WO2019242212A1 - 一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法 - Google Patents

一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法 Download PDF

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WO2019242212A1
WO2019242212A1 PCT/CN2018/114746 CN2018114746W WO2019242212A1 WO 2019242212 A1 WO2019242212 A1 WO 2019242212A1 CN 2018114746 W CN2018114746 W CN 2018114746W WO 2019242212 A1 WO2019242212 A1 WO 2019242212A1
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solution
parecoxib sodium
related substances
liquid chromatography
chromatography method
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PCT/CN2018/114746
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French (fr)
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张鑫
杜芳
周亚兵
汤征
丁云晖
汪晓铭
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上药东英（江苏）药业有限公司
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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
    • G01N30/89—Inverse chromatography
    • 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
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

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  • the invention belongs to the field of medical analysis, and specifically relates to a method for the simultaneous detection of 12 related substances, especially 5 positional isomers, in parecoxib sodium and its synthetic intermediates by liquid chromatography.
  • non-steroidal anti-inflammatory drugs is to inhibit prostaglandin synthesis by inhibiting the activity of cyclooxygenase (COX), thereby exerting anti-inflammatory, antipyretic and analgesic effects.
  • COX cyclooxygenase
  • Conventional non-steroidal anti-inflammatory drugs inhibit constitutively expressed COX-1 while inhibiting inflammation-related or inducible COX-2.
  • COX-1 will catalyze the production of prostaglandins required for normal cell function, so the application of conventional NSAIDs will inhibit COX-1 and cause certain toxic and side effects.
  • Parecoxib Sodium is a water-soluble prodrug of the selective COX-2 inhibitory drug vardecoxib and is the first COX-2 inhibitor for injection. Parecoxib sodium is hydrolyzed by liver enzymes after intravenous or intramuscular injection, and is quickly converted into vardecoxib, a pharmacologically active substance. Parecoxib can be used for the treatment of moderate to severe acute pain after surgery. Its clinical efficacy has been confirmed in various analgesic treatments such as stomatology, gynecology, orthopedics, and intravenous administration of this product after surgery can reduce the amount of morphine, thereby improving the quality of postoperative analgesia.
  • the chemical name of parecoxib sodium is N-[[4- (5-methyl-3-phenyl-4-isoxazolyl) phenyl] sulfonyl] propionamide sodium salt, the structural formula is as follows:
  • the invention can effectively separate 12 potential substances related to the synthesis of parecoxib sodium, especially has strong separation ability for the 5 positional isomers existing in the synthesis process.
  • the present invention provides a liquid chromatography method for detecting related substances in parecoxib sodium and synthetic intermediates.
  • a liquid chromatography method for detecting related substances in parecoxib sodium and synthetic intermediates including the following steps:
  • Step 1 System suitability experiment: (1) Take each impurity and the reference of parecoxib sodium separately, dilute with methanol to a mixed solution as the system suitability solution; (2) Set up the liquid chromatograph, and The system suitability solution is injected into a liquid chromatograph and the chromatogram is recorded until the resolution between each impurity peak and the parecoxib sodium peak meets the requirements;
  • Step 2 Measurement experiment: (1) take the test parecoxib sodium in a container, add methanol to dissolve and dilute it as a test solution; (2) take each impurity, dilute it with methanol, and use it as a control solution; (3) Take the test solution and the reference solution into a liquid chromatograph and record the chromatogram; (4) Calculate according to the external standard method based on the peaks in the chromatograms of the control solution and the test solution.
  • the system suitability solution is a solution containing 0.5 mg of parecoxib sodium per milliliter and each impurity is 0.75 ⁇ g.
  • the injection solution to be tested is 10 ⁇ l.
  • the concentration of the test solution is 0.5 mg / ml
  • the concentration of the control solution is 0.75 ⁇ g / ml.
  • the solvent is methanol.
  • the instrument used in the liquid chromatography analysis method includes a liquid chromatograph, a chromatographic data processing system, and a chromatographic column.
  • a 250 mm ⁇ 4.6 mm silica gel column is used as the chromatographic column, and a pentafluorophenyl-bonded silica gel and an octadecylsilane-bonded silica gel filler are installed therein, and the particle diameter of the filler is 5 ⁇ m.
