CN109490440A - A method of detection Cefixime related impurities - Google Patents
A method of detection Cefixime related impurities Download PDFInfo
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- CN109490440A CN109490440A CN201811395173.2A CN201811395173A CN109490440A CN 109490440 A CN109490440 A CN 109490440A CN 201811395173 A CN201811395173 A CN 201811395173A CN 109490440 A CN109490440 A CN 109490440A
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Abstract
The invention discloses the methods that Cefixime related impurities are detected from Cefixime drug, carry out qualitative or/and quantitative detection to Cefixime impurity A, Cefixime impurity B, Cefixime impurity D and Cefixime impurity E simultaneously using the method for high performance liquid chromatography.Cefixime impurity A, Cefixime impurity B, Cefixime impurity D and the Cefixime impurity E in bulk pharmaceutical chemicals disposably can be effectively detected using method of the invention, applicable dosage form range is wide, and impurity Detection capability is excellent, content is accurate.
Description
Technical field
The present invention relates to detection method fields, more particularly to the method for checked for impurities in drug.
Background technique
Cefixime is third generation oral cephalosporin, and about the detection of its raw material and its preparation impurity, pharmacopoeia of each country is received
The standard method and Control of Impurities limit of load, pharmacopoeia of each country chromatographic condition are similar: mobile phase is that tetrabutylammonium hydroxide is molten
The solution that liquid and acetonitrile are prepared by a certain percentage is all made of HPLC-UV detection, and wavelength is 254nm；It is all made of C18 chromatography
Column.In addition to European Pharmacopoeia makees dilution using mobile phase, remaining pharmacopeia is diluted using the phosphoric acid or phosphate buffer of pH7.0
Liquid.Japanese Pharmacopoeia and USP standard are calculated using area normalization method, remaining is all made of Self-control method calculating.
Situation is recorded by pharmacopeia and literature survey is learnt, Cefixime has 5 known impurities, respectively impurity A, impurity
B, impurity C, impurity D and impurity E.Wherein impurity A, B, D are not only process impurity, but also are degradation impurity, in Material synthesis, preparation
It can produce in technique and storage；Impurity C is process byproducts, only generates, was storing during Material synthesis
It will not increase in journey；Impurity E is degradation impurity, in storage there may be.Therefore, Cefixime degradation impurity A, B, D,
The content of E should be the emphasis of concern.
Since impurity analysis method of the pharmacopoeia of each country to Cefixime raw material and preparation is similar, my company initially exists
The method suitable for the related substance detection of this product is established on the basis of the chromatographic condition of Chinese Pharmacopoeia, is shown in Table 1.Actually detected
Shi Faxian, under the chromatographic condition, impurity A peak shape is poor (see Fig. 1), and impurity A is in three peaks, and retention time is
17.208min, 18.258min, 19.989min are unable to accurate statistics peak area.By to impurity further study show that, it is miscellaneous
Matter A and impurity B are mixture, and correction factor is in 0.9~1.1 range, but the standard method that pharmacopoeia of each country records
In none using be added correction factor calculation method.Test agent in self-control preparation is put using Chinese Pharmacopoeia chromatographic process
Row.Since in made products, the content of degradation impurity A and B are much smaller than limit 1.0%, the calculation of correction factor is not added
It is influenced smaller.But during study on the stability, the trend gradually increased is presented in impurity A and impurity B.As it can be seen that for head
The detection method of related impurities also needs further to explore optimization in spore gram oximes drug.
Related substance original method detects in 1 our company of table
Summary of the invention
The present invention provides a kind of methods for detecting Cefixime related impurities, being capable of Accurate Determining Cefixime impurity A
Content, while can also be successfully by main peak, Cefixime impurity A, Cefixime impurity B, Cefixime impurity D and cephalo gram
Oxime impurity E efficiently separates, and separating degree meets regulation, and impurity A is unimodal.
