WO2022252731A1 - Pretreatment method for determination of tacrolimus ointment isomer - Google Patents
Pretreatment method for determination of tacrolimus ointment isomer Download PDFInfo
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- WO2022252731A1 WO2022252731A1 PCT/CN2022/079968 CN2022079968W WO2022252731A1 WO 2022252731 A1 WO2022252731 A1 WO 2022252731A1 CN 2022079968 W CN2022079968 W CN 2022079968W WO 2022252731 A1 WO2022252731 A1 WO 2022252731A1
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- tacrolimus
- tacrolimus ointment
- isomer
- acetonitrile
- isomers
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- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 title claims abstract description 82
- 229960001967 tacrolimus Drugs 0.000 title claims abstract description 72
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 title claims abstract description 72
- 239000002674 ointment Substances 0.000 title claims abstract description 64
- 238000002203 pretreatment Methods 0.000 title claims abstract description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 75
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 45
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 239000012085 test solution Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000008014 freezing Effects 0.000 claims abstract description 11
- 238000007710 freezing Methods 0.000 claims abstract description 11
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 4
- 239000002480 mineral oil Substances 0.000 claims description 4
- 235000010446 mineral oil Nutrition 0.000 claims description 4
- 239000012188 paraffin wax Substances 0.000 claims description 4
- RUOJZAUFBMNUDX-UHFFFAOYSA-N propylene carbonate Chemical group CC1COC(=O)O1 RUOJZAUFBMNUDX-UHFFFAOYSA-N 0.000 claims description 4
- 239000003871 white petrolatum Substances 0.000 claims description 4
- 229940045860 white wax Drugs 0.000 claims description 4
- 229940042472 mineral oil Drugs 0.000 claims description 3
- 229940056211 paraffin Drugs 0.000 claims description 3
- 235000019271 petrolatum Nutrition 0.000 claims description 3
- -1 white wax Substances 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 14
- 238000003908 quality control method Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 238000005191 phase separation Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 23
- 238000001514 detection method Methods 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- DWZNQQMVUFPQMQ-UHFFFAOYSA-N acetonitrile;2-methoxy-2-methylpropane Chemical compound CC#N.COC(C)(C)C DWZNQQMVUFPQMQ-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000006317 isomerization reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Definitions
- the invention belongs to the field of detection of pharmaceutical products, and in particular relates to a pretreatment method for the determination of isomers of tacrolimus ointment.
- tacrolimus ointment The quality control of tacrolimus ointment currently only has the control on the content of related substances in tacrolimus ointment, and there is no control on the content of isomers in tacrolimus ointment. Due to the oily viscous matrix contained in tacrolimus ointment and the instability of tacrolimus isomers, the extraction of tacrolimus ointment isomers is quite difficult, which further affects the subsequent tacrolimus isomers. The effective determination of tacrolimus ointment isomers, so pre-treatment is the key to the detection of tacrolimus ointment isomers.
- the present invention provides a pretreatment method for the determination of isomers of tacrolimus ointment.
- adding n-hexane water bath to dissolve tacrolimus ointment then add acetonitrile or methanol, let stand to separate layers, freeze, take the acetonitrile layer or methanol layer as the test solution.
- the content of tacrolimus isomers was determined according to high performance liquid chromatography.
- the pretreatment method is simple to operate, makes tacrolimus isomers easy to extract from tacrolimus ointment, further satisfies the determination of high performance liquid chromatography, and provides research for the quality control of tacrolimus ointment.
- a pretreatment method for the determination of isomers of tacrolimus ointment adding n-hexane water bath to dissolve tacrolimus ointment, then adding acetonitrile or methanol, standing to separate layers, freezing, taking the acetonitrile layer or methanol layer, as The test solution.
- the volume ratio of n-hexane to acetonitrile or methanol is 1:1, preferably n-hexane and acetonitrile solvent system.
- the isomers of tacrolimus can be dissolved in the acetonitrile layer or the methanol layer as completely as possible from the tacrolimus ointment, and the n-hexane layer and the acetonitrile layer or the methanol layer can be completely dissolved.
