CN108802212A - A kind of liquid phase detection method of lactation period Cefquinome sulfate breast injection related substances - Google Patents
A kind of liquid phase detection method of lactation period Cefquinome sulfate breast injection related substances Download PDFInfo
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Abstract
The present invention provides the liquid phase detection methods of lactation period Cefquinome sulfate breast injection related substances.In the method for the present invention, establish the special related substance of a Cefquinome sulfate breast injection (lactation period) (2,3- cyclohexylmethylpyridines) appearance time can more be bonded the related substance detecting method of the retention time determined using main ingredient Self-control method, and the method tolerance, reproducibility and specificity are more preferable.Meanwhile detection method enables to each impurity peaks separating effect more preferable, testing result is more acurrate.
Description
Technical field
The present invention relates to drug tests, in particular to a kind of lactation period Cefquinome sulfate breast injection
The liquid phase detection method of related substances.
Background technology
Cefquinome sulfate is that a kind of the 4th generation cephalosporins of animal specific, the medicine has a broad antifungal spectrum, and antibacterial are lived
Property is strong, for the various G being clinically separated+Bacterium, G-The MIC of bacterium50、MIC90Value is smaller.Cefquinome sulfate is suitable for being administered to
Medicine, absorb it is fast, reach that time to peak is short, and bioavilability is higher, it is dense that higher tissue can be reached in tissues such as lung, mammary gland
Degree, and be widely used in the clinical treatment of pig, ox infection in respiratory system and mastitis for milk cows.
It is special related in existing liquid phase detection method of the Cefquinome sulfate breast injection (lactation period) in relation to substance
The retention time of substance (2,3- cyclohexylmethylpyridine) determines by carrying out own control with main ingredient (Cefquinome) peak, but
When actually detected, 2,3- cyclohexylmethylpyridines peak retention time and the theoretical retention time in method isolated have a little difference,
And its retention time is easily by different brands, the liquid chromatograph of model, chromatographic column, the difference of mobile phase preformulation situation and shadow
Ring, also resulting in each impurity peaks separating effect sometimes is also influenced, though separating degree meet regulation it is relatively less than normal.
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 of lactation period Cefquinome sulfate breast injection related substances
Phase detection method, liquid phase detection method tolerance, reproducibility and specificity of the present invention are good, each impurity peaks good separating effect, detection
As a result accurate.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
A kind of liquid phase detection method of lactation period Cefquinome sulfate breast injection related substances, including：
Kind of a lactation period Cefquinome sulfate breast injection related substances are detected using high performance liquid chromatography, and
Gradient elution is carried out according to following condition：
Mobile phase A is the mixed liquor of citrate buffer and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile
For：80~90:10~20；
Mobile phase B is the mixed liquor of citrate buffer solution and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile is：
50~70:30~50.
Preferably, liquid phase detection method of the present invention carries out gradient elution according to following condition：
Mobile phase A be citrate buffer and acetonitrile mixed liquor, wherein the pH of citrate buffer solution be 3.6~
4.0, the ratio of citrate buffer solution and acetonitrile is：85~90:10~15；
Mobile phase B is the mixed liquor of citrate buffer solution and acetonitrile, wherein the pH of citrate buffer solution is 3.6~4.0,
The ratio of citrate buffer solution and acetonitrile is：60~70:30~40.
Preferably, liquid phase detection method of the present invention carries out gradient elution according to following condition：
Mobile phase A is the mixed liquor of citrate buffer and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile
For：85:15；
Mobile phase B is the mixed liquor of citrate buffer solution and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile is：
60:40。
Preferably, in liquid phase detection method of the present invention, the high-efficient liquid phase chromatogram condition is as follows：Flow velocity：0.8~
1.2ml/s；Column temperature：25~35 DEG C；Detection wavelength：230~280nm.
Preferably, in liquid phase detection method of the present invention, the high-efficient liquid phase chromatogram condition is as follows：Flow velocity：
1.0ml/s；Column temperature：30℃；Detection wavelength：254nm；Number of theoretical plate is calculated by Cefquinome sulfate is not less than 2000.
Preferably, in liquid phase detection method of the present invention, the liquid phase detection method includes：Test solution is noted
Enter in liquid chromatograph, 2 times of record chromatogram to main peak retention time；
2,3- cyclohexylmethylpyridines peak is 0.15~0.25 relative to the retention time at Cefquinome sulfate peak.
