达肝素钠标准(USP 39)

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肝素检测细则

肝素盐检测细则一．效价F：（标准≥150IU/mg），环境温度要求20~30℃。

F=标品微升数*预估效价/样品微升数1.1试剂：1羊血浆（冷冻保存，使用时室温解冻，过滤）28IU/mg肝素钠标准品30.9%生理羊水：9g氯化钠（精确至0.01g）溶解于100ml去离子水中，并定容至1000ml，待用。

40.25%氯化钙溶液：用0.9%生理盐水配制，取1.25gCaCl2（精确至0.0001g）溶于100ml生理盐水中，溶解并定容至500ml，待用。

58%柠檬酸钠溶液：用0.9%生理盐水配制，取20g柠檬酸钠（精确至0.0001g）溶于100ml生理盐水中，溶解并定容至250ml，待用。

1.2测定8%柠檬酸钠溶液用量：取3支试管分1、2、3，分别加入标准品溶液110ul，再依次加入8%柠檬酸钠溶液10ul、20ul、30ul，→分别加入0.25%氯化钙溶液0.8ml，加入过滤好的羊血浆1ml，摇匀，置于37℃水浴中10min，观察凝固情况，取半凝固管的柠檬酸钠量xul作为后续的加量标准。

2.1待测品配制：M=20/样品预估效价（g）称取Mg待测样品（精确至0.0001g），溶解于生理盐水中，并定容至100ml，取溶液2ml溶液溶解定容至50ml，取5ml样品溶液于试管中剩余样液冷藏保存。

2.2测定：标准管：取10支试管分别加标品溶液100ul、110ul、120ul、……190ul，加入8%柠檬酸钠溶液xul，0.25%氯化钙溶液0.8ml，过滤好的羊血浆1ml，37°C水浴1h，记录半凝固点。

待测管：取10支试管分别加样品溶液100ul、110ul、120ul、……190ul，加入8%柠檬酸钠溶液xul，0.25%氯化钙溶液0.8ml，过滤好的羊血浆1ml，37°C水浴1h，记录半凝固点。

二．PH值精确称取0.4g样品（精确至0.01g）溶于40ml去离子水中，测定其PH值，记录室温，湿度。

低分子肝素药物的质量标准综述

低分子肝素药物的质量标准综述作者：张利赟来源：《科技传播》2010年第16期摘要本文通过对国内外低分子肝素质量标准、生产工艺、结构特点、药理作用等比较和总结,为我国低分子肝素产业的进一步发展提供参考。

关键词低分子肝素;质量标准;生产工艺;结构;药理中图分类号R9 文献标识码A 文章编号 1674-6708(2010)25-0091-02肝素是临床常用的抗凝血药,但在临床使用中暴露出很多缺点,如:剂量个体差异大,自发性出血倾向,停药后血栓易复发等,还有可引起血小板减少症,长期应用可产生暂时性脱发、骨质疏松和自发性骨折等不良反应[1]。

随着对肝素的深入研究,特别是对以往认为无活性的低分子组分对Xa因子和凝血酶(thrombin)的作用差异的认识,为开发LMWH的提供了理论依据,20世纪70年代末,LMWH进入临床。

低分子量肝素(low-molecular-weight heparin,LMWH)是未分级肝素(unfractionated heparin,UFH)中具有较低分子量的组分或片段,具有与UFH相同的母体结构。

UFH是一种高度硫酸化的糖胺聚糖,分子量范围为3 000~30 000。

LMWH是近20年发展起来的新一代肝素类抗血栓药物,是采用适当方法将肝素分级或降解得到的具有较低分子量的肝素组分或片段3 000~8 000。

在临床使用中,与肝素比较,LMWH抗血栓作用强,抗凝血作用弱于肝素,且具有皮下注射吸收良好、生物利用度高、体内半衰期长、出血倾向小,按体重给药,抗凝效果可以预测等优点[2],目前已逐步取代肝素应用于临床。