- the n-hexane, acetonitrile and methanol are chromatographically pure.
- the temperature of the water bath is 60°C-65°C, preferably 60°C. If the temperature of the water bath is too low, it will lead to insufficient dissolution of tacrolimus ointment, which will affect the dissolution of subsequent isomers, thereby affecting the detection of isomer content; if the temperature of the water bath is too high, it will lead to isomerization of tacrolimus The content of isomers in tacrolimus ointment increased, and the subsequent detection of isomers in tacrolimus ointment was inaccurate.
- the freezing conditions the freezing temperature is minus 18° C. to minus 22° C., and the freezing time is 20 minutes to 25 minutes.
- the freezing temperature is minus 20°C and the freezing time is 20 minutes, so that the tacrolimus isomer components can be dissolved in the acetonitrile layer as completely as possible, and the matrix components in the tacrolimus ointment can be dissolved in the n-hexane layer.
- the pretreatment method for the determination of the isomers of tacrolimus ointment add n-hexane to the tacrolimus ointment in a 60°C water bath to dissolve, then add acetonitrile, stand to separate layers, and place at minus 20°C for 20 minutes, Take the acetonitrile layer as the test solution; the volume ratio of the n-hexane to acetonitrile is 1:1.
- the need testing solution that adopts the pretreatment method of the present invention to obtain is placed at room temperature for 3 hours, and the stability is better.
- the test solution was injected into a liquid chromatograph, and the tacrolimus isomer content was determined according to the chromatographic conditions described in Tacrolimus USP 40.
- Tacrolimus is prone to isomerization into isomer I and isomer II.
- the pretreatment method for the determination of tacrolimus ointment isomers of the present invention is simple to operate, so that tacrolimus isomers are easily extracted from tacrolimus ointment, and further meet the determination of high performance liquid chromatography, It can better monitor the content of isomers of tacrolimus ointment, and provide research for the quality control of tacrolimus ointment.
- Fig. 1 is the HPLC spectrogram of reference preparation tacrolimus ointment isomer
- Fig. 2 is the HPLC spectrogram of the tacrolimus ointment isomer described in Example 1 of the present invention
- Fig. 3 is the HPLC spectrogram of the tacrolimus ointment isomer described in Example 2 of the present invention
- Fig. 4 is the HPLC spectrogram of the tacrolimus ointment isomer described in Example 3 of the present invention.
- Fig. 5 is the HPLC spectrogram of the tacrolimus ointment isomer when the test solution described in Example 1 of the present invention is left for 0 h;
- Fig. 6 is the HPLC spectrogram of the tacrolimus ointment isomer when the test solution described in Example 1 of the present invention is placed for 1 h;
- Fig. 7 is the HPLC spectrogram of tacrolimus ointment isomer when need testing solution described in the embodiment of the present invention 1 is placed 2h;
- Fig. 8 is the HPLC spectrogram of the isomers of tacrolimus ointment when the test solution described in Example 1 of the present invention was placed for 3 hours.
- the content of tacrolimus in the tacrolimus ointment of the present invention is 0.1% w/w, and the matrix is propylene carbonate, white petrolatum, mineral oil, white wax and paraffin.
- the tacrolimus, propylene carbonate, white petrolatum, mineral oil, white wax, and paraffin used in the homemade tacrolimus ointment can all be purchased from the manufacturers announced on the CDE official website.
- the reference preparation used was tacrolimus ointment produced by Astellas Pharmaceuticals (China) Co., Ltd.
- tacrolimus ointment of the present invention tacrolimus 0.1%w/w, propylene carbonate 0.4g, white vaseline 10g, mineral oil 1.5g, white wax 1.5g, paraffin 1.5g.
- Chromatographic conditions used use octadecylsilane bonded silica gel as filler; use 6mmol/L phosphoric acid as solution A, use acetonitrile-tert-butyl methyl ether (81:19) as solution B, use solution A-solution B ( Volume ratio 4:1) is mobile phase A, solution A-solution B (volume ratio 1:4) is mobile phase B; detection wavelength is 220nm; flow rate is 1.5mL per minute; column temperature is 60°C; according to Table 1 Perform gradient elution.
