CN104569264B - A kind of assay method of Etomidate related substance - Google Patents
A kind of assay method of Etomidate related substance Download PDFInfo
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Abstract
The present invention relates to measure the technical field of Drug-related Substances, a kind of assay method of Etomidate related substance is disclosed, described method adopts high performance liquid chromatography to measure, and the chromatographic condition of described high performance liquid chromatography comprises: chromatographic column adopting octadecylsilane chemically bonded silica is as filler; Mobile phase is the mixed solution of phosphate buffer and acetonitrile, and wherein, the pH value of phosphate buffer is 7.8～8.2, and the volume ratio of phosphate buffer and acetonitrile is 50:50～90:10; Detection wavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 29 DEG C～31 DEG C. The assay method of Etomidate related substance provided by the present invention, making support miaow in chromatogram acid peak reach good with solvent peak separates, and rely on miaow acid peak also can separate preferably with main peak with other impurity peaks, and then can rely on the peak area of miaow acid to obtain relying in Etomidate the content of miaow acid by calculating.
Description
Technical field
The present invention relates to measure the technical field of Drug-related Substances, particularly the relevant thing of a kind of EtomidateThe assay method of matter.
Background technology
Etomidate is a kind of hypnosis intravenous anesthetics, belongs to imidazole derivative, is the hydroxylation salt of imidazoles,Molecular formula is C14H16N2O2, molecular weight is 244.29, security is large, be the conventional medicine of anesthesia induction itOne, the clinical practice history of existing 30 years of Etomidate.
Etomidate often adopts the form of preparation, for example Etomidate Injection, and Etomidate is in parenteral solutionEasily degraded produces impurity (related substance) and relies on miaow acid, in 2010 editions Chinese pharmacopoeia, has recorded supportThe assay method of miaow ester related substance, it has adopted high performance liquid chromatography, the correction that wherein relies on miaow acid because ofSon is 1.59, relies on miaow acid to go out peak early, substantially overlaps with solvent peak, therefore cannot measure in EtomidateRelated substance relies on miaow acid.
Summary of the invention
In order to solve the above-mentioned defect existing in prior art, the invention provides a kind of Etomidate related substanceAssay method, adopt the method can be well the related substance in Etomidate to be relied on to miaow acid peak and moltenAgent peak separates, and then can obtain the content that relies on miaow acid.
First the embodiment of the present invention provides a kind of assay method of Etomidate related substance, and described method adoptsHigh performance liquid chromatography is measured, and the chromatographic condition of described high performance liquid chromatography comprises:
Chromatographic column adopting octadecylsilane chemically bonded silica is as filler;
Mobile phase is the mixed solution of phosphate buffer and acetonitrile, wherein, and the pH value of phosphate bufferBe 7.8～8.2, the volume ratio of phosphate buffer and acetonitrile is 50:50～90:10;
Detection wavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 29 DEG C～31 DEG C.
In technical solution of the present invention, high-efficient liquid phase chromatogram condition is optimized, make complying with in chromatogramHolder miaow acid peak has reached good with solvent peak and has separated, and rely on miaow acid peak also with other impurity peaks and main peakCan separate preferably, and then can rely on the peak area of miaow acid to obtain relying in Etomidate by calculatingThe content of miaow acid.
In technical solution of the present invention, the form of Etomidate is not limited, as long as Etomidate meetsEtomidate raw material and the preparation of chromatographic condition can use, and Etomidate preparation can be Etomidate notePenetrate liquid, Etomidate fatty emulsion parenteral solution.
Preferably, described method comprises:
Get the acetonitrile solution dilution of Etomidate fat raw material or preparation employing 25% for containing EtomidateThe solution of 0.5mg/ml, as need testing solution;
Get described need testing solution and adopt 30% acetonitrile solution dilution, solution in contrast;
Get and rely on the acetonitrile solution of miaow acid, metomidate and Etomidate employing 30% to dilute for containingThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
Adopt high performance liquid chromatography respectively to described system suitability solution, contrast solution and test sampleSolution is measured, and records chromatogram, and the chromatographic condition of described high performance liquid chromatography comprises:
Chromatographic column adopting octadecylsilane chemically bonded silica is as filler;
Mobile phase is the mixed solution of phosphate buffer and acetonitrile, wherein, and the pH value of phosphate bufferBe 7.8～8.2, the volume ratio of phosphate buffer and acetonitrile is 50:50～90:10;
According to gradient elution, detection wavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 30 DEG C.
