158硫酸软骨素钠(供口服用)成品、半成品质量标准修订.doc

文件名称:硫酸软骨素钠（供口服用）成品、半成品质量标准 文件编码:TC0158-02Doc.Name:Quality Standard of Finished & Bulk Product of Chondroitin Sulfate Sodium (for oral) Doc.No.: TC0158-02硫酸软骨素钠（供口服用）成品、半成品质量标准Quality Standard of Finished & Bulk Product ofChondroitin Sulfate Sodium (for oral)【中文名】硫酸软骨素钠（供口服用） 【Chinese name】硫酸软骨素钠（供口服用）【汉语拼音名】Liusuan Ruangusuna（Gongkoufuyong）【Chinese Phonetic Alphabet】Liusuan Ruangusuna（Gongkoufuyong）【英文名】Chondroitin Sulfate Sodium(for oral) 【English name】Chondroitin Sulfate Sodium(for oral)【曾用名】硫酸软骨素、硫酸软骨素A钠【Former name】Chondroitin Sulfate、Chondroitin Sulfate A Sodium【物料编码】半成品：B04，成品：C04【Material code】Bulk product: B04,Finished product ：C04. A.中国标准CHINESE STANDARD本品系自猪的喉骨、鼻中骨、气管等软骨组织提取制得的硫酸化链状粘多糖钠盐。硫酸软骨素钠主要为N-乙酰半乳糖胺（2-乙酰胺-2脱氧-D-吡喃半乳糖）和D-葡萄糖醛酸的共聚物的硫酸酯钠盐，共聚物内己糖通过-1，3及-1，4糖苷键交替连接。按干燥品计算，含硫酸软骨素钠法定标准应为90.0 %105.0 %；内控标准应为92.0 %102.0 %。Chondroitin Sulfate Sodium(for oral) is the sodium salt of the sulfated linear glycosaminoglycan obtained from cartilage system of pig such as throat bone, nasal bone and trachea and so on . Chondroitin Sulfate Sodium consists mostly of the sodium salt of the sulfate ester of N-acetylchondrosamine (2-acetamido-2-deoxy-D- galactopyranose) and D-glucuronic acid copolymer. These hexoses are alternately linked -1, 4 and -1, 3 in the polymer. Calculated on the dried basis, the official standard is 90.0 %105.0 %of chondroitin sulfate sodium, the internal standard is 92.0 %102.0 % of chondroitin sulfate sodium.【性状】CHARACTERS本品为白色或类白色粉末；略带咸味；无臭；有引湿性。本品的水溶液具粘稠性，加热不凝结。本品在水中易溶，在乙醇、丙酮或冰醋酸中不溶。White or off-white powder , saltish , odourless and hygroscopic. The aqueous solution is ropy , no clotting.Freely soluble in water, insoluble in ethanol , acetone and glacial acetic acid.【鉴别】IDENTIFICATION（1） 本品的红外光吸收图谱应与硫酸软骨素钠对照品的图谱一致。照红外分光光度法操作规程（WC0201-01）。Infra-red spectra of this substance should be corresponding to the infra-red reference spectra of Chondroitin Sulfate Sodium. Following the SOP of Infrared Spectrophotometry（WC0201-01）.（2） 本品含量测定项下硫酸软骨素A与C的峰高比应为5.06.0。Under the item of Content Determination ,the peak height of the Chondroitin Sulfate A and C should be 5.06.0. （3）本品的水溶液显钠盐的鉴别（1）反应（中国药典2010年版二部附录II ）。The identification of sodium of aqueous solution.（Chinese pharmacopoeia P.2010 edition, second part，appendix II）。【检查】TESTS 比旋度 取本品，精密称定，用水溶解并定量稀释制成每1 mL中含40 mg的溶液。照旋光度测定法操作规程（WC0173-01）测定，比旋度法定标准应为28°至33°，内控标准应为29°至32°。Specific Rotation: Accurately weigh the substance ,dissolve with water and dilute to the solution of 40mg in 1ml. Test following the SOP for Optical Rotation Test（WC0173-01）.The official standard of Specific Rotation is 28°to 33°,the internal standard is 29°to 32°.酸度 取本品0.5 g，用水10 mL溶解后，照PH值测定法操作规程（WC0152-01）测定，PH值法定标准应为6.07.0，内控标准应为6.26.8。PH Sample 0.5g the product, dissolve with 10ml water .Test following the SOP of PH Test, the official standard is 6.07.0,the internal standard is 6.26.8.