国外已有超低分子肝素研究[3],其平均分子量为2 000~3 000,抗FXa/FIIa>30,已经进入III期临床研究。

1 国内外几种LMWH的质量标准综述目前,国外LMWH商品约有十多种,我国进口的有6种,国内仿制的四类新药生产企业约有十几家。

我国有低分子肝素钠部颁标准。

肝素钠_usp33

1
Record the H NMR spectra of the Standard solution and System suitability solution at 25 . 1 Collect the H NMR spectrum with a spectral window of at least 10 to 2 ppm and without spinning. The number of transients should be adjusted until the signal-to-noise ratio of the Nacetyl heparin signal in the Standard solution is at least 1000/1 in the region near 2 ppm. The Standard solution shall be run at least daily when Sample solutions are being run. For all samples, the TSP methyl signal should be set to 0.00 ppm. The chemical shift for the Nacetyl resonance of heparin and oversulfated chondroitin sulfate in the System suitability solution should be observed at 2.05 ± 0.02 and 2.16 ± 0.03 ppm, respectively. Record the H NMR spectrum of the Sample solution at 25 . Draw a baseline from 8.00 ppm to 0.10 ppm. The ppm values for H1 of GlcNAc/GlcNS, 6S (signal 1), H1 of IdoA2S (signal 2), the H2 of GlcNS (signal 3), and the methyl of GlcNAc (signal 4) of heparin are present at 5.42, 5.21, 3.28 (doublet centered at 3.28 ppm), and 2.05 ppm, respectively.1 The ppm values of these signals do not differ by more than ±0.03 ppm. Measure the signal heights above the baseline of signal 1 and signal 2, and calculate the mean of these signal heights. Other signals of variable heights and ppm values, attributable to heparin and HOD, may be seen between signal 2 and 4.55 ppm. Residual solvent signals may be observed in the 0.10–3.00 range. Heparin Sodium must meet the requirements stated in Residual Solvents 467 . Suitability requirements Number of transients: Adjust until the signal-to-noise ratio of the N-acetyl heparin signal in the Standard solution is at least 1000/1 in the region near 2 ppm. Chemical shift: The TSP methyl signal should be set to 0.00 ppm for all samples. Chemical shifts (for the N-acetyl resonance of heparin and oversulfated chondroitin sulfate): Should be observed at 2.05 ± 0.02 and 2.16 ± 0.03 ppm, respectively, System suitability solution Analysis Sample: Sample solution Acceptance criteria: No unidentified signals greater than 4% of the mean of signal height of 1 and 2 are present in the following ranges: 0.10–2.00, 2.10–3.20, and 5.70–8.00 ppm. No signals greater than 200% signal height of the mean of the signal height of 1 and 2 are present in the 3.35–4.55 ppm for porcine heparin . • B. CHROMATOGRAPHIC IDENTITY Solution A: Dissolve 0.8 g of monobasic sodium phosphate dihydrate in 2 L of water and adjust with phosphoric acid to a pH of 3.0. Pass the solution through a filter membrane with pore sizes of 0.45 µm and degas before use. Solution B: Dissolve 0.8 g of monobasic sodium phosphate dihydrate and 280 g of sodium perchlorate monohydrate in 2 L of water and adjust with phosphoric acid to a pH of 3.0. Pass the solution through a filter membrane with pore sizes of 0.45 µm and degas before use. Mobile phase: See the gradient table below. Time (min) 0 60 61 Solution A (%) 80 10 80 Solution B (%) 20 90 20 Elution Equilibration Linear gradient Linear gradient

肝素钠_中国药典2015版公示标准

肝素钠_中国药典2015版公示标准肝素钠GansunaHeparin Sodium■本品系自猪或牛的肠黏膜中提取的硫酸氨基葡聚糖的钠盐，属黏多糖类物质，是由不同分子量的糖链组成的混合物，由α-D-氨基葡萄糖（N-硫酸化，O-硫酸化，或N-乙酰化）和O-硫酸化糖醛酸（α-L-艾杜糖醛酸或β-D葡萄糖醛酸）交替连接形成聚合物，具有延长血凝时间的作用。

按干燥品计算，每1mg中抗Ⅱa因子效价不得少于180 IU，抗Xa因子效价与抗IIa因子效价比为0.9～1.1。

■[修订] ■核酸取本品，加水溶解并稀释制成每1ml中含4mg的溶液，照紫外-可见分光光度法（附录ⅣA）测定，在260nm的波长处，吸光度不得大于0.10。

■[增订]■蛋白质取本品适量，精密称定，加水溶解并稀释制成每1ml中约含30mg的溶液，作为供试品溶液；另取牛血清白蛋白对照品适量，分别加水制成每1ml中各含0、10μg、20μg、30μg、40μg与50μg 的溶液，作为对照品溶液，照蛋白质含量测定法（附录ⅦM 第二法）测定。

