CN104198609B - The method of the compound with pregnane mother nucleus structure is extracted from composition - Google Patents
The method of the compound with pregnane mother nucleus structure is extracted from composition Download PDFInfo
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Abstract
The invention belongs to technical field of analytical chemistry, be specifically related to extract the method for the compound with pregnane mother nucleus structure from composition. The method acetonitrile or acetonitrile solution are made basic solvent, and add inorganic salts to improve the Determination of oil-water partition coefficient of medicine, also separating out of the auxiliary material/matrix in reduction temperature acceleration composition, by component separation of not dissolving each other such as CENTRIFUGAL ACCELERATING profits. The method versatility is wide, this compounds effectively can be extracted and can not it be produced and be destroyed, the analyzing and testing that solvent used also can not interfere with subsequent; The solution of direct acquisition carries out high-efficient liquid phase analysis, and the collection of illustrative plates baseline that obtains is clean, peak shape is symmetrical, theoretical cam curve is high; Meanwhile, sample solution is also more stable, and more related substance can be detected; The quality control of the composition that contain adrenocortical hormone composition can be widely used in.
Description
Technical field
The invention belongs to technical field of analytical chemistry, be specifically related to extract and there is pregnane parent nucleus from compositionThe method of the compound of structure.
Background technology
Cortex hormone of aadrenaline is the steroid hormone that is synthesized by adrenal cortex and secreted, and has pregnane parent nucleus.Tens of kind sterols crystallizations can be extracted from adrenal cortex. This compounds major function is to regulate animalWater-electrolyte metabolism and glycometabolism in body, ubiquity in various vertebrates. About there are 30 kinds of adrenal cortexsHormone is isolated and identified, and wherein about 10 kinds have biologically active. Activity with cortisol is the strongest, has tuneJoint sugar, protein and lipometabolic function, can affecting glucose synthetic and utilize, the mobilization of fat andFlooding dose. At present, the application of cortex hormone of aadrenaline in medicine mainly comprises following a few class: weak effectHave hydrocortisone acetate; Middle effect have dexamethasone acetate (Compound Dexamethasone Acetate), butyric acid Clobetasol, acetic acidTriamcinolone acetonide (triamcinolone), butyric acid hydrocortisone (You Zhuoer), momestasone furoate (Eloson), acetic acidFluocinolone acetonide; Potent has Halometasone, beclomeasone propionate, chlorine flumethasone (Le-Fu-Ye bacteriostatic or happiness are happy); The most potentHave clobetasol propionate (dermovate or clobetasol or PIKANGWANG), sicorten see halometasone (sicorten or difficult to understand can ointment),Clobetasone butyrate (Mupirocin Ointment), BDP, etc. The derived structure signal of such medicine asUnder:
On market, these adrenal cortex hormones drugs are widely used as local application, are used for the treatment of skin disease,Therefore the composition that contains cortex hormone of aadrenaline composition in product is widely used. How fast from compositionMiddle separated adrenocortical hormone compound and derivative (degradation impurity) thereof, and carry out next step smoothlyTest, to the production of product and storage, and the sampling observation in market all has great importance.
But because adrenal cortex hormones drug is of a great variety, the auxiliary material that different pharmaceutical uses again not to the utmostIdentical, generally adopt different extracting methods for concrete cortex hormone of aadrenaline composition. Existing method is logicalThe property used difference, for the extraction of polytype medicine, just need to prepare different reagent, carry out different operations,Tester easily obscures, and extraction effect difference, the most serious problem be also can interfere with subsequent test.For example:
Clobetasone butyrate emulsifiable paste import registered standard (JX20070012), it chooses clobetasol propionate,Add ethanol and make inner mark solution. Take sample, put in tool plug conical flask, add inner mark solution 5ml and ethanol 15ml,In 98-100 DEG C of water-bath, heat, shake well makes emulsifiable paste dissolve completely, lets cool, put in ice bath cooling 1 hour withUpper, take out, centrifugal, cross leaching filtrate, obtain test solution. This extracting method adds in 98-100 DEG C of water-bathHot clobetasone butyrate emulsifiable paste, solvent volatilization is more, and complex operation is dangerous; Selection adds interior mark, noEconomical; Clobetasone butyrate, at 98-100 DEG C of heating water bath, can be hydrolyzed, and its catabolite is as shown in the formula instituteShow. In addition, general cortex hormone of aadrenaline compounds content and determination of related substances use HPLC to measure, secondAlcohol has larger solvent effect as Extraction solvent, measures peak shape poor.
