CN103412060B - Method for separating and measuring tebipenem pivoxil related substances - Google Patents
Method for separating and measuring tebipenem pivoxil related substances Download PDFInfo
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- CN103412060B CN103412060B CN201310259272.9A CN201310259272A CN103412060B CN 103412060 B CN103412060 B CN 103412060B CN 201310259272 A CN201310259272 A CN 201310259272A CN 103412060 B CN103412060 B CN 103412060B
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Abstract
The invention provides a method for separating and measuring tebipenem pivoxil related substances. According to the method, a chromatographic column adopts a C18 chromatographic column; a mobile phase is a mixed solution consisting of acetonitrile and a phosphate solution with the pH value being 3.0 to 6.0; column temperature is 38 to 40 DEG C; detection wavelength is 220 nm; flow velocity is 0.8 to 1.2 ml/min; gradient elution is performed by changing the ratio of the acetonitrile to the phosphate solution in the mobile phase. The method can effectively separate and measure six impurities of tebipenem pivoxil, has good separation degree and good peak shape, and can be applied to quality control of tebipenem pivoxil raw material medicines and tebipenem pivoxil medicine preparations.
Description
Technical field
The present invention relates to Pharmaceutical Analysis field, what be specifically related to is the high performance liquid chromatography method of separating and assaying of carbapenem antibiotic tebipenem pivoxil related substance.
Background technology
Tebipenem pivoxil (formula I) is Tebipenem Pivoxil again, being the prodrug that training southern class (Carbapenems) medicine tebipenem C2 position carboxylic esterification is formed, is a kind of novel oral Carbapenems medicine researched and developed by Wyeth Pharmaceuticals.Tebipenem has a broad antifungal spectrum, all show and cephalo serial stronger antibiotic property more serial than penicillin, and compared with the carbapenem antibiotic of other injections, tebipenem also shows same degree or stronger antibacterial effect to the bacterial strain of most of clinical separation.Particularly for causing the PRSP of childhood infection main cause (penicillin resistance pneumococcus), MRSP (resistance to erythromycin streptococcus pneumonia) and Haemophilus influenzae (haemophilus influenzae) to show extremely strong antibacterial effect in recent years.As the prodrug of tebipenem, tebipenem pivoxil has better absorption dynamics compared to tebipenem, and has good stability.Its structural formula and reaction scheme as follows:
Containing the unreacted related substance such as initiation material, intermediate and catabolite completely, through structure analysis, related substance can be mainly contained and comprises following in the product that the synthesis of above-mentioned tebipenem pivoxil obtains:
In order to strictly control drug quality, ensure the safe and effective of clinical application, need the inspection method setting up tebipenem pivoxil related substance, and the related substance quantity of tebipenem pivoxil is more, wherein impurity F and tebipenem pivoxil body polarity similar, be that the chromatographic column of filling agent has identical chromatographic behavior with tebipenem pivoxil and is difficult to separately at general octadecylsilane chemically bonded silica, and impurity A is the material that a polarity is very strong, at general octadecylsilane chemically bonded silica be filling agent chromatographic column on basic not retain, and exist with the inseparable situation of solvent peak, the existence of these problems, make, with HPLC analytical method, the separation of the chromatographic peak of each related substance is had certain difficulty, thus can not control the related substance that may exist in sample effectively accurately.
Summary of the invention
The technical matters that the present invention solves there is provided a kind of method of efficient liquid-phase chromatography method separation determination tebipenem pivoxil related substance.
The method of high efficiency liquid chromatography for separating and determining tebipenem pivoxil related substance provided by the invention, it is characterized in that chromatographic condition is that chromatographic column adopts C18 chromatographic column, mobile phase is acetonitrile and pH is the mixed solution that 3.0 ~ 6.0 phosphate solutions form, column temperature is 38 DEG C ~ 40 DEG C, determined wavelength is 220nm, flow velocity is 0.8 ~ 1.2ml/min, carries out gradient elution by the ratio of acetonitrile and phosphate solution in conversion mobile phase.