含氮量 取本品，照氮测定法操作规程（WC0156-02第二法）进行测定，按干燥品计算，含氮（N）量法定标准应为2.53.5 %，内控标准应为2.63.4 %。Nitrogen content :Sample and test following the SOP of Nitrogen Test（WC0156-02 sencond method ）.The official standard of nitrogen content is 2.53.5 %,the internal standard is 2.63.4 %.氯化物 取本品约0.01 g，照氯化物检查法操作规程（WC0159-01）测定，与标准氯化钠溶液5 mL制成的对照液比较，不得更浓（ 0.5 %）。Chloride Weigh 0.01g the product ,test following the SOP of Chloride Test（WC0159-01）。Compare to the reference solution prepared from 5ml standard solution of NaCl,NMT 0.50 %.硫酸盐 称取本品0.1 g，照硫酸盐检查法操作规程（WC0172-01）测定，与标准硫酸钾溶液2.4 mL制成的对照液比较，不得更浓（ 0.24 %）。Weigh 0.1g this substance , test following SOP for Sulfate Test （WC0172-01），Compare to the reference solution prepared from 2.4ml standard solution of potassium sulfate,NMT 0.24 %. 干燥失重 取本品，照干燥失重检查法操作规程（WC0160-01），在105干燥4小时，减失重量法定标准不得过10.0 %，内控标准不得过8.0 %。Loss on drying: Sample and test the substance following the SOP for Loss On Drying Test （WC0160-01）。Dry at 105 for 4 h, the official standard of loss is NMT 10.0 % of its weight, internal standard is NMT 8.0 %.炽灼残渣 取本品1.0 g，照炽灼残渣检查法操作规程（WC0153-01）测定，按干燥品计算，遗留残渣法定标准应为20.0 %30.0 %，内控标准应为22.0 %28.0 %。Residue on ignition: Sample 1.0g the substance ,test following the SOP of Residue On Ignition Test （WC0153-01）.Calculate on the dried basis ,the official standard of the residue is 20.0 %30.0 %,the internal standard is 22.0 %28.0 % 重金属 取炽灼残渣项下遗留的残渣，照重金属检查法操作规程（WC0154-01第二法）测定，含重金属不得过百万分之二十。Heavy Metal Sample the residue under the item of Residue On Ignition,Test following the SOP of Heavy Metal Test（WC0154-01 the second method）.The content of heavy metal is NMT 20 ppm.残留溶剂 取本品，照残留溶剂测定法操作规程（WC0170-01）测定。所含乙醇量应不超过0.5 %。Residual solvent Sample the substance ,test following the SOP of Residual Solvent （WC0170-01）. The content of ethanol is NMT 0.5 %.【含量测定】Content 照高效液相色谱法操作规程（WC0194-01）测定。Test following the SOP for High Performance Liquid Chromatography Method（WC0194-01）。色谱条件与系统适用性试验：用强阴离子交换硅胶为填充剂，以pH 3.5的水为流动相A，以pH 3.5的2 mol/L，氯化钠溶液为流动相B，按下表进行线性梯度洗脱，流速为1.0 mL/min，Chromatographic condition and system suitability Test: Strong anion exchange silica gel as filler and the water of pH 3.5 as mobile phase A , 2 mol/L NaCl solution of pH 3.5 as mobile phase B, perform linear gradient elution corresponding to the table above, the flow rate is 1.0 mL/min,时间（分）Time(minutes)流动相A Mobile PhaseA（%）流动相B Mobile PhaseB（%）0100041000455050检测波长为232 nm。硫酸软骨素B、硫酸软骨素C和硫酸软骨素A的分离度均应符合要求（组分流出顺序为硫酸软骨素B、硫酸软骨素C和硫酸软骨素A）。The determine wavelength is 232 nm. The degrees of separation of chondroitin sulfate A , chondroitin sulfate B and chondroitin sulfate C should meet requirements(The elution order of the components is chondroitin sulfate B, chondroitin sulfate C ,chondroitin sulfate A).对照品溶液的制备：精密称取经105干燥至恒重的硫酸软骨素钠对照品0.1 g，置10 mL的量瓶中，用水溶解并稀释至刻度，摇匀，0.45 µm的滤膜过滤，作为对照品溶液。