CN104198609A - Method for extracting compounds with pregnane mother nucleus structure from compositions - Google Patents
Method for extracting compounds with pregnane mother nucleus structure from compositions Download PDFInfo
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Abstract
The invention particularly relates to a method for extracting compounds with a pregnane mother nucleus structure from compositions, belonging to the technical field of analytical chemistry. According to the method, inorganic salt is added into acetonitrile or an acetonitrile water solution which serves as a basic solvent so as to improve oil-water partition coefficients, the temperature is lowered to accelerate separation of auxiliary materials/matrixes in the compositions, and the separation of immiscible components such as oil and water is accelerated by centrifugation. The method has the beneficial effects that the universality is wide, the compounds can be effectively extracted without being damaged, and the subsequent analysis and detection cannot be interfered by the used solvent; the obtained solution can be directly utilized for carrying out high performance liquid phase analysis, and an obtained atlas has clean base lines, symmetric peak shapes and high number of theoretical plates; meanwhile, a sample solution is relatively stable, and more related substances can be detected; the method can be widely applied to the quality control of the compositions containing adrenocortical hormone components.
Description
Technical field
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.
Background technology
Cortex hormone of aadrenaline is the steroid hormone that is synthesized and secreted by adrenal cortex, has pregnane parent nucleus.From adrenal cortex, can extract tens of kinds of sterols crystallizations.This compounds major function is water-electrolyte metabolism and the glycometabolism regulating in animal body, ubiquity in various vertebrates.Approximately have 30 kinds of cortex hormone of aadrenaline to be isolated and identified, wherein approximately 10 kinds have biologically active.Activity with cortisol is the strongest, has the sugar of adjusting, protein and lipometabolic function, can affecting glucose synthetic and utilize, mobilization and the protein of fat is synthetic.At present, the application of cortex hormone of aadrenaline in medicine mainly comprises following a few class: weak effect have a hydrocortisone acetate; Middle effect have dexamethasone acetate (Compound Dexamethasone Acetate), butyric acid Clobetasol, triamcinolone acetonide acetate (triamcinolone), butyric acid hydrocortisone (You Zhuoer), momestasone furoate (Eloson), a fluocinonide; Potent have Halometasone, beclomeasone propionate, a chlorine flumethasone (Le-Fu-Ye bacteriostatic or happiness are happy); The most potent have clobetasol propionate (dermovate or clobetasol or PIKANGWANG), sicorten see halometasone (sicorten or difficult to understand can ointment), clobetasone butyrate (Mupirocin Ointment), a BDP, etc.The derived structure of such medicine is schematically as follows:
On market, these adrenal cortex hormones drugs, extensively as local application, are used for the treatment of skin disease, and the composition that therefore contains cortex hormone of aadrenaline composition in product is widely used.How from composition, to separate fast adrenocortical hormone compound and derivant thereof (degradation impurity), and carry out smoothly next step test, the production to 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 is not quite similar again, generally adopts different extracting method for concrete cortex hormone of aadrenaline composition.Existing method versatility is poor, for the extraction of polytype medicine, just need to prepare different reagent, carries out different operations, and tester easily obscures, and extraction effect is poor, 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, adds ethanol and makes 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 dissolves emulsifiable paste completely, lets cool, and puts in ice bath coolingly more than 1 hour, takes out, centrifugal, crosses leaching filtrate, obtains test solution.This extracting method is in 98-100 DEG C of heating water bath clobetasone butyrate emulsifiable paste, and solvent evaporates is more, and complex operation is dangerous; Selection adds interior mark, uneconomical; Clobetasone butyrate, at 98-100 DEG C of heating water bath, can be hydrolyzed, and its catabolite is shown below.In addition, general cortex hormone of aadrenaline compounds content and determination of related substances are used HPLC to measure, and ethanol is made to extract solvent and had larger solvent effect, measures peak shape poor.
Separately there is Halometasone Cream import registered standard (JX20010092), get Halometasone Cream and put in right amount in beaker, add tetrahydrofuran 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 tetrahydrofuran reagent of use is cyclic ethers, and easily oxidation by air, produces certain impurity, often adds BHT (2,6-di-t-butyl is to this phenol of first); In addition, tetrahydrofuran all dissolves the matrix in sample, not removed matrix when ice bath, and therefore, the chromatographic peak baseline obtaining is poor, disturbs greatly, and peak shape is poor, and impurity also increases.