Preferably, described Etomidate preparation is Etomidate fatty emulsion parenteral solution. Due to Etomidate often withThe form of Etomidate fatty emulsion parenteral solution exists, and in Etomidate fatty emulsion parenteral solution EtomidateEasily degraded generates and relies on miaow acid impurity, and therefore, the assay method of Etomidate related substance of the present invention is specialBe applicable to being applied in the mensuration of Etomidate fatty emulsion parenteral solution related substance.
Preferably, described method comprises:
Get Etomidate fatty emulsion parenteral solution 5ml and be placed in 10ml measuring bottle and place 5 minutes in ice bath, addEnter dilution in acetonitrile that ice bath crosses to scale, violent jolting, first uses the filter membrane mistake of 0.45 μ m again with Filter paper filteringFilter, gets the subsequent filtrate 5ml 5ml that adds water and shakes up, as need testing solution;
Precision measures 1ml need testing solution and is placed in 100ml measuring bottle, is diluted to 30% acetonitrile solutionScale, shakes up, in contrast solution;
Get and rely on miaow acid, metomidate and Etomidate, add 30% acetonitrile solution and dissolve and be diluted to and containThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
System suitability solution, the need testing solution of 10 μ l and the contrast of 10 μ l of getting successively 10 μ l are moltenLiquid, injects respectively high performance liquid chromatograph, records chromatogram, wherein, and the chromatogram of described high performance liquid chromatographyCondition comprises: chromatographic column adopting octadecylsilane chemically bonded silica is filler; Mobile phase is phosphate-bufferedThe mixed solution of liquid and acetonitrile, wherein, the pH value of phosphate buffer is 8.0, according to gradient elution, 0～In 5 minutes, the volume ratio of phosphate buffer and acetonitrile is for to transit to 50:50 by 90:10, after 5 minutesKeeping the volume ratio of phosphate buffer and acetonitrile is 50:50, and retention time is not less than 15 minutes; DetectWavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 30 DEG C.
Preferably, the preparation method of described phosphate buffer comprises: get potassium dihydrogen phosphate 6.8g and hydroxideSodium 1.87g, adds the water-soluble solution of 1000ml, and adopting phosphoric acid or NaOH to regulate pH value is 7.8～8.2.
Brief description of the drawings
Fig. 1 is the unbroken support miaow that adopts the assay method of Etomidate related substance of the present invention to measureEster chromatogram;
Fig. 2 is the support miaow that adopts the photo damage of the assay method mensuration of Etomidate related substance of the present inventionEster chromatogram;
Fig. 3 is the support miaow that adopts the oxygen of the assay method mensuration of Etomidate related substance of the present invention to destroyEster chromatogram;
Fig. 4 is the support miaow that adopts the acid of the assay method mensuration of Etomidate related substance of the present invention to destroyEster chromatogram;
Fig. 5 is the support miaow that adopts the alkali of the assay method mensuration of Etomidate related substance of the present invention to destroyEster chromatogram;
Fig. 6 is the support that adopts the high temperature of the assay method mensuration of Etomidate related substance of the present invention to destroyMiaow ester chromatogram;
Fig. 7 is the UV scanning figure of the unknown impuritie 1 of the chromatogram shown in Fig. 4;
Fig. 8 is the UV scanning figure of the support miaow acid of the chromatogram shown in Fig. 1;
Fig. 9 is the UV scanning figure of the unknown impuritie 2 of the chromatogram shown in Fig. 5;
Figure 10 is the UV scanning figure of the metomidate of the chromatogram shown in Fig. 1;
Figure 11 is the UV scanning figure of the Etomidate of the chromatogram shown in Fig. 1;
Figure 12 is the system suitability that adopts the assay method of the Etomidate related substance of Chinese pharmacopoeia to measureThe chromatogram of testing liquid;
Figure 13 is the need testing solution that adopts the assay method of the Etomidate related substance of Chinese pharmacopoeia to measureChromatogram;
Figure 14 is the contrast solution that adopts the assay method of the Etomidate related substance of Chinese pharmacopoeia to measureChromatogram;
Figure 15 is the chromatogram that adopts the empty needle of the assay method mensuration of the Etomidate related substance of British PharmacopoeiaFigure;
Figure 16 is the system suitability that adopts the assay method of the Etomidate related substance of British Pharmacopoeia to measureThe chromatogram of testing liquid;
Figure 17 is the need testing solution that adopts the assay method of the Etomidate related substance of British Pharmacopoeia to measureChromatogram;
Figure 18 is the contrast solution that adopts the assay method of the Etomidate related substance of British Pharmacopoeia to measureChromatogram;
Figure 19 is the system suitability that adopts the assay method of the Etomidate related substance of American Pharmacopeia to measureThe chromatogram of testing liquid;
Figure 20 is the need testing solution that adopts the assay method of the Etomidate related substance of American Pharmacopeia to measureChromatogram;
Figure 21 is the contrast solution that adopts the assay method of the Etomidate related substance of American Pharmacopeia to measureChromatogram;
Figure 22 is the system suitability examination that adopts the assay method of Etomidate related substance of the present invention to measureTest the chromatogram of solution;
Figure 23 is the need testing solution that adopts the assay method of Etomidate related substance of the present invention to measureChromatogram;
Figure 24 is the look that adopts the contrast solution of the assay method mensuration of Etomidate related substance of the present inventionSpectrogram.