In tebipenem pivoxil, impurity is more, and the determination of determined wavelength needs to carry out experiment screening, prevents related substance from being underestimated even undetected.Inventor measures tebipenem pivoxil raw material and impurity A respectively, B, C, D, E, F solution is appropriate, injection liquid chromatography diode array detector scans, tebipenem pivoxil has maximum absorption band at 323nm place, but impurity A, B, C almost do not absorb under the wavelength being greater than 250nm; Tebipenem pivoxil raw material and impurity A, B, C, D, E, F increase influx and translocation along with wavelength; Precision takes tebipenem pivoxil raw material and impurity A, B, C, D, E, F make the sample solution of every 1ml containing sample 10 μ g with mobile phase dissolved dilution in right amount, scan with ultraviolet spectrophotometer, confirm under 220nm, the relative response factor of impurity A, B, C, D, E, F and tebipenem pivoxil is all in 0.9 ~ 1.1 scope.Therefore, confirm that 220nm is determined wavelength.
Further optimization, the preferred model of C18 chromatographic column of the present invention is AQ type, AQ-C18 chromatographic column is applicable to the watr-proportion of in mobile phase 0 ~ 100%, long-time use under pure water phase condition and can not causing is subsided mutually, inventor once investigated other C18 chromatographic columns, there will be chromatographic peak symmetry bad, and the problem that between one or several adjacent peak, degree of separation is bad.
Described phosphate be sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate (ADP) wherein one or more, most preferably potassium dihydrogen phosphate; The preferred 0.025mol/L of phosphate concn is 6.0 with potassium hydroxide solution adjust pH; Column temperature preferably 40 DEG C; The preferred 1.0ml/min of elution flow rate.
Because related substance quantity is more, mobile phase need convert ratio and carry out gradient elution, otherwise each peak can not be separated and possess good degree of separation, and particularly impurity A is overlapping with solvent peak, is difficult to separately.Inventor is with 0.025M potassium dihydrogen phosphate (pH=6.0) for A phase, and using acetonitrile as B phase, flow velocity 1.0ml/min, column temperature is 40 DEG C, screens the program of gradient elution.
By when such as Gradient carries out wash-out:
| Time (min) | A％ | B％ |
| 0～10 | 95 | 5 |
| 10～25 | 95～60 | 5～40 |
| 50 | 60 | 40 |
| 50.1 | 95 | 5 |
| 60 | 95 | 5 |
Result: impurity A Rt=3.65min, impurity A appearance time too early, can not effectively be separated with solvent peak; Tebipenem pivoxil Rt=39.98min, impurity F Rt=43.04min, tebipenem pivoxil, impurity F appearance time are too late.
By when such as Gradient carries out wash-out:
| Time (min) | A％ | B％ |
| 0～15 | 95～60 | 5～40 |
| 50 | 60 | 40 |
| 50.1 | 95 | 5 |
| 60 | 95 | 5 |
Result: impurity A Rt=3.58min, impurity A appearance time too early, can not effectively be separated with solvent peak; Tebipenem pivoxil Rt=26.92min, impurity F Rt=28.72min, tebipenem pivoxil, impurity F appearance time are a little later.
By when such as Gradient carries out wash-out:
| Time (min) | A％ | B％ |
| 0～10 | 80～60 | 20～40 |
| 50 | 60 | 40 |
| 50.1 | 80 | 20 |
| 60 | 80 | 20 |
Result: impurity A, impurity B Rt=3.14min, impurity A, impurity B appearance time be morning too, and solvent peak can not effectively be separated; Tebipenem pivoxil Rt=17.6min.
By when such as Gradient carries out wash-out:
| Time (min) | A％ | B％ |
| 0～10 | 85～60 | 15～40 |
| 50 | 60 | 40 |
| 50.1 | 85 | 15 |
| 60 | 85 | 15 |
Result: impurity A, impurity B=3.32min.Impurity A, impurity B appearance time too early, and can not effectively be separated with solvent peak.