- Chromatographic conditions used use octadecylsilane bonded silica gel as filler; use 6mmol/L phosphoric acid as solution A, use acetonitrile-tert-butyl methyl ether (81:19) as solution B, use solution A-solution B ( Volume ratio 4:1) is mobile phase A, solution A-solution B (volume ratio 1:4) is mobile phase B; detection wavelength is 220nm; flow rate is 1.5mL per minute; column temperature is 60°C; according to Table 1 Perform gradient elution.
- Chromatographic conditions used use octadecylsilane bonded silica gel as filler; use 6mmol/L phosphoric acid as solution A, use acetonitrile-tert-butyl methyl ether (81:19) as solution B, use solution A-solution B ( Volume ratio 4:1) is mobile phase A, solution A-solution B (volume ratio 1:4) is mobile phase B; detection wavelength is 220nm; flow rate is 1.5mL per minute; column temperature is 60°C; according to Table 1 Perform gradient elution.
- test solution prepared in step (1) of Example 1 is placed at room temperature for 0h, 1h, 2h, and 3h, and then sampled according to step (2) respectively, and the stability of the test solution is investigated as shown in Figure 5-- 8 HPLC spectrogram, its investigation result sees Table 2, calculates the sum of its peak area and main peak area, test result shows that need testing solution room temperature is placed 3 hours, and the content determination result of the isomer in the tacrolimus ointment and 0 There is little change in the hour comparison.
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Abstract
Disclosed in the present invention is a pretreatment method for determination of a tacrolimus ointment isomer. According to the physical and chemical properties of tacrolimus ointment itself, n-hexane is added to the tacrolimus ointment for dissolving in a water bath, then acetonitrile or methanol is added, leaving same to stand is carried out for phase separation, freezing is carried out, and an acetonitrile layer or a methanol layer is taken as a test solution; and then the content of a tacrolimus isomer is determined according to high performance liquid chromatography. The pretreatment method is simple to operate, so that the tacrolimus isomer can be easily extracted from a tacrolimus ointment isomer, thereby further meeting the determination of high performance liquid chromatography, and providing research for the quality control of the tacrolimus ointment.
Description
本发明属于医药产品检测领域，具体涉及一种他克莫司软膏异构体测定的前处理方法。The invention belongs to the field of detection of pharmaceutical products, and in particular relates to a pretreatment method for the determination of isomers of tacrolimus ointment.
他克莫司软膏的质量控制，目前仅仅有对他克莫司软膏有关物质含量的控制，还未有他克莫司软膏异构体含量的控制。由于他克莫司软膏中含有油性黏性基质，并且他克莫司异构体的不稳定性，导致他克莫司软膏异构体的提取相当困难，进一步影响后续他克莫司异构体的有效测定，所以他克莫司软膏异构体的测定中前处理是检测的关键。The quality control of tacrolimus ointment currently only has the control on the content of related substances in tacrolimus ointment, and there is no control on the content of isomers in tacrolimus ointment. Due to the oily viscous matrix contained in tacrolimus ointment and the instability of tacrolimus isomers, the extraction of tacrolimus ointment isomers is quite difficult, which further affects the subsequent tacrolimus isomers. The effective determination of tacrolimus ointment isomers, so pre-treatment is the key to the detection of tacrolimus ointment isomers.
发明内容Contents of the invention
针对现有技术的不足，本发明提供了一种他克莫司软膏异构体测定的前处理方法，根据他克莫司软膏本身的理化性质，在他克莫司软膏中加入正己烷水浴溶解，然后加入乙腈或甲醇，静置分层，冷冻，取乙腈层或甲醇层，作为供试品溶液。然后按照高效液相色谱法进行测定他克莫司异构体的含量。所述前处理方法操作简单，使得他克莫司异构体易于从他克莫司软膏中提取出来，进一步满足高效液相色谱法的测定，为他克莫司软膏的质量控制提供了研究。Aiming at the deficiencies of the prior art, the present invention provides a pretreatment method for the determination of isomers of tacrolimus ointment. According to the physical and chemical properties of tacrolimus ointment itself, adding n-hexane water bath to dissolve tacrolimus ointment , then add acetonitrile or methanol, let stand to separate layers, freeze, take the acetonitrile layer or methanol layer as the test solution. Then, the content of tacrolimus isomers was determined according to high performance liquid chromatography. The pretreatment method is simple to operate, makes tacrolimus isomers easy to extract from tacrolimus ointment, further satisfies the determination of high performance liquid chromatography, and provides research for the quality control of tacrolimus ointment.