Separately there are two version clobetasol propionate cream content determinations in 2010 of Chinese Pharmacopoeia, add Fluocinonide to do interior mark, do solvent extraction with methyl alcohol and measure content.The method also adds interior mark, and complex operation is uneconomical, and 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, in the document of existing adrenocortical hormone quality analysis, the document not extracting about the related substance aspect of Aeroseb-Dex.The concentration of determination of related substances is generally 10-100 times of assay, therefore determination of related substances is tighter than assay requirement, requires baseline noise noiseless to impurity determination, just sample pre-treatments is had higher requirement.In existing document, just the main ingredient in sample is dissolved, carry out assay, not too take notice of that the matrix of dissolving is on the impact of measuring.Therefore extract the method for adrenocortical hormone composition while adopting existing assay, extract the test of carrying out related substance after its degradation product, test effect is undesirable, and noise is many, interference is many, causes testing unstable, inaccurate.
For these reasons, the present invention has explored a kind of for carry out effective cortex hormone of aadrenaline composition extraction containing the composition of cortex hormone of aadrenaline composition, and is beneficial to the method for aftermentioned test.
Summary of the invention
In view of this, the invention provides a kind of method of extracting the compound with pregnane mother nucleus structure from composition, the method versatility is wide, can effectively extract this compounds and can it not produced and be destroyed, the analyzing and testing that solvent used can interfere with subsequent yet.
For achieving the above object, technical scheme of the present invention is:
From composition, extract the method for compound with pregnane mother nucleus structure, use the aqueous solutions of organic solvent that is added with the organic solvent of inorganic salts or is added with inorganic salts for extracting solvent, composition to be extracted.Cortex hormone of aadrenaline is exactly the compound that a class has pregnane mother nucleus structure, and pregnane parent nucleus has the basic structure of cyclopentanoperhy drophenanthrene (structural formula is as follows), cyclopentanoperhy drophenanthrene structure is again the parent nucleus of steroid hormone, therefore, with adrenal hormone and belong to the sex hormone (being subdivided into androgen, estrogen and progestational hormone) under steroid hormone, do not get rid of yet and be suitable for method of the present invention 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 one or more: sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, sodium sulphate and potassium sulfate, preferably potassium chloride.
Further, described inorganic salts are 0.5%-5.0% at the mass percent extracting in solvent, preferably 2.0%.
Further, described composition is pharmaceutical composition, contains but is not limited to one or more in following compound and its derivant: 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, clobetasone butyrate.
Described derivant refers to that hydrogen atom or the atomic group in a kind of simple compounds replaced and derivative more complicated product by other atoms or atomic group.Specifically, in pharmaceutical composition, these derivants comprise the catabolite of these compounds; When drug quality monitoring, need to extract these compounds and degradation product thereof, to obtain respectively the test result of effective constituent and its related substances simultaneously.
Further, the form of described pharmaceutical composition is gel or emulsifiable paste.Described pharmaceutical composition specifically can be including but not limited to one or more in following auxiliary material: the carboxylate of carbomer, hexadecanol and carboxylate thereof, 18 alcohol and carboxylate thereof, polyglycol and stearic acid different saturation.
Further, described leaching process comprises the following steps: at 45-88 DEG C of temperature, and preferably 60 DEG C, with described dissolution with solvents composition; After dissolution with solvents composition, then separate out matrix at-20-4 DEG C temperature, preferably carry out ice bath at 0 DEG C, Separation of Solid and Liquid obtains filtrate, obtains the solution that contains pregnane parent nucleus compound.The mode that described Separation of Solid and Liquid adopts comprise filtrations, centrifugal, leave standstill and clarify etc.
Embodiments of the invention confirm, use solvent system of the present invention can effectively extract adrenocortical hormone compound and degradation product thereof, and effectively remove auxiliary material/matrix in ointment etc., directly carry out the analyzing and testing such as high efficiency liquid phase with the solution obtaining, 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, can detect more related substance.The pharmaceutic adjuvant composition of gel and ointment is similar, uses the inventive method also to have same good effect.