Preferably, in liquid phase detection method of the present invention, if any impurity peaks in the chromatogram of test solution, by peak
Area normalization method calculates, and the cyclohexylmethylpyridine containing 2,3- must not exceed 2.0%, and other single contaminants must not exceed 1.0%, each impurity
And must not exceed 4.0%.
Preferably, in liquid phase detection method of the present invention, the preparation of the test solution includes the following steps：To
In lactation period Cefquinome sulfate breast injection, the extraction solution being saturated with Cefquinome sulfate reference substance is added, after shaking up
It centrifuges, is extracted 1~3 time under gained sediment the same terms；
After last time gained sediment is dried, to flow phased soln, test solution is obtained.
Preferably, in liquid phase detection method of the present invention, the extraction of the Cefquinome sulfate reference substance saturation is molten
Liquid is the hexane solution of Cefquinome sulfate reference substance saturation.
Preferably, in liquid phase detection method of the present invention, the preparation of the test solution includes the following steps：
Into lactation period Cefquinome sulfate breast injection, it is molten that the hexane being saturated with Cefquinome sulfate reference substance is added
Liquid centrifuges after shaking up, and is extracted 1 time under gained sediment the same terms, after dry plus flows phased soln, ultrasound, then filters, take
Subsequent filtrate injects in liquid chromatograph；
It is furthermore preferred that sample size is 20 μ L.
Compared with prior art, beneficial effects of the present invention are：
1. the present invention establishes special related substance (the 2,3- hexamethylenes of a Cefquinome sulfate breast injection (lactation period)
Yl pyridines) appearance time can more be bonded the related substance detecting method of the retention time determined using main ingredient Self-control method, this
Method tolerance, reproducibility and specificity are more preferable.
2. detection method can make each impurity peaks separating effect more preferable, testing result is more acurrate.
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 existing lactation period Cefquinome sulfate injectant in related substances content detection, especially 2,3- hexamethylenes
In the presence of the defects inspectings such as yl pyridines preparation in each ingredient inferior separating effect, impurity peaks the shortcomings of can not efficiently separating,
The present invention provides a kind of liquid phase detection methods of new lactation period Cefquinome sulfate breast injection related substances.
Specifically, detection method provided by the present invention includes：
It is prepared by test solution：Lactation period Cefquinome sulfate injectant sample is taken, is added in centrifuge tube, it is preferred that secrete
The quality milligram number of newborn phase Cefquinome sulfate injectant sample (in terms of Cefquinome sulfate) and the calibration volume milli of centrifuge tube
It is (1~2) to rise the ratio between number：(4~6)；It is furthermore preferred that lactation period, Cefquinome sulfate injectant sample was (with Cefquinome sulfate
Meter) quality milligram number and the ratio between the calibration volume ml of centrifuge tube be 1:5；
Then, the hexane solution that addition is saturated with Cefquinome sulfate standard items into centrifuge tube is (that is, into n-hexane
Cefquinome sulfate is added, until Cefquinome sulfate is saturated)；
Wherein, it is preferred that the quality milligram of lactation period Cefquinome sulfate injectant sample (in terms of Cefquinome sulfate)
Number is (10~20) with the ratio between the volume ml of hexane solution being saturated with Cefquinome sulfate standard items：(20~30)；
It is furthermore preferred that the quality milligram number of lactation period Cefquinome sulfate injectant sample (in terms of Cefquinome sulfate) and use sulfuric acid head
The ratio between the volume ml of hexane solution of spore quinoline oxime standard items saturation is 10:25；
Then, mixing, centrifugation (preferred, the rotating speed of centrifugation is 4500r/min, and the time of centrifugation is 10~30min), is removed
Remove supernatant；
Then, the hexane solution by the precipitation after centrifugation according to method as above to be saturated with Cefquinome sulfate standard items
It extracts again 1~3 time (preferably extracting again 1 time)；
Will last time extract after supernatant in centrifuge tube discard, and precipitation will be evaporated at room temperature it is dry, then,
Mobile phase is added into centrifuge tube；
Wherein, it is preferred that the quality milligram of lactation period Cefquinome sulfate injectant sample (in terms of Cefquinome sulfate)
The ratio between number and the volume ml of mobile phase are (1~2)：(1~2)；It is furthermore preferred that lactation period Cefquinome sulfate injectant sample
The ratio between the quality milligram number of product (in terms of Cefquinome sulfate) and the volume ml of mobile phase are 1:1；
Then, centrifuge tube is placed in supersound process (being preferably ultrasonically treated 10min) in cold bath so that sulfuric acid therein
Cefquinome dissolves, filtration, as test solution.