Specifically, the present invention provides a kind of method for detecting Cefixime related impurities, which is characterized in that use efficient liquid
Phase chromatography carries out qualitative or/and quantitative detection Cefixime related impurities, the testing conditions of liquid chromatogram simultaneously
Chromatographic column: alkyl linked silicagel column is further selected from C18 chromatographic column；
Mobile phase: water phase, organic phase；
Wherein, water phase is tetrabutylammonium hydroxide solution；Organic phase is selected from acetonitrile or/and methanol, is not individually for methanol；Stream
It is dynamic mutually to use Gradient elution program as shown in table 2:
Table 2
| Time (min) | Water phase (vol.%) | Organic phase (vol.%) |
| 0 | 85~90 | 10~15 |
| 5 | 80~90 | 10~20 |
| 25 | 70~85 | 15~30 |
| 50 | 70~85 | 15~30 |
| 70 | 80~90 | 10~20 |
| 85 | 80~90 | 10~20 |
| 86 | 85~90 | 10~15 |
| 100 | 85~90 | 10~15 |
The impurity is the impurity that synthesis etc. introduces in technical process, including reactant, intermediate, by-product, reagent, is urged
Agent etc..
The organic phase is selected from acetonitrile or/and methanol, is not individually for methanol, refers to that organic phase can be individually for acetonitrile,
It can be the mixed solvent of acetonitrile and methanol, but not select methanol individually.
Further, the impurity is selected from Cefixime impurity A, Cefixime impurity B, Cefixime impurity D, cephalo gram
One or more of oxime impurity E.
Further, the tetrabutylammonium hydroxide solution ph range of the water phase is 4.9~7.0, and mass concentration is
0.4%~0.5%.
When further, using gradient elution, the organic phase is acetonitrile, and mobile phase uses the gradient elution as shown in table 3
Program:
Table 3
Further, the liquid chromatographic detection condition further includes one or more in i~iii below:
I chromatographic column specification: 4.6 × 250mm, 5 μm；
Ii column temperature: 45~55 DEG C；
Iii flow velocity: 1.2~0.8ml/min.
The present invention also provides a kind of methods for detecting Cefixime related impurities, which is characterized in that uses high-efficient liquid phase color
It composes while carrying out qualitative or/and quantitative detection Cefixime impurity A, the testing conditions of liquid chromatogram and include:
Chromatographic column: alkyl linked silicagel column is further selected from C18 chromatographic column；
Mobile phase: water phase, organic phase；
Wherein, water phase is tetrabutylammonium hydroxide solution；Organic phase is selected from acetonitrile or/and methanol, is not individually for methanol；Stream
It is dynamic mutually to use gradient elution program as shown in table 4:
Table 4
| Time (min) | Water phase (vol.%) | Organic phase (vol.%) |
| 0 | 85~90 | 10~15 |
| 5 | 80~90 | 10~20 |
| 25 | 70~85 | 15~30 |
| 50 | 70~85 | 15~30 |
| 70 | 80~90 | 10~20 |
| 85 | 80~90 | 10~20 |
| 86 | 85~90 | 10~15 |
| 100 | 85~90 | 10~15 |
Further, the tetrabutylammonium hydroxide solution ph range of the water phase is 4.9~7.0, and mass concentration is
0.25%~10%.
In a specific embodiment of the invention, the liquid chromatographic detection condition further includes in i~iii below
It is one or more:
I chromatographic column specification: 4.6 × 250mm, 5 μm；
Ii column temperature: 40~55 DEG C；
Iii flow velocity: 1.2~0.8ml/min.
Chromatographic column trade name Agilent Zorbax Eclipse plus used in the specific embodiment of the invention
C18 still as long as the chromatographic column for meeting foregoing description can operate with detection method of the invention, is not limited to above-mentioned commodity.
Further, above-mentioned detection method includes the following contents:
(1) test solution is prepared；
(2) contrast solution is prepared；
(3) by contrast solution sample detection；
(4) by test solution sample detection.
The analysis of the methods of area normalization method, Self-control method, internal standard method, external standard method, meter can be used in the detection method
Calculate testing result.