Thus, another object of the present invention is to provide a kind of method in pharmaceutical composition with the compound of pregnane mother nucleus structure of analyzing, the technical scheme adopting is: adopt the above-mentioned method of extracting the compound with pregnane mother nucleus structure from composition to carry out the extraction of compound, test as need testing solution with extracting the solution obtaining.
Certainly, in concrete test process, need different test parameters be set according to different adrenocortical hormone types, also comprise the conventional processing step of the front need testing solution of test.For example, carrying out before high performance liquid chromatography test, the described need testing solution obtaining is carried out to the operations such as membrane filtration.
Particularly, a kind of method of effective constituent and related substance in analytical test cortex hormone of aadrenaline ointment, comprises the following steps: get described ointment appropriate (being approximately equivalent to main ingredient composition 5-10mg); Add acetonitrile, add inorganic salts and dissolve that (be mixed with every 1ml approximately containing main ingredient composition 10-1000 μ g), 45-88 DEG C of water-bath makes to dissolve; In put again-20-4 DEG C ice bath, place 0.5-1 hour; Take out, Separation of Solid and Liquid, obtains filtrate; Filtrate, again through micro porous filtration, is got subsequent filtrate as need testing solution; Enter liquid chromatography and carry out the mensuration of cortex hormone of aadrenaline (main ingredient composition) and related substance.
Further, in the time that described pharmaceutical composition is Halometasone Cream, carry out the test of related substance by following chromatographic condition:
Chromatographic column: Shimpack VP-ODS, 250mm × 4.6mm, 5 μ m;
UV detecting device: 254nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: A is 0.1% glacial acetic acid solution mutually, and B is acetonitrile mutually, 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, in the time that described pharmaceutical composition is clobetasone butyrate emulsifiable paste, carry out the test of related substance by following chromatographic condition:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μ m;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Detect wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A of mobile phase is water mutually, and B is acetonitrile mutually, 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, in the time that described pharmaceutical composition is betamethasone dipropionate cream, carry out the test of related substance by following chromatographic condition:
Chromatographic column: Agilent XDB-C18,250mm × 4.6mm, 5 μ m;
UV detecting device: 240nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: 45% acetonitrile;
Further, in the time that described pharmaceutical composition is clobetasol propionate cream, carry out the test of related substance by following chromatographic condition:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μ m;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Detect wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A of mobile phase is water mutually, and B is acetonitrile mutually, 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 chromatography is tested, if be the compound of one-component after the present invention extracts, just can use ultraviolet spectrometer (UVS) to carry out assay.Extract liquid of the present invention also can carry out the qualitative discriminating of material by infrared scanner, also can be used for tlc scanning and qualitatively or quantitatively determines.High performance liquid chromatograph is not limit and is used UV-detector, the loose look device of evaporative light, mass detector etc.
The present invention also provides that a kind of this solvent can effectively extract this compounds for extract the solvent of compound with pregnane mother nucleus structure from composition, and the extraction solution of acquisition is also convenient to, for follow-up analytical test, not produce interference.The concrete technical scheme adopting is:
For extract a solvent for the compound with pregnane mother nucleus structure from composition, described solvent is the aqueous solutions of organic solvent that is added with the organic solvent of inorganic salts or is added with inorganic salts; Described organic solvent is acetonitrile; Described inorganic salts are one or more in sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, sodium sulphate and potassium sulfate.
The compound further, with pregnane mother nucleus structure is one or more in following compound and its derivant: 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.
Useful technique effect of the present invention is:
The method of extracting the compound with pregnane mother nucleus structure from composition provided by the invention, select acetonitrile or acetonitrile solution to make basic solvent, and add inorganic salts to improve the Determination of oil-water partition coefficient of medicine, also reduce temperature and accelerate separating out of auxiliary material/matrix in composition, separate by the component that do not dissolve each other such as centrifugation accelerates profit.The method versatility is wide, can effectively extract adrenocortical hormone compound and degradation product thereof and can not produce destruction, can effectively remove auxiliary material/matrix in ointment etc., the analyzing and testing that solvent used can interfere with subsequent yet; Directly carry out high-efficient liquid phase analysis with the solution of acquisition, 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, can detect more related substance.Therefore, can be widely used in the quality control of the composition that contains adrenocortical hormone composition.