By when such as Gradient carries out wash-out:
| Time (min) | A％ | B％ |
| 0 | 98 | 2 |
| 20 | 50 | 50 |
| 50 | 50 | 50 |
| 50.1 | 98 | 2 |
| 60 | 98 | 2 |
Result: impurity A Rt=6.705min, tebipenem pivoxil Rt=18.597min.Impurity A is effectively separated with solvent peak energy (degree of separation is 2.02).Therefore this gradient elution method is selected to carry out the separation of tebipenem pivoxil related substance.
The related substance of employing HPLC analytical method separation determination tebipenem pivoxil provided by the invention, the method can six impurity of effective separation determination tebipenem pivoxil, degree of separation is good, peak shape is good, may be used for the quality control of tebipenem pivoxil bulk drug and tebipenem pivoxil pharmaceutical preparation.
Below in conjunction with embodiment and Figure of description, the present invention will be further described.
Figure of description
Accompanying drawing 1 tebipenem pivoxil reference substance chromatogram
Accompanying drawing 2 degree of separation test chromatogram
Accompanying drawing 3 impurity A and solvent peak separation case amplify contrast colors spectrogram
Accompanying drawing 4 tebipenem pivoxil bulk drug chromatogram
Accompanying drawing 5 tebipenem pivoxil granule chromatogram
Embodiment
Embodiment 1 degree of separation is tested
Instrument and reagent:
Instrument: Waters2695 high performance liquid chromatograph, chromatographic grade acetonitrile (Merck company), potassium dihydrogen phosphate (analyzing pure).
Reagent: tebipenem pivoxil reference substance, impurity A, B, C, D, E, F reference substance
Chromatographic condition:
Chromatographic column: Ultimate AQ5 μ l250mm × 4.6mm (Yue Xu Science and Technology Ltd.)
Detecting device and determined wavelength: UV-220nm
Mobile phase:
A:0.025mol/L potassium dihydrogen phosphate (pH6.0)
B: acetonitrile
Linear eluent gradient:
| Time (min) | A％ | B％ |
| 0 | 98 | 2 |
| 20 | 50 | 50 |
| 50 | 50 | 50 |
| 50.1 | 98 | 2 |
| 60 | 98 | 2 |
Flow velocity: 1.0ml/min, sample size: 10 μ l
Column temperature: 40 DEG C
The preparation of solution:
Contrast solution precision takes tebipenem pivoxil raw material and is about 12.5mg, put in 25ml volumetric flask, add mobile phase (acetonitrile-0.025mol/L potassium dihydrogen phosphate (pH6.0)=50: 50) and make dissolving in right amount, and be diluted to every 1ml containing tebipenem pivoxil 0.5mg; Precision pipettes in above-mentioned solution 1ml to 100ml volumetric flask, uses mobile phase constant volume, obtains contrast solution.
Test solution takes tebipenem pivoxil reference substance and impurity A respectively, B, C, D, E, F are appropriate, dissolves the sample solution being configured to concentration and being about 0.5mg/ml, draw above each sample solution respectively appropriate, mix as need testing solution with mobile phase.
Experimental implementation:
Get contrast solution 10 μ l, injection liquid chromatography, under above-mentioned chromatographic condition, record chromatogram (see accompanying drawing 1); Get test solution 10 μ l, injection liquid chromatography, under above-mentioned chromatographic condition, separation determination is carried out to tebipenem pivoxil raw material and each impurity, record chromatogram, adjacent peak-to-peak degree of separation meets the requirements (see accompanying drawing 2), through contrasting the peak position of known tebipenem pivoxil with accompanying drawing 1, be respectively impurity A, impurity B, impurity C, impurity D, impurity E, tebipenem pivoxil, impurity F by peak sequence.Adjacent peak-to-peak degree of separation is as follows:
Each adjacent peak-to-peak degree of separation is all greater than 1.5.
Amplified by impurity A chromatographic peak in accompanying drawing 2 and contrast known with independent by the collection of illustrative plates that the mobile phase sample introduction of sample dissolution records, impurity A chromatographic peak and solvent peak separate (see accompanying drawing 3).