Further, prepare solvent that test solution or/and contrast solution use be selected from tetrabutylammonium hydroxide solution,
The mixed solvent of one or more of pH7.0 phosphate buffer, methanol, acetonitrile.
It prepares test solution or/and solvent that reference substance solution uses includes dissolution solvent and diluent.Wherein: dissolution
Solvent is further selected from one or more of pH7.0 phosphate buffer, methanol, acetonitrile, further selected from methanol and/or
Acetonitrile, preferably methanol；Diluent is further selected from tetrabutylammonium hydroxide solution and/or acetonitrile, and preferably pH is the 0.4% of 5.00
The solution that tetrabutylammonium hydroxide solution and acetonitrile volume ratio are 90: 10.
Prepare the more excellent operating procedure of test solution are as follows: 4%~60% volume dissolution solvent, ultrasound are added into sample
0.5~5min, then it is settled to concentration to be measured with diluent, 0~4mL of primary filtrate is discarded after centrifugal filtration.Wherein: dissolution solvent
The 4%~40% of the preferred test solution total volume of dosage, the 10% of further preferred test solution total volume；Ultrasonic time
It is preferred that 0.5~2min, further preferred 0.5min；Primary filtrate discards the preferred 2mL of volume.
The beneficial effects of the present invention are:
(1) present invention can effectively detect Cefixime impurity A, be allowed to big with main peak and other impurities separating degree, peak shape
It is good, solve the disadvantages of, bad calculating peak area poor to peak shape in the detection of Cefixime impurity A in the prior art.
(2) detection method of the invention can in one test simultaneously effectively detection drug in Cefixime impurity A,
Cefixime impurity B, Cefixime impurity D and Cefixime impurity E, separating degree meets measurement and requires, high-efficient, impurity detection
Ability is excellent, and content is accurate, can meet the impurity monitoring in technical process and the Control of Impurities requirement in finished product well.
(3) present invention can be used in the detection of related impurities in all kinds of drug forms of Cefixime, including Cefixime glue
Capsule, Cefixime granule, cefixime dispersible tablet etc., it is applied widely.
Detailed description of the invention
Fig. 1 is the related spectrogram of our company's original detection method；
Fig. 2 is the spectrogram of mobile phase pH screening-pH4.0 of the present invention；
Fig. 3 is the spectrogram of mobile phase pH screening-pH5.0 of the present invention；
Fig. 4 is the spectrogram of mobile phase pH screening-pH6.0 of the present invention；
Fig. 5 is the spectrogram of mobile phase pH screening-pH8.0 of the present invention；
Fig. 6 is the spectrogram of the embodiment of the present invention 3 (1)；
Fig. 7 is the spectrogram of the embodiment of the present invention 3 (2)；
Fig. 8 is the spectrogram of the embodiment of the present invention 3 (3)；
Fig. 9 is the spectrogram of the embodiment of the present invention 4 (1)；
Figure 10 is the spectrogram of the embodiment of the present invention 4 (2)；
Figure 11 is the spectrogram of the embodiment of the present invention 4 (3)；
Figure 12 is the spectrogram of the embodiment of the present invention 5 (1)；
Figure 13 is the spectrogram of the embodiment of the present invention 5 (2)；
Figure 14 is the spectrogram of the embodiment of the present invention 5 (3)；
Figure 15 is the spectrogram of the embodiment of the present invention 6 (1)；
Figure 16 is the spectrogram of the embodiment of the present invention 6 (2)；
Figure 17 is original method detection spectrogram；
Figure 18 is the method for the present invention optimum condition detection spectrogram.