Brief description of the drawings
Fig. 1 is the related substance HPLC figure (in figure: the peak before 9.175min is matrix peak, and 58.159min is the antioxidant peak in matrix, and 19.292min is Halometasone, and other peaks are the impurity peaks of Halometasone) that measures Halometasone Cream in embodiment 1;
Fig. 2 is the HPLC figure that measures the blank matrix of Halometasone Cream in embodiment 1;
Fig. 3 is that the related substance HPLC that measures Halometasone Cream in comparative example 1 schemes (in figure: the peak before 11.478min is matrix peak, 58.245min is the antioxidant peak in matrix, 54.923min is the BHT extracting in solvent THF, 19.259min is Halometasone, and other peaks are the impurity peaks of Halometasone);
Fig. 4 is the HPLC figure that measures the blank matrix of Halometasone Cream in comparative example 1;
Fig. 5 is the related substance HPLC figure (in figure: 28.505min is clobetasone butyrate peak, 9.964min is clobetasone peak, and 24.422min is related substance 1) that measures clobetasone butyrate emulsifiable paste in embodiment 2;
Fig. 6 is the HPLC figure that measures the blank matrix of clobetasone butyrate emulsifiable paste in embodiment 2;
Fig. 7 is that the related substance HPLC that measures clobetasone butyrate emulsifiable paste in comparative example 2 schemes (in figure: 28.915 is clobetasone butyrate peak, 10.572min is clobetasone (clobetasone butyrate hydrolysis produces), and 30.938min is 21-OH impurity (clobetasone butyrate hydrolysis produces));
Fig. 8 is the HPLC figure that measures the blank matrix of clobetasone butyrate emulsifiable paste in comparative example 2;
Fig. 9 is the related substance HPLC figure (in figure: 14.458min is BDP peak, 5.249min is betamethasone 17-ester peak, and 9.421 is betamethasone 21-ester peak) that measures betamethasone dipropionate cream in embodiment 3;
Figure 10 is the HPLC figure that measures the blank matrix of betamethasone dipropionate cream 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 that measures the blank extract of clobetasol propionate cream in embodiment 4;
Figure 13 is the HPLC figure (in figure: 4.512min is 2-phenoxy group plinth ethanol, and 16.123min is Halometasone) that measures Halometasone Cream content in embodiment 5.
Embodiment
Referring to accompanying drawing, embodiments of the invention and comparative example are described in detail, the wherein experimental technique of unreceipted actual conditions, carries out according to Chinese Pharmacopoeia or import registered standard.
Impurity for 0.05-1% in medicine when determination of related substances can quantitatively be detected, determination of related substances concentration is 10-100 times of assay concentration, therefore the concentration of related substance matrix be content substrate concentration 10-100 doubly, therefore use the extracting method of related substance applicable equally when assay.
The mensuration of embodiment 1 Halometasone Cream related substance
(1) chromatographic process
Chromatographic column: (250mm × 4.6mm, 5 μ m) for Shimpack VP-ODS
UV detecting device: 254nm
Column temperature: 25 DEG C
Flow velocity: 1.5ml/min
Sample size: 20 μ l
System flexibility: the degree of separation of Halometasone peak and adjacent impurity peaks should reach 1.5.
Mobile phase: A is 0.1% glacial acetic acid solution mutually, and B is acetonitrile mutually, and according to the form below carries out gradient elution:
(2) extract and test
(Chongqing Huapont Pharmaceutical Co., Ltd. produces to get Halometasone Cream, specification 0.05%) the about 10g of content (being approximately equivalent to Halometasone 5mg), put in tool plug conical flask, add acetonitrile 10ml and sodium chloride 0.2g, 60 DEG C of water-baths make to dissolve, and put in ice-water bath and place 30 minutes, taking-up shakes up, put to room temperature, get clear liquid and filter, get subsequent filtrate as need testing solution.Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds dilution in acetonitrile to scale, shakes up, in contrast solution.Get blank matrix 10g, according to need testing solution same treatment, as vehicle solution.
By above-mentioned chromatographic condition, get contrast solution 20 μ l injection liquid chromatographies, regulate the sensitivity of instrument, make the peak height at Halometasone peak be about the 20%-25% of full scale; Precision measures need testing solution and the each 20 μ l of vehicle solution again, and injection liquid chromatography, records chromatogram respectively.