Detailed description of the invention
Rely on miaow acid in order to measure in Etomidate raw material or preparation, the invention provides a kind of Etomidate hasThe assay method of related substance. In this technical scheme, by optimizing high-efficient liquid phase chromatogram condition, make to rely onMiaow acid peak and solvent peak are separated, and then can obtain and rely on containing of miaow acid according to the peak area that relies on miaow acidAmount. Illustrate technical scheme of the present invention below in conjunction with accompanying drawing.
In the following embodiment of the present invention, the high performance liquid chromatograph that high performance liquid chromatography adopts is peace victoryHuman relations 1200 type chromatographs, sample and river that the Etomidate of use is produced for Zhejiang Province Jiuxu Pharmaceutical Co., LtdThe sample that Su Enhua medicine company limited company produces.
First the embodiment of the present invention provides a kind of assay method of Etomidate related substance, and described method adoptsHigh performance liquid chromatography is measured, and the chromatographic condition of described high performance liquid chromatography comprises:
Chromatographic column adopting octadecylsilane chemically bonded silica is as filler;
Mobile phase is the mixed solution of phosphate buffer and acetonitrile, wherein, and the pH value of phosphate bufferBe 7.8～8.2, the volume ratio of phosphate buffer and acetonitrile is 50:50～90:10;
Detection wavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 29 DEG C～31 DEG C.
In technical solution of the present invention, high-efficient liquid phase chromatogram condition is optimized, make complying with in chromatogramHolder miaow acid peak has reached good with solvent peak and has separated, and rely on miaow acid peak also with other impurity peaks and main peakCan separate preferably, and then can rely on the peak area of miaow acid to obtain relying in Etomidate by calculatingThe content of miaow acid, more can react the quality of Etomidate raw material and preparation truly.
As long as in fact column temperature is no more than 60 DEG C of the highest tolerable temperatures of chromatogram, usually, column temperature can beBetween 10-40 DEG C, change, preferred, column temperature can change between 29 DEG C～31 DEG C, for example column temperature be 29 DEG C,29.5 DEG C, 30 DEG C, 30.5 DEG C or 31 DEG C. Preferably, column temperature is 30 DEG C. When column temperature 29 DEG C～31 DEG C itBetween while changing, can reduce column temperature and change the impact on retention time, make the detection of Etomidate sample moreAccurately.
Actual buffer solution is more than 7.0 all feasible in pH value, and preferred, the pH value of phosphate buffer canBetween 7.8～8.2, change, the pH value of for example phosphate buffer is 7.8,8.0,8.1 or 8.2. ExcellentChoosing, the pH value of phosphate buffer solution is preferably 8.0. In fact get potassium dihydrogen phosphate 6.8g and hydroxideSodium 1.87g, add the water-soluble solution of 1000ml after pH value substantially 8.0, because solution is for this reason buffer solution, soPH value is fixed substantially.
