CN110514604B - Method for measuring anisidine value of alprostadil freeze-dried lipid emulsion - Google Patents
Method for measuring anisidine value of alprostadil freeze-dried lipid emulsion Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 65
- BHAAPTBBJKJZER-UHFFFAOYSA-N p-anisidine Chemical compound COC1=CC=C(N)C=C1 BHAAPTBBJKJZER-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 239000002960 lipid emulsion Substances 0.000 title claims abstract description 62
- GMVPRGQOIOIIMI-UHFFFAOYSA-N (8R,11R,12R,13E,15S)-11,15-Dihydroxy-9-oxo-13-prostenoic acid Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CCCCCCC(O)=O GMVPRGQOIOIIMI-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 229960000711 alprostadil Drugs 0.000 title claims abstract description 56
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 title claims abstract description 56
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 174
- 239000000839 emulsion Substances 0.000 claims abstract description 73
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000012360 testing method Methods 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 21
- 235000000346 sugar Nutrition 0.000 claims abstract description 19
- -1 alkali metal salt Chemical class 0.000 claims abstract description 17
- 229910052783 alkali metal Inorganic materials 0.000 claims abstract description 16
- 238000001704 evaporation Methods 0.000 claims abstract description 9
- 239000012071 phase Substances 0.000 claims description 72
- MHZZQFJGUZUBCN-UHFFFAOYSA-N propan-2-ol;2,2,4-trimethylpentane Chemical compound CC(C)O.CC(C)CC(C)(C)C MHZZQFJGUZUBCN-UHFFFAOYSA-N 0.000 claims description 35
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 30
- 239000003921 oil Substances 0.000 claims description 27
- 239000012046 mixed solvent Substances 0.000 claims description 24
- 235000002639 sodium chloride Nutrition 0.000 claims description 21
- 239000000523 sample Substances 0.000 claims description 18
- 239000012085 test solution Substances 0.000 claims description 18
- 229960000583 acetic acid Drugs 0.000 claims description 15
- 239000012362 glacial acetic acid Substances 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 15
- 238000003556 assay Methods 0.000 claims description 14
- 238000002347 injection Methods 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 14
- 238000002835 absorbance Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 238000002390 rotary evaporation Methods 0.000 claims description 12
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000003223 protective agent Substances 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 4
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 4
- 235000011151 potassium sulphates Nutrition 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 4
- 235000011152 sodium sulphate Nutrition 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 5
- 235000019198 oils Nutrition 0.000 description 24
- NSBIQPJIWUJBBX-UHFFFAOYSA-N n-methoxyaniline Chemical compound CONC1=CC=CC=C1 NSBIQPJIWUJBBX-UHFFFAOYSA-N 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000003549 soybean oil Substances 0.000 description 7
- 235000012424 soybean oil Nutrition 0.000 description 7
- 238000011084 recovery Methods 0.000 description 6
- 239000003995 emulsifying agent Substances 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 5
- OLBCVFGFOZPWHH-UHFFFAOYSA-N propofol Chemical compound CC(C)C1=CC=CC(C(C)C)=C1O OLBCVFGFOZPWHH-UHFFFAOYSA-N 0.000 description 5
- 229960004134 propofol Drugs 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 2
- ZLUXDGOKSONZQR-UHFFFAOYSA-N acetic acid 4-methoxyaniline Chemical compound COC1=CC=C(C=C1)N.C(C)(=O)O ZLUXDGOKSONZQR-UHFFFAOYSA-N 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 229910052751 metal Chemical class 0.000 description 2
- 239000002184 metal Chemical class 0.000 description 2
- OEHAYUOVELTAPG-UHFFFAOYSA-N methoxyphenamine Chemical compound CNC(C)CC1=CC=CC=C1OC OEHAYUOVELTAPG-UHFFFAOYSA-N 0.000 description 2
- 229960005405 methoxyphenamine Drugs 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- QWLISCJHYITNQF-UHFFFAOYSA-N n-methoxy-1-phenylmethanamine Chemical compound CONCC1=CC=CC=C1 QWLISCJHYITNQF-UHFFFAOYSA-N 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical group O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Cosmetics (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention relates to a method for measuring a anisidine value of an alprostadil freeze-dried lipid emulsion, which comprises the following steps: carrying out multiple emulsion treatment on the alprostadil freeze-dried lipid emulsion to obtain multiple emulsion; adding absolute ethyl alcohol and alkali metal salt into the multiple emulsion to demulsify; adding anhydrous ether for extraction, collecting ether phase, and then adding water to wash residual sugar and alkali metal salt in the ether phase to obtain ether phase and water phase; evaporating the ether phase under vacuum at 40 deg.C to obtain residue I; dissolving the residue I with anhydrous ethanol, and rotary evaporating to remove anhydrous ethanol under vacuum at below 60 deg.C to obtain residue II as test sample for determining anisidine value. The method has the advantages of complete oil phase extraction, high accuracy, capability of eliminating the interference of sugar on the measurement, capability of effectively controlling the product quality, strong specificity, good repeatability and accurate measurement result, and improves the quality detection standard of the alprostadil freeze-dried lipid emulsion.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining a anisidine value of an alprostadil freeze-dried lipid emulsion.