本发明所述技术方案是这样实现的：The technical scheme of the present invention is realized like this:
一种他克莫司软膏异构体测定的前处理方法：在他克莫司软膏中加入正己烷水浴溶解，然后加入乙腈或甲醇，静置分层，冷冻，取乙腈层或甲醇层，作为供试品溶液。A pretreatment method for the determination of isomers of tacrolimus ointment: adding n-hexane water bath to dissolve tacrolimus ointment, then adding acetonitrile or methanol, standing to separate layers, freezing, taking the acetonitrile layer or methanol layer, as The test solution.
所述正己烷与乙腈或甲醇的体积比为1：1，优选正己烷与乙腈溶剂体系。在这样的溶剂体系中，既可以使得他克莫司异构体从他克莫司软膏中尽可能完全的溶出于乙腈层或甲醇层中，又能使得正己烷层与乙腈层或甲醇层完全分层。所述正己烷、乙腈、甲醇均为色谱纯。The volume ratio of n-hexane to acetonitrile or methanol is 1:1, preferably n-hexane and acetonitrile solvent system. In such a solvent system, the isomers of tacrolimus can be dissolved in the acetonitrile layer or the methanol layer as completely as possible from the tacrolimus ointment, and the n-hexane layer and the acetonitrile layer or the methanol layer can be completely dissolved. layered. The n-hexane, acetonitrile and methanol are chromatographically pure.
所述水浴温度为60℃-65℃，优选60℃。如果水浴温度过低，将会导致他克莫司软膏溶解不充分，影响后续异构体的溶出，进而影响异构体含量的检测；如果水浴温度过高，则会导致他克莫司异构体的 含量增加，后续对于他克莫司软膏中异构体的含量检测不准确。The temperature of the water bath is 60°C-65°C, preferably 60°C. If the temperature of the water bath is too low, it will lead to insufficient dissolution of tacrolimus ointment, which will affect the dissolution of subsequent isomers, thereby affecting the detection of isomer content; if the temperature of the water bath is too high, it will lead to isomerization of tacrolimus The content of isomers in tacrolimus ointment increased, and the subsequent detection of isomers in tacrolimus ointment was inaccurate.
所述冷冻条件：冷冻温度零下18℃-零下22℃，冷冻时间20min-25min。优选地，冷冻温度零下20℃，冷冻时间20min，能够使得他克莫司异构体成分尽可能完全地溶出在乙腈层，他克莫司软膏中的基质成分溶出在正己烷层。The freezing conditions: the freezing temperature is minus 18° C. to minus 22° C., and the freezing time is 20 minutes to 25 minutes. Preferably, the freezing temperature is minus 20°C and the freezing time is 20 minutes, so that the tacrolimus isomer components can be dissolved in the acetonitrile layer as completely as possible, and the matrix components in the tacrolimus ointment can be dissolved in the n-hexane layer.
优选地，所述他克莫司软膏异构体测定的前处理方法：他克莫司软膏中加入正己烷60℃水浴溶解，然后加入乙腈，静置分层，置于零下20℃放置20min，取乙腈层，作为供试品溶液；所述所述正己烷与乙腈的体积比为1：1。Preferably, the pretreatment method for the determination of the isomers of tacrolimus ointment: add n-hexane to the tacrolimus ointment in a 60°C water bath to dissolve, then add acetonitrile, stand to separate layers, and place at minus 20°C for 20 minutes, Take the acetonitrile layer as the test solution; the volume ratio of the n-hexane to acetonitrile is 1:1.