  • the detection wavelength of the liquid chromatograph is 215 nm
  • 0.01 mol / l KH 2 PO 4 buffer solution is used as mobile phase A
  • methanol is used as mobile phase B
  • gradient elution is performed, wherein the buffer pH range It is 2.5 to 3.5.
  • Step 1 System suitability experiment: (1) Take the reference substance of each impurity and a substance separately, use methanol as the mixed solution as the system suitability solution; (2) Set up the liquid chromatograph, and apply the system The solution is injected into the liquid chromatograph, and the chromatogram is recorded until the resolution between each impurity peak and a substance meets the requirements, and the setting is fixed;
  • Step 2 Measurement experiment: (1) take a substance to be tested in a container, dissolve it with a solvent and dilute it as a test solution; (2) take an appropriate amount of each single impurity, dilute it with a solvent, and use it as a control solution; 3) Take the test solution and the reference solution into a liquid chromatograph respectively, and record the chromatogram; (4) Calculate according to the external standard method based on the peak in the chromatogram of the test solution.
  • the present invention adopts reversed-phase high-performance liquid chromatography, and uses pentafluorophenyl bonded silica and octadecylsilane bonded silica as column fillers, adopts a UV detector, and uses a mobile phase containing potassium dihydrogen phosphate. Elution, divided into two determinations of related substances in parecoxib sodium intermediate vardecoxib and parecoxib, that is, related substance I and related substance II; determination of the flow of said related substance I and related substance II
  • the phases are a mixed solution of methanol and an aqueous solution of potassium dihydrogen phosphate.
  • the method of the present invention is applicable to the detection of parecoxib sodium intermediates, raw materials and degradation products of vardecoxib, process impurities, and positional isomers of target intermediates, which can help control parecoxib more effectively Sodium quality.
  • FIG. 1 is a schematic diagram showing the separation of each impurity peak of a system suitability solution under the conditions of the relevant substance I in the present invention
  • FIG. 2 is a schematic diagram showing the separation of each impurity peak of a system suitability solution under the conditions of the related substance II in the present invention.
  • the invention adopts reversed-phase high-performance liquid chromatography, uses pentafluorophenyl bonded silica gel and octadecylsilane bonded silica gel as chromatographic column packing, adopts a UV detector, and selects a mobile phase containing potassium dihydrogen phosphate for elution. , Divided into two determinations of related substances in parecoxib sodium intermediate vardecoxib and parecoxib, that is, related substance I and related substance II; the mobile phase of the related substance I and related substance II was determined It is a mixed solution of methanol and aqueous potassium dihydrogen phosphate.
  • the method for detecting positional isomers in the intermediate of parecoxib sodium synthesis comprises the following steps:
  • the gradient is as follows:
  • Detection wavelength 210 ⁇ 220nm (preferably 215nm)
  • Injection volume 2 to 20 ⁇ l (preferably 10 ⁇ l)
  • Test solution Precisely weigh parecoxib sodium (about 25mg is placed in a 50ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, cool, dilute the solvent to the mark, and shake to obtain.
  • Cloth sodium concentration is about: 0.5mg / ml.
  • Impurity mother liquor Precisely weigh the impurities IM9, IM10, IM11, IM2, IM2-1, IM2-2, IM2-3, IM2-4, IM3-1, IM3-2, IM3-3 and IM3-4. About 10mg is placed in a 20ml volumetric flask, diluted with solvent to the mark, and shaken. Transfer 1ml to a 20ml volumetric flask and dilute to the mark with solvent. (IM11, IM2, IM2-1, IM2-2, IM2-3, IM2-4, IM3-1, IM3-2, IM3-3 and IM3-4 concentrations are about: 25 ⁇ g / ml).
  • the gradient is as follows:
  • Detection wavelength 210 ⁇ 220nm (preferably 215nm)
  • Injection volume 2 to 20 ⁇ l (preferably 10 ⁇ l)
  • Test solution accurately weigh API (approximately 25mg is placed in a 50ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, cool, dilute the solvent to the mark, and shake to obtain. (API concentration: 0.5 mg / ml).