Background
An alprostadil freeze-dried lipid emulsion belongs to a fat emulsion for intravenous injection, which is prepared by stirring and uniformly mixing alprostadil and oil for injection to obtain an oil phase; adding the freeze-drying protective agent into the water phase, adding the emulsifier into the oil phase or the water phase according to the solubility of the emulsifier, preparing the emulsion from the two phases by a high-pressure homogenization method, and freeze-drying the emulsion by a freeze-drying method.
The anisidine value is an index for reflecting the content of aldehyde and ketone which are secondary decomposition products of peroxide in the grease, the decomposition products can destroy the normal physiological function of human cell membranes to cause the occurrence and the development of diseases, and the liver function of a human body can be damaged due to overhigh anisidine value. Meanwhile, the anisidine value is also a key index for controlling the quality of the fat emulsion injection preparation.
During the preparation process, the oil components in the alprostadil freeze-dried lipid emulsion may be oxidized and decomposed into aldehyde, ketone and other substances with certain toxicity, thereby causing the potential safety hazard of clinical medication. Therefore, the methoxyphenylamine value of the alprostadil freeze-dried lipid emulsion needs to be controlled. In the determination process of the anisidine value of the alprostadil freeze-dried lipid emulsion by referring to the determination method of the anisidine value item in the fat emulsion injection (C14-C24) quality standard (survey opinion draft) and the propofol emulsion injection quality standard (survey opinion draft) issued by the Chinese pharmacopoeia Committee, the biggest problem exists that the interference of the freeze-drying protective agent sugar in a sample cannot be effectively removed, so that the dissolution uniformity of the sample solution is influenced, and the determination result is unstable and inaccurate.
Therefore, before the methoxyphenamine value of the alprostadil freeze-dried lipid emulsion is determined, a pretreatment method for effectively removing sugar from a sample must be found so as to improve the effectiveness of the methoxyphenamine value determination of the alprostadil freeze-dried lipid emulsion. Obviously, solving the problem of sugar removal is a key problem for measuring the anisidine value of the alprostadil freeze-dried lipid emulsion.
The determination method of anisidine value in fat emulsion injection (C14-C24) quality standard (solicited comments on.
Chinese patent CN103712936A adopts an extraction method to extract oil phase to determine the anisidine value of the fat emulsion, when the method is used for determining the alprostadil freeze-dried lipid emulsion, a sample solution reacts with 4-anisidine to generate flocculent precipitate, so that the solution to be determined is not uniform, the determination result is influenced, and the determination value is large.
Therefore, at present, research and development of a method which is feasible to operate, can effectively remove sugar and has controllable quality are urgently needed to determine the anisidine value of the alprostadil freeze-dried lipid emulsion.
Disclosure of Invention
The invention aims to make up the defects of the prior art and provide a method for effectively determining the anisidine value of the alprostadil freeze-dried lipid emulsion with feasible operation, thereby eliminating the interference of sugar serving as a freeze-drying protective agent, completely extracting an oil phase and effectively controlling the product quality.
The purpose of the invention is realized by the following technical scheme:
a method for measuring the anisidine value of an alprostadil freeze-dried lipid emulsion comprises the following steps:
(1) carrying out multiple emulsion treatment on the alprostadil freeze-dried lipid emulsion to obtain multiple emulsion, taking the multiple emulsion, and then adding anhydrous ethanol and alkali metal salt into the taken multiple emulsion to carry out emulsion breaking;
(2) adding anhydrous ether for extraction, collecting ether phase, and then adding water to wash residual sugar and alkali metal salt in the ether phase to obtain ether phase and water phase;
(3) evaporating the ether phase under vacuum at 40 deg.C to obtain residue I;
(4) dissolving the residue I with anhydrous ethanol, and vacuum rotary evaporating to remove anhydrous ethanol at below 60 deg.C to obtain residue II as test sample for determining anisidine value.
The determination method is suitable for various formulations of alprostadil freeze-dried lipid emulsions, and is particularly suitable for removing the interference of freeze-drying protective agent sugar contained in the alprostadil freeze-dried lipid emulsions. In general, an emulsifier (e.g., egg yolk lecithin, soybean lecithin, etc.), a sugar (e.g., sucrose, maltose, lactose, glucose, trehalose, etc.) as a lyoprotectant, an oil for injection (e.g., soybean oil, medium chain triglyceride, medium chain diglyceride, medium chain monoglyceride, olive oil, etc.), and the like may be included in the fat emulsion. The emulsifier in the fat emulsion has strong emulsibility, and the alkali metal salt (such as potassium chloride, sodium chloride, potassium sulfate, sodium sulfate and the like) is used as a good demulsifier, so that the balance of the emulsion can be destroyed, and the sample can be prevented from emulsifying in the extraction process; because the alprostadil freeze-dried lipid emulsion after the multiple emulsion belongs to O/W type emulsion, the water layer on the outer layer of the emulsion can be properly destroyed by adding a proper amount of absolute ethyl alcohol, so that the oil layer is exposed, and the extraction of the oil phase in the emulsion by ethyl ether is facilitated. Because the boiling point of the ether is low, the sugar is not dissolved in the ether, the grease is dissolved in the ether but not dissolved in the ether, and the water is slightly dissolved in the ether, the anhydrous ether is adopted as the extractant, so that the extraction solvent can be quickly removed, the oil phase and the freeze-drying protective agent can be effectively separated, and new water can be prevented from being brought into the extractant; during the extraction process, a small amount of water remained in the oil phase can be removed by rotary evaporation after the residue I is dissolved by absolute ethyl alcohol.