采用本发明所述前处理方法得到的供试品溶液在室温下放置3小时，稳定性较好。取所述供试品溶液注入液相色谱仪，所述他克莫司异构体含量的测定是按照Tacrolimus USP 40中所述色谱条件进行检测。The need testing solution that adopts the pretreatment method of the present invention to obtain is placed at room temperature for 3 hours, and the stability is better. The test solution was injected into a liquid chromatograph, and the tacrolimus isomer content was determined according to the chromatographic conditions described in Tacrolimus USP 40.
他克莫司易发生异构化成为异构体I和异构体Ⅱ，为了保证药品质量的均一性，需要将他克莫司异构体含量的控制订入他克莫司软膏的质量标准中，异构体I和异构体Ⅱ的峰面积之和不得超过主峰面积的8.0％。Tacrolimus is prone to isomerization into isomer I and isomer II. In order to ensure the uniformity of drug quality, it is necessary to set the control of tacrolimus isomer content in the quality standard of tacrolimus ointment Among them, the sum of the peak areas of isomer I and isomer II shall not exceed 8.0% of the main peak area.
本发明所述他克莫司软膏异构体测定的前处理方法，操作简单，使得他克莫司异构体易于从他克莫司软膏中提取出来，进一步满足高效液相色谱法的测定，能更好地监测他克莫司软膏异构体的含量，为他克莫司软膏的质量控制提供了研究。The pretreatment method for the determination of tacrolimus ointment isomers of the present invention is simple to operate, so that tacrolimus isomers are easily extracted from tacrolimus ointment, and further meet the determination of high performance liquid chromatography, It can better monitor the content of isomers of tacrolimus ointment, and provide research for the quality control of tacrolimus ointment.
图1是参比制剂他克莫司软膏异构体的HPLC谱图；Fig. 1 is the HPLC spectrogram of reference preparation tacrolimus ointment isomer;
图2是本发明实施例1所述他克莫司软膏异构体的HPLC谱图；Fig. 2 is the HPLC spectrogram of the tacrolimus ointment isomer described in Example 1 of the present invention;
图3是本发明实施例2所述他克莫司软膏异构体的HPLC谱图；Fig. 3 is the HPLC spectrogram of the tacrolimus ointment isomer described in Example 2 of the present invention;
图4是本发明实施例3所述他克莫司软膏异构体的HPLC谱图；Fig. 4 is the HPLC spectrogram of the tacrolimus ointment isomer described in Example 3 of the present invention;
图5是本发明实施例1所述供试品溶液放置0h时他克莫司软膏异构体的HPLC谱图；Fig. 5 is the HPLC spectrogram of the tacrolimus ointment isomer when the test solution described in Example 1 of the present invention is left for 0 h;
图6是本发明实施例1所述供试品溶液放置1h时他克莫司软膏异构体的HPLC谱图；Fig. 6 is the HPLC spectrogram of the tacrolimus ointment isomer when the test solution described in Example 1 of the present invention is placed for 1 h;
图7是本发明实施例1所述供试品溶液放置2h时他克莫司软膏 异构体的HPLC谱图；Fig. 7 is the HPLC spectrogram of tacrolimus ointment isomer when need testing solution described in the embodiment of the present invention 1 is placed 2h;
图8是本发明实施例1所述供试品溶液放置3h时他克莫司软膏异构体的HPLC谱图。Fig. 8 is the HPLC spectrogram of the isomers of tacrolimus ointment when the test solution described in Example 1 of the present invention was placed for 3 hours.
本发明所述他克莫司软膏中他克莫司的含量为0.1％w/w，基质为碳酸丙烯酯、白凡士林、矿物油、白蜡、石蜡。自制他克莫司软膏中所用的他克莫司、碳酸丙烯酯、白凡士林、矿物油、白蜡、石蜡均可以在CDE官方网站上公布的厂家购买得到。所用参比制剂是安斯泰来制药(中国)有限公司生产的他克莫司软膏。The content of tacrolimus in the tacrolimus ointment of the present invention is 0.1% w/w, and the matrix is propylene carbonate, white petrolatum, mineral oil, white wax and paraffin. The tacrolimus, propylene carbonate, white petrolatum, mineral oil, white wax, and paraffin used in the homemade tacrolimus ointment can all be purchased from the manufacturers announced on the CDE official website. The reference preparation used was tacrolimus ointment produced by Astellas Pharmaceuticals (China) Co., Ltd.