As depicted in figs. 1 and 2, the related substance HPLC that Fig. 1 is need testing solution schemes test findings, the HPLC figure that Fig. 2 is vehicle solution.Comparison diagram 1 and Fig. 2 are known, and the extraction solvent of Halometasone Cream does not disturb the mensuration of Halometasone Cream related substance and blank auxiliary material thereof, and baseline is level and smooth.
In follow-up related substance methodology checking, add the known impurities of about 0.05%-3% and Halometasone to carry out recovery test in blank matrix, the recovery is between 98.5%-103.2%.Test findings shows, this extracting method extracts completely Halometasone and impurity thereof, removes matrix thorough.
Comparative example 1 import registered standard (JX20010092) method is extracted Halometasone Cream and is carried out determination of related substances
Get Halometasone Cream (Chongqing Huapont Pharmaceutical Co., Ltd. produces, specification 0.05%) appropriate (the about 5mg of about Halometasone), put in conical flask, add tetrahydrofuran 10ml, make paste dissolves, shake up.Put in ice bath and place 30 minutes, filter, get subsequent filtrate and make test liquid.Get this solution injection liquid chromatography, measure by the chromatographic condition of embodiment 1, record chromatogram, the results are shown in Figure 3.Separately get the blank matrix 10g of Halometasone Cream and operate with method, 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 respectively 15609 and 4603; Symmetrical factor is respectively 0.98 and 1.21; The theoretical cam curve of example 1 is higher, and peak shape is more symmetrical, and 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 inclusion-free peak main peak.
Comparative example 1 proves, import registered standard (JX20010092) extracting method detects the ability of known impurities, not as the inventive method antijamming capability strong.
The mensuration of embodiment 2 clobetasone butyrate emulsifiable paste related substances
(1) chromatographic process
Chromatographic column: YMC-Pack ODS-A 150mm × 4.6mm, 3.0 μ m
Flow velocity: 1.5ml/min column temperature: 40 DEG C
Detect wavelength: 241nm
Sample size: 30 μ l
Mobile phase: the A of mobile phase is water mutually, and B is acetonitrile mutually, and according to the form below carries out gradient elution:
(2) extract and test
Lucifuge operation, gets clobetasone butyrate emulsifiable paste (Sino-America Tianjin Shike Pharmaceutical Co., Ltd. produces, specification 0.05%) content appropriate (being approximately equivalent to clobetasone butyrate 10mg), put in tool plug conical flask, add acetonitrile 16ml and potassium chloride 0.2g, 60 DEG C of water-baths make to dissolve, and put in ice bath and place 60 minutes, taking-up shakes up, then be placed in hydro-extractor, centrifugal 10 minutes of 4000rpm, puts to room temperature, get clear liquid PTFE filter membrane and filter, get subsequent filtrate as need testing solution.Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds 40% second eyeball and is diluted to scale, shakes up, in contrast solution.Get blank matrix 20g, according to need testing solution same treatment, as vehicle solution.
By above-mentioned chromatographic condition, get contrast solution 30 μ l injection liquid chromatographies, regulate the sensitivity of instrument, make the peak height at clobetasone butyrate peak be about the 20%-25% of full scale; Precision measures need testing solution and the each 30 μ l of vehicle solution again, and injection liquid chromatography, records chromatogram respectively.
As shown in Figure 5 and Figure 6, the related substance HPLC that Fig. 5 is need testing solution schemes test findings, the HPLC figure that Fig. 6 is vehicle solution.Comparison diagram 5 and Fig. 6 are known, and the extraction solvent of clobetasone butyrate emulsifiable paste does not disturb the mensuration of clobetasone butyrate emulsifiable paste related substance and blank auxiliary material thereof, and baseline is level and smooth.
Get the each about 10mg of impurity 1, impurity 2, impurity 3, impurity 4 of clobetasone butyrate, be mixed with storing solution with 40% acetonitrile solution, get respectively storing solution 0.25ml, 1.0ml again, 1.5ml joins in sample, operates by example 2, its recovery all reaches 90%～110%.
By sample continuous sample introduction 10 pins, the impurity RSD of related substance reaches 3.1%, and baseline, without the baseline of the unknown epirelief of comparative example, illustrates that extracting method of the present invention extracts sample complete, removes matrix thorough.