Preferably, the assay method of the present invention further comprises the steps of:
(a) dissolving the residue II with isopropanol-isooctane mixed solvent at volume ratio of 2: 8 to obtain sample solution I, and measuring absorbance value A at 350nm wavelength by ultraviolet-visible spectrophotometry with isopropanol-isooctane mixed solvent at volume ratio of 2: 8 as blank control 0 ；
(b) Accurately measuring equal volumes of a test solution I to be tested and an isopropanol-isooctane mixed solvent with the volume ratio of 2: 8, respectively placing the test solution I to be tested and the isopropanol-isooctane mixed solvent with the volume ratio of 2: 8 into a test tube A and a test tube B with plugs, respectively adding 0.25 percent (g/ml) of 4-methoxyaniline glacial acetic acid solution with the same volume to obtain a test solution II and an isopropanol-isooctane mixed solution containing 4-methoxyaniline glacial acetic acid, respectively measuring the absorbance values of the test solution II and the isopropanol-isooctane mixed solution containing 4-methoxyaniline glacial acetic acid at a wavelength of 350nm according to an ultraviolet-visible spectrophotometry by taking the isopropanol-isooctane mixed solvent with the volume ratio of 2: 8 as a blank reference, and respectively marking the absorbance values as A1 and A2.
In certain embodiments of the invention, in step (1) of the assay method, the alkali metal salt is selected from one or more of potassium chloride, sodium chloride, potassium sulfate or sodium sulfate; preferably, the ratio of the amount (g) of alkali metal salt to the amount (ml) of the multiple emulsion taken is 2: 100 to 1000 in g/ml.
In some embodiments of the invention, in the step (2), the volume ratio of the anhydrous ether to the taken multiple emulsion is 3-7: 1, and the volume ratio of the water to the taken multiple emulsion is 7-12: 1.
In certain embodiments of the present invention, step (2) of the assay method further comprises performing a second extraction with anhydrous diethyl ether, collecting the diethyl ether phase, and washing the residual sugars and metal salts in the diethyl ether phase with water to obtain a diethyl ether phase and an aqueous phase; preferably, the water phase obtained by the second anhydrous ether extraction is subjected to third anhydrous ether extraction, the ether phase is collected, and then water is added to wash residual sugar and metal salt in the oil phase, so that the ether phase and the water phase are obtained; wherein the volume ratio of the anhydrous ether to the taken multiple emulsion is 1-5: 1, and the volume ratio of the water to the taken multiple emulsion is 3-10: 1 when the ether phase is washed by adding water.
In certain embodiments of the invention, in step (2) of the assay method, after each extraction with anhydrous ether, the ether phase and the aqueous phase are allowed to stand for a layering time of 20-30 min, preferably 30 min.
In certain embodiments of the invention, in step (3) of the assay method, the diethyl ether is rotary evaporated in vacuo at below 30 ℃; preferably, the time for evaporating the diethyl ether by vacuum rotary evaporation is 5-10 min.
In certain embodiments of the invention, in step (4) of the assay method, the absolute ethanol is rotary evaporated in vacuo at below 50 ℃; preferably, dissolving the residue I with anhydrous ethanol, and then evaporating the anhydrous ethanol by vacuum rotation at below 60 ℃, repeating the operation for 3 times so as to fully remove the water; preferably, the time for vacuum rotary evaporation of the absolute ethyl alcohol is 10-15 min.