本发明所述他克莫司软膏的配方：他克莫司0.1％w/w、碳酸丙烯酯0.4g、白凡士林10g、矿物油1.5g、白蜡1.5g、石蜡1.5g。The formula of tacrolimus ointment of the present invention: tacrolimus 0.1%w/w, propylene carbonate 0.4g, white vaseline 10g, mineral oil 1.5g, white wax 1.5g, paraffin 1.5g.
实施例1Example 1
他克莫司软膏异构体含量的测定：Determination of isomer content of tacrolimus ointment:
(1)他克莫司软膏的前处理(1) Pretreatment of tacrolimus ointment
取本品约2g，置10mL具塞玻璃管中，加入正己烷4.0mL，60℃水浴使溶解，精密量取乙腈4.0mL，置同一具塞玻璃管中，静置分层，置冷冻冰箱-20℃放置20分钟，取乙腈层，作为供试品溶液；Take about 2g of this product, put it into a 10mL stoppered glass tube, add 4.0mL of n-hexane, dissolve it in a 60°C water bath, accurately measure 4.0mL of acetonitrile, put it into the same stoppered glass tube, let it stand for layers, and put it in a freezer- Stand at 20°C for 20 minutes, take the acetonitrile layer as the test solution;
(2)他克莫司软膏异构体含量的测定(2) Determination of tacrolimus ointment isomer content
精密量取供试品溶液20μL，注入液相色谱仪，记录色谱图，各异构体的峰面积之和不得过主峰面积的8.0％。Precisely measure 20 μL of the test solution, inject it into a liquid chromatograph, record the chromatogram, the sum of the peak areas of each isomer must not exceed 8.0% of the main peak area.
所用色谱条件：用十八烷基硅烷键合硅胶为填充剂；以6mmol/L的磷酸为溶液A，以乙腈-叔丁基甲基醚(81︰19)为溶液B，以溶液A-溶液B(体积比4︰1)为流动相A，以溶液A-溶液B(体积比1︰4)为流动相B；检测波长为220nm；流速为每分钟1.5mL；柱温为60℃；按照表1进行梯度洗脱。Chromatographic conditions used: use octadecylsilane bonded silica gel as filler; use 6mmol/L phosphoric acid as solution A, use acetonitrile-tert-butyl methyl ether (81:19) as solution B, use solution A-solution B ( Volume ratio 4:1) is mobile phase A, solution A-solution B (volume ratio 1:4) is mobile phase B; detection wavelength is 220nm; flow rate is 1.5mL per minute; column temperature is 60°C; according to Table 1 Perform gradient elution.
表1 梯度洗脱条件Table 1 Gradient elution conditions
| 时间(min)time (min) | 流动相A(％)Mobile phase A(%) | 流动相B(％)Mobile phase B(%) |
| 00 | 7272 | 2828 |
| 3030 | 7272 | 2828 |
| 5353 | 1515 | 8585 |
| 5454 | 7272 | 2828 |
| 6060 | 7272 | 2828 |
实施例2Example 2
他克莫司软膏异构体含量的测定：Determination of isomer content of tacrolimus ointment:
(1)他克莫司软膏的前处理(1) Pretreatment of tacrolimus ointment
取本品约2g，置10mL具塞玻璃管中，加入正己烷4.0mL，65℃水浴使溶解，精密量取甲醇4.0mL，置同一具塞玻璃管中，静置分层，置冷冻冰箱-18℃放置25分钟，取甲醇层，作为供试品溶液；Take about 2g of this product, put it into a 10mL stoppered glass tube, add 4.0mL of n-hexane, dissolve in a water bath at 65°C, accurately measure 4.0mL of methanol, put it into the same stoppered glass tube, let it stand for layers, and put it in a freezer- Place at 18°C for 25 minutes, take the methanol layer as the test solution;
(2)他克莫司软膏异构体含量的测定(2) Determination of tacrolimus ointment isomer content
精密量取供试品溶液20μL，注入液相色谱仪，记录色谱图，各异构体的峰面积之和不得过主峰面积的8.0％。Precisely measure 20 μL of the test solution, inject it into a liquid chromatograph, record the chromatogram, the sum of the peak areas of each isomer must not exceed 8.0% of the main peak area.