Then, 20 μ L test solutions are taken, liquid chromatographic detection is carried out.
Liquid phase chromatogram condition is then one of the key point of the present invention, and by adjustment for Traditional liquid phase chromatographic condition and
Optimization, but also in detection method, it is special in Cefquinome sulfate breast injection (lactation period) sample to enable to
The appearance time of related substance (2,3- cyclohexylmethylpyridine), can more be bonded the retention time determined using main ingredient Self-control method.
Specifically, in liquid phase detection method of the present invention, chromatographic condition 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；
Flow velocity：1.0ml/min；
Column temperature：30℃；
Detection wavelength：254nm；
Number of theoretical plate is calculated by Cefquinome sulfate peak is not less than 2000；
Meanwhile in liquid phase detection method of the present invention, without carrying out system suitability experiment.And this is but also the method for the present invention
With higher detection efficiency.
Further, it unlike the isocratic elution method employed in existing detection, is examined in liquid chromatogram in the present invention
It is the method using gradient elution, condition of gradient elution and mobile phase used during survey, then is the another key point institute of the present invention
?.
Specifically, in the present invention, mobile phase used includes two components of mobile phase A and Mobile phase B：
Wherein, mobile phase A is preferably the mixed liquor of citric acid solution-acetonitrile of pH4.0, and the ratio of the two is preferred
It is 85:15(v/v)；
In mobile phase A, the citrate buffer solution of pH4.0 is referred to following method and prepares：Take 0.1mol/L lemons
Acid solution and 0.1mol/L sodium citrate solutions, according to 13.1:6.9 (v/v) mixing is to get to the Citrate buffer of pH4.0
Liquid.
Wherein, Mobile phase B is preferably the mixed liquor of citric acid solution-acetonitrile of pH4.0, and the ratio of the two is preferred
It is 60:40(v/v)；
In Mobile phase B, the citrate buffer solution of pH4.0 is referred to following method and prepares：Take 0.1mol/L lemons
Acid solution and 0.1mol/L sodium citrate solutions, according to 13.1:6.9 (v/v) mixing is to get to the Citrate buffer of pH4.0
Liquid.
Preferably, the condition of gradient elution is as follows：
| Time (minute) | 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, chromatogram is recorded, until 2 times of main peak retention time, 2,3- cyclohexylmethylpyridine peaks are relative to Cefquinome peak
Retention time be about 0.20；
Meanwhile it being calculated by areas of peak normalization method, the cyclohexyl containing 2,3- if any impurity peaks in the chromatogram of test solution
Pyridine must not cross 2.0%, and other single contaminants must not cross 1.0%, each impurity and 4.0% must not be crossed.
And in detection method, by the optimization for each chromatographic condition so that 2,3- cyclohexylmethylpyridine appearances
Time is more bonded the retention time determined using main ingredient Self-control method, and more stable, and tolerance, reproducibility are more preferable, while
Improving the separating effect of each ingredient in preparation, it is ensured that each impurity peak energy is preferably detached, to keep testing result more acurrate,
Specificity is stronger.
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；It is slow with citrate
Fliud flushing (ph4.0) (takes 0.1mol/L citric acid solutions with 0.1mol/L sodium citrate solutions by (V/V) 13.1:6.9 mixing, i.e.,
)-acetonitrile (85:15) it is mobile phase A, citrate buffer (ph4.0)-acetonitrile (60:40) be Mobile phase B, according to the form below into
Row linear gradient elution；Flow velocity is 1.0ml/min；Column temperature is 30 DEG C；Detection wavelength is 254nm.Number of theoretical plate presses sulfuric acid cephalo
Quinoline oxime peak, which calculates, is not less than 2000.Linear gradient elution is carried out according to method shown in 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 |
Assay method：The lactation period Cefquinome sulfate breast injection sample after mixing is taken (to be approximately equivalent to cephalo in right amount
Quinoline oxime 10mg), it sets in 50ml centrifuge tubes, the n-hexane 25ml that addition Cefquinome sulfate is saturated, mixing, centrifuges 10 minutes
(4500 turns per minute), discard supernatant liquid, repeat extraction 1 time as stated above, and precipitation evaporates into dry at room temperature.Precision is added
Mobile phase 10ml sets in cold bath and is ultrasonically treated 10 minutes, Cefquinome sulfate is made to dissolve, and filtration, it is continuous that precision measures test sample
20 μ l of filtrate, inject liquid chromatograph, record chromatogram to 2 times of main peak retention time, 2,3- cyclohexylmethylpyridine peaks relative to
The retention time at Cefquinome peak be about 0.20. test solutions chromatogram in if any impurity peaks, by areas of peak normalization method
Calculate, 2.0% must not be crossed containing 2,3- cyclohexylmethylpyridines, other single contaminants must not cross 1.0%, each impurity and must not mistake
4.0%.