Preparation of reference solution :Accurately weigh 0.1g chondroitin sulfate sodium dried to constant weight at 105, add 10ml to measuring flask ,dissolve with water and dilute to the scale, mix ,then filter with 0.45 µm filter membrane供试品溶液的制备：取本品约0.1g，精密称定，置10 mL的量瓶中，用水溶解并稀释至刻度，摇匀，0.45 µm的滤膜过滤，作为供试品溶液。Preparation of sample solution: Accurately weigh 0.1g of the substance, add to 10ml measuring flask , dissolve with water and dilute to the scale, mix ,then filter with 0.45 µm filter membrane.测定法Determination Method 量取对照品溶液与供试品溶液各100 µL，各取两份，分别置具塞试管中，各加入50 mmol/l三羟甲基氨基甲烷缓冲液（取三羟甲基氨基甲烷6.06 g与三水乙酸钠8.17 g，用水溶解成900 mL，用盐酸试液调节pH至8.0，加水至1000 mL，即得）800 µL，充分混匀后，加入软骨素ABC酶液（称取适量，用50 mmol/l三羟甲基氨基甲烷缓冲液稀释成每1 µL含0.001单位后使用）100 µL后，摇匀，置于37水浴中反应1小时，取出，在100加热5分钟后，用冷水冷却至室温，以10000转/分钟离心20分钟，取上清液，0.45 µm的滤膜过滤，分别得对照品溶液和供试品溶液的测定液，精密量取20 µL注入液相色谱仪，记录色谱图。按外标法以硫酸软骨素A、B和C的面积之和计算硫酸软骨素的含量。Measure 100 µL reference solution and sample solution respectively , sample duplicates respectively , add to scale test tubes separately ,add 800 µL of 50 mmol/l TRIS buffer(dissolve 6.06g TRIS and 8.17g Sodium acetate trihydrate with water ,adjust PH to 8.0 by HCl ,then add water to 1000 mL) to each tubes. Mix completely, add 100 µL chondroitin ABC enzyme solution (weigh an appropriate quantity, dilute with 50 mmol/l TRIS buffer to the solution which contain 0.001unit per 1µL ),mix , 37 water bath for 1 hour ,take it out ,heat at 100 for 5 minutes, cool down to room temperature with cold water .Centrifuge at the speed of 10000 r/min for 30 minites. Take the clear liquid to filter with 0.45 µm membrane, get the test solution of reference solution and sample solution respectively ,accurately measure 20µL and inject into liquid chromatograph,record the chromatograpm.On the basis of external standard method ,calculate the content of the chondroitin sulfate sodium by the sum of areas of chondroitin sulfate sodium A、B、C.微生物限度检查Microbial Limit Test按微生物限度检查法操作规程（WC0151-01）中的中国标准进行检定，应达到如下要求：Test on the basis of Chinese standard in SOP for Microbial Limit Test（WC0151-01）, the requirement below should be met. 法定标准：细菌总数1000个/g；霉菌和酵母菌100个/g；大肠埃希菌每g不得检出； 沙门菌每10 g不得检出。Official standard: The total bacterial count does not exceed 1000 cfu/g; and the total combined molds and yeasts count does not exceed 100 cfu/g; Escherichia coil is absence per gram; salmonella is absence per 10 grams.内控标准：细菌总数800个/g；霉菌和酵母菌80个/g；大肠埃希菌每g不得检出； 沙门菌每10 g不得检出。Internal standard : The total bacterial count does not exceed 800 cfu/g; and the total combined molds and yeasts count does not exceed 80 cfu/g; Escherichia coil is absence per gram; salmonella is absence per 10 grams.【取样方法】取样按照取样管理规程（MC0002-01）进行。【Sampling Method】Sample following the SMP for Sampling（MC0002-01）【检验方法】检验按照硫酸软骨素钠（供口服用）成品、半成品检验操作规程（WC0108-01）进行。【Test Method】Test following the SOP of Test for Finished & Bulk Product of Chondroitin Sulfate Sodium (for oral)【规格】25kg/桶【Size】25kg/barrel【包装】内层为双层药用低密度聚乙烯塑料袋，外层为方纸桶。【Package】The inlayer is 2 layers of plastic bag made by LDPE for medicinal use,the outer layer is paper babrrel.