按干燥品计，含蛋白质不得过0.5%。

■[增订]■有关物质取本品适量，精密称定，加水溶解并定量稀释制成每1ml中约含100mg的溶液，涡旋混合至完全溶解，取0.5ml，加入1 mol/L盐酸溶液0.25ml和25%亚硝酸钠溶液0.05ml，振摇混匀，反应40分钟，加入1mol/L氢氧化钠溶液0.2ml终止反应，作为供试品溶液；取肝素对照品250mg，加水2ml，涡旋混匀至完全溶解，作为对照品溶液（1）；取对照品溶液（1）1.2ml，加2%硫酸皮肤素对照品0.15ml与2%多硫酸软骨素对照品0.15ml，作为对照品溶液（2）；取对照品溶液（2）0.1ml，加水稀释至1ml，作为对照品溶液（3）；取对照品溶液（1）0.4ml，加水0.1ml，混匀，加1 mol/L盐酸溶液0.25ml和25%亚硝酸钠溶液0.05ml，振摇混匀，反应40分钟，加1mol/L氢氧化钠溶液0.2ml终止反应，作为对照品溶液（4）；取对照品溶液（2）0.5ml，加1 mol/L盐酸溶液0.25ml和25%亚硝酸钠溶液0.05ml，振摇混匀，反应40分钟，加1mol/L氢氧化钠溶液0.2ml终止反应，作为对照品溶液（5）。

64-0015 肝素钠抗FIIa效价检测EP方法与USP方法对比验证方案0000

G：美国标准品质量g；
365：美国标准品浓度USP U/ml。
6.4.1.3.用滴管加入质量为W的新鲜放冷的注射用水到50ml比色管中，盖紧瓶塞轻摇混合，待全部溶解混匀后，按1ml/支的装量，分装于2ml的洁净安瓿中，火焰封口；放入冰箱内2~8℃下保存3个月，使用时放至室温。
6.4.1.4.配制/分装记录填写附件3。
8.00
7.65
总体积（ml）
10.00
10.00
10.00
6.5.2.1.7.样品检测
取12支试管，标记为S1、S2、S3、S1、S2、S3、T1、T2、T3、T1、T2、T3每个浓度做2平行管。做将试管置于冰水浴中，用100-1000ul可调式微量移液器分别加入绵羊血浆1.0ml和相应浓度的标准品溶液、样品溶液1.0ml，混匀，避免产生气泡。将试管按S1、S2、S3、T1、T2、T3的顺序放入恒温水浴箱中，37℃下恒温水浴15分钟；向每支试管中加入1.0ml脑磷脂溶液，37℃下恒温水浴2分钟两分钟后，加入3.7g/L (w/v)氯化钙溶液1.0ml，混匀后以秒计时，记录绵羊血浆凝固所用时间。
质量控制部
刘家全
会审批准签名表
姓名
部门/职务
签名
日期
许万玲
质量控制部
生测Ⅰ组检验员
钱欣
质量保证部经理
李坦
质量总监
注：参加会审的人员：
1.海普瑞公司验证委员会的成员；
2.与该验证方案相关部门的主管（班组长）以上成员。
1.
1.1.对肝素钠抗FⅡa效价检测一般有两种方法：
1.1.1.按照《美国药典》（现行版）中“肝素钠专论”提供的方法，以USP的标准品为基准标准品，其检测结果的单位用USP U/mg表示；

依诺肝素钠标准(USP 39)