Comparative example 2 import registered standard (JX20070012) methods are extracted clobetasone butyrate emulsifiable paste determination of related substances
Lucifuge operation, (Sino-America Tianjin Shike Pharmaceutical Co., Ltd. produces to get clobetasone butyrate emulsifiable paste, specification 0.05%) 20g, put in tool plug conical flask, precision adds clobetasol propionate inner mark solution 10ml and ethanol 30ml, and jam-pack bottle stopper is put 98 DEG C of water-baths with upper heating, shake well dissolves emulsifiable paste completely, let cool, put in ice bath cooling more than 1 hour, take out, centrifugal, get supernatant 30 μ l injection liquid chromatographies, by the chromatographic condition of embodiment 2, be measured in the same method, record chromatogram, obtain Fig. 7.The blank matrix 20g that separately gets clobetasone butyrate emulsifiable paste operates with method, the results are shown in Figure 8.
Embodiment 2 is compared with comparative example 2: embodiment 2 adds solvent acetonitrile, with 60 DEG C of heating water baths just can sample dissolution; Comparative example 2 adds ethanol to need about 95 DEG C ability sample dissolution, and security is poor, and clobetasone butyrate is in ethanolic solution, and under 98 DEG C of high temperature, hydrolysis produces two impurity such as clobetasone, poor stability.In embodiment 2, added inorganic salts, by centrifugal, phenomenon is that obvious solid-liquid is two-layer, gets solution layer and is easy to filter; And comparative example 2, the pasty state of cooling rear one-tenth homogeneous, is not easy to filter, and the solution obtaining is little, operates very loaded down with trivial details.The solvent acetonitrile that the present invention uses is more conventional in stratographic analysis, and owing to there is no comparative example 2 solvent effects, peak shape is better.Method of the present invention is more more convenient, more convenient than comparative example import registered standard (JX20070012) method, and chromatographic peak profile is better, and sample solution is more stable, and it is more accurate to measure.
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, can not disturb the mensuration of related substance.
The mensuration of embodiment 3 betamethasone dipropionate cream related substances
(1) chromatographic process
Chromatographic column: (250mm × 4.6mm, 5 μ m) for Agilent XDB-C18
UV detecting device: 240nm
Column temperature: 25 DEG C
Flow velocity: 1.5ml/min
Sample size: 20 μ l
Mobile phase: 45% acetonitrile
(2) extract and test
(Chongqing Huapont Pharmaceutical Co., Ltd. produces to get betamethasone dipropionate cream, specification 0.1%) the about 10g of content (being approximately equivalent to BDP 10mg), put in tool plug conical flask, add acetonitrile 10ml and sodium chloride 0.2g, 60 DEG C of water-baths make to dissolve, and put in ice-water bath and place 30 minutes, taking-up shakes up, put to room temperature, get clear liquid and filter, get subsequent filtrate as need testing solution.Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds dilution in acetonitrile to scale, shakes up, in contrast solution.Get blank matrix 10g, according to need testing solution same treatment, as vehicle solution.
By above-mentioned chromatographic condition, get contrast solution 20 μ l injection liquid chromatographies, regulate the sensitivity of instrument, make the peak height at BDP peak be about the 20%-25% of full scale; Precision measures need testing solution and the each 20 μ l of vehicle solution again, and injection liquid chromatography, records chromatogram respectively.
As shown in Figure 9 and Figure 10, the related substance HPLC that Figure 10 is need testing solution schemes test findings, the HPLC figure that Fig. 9 is vehicle solution.Comparison diagram 9 and Figure 10 are known, use the blank auxiliary material of the extraction solvent extraction of betamethasone dipropionate cream not disturb the mensuration of betamethasone dipropionate cream related substance, and Fig. 9 and Figure 10 baseline are level and smooth.