Specific embodiment
Instrument and equipment:
Table 5
Reagent
Table 6
| Reagent | Rank | Manufacturer | Lot number |
| Acetonitrile | Chromatographically pure | Shanghai Xingke High Purity Solvent Co., Ltd. | 0114180102 etc. |
| Methanol | Chromatographically pure | Shanghai Xingke High Purity Solvent Co., Ltd. | 021271201 etc. |
| 10% tetrabutylammonium hydroxide | It analyzes pure | Shanghai Ke Feng chemical reagent Co., Ltd | 20171016 etc. |
| Phosphoric acid | It analyzes pure | Chengdu section dragon reagent chemical plant | 2016091901 etc. |
Reference substance and sample
Table 7
The preparation of each impurity stock solution of embodiment 1
Impurity A stock solution: weighing impurity A 4mg to 20ml measuring bottle, after adding phosphate buffer (pH7.0) 4ml to dissolve,
Add mobile phase to be settled to scale, shake up to obtain the final product.
Impurity B stock solution: impurity B 5mg is weighed to 25ml measuring bottle, phosphate buffer (pH7.0) dissolution is added to be settled to
Scale shakes up to obtain the final product.
Impurity C stock solution: weighing impurity C 2mg to 10ml measuring bottle, after adding phosphate buffer (pH7.0) 2ml to dissolve,
It is settled to scale with mobile phase, shakes up to obtain the final product.
Impurity D stock solution: impurity D 5mg is weighed to 25ml measuring bottle, phosphate buffer (pH7.0) dissolution is added to be settled to
Scale shakes up to obtain the final product.
Impurity E stock solution: impurity E 5mg is weighed to 25ml measuring bottle, phosphate buffer (pH7.0) dissolution is added to be settled to
Scale shakes up to obtain the final product.
7-AVCA stock solution: impurity 7-AVCA 4mg is weighed to 20ml measuring bottle, adds phosphate buffer (pH7.0) 4ml molten
Xie Hou adds mobile phase to be settled to scale, shakes up to obtain the final product.
MICA stock solution: weighing impurity MICA 4mg to 20ml measuring bottle, after adding phosphate buffer (pH7.0) 4ml to dissolve,
Add mobile phase to be settled to scale, shake up to obtain the final product.
Cefixime methyl esters stock solution: impurity Cefixime methyl esters 4mg is weighed to 20ml measuring bottle, adds phosphate buffer
(pH7.0) after 4ml dissolution, add mobile phase to be settled to scale, shake up to obtain the final product.
The screening of 2 flowing phase pH value of embodiment
Mobile phase: 0.25% tetrabutylammonium hydroxide (pH is respectively 4.0,5.0,6.0,8.0)-acetonitrile (72: 28)；
Chromatographic column: Agilent Zorbax Eclipse plus C18 250*4.6mm, 5 μm, INT-Col-038；
Mark-on test solution: 60 DEG C of -30 days content 12mg of Cefixime Capsules (041702 batch) high temperature are taken, until 10ml
Measuring bottle takes each impurity stock solution 0.5ml to same 10ml measuring bottle respectively, adds phosphate buffer (pH7.0) 2ml, ultrasonic dissolution
Afterwards, add mobile phase to be settled to scale, shake up to obtain the final product.
Flow velocity: 1.0ml/min；
Detection wavelength: 254nm；
Column temperature: 40 DEG C；
The selection result is as follows:
(1) when pH=4.0, detection spectrogram is shown in Fig. 2.
By testing result it is found that impurity B (first peak RT12.004min) and impurity is not up to baseline point under this condition
From；It is inner that impurity A is wrapped in main peak (RT26.152min), does not separate；Impurity D is in bifurcated peak (22.054min, 22.524min),
Baseline separation is not reached.
(2) when pH=5.0, detection spectrogram is shown in Fig. 3.
By testing result it is found that main peak and other impurities reach baseline separation under this condition；Impurity A be in two peaks, two
Peak and intermediate unknown impuritie (RT33.066min) separating degree are respectively 1.22,1.35；Second peak of impurity A is overlapped with impurity C
(RT36.057min)；First peak of impurity B (RT 11.716min) is inner package.
(3) when pH=6.0, detection spectrogram is shown in Fig. 4.