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, the results show that the head at wavelength 254nm
Spore quinoline oxime has absorption maximum.
2. the determination of impurity computational methods
Same batch of sample is taken to investigate retention time and same sample repetition into 3 needles investigation impurity accounting content (peak face
Product %), area normalization method and Self-control method are compared respectively, and preferably more suitably impurity computational methods, experimental result are specific
It is as follows：
From experimental result as above：When with area normalization method, 3 sample rooms 2 of same batch of sample, 3- cyclohexyl pyrroles
Pyridine and other maximum singly miscellaneous retention time RSD are larger (> 2%), and when use Self-control method, the equal < of retention time RSD
0.5%.
It can be seen that single impurity appearance time can be made more stable using Self-control method, tolerance, the reproducibility of method
More preferably；And when using area normalization method, even same sample is repeated into 3 needles, it is also possible to occur 2,3- cyclohexylmethylpyridines and
Situation bigger than normal other maximum single miscellaneous accounting content (peak area %) RSD, total miscellaneous accounting content (peak area %) RSD are even as high as
4.97%, the result of Self-control method is obviously good compared with area normalization method, it was demonstrated that can make single impurity using Self-control method
Accounting content repeatability is more preferable, while also substantially improving the repeatability of total miscellaneous detection.It is therefore preferable that Self-control method is this hair
Bright impurity computational methods.
3. specificity is tested
The preparation of blank auxiliary solution：Cefquinome sulfate breast injection (lactation period) 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.The result shows that there is color on a corresponding position with reference substance in test sample
Spectral peak, and baseline is steady in test sample chromatographic peak, each impurity peaks separating degree is good；Blank auxiliary solution, mobile phase are in each impurity
Occur without chromatographic peak on position.It can be seen that mobile phase and negative control are noiseless to each impurity retention time.
4. sample solution stability experiment
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 is 0.88% < 2.0%, it can be seen that, reference substance and test solution are steady in 10h
It is qualitative good.
Solution stability testing result is as follows：
5. linear relationship
Precision weighs Cefquinome reference substance 20mg and is placed in 200ml measuring bottles, and mobile phase is added and is allowed to after being completely dissolved fixed
Hold, shakes up, as storing solution.The storing solution 1,5,10,20,40,60ml 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 0.001mg/ml, 0.005mg/ml, 0.01mg/ml, 0.02mg/
Ml, 0.04mg/ml, 0.06mg/ml Series Measurement strength solution.By the chromatographic condition that the present invention establishes, 20 μ l of sample introduction measure sample
Product chromatographic peak area.Linear test result is 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 |
With peak area and a concentration of coordinate, standard curve is established, obtaining equation is：Y=7385.6X+0.3086 (R2=
0.9999).It can be seen that in Cefquinome sulfate in relation to material concentration within the scope of 0.001mg/ml~0.06mg/ml and peak
There are good linear relationships for area.
6. precision test
By the test solution of 0h in " sample solution stability " item, (i.e. a concentration of 1.0mg/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 0.55% (< 2.0%) it can be seen that originally
It is good to invent the related substance-measuring method precision established.
Precision test result is specific as follows：：
7. repetitive test
Selection is with 6 parts of lot number Cefquinome sulfate breast injection (lactation period), 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 1.07% (< 2.0%).It can be seen that the related substance-measuring method repeatability that the present invention establishes is good.
Repetitive test result is specific as follows：
8. reappearance test
Selection is with 6 parts of lot number Cefquinome sulfate breast injection (lactation period), 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, prompts the related substance-measuring method reproducibility that the present invention establishes good.
9. accuracy test
It is accurately weighed by 80%, 100%, 120% difference of Cefquinome sulfate breast injection (lactation period) labelled amount
Known each 3 parts of Cefquinome sulfate breast injection (lactation period) sample in relation to amount of substance is respectively placed in 9 100ml centrifugations
Guan Zhong prepares test sample and contrast solution according to experimental implementation, is measured by the related substance-measuring method that the present invention establishes related
Amount of substance calculates the rate of recovery.