•L IMIT OF F LUORIDE ADDITIONAL REQUIREMENTS [N OTE—Use plasticware throughout this test.]•P ACKAGING AND S TORAGE: Preserve in tight, light-resistant Buffer: Dissolve 110g of sodium chloride and 1g of containers, and avoid exposure to excessive heat.sodium citrate in 700mL of water in a 2000-mL volu-•USP R EFERENCE S TANDARDS〈11〉metric flask. Cautiously add 150g of sodium hydroxide,USP Enflurane RSand dissolve with shaking. Cool to room temperature,and, while stirring, cautiously add 450mL of glacialacetic acid to the cooled solution. Cool, add 600mL ofisopropyl alcohol, and dilute with water to volume; thepH of this solution is 5.0–5.5.Enoxaparin SodiumStandard stock solution: 1mg/mL of fluoride ion, pre-pared as follows. Transfer 221mg of sodium fluoride, previously dried at 150° for 4 h, to a 100-mL volumet-ric flask. Add 20mL of water, and mix to dissolve. Add 1.0mL of sodium hydroxide solution (1 in 2500), and dilute with water to volume. [N OTE—Store in a tightly closed plastic container.]Standard solution A: 100mL of a solution containing 1µg/mL of fluoride ion in Buffer, from Standard stock solutionStandard solution B: 100mL of a solution containing 3µg/mL of fluoride ion in Buffer, from Standard stock solutionStandard solution C: 100mL of a solution containing 5µg/mL of fluoride ion in Buffer, from Standard stock solutionStandard solution D: 100mL of a solution containing 10µg/mL of fluoride ion in Buffer, fromStandard stocksolution[9041-08-1].Sample stock solution: Enflurane and water (1:1).Shake the Sample stock solution for 5 min, allow theDEFINITIONliquids to separate completely, and use the water layer.Enoxaparin Sodium is the sodium salt of a depolymerized Sample solution: Sample stock solution and Bufferheparin. It is obtained by alkaline depolymerization of (1:1). Use volumetric glassware.heparin benzyl ester. The starting material, heparin, is ob-Electrode system: Use a pH meter capable of a mini-tained exclusively from porcine intestinal mucosa. Heparin mum reproducibility of ±0.2 mV, equipped with a glass-source material used in the manufacture of Enoxaparin sleeved, calomel-fluoride, specific-ion electrode systemSodium complies with the compendial requirements (see pH 〈791〉).stated in the Heparin Sodium monograph. Enoxaparin So-Analysisdium consists of a complex set of oligosaccharides that Samples: Standard solutions and Sample solutionhave not yet been completely characterized. The majority Transfer the solution to a 150-mL beaker, add aof the components have a 4-enopyranose uronate struc-polytef-coated stirring bar, and immerse the elec-ture at the nonreducing end of their chain. About 20% of trodes in the solution. Stir with a magnetic stirrerthe materials contain a 1,6-anhydro derivative on the re-having an insulated top until equilibrium is obtainedducing end of the chain, the range being between 15% (1–2 min), and record the potential, in mv. [N OTE—and 25%. The weight-average molecular weight of Enox-Rinse and dry the electrodes between measurements,aparin Sodium is 4500 Da, the range being between taking care to avoid damaging the crystal of the spe-3800 and 5000 Da; about 16% have a molecular weight cific-ion electrode.]of less than 2000 Da, the range being between 12.0% Plot the logarithm of the fluoride-ion concentrations,and 20.0%; about 74% have a molecular weight between in µg/mL, of the Standard solutions versus the poten-2000 and 8000 Da, the range being between 68.0% and tial, in mV. From the measured potential of the Sam-82.0%. NMT 18.0% have a molecular weight higher thanple solution and the standard curve, determine the8000 Da. When prepared as a solution, the solution is concentration, in µg/mL, of fluoride ions in the Sam-analyzed for clarity and degree of color using a validated ple solution.method. The degree of sulfation is NLT 1.8per disaccha-Acceptance criteria: NMT 10µg/mLride unit. It has a potency of NLT 90 and NMT 125 Anti-•L IMIT OF N ONVOLATILE R ESIDUEFactor Xa International Units (IU)/mg, and NLT 20.0 and Analysis: Allow 10.0mL to evaporate at room tempera-NMT 35.0 Anti-Factor IIa IU/mg, calculated on the dried ture in a tared evaporating dish, dry the residue at 50°basis. The ratio of Anti-Factor Xa activity to Anti-Factor IIa for 2 h, and weigh.activity is between 3.3 and 5.3.Acceptance criteria: NMT 2mg of residue remains.IDENTIFICATIONSPECIFIC TESTS•A. U LTRAVIOLET A BSORPTION〈197U〉•S PECIFIC G RAVITY〈841〉: 1.516–1.519Medium: 0.01 N hydrochloric acid•R EFRACTIVE I NDEX〈831〉: 1.3020–1.3038 at 20°Sample solution: 500µg/mL•W ATER D ETERMINATION, Method I〈921〉: NMT 0.14%Acceptance criteria: The spectra exhibit maxima at •A CIDITY OR A LKALINITY231±2 nm.Sample: Shake 20mL of Enflurane with 20mL of car-bon dioxide-free water for 3 min, and allow the layersto separate. Draw off the water layer, add bromocresol Change to read:purple TS as the indicator, and titrate with 0.010 Nsodium hydroxide or 0.010 N hydrochloric acid.