Separately there is Halometasone Cream import registered standard (JX20010092), get Halometasone Cream and put beaker in right amountIn, add oxolane appropriate, be stirred to dissolve and shake up, put in ice bath and place 30 minutes, filter and get filtrate,Make test liquid. In this extracting method, the oxolane reagent of use is cyclic ethers, easily oxidation by air, producesRaw certain impurity, often adds BHT (2,6-di-t-butyl is to this phenol of first); In addition, oxolane is by sampleMatrix in product is all dissolved, not removed matrix during ice bath, and therefore, the chromatographic peak baseline obtaining is poor, dryDisturb large, peak shape is poor, and impurity also increases.
Separately there are Chinese Pharmacopoeia two version clobetasol propionate cream content determinations in 2010, add acetic acid fluorine lightPine is cooked interior mark, does solvent extraction measure content with methyl alcohol. The method also adds interior mark, complex operation, withoutJi, matrix is removed not thorough.
Not deep enough to the research of external application drug product related substance both at home and abroad a few years ago, existing adrenal gland skinIn the document of matter steroids quality analysis, do not carry about the related substance aspect of Aeroseb-DexThe document of getting. The concentration of determination of related substances is generally 10-100 times of assay, therefore determination of related substancesRequire tighter than assay, require baseline noise noiseless to impurity determination, just sample pre-treatments is proposedHigher requirement. In existing document, just the main ingredient in sample is dissolved, carry out assay, not tooTake notice of that the matrix of dissolving is on the impact of measuring. Therefore extract adrenocortical hormone when adopting existing assayThe method of composition, carries out the test of related substance after extracting its degradation product, test effect is undesirable, noise is many,Interference is many, causes test unstable, inaccurate.
For these reasons, the present invention explores a kind of for entering containing the composition of cortex hormone of aadrenaline compositionThe effective cortex hormone of aadrenaline constituents extraction of row, and the method that is beneficial to aftermentioned test.
Summary of the invention
In view of this, a kind of compound with pregnane mother nucleus structure that extracts from composition is the invention providesMethod, the method versatility is wide, this compounds effectively can be extracted and can not it be produced and be destroyed, instituteSolvent also can not interfere with subsequent analyzing and testing.
For achieving the above object, technical scheme of the present invention is:
From composition, extract a method for the compound with pregnane mother nucleus structure, use is added with inorganicThe organic solvent of salt or the aqueous solutions of organic solvent that is added with inorganic salts are that Extraction solvent extracts composition.Cortex hormone of aadrenaline is exactly the compound that a class has pregnane mother nucleus structure, and pregnane parent nucleus hasThe basic structure of cyclopentanoperhy drophenanthrene (structural formula is as follows), cyclopentanoperhy drophenanthrene structure is again steroid hormoneParent nucleus, therefore, the sex hormone with adrenal hormone and under belonging to steroid hormone (is subdivided into androgen, female sharpElement and progestational hormone), do not get rid of applicable method of the present invention yet and extract and detect.
Further, described organic solvent is acetonitrile.
Further, described inorganic salts are non-oxidizing inorganic salts, are specifically selected from but are not limited to following oneOr multiple: sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, sodium sulphate and potassium sulfate, preferablyPotassium chloride.
Further, the mass percent of described inorganic salts in Extraction solvent is 0.5%-5.0%, preferably 2.0%.
Further, described composition is pharmaceutical composition, contains but is not limited to following compound and its derivativeIn one or more: hydrocortisone acetate, dexamethasone acetate, butyric acid Clobetasol, acetic acid Qu AnNai De, butyric acid hydrocortisone, momestasone furoate, fluocinonide, beclomeasone propionate, dipropionic acid are doublyTa meter Song, chlorine flumethasone, clobetasol propionate, Halometasone, diflorasone diacetate, clobetasone butyrate.
Described derivative refers to that hydrogen atom or the atomic group in a kind of simple compounds got by other atoms or atomic groupGeneration and derivative more complicated product. Specifically in pharmaceutical composition, these derivatives comprise these compoundsCatabolite; During drug quality monitoring, these compounds and degradation product thereof need to be extracted simultaneously, with differenceObtain the test result of active ingredient and its related substances.
Further, the form of described pharmaceutical composition is gel or emulsifiable paste. Described pharmaceutical composition specifically can wrapContaining but be not limited to one or more in following auxiliary material: carbomer, hexadecanol and carboxylate thereof, octadecyl alcolol andThe carboxylate of its carboxylate, polyethylene glycol and stearic acid different saturation.
Further, described leaching process comprises the following steps: at 45-88 DEG C of temperature, preferably 60 DEG C, usesDescribed dissolution with solvents composition; After dissolution with solvents composition, then separate out matrix at-20-4 DEG C temperature, preferablyCarry out ice bath at 0 DEG C, Separation of Solid and Liquid obtains filtrate, namely obtains containing the solution of pregnane parent nucleus compound. InstituteThe mode of stating Separation of Solid and Liquid employing comprises filtration, centrifugal, standing clarification.