【类别】降血脂及骨关节疾病治疗药。【Category】Hypolipidemic and treatment for OAD【贮藏】密封，遮光，在干燥处保存。【Storage】Keep away from sunlight, sealed in dry place.【制剂】（1）硫酸软骨素钠片 （2）硫酸软骨素钠胶囊 （3）硫酸软骨素钠滴眼液 （4）硫酸软骨素钠注射液 【Dosage Form】（1）Tablet of chondroitin sulfate sodium (2)Capsule of chondroitin sulfate sodium (3)Eye drop of chondroitin sulfate sodium (4)Injection of chondroitin sulfate sodium 【标准来源】中国药典2010年版第一增补本【Original Standard】Chinese pharmacopoeia P.2010 edition, First Supplement【半成品复验期】一年【Time for reinspection of bulk product】One year.【成品有效期】二年【Expiry Date】Two year.B. 美国标准 UNITED STATE STANDARD硫酸软骨素钠是线性粘多糖硫酸钠盐，是从健康的，被用于人类食用的如牛、猪和禽的软骨中获得。硫酸软骨素钠主要为N-乙酰半乳糖胺（2-乙酰氨基-2-脱氧-D-吡喃半乳糖）和D-葡萄糖醛酸的共聚物硫酸酯钠盐。这些己糖在聚合物中由-1,4 和 -1,3交替连接。糖胺聚糖中的氨基半乳糖基团的单硫酸化主要在第4位，少量在第6位。按干燥品计算，含硫酸软骨素钠在90.0 %105.0 %之间。注：硫酸软骨素钠干燥后极易受潮，应迅速称量，避免暴露在空气中。Chondroitin Sulfate Sodium is the sodium salt of the sulfated linear glycosaminoglycan obtained from bovine, porcine, or avian cartilages of healthy and domestic animals used for food by humans. Chondroitin Sulfate Sodium consists mostly of the sodium salt of the sulfate ester of N-acetylchondrosamine (2-acetamido-2-deoxy-D- galactopyranose) and D-glucuronic acid copolymer. These hexoses are alternately linked -1, 4 and -1, 3 in the polymer. Chondrosamine moieties in the prevalent glycosaminoglycan are monosulfated primarily on position 4 and less so on position 6. It contains NLT 90.0 % and NMT 105.0 % of chondroitin sulfate sodium, calculated on the dried basis.NOTE: Chondroitin Sulfate Sodium is extremely hygroscopic once dried. Avoid exposure to the atmosphere, and weigh promptly. 【鉴定】 IDENTIFICATION1. 红外吸收(USP 197K)：符合规定Infrared absorption (USP 197K): Meets the requirements2. 钠：符合规定Sodium: Meets the requirements测试溶液：本品0.5 g溶解于10 mL水中Sample solution: 0.5 g in 10 mL of water【检查】TESTS炽灼残渣: 按干燥品计算，20.0 %30.0 %。Residue on ignition: 20.0 %30.0 % on the dried basis氯化物：不超过0.50 %，取本品0.01 g与0.020 N盐酸0.7 mL对照液比较，不得更浓（0.50 %）。Chloride: NMT 0.50 %; a 0.10 g portion shows no more chloride than corresponds to 0.7 mL of 0.020 N hydrochloric acid.硫酸盐Sulfate：供试品溶液：取本品200 mg溶解于40 mL 水中，加入30 mg/mL氯化十六烷吡啶溶液10 mL，过滤。取25 mL滤液备用Sample solution: Dissolve 200 mg in 40 mL of water. Add 10 mL of a solution of cetylpyridinium chloride having a concentration of 30 mg/mL, and pass through a filter. Use a 25 mL portion of the filtrate.接受标准：不超过0.24 %；25 mL过滤液与0.020 N 硫酸0.25 mL对照液比较，不得更浓（0.24 %）Acceptance criteria: NMT 0.24 %; the Sample solution shows no more sulfate than corresponds to 0.25 mL of 0.020 N sulfuric acid.电泳纯度Electrophoretic Purity：醋酸钡缓冲液：将25.24 g乙酸钡溶于900 mL水中，乙酸调pH至5.0，稀释至1000 mL.Barium acetate buffer: Dissolve 25.24 g of barium acetate in 900 mL of water. Adjust with acetic acid to a pH of 5.0, and dilute with water to 1000 mL. 染色剂：将1 g甲苯胺蓝溶解于1000 mL 0.1 M乙酸中。Staining reagent: Dissolve 1 g of toluidine blue in 1000 mL of 0.1 M acetic acid.