The screening of chromatographic condition
The condition of the high performance liquid chromatography adopting in the present invention obtains through screening. With reference to Chinese pharmacopoeia(Chp), the chromatographic condition of American Pharmacopeia (USP) and British Pharmacopoeia (BP) standard, table 1 is complied with for measuringChp standard and the BP standard of holder miaow ester related substance; Table 2 is for measuring the USP of Etomidate related substanceStandard and standard of the present invention; By the USP in Chp standard and BP standard and table 2 in table 1Standard is found, the relatively positive divisor that in Chinese pharmacopoeia standard, the main degradation impurity of Etomidate is relied on miaow acidBe 1.59 and rely on miaow acid to go out peak almost to overlap with solvent peak very early, in USP standard Etomidate mainlyIt is 8.9 left and right that degradation impurity is relied on the relatively positive divisor of miaow acid, and this standard cannot detect support miaow at allThe related substance of ester, only has the correction factor of known impurities in the chromatographic condition of BP standard in 0.8-1.2 scopeIn, but in the chromatographic condition of BP standard, also there are problems: gradient peak serious interference; Mobile phase pHHigher, seriously shorten service life of chromatographic column; In the time that gradient moves to 6-10 minute, due to organic phaseLess and salt face is more, cause occurring a large amount of salt out and high performance liquid chromatograph during this period of time showsPressure is 0. Therefore chromatographic condition of the present invention is taking BP standard as basis, and preferred chromatographic condition, has adoptedLower sample introduction concentration, selects the mobile phase that example is suitable, and the mobile phase using has relatively low pHValue, in addition reasonably optimizing gradient time and gradient ratio, make to rely on miaow acid peak and solvent peak to divide preferablyFrom, and avoided the above-mentioned defect in BP method.
Table 1 is measured Chp standard and the BP standard of Etomidate related substance
Table 2 is measured USP standard and the standard of the present invention of Etomidate related substance
Shakedown test
Fig. 1 is the unbroken support miaow that adopts the assay method of Etomidate related substance of the present invention to measureEster chromatogram; In Fig. 1, the preparation method of unbroken Etomidate related substance is as follows:
Precision measures Etomidate fatty emulsion parenteral solution 5ml, is placed in 10ml measuring bottle, adds acetonitrile and is settled toScale, strong jolting, filters, and precision measures subsequent filtrate 5ml, is placed in 10ml measuring bottle, is diluted with waterTo scale, shake up, to obtain final product.
Fig. 2 is the support miaow that adopts the photo damage of the assay method mensuration of Etomidate related substance of the present inventionEster chromatogram; In Fig. 2, the preparation method of the Etomidate related substance of photo damage is as follows:
Precision measures Etomidate fatty emulsion parenteral solution 5ml, is placed in 10ml measuring bottle, adds acetonitrile and is settled toScale was placed after 48 hours under 4500LX high light, and strong jolting, filters, and precision measures subsequent filtrate5ml, is placed in 10ml measuring bottle, is diluted with water to scale, shakes up, and to obtain final product.
Fig. 3 is the support miaow that adopts the oxygen of the assay method mensuration of Etomidate related substance of the present invention to destroyEster chromatogram; The assay method of the Etomidate related substance that in Fig. 3, oxygen destroys is as follows:
Precision measures Etomidate fatty emulsion parenteral solution 5ml, is placed in 10ml measuring bottle, adds hydrogen peroxide 0.1ml,Shake up, be placed in 100 DEG C of water-baths and heat 30min, take out, let cool, add acetonitrile and be settled to scale, strongJolting, filters, and precision measures subsequent filtrate 5ml, is placed in 10ml measuring bottle, is diluted with water to scale, shakesEven, to obtain final product.
Fig. 4 is the support miaow that adopts the acid of the assay method mensuration of Etomidate related substance of the present invention to destroyEster chromatogram; The preparation method of the Etomidate related substance that in Fig. 4, acid destroys is as follows:
Precision measures Etomidate fatty emulsion parenteral solution 5ml, is placed in 10ml measuring bottle, adds the salt of 10mol/LAcid 0.1ml, shakes up, and is placed in 100 DEG C of water-baths and heats 30min, takes out, and lets cool, and adds the hydrogen of 10mol/LSodium oxide molybdena 0.1ml, adds acetonitrile and is settled to scale, and strong jolting, filters, and precision measures subsequent filtrate 5ml,Be placed in 10ml measuring bottle, be diluted with water to scale, shake up, to obtain final product.
In the Etomidate related substance destroying in acid, find unknown impuritie 1, its relative retention time(RelativeRetentionTime is called for short RRT), i.e. peak position RRT=0.38, UV scanning figure is as figureShown in 7, the ultraviolet maximum absorption wavelength of visible unknown impuritie 1 is about 235nm.
Fig. 5 is the support miaow that adopts the alkali of the assay method mensuration of Etomidate related substance of the present invention to destroyEster chromatogram; The preparation method of the Etomidate related substance that in Fig. 5, alkali destroys is as follows:
Precision measures Etomidate fatty emulsion parenteral solution 5ml, is placed in 10ml measuring bottle, adds the hydrogen of 10mol/LSodium oxide molybdena 0.1ml, shakes up, and places 10min, adds the hydrochloric acid 0.1ml of 10mol/L, adds acetonitrile and is settled to quarterDegree, strong jolting, filters, and precision measures subsequent filtrate 5ml, is placed in 10ml measuring bottle, is diluted with water toScale, shakes up, and to obtain final product.