  • Impurity mother liquor Precisely weigh impurities IM9, IM10, IM11, IM2, IM2-1, IM2-2, IM2-3, IM2-4, IM3-1, IM3-2, IM3-3 and IM3-4 About 10mg is placed in a 20ml volumetric flask, diluted with solvent to the mark, and shaken. Transfer 5ml to a 100ml volumetric flask and dilute to the mark with solvent. (IM11, IM2, IM2-1, IM2-2, IM2-3, IM2-4, IM3-1, IM3-2, IM3-3 and IM3-4 concentrations are about: 25 ⁇ g / ml).
  • System suitability solution Precisely weigh about 25mg of API into a 50ml volumetric flask, add 1.5ml of "control mother solution under 2.2 control solution", dilute to the mark with the solvent, and shake to obtain.
  • the API concentration is about 0.5mg / ml, and the IM9 and IM10 concentrations are about: 0.75ug / ml).
  • the system suitability map is shown in Figure 2 of the accompanying drawings.
  • Control solution Precisely weigh about 10 mg of each of the impurities IM9, IM10, and IM11 and place them in the same 20ml volumetric flask. Dilute with solvent to the mark and shake. Transfer 1ml to a 20ml volumetric flask, dilute to the mark with solvent, and shake well. Transfer 1.5ml to a 50ml volumetric flask and dilute to the mark with solvent. (IM9 and IM10 concentrations are approximately: 0.75 ⁇ g / ml).
  • the impurity IM9 in the related substance method I coincides with the intermediate IM2, the impurity IM10 and the parecoxib peak position, so the related substance method I is used for the parixib sodium related substances IM2-1, IM2-2, IM2-3 , IM3-1, IM3-2, IM3-3 and positional isomers IM2-4 and IM3-4;
  • the impurity IM2-4 and the intermediate IM2 peak position coincide, so the method II for the related substances is used Detection of related substances of parecoxib sodium IM9, IM10, IM11.
  • Thermo U3000 high performance liquid chromatograph pentafluorophenyl bonded silica gel (Thermo Hypersil GOLD PFP 4.6 * 250mm / 5um) and octadecylsilane bonded silica gel as filler (Thermo Hypersil GOLD 4.6 * 250mm / 5um ),
  • the column temperature is 30 ° C
  • the flow rate is 1.0ml / min