The mensuration of embodiment 4 clobetasol propionate cream related substances
(1) chromatographic process
Chromatographic column: YMC-Pack ODS-A 150mm × 4.6mm, 3.0 μ m
Flow velocity: 1.5ml/min
Column temperature: 40 DEG C
Detect wavelength: 241nm
Sample size: 30 μ l
Mobile phase: the A of mobile phase is water mutually, and B is acetonitrile mutually, and according to the form below carries out gradient elution:
(2) extract and test
Lucifuge operation, gets clobetasol propionate cream (Shanghai General Pharmaceutical Co., ltd. produces, specification 0.02%) content appropriate (being approximately equivalent to clobetasone butyrate 5mg), put in tool plug conical flask, add acetonitrile 16ml and potassium chloride 0.2g, 60 DEG C of water-baths make to dissolve, and put in ice bath and place 60 minutes, taking-up shakes up, then be placed in hydro-extractor, centrifugal 10 minutes of 4000rpm, puts to room temperature, get clear liquid PTFE filter membrane and filter, get subsequent filtrate as need testing solution.Precision measures need testing solution 1.0ml, puts in 100ml measuring bottle, adds 40% second eyeball and is diluted to scale, shakes up, in contrast solution.Get blank solution, according to need testing solution same treatment, as blank solution.
By above-mentioned chromatographic condition, get contrast solution 30 μ l injection liquid chromatographies, regulate the sensitivity of instrument, make the peak height at clobetasol propionate peak be about the 20%-25% of full scale; Precision measures need testing solution and the each 30 μ l of blank solution again, and injection liquid chromatography, records chromatogram respectively.
Test findings is as shown in Figure 11 and Figure 12, and the related substance HPLC that Figure 11 is need testing solution schemes, the HPLC figure that Figure 12 is blank solution.Contrast Figure 11 and Figure 12 figure are known, use this extraction solvent extraction not disturb the mensuration of clobetasol propionate cream related substance, and baseline is level and smooth, remove matrix thorough.
The mensuration of embodiment 5 Halometasone Cream content
(1) chromatographic process
Chromatographic column: (250mm × 4.6mm, 5 μ m) for Shimadzu VP ODS-C18
Flow velocity: 1.5ml/min
Column temperature: 25 DEG C
Detect wavelength: 254nm
Sample size: 20 μ l
Mobile phase: 0.1% glacial acetic acid solution-acetonitrile (65:35)
System flexibility: measure reference substance solution 20 μ l, injection liquid chromatography, records chromatogram; Be followed successively by 2-phenoxetol and Halometasone by peak elution order; Number of theoretical plate calculates and should be not less than 5000 by Halometasone peak; The degree of separation at Halometasone peak and 2-phenoxetol peak should meet the requirements.
(2) extract and test
(Australia can to get Halometasone Cream, the U.S. pharmaceutical manufacturing of Hong Kong Australia, specification 0.05%) the about 2.0g of content (being approximately 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 are dissolved Halometasone, and cool to room temperature, shakes up to scale by 50% dilution in acetonitrile; Put in ice bath and place 30 minutes, take out and put to room temperature, filter, get subsequent filtrate as test sample liquid.Separately get the about 200mg of 2-phenoxetol reference substance and the about 10mg of Halometasone reference substance, accurately weighed, put in 50ml measuring bottle, add acetonitrile and dissolve and be diluted to scale, shake up.Precision measures 5ml, puts in 50ml measuring bottle, adds 50% dilution in acetonitrile to scale, shakes up.Precision measures the each 20 μ l of above-mentioned two kinds of solution, and injection liquid chromatography, records chromatogram respectively,, to obtain final product with calculated by peak area by external standard method.
Test findings is as Figure 13, and the content HPLC of need testing solution schemes, Halometasone peak theoretical cam curve 8700, and Halometasone peak symmetrical factor is 1.01, baseline is level and smooth.
Follow-up content method is learned in checking, recovery test, and the recovery is between 99.5%-101.2%; The quantitative limit that joins matrix of sample reaches 8.7 × 10
-8g/ml.Test findings shows, this extracting method extracts completely Halometasone and impurity thereof, removes matrix thorough.
From above-described embodiment and comparative example, the inventive method is specially adapted to the extraction before the test of paste body shape adrenal cortex hormones drug, to the derivant of cortex hormone of aadrenaline composition, and degradation impurity all can effectively extract, compared with prior art, improve separation efficiency, more convenient, chromatographic peak profile is better, noiseless, and sample solution is more stable.In preparation is produced, energy effective control for product quality, safe and effective, more economical.There is significant progressive compared with import registered standard (JX20070012, JX20010092 etc.).