In the Etomidate related substance destroying in acid, find unknown impuritie 2, its peak position RRT=0.62,UV scanning figure as shown in Figure 9, visible, and the ultraviolet maximum absorption wavelength of unknown impuritie 2 is about 240nm.
Fig. 6 is the support that adopts the high temperature of the assay method mensuration of Etomidate related substance of the present invention to destroyMiaow ester chromatogram; The preparation method of the Etomidate related substance that in Fig. 6, high temperature destroys is as follows:
Get Etomidate fatty emulsion parenteral solution and be placed in 100 DEG C of water-baths and heat 5h, take out, let cool, accurate amountGet 5ml, be placed in 10ml measuring bottle, add acetonitrile and be settled to scale, strong jolting, filters, and precision measuresSubsequent filtrate 5ml, is placed in 10ml measuring bottle, is diluted with water to scale, shakes up, and to obtain final product.
Etomidate sample all shows impurity is relied on to miaow acid, metomidate after various destructive testingsMeasure and do not produce interference, it is equal in 15 minutes more than 990 and after various destruction that each main peak purity all can reachGo out peak complete. Detect through UV absorption, record unknown impuritie 1, rely on miaow acid, unknown impuritie 2, U.S.The wavelength of holder miaow ester and Etomidate is respectively as shown in Fig. 7, Fig. 8, Fig. 9, Figure 10 and Figure 11, visible,Destroy the unknown impuritie 1 producing and the ultraviolet maximum absorption wavelength that relies on miaow acid through acid and be all about 235nm; WarpAlkali destroys the unknown impuritie 2 producing and is all about with the ultraviolet maximum absorption wavelength of metomidate, Etomidate240nm. In chromatographic condition screening, verified the correction factor that relies on miaow acid and metomidate 0.8-1.2 itBetween, therefore the content confidence level of chromatographic condition mensuration Etomidate related substance impurity of the present invention is higher, speciallyAttribute is good.
Comparative example 1
The method that adopts Chinese pharmacopoeia 2010 editions two Etomidate raw materials the ChP standard in table 1 to complying withThe related substances of holder miaow ester fat emulsion injection is measured, and obtains the chromatogram shown in Figure 12 and Figure 13,Concrete assay method is as follows:
Get Etomidate fatty emulsion parenteral solution 5ml and be placed in 10ml measuring bottle and place 5 minutes in ice bath, addEnter dilution in acetonitrile that ice bath crosses to scale, violent jolting, first uses the filter membrane mistake of 0.45 μ m again with Filter paper filteringFilter, gets the subsequent filtrate 5ml 5ml that adds water and shakes up, as need testing solution;
Precision measures 1ml need testing solution and is placed in 100ml measuring bottle, is diluted to 30% acetonitrile solutionScale, shakes up, in contrast solution;
Get and rely on miaow acid, metomidate and Etomidate, add 30% acetonitrile solution and dissolve and be diluted to and containThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
Chromatographic condition according to the Chp2010 standard in table 1 carries out high performance liquid chromatography detection, and the system of getting is suitableWith property testing liquid 5 μ l, injection liquid chromatography, peak sequence is for relying on miaow acid, metomidate and supportMiaow ester; Get contrast solution 5 μ l, injection liquid chromatography, regulates detection sensitivity, makes principal component chromatographic peakPeak height be about the 20%-25% of full scale; Precision measures need testing solution and the each 5 μ l of contrast solution again, pointOther injection liquid chromatography, records chromatogram.
Figure 12 is the system suitability that adopts the assay method of the Etomidate related substance of Chinese pharmacopoeia to measureThe chromatogram of testing liquid; Figure 13 adopts the assay method of the Etomidate related substance of Chinese pharmacopoeia to surveyThe chromatogram of fixed need testing solution; Figure 14 is the mensuration that adopts the Etomidate related substance of Chinese pharmacopoeiaThe chromatogram of the contrast solution that method is measured; From Figure 12 to Figure 14, can find out in need testing solution and rely onMiaow acid peak and solvent peak overlap, and cannot distinguish and rely on miaow acid.