Accuracy test result is as follows：
From experimental result as above：The rate of recovery and average recovery rate are in 92%~105%, RSD all within 2%.
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 (lactation period) in right amount, investigates change in flow ± 0.2ml/ respectively
Citrate buffer pH variations ± 0.2 in acetonitrile ratio variation ± 2%, mobile phase in min, ± 5 DEG C of column temperature variation, mobile phase
(i.e. 0.1mol/L citric acid solutions：0.1mol/L sodium citrate solutions (V/V)=14.0:6.0 or 12.3:7.7), different chromatographies
The variation of instrument chromatographic behavior when column, observation base detach situation, the average value and RSD values of the data obtained under the conditions of calculating respectively,
Test result is as follows：
Test result shows the related substance-measuring side of Cefquinome sulfate breast injection (lactation period) Cefquinome sulfate
In method serviceability test, flow velocity, column temperature, chromatographic column, flowing phase composition, PH small variations, each impurity peaks reach baseline point
From surveying 2,3- cyclohexylmethylpyridines, other maximum singly miscellaneous, total equal < of miscellaneous RSD values in relation to substance 2% under the conditions of each.Thus may be used
See, the related substance-measuring method good tolerance that the present invention establishes.
11. detection limit
It takes Cefquinome reference substance to be made into the testing liquid of 0.01mg/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 3 μ g/ml, and
It is provided in method " ignoring at peak of 0.5 times less than contrast solution main peak area " (ignoring at the peak for being less than 5 μ g/ml).
It can be seen that 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 injection (lactations respectively
Phase) in the related substance of Cefquinome sulfate, it is as a result as follows：
It can be seen 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；It is slow with sodium perchlorate
Fliud flushing (takes a perchloric acid hydrate sodium 3.45g, water 1000ml is added to make dissolving, phosphorate sour 12ml and acetonitrile 115ml, with triethylamine tune
Save pH to 3.6 ± 0.1) it is mobile phase, flow velocity 1.0ml/min；Column temperature is 30 DEG C；Detection wavelength is 270nm.
Cefotaxime about 25mg is taken, mobile phase 100ml is added to make dissolving, separately takes Cefquinome about 25mg, in addition stating cephalo thiophene
Oxime solution 1ml, is diluted to 25ml with mobile phase, shakes up, as system suitability solution.Take 20 μ l injection liquid chromatograies
Instrument, records chromatogram, and the separating degree between Cefquinome peak and cefotaxime peak should be greater than 1.0.
Assay method：
Take the lactation period Cefquinome sulfate breast injection sample after mixing appropriate (being approximately equivalent to Cefquinome 10mg),
It sets in 50ml centrifuge tubes, the n-hexane 25ml that addition Cefquinome sulfate is saturated, mixing, centrifuges 10 minutes (per minute 4500
Turn), liquid is discarded supernatant, repeats extraction 1 time as stated above, precipitation evaporates into dry at room temperature.Mobile phase 10ml is added in precision,
It sets in cold bath and is ultrasonically treated 10 minutes, Cefquinome sulfate is made to dissolve, filter.Precision measures 20 μ l of subsequent filtrate, injects liquid phase
Chromatograph, record chromatogram is to 2 times of main peak retention time, when reservation of 2, the 3- cyclohexylmethylpyridine peaks relative to Cefquinome peak
Between about 0.3.If any impurity peaks in the chromatogram of test solution, calculated by areas of peak normalization method, the cyclohexyl pyrrole containing 2,3-
Pyridine must not cross 2.0%, and other single contaminants must not cross 1.0%, each impurity and 4.0% must not be crossed.
1. specificity is tested
(1) preparation of blank auxiliary solution：Cefquinome sulfate breast injection (lactation 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.
(2) comparative example 1 and comparative example 1 record chromatogram, observe separating degree, 2, the 3- cyclohexyl pyrroles of each impurity peaks
The ratio of pyridine and Cefquinome retention time.
Testing result of the Cefquinome sulfate breast injection (lactation period) under embodiment 1, comparative example 1 is as follows：
The result shows that：1 chromatographic condition of embodiment more can guarantee more preferably separating degree, 2,3- cyclohexylmethylpyridines and Cefquinome
The ratio of retention time is more stable, it was demonstrated that 2,3- cyclohexylmethylpyridine appearance times can be more bonded to be determined using main ingredient Self-control method
Retention time.