In a specific embodiment of the determination method, the method for determining the anisidine value of the alprostadil freeze-dried lipid emulsion comprises the following steps:
(1) taking 5 bottles of 10 microgram/bottle or 10 bottles of 5 microgram/bottle of alprostadil freeze-dried lipid emulsion, adding 10ml of water to dissolve the alprostadil freeze-dried lipid emulsion into emulsion to obtain multiple emulsion, and putting the multiple emulsion into a separating funnel; or taking 6 bottles (10 microgram/bottle) or 12 bottles (5 microgram/bottle) of the alprostadil freeze-dried lipid emulsion, adding 12ml of water to dissolve the mixture into emulsion to obtain multiple emulsion, taking 10ml of the multiple emulsion, and then placing the multiple emulsion in a separating funnel;
(2) adding 5ml of absolute ethyl alcohol into a separating funnel, uniformly mixing, adding 0.02-0.2 g of alkali metal salt and 30-70 ml of absolute ethyl ether, violently shaking, collecting an ethyl ether phase, and adding 70-120 ml of water to violently shake;
(3) standing until complete layering, transferring the ether oil phase at the upper layer into a flask, placing the water phase at the lower layer into a separating funnel, extracting twice with anhydrous ether, adding 10-50 ml of anhydrous ether each time, shaking violently, standing until complete layering, combining the three ether phases, and discarding the water layer;
(4) collecting an ether phase obtained by three times of ether extraction, placing the ether phase in a separating funnel, then adding 30-100 ml of water, violently shaking, standing until complete layering, and removing a water layer to obtain the ether phase;
(5) carrying out vacuum rotary evaporation on the ether phase at the temperature lower than 40 ℃ to recover the solvent to obtain a residue I;
(6) dissolving the residue I with 20ml of anhydrous ethanol to obtain an anhydrous ethanol phase, performing vacuum rotary evaporation at a temperature lower than 60 ℃, and repeating for 3 times to obtain a residue II as a test sample;
(7) adding an isopropanol-isooctane mixed solvent with the volume ratio of 2: 8 into the residue II, dissolving the residue II, quantitatively transferring the dissolved residue II into a 25ml volumetric flask, adding the isopropanol-isooctane mixed solvent with the volume ratio of 2: 8, diluting to a scale, shaking uniformly, filtering by using a 0.45 mu m syringe filter, and taking the filtrate as a sample solution I;
(8) taking a sample solution I, taking an isopropanol-isooctane mixed solvent with the volume of 2: 8 as a blank control, and measuring an absorbance value A0 at the wavelength of 350nm by adopting an ultraviolet-visible spectrophotometry;
(9) precisely measuring 5ml of a test sample solution I and 5ml of an isopropanol-isooctane mixed solvent with the volume of 2: 8 respectively, placing the test sample solution I and the isopropanol-isooctane mixed solvent respectively in a test tube with a plug A and a test tube with a plug B, precisely adding 1ml of a newly prepared 4-methoxyaniline glacial acetic acid solution with a plug, shaking, and accurately placing for 10 minutes in a dark place to respectively obtain a test sample solution II and an isopropanol-isooctane mixed solution containing 4-methoxyaniline glacial acetic acid, taking the isopropanol-isooctane mixed solvent with the volume ratio of 2: 8 as a blank control, and respectively measuring absorbances A1 and A2 at the wavelength of 350nm according to an ultraviolet-visible spectrophotometry;
(10) the anisidine value is calculated as follows:
in the formula: v is the amount of the multiple emulsion taken in the step (1), and the unit is ml; c is the amount of the oil for injection in the multiple emulsion taken in the step (1), and the unit is g/ml;
wherein, the value of C can be obtained by calculation according to the amount of the compound emulsion taken and the marked amount of the prescription;
the value 1.2 is the dilution factor of the solution after addition of the 4-methoxyaniline acetic acid solution.
The invention adopts alkali metal salt to carry out demulsification on the freeze-dried lipid emulsion after double emulsification, adopts anhydrous ether to extract the oil phase, washes residual sugar and alkali metal salt in the oil phase, uses anhydrous ethanol to finally remove water in a test sample, and uses an ultraviolet spectrophotometer method to determine the anisidine value. Proved by methodology, the determination method has strong specificity, good repeatability, high recovery rate and more uniform and stable test solution. In the measuring process, the oil phase and the sugar can be effectively and completely separated, and the auxiliary materials in the recovery process can be prevented from being oxidized. The test result shows that the sample measured by the method has more uniform solution of the test sample and more accurate and reasonable measurement result, which shows that the method can truly reflect the result of the anisidine value in the sample and can effectively control the quality of the product.
The invention fills the blank of the prior art, provides a feasible, stable and accurate determination method of the anisidine value for the freeze-dried lipid emulsion, and is convenient for controlling the safety quality index of the freeze-dried lipid emulsion. The method has the advantages of complete oil phase extraction, high accuracy, capability of eliminating the interference of sugar on the measurement, effective control of the product quality and improvement of the quality detection standard of the alprostadil freeze-dried lipid emulsion.
Detailed Description
The method for detecting the anisidine value of the alprostadil lyophilized lipid emulsion according to the present invention will be further described with reference to the following specific examples, but the present invention is not limited to the scope of the present embodiment.
Example 1
Determination of methoxyphenylamine value of alprostadil freeze-dried lipid emulsion
1. Instruments, drugs and reagents:
thermo corporation Evolution 220 type ultraviolet-visible spectrophotometer
N-1100 type rotary evaporator and SB-1100 type water bath of Shanghai Ailang apparatus Limited
Alprostadil freeze-dried lipid emulsion: self-made
The preparation method comprises the following steps: referring to the components in table 1, 1 batch of lyophilized lipid emulsion samples of alprostadil was prepared. Respectively adding 5mg of alprostadil and 20g of emulsifier into 10g of oil for injection, stirring and uniformly mixing to obtain an oil phase; adding 200g of freeze-drying protective agent into 500ml of water for injection, stirring and uniformly mixing to obtain a water phase, mixing the two phases, fixing the volume to 1000ml, preparing an emulsion by a high-pressure homogenization method, and freeze-drying to obtain the alprostadil freeze-dried lipid emulsion.