Comparative example 2
The method that adopts British Pharmacopoeia i.e. relevant to Etomidate fatty emulsion parenteral solution of BP standard in table 1Material is measured, and obtains the chromatogram shown in Figure 15 to Figure 18, and concrete assay method is as follows:
Get Etomidate fatty emulsion parenteral solution 5ml and be placed in 10ml measuring bottle and place 5 minutes in ice bath, addEnter dilution in acetonitrile that ice bath crosses to scale, violent jolting, first uses the filter membrane mistake of 0.45 μ m again with Filter paper filteringFilter, gets the subsequent filtrate 5ml 5ml that adds water and shakes up, as need testing solution;
Precision measures 1ml need testing solution and is placed in 100ml measuring bottle, is diluted to 30% acetonitrile solutionScale, shakes up, in contrast solution;
Get and rely on miaow acid, metomidate and Etomidate, add 30% acetonitrile solution and dissolve and be diluted to and containThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
Chromatographic condition according to the BP2013 standard in table 1 carries out high performance liquid chromatography detection, and the system of getting is suitableWith property testing liquid 100 μ l, injection liquid chromatography, peak sequence is for relying on miaow acid, metomidate and complying withHolder miaow ester; Get contrast solution 100 μ l, injection liquid chromatography, regulates detection sensitivity, makes principal component lookThe peak height at spectrum peak is about the 20%-25% of full scale; Precision measures need testing solution and contrast solution is each again100 μ l, injection liquid chromatography, records chromatogram respectively.
Figure 15 is the chromatogram that adopts the empty needle of the assay method mensuration of the Etomidate related substance of British PharmacopoeiaFigure; Figure 16 is the system suitability that adopts the assay method of the Etomidate related substance of British Pharmacopoeia to measureThe chromatogram of testing liquid; Figure 17 adopts the assay method of the Etomidate related substance of British Pharmacopoeia to surveyThe chromatogram of fixed need testing solution; Figure 18 is the mensuration that adopts the Etomidate related substance of British PharmacopoeiaThe chromatogram of the contrast solution that method is measured; From Figure 15 to Figure 18, can find out, retention time is at 6-10Minute occur more embodying on Interference Peaks, especially Figure 15 empty needle figure.
Comparative example 3
Adopt the i.e. phase of USP standard in table 2 to Etomidate fatty emulsion parenteral solution of method of American PharmacopeiaRelated substance is measured, and obtains the chromatogram shown in Figure 19 and Figure 21, and concrete assay method is as follows:
Get Etomidate fatty emulsion parenteral solution 5ml and be placed in 10ml measuring bottle and place 5 minutes in ice bath, addEnter dilution in acetonitrile that ice bath crosses to scale, violent jolting, first uses the filter membrane mistake of 0.45 μ m again with Filter paper filteringFilter, gets the subsequent filtrate 5ml 5ml that adds water and shakes up, as need testing solution;
Precision measures 1ml need testing solution and is placed in 100ml measuring bottle, is diluted to 30% acetonitrile solutionScale, shakes up, in contrast solution;
Get and rely on miaow acid, metomidate and Etomidate, add 30% acetonitrile solution and dissolve and be diluted to and containThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
Chromatographic condition according to the USP35-NF30 standard in table 2 carries out high performance liquid chromatography detection, gets to beSystem employment and suitability test (E & ST) solution 50 μ l, injection liquid chromatography, peak sequence for rely on miaow acid, metomidate andEtomidate; Get contrast solution 50 μ l, injection liquid chromatography, regulates detection sensitivity, makes principal component lookThe peak height at spectrum peak is about the 20%-25% of full scale; Precision measures need testing solution and contrast solution is each again50 μ l, injection liquid chromatography, records chromatogram respectively.
Figure 19 is the system suitability that adopts the assay method of the Etomidate related substance of American Pharmacopeia to measureThe chromatogram of testing liquid; Figure 20 adopts the assay method of the Etomidate related substance of American Pharmacopeia to surveyThe chromatogram of fixed need testing solution; Figure 21 is the mensuration that adopts the Etomidate related substance of American PharmacopeiaThe chromatogram of the contrast solution that method is measured; Can be found out by Figure 19 to Figure 21, cannot examine in the methodThe main degradation material that measures Etomidate relies on miaow acid.