所用色谱条件：用十八烷基硅烷键合硅胶为填充剂；以6mmol/L的磷酸为溶液A，以乙腈-叔丁基甲基醚(81︰19)为溶液B，以溶液A-溶液B(体积比4︰1)为流动相A，以溶液A-溶液B(体积比1︰4)为流动相B；检测波长为220nm；流速为每分钟1.5mL；柱温为60℃；按照表1进行梯度洗脱。Chromatographic conditions used: use octadecylsilane bonded silica gel as filler; use 6mmol/L phosphoric acid as solution A, use acetonitrile-tert-butyl methyl ether (81:19) as solution B, use solution A-solution B ( Volume ratio 4:1) is mobile phase A, solution A-solution B (volume ratio 1:4) is mobile phase B; detection wavelength is 220nm; flow rate is 1.5mL per minute; column temperature is 60°C; according to Table 1 Perform gradient elution.
实施例3Example 3
他克莫司软膏异构体含量的测定：Determination of isomer content of tacrolimus ointment:
(1)他克莫司软膏的前处理(1) Pretreatment of tacrolimus ointment
取本品约2g，置10mL具塞玻璃管中，加入正己烷4.0mL，65℃水浴使溶解，精密量取乙腈4.0mL，置同一具塞玻璃管中，静置分层，置冷冻冰箱-22℃放置22分钟，取乙腈层，作为供试品溶液；Take about 2g of this product, put it into a 10mL stoppered glass tube, add 4.0mL of n-hexane, dissolve it in a water bath at 65°C, accurately measure 4.0mL of acetonitrile, put it into the same stoppered glass tube, let it stand for layers, and put it in a freezer- Place at 22°C for 22 minutes, take the acetonitrile layer as the test solution;
(2)他克莫司软膏异构体含量的测定(2) Determination of tacrolimus ointment isomer content
精密量取供试品溶液20μL，注入液相色谱仪，记录色谱图，各异构体的峰面积之和不得过主峰面积的8.0％。Precisely measure 20 μL of the test solution, inject it into a liquid chromatograph, record the chromatogram, the sum of the peak areas of each isomer must not exceed 8.0% of the main peak area.
所用色谱条件：用十八烷基硅烷键合硅胶为填充剂；以6mmol/L的磷酸为溶液A，以乙腈-叔丁基甲基醚(81︰19)为溶液B，以溶液A-溶液B(体积比4︰1)为流动相A，以溶液A-溶液B(体积比1︰4)为流动相B；检测波长为220nm；流速为每分钟1.5mL；柱温为60℃；按照表1进行梯度洗脱。Chromatographic conditions used: use octadecylsilane bonded silica gel as filler; use 6mmol/L phosphoric acid as solution A, use acetonitrile-tert-butyl methyl ether (81:19) as solution B, use solution A-solution B ( Volume ratio 4:1) is mobile phase A, solution A-solution B (volume ratio 1:4) is mobile phase B; detection wavelength is 220nm; flow rate is 1.5mL per minute; column temperature is 60°C; according to Table 1 Perform gradient elution.