标准溶液A：30 mg/mL USP硫酸软骨素钠RSStandard solution A: 30 mg/mL of USP Chondroitin Sulfate Sodium RS in water标准溶液B：1 mL标准溶液A加水稀释至50 mL。Standard solution B: Dilute 1 mL of Standard solution A with water to 50 mL.供试品溶液：30 mg/mL 硫酸软骨素钠水溶液Sample solution: 30 mg/mL of Chondroitin Sulfate Sodium in water.方法Analysis：用醋酸钡缓冲液填充用于醋酸纤维膜分离的电泳仪的电泳槽（小型凝胶室或专门的膜介质室）。用醋酸钡缓冲液浸泡醋酸纤维膜（56 cm ×1214 cm）10分钟或至均匀湿润，将膜置于两张吸水纸之间吸干。用适于电泳的涂药器，分别涂等体积（0.5 L）的供试品溶液,、标准溶液A、标准溶液B在放置于涂药器台/分离桥上的醋酸纤维膜的亮面。保证膜的两端浸泡在缓冲液中至少0.51.0 cm深。保持电压60伏特（开始时6毫安）2小时（注：5分钟内完成涂样和电压设置，过度的干燥会降低膜的敏感性）。将膜置于塑料染色盘中，涂样面朝下，浮于或轻轻浸入染色剂5分钟，然后轻轻搅拌溶液1分钟，移出膜，用5 %醋酸使其退色直到背景清晰，比较条带。（注：在脱色后15分钟内完成拍照，作为记录结果。）Fill the chambers of an electrophoresis apparatus suitable for separations on cellulose acetate membranes (a small submarine gel chamber or one dedicated to membrane media) with Barium acetate buffer. Soak a cellulose acetate membrane, 56 cm × 1214 cm, in Barium acetate buffer for 10 min, or until evenly wetted, then blot dry between two sheets of absorbent paper. Using an applicator suitable for electrophoresis, apply equal volumes (0.5 L) of the Sample solution, Standard solution A, and Standard solution B to the brighter side of the membrane held in position in an appropriate applicator stand or on a separating bridge in the chamber. Ensure that both ends of the membrane are dipped at least 0.51.0 cm deep into the buffer chambers. Apply a constant 60 volts (6 mA at the start) for 2 h. NOTEPerform the application of solutions and voltage within 5 min because further drying of the blotted paper reduces sensitivity. Place the membrane in a plastic staining tray, and with the application side down, float or gently immerse in Staining reagent for 5 min. Then stir the solution gently for 1 min. Remove the membrane, and destain in 5 % acetic acid until the background clears. Compare the bands. NoteDocument the results by taking a picture within 15 min of completion of destaining.接受标准：电泳获得的供试品溶液主带与标准溶液A的条带位置相同。标准溶液B的条带清晰可见，且其移动性与标准溶液A的条带相似。供试品溶液的任何次要电泳条带不能比标准溶液B条带更加明显。没有超过2 %的单个杂质被发现。（注：在脱色后15分钟内完成拍照，作为记录结果。）Acceptance criteria: The electropherogram from the Sample solution exhibits a major band that is identical in position to the band from Standard solution A. The band from Standard solution B is clearly visible at a mobility similar to the band from Standard solution A. Any secondary band in the electropherogram of the Sample solution is not more intense than the band from Standard solution B. NMT 2 % of any individual impurity is found. NoteDocument the results by taking a picture within 15 min of completion of destaining.蛋白质限度Limit of protein：溶液A：20 mg/mL酒石酸钠Solution A: 20 mg/mL of sodium tartrate dehydrate溶液B：10 mg/mL硫酸铜Solution B: 10 mg/mL of cupric sulfate溶液C：用0.1M氢氧化钠溶解为10 mg/mL的无水碳酸钠Solution C: 10 mg/mL of anhydrous sodium carbonate in 0.1 M sodium hydroxide Dilute 福林酚试剂：用水稀释福林酚TS 5倍（现配）Folin-Ciocalteu reagent: Dilute Folin-Ciocalteu phenol TS with water