Embodiment
Adopt i.e. relevant to Etomidate fatty emulsion parenteral solution of standard of the present invention in table 2 of method of the present inventionMaterial is measured, and obtains the chromatogram shown in Figure 19 and Figure 20, and concrete assay method is as follows:
Get Etomidate fatty emulsion parenteral solution 5ml and be placed in 10ml measuring bottle and place 5 minutes in ice bath, addEnter dilution in acetonitrile that ice bath crosses to scale, violent jolting, first uses the filter membrane mistake of 0.45 μ m again with Filter paper filteringFilter, gets the subsequent filtrate 5ml 5ml that adds water and shakes up, as need testing solution;
Precision measures 1ml need testing solution and is placed in 100ml measuring bottle, is diluted to 30% acetonitrile solutionScale, shakes up, in contrast solution;
Get and rely on miaow acid, metomidate and Etomidate, add 30% acetonitrile solution and dissolve and be diluted to and containThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
Carry out high performance liquid chromatography detection according to the chromatographic condition of the standard of the present invention in table 2, the system of getting is suitable forProperty testing liquid 10 μ l, injection liquid chromatography, peak sequence is for relying on miaow acid, metomidate and support miaowEster, the separating degree at metomidate peak and Etomidate peak should be greater than 2.0; Get contrast solution 10 μ l, injection liquidChromatography, regulates detection sensitivity, makes the peak height of principal component chromatographic peak be about the 20%-25% of full scale;Precision measures need testing solution and the each 10 μ l of contrast solution again, and injection liquid chromatography, records chromatogram respectively.
Figure 22 is the system suitability examination that adopts the assay method of Etomidate related substance of the present invention to measureTest the chromatogram of solution; Figure 23 adopts the assay method of Etomidate related substance of the present invention to measureThe chromatogram of need testing solution; Figure 24 adopts the assay method of Etomidate related substance of the present invention to surveyThe chromatogram of fixed contrast solution; Can find out according to Figure 22 to Figure 24, adopt assay method of the present inventionThe peak that relies on miaow acid can be separated with other impurity peaks with solvent peak, main peak (being Etomidate peak) respectivelyOpen, and then convenient detection relied on miaow acid and content thereof.
In addition, the present invention also provides criterion: in test sample chromatogram, if any impurity peaks, rely on miaow acid not1.4 times (1.4%) that must be greater than contrast solution main peak area, metomidate must not be greater than contrast solution main peak0.2 times (0.2%) of area, other any lists must not mix and are greater than 0.2 times of contrast solution main peak area(0.2%), 1.6 times (1.6%) each impurity peak area and that must not be greater than contrast solution main peak area.
Detectability is measured
Precision takes Etomidate 10.30g, relies on miaow acid 10.26g, metomidate 10.14g, adds 30%Acetonitrile dissolves, and after multistep dilution, as need testing solution, gets 10ul and inject high performance liquid chromatograph,Recording chromatogram, is 3 times or 10 times of baseline noise to main peak peak height, records the detectability of Etomidate(Limitofdetection is called for short LOD) is 0.49ng, relies on the quantitative limit (Limitof of miaow acidQuantity, is called for short LOQ) LOQ of 1.64ng, metomidate is 1.75ng.
Linear
Rely on the content of miaow acid to be good line in the scope of 0.04%～2.2% (LOQ～150% limit)Sexual intercourse, linear equation: Y=20.502X+0.5289, coefficient R2＝1。
The content of metomidate is good in the scope of 0.04%～0.32% (LOQ～150% limit)Linear relationship, linear equation: Y=25.845X+0.4308, coefficient R2＝1。
Therefore, can determine the content that relies on miaow acid and metomidate according to the range of linearity.
Obviously, those skilled in the art can carry out various changes and modification and not depart from this present inventionBright design and scope. Like this, if of the present invention these amendment and modification belong to the claims in the present invention andWithin the scope of its equivalent technologies, the present invention be also intended to comprise these change and modification interior.
Claims (4)
1. an assay method for Etomidate related substance, is characterized in that, described method adopts efficientLiquid chromatography is measured, and the chromatographic condition of described high performance liquid chromatography comprises:
Chromatographic column adopting octadecylsilane chemically bonded silica is as filler;
Mobile phase is the mixed solution of phosphate buffer and acetonitrile, wherein, and the pH value of phosphate bufferBe 7.8～8.2, the volume ratio of phosphate buffer and acetonitrile is 50:50～90:10, according to gradient elution,In 0～5 minute, the volume ratio of phosphate buffer and acetonitrile transits to 50:50 by 90:10,5 minutes withThe volume ratio of rear maintenance phosphate buffer and acetonitrile is 50:50, and retention time is not less than 15 minutes;
The preparation method of described phosphate buffer comprises: get potassium dihydrogen phosphate 6.8g and NaOH 1.87g,Add the water-soluble solution of 1000ml, adopting NaOH or phosphorus acid for adjusting pH value is 7.8～8.2;
Detection wavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 29 DEG C～31 DEG C.