By testing result it is found that main peak and other impurities reach baseline separation under this condition；Impurity A be in two peaks, two
Peak and intermediate unknown impuritie (RT20.842min) separating degree are respectively 1.11,1.38；Second peak of impurity A is overlapped with impurity C
(RT22.377min)；First peak of impurity B (RT 8.871min) is inner package.Unknown impuritie (RT 7.584min) with it is unknown
Impurity (RT 7.319min) does not reach baseline separation.
(4) when pH=8.0, detection spectrogram is shown in Fig. 5.
By testing result it is found that unknown impuritie (RT6.505) and two neighboring impurity do not reach baseline separation under this condition；
Impurity B (RT7.824min) is in 1 peak, there is package；Two peaks of impurity A and impurity C are even in " mountain " type, do not reach baseline separation.
From the above results, under conditions of pH=5.0, main peak and other impurities reach baseline separation；Impurity A is in two
There is a unknown impuritie (RT33.066min) at peak between two peaks, and separating degree is respectively 1.22,1.35；Second peak of impurity A
(RT36.057min) is overlapped with impurity C；First peak of impurity B (RT 11.716min) is inner package.Impurity C is raw material process
Impurity is not detected in more batches of raw materials, therefore impurity C is overlapped not the quantitative detection for influencing impurity A with impurity A.Other are known miscellaneous
Matter and degradation impurity are in good separation.Therefore the pH value of detection method mobile phase is chosen to be 5.0.
The screening of the preliminary gradient of embodiment 3
Column temperature: 50 DEG C；
Chromatographic column: Agilent Zorbax Eclipse plus C18 250*4.6mm, 5 μm, INT-Col-038；
Mark-on test solution and other chromatographic conditions in addition to mobile phase are the same as embodiment 2.
(1) mobile phase A: 0.25% tetrabutylammonium hydroxide solution (pH=5.0)；
Mobile phase B: acetonitrile；Using gradient elution as shown in table 8:
Table 8
By testing result (Fig. 6) it is found that impurity D and main peak separating degree are 0.77 under this condition, impurity A is in two peaks,
Have between two peaks a unknown impuritie (RT 37.713min), the separating degree at unknown impuritie and two peaks of impurity A is respectively 0.97,
1.14, other each peak separating degrees are good.
(2) mobile phase A: 0.25% tetrabutylammonium hydroxide solution (pH=5.0)-acetonitrile (90: 10)；
Mobile phase B: 0.25% tetrabutylammonium hydroxide solution (pH=5.0)-acetonitrile (20: 80)；
Using gradient elution as shown in table 9:
Table 9
By testing result (Fig. 7) it is found that without separating degree, main peak is separated with impurity D for impurity B and other impurities under this condition
Degree is 0.84, and impurity A is in two peaks, and for the unknown peak and impurity A between two peaks without separating degree, other each peak separating degrees are good.
(3) mobile phase A: 0.25% tetrabutylammonium hydroxide solution (pH=5.0)；
Mobile phase B: acetonitrile；Mobile phase C: methanol；Using gradient elution as shown in table 10:
Table 10
By testing result (Fig. 8) it is found that increasing methanol in mobile phase, impurity A is in two peaks, is wrapped up not between two peaks
Know that impurity and impurity A separating degree are respectively 0.94,1.06, while baseline interference is big.
From the above it is found that when salinity is 0.25%, impurity A is in two peaks under each gradient condition, two peaks it
Between have a unknown impuritie, the unknown impuritie and impurity A separating degree do not reach baseline separation, and impurity A is caused to be difficult to quantitative statistics.Increase
After adding methanol, although the unknown impuritie and impurity A separating degree between two peaks of impurity A slightly improve, baseline interference is big.
The screening of the further gradient of embodiment 4
The test data obtained by analyzing embodiment 3, inventor improve tetrabutylammonium hydroxide solution concentration extremely
0.5%, continue to optimize gradient.
Salinity in mobile phase is improved to 0.5%, different gradient systems are respectively adopted, to each in mark-on test solution
The separating degree between separating degree and impurity and main peak between impurity is investigated, and the peak of the separation and impurity A to each ingredient
Shape comparative analysis.