Embodiments of the invention confirm, use solvent system of the present invention that adrenal cortex effectively can be extracted and swashChlorins compound and degradation product thereof, and effectively remove auxiliary material/matrix in ointment etc., directly molten with acquisitionLiquid carries out the equal analyzing and testing of high-efficient liquid, and the collection of illustrative plates baseline that obtains is clean, peak shape is symmetrical, theoretical cam curve is high,Sample solution is also more stable simultaneously, and more related substance can be detected. Gel and ointment medicinal auxiliaryMaterial composition is similar, uses the inventive method also to have same good effect.
Thus, another object of the present invention is to provide in a kind of analysis pharmaceutical composition and has pregnane parent nucleus knotThe method of the compound of structure, the technical scheme of employing is: adopt above-mentioned extracting and have pregnant steroid from compositionThe method of the compound of alkane mother nucleus structure is carried out the extraction of compound, and the solution obtaining with extraction is as test sampleSolution is tested.
Certainly, in concrete test process, need difference be set according to different adrenocortical hormone typesTest parameter, also comprise test before the Conventional processing steps of need testing solution. For example, high-efficient liquid is being carried outBefore the test of phase chromatogram, the operations such as membrane filtration are carried out to the described need testing solution obtaining.
Particularly, the side of active ingredient and related substance in a kind of analytical test cortex hormone of aadrenaline ointmentMethod, comprises the following steps: get described ointment appropriate (being about equivalent to main ingredient composition 5-10mg); Add acetonitrile,Add inorganic salts and dissolve (being mixed with every 1ml about containing main ingredient composition 10-1000 μ g), 45-88 DEG C of water-bath makes dissolving;0.5-1 hour is placed in put again-20-4 DEG C ice bath; Take out, Separation of Solid and Liquid, obtains filtrate; Filtrate is again through microporeFilter, get subsequent filtrate as need testing solution; Enter liquid chromatogram and carry out cortex hormone of aadrenaline (main ingredient composition)Mensuration with related substance.
Further, when described pharmaceutical composition is Halometasone Cream, carry out relevant thing by following chromatographic conditionThe test of matter:
Chromatographic column: Shimpack VP-ODS, 250mm × 4.6mm, 5 μm;
UV detector: 254nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: A phase is 0.1% glacial acetic acid solution, B phase is acetonitrile, and according to the form below carries out gradient elution:
| Time, min | Mobile phase A, % | Mobile phase B, % |
| 0 | 65 | 35 |
| 22 | 65 | 35 |
| 45 | 30 | 70 |
| 60 | 30 | 70 |
| 61 | 65 | 35 |
| 66 | 65 | 35 |
Further, when described pharmaceutical composition is clobetasone butyrate emulsifiable paste, undertaken by following chromatographic conditionThe test of related substance:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μm;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Determined wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A phase of mobile phase is water, B phase is acetonitrile, and according to the form below carries out gradient elution:
| Time, min | Mobile phase A, % | Mobile phase B, % |
| 0 | 60 | 40 |
| 3 | 60 | 40 |
| 30 | 50 | 50 |
| 38 | 50 | 50 |
| 39 | 60 | 60 |
| 44 | 60 | 40 |
Further, when described pharmaceutical composition is betamethasone dipropionate cream, enter by following chromatographic conditionThe test of row related substance:
Chromatographic column: Agilent XDB-C18,250mm × 4.6mm, 5 μm;
UV detector: 240nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: 45% acetonitrile;
Further, when described pharmaceutical composition is clobetasol propionate cream, undertaken by following chromatographic conditionThe test of related substance:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μm;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Determined wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A phase of mobile phase is water, B phase is acetonitrile, and according to the form below carries out gradient elution:
| Time, min | Mobile phase A, % | Mobile phase B, % |
| 0 | 60 | 40 |
| 3 | 60 | 40 |
| 30 | 50 | 50 |
| 38 | 50 | 50 |
| 39 | 60 | 60 |
| 44 | 60 | 40 |
" % " is wherein percent by volume.
For extract the extract obtaining by the inventive method, except the method for above-mentioned employing liquid chromatogram is surveyedOutside examination, if be the compound of one-component after the present invention extracts, ultraviolet spectrometer just can be used to containQuantitative determination. Extract liquid of the present invention also can infrared scanner carries out the Qualitive test of material, also can be used for thin layerChromatographic scan qualitatively or quantitatively determines. High performance liquid chromatograph is not limit and is used UV-detector, evaporative light to fall apartLook device, mass detector etc.