The mensuration of embodiment 2 tebipenem pivoxil bulk drug
Instrument and reagent:
Instrument: Waters2695 high performance liquid chromatograph, chromatographic grade acetonitrile (Merck company), potassium dihydrogen phosphate (analyzing pure).
Reagent: tebipenem pivoxil raw material 20120627 (Wanle Pharmaceutical Co Ltd, Shenzhen)
Chromatographic condition:
Chromatographic column: Ultimate AQ5 μ l250mm × 4.6mm (Yue Xu Science and Technology Ltd.)
Detecting device and determined wavelength: UV-220nm
Mobile phase:
A:0.025mol/L potassium dihydrogen phosphate (pH6.0)
B: acetonitrile
Linear eluent gradient:
| Time (min) | A％ | B％ |
| 0 | 98 | 2 |
| 20 | 50 | 50 |
| 50 | 50 | 50 |
| 50.1 | 98 | 2 |
| 60 | 98 | 2 |
Flow velocity: 1.0ml/min, sample size: 10 μ l
Column temperature: 40 DEG C
The preparation of sample solution: it is appropriate that precision takes tebipenem pivoxil bulk drug, makes every 1ml about containing the sample solution of tebipenem pivoxil 0.5mg/ml with mobile phase dissolved dilution
Experimental implementation: precision measures sample solution 10 μ l injection liquid chromatography, record chromatogram (see accompanying drawing 4).Impurity A, B, C, D, E, F is not detected in result show sample.
The mensuration of embodiment 3 tebipenem pivoxil granule
Instrument and reagent:
Instrument: Waters2695 high performance liquid chromatograph, chromatographic grade acetonitrile (Merck company), potassium dihydrogen phosphate (analyzing pure).
Reagent: tebipenem pivoxil granule 120801 (Wanle Pharmaceutical Co Ltd, Shenzhen)
Chromatographic condition:
Chromatographic column: Ultimate AQ5 μ l250mm × 4.6mm (Yue Xu Science and Technology Ltd.)
Detecting device and determined wavelength: UV-220nm
Mobile phase:
A:0.025mol/L potassium dihydrogen phosphate (pH6.0)
B: acetonitrile
Linear eluent gradient:
| Time (min) | A％ | B％ |
| 0 | 98 | 2 |
| 20 | 50 | 50 |
| 50 | 50 | 50 |
| 50.1 | 98 | 2 |
| 60 | 98 | 2 |
Flow velocity: 1.0ml/min, sample size: 10 μ l
Column temperature: 40 DEG C
The preparation of sample solution: it is appropriate that precision takes tebipenem pivoxil granule, makes every 1ml about containing the sample solution (wherein every 1ml is about containing tebipenem pivoxil bulk drug 0.5mg) of tebipenem pivoxil granule 5mg with mobile phase dissolved dilution.
Experimental implementation: precision measures sample solution 10 μ l injection liquid chromatography, records chromatogram (see accompanying drawing 5) under above-mentioned chromatographic condition.Impurity A, B, C, D, E, F is not detected in result show sample.
Claims (5)
1., by a method for high performance liquid chromatography separation determination tebipenem pivoxil related substance, described related substance comprises following:
It is characterized in that, chromatographic column is C18 chromatographic column, described C18 chromatographic column model is AQ, mobile phase to be acetonitrile and pH be 6.0 the mixed solution of 0.025mol/L phosphate solution composition, column temperature is 38 DEG C ~ 40 DEG C, determined wavelength is 220nm, and flow velocity is 0.8 ~ 1.2ml/min, and mobile phase carries out wash-out by such as Gradient:
Wherein A phase is phosphate solution, and B phase is acetonitrile.
2. method according to claim 1, it is characterized in that described phosphate be sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonium dihydrogen phosphate (ADP) wherein one or more.
3. method according to claim 1, is characterized in that described phosphate is potassium dihydrogen phosphate.
4. method according to claim 1, is characterized in that column temperature is 40 DEG C.
5. method according to claim 1, is characterized in that flow velocity is 1.0ml/min.
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