(1) 50 DEG C of column temperature；
Mobile phase A: 0.5% tetrabutylammonium hydroxide solution (pH=5.0)；
Mobile phase B: acetonitrile；Mobile phase C: methanol；Using gradient elution as shown in table 11:
Table 11
Mark-on test solution and other chromatographic conditions are the same as embodiment 2.
By testing result (Fig. 9) it is found that improving the concentration of salting liquid, impurity A and impurity C reach baseline under this condition
Separation, other each peak separating degrees are good, but baseline interference is big.
(2) 50 DEG C of column temperature；
Mobile phase A: 0.5% tetrabutylammonium hydroxide solution (pH5.0)；
Mobile phase B: acetonitrile；Mobile phase C: methanol；Using gradient elution as shown in table 12:
Table 12
Mark-on test solution and other chromatographic conditions are the same as embodiment 2.
By testing result (Figure 10) it is found that each peak separating degree is good under this condition, but baseline interference is big.
(3) 50 DEG C of column temperature；
Mobile phase A: 0.5% tetrabutylammonium hydroxide solution (pH5.0)；
Mobile phase B: acetonitrile；Using gradient elution as shown in table 13:
Table 13
Mark-on test solution and other chromatographic conditions are the same as embodiment 2.
By testing result (Figure 11) it is found that each known impurities, main peak and other impurities and principal degradation are miscellaneous under this condition
Matter efficiently separates.
From the above it is found that improving tetrabutylammonium hydroxide concentration to after 0.5%, can effectively divide under each gradient condition
From each ingredient, when having methanol in flow phase system, baseline interference is larger, influences impurity detection.It is thus determined that mobile phase of the present invention
System is tetrabutylammonium hydroxide solution-acetonitrile.
The screening of 5 column temperature of embodiment
On the basis of embodiment 4 (3) condition, adjustment mobile phase A, B make not for pure salt and pure organic phase, optimize column temperature and
Gradient.Different chromatographic conditions is respectively adopted and carries out gradient elution, to the separating degree between each impurity in mark-on test solution
And the separating degree between impurity and main peak is investigated, and the peak shape comparative analysis to impurity A.
Mobile phase A: 0.5% tetrabutylammonium hydroxide solution (pH5.0)-acetonitrile (90: 10)；
Mobile phase B: 0.5% tetrabutylammonium hydroxide solution (pH5.0)-acetonitrile (50: 50)；
(1) mark-on test solution and other chromatographic conditions are the same as embodiment 2.
55 DEG C of column temperature；Using gradient elution as shown in table 14:
Table 14
By testing result (Figure 12) it is found that impurity A is in two peaks under this condition, have between two peaks of impurity A one unknown
Impurity is good with two peak separating degrees of impurity A；Each known impurities and degradation impurity and main peak and other impurities are up to baseline point
From.
(2) mark-on test solution and other chromatographic conditions are the same as embodiment 2.
45 DEG C of column temperature；Using gradient elution as shown in Table 15:
Table 15
By testing result (Figure 13) it is found that impurity A is in two peaks under this condition, have between two peaks of impurity A one unknown
Impurity is good with two peak separating degrees of impurity A；Each known impurities and degradation impurity and main peak and other impurities are up to baseline point
From.
(3) mark-on test solution and other chromatographic conditions are the same as embodiment 2.
40 DEG C of column temperature；Using gradient elution as shown in table 16:
Table 16
By testing result (Figure 14) it is found that impurity A is in two peaks under this condition, have between two peaks of impurity A one unknown
Impurity is good with two peak separating degrees of impurity A；Second peak (RT26.823min) of impurity B and unknown impuritie (RT
27.380min) separating degree is 0.80, other each impurity and main peak meet the requirements with other impurities separating degree.
In aforementioned four test, 0~50min gradient is consistent, as can be known from the results, it is each under the conditions of impurity A be in two peaks,
Impurity A has a unknown impuritie between two peaks, good with two peak separating degrees of impurity A；Under the conditions of 45 DEG C, 55 DEG C of column temperature, impurity B
It is separated with other impurities more preferably, takes into account using life of chromatographic column and peak separation, select 45 DEG C of colors as detection method
Spectral condition.