The present invention also provides a kind of for extracting compound molten with pregnane mother nucleus structure from compositionAgent, this solvent can effectively extract this compounds, and the extraction solution of acquisition is also convenient to survey for follow-up analysisExamination, does not produce interference. The concrete technical scheme adopting is:
For extracting a solvent for the compound with pregnane mother nucleus structure, described solvent from compositionFor the organic solvent that is added with inorganic salts or the aqueous solutions of organic solvent that is added with inorganic salts; Described organic solvent is secondNitrile; Described inorganic salts are sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, sodium sulphate and sulfuric acidOne or more in potassium.
Further, the compound described in pregnane mother nucleus structure is in following compound and its derivativeOne or more: hydrocortisone acetate, dexamethasone acetate, butyric acid Clobetasol, triamcinolone acetonide acetate,Butyric acid hydrocortisone, momestasone furoate, fluocinonide, beclomeasone propionate, BDP,Chlorine flumethasone, clobetasol propionate, Halometasone, diflorasone diacetate and clobetasone butyrate.
Advantageous Effects of the present invention is:
The method of compound with pregnane mother nucleus structure of extracting from composition provided by the invention, selectsAcetonitrile or acetonitrile solution are made basic solvent, and add inorganic salts to improve the Determination of oil-water partition coefficient of medicine, also fallLow temperature accelerates separating out of auxiliary material/matrix in composition, is divided by components that do not dissolve each other such as CENTRIFUGAL ACCELERATING profitsFrom. The method versatility is wide, and adrenocortical hormone compound and degradation product thereof and not effectively can be extractedCan produce and destroy, auxiliary material/matrix in ointment etc. effectively can be removed, and solvent used also can not interfere with subsequentAnalyzing and testing; The solution of direct acquisition carries out high-efficient liquid phase analysis, and the collection of illustrative plates baseline that obtains is clean, peakShape symmetry, theoretical cam curve are high; Meanwhile, sample solution is also more stable, and more related substance can be detected.Therefore, the quality control of the composition that contain adrenocortical hormone composition can be widely used in.
Accompanying drawing explanation
Fig. 1 is that the related substance HPLC that measures Halometasone Cream in embodiment 1 schemes (in figure: 9.175minPeak is before matrix peak, and 58.159min is the antioxidant peak in matrix, and 19.292min is Halometasone, itsHis peak is the impurity peaks of Halometasone);
Fig. 2 is the HPLC figure that measures Halometasone Cream bare substrate in embodiment 1;
Fig. 3 is that the related substance HPLC that measures Halometasone Cream in comparative example 1 schemes (in figure: 11.478minPeak is before matrix peak, and 58.245min is the antioxidant peak in matrix, and 54.923min is Extraction solvent THFIn BHT, 19.259min is Halometasone, and other peaks are the impurity peaks of Halometasone);
Fig. 4 is the HPLC figure that measures Halometasone Cream bare substrate in comparative example 1;
Fig. 5 be in embodiment 2, measure clobetasone butyrate emulsifiable paste related substance HPLC figure (in figure:28.505min is clobetasone butyrate peak, and 9.964min is clobetasone peak, and 24.422min is related substance
1)；
Fig. 6 is the HPLC figure that measures clobetasone butyrate emulsifiable paste bare substrate in embodiment 2;
Fig. 7 is that the related substance HPLC that measures clobetasone butyrate emulsifiable paste in comparative example 2 schemes (in figure: 28.915For clobetasone butyrate peak, 10.572min is clobetasone (clobetasone butyrate hydrolysis produces), 30.938minFor 21-OH impurity (clobetasone butyrate hydrolysis produces));
Fig. 8 is the HPLC figure that measures clobetasone butyrate emulsifiable paste bare substrate in comparative example 2;
Fig. 9 be in embodiment 3, measure betamethasone dipropionate cream related substance HPLC figure (in figure:14.458min is BDP peak, and 5.249min is betamethasone 17-ester peak, and 9.421 is his rice doublyPine 21-ester peak);
Figure 10 is the HPLC figure that measures betamethasone dipropionate cream bare substrate in embodiment 3;
Figure 11 is the related substance HPLC figure that measures clobetasol propionate cream in embodiment 4;
Figure 12 is the HPLC figure of the blank extract of measuring clobetasol propionate cream in embodiment 4;
Figure 13 is that the HPLC that measures Halometasone Cream content in embodiment 5 schemes (in figure: 4.512min is 2-benzeneOxygen basis ethanol, 16.123min is Halometasone).
Detailed description of the invention
Referring to accompanying drawing, embodiments of the invention and comparative example are described in detail, wherein unreceipted concreteThe experimental technique of condition, carries out according to Chinese Pharmacopoeia or import registered standard.
Impurity for 0.05-1% in medicine during determination of related substances can quantitatively be detected, determination of related substancesConcentration is 10-100 times of assay concentration, therefore the concentration of related substance matrix is content substrate concentration10-100 is doubly, therefore use the extracting method of related substance applicable equally during assay.