By experimental result as above it is recognized that while embodiment 1 and comparative example 1 can make Cefquinome peak not by mobile phase, sky
The interference of white preparation control meets specificity basic demand, but the related substance detecting method of embodiment 1 is special compared with comparative example 1
Attribute, reproducibility are stronger, when 2,3- cyclohexylmethylpyridine appearance times can more be bonded the reservation determined using main ingredient Self-control method
Between.
2. the related substance-measuring result of Cefquinome in three batches of samples：
By experiment contrast result as above it is found that the related substance RSD of three batches of samples is respectively less than 2%, but 1 reproducibility of embodiment
As a result more preferable compared with comparative example 1.
To sum up result as it can be seen that the present invention optimization after the related substance of Cefquinome sulfate breast injection (lactation period) liquid phase
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. a kind of liquid phase detection method of lactation period Cefquinome sulfate breast injection related substances, which is characterized in that including：
Kind of a lactation period Cefquinome sulfate breast injection related substances are detected using high performance liquid chromatography, and according to
Following condition carries out gradient elution：
Mobile phase A is the mixed liquor of citrate buffer and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile is：80
~90:10~20；
Mobile phase B is the mixed liquor of citrate buffer solution and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile is：50~
70:30~50.
2. liquid phase detection method according to claim 1, which is characterized in that the liquid phase detection method is according to following condition
Carry out gradient elution：
Mobile phase A is the mixed liquor of citrate buffer and acetonitrile, wherein the pH of citrate buffer solution is 3.6~4.0, lemon
The ratio of lemon acid buffer and acetonitrile is：85~90:10~15；
Mobile phase B is the mixed liquor of citrate buffer solution and acetonitrile, wherein the pH of citrate buffer solution is 3.6~4.0, lemon
The ratio of acid buffer and acetonitrile is：60~70:30~40.
3. liquid phase detection method according to claim 2, which is characterized in that the liquid phase detection method is according to following condition
Carry out gradient elution：
Mobile phase A is the mixed liquor of citrate buffer and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile is：85:
15；
Mobile phase B is the mixed liquor of citrate buffer solution and acetonitrile, wherein the ratio of citrate buffer solution and acetonitrile is：60:
40。
4. liquid phase detection method according to claim 1, which is characterized in that the high-efficient liquid phase chromatogram condition is as follows：
Flow velocity：0.8~1.2ml/s；
Column temperature：25~35 DEG C；
Detection wavelength：230~280nm.
5. liquid phase detection method according to claim 4, which is characterized in that the high-efficient liquid phase chromatogram condition is as follows：
Flow velocity：1.0ml/s；
Column temperature：30℃；
Detection wavelength：254nm；
Number of theoretical plate is calculated by Cefquinome sulfate is not less than 2000.
6. liquid phase detection method according to claim 1, which is characterized in that the liquid phase detection method includes：
Test solution is injected in liquid chromatograph, 2 times of record chromatogram to main peak retention time；
2,3- cyclohexylmethylpyridines peak is 0.15~0.25 relative to the retention time at Cefquinome sulfate peak.
7. liquid phase detection method according to claim 6, which is characterized in that if any impurity in the chromatogram of test solution
Peak is calculated by peak area normalization method, and the cyclohexylmethylpyridine containing 2,3- must not exceed 2.0%, and other single contaminants must not exceed
1.0%, each impurity and must not exceed 4.0%.
8. liquid phase detection method according to claim 6, which is characterized in that the preparation of the test solution includes as follows
Step：Into lactation period Cefquinome sulfate breast injection, the extraction solution being saturated with Cefquinome sulfate reference substance is added,
It centrifuges after shaking up, is extracted 1~3 time under gained sediment the same terms；
After last time gained sediment is dried, to flow phased soln, test solution is obtained.
9. liquid phase detection method according to claim 8, which is characterized in that the Cefquinome sulfate reference substance saturation
Extract the hexane solution that solution is Cefquinome sulfate reference substance saturation.
10. liquid phase detection method according to claim 9, which is characterized in that the preparation of the test solution includes such as
Lower step：
Into lactation period Cefquinome sulfate breast injection, the hexane solution being saturated with Cefquinome sulfate reference substance is added,
It centrifuges after shaking up, is extracted 1 time under gained sediment the same terms, after dry plus flow phased soln, ultrasound, then filter, take continuous
Filtrate is injected in liquid chromatograph；
Preferably, sample size is 20 μ L.
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