For Cefixime granule kind, its high temp samples has nonpolarity at longer retention time in related substance detection
Impurity generate, to combine 2 kinds, draft and improve Mobile phase B after 50min in chromatographic condition (2) gradient design
Ratio.
The optimization of 6 mobile phase salinity of embodiment
On the basis of embodiment 5 (2) condition, mobile phase salinity is optimized and revised, while gradient design mentions after 50min
High Mobile phase B ratio is accelerated nonpolarity element and is washed out, designs different salinity chromatographic conditions to Cefixime particle high-temperature sample
The separating degree between separating degree and impurity and main peak in mark-on test solution between each impurity is investigated.
Column temperature: 45 DEG C；
Test solution: 60 DEG C of -30 days samples of Cefixime granule high temperature are formulated with each known impurities solution.
(1) mobile phase A: 0.5% tetrabutylammonium hydroxide solution (pH5.0)-acetonitrile (90: 10)；
Mobile phase B: 0.5% tetrabutylammonium hydroxide solution (pH5.0)-acetonitrile (50: 50)；
Using gradient elution as shown in table 17:
Table 17
By testing result (Figure 15) it is found that each known impurities and degradation impurity, main peak and other impurities are equal under this condition
Up to baseline separation, mpB is up to 100% after 70min, but baseline interference is larger.
(2) mobile phase A: 0.4% tetrabutylammonium hydroxide solution (pH5.0)-acetonitrile (90: 10)；
Mobile phase B: 0.4% tetrabutylammonium hydroxide solution (pH5.0)-acetonitrile (50: 50)；
Using gradient elution as shown in table 18:
Table 18
By testing result (Figure 16) it is found that each known impurities and degradation impurity, main peak and other impurities are equal under this condition
Up to baseline separation, for mpB up to 100%, baseline is more steady after 70min.
From said determination result it is found that when mobile phase salinity is respectively 0.4% and 0.5%, each known impurities and drop
It is good to solve impurity, main peak and other impurities separating degree, and 70min Mobile phase B is up to after 100%, mobile phase salinity is 0.4%
Baseline is more stable, therefore selective flow phase salinity is 0.4% as chromatographic condition of the invention.It finally fixes tentatively and uses embodiment
7 (2) chromatographic conditions are as related substance-measuring method.
7 system suitability of embodiment
From detection method development process it is found that main peak with impurity D (i.e. (E) isomers) is more difficult separates, in order to true
It protects each impurity and main peak and can reach under the chromatographic condition and efficiently separate, " system is applicable in reference in related substance original method (table 1)
Property test " draft system suitability, determine quality standard system suitability solution and its requirement are as follows:
System suitability solution: taking Cefixime reference substance appropriate, dissolved with water and dilute be made in every 1ml containing about
The solution of 1mg cools down, in heating 45 minutes on boiling water bath as system suitability solution.
System suitability requirement: (E) isomers peak and the peak-to-peak separating degree of Cefixime answer >=1.5.
8 chromatographic condition of embodiment determines
The related substance chromatographic condition that table 19 determines
The method of the present invention and original method comparison of the related substance of embodiment 9 detection
Comparison in relation to substance original method and new method is shown in Table 20.
The method of the present invention and original method contrast table of the related substance of table 20 detection
Respectively according to related substance-measuring method original method and the method for the present invention centering test agent stability June, each batch of ginseng
It is measured than preparation and reference preparation stability sample in March.The map of two methods measurement mark-on test solution is shown in figure
17~18.
By said determination result it is found that impurity A peak shape is poor under original pass substance detecting method (Figure 17) chromatographic condition,
In multiple chromatographic peaks, unknown degradation impurity is also wrapped up in multiple chromatographic peaks of impurity A, baseline separation cannot be reached；Present invention inspection
Survey method (Figure 18) optimizes chromatographic condition by the way of eluent gradient elution, and by changing diluent type, impurity A is in
It is unimodal, reach baseline separation with unknown each impurity, true feelings of this product in relation to substance can be reflected by illustrating detection method more
Condition.