The mensuration of embodiment 1 Halometasone Cream related substance
(1) chromatographic process
Chromatographic column: Shimpack VP-ODS (250mm × 4.6mm, 5 μm)
UV detector: 254nm
Column temperature: 25 DEG C
Flow velocity: 1.5ml/min
Sample size: 20 μ l
System suitability: the separating degree at Halometasone peak and other impurities peak should reach 1.5.
Mobile phase: A phase is 0.1% glacial acetic acid solution, B phase is acetonitrile, and according to the form below carries out gradient elution:
(2) extract and test
Get Halometasone Cream (Chongqing Huapont Pharmaceutical Co., Ltd. produces, specification 0.05%) content and be about 10g (aboutBe equivalent to Halometasone 5mg), put in tool plug conical flask, add acetonitrile 10ml and sodium chloride 0.2g, 60 DEG C of water-baths makeDissolve, put in ice-water bath and place 30 minutes, taking-up shakes up, and puts to room temperature, gets clear liquid and filters, and gets subsequent filtrate and doesFor need testing solution. Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds dilution in acetonitrile to carvingDegree, shakes up, in contrast solution. Get bare substrate 10g, according to need testing solution same treatment, as blankMatrix solution.
By above-mentioned chromatographic condition, get contrast solution 20 μ l injection liquid chromatography, the sensitivity of conditioning instrumentation, makesThe peak height at Halometasone peak is about the 20%-25% of full scale; Precision measures need testing solution and vehicle solution againEach 20 μ l, respectively injection liquid chromatography, record chromatogram.
As depicted in figs. 1 and 2, the related substance HPLC that Fig. 1 is need testing solution schemes result of the test, Fig. 2For the HPLC figure of vehicle solution. Comparison diagram 1 and Fig. 2 are known, and the Extraction solvent of Halometasone Cream is not dryDisturb the mensuration of Halometasone Cream related substance and blank auxiliary thereof, baseline is level and smooth.
In follow-up related substance Method validation, in bare substrate, add the about known impurities of 0.05%-3%Carry out recovery test with Halometasone, the rate of recovery is between 98.5%-103.2%. Result of the test shows, this extractionMethod is extracted completely Halometasone and impurity thereof, removes matrix thorough.
Comparative example 1 import registered standard (JX20010092) method is extracted Halometasone Cream and is carried out related substanceMeasure
Get appropriate (the about Halometasone of Halometasone Cream (Chongqing Huapont Pharmaceutical Co., Ltd. produces, specification 0.05%)About 5mg), put in conical flask, add oxolane 10ml, make paste dissolves, shake up. Put in ice bath and place 30Minute, filter, get subsequent filtrate and make test liquid. Get this solution injection liquid chromatography, by the chromatogram of embodiment 1Condition is measured, and record chromatogram, the results are shown in Figure 3. Separately get Halometasone Cream bare substrate 10g to grasp with methodDo, the results are shown in Figure 4.
In embodiment 1, Fig. 1 compares with comparative example 1 Fig. 3, and example 1 Fig. 1 and comparative example 1 Fig. 3 theoretical cam curve are respectivelyBe 15609 and 4603; Symmetrical factor is respectively 0.98 and 1.21; The theoretical cam curve of example 1 is higher, and peak shape moreSymmetry, baseline is better. In embodiment 1, Fig. 2 compares with comparative example 1 Fig. 4, and the blank baseline of embodiment 1 is level and smooth,Near free from admixture peak main peak.
Comparative example 1 proves, import registered standard (JX20010092) extracting method detects the energy of known impuritiesPower, is not so good as the inventive method antijamming capability strong.
The mensuration of embodiment 2 clobetasone butyrate emulsifiable paste related substance
(1) chromatographic process
Chromatographic column: YMC-Pack ODS-A 150mm × 4.6mm, 3.0 μm
Flow velocity: 1.5ml/min column temperature: 40 DEG C
Determined wavelength: 241nm
Sample size: 30 μ l
Mobile phase: the A phase of mobile phase is water, B phase is acetonitrile, and according to the form below carries out gradient elution:
(2) extract and test
Lucifuge operation, (Sino-America Tianjin Shike Pharmaceutical Co., Ltd. produces, specification to get clobetasone butyrate emulsifiable paste0.05%) content appropriate (being about equivalent to clobetasone butyrate 10mg), puts in tool plug conical flask, adds acetonitrile16ml and potassium chloride 0.2g, 60 DEG C of water-baths make dissolving, and put in ice bath and place 60 minutes, taking-up shakes up, thenBe placed in centrifuge, centrifugal 10 minutes of 4000rpm, puts to room temperature, gets clear liquid PTFE filter membrane and filters, getSubsequent filtrate is as need testing solution. Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds 40%Second eyeball is diluted to scale, shakes up, in contrast solution. Get bare substrate 20g, shine need testing solution with method placeReason, as vehicle solution.