  • the detection wavelength is 215nm.
  • An appropriate amount of 4 and IM10 reference materials were dissolved in methanol and diluted to make a solution containing about 0.5 mg of parecoxib sodium and about 0.75 ⁇ g per 1 ml as a system suitability solution.
  • the impurity IM9 in method I coincides with the intermediate IM2, the impurity IM10 and the parecoxib peak location; the impurity II2-4 in the method II coincides with the intermediate IM2 peak location.
  • the relevant substance method I was used for the reaction control, intermediate and final product detection.

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Abstract

一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法。本发明采用反相高效液相色谱法，以五氟苯基键合硅胶和十八烷基硅烷键合硅胶为色谱柱填料，采用紫外检测器，选用含有磷酸二氢钾的流动相进行洗脱，分为两次测定帕瑞昔布钠中间体伐地昔布和帕瑞昔布中的有关物质，即有关物质Ⅰ和有关物质Ⅱ；测定有关物质Ⅰ和有关物质Ⅱ的流动相均为甲醇与磷酸二氢钾水溶液的混合溶液。本发明采用液相色谱法两种梯度方法结合对11种帕瑞昔布钠及其中间体的有关物质完成了同时检测，其中包括了较难分离的5种位置异构体杂质，方法灵敏度高，重复性好，精密度高。

Description

一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法 技术领域
本发明属于医药分析领域，具体涉及的是液相色谱同时检测帕瑞昔布钠及其合成中间体中12种有关物质，尤其是5种位置异构体的检测方法。
背景技术
非甾体抗炎药(NSAID)的作用机制是通过抑制环加氧酶(COX)的活性而抑制前列腺素合成，从而发挥抗炎、退热和镇痛效应。常规非甾体抗炎药抑制组成型表达的COX-1，同时抑制炎症相关的或诱导型的COX-2。COX-1会催化产生正常细胞功能所需的前列腺素，因而应用常规NSAID会抑制COX-1从而引起某些毒副作用。与此不同，若药物能够选择性抑制COX-2而基本上不抑制COX-1，则会获得抗炎、退热、镇痛和其它有用的治疗效果，同时降低或消除所述毒副作用。因此，选择性COX-2抑制药物是本领域内一大进步。
帕瑞昔布钠(Parecoxib Sodium)是选择性COX-2抑制药物伐地昔布的水溶性前体药物，是第一个注射用COX-2抑制剂。帕瑞昔布钠在静注或肌注后经肝脏酶水解，迅速转化为有药理学活性的物质伐地昔布。帕瑞昔布可用于术后中重度急性疼痛的治疗。其临床疗效已经在口腔科、妇科、骨科等多种手术术后的止痛治疗中得到证实，并且术后静脉给予本品可减少吗啡的用量，进而提高术后镇痛的质量。帕瑞昔布钠的化学名称为N-[[4-(5-甲基-3-苯基-4-异恶唑基)苯基]磺酰基]丙酰胺钠盐，结构式见下：
Figure PCTCN2018114746-appb-000001
尽管目前已经可以大规模地生产帕瑞昔布钠，但帕瑞昔布钠有关物质的分析检测方法仍未被收录于各国药典，而注射用帕瑞昔布钠进口注册标准中主要关注帕瑞昔布钠的降解杂质，对原料药及原料药合成中间体中一些工艺杂质无法有效地分离，更无法准确定量检测这些杂质，所以无法有效保证原料药及制剂产品的质量。经检索，目前针对帕瑞昔布钠有关物质分析方法的相关专利(如CN201510181846.4)即使对部分工艺杂质能够有效分离定量，但对于合成过程中产生的位置异构体的控制不够充分。
本发明能够有效分离帕瑞昔布钠合成过程中潜在的12个有关物质，尤其对合成过程中存在的5个位置异构体有较强的分离能力。
发明内容
发明目的：为了解决现有技术的不足，本发明提供了一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法。
技术方案：一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，包括如下步骤：