2. the assay method of Etomidate related substance according to claim 1, is characterized in that instituteThe method of stating comprises:
Get the acetonitrile solution dilution of Etomidate fat raw material or preparation employing 25% for containing EtomidateThe solution of 0.5mg/ml, as need testing solution;
Get described need testing solution and adopt 30% acetonitrile solution dilution, solution in contrast;
Get and rely on the acetonitrile solution of miaow acid, metomidate and Etomidate employing 30% to dilute for containingThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
Adopt high performance liquid chromatography respectively to described system suitability solution, contrast solution and test sampleSolution is measured, and records chromatogram, and the chromatographic condition of described high performance liquid chromatography comprises:
Chromatographic column adopting octadecylsilane chemically bonded silica is as filler;
Mobile phase is the mixed solution of phosphate buffer and acetonitrile, wherein, and the pH value of phosphate bufferBe 7.8～8.2, the volume ratio of phosphate buffer and acetonitrile is 50:50～90:10;
According to gradient elution, detection wavelength is 235nm, and flow velocity is 1.0ml/min, and column temperature is 30 DEG C.
3. the assay method of Etomidate related substance according to claim 2, is characterized in that instituteStating Etomidate preparation is Etomidate fatty emulsion parenteral solution.
4. the assay method of Etomidate related substance according to claim 3, is characterized in that instituteThe method of stating comprises:
Get Etomidate fatty emulsion parenteral solution 5ml and be placed in 10ml measuring bottle and place 5 minutes in ice bath, addEnter dilution in acetonitrile that ice bath crosses to scale, violent jolting, first uses the filter membrane mistake of 0.45 μ m again with Filter paper filteringFilter, gets the subsequent filtrate 5ml 5ml that adds water and shakes up, as need testing solution;
Precision measures 1ml need testing solution and is placed in 100ml measuring bottle, is diluted to 30% acetonitrile solutionScale, shakes up, in contrast solution;
Get and rely on miaow acid, metomidate and Etomidate, add 30% acetonitrile solution and dissolve and be diluted to and containThe mixing of the acid of support miaow, the metomidate of 0.02mg/ml and the Etomidate of 0.02mg/ml of 0.02mg/mlSolution, as system suitability solution;
System suitability solution, the need testing solution of 10 μ l and the contrast of 10 μ l of getting successively 10 μ l are moltenLiquid, injects respectively high performance liquid chromatograph, records chromatogram, wherein, and the chromatogram of described high performance liquid chromatographyCondition comprises: chromatographic column adopting octadecylsilane chemically bonded silica is filler; Mobile phase is phosphate-bufferedThe mixed solution of liquid and acetonitrile, wherein, the pH value of phosphate buffer is 8.0, according to gradient elution, 0～In 5 minutes, the volume ratio of phosphate buffer and acetonitrile transits to 50:50 by 90:10, within 5 minutes, protects laterThe volume ratio of holding phosphate buffer and acetonitrile is 50:50, and retention time is not less than 15 minutes; Detect rippleLong is 235nm, and flow velocity is 1.0ml/min, and column temperature is 30 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1399959A (en) * | 2002-03-25 | 2003-03-05 | 徐州颐华医药科技有限公司 | Etomidate fatty emulsion injection preparation method |
| WO2010147632A1 (en) * | 2009-06-14 | 2010-12-23 | Children's Medical Center Corporation | Microgel compositions and methods |
| CN102046607A (en) * | 2008-03-31 | 2011-05-04 | 通用医疗公司 | Etomidate analogues with improved pharmacokinetic and pharmacodynamic properties |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1399959A (en) * | 2002-03-25 | 2003-03-05 | 徐州颐华医药科技有限公司 | Etomidate fatty emulsion injection preparation method |
| CN102046607A (en) * | 2008-03-31 | 2011-05-04 | 通用医疗公司 | Etomidate analogues with improved pharmacokinetic and pharmacodynamic properties |
| WO2010147632A1 (en) * | 2009-06-14 | 2010-12-23 | Children's Medical Center Corporation | Microgel compositions and methods |
Non-Patent Citations (2)
| Title |
|---|
| Manuela Hammitzsch et al.Development and validation of a robust capillary electrophoresis method for impurity profiling of etomidate including the determination of chiral purity using a dual cyclodextrin system.《Electrophoresis》.2006,第27卷(第21期),4334-4344. * |
| 依托咪酯微乳注射液中依托咪酯及其依托咪酸的测定;宋景政 等;《药物分析杂志》;19981231;第18卷(第S1期);300-301 * |
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