By above-mentioned chromatographic condition, get contrast solution 30 μ l injection liquid chromatography, the sensitivity of conditioning instrumentation, makesThe peak height at clobetasone butyrate peak is about the 20%-25% of full scale; Precision measures need testing solution and blank base againThe each 30 μ l of matter solution, respectively injection liquid chromatography, record chromatogram.
As shown in Figure 5 and Figure 6, the related substance HPLC that Fig. 5 is need testing solution schemes result of the test, Fig. 6For the HPLC figure of vehicle solution. Comparison diagram 5 and Fig. 6 are known, and the extraction of clobetasone butyrate emulsifiable paste is moltenThe mensuration of clobetasone butyrate emulsifiable paste related substance and blank auxiliary thereof is not disturbed in agent, and baseline is level and smooth.
Respectively 10mg about of the impurity 1, impurity 2, impurity 3, impurity 4 of getting clobetasone butyrate, uses 40% acetonitrileSolution preparation becomes storing solution, then gets respectively storing solution 0.25ml, 1.0ml, 1.5ml and join in sample, by realExample 2 operates, and its rate of recovery all reaches 90% ~ 110%.
By sample continuous sample introduction 10 pin, the impurity RSD of related substance reaches 3.1%, and baseline is without the unknown of comparative exampleThe baseline of epirelief, illustrates that extracting method of the present invention is extracted sample complete, removes matrix thorough.
It is relevant that comparative example 2 import registered standard (JX20070012) method is extracted clobetasone butyrate emulsifiable pasteSubstance-measuring
Lucifuge operation, (Sino-America Tianjin Shike Pharmaceutical Co., Ltd. produces, specification to get clobetasone butyrate emulsifiable paste0.05%) 20g, put in tool plug conical flask, precision adds clobetasol propionate inner mark solution 10ml and ethanol 30ml,Jam-pack bottle stopper, put 98 DEG C of water-baths with upper heating, shake well makes emulsifiable paste dissolve completely, lets cool, and puts ice bathMiddle cooling more than 1 hour, takes out, centrifugal, gets supernatant 30 μ l injection liquid chromatography, by embodiment's 2Chromatographic condition, is measured in the same method, and record chromatogram, obtains Fig. 7. Separately get the bare substrate of clobetasone butyrate emulsifiable paste20g operates with method, the results are shown in Figure 8.
Embodiment 2 is compared with comparative example 2: embodiment 2 adds solvent acetonitrile, just can with 60 DEG C of heating water bathsWith sample dissolution; Comparative example 2 adds ethanol then to need about 95 DEG C ability sample dissolution, and security is poor,Clobetasone butyrate is in ethanolic solution, and under 98 DEG C of high temperature, hydrolysis produces two impurity such as clobetasone, surelyQualitative difference. In embodiment 2, add inorganic salts, by centrifugal, phenomenon is that obvious solid-liquid is two-layer, gets moltenLiquid layer is easy to filter; And comparative example 2, become the pasty state of homogeneous after cooling, be not easy to filter, what obtain is moltenLiquid is little, operates very loaded down with trivial details. The solvent acetonitrile that the present invention uses is more conventional in chromatography, owing to not havingHave comparative example 2 solvent effect, peak shape is better. Method of the present invention is than comparative example import registered standard(JX20070012) method is more convenient, more convenient, and chromatographic peak profile is better, and sample solution is more stable, measuresMore accurate.
Comparison diagram 6 and Fig. 8, the blank baseline of embodiment 2 is smoothly more a lot of than the blank baseline of embodiment 2, noThe mensuration of related substance can be disturbed.
The mensuration of embodiment 3 betamethasone dipropionate cream related substance
(1) chromatographic process
Chromatographic column: Agilent XDB-C18 (250mm × 4.6mm, 5 μm)
UV detector: 240nm
Column temperature: 25 DEG C
Flow velocity: 1.5ml/min
Sample size: 20 μ l
Mobile phase: 45% acetonitrile
(2) extract and test
Content about to get betamethasone dipropionate cream (Chongqing Huapont Pharmaceutical Co., Ltd. produces, specification 0.1%)10g (being about equivalent to BDP 10mg), puts in tool plug conical flask, adds acetonitrile 10ml and sodium chloride0.2g, 60 DEG C of water-baths make dissolving, and put in ice-water bath and place 30 minutes, taking-up shakes up, and puts to room temperature, gets clear liquidFilter, get subsequent filtrate as need testing solution. Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle,Add dilution in acetonitrile to scale, shake up, in contrast solution. Get bare substrate 10g, according to the same method of need testing solutionProcess, as vehicle solution.
