast solution 1 and contrast solution 2 are injected separately into high performance liquid chromatography, and record chromatography.
8. liquid phase detection method according to claim 7, which is characterized in that described to be saturated with Cefquinome sulfate reference substance
Extraction solution be the petroleum ether solution being saturated with Cefquinome sulfate reference substance；
Preferably, the boiling range of petroleum ether is 30~60 DEG C.
9. liquid phase detection method according to claim 7, which is characterized in that prepared by contrast solution 1 includes：
1ml test solutions are placed in 100ml measuring bottles, then to flow phase dilution constant volume, contrast solution 1 is obtained after shaking up.
10. liquid phase detection method according to claim 9, which is characterized in that including：In test solution chromatogram if any
Impurity peaks, the cyclohexylmethylpyridine containing 2,3- are not greater than 3 times of contrast solution main peak area, and other single contaminants are not greater than control
Solution main peak area, total impurities are not greater than 4 times of contrast solution main peak area；
It ignores at peak in test solution chromatogram less than 0.5 times of contrast solution main peak area.
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