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
1. from composition, extract the method for compound with pregnane mother nucleus structure, it is characterized in that: use the aqueous solutions of organic solvent that is added with the organic solvent of inorganic salts or is added with inorganic salts for extracting solvent, composition to be extracted.
2. the method for extracting the compound with pregnane mother nucleus structure from composition according to claim 1, is characterized in that: described organic solvent is acetonitrile; Described inorganic salts are one or more in sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, sodium sulphate and potassium sulfate.
3. the method for extracting the compound with pregnane mother nucleus structure from composition according to claim 2, is characterized in that: described inorganic salts are 0.5%-5.0% at the mass percent extracting in solvent.
4. the method for extracting the compound with pregnane mother nucleus structure from composition according to claim 1, it is characterized in that: described composition is pharmaceutical composition, contain one or more in following compound and its derivant: 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.
5. the method for extracting the compound with pregnane mother nucleus structure from composition according to claim 4, is characterized in that: the form of described pharmaceutical composition is gel or emulsifiable paste; Described pharmaceutical composition comprises one or more in following auxiliary material: the carboxylate of carbomer, hexadecanol and carboxylate thereof, 18 alcohol and carboxylate thereof, polyglycol and stearic acid different saturation.
6. the method for extracting the compound with pregnane mother nucleus structure from composition according to claim 5, is characterized in that, the process of described extraction comprises the following steps: at 45-88 DEG C of temperature, with described dissolution with solvents composition; After dissolution with solvents composition, then separate out matrix at-20-4 DEG C temperature, Separation of Solid and Liquid obtains filtrate, obtains the solution that contains pregnane parent nucleus compound.
7. analyze the method in pharmaceutical composition with the compound of pregnane mother nucleus structure for one kind, it is characterized in that: the method for extracting the compound with pregnane mother nucleus structure from composition described in employing claim 1 to 6 any one is carried out the extraction of compound, test as need testing solution with extracting the solution obtaining.
8. the method in analysis pharmaceutical composition according to claim 7 with the compound of pregnane mother nucleus structure, is characterized in that: described test adopts high performance liquid chromatography to carry out;
In the time that described pharmaceutical composition is Halometasone Cream, carry out the test of related substance by following chromatographic condition:
Chromatographic column: Shimpack VP-ODS, 250mm × 4.6mm, 5 μ m;
UV detecting device: 254nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: A is 0.1% glacial acetic acid solution mutually, and B is acetonitrile mutually, and according to the form below carries out gradient elution:
In the time that described pharmaceutical composition is clobetasone butyrate emulsifiable paste, carry out the test of related substance by following chromatographic condition:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μ m;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Detect wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A of mobile phase is water mutually, and B is acetonitrile mutually, and according to the form below carries out gradient elution:
In the time that described pharmaceutical composition is betamethasone dipropionate cream, carry out the test of related substance by following chromatographic condition:
Chromatographic column: Agilent XDB-C18,250mm × 4.6mm, 5 μ m;
UV detecting device: 240nm;
Column temperature: 25 DEG C;
Flow velocity: 1.5ml/min;
Sample size: 20 μ l;
Mobile phase: 45% acetonitrile;
In the time that described pharmaceutical composition is clobetasol propionate cream, carry out the test of related substance by following chromatographic condition:
Chromatographic column: YMC-Pack ODS-A, 150mm × 4.6mm, 3.0 μ m;
Flow velocity: 1.5ml/min;
Column temperature: 40 DEG C;
Detect wavelength: 241nm;
Sample size: 30 μ l;
Mobile phase: the A of mobile phase is water mutually, and B is acetonitrile mutually, and according to the form below carries out gradient elution:
" % " is wherein percent by volume.
9. for extract a solvent for the compound with pregnane mother nucleus structure from composition, it is characterized in that: described solvent is the aqueous solutions of organic solvent that is added with the organic solvent of inorganic salts or is added with inorganic salts; Described organic solvent is acetonitrile; Described inorganic salts are one or more in sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, sodium sulphate and potassium sulfate.
10. according to claim 9 for extract the solvent of compound with pregnane mother nucleus structure from composition, it is characterized in that: described in there is pregnane mother nucleus structure compound be one or more in following compound and its derivant: 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, clobetasone butyrate.
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