By above-mentioned chromatographic condition, get contrast solution 20 μ l injection liquid chromatography, the sensitivity of conditioning instrumentation, makesThe peak height at BDP peak is about the 20%-25% of full scale; Precision measures need testing solution with blank againThe each 20 μ l of matrix solution, respectively injection liquid chromatography, record chromatogram.
As shown in Figure 9 and Figure 10, the related substance HPLC figure that Figure 10 is need testing solution, schemes result of the test9 is the HPLC figure of vehicle solution. Comparison diagram 9 and Figure 10 are known, use betamethasone dipropionate creamThe blank auxiliary extracted of Extraction solvent do not disturb the mensuration of betamethasone dipropionate cream related substance, Fig. 9 andFigure 10 baseline is level and smooth.
The mensuration of embodiment 4 clobetasol propionate cream related substance
(1) chromatographic process
Chromatographic column: YMC-Pack ODS-A 150mm × 4.6mm, 3.0 μm
Flow velocity: 1.5ml/min
Column temperature: 40 DEG C
Determined wavelength: 241nm
Sample size: 30 μ l
Mobile phase: the A phase of mobile phase is water, B phase is acetonitrile, and according to the form below carries out gradient elution:
(2) extract and test
Lucifuge operation, (Shanghai General Pharmaceutical Co., ltd. produces, specification to get clobetasol propionate cream0.02%) content appropriate (being about equivalent to clobetasone butyrate 5mg), puts in tool plug conical flask, adds acetonitrile 16mlWith potassium chloride 0.2g, 60 DEG C of water-baths make dissolving, and put in ice bath and to place 60 minutes, taking-up shakes up, be then placed in fromIn scheming, centrifugal 10 minutes of 4000rpm, puts to room temperature, gets clear liquid PTFE filter membrane and filters, get subsequent filtrateAs need testing solution. Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds 40% second eyeball rareRelease to scale, shake up, in contrast solution. Get blank solution, according to need testing solution same treatment, as skyWhite solution.
By above-mentioned chromatographic condition, get contrast solution 30 μ l injection liquid chromatography, the sensitivity of conditioning instrumentation, makesThe peak height at clobetasol propionate peak is about the 20%-25% of full scale; Precision measures need testing solution with blank molten againThe each 30 μ l of liquid, respectively injection liquid chromatography, record chromatogram.
As is illustrated by figs. 11 and 12, the related substance HPLC that Figure 11 is need testing solution schemes result of the test,Figure 12 is the HPLC figure of blank solution. Contrast Figure 11 and Figure 12 figure is known, uses this Extraction solvent to extractDo not disturb the mensuration of clobetasol propionate cream related substance, baseline is level and smooth, removes matrix thorough.
The mensuration of embodiment 5 Halometasone Cream content
(1) chromatographic process
Chromatographic column: Shimadzu VP ODS-C18 (250mm × 4.6mm, 5 μm)
Flow velocity: 1.5ml/min
Column temperature: 25 DEG C
Determined wavelength: 254nm
Sample size: 20 μ l
Mobile phase: 0.1% glacial acetic acid solution-acetonitrile (65:35)
System suitability: measure reference substance solution 20 μ l, injection liquid chromatography, record chromatogram; Press peak streamGo out order and be followed successively by 2-phenoxetol and Halometasone; Number of theoretical plate calculates 5000 should be not less than by Halometasone peak;The separating degree at Halometasone peak and 2-phenoxetol peak should meet the requirements.
(2) extract and test
Get Halometasone Cream (Australia's energy, the U.S. pharmaceutical manufacturing of Hong Kong Australia, specification 0.05%) content and be about 2.0g (aboutBe equivalent to Halometasone 1mg), accurately weighed, put in 50ml volumetric flask, add acetonitrile 20ml and magnesium chloride 0.5g,60 DEG C of water-baths make Halometasone dissolve, and cool to room temperature, shakes up to scale by 50% dilution in acetonitrile; Put in ice bath and putPut 30 minutes, take out and put to room temperature, filter, get subsequent filtrate as test sample liquid. Separately get 2-phenoxetol pair200mg is about and Halometasone reference substance is about 10mg according to product, accurately weighed, put in 50ml measuring bottle, add acetonitrile and dissolveAnd be diluted to scale, shake up. Precision measures 5ml, puts in 50ml measuring bottle, adds 50% dilution in acetonitrile to scale,Shake up. Precision measures the each 20 μ l of above-mentioned two kinds of solution, respectively injection liquid chromatography, and record chromatogram, outside pressingMark method, with calculated by peak area, to obtain final product.
Result of the test is as Figure 13, and the content HPLC of need testing solution schemes, Halometasone peak theoretical cam curve 8700,Halometasone peak symmetrical factor is 1.01, and baseline is level and smooth.