步骤1：系统适用性实验：(1)分别取各杂质和帕瑞昔布钠的对照品，以甲醇稀释为混合溶液，作为系统适用性溶液；(2)设定液相色谱仪，并将所述系统适用性溶液注入液相色谱仪，记录色谱图，直至各杂质峰与帕瑞昔布钠峰之间的分离度符合规定；
步骤2：测定实验：(1)取待测帕瑞昔布钠于容器中，加甲醇溶解并稀释，作为供试品溶液；(2)取所述各杂质，加甲醇稀释，作为对照溶液；(3)分别取所述供试品溶液和对照溶液注入液相色谱仪，记录色谱图；(4)根据对照溶液和供试品溶液的色谱图中的峰，按外标法计算。
作为优化：所述系统适用性溶液为每毫升含有帕瑞昔布钠0.5mg、各杂质均为0.75μg的溶液。
作为优化：在使用液相色谱仪时，注入待测溶液为10μl。
作为优化：所述供试品溶液的浓度为0.5mg/ml，所述对照溶液的浓度为0.75μg/ml。
作为优化：所述溶剂为甲醇。
作为优化：所述的液相色谱分析方法使用的仪器包括液相色谱仪、色谱数据处理系统与色谱柱。
作为优化：所述的色谱柱选用250mm×4.6mm硅胶柱，内装五氟苯基键合硅胶和十八烷基硅烷键合硅胶填料，所述填料粒径为5μm。
作为优化：所述的液相色谱仪的检测波长为215nm，以0.01mol/l KH 2PO 4缓冲液为流动相A，以甲醇为流动相B，并进行梯度洗脱，其中缓冲液pH范围为2.5～3.5。
一种所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法在其他物质的检测分析中的应用：
步骤1：系统适用性实验：(1)分别取各杂质和某物质的对照品，以甲醇为混合溶液，作为系统适用性溶液；(2)设定液相色谱仪，并将所述系统适用性溶液注入液相色谱仪，记录色谱图，直至各杂质峰与某物质之间的分离度符合规定，固定所述设定；
步骤2：测定实验：(1)取待测某物质于容器中，加溶剂溶解并稀释，作为供试品溶液；(2)取所述各单个杂质适量，加溶剂稀释，作为对照溶液；(3)分别取所述供试品溶液和对照溶液注入液相色谱仪，记录色谱图；(4)根据供试品溶液的色谱图中的峰，按外标法计算。
有益效果：本发明的具体优势如下：
1、本发明采用反相高效液相色谱法，以五氟苯基键合硅胶和十八烷基硅烷键合硅胶为色谱柱填料，采用紫外检测器，选用含有磷酸二氢钾的流动相进行洗脱，分为两次测定帕瑞昔布钠中间体伐地昔布和帕瑞昔布中的有关物质，即有关物质Ⅰ和有关物质Ⅱ；测定所述有关物质Ⅰ和有关物质Ⅱ的流动相均为甲醇与磷酸二氢钾水溶液的混合溶液。
2、本发明采用液相相色谱法两种梯度方法结合对11种帕瑞昔布钠及其中间体的有关物质完成了同时检测，其中包括了较难分离的5种位置异构体杂质，方法灵敏度高，重复性好，精密度高。
3、本发明方法适用于帕瑞昔布钠中间体、原料药及制剂中降解产物伐地昔布、工艺杂质及目标中间体位置异构体的检测，能够帮助更有效地控制帕瑞昔布钠质量。
附图说明
图1为本发明中有关物质Ⅰ条件下系统适用性溶液各杂质峰分离情况示意图；
图2为本发明中有关物质Ⅱ条件下系统适用性溶液各杂质峰分离情况示意图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，以使本领域 的技术人员能够更好的理解本发明的优点和特征，从而对本发明的保护范围做出更为清楚的界定。本发明所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
本发明采用反相高效液相色谱法，以五氟苯基键合硅胶和十八烷基硅烷键合硅胶为色谱柱填料，采用紫外检测器，选用含有磷酸二氢钾的流动相进行洗脱，分为两次测定帕瑞昔布钠中间体伐地昔布和帕瑞昔布中的有关物质，即有关物质Ⅰ和有关物质Ⅱ；测定所述有关物质Ⅰ和有关物质Ⅱ的流动相均为甲醇与磷酸二氢钾水溶液的混合溶液。
帕瑞昔布钠中间体有关物质见下：
Figure PCTCN2018114746-appb-000002
Figure PCTCN2018114746-appb-000003
Figure PCTCN2018114746-appb-000004
本发明所述的一种检测帕瑞昔布钠合成中间体中位置异构体的方法，包括如下步骤：
1.有关物质I
1.1 色谱条件
色谱柱：五氟苯基键合硅胶为填充剂的色谱柱(优选Thermo Hypersil GOLD PFP 4.6*250mm 5um)
流动相A：0.01mol/l KH 2PO 4缓冲液(pH2.5～3.5，优选pH3.0)
流动相B：甲醇
梯度如下：
时间(min) A(％) B(％)
0 55 45
20 55 45
49 20 80
49.1 55 80
55 55 45
柱温：30～40℃(优选35℃)
流速：0.5～1.5ml/min(优选1.0ml/min)