TABLE 1 ingredient table of alprostadil freeze-dried lipid emulsion
The absolute ethyl alcohol, the absolute ethyl ether, the isopropanol, the isooctane, the potassium chloride, the sodium chloride, the potassium sulfate, the sodium sulfate, the 4-methoxyaniline and the glacial acetic acid used in the invention are all provided by chemical reagents of national medicine group, Inc. (analytically pure); the water is purified water for drinking from Shenyang Waha Ha Co.
2. Detection conditions
The detection method comprises the following steps: ultraviolet-visible spectrophotometry
Detection wavelength: 350 nm.
3. Preparation of the solution
(1) Preparation of test solution
Preparing an isopropanol-isooctane (volume ratio of 2: 8) mixed solvent: and mixing and shaking uniformly 200ml of isopropanol and 800ml of isooctane to obtain the finished product.
Preparation of 0.25% 4-methoxyaniline glacial acetic acid solution: before measurement, 0.025g of 4-anisidine is precisely weighed into a 10ml brown volumetric flask, and glacial acetic acid is dissolved and diluted to the scale, thus obtaining the product.
(2) Preparation of test solution
Dissolving 10 bottles (5 mu g/bottle) of alprostadil freeze-dried lipid emulsion into 10ml of water to form emulsion, obtaining multiple emulsion, taking all multiple emulsion, and then placing the multiple emulsion in a separating funnel;
adding 5ml of absolute ethyl alcohol into a separating funnel, uniformly mixing, adding 0.1g of sodium chloride and 50ml of absolute ethyl ether, violently shaking, collecting an ethyl ether phase, adding 80ml of water, violently shaking;
standing until complete layering, transferring the upper diethyl ether phase into a flask, placing the lower water phase into a separating funnel, extracting with anhydrous diethyl ether twice, adding 25ml of anhydrous diethyl ether each time, shaking vigorously, standing until complete layering, combining the three diethyl ether phases, and discarding the water layer;
placing the collected ether phase in a separating funnel, adding 50ml of water, violently shaking, standing until the ether phase is completely layered, and removing a water layer to obtain the ether phase;
carrying out vacuum rotary evaporation on the ether phase at 38 ℃ to recover the solvent to obtain a residue I;
dissolving the residue I with 20ml of anhydrous ethanol to obtain an anhydrous ethanol phase, performing vacuum rotary evaporation at 55 deg.C for 3 times to obtain residue II as a sample;
adding isopropanol-isooctane (volume ratio of 2: 8) into the residue II to dissolve the residue II, quantitatively transferring to a 25ml volumetric flask, adding the above solvent to dilute to scale, shaking, and filtering with 0.45 μm syringe filter to obtain filtrate I.
4. Methoxyphenylamine value determination method
Taking a sample solution I, taking isopropanol-isooctane (volume ratio of 2: 8) as a blank control, and measuring an absorbance value A0 at the wavelength of 350nm by adopting an ultraviolet-visible spectrophotometry;
precisely measuring 5ml of each of a test solution I and isopropanol-isooctane (volume ratio is 2: 8), respectively placing the test solution I and the isopropanol-isooctane into a test tube with a plug A and a test tube with a plug B, respectively and precisely adding 1ml of a 0.25% 4-methoxyaniline glacial acetic acid solution (prepared by clinical new preparation), plugging, shaking, and accurately placing for 10 minutes in a dark place to respectively obtain a test solution II and an isopropanol-isooctane mixed solution containing 4-methoxyaniline glacial acetic acid, and respectively measuring absorbance A1 and absorbance A2 at a wavelength of 350nm by using an isopropanol-isooctane (volume ratio is 2: 8) as a blank control according to an ultraviolet-visible spectrophotometry;
the anisidine value is calculated as follows:
in the formula: v is the amount of the multiple emulsion taken in the step (1), and the unit is ml;
c is the amount of the oil for injection in the multiple emulsion taken in the step (1), and the unit is g/ml;
the value 1.2 is the dilution factor of the solution after addition of the 4-methoxyaniline acetic acid solution.
5. Repeatability survey
6 parts (first to sixth parts) of the test solutions were prepared simultaneously in accordance with the above method, and the anisidine values were measured, respectively, to examine the reproducibility of the measurement method of the sample solutions.
6. Results
TABLE 2 determination of the Methoxyphenylamine value of Alprostadil lyophilized lipid emulsion and the results of repeated investigation
The results show that the interference of the freeze-drying protective agent can be well removed by adopting the method disclosed by the invention, the method is used for determining the anisidine value of the alprostadil freeze-dried lipid emulsion, the result is stable, and the repeatability is good.