During follow-up content method is verified, recovery test, the rate of recovery is between 99.5%-101.2%; SampleThe quantitative limit that joins matrix reach 8.7 × 10-8G/ml. Result of the test shows, this extracting method to Halometasone andIts impurity extracts completely, removes matrix thorough.
From above-described embodiment and comparative example, the inventive method is specially adapted to paste body shape adrenal cortex and swashsExtraction before the drug test of element class, to the derivative of cortex hormone of aadrenaline composition, and the equal energy of degradation impurityEffectively extract, compared with prior art, improve separative efficiency, more convenient, chromatographic peak profile is better, without dryDisturb, sample solution is more stable. In preparation is produced, energy effective control for product quality, safe and effective, more economical.Significant progressive is there is compared with import registered standard (JX20070012, JX20010092 etc.).
Finally illustrate, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, althoughWith reference to preferred embodiment, to invention has been detailed description, those of ordinary skill in the art should be appreciated thatCan modify to technical scheme of the present invention or be equal to replacement, and not depart from technical solution of the present inventionAim and scope, it all should be encompassed in the middle of right of the present invention.
Claims (2)
1. analyze a method for related substance in pharmaceutical composition, it is characterized in that: use and be added with inorganic saltsOrganic solvent or the aqueous solutions of organic solvent that is added with inorganic salts be that Extraction solvent extracts composition;Described organic solvent is acetonitrile; Described inorganic salts are sodium chloride, potassium chloride, magnesium chloride, sodium acetate, secondOne or more in acid potassium, sodium sulphate and potassium sulfate; The quality hundred of described inorganic salts in Extraction solventMark is 0.5%-5.0%; Extract with said method the solution testing that obtains and analyze relevant in pharmaceutical compositionMaterial, described test adopts high performance liquid chromatography to carry out; Described pharmaceutical composition be Halometasone Cream orClobetasone butyrate emulsifiable paste or betamethasone dipropionate cream or clobetasol propionate cream;
When described pharmaceutical composition is Halometasone Cream, carry out the survey of related substance by following chromatographic conditionExamination:
Chromatographic column: Shimpack VP-ODS, 250mm × 4.6mm, 5 μm;
UV detector: 254nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: A phase is 0.1% glacial acetic acid solution, B phase is acetonitrile, and according to the form below carries out gradient elution:
When described pharmaceutical composition is clobetasone butyrate emulsifiable paste, carry out relevant thing by following chromatographic conditionThe test of matter:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μm;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Determined wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A phase of mobile phase is water, B phase is acetonitrile, and according to the form below carries out gradient elution:
When described pharmaceutical composition is betamethasone dipropionate cream, carry out relevant thing by following chromatographic conditionThe test of matter:
Chromatographic column: Agilent XDB-C18,250mm × 4.6mm, 5 μm;
UV detector: 240nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: 45% acetonitrile;
When described pharmaceutical composition is clobetasol propionate cream, carry out related substance by following chromatographic conditionTest:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μm;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Determined wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A phase of mobile phase is water, B phase is acetonitrile, and according to the form below carries out gradient elution:
" % " is wherein percent by volume.
2. the method for related substance in analysis pharmaceutical composition according to claim 1, is characterized in that,The process of described extraction comprises the following steps: at 45-88 DEG C of temperature, dissolves combine with described Extraction solventThing; After dissolution with solvents composition, then separate out matrix at-20-4 DEG C temperature, Separation of Solid and Liquid obtains filtrate.
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| CN105651900A (en) * | 2016-04-08 | 2016-06-08 | 重庆华邦制药有限公司 | Method for separating and measuring process impurities in Clobetasone butyrate and preparation of Clobetasone butyrate |
| CN108226340B (en) * | 2017-12-29 | 2020-08-11 | 重庆华邦制药有限公司 | Method for separating and measuring diflucortolone and 6 beta diflucortolone and 16 beta diflucortolone thereof |
| CN108445123B (en) * | 2018-03-20 | 2020-06-23 | 广西壮族自治区食品药品检验所 | HPLC detection method for related substances in triamcinolone acetonide emulsifiable paste |
| CN114720570B (en) * | 2020-12-22 | 2023-08-29 | 上海市环境科学研究院 | Method for detecting 8 estrogens in fish meat |
| CN113203807A (en) * | 2021-04-25 | 2021-08-03 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Detection method of mometasone furoate cream related substances |
| CN113109488A (en) * | 2021-05-18 | 2021-07-13 | 广州白云山医药集团股份有限公司白云山何济公制药厂 | Method for extracting and detecting mometasone furoate in mometasone furoate gel |
| CN114184716B (en) * | 2021-11-18 | 2024-01-05 | 江苏吉贝尔药业股份有限公司 | High performance liquid chromatography analysis method for determining related substance components in halominosone cream |
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