In conclusion detection method carries out related substance detection to Cefixime Capsules using HPLC method, use
The Self-control method that correction factor is not added controls impurity D, E, single unknown impuritie, using the correction up factor itself is right
Known impurities A, B and total impurities are controlled according to method.In preliminary screening Detection wavelength, mobile phase, chromatographic column, test sample solvent
With excellent chromatographic condition is established on the basis of extracting mode and filter membrane Absorbability etc..Compared with original method, the present invention
The separation of method impurity, Detection capability are more excellent, can once checked for impurities A, B, D, E, detection efficiency are high simultaneously.The present invention simultaneously
Method can be used in the detection of related impurities in all kinds of drug forms of Cefixime, including Cefixime Capsules, Cefixime
Grain, cefixime dispersible tablet etc., it is applied widely.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright specification and accompanying drawing content is applied directly or indirectly in other relevant skills
Art field, is included within the scope of the present invention.
Claims (10)
1. a kind of method for detecting Cefixime related impurities, which is characterized in that carried out simultaneously using high performance liquid chromatography qualitative
Or/and quantitative detection Cefixime related impurities, the testing conditions of liquid chromatogram include:
Chromatographic column: alkyl linked silicagel column is further selected from C18 chromatographic column；
Mobile phase: water phase, organic phase；
Wherein, water phase is tetrabutylammonium hydroxide solution；Organic phase is selected from acetonitrile or/and methanol, is not individually for methanol；Mobile phase
Using following gradient elution program:
。
2. detection method according to claim 1, which is characterized in that the impurity is selected from Cefixime impurity A, cephalo gram
One or more of oxime impurity B, Cefixime impurity D, Cefixime impurity E.
3. detection method according to claim 1, which is characterized in that the tetrabutylammonium hydroxide solution ph of the water phase
Range is 4.9~7.0, and mass concentration is 0.4%~0.5%.
4. detection method according to claim 1, which is characterized in that the organic phase is acetonitrile, and mobile phase is using as follows
Gradient elution program:
5. detection method according to claim 1, which is characterized in that the liquid chromatographic detection condition further includes below
It is one or more in i~iii:
I chromatographic column specification: 4.6 × 250mm, 5 μm；
Ii column temperature: 45~55 DEG C；
Iii flow velocity: 1.2~0.8ml/min.
6. a kind of method for detecting Cefixime related impurities, which is characterized in that carried out simultaneously using high performance liquid chromatography qualitative
Or/and quantitative detection Cefixime impurity A, the testing conditions of liquid chromatogram include:
Chromatographic column: alkyl linked silicagel column is further selected from C18 chromatographic column；
Mobile phase: water phase, organic phase；
Wherein, water phase is tetrabutylammonium hydroxide solution；Organic phase is selected from acetonitrile or/and methanol, is not individually for methanol；Mobile phase
Using following gradient elution program:
。
7. detection method according to claim 6, which is characterized in that the tetrabutylammonium hydroxide solution ph of the water phase
Range is 4.9~7.0, and mass concentration is 0.25%~10%.
8. detection method according to claim 6, which is characterized in that the liquid chromatographic detection condition further includes below
It is one or more in i~iii:
I chromatographic column specification: 4.6 × 250mm, 5 μm；
Ii column temperature: 40~55 DEG C；
Iii flow velocity: 1.2~0.8ml/min.
9. detection method according to claim 1 or 6, which is characterized in that the detection method includes the following contents:
(1) test solution is prepared；
(2) contrast solution is prepared；
(3) by contrast solution sample detection；
(4) by test solution sample detection.
10. detection method according to claim 9, which is characterized in that prepare used in test solution or/and contrast solution
Solvent is selected from the mixed of one or more of tetrabutylammonium hydroxide solution, pH7.0 phosphate buffer, methanol, acetonitrile
Bonding solvent.
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