Examples 2 to 5
Determination of methoxyphenylamine value of alprostadil freeze-dried lipid emulsion
Examples 2-5 the multiple emulsion volumes and emulsion breaker types used in example 1 were varied in terms of emulsion breaker, absolute ethanol, first extractant absolute ether, first water addition, combined oil phase and water wash usage as shown in table 3. The remaining procedure was as in example 1.
TABLE 3 dosage of demulsifier, absolute ethanol, first extractant, first water addition, and combined oil phase and water washing amount in examples 2-5
And (3) measuring results: the anisidine values measured for examples 2-5 were 3.21, 3.27, 3.19 and 3.34 respectively. And (4) conclusion: the dosage of the demulsifier, the absolute ethyl alcohol, the first extractant, the first water addition amount and the washing amount after the oil phase is combined can accurately determine the anisidine value within a certain application range.
Examples 6 to 9
Determination of methoxyphenylamine value of alprostadil freeze-dried lipid emulsion
The operating parameters and procedures in examples 6-9 were the same as in example 1 except that the volume of the multiple emulsion taken was different from that of example 1, as shown in table 4.
TABLE 4 multiple breast volumes taken in examples 6-9
| Examples for use with | Example 6 | Example 7 | Example 8 | Example 9 |
| Compound milk volume (ml) | 5 | 8 | 15 | 20 |
| Anisidine number (mean value) | 3.15 | 3.21 | 3.18 | 3.23 |
The method of the invention is adopted to determine the anisidine value of the alprostadil freeze-dried lipid multiple emulsion with different volumes. The results show that taking different multiple emulsion volumes (V) is in linear relation with the calculated value of 1.2(A1-A2) -A0, and the correlation coefficient R 2 Therefore, in the linear range, the use of different amounts of multiple emulsion volume does not affect the determination of anisidine value, indicating that the determination method of the present invention is feasible.
Example 10
Determination of methoxyphenylamine value of alprostadil freeze-dried lipid emulsion
The operating parameters and procedure of example 10 are the same as in example 1, except that the temperature at which the diethyl ether is recovered is different from that of example 1. Example 10 was run at less than 20 ℃ (18 ℃).
And (3) measuring results: the results of parallel determination of the anisidine values of 6 1706012 batches were: 3.10, 3.12, 3.09, 3.08, 3.11, 3.09. And (4) conclusion: the anhydrous ether is recovered at a temperature lower than 20 ℃, and the anisidine value can be accurately determined.
Example 11
Determination of methoxyphenylamine value of alprostadil freeze-dried lipid emulsion
The operating parameters and procedure of example 11 are the same as in example 1, except that the temperature at which absolute ethanol is recovered differs from that of example 1, and example 11 is operated at a temperature below 40 ℃ (38 ℃).
And (3) measuring results: the results of parallel determination of the anisidine values of 6 1706012 batches were: 3.07, 3.10, 3.05, 3.09, 3.12, 3.10. And (4) conclusion: the anhydrous ethanol is recovered at the temperature lower than 40 ℃, and the anisidine value can be accurately determined.
Examples 12 to 14
Determination of methoxyphenylamine value of alprostadil freeze-dried lipid emulsion
The operating parameters and procedures for examples 12-14 were the same as in example 1, except that the demulsifier used was different from that of example 1, as shown in table 5,
TABLE 5 demulsifier types used in examples 12-14
| Amount of examples | Example 12 | Example 13 | Example 14 |
| Demulsifier | Potassium chloride | Potassium sulfate | Sodium sulfate |
| Anisidine number (mean value) | 3.22 | 3.17 | 3.13 |
And (3) measuring results: the anisidine values measured for examples 12-14 were 3.22, 3.17 and 3.13 respectively. The results show that the anisidine value can be accurately determined by adopting different alkali metal salts as demulsifiers.
Example 15
Examination of accuracy of measurement method
The method for measuring the sample adding recovery rate of the method by respectively using soybean oil and yolk lecithin and investigating the accuracy of the method comprises the following steps:
(1) taking 10ml of emulsion after re-emulsification of the alprostadil freeze-dried lipid emulsion, operating according to the method of the embodiment 1, simultaneously measuring the anisidine values of 6 samples, and calculating the average value A1;
(2) taking 1g of soybean oil, operating according to the method of example 1, and measuring the anisidine value A2;
(3) 0.12g of phospholipid was collected and measured for its anisidine value A3 by the method of example 1;
(4) taking the alprostadil freeze-dried lipid emulsion, taking 10ml of emulsion after multiple emulsion, adding 1g of soybean oil test solution, operating according to the method of example 1, simultaneously measuring the anisidine values of 6 samples, and calculating the average value A4;
(5) taking the alprostadil freeze-dried lipid emulsion, taking 10ml of emulsion after re-emulsification, adding 0.12g of phospholipid test solution, operating according to the method of example 1, simultaneously measuring the anisidine values of 6 samples, and calculating the average value A5;
(6) the results of the samples directly measured according to the method of example 1 were compared with the results of the samples added with soybean oil or phospholipid, respectively, and calculated according to the following formula:
the soybean oil loading recovery rate is (A4-A1)/A2;
the phospholipid sample recovery rate was (a5-a 1)/A3.