实施例1 所述供试品溶液的稳定性试验：The stability test of need testing solution described in embodiment 1:
将实施例1步骤(1)制备好供试品溶液在室温下放置0h、1h、2h、3h后按照步骤(2)分别进样，考察所述供试品溶液的稳定性见 图5-图8的HPLC谱图，其考察结果见表2，计算其峰面积与主峰面积的和，试验结果表明供试品溶液室温放置3小时，他克莫司软膏中异构体的含量测定结果与0小时比较变化不大。The test solution prepared in step (1) of Example 1 is placed at room temperature for 0h, 1h, 2h, and 3h, and then sampled according to step (2) respectively, and the stability of the test solution is investigated as shown in Figure 5-- 8 HPLC spectrogram, its investigation result sees Table 2, calculates the sum of its peak area and main peak area, test result shows that need testing solution room temperature is placed 3 hours, and the content determination result of the isomer in the tacrolimus ointment and 0 There is little change in the hour comparison.
表2 实施例1所述供试品溶液稳定性考察结果The investigation result of need testing solution stability described in table 2 embodiment 1
| 规格(0.1％)Specifications (0.1%) | 0h峰面积(V)0h peak area (V) | 1h峰面积(V)1h peak area (V) | 2h峰面积(V)2h peak area (V) | 3h峰面积(V)3h peak area (V) |
| 异构体IIIsomer II | 7383373833 | 7520175201 | 8744987449 | 8665986659 |
| 主峰main peak | 18861581886158 | 19203961920396 | 22136862213686 | 22119692211969 |
| 含量％content% | 3.913.91 | 3.923.92 | 3.953.95 | 3.923.92 |
Claims (6)
- 一种他克莫司软膏异构体测定的前处理方法，其特征在于：他克莫司软膏中加入正己烷水浴溶解，然后加入乙腈或甲醇，静置分层，冷冻，取乙腈层或甲醇层，作为供试品溶液。A pretreatment method for the determination of isomers of tacrolimus ointment, characterized in that: adding n-hexane water bath to dissolve tacrolimus ointment, then adding acetonitrile or methanol, standing to separate layers, freezing, taking the acetonitrile layer or methanol Layer, as the test solution.
- 根据权利要求1所述他克莫司软膏异构体测定的前处理方法，其特征在于：所述正己烷与乙腈或甲醇的体积比为1：1。The pretreatment method for determining isomers of tacrolimus ointment according to claim 1, wherein the volume ratio of the n-hexane to acetonitrile or methanol is 1:1.
- 根据权利要求2所述他克莫司软膏异构体测定的前处理方法，其特征在于：所述水浴温度为60℃-65℃。The pretreatment method for determining isomers of tacrolimus ointment according to claim 2, characterized in that: the temperature of the water bath is 60°C-65°C.
- 根据权利要求3所述他克莫司软膏异构体测定的前处理方法，其特征在于：所述冷冻条件：冷冻温度零下18℃-零下22℃，冷冻时间20min-25min。The pretreatment method for determining isomers of tacrolimus ointment according to claim 3, characterized in that: the freezing conditions: freezing temperature minus 18°C-minus 22°C, freezing time 20min-25min.
- 根据权利要求1所述他克莫司软膏异构体测定的前处理方法，其特征在于：他克莫司软膏中加入正己烷60℃水浴溶解，然后加入乙腈，静置分层，置于零下20℃放置20min，取乙腈层，作为供试品溶液；According to the pretreatment method for the determination of isomers of tacrolimus ointment according to claim 1, it is characterized in that: n-hexane is added to the tacrolimus ointment to dissolve in a 60°C water bath, then acetonitrile is added, the layers are left to stand, and placed below zero Place at 20°C for 20 minutes, take the acetonitrile layer as the test solution;所述正己烷与乙腈的体积比为1：1。The volume ratio of n-hexane to acetonitrile is 1:1.
- 根据权利要求5所述他克莫司软膏异构体测定的前处理方法，其特征在于：所述他克莫司软膏中他克莫司的含量为0.1％w/w，基质为碳酸丙烯酯、白凡士林、矿物油、白蜡、石蜡。According to the pretreatment method for determining the isomers of tacrolimus ointment according to claim 5, it is characterized in that: the content of tacrolimus in the tacrolimus ointment is 0.1% w/w, and the matrix is propylene carbonate , white petrolatum, mineral oil, white wax, paraffin.
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