检测波长：210～220nm(优选215nm)
进样量：2～20μl(优选10μl)
溶剂：甲醇
1.2 样品测定
采用外标法，其具体步骤为：
1.2.1 供试品溶液：精密称取帕瑞昔布钠(约25mg置50ml量瓶中，加溶剂适量，超声使溶解，冷却，加溶剂稀释至刻度，摇匀，即得。帕瑞昔布钠浓度约为：0.5mg/ml。
1.2.2 杂质母液：精密称取杂质IM9、IM10、IM11、IM2、IM2-1、IM2-2、IM2-3、IM2-4、IM3-1、IM3-2、IM3-3和IM3-4各约10mg置20ml量瓶中，加溶剂稀释至刻度，摇匀。移取1ml于20ml量瓶中，加溶剂稀释至刻度即得。(IM11、IM2、IM2-1、IM2-2、IM2-3、IM2-4、IM3-1、IM3-2、IM3-3和IM3-4浓度约为：25μg/ml)。
1.2.3 系统适用性溶液：精密称取帕瑞昔布钠约25mg置50ml量瓶中，分别加入“2.2对照溶液项下对照母液”1.5ml，再加溶剂稀释至刻度，摇匀，即得。(帕瑞昔布钠浓度约为：0.5mg/ml，IM11、IM2、IM2-1、IM2-2、IM2-3、IM2-4、IM3-1、IM3-2、IM3-3和IM3-4浓度约为：浓度约为0.75ug/ml)，系统适用性溶液图谱见附图1。
2.1.4 对照溶液：精密称取杂质IM2、IM2-1、IM2-2、IM2-3、IM2-4、IM3-1、IM3-2、IM3-3和IM3-4各约10mg置20ml量瓶中，加溶剂稀释至刻度，摇匀。移取1ml于20ml量瓶中，加溶剂稀释至刻度，摇匀。移取1.5ml于50ml量瓶中，加溶剂稀释至刻度即得。(IM9和IM10浓度约为：0.75μg/ml)。
2 有关物质Ⅱ
2.1 色谱条件
色谱柱：十八烷基硅烷键合硅胶为填充剂色谱柱(优选Thermo Hypersil GOLD aQ 4.6*250mm 5um)
流动相A：0.01mol/l KH 2PO 4缓冲液(pH2.5～3.5，优选pH3.0)
流动相B：甲醇
梯度如下：
时间(min) A(％) B(％)
0 65 35
20 20 80
25 20 80
25.1 65 35
30 65 35
柱温：30～40℃(优选35℃)
流速：0.5～1.5ml/min(优选1.0ml/min)
检测波长：210～220nm(优选215nm)
进样量：2～20μl(优选10μl)
溶剂：甲醇
2.1.1 供试品溶液：精密称取API(约25mg置50ml量瓶中，加溶剂适量，超声使溶解，冷却，加溶剂稀释至刻度，摇匀，即得。(API浓度约为：0.5mg/ml)。
2.1.2 杂质母液：精密称取杂质IM9、IM10、IM11、IM2、IM2-1、IM2-2、IM2-3、IM2-4、IM3-1、IM3-2、IM3-3和IM3-4各约10mg置20ml量瓶中，加溶剂稀释至刻度，摇匀。移取5ml于100ml量瓶中，加溶剂稀释至刻度即得。(IM11、IM2、IM2-1、IM2-2、IM2-3、IM2-4、IM3-1、IM3-2、IM3-3和IM3-4浓度约为：25μg/ml)。
2.1.3 系统适用性溶液：精密称取API约25mg置50ml量瓶中，分别加入“2.2对照溶液项下对照母液”1.5ml，再加溶剂稀释至刻度，摇匀，即得。(API 浓度约为：0.5mg/ml，IM9和IM10浓度约为：浓度约为0.75ug/ml)，系统适用性图谱见附图2。
2.1.4 对照溶液：精密称取杂质IM9、IM10和IM11各约10mg置同一20ml量瓶中，加溶剂稀释至刻度，摇匀。移取1ml于20ml量瓶中，加溶剂稀释至刻度，摇匀。移取1.5ml于50ml量瓶中，加溶剂稀释至刻度即得。(IM9和IM10浓度约为：0.75μg/ml)。
其中有关物质方法I中杂质IM9与中间体IM2，杂质IM10和帕瑞昔布出峰位置重合，故有关物质方法I用于帕瑞昔布钠有关物质IM2-1、IM2-2、IM2-3、IM3-1、IM3-2、IM3-3及位置异构体IM2-4及IM3-4的检测；方法II中杂质IM2-4与中间体IM2出峰位置重合，故有关物质方法II用于帕瑞昔布钠有关物质IM9、IM10、IM11的检测。
本发明的帕瑞昔布钠合成中间体及其有关物质检测如下：
仪器及色谱条件：
采用Thermo U3000高效液相色谱仪，以五氟苯基键合硅胶(Thermo Hypersil GOLD PFP 4.6*250mm/5um)和十八烷基硅烷键合硅胶为填充剂(Thermo Hypersil GOLD aQ 4.6*250mm/5um)，柱温为30℃；流速为1.0ml/min；检测波长为215nm。
有关物质Ⅰ以0.01mol/l的磷酸二氢钾水溶液作为流动相A；以甲醇作为流动相B；按下表进行梯度洗脱：
时间(min) A(％) B(％)
0 55 45