According to the formula, the sample recovery rates of the soybean oil and the phospholipid are respectively 95.2% and 96.7%, and the RSD is respectively 4.2% and 3.7%. The accuracy of the assay method of the invention is demonstrated to be high.
Comparative example 1
The method of the invention and the method described in Propofol emulsion injection quality Standard issued by the Committee of Chinese pharmacopoeia are adopted to determine the anisidine value of the alprostadil freeze-dried lipid emulsion.
The process of the invention is described in the extraction method of example 1.
The method (standard method) described in propofol emulsion injection quality standard directly measures the freeze-dried lipid double emulsion of alprostadil after removing water by rotary evaporation of absolute ethyl alcohol and dissolving residue by mixed solvent.
The rest is the same as example 1.
And (3) measuring results: see table 6.
TABLE 6 comparison of results of determination of methoxybenzylamine values of alprostadil lyophilized lipid emulsions
By comparing the measurement results of the method disclosed by the invention and the Propofol emulsion injection quality standard, the same product is measured by the method disclosed by the invention, and the sample residue is semitransparent oily after the solvent is recovered, can be completely dissolved in the mixed solvent, is clear and transparent in state, and has the advantages of stable measurement result, good repeatability and good accuracy; the methoxyaniline value measured by the method of propofol emulsion injection quality standard is remarkably high in result, the reproducibility of sample operation is poor, sample residues are plastic and incompletely dissolved in the solvent after the solvent is recovered, and the measurement result is inaccurate. The determination method is more reasonable, feasible and accurate.
Claims (15)
1. A method for measuring a anisidine value of an alprostadil freeze-dried lipid emulsion is characterized by comprising the following steps:
(1) carrying out multiple emulsion treatment on the alprostadil freeze-dried lipid emulsion to obtain multiple emulsion, taking the multiple emulsion, and adding absolute ethyl alcohol and alkali metal salt into the taken multiple emulsion to carry out emulsion breaking;
(2) adding anhydrous ether for extraction, wherein the anhydrous ether is adopted as an extractant, so that the extraction solvent can be quickly removed, and the oil phase and the freeze-drying protective agent can be effectively separated due to the characteristics that the ether is low in boiling point, the sugar is insoluble in the ether, the oils are soluble in the ether but insoluble in water, and the water is slightly soluble in the ether; collecting the ether phase, and then adding water to wash residual sugar and alkali metal salt in the ether phase to obtain an ether phase and a water phase;
(3) evaporating the ether phase under vacuum at 40 deg.C to obtain residue I;
(4) dissolving the residue I with anhydrous ethanol, and vacuum rotary evaporating to remove anhydrous ethanol at 60 deg.C to obtain residue II as test sample for determining anisidine value.
2. The assay of claim 1, further comprising the steps of:
(a) dissolving the residue II with isopropanol-isooctane mixed solvent at volume ratio of 2: 8 to obtain sample solution I, and measuring absorbance value A at 350nm wavelength by ultraviolet-visible spectrophotometry with isopropanol-isooctane mixed solvent at volume ratio of 2: 8 as blank control 0 ；
(b) Accurately measuring equal volumes of a test solution I and an isopropanol-isooctane mixed solvent with a volume ratio of 2: 8, respectively placing the test solution I and the isopropanol-isooctane mixed solvent into a test tube with a plug A and a test tube with a plug B, respectively adding 0.25% of a 4-methoxyaniline glacial acetic acid solution with the same volume and a unit of g/ml to obtain a test solution II and an isopropanol-isooctane mixed solution containing 4-methoxyaniline glacial acetic acid, respectively measuring the absorbance values of the test solution II and the isopropanol-isooctane mixed solution containing 4-methoxyaniline glacial acetic acid at a wavelength of 350nm by using the isopropanol-isooctane mixed solvent with the volume ratio of 2: 8 as a blank control according to an ultraviolet-visible spectrophotometry, and respectively marking the absorbance values as A1 and A2.
3. The assay method according to claim 1 or 2, wherein in step (1), the alkali metal salt is selected from one or more of potassium chloride, sodium chloride, potassium sulfate or sodium sulfate.
4. The method according to claim 1 or 2, wherein in the step (1), the ratio of the amount of the alkali metal salt to the amount of the double emulsion taken is 2 g: 100 to 1000 ml.
5. The method according to claim 1 or 2, wherein in the step (2), the volume ratio of the anhydrous ether to the taken multiple emulsion is 3-7: 1, and the volume ratio of the water to the taken multiple emulsion is 7-12: 1.
6. The method according to claim 1 or 2, wherein in the step (2), the second and third extraction with anhydrous ether is carried out, wherein the volume ratio of anhydrous ether to the taken multiple emulsion is 1 to 5: 1, respectively, and the volume ratio of water to the taken multiple emulsion when the ether phase is washed with water is 3 to 10: 1.
7. The method according to claim 6, wherein in the step (2), after each extraction with anhydrous ether, the ether phase and the aqueous phase are allowed to stand for a layering time of 20 to 30 min.
8. The method according to claim 6, wherein in the step (2), the time for the ether phase to stand still with the aqueous phase for demixing is 30min after each extraction with anhydrous ether.
9. An assay as claimed in claim 1 or claim 2 wherein in step (3) the ether phase is rotary evaporated under vacuum at below 30 ℃.
10. The method according to claim 1 or 2, wherein in the step (3), the time for evaporating the ether by vacuum rotary evaporation is 5-10 min.
11. The assay of claim 1 or 2, wherein in step (4) the absolute ethanol is rotary evaporated under vacuum at below 50 ℃.
12. The method according to claim 1 or 2, wherein in the step (4), the residue I is dissolved in absolute ethanol, and then the absolute ethanol is rotary evaporated under vacuum at 60 ℃ or lower, and the process is repeated 3 times.
13. The method according to claim 1 or 2, wherein in the step (4), the time for rotary evaporation of the absolute ethanol under vacuum is 10-15 min.
14. The assay of claim 1, wherein the assay comprises the steps of:
(1) taking 5 bottles of 10 microgram/bottle or 10 bottles of 5 microgram/bottle of alprostadil freeze-dried lipid emulsion, adding 10ml of water to dissolve the alprostadil freeze-dried lipid emulsion into emulsion to obtain multiple emulsion; putting the multiple emulsion into a separating funnel; or taking 6 bottles of 10 microgram/bottle or 12 bottles of 5 microgram/bottle alprostadil freeze-dried lipid emulsion, adding 12ml of water to dissolve the alprostadil freeze-dried lipid emulsion into emulsion to obtain multiple emulsion, taking 10ml of the multiple emulsion, and then placing the multiple emulsion in a separating funnel;
(2) adding 5ml of absolute ethyl alcohol into a separating funnel, uniformly mixing, adding 0.02-0.2 g of alkali metal salt and 30-70 ml of absolute ethyl ether, violently shaking, collecting an ethyl ether phase, and adding 70-120 ml of water to violently shake;
(3) standing until complete layering, transferring the upper diethyl ether phase into a flask, placing the lower water phase into a separating funnel, extracting twice with anhydrous diethyl ether, adding 10-50 ml of anhydrous diethyl ether each time, shaking violently, standing until complete layering, combining three diethyl ether phases, and removing a water layer;
(4) collecting an ether phase obtained by three times of ether extraction, placing the ether phase in a separating funnel, then adding 30-100 ml of water, violently shaking, standing until complete layering, and removing a water layer to obtain the ether phase;
(5) carrying out vacuum rotary evaporation on the ether phase at the temperature lower than 40 ℃ to recover the solvent to obtain a residue I;
(6) dissolving the residue I with 20ml of anhydrous ethanol to obtain an anhydrous ethanol phase, performing vacuum rotary evaporation at a temperature lower than 60 ℃, and repeating for 3 times to obtain a residue II as a test sample;
(7) adding a mixed solvent of isopropanol-isooctane with the volume ratio of 2: 8 into the residue II, dissolving the residue II, quantitatively transferring the dissolved residue II into a 25ml volumetric flask, adding the mixed solvent of isopropanol-isooctane with the volume ratio of 2: 8, diluting to a scale, shaking up, filtering by using a 0.45 mu m syringe filter, and taking the filtrate as a test solution I;
(8) taking the sample solution I, taking an isopropanol-isooctane mixed solvent with the volume of 2: 8 as a blank control, and measuring an absorbance value A0 at the wavelength of 350nm by adopting an ultraviolet-visible spectrophotometry;
(9) precisely measuring 5ml of a test sample solution I and 5ml of an isopropanol-isooctane mixed solvent with a volume ratio of 2: 8 respectively, placing the test sample solution I and the isopropanol-isooctane mixed solvent into a test tube A and a test tube B respectively with plugs, precisely adding 0.25% of a newly prepared 4-methoxyaniline glacial acetic acid solution with the unit of g/ml respectively, adding the plugs, shaking, and accurately placing for 10 minutes in a dark place to obtain a test sample solution II and an isopropanol-isooctane mixed solution containing the 4-methoxyaniline glacial acetic acid respectively, taking the isopropanol-isooctane with the volume ratio of 2: 8 as a blank control, and respectively measuring absorbances A1 and A2 at a wavelength of 350nm according to an ultraviolet-visible spectrophotometry;
(10) the anisidine value is calculated as follows:
in the formula: v is the amount of the multiple emulsion taken in the step (1), and the unit is ml;
c is the amount of the oil for injection in the multiple emulsion taken in the step (1) and the unit is g/ml.
15. Use of the assay of any one of claims 1-14 in the determination of the anisidine value of an alprostadil lyophilized lipid emulsion with a sugar as lyoprotectant.
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