20 55 45
49 20 80
49.1 55 80
55 55 45
精密量取系统适用性溶液和供试品溶液各10μl，分别注入液相色谱仪，记录色谱图；
有关物质Ⅱ以0.01mol/l的磷酸二氢钾水溶液作为流动相A；以甲醇作为流动相B；按下表进行梯度洗脱：
时间(min) A(％) B(％)
0 65 35
20 20 80
25 20 80
25.1 65 35
30 65 35
精密量取系统适用性溶液、供试品溶液和对照溶液各10μl，分别注入液相色谱仪，记录色谱图；
实验步骤：
取帕瑞昔布钠适量，用甲醇溶解并稀释制成每1ml中约含0.5mg的溶液，作为供试品溶液；精密量取供试品溶液适量。
另精密称定帕瑞昔布钠、伐地昔布、杂质IM2-1、IM2-2、IM2-3、IM2-4、IM9、IM11、IM3-1、IM3-2、IM3-3、IM3-4及IM10对照品各适量，用甲醇溶解并稀释制成每1ml中含帕瑞昔布钠约0.5mg、约0.75μg的溶液，作为系统适用性溶液。
其中方法I中杂质IM9与中间体IM2，杂质IM10和帕瑞昔布出峰位置重合；方法II中杂质IM2-4与中间体IM2出峰位置重合。通过对磺酰化反应样品的检测，确认反应过程中主要杂质为IM2-4，无IM9杂质生成。因此反应中控、中间体和成品检测采用有关物质方法I。

Claims (9)

  1. 一种检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：包括如下步骤：
    步骤1：系统适用性实验：(1)分别取各杂质和帕瑞昔布钠的对照品，以甲醇稀释为混合溶液，作为系统适用性溶液；(2)设定液相色谱仪，并将所述系统适用性溶液注入液相色谱仪，记录色谱图，直至各杂质峰与帕瑞昔布钠峰之间的分离度符合规定；
    步骤2：测定实验：(1)取待测帕瑞昔布钠于容器中，加甲醇溶解并稀释，作为供试品溶液；(2)取所述各杂质，加甲醇稀释，作为对照溶液；(3)分别取所述供试品溶液和对照溶液注入液相色谱仪，记录色谱图；(4)根据对照溶液和供试品溶液的色谱图中的峰，按外标法计算。
  2. 根据权利要求1所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：所述系统适用性溶液为每毫升含有帕瑞昔布钠0.5mg、各杂质均为0.75μg的溶液。
  3. 根据权利要求1所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：在使用液相色谱仪时，注入待测溶液为10μl。
  4. 根据权利要求1所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：所述供试品溶液的浓度为0.5mg/ml，所述对照溶液的浓度为0.75μg/ml。
  5. 根据权利要求1所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：所述溶剂为甲醇。
  6. 根据权利要求1所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：所述的液相色谱分析方法使用的仪器包括液相色谱仪、色谱数据处理系统与色谱柱。
  7. 根据权利要求6所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：所述的色谱柱选用250mm×4.6mm硅胶柱，内装五氟苯基键合硅胶和十八烷基硅烷键合硅胶填料，所述填料粒径为5μm。
  8. 根据权利要求6所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法，其特征在于：所述的液相色谱仪的检测波长为215nm，以0.01mol/l KH 2PO 4缓冲液为流动相A，以甲醇为流动相B，并进行梯度洗脱，其中缓冲液pH范围为2.5～3.5。
  9. 一种权利要求1-8任一项所述的检测帕瑞昔布钠及合成中间体中有关物质的液相色谱方法在其他物质的检测分析中的应用，其特征在于：
    步骤1：系统适用性实验：(1)分别取各杂质和某物质的对照品，以甲醇为混合溶液，作为系统适用性溶液；(2)设定液相色谱仪，并将所述系统适用性溶液注入液相色谱仪，记录色谱图，直至各杂质峰与某物质之间的分离度符合规定，固定所述设定；
    步骤2：测定实验：(1)取待测某物质于容器中，加溶剂溶解并稀释，作为供试品溶液；(2)取所述各单个杂质适量，加溶剂稀释，作为对照溶液；(3)分别取所述供试品溶液和对照溶液注入液相色谱仪，记录色谱图；(4)根据供试品溶液的色谱图中的峰，按外标法计算。
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