•B.13C NMR S PECTRUMAcceptance criteria: NMT 0.10mL of 0.010 N sodium(See •Nuclear Magnetic Resonance Spectroscopy 〈761〉•(CN hydroxide or NMT 0.60mL of 0.010 N hydrochloric1-May-2016).)acid is required for neutralization.Standard solution: Dissolve 200mg of USP EnoxaparinSodium RS in a mixture of 0.2mL of deuterium oxideand 0.8mL of water. Add 0.05mL of deuterated meth-Acceptance criteria:M2000 is between 12.0% and anol to serve as an internal reference.20.0%, M2000–8000 is between 68.0% and 82.0%, and Sample solution: Dissolve 200mg of Enoxaparin So-M8000 is NMT 18.0%.dium in a mixture of 0.2mL of deuterium oxide and•E. I DENTIFICATION T ESTS—G ENERAL, Sodium〈191〉: Meets0.8mL of water. Add 0.05mL of deuterated methanol.the requirementsAnalysis: Transfer the Standard solution and the SampleASSAYsolution to NMR tubes of 5-mm diameter. Using apulsed (Fourier transform) NMR spectrometer operatingat NLT 75MHz for 13C, record the 13C NMR spectra of Change to read:the Standard solution and the Sample solution at 40°.Acceptance criteria: The spectra are similar.•ANTI-F ACTOR X a A CTIVITY•C. The ratio of the numerical value of the Anti-Factor Xa Acetic acid solution: Glacial acetic acid and water activity, in Anti-Factor Xa IU/mg, to the numerical value(42:58)of the Anti-Factor IIa activity, in Anti-Factor IIa IU/mg, as pH 7.4polyethylene glycol 6000 buffer: Dissolve determined by the Assay (Anti-Factor Xa Activity) and Im- 6.08g of tris(hydroxymethyl)aminomethane and 8.77g purities (Anti-Factor IIa Activity), respectively, is NLT 3.3of sodium chloride in 500mL of water. Add 1.0g of and NMT 5.3.polyethylene glycol 6000, adjust with hydrochloric acid •D. M OLECULAR W EIGHT D ISTRIBUTION AND W EIGHT-A VERAGE to a pH of 7.4, and dilute with water to 1000mL.M OLECULAR W EIGHT pH 7.4 buffer: Dissolve 6.08g of tris(hydroxymethyl)-Mobile phase: Prepare a 0.5 M lithium nitrate solution.aminomethane and 8.77g of sodium chloride in Pass through a membrane filter of 0.45-µm or smaller500mL of water. Adjust with hydrochloric acid to a pH pore size, and degas with helium.of 7.4, and dilute with water to 1000mL.Standard solution: 10mg/mL of USP Enoxaparin So-pH 8.4 buffer: Dissolve 3.03g of tris(hydroxymethyl)-dium RS in Mobile phase aminomethane, 5.12g of sodium chloride, and 1.40g Sample solution: 10mg/mL of Enoxaparin Sodium in of edetate sodium in 250mL of water. Adjust with hy-Mobile phase drochloric acid to a pH of 8.4, and dilute with water to Chromatographic system500mL.(See Chromatography 〈621〉, System Suitability.)Human antithrombin III solution: Reconstitute a vial Mode: Size exclusion LC of antithrombin III (see Reagents, Indicators, and Solu-Detector: Differential refractive index tions—Reagent Specifications) in water to obtain a solu-Column tion containing 5 Antithrombin III Units/mL. Dilute this Guard: 6-mm × 40-mm; packing L59solution with pH 7.4polyethylene glycol 6000 buffer to Analytical: Two 7.8-mm × 300-mm columns in series;obtain a solution having a concentration of 1.0 Anti-packing L59thrombin III Unit/mL.Temperature: Room temperature Factor Xa solution: Reconstitute a weighed quantity of Flow rate: 0.6mL/min maintained constant to ± 1.0%bovine factor Xa (see Reagents, Indicators, and Solu-Analysis: Reconstitute 1 vial each of USP Enoxaparin tions—Reagent Specifications) in pH 7.4polyethylene gly-Sodium Molecular Weight Calibrant A RS and USP col 6000 buffer to obtain a solution that gives an in-Enoxaparin Sodium Molecular Weight Calibrant B RS in crease in absorbance value at 405 nm of NMT 0.20 1mL of Mobile phase. Separately inject 20µL of USP absorbance units/min when assayed as described below Enoxaparin Sodium Molecular Weight Calibrant A RS but using as an appropriate volume, V, the volume in and USP Enoxaparin Sodium Molecular WeightµL of pH 7.4 buffer instead of VµL of the enoxaparin Calibrant B RS, record the chromatograms, and meas-solution.ure the retention times. Inject in duplicate 20µL each Chromogenic substrate solution: Prepare a solution of of the Standard solution and the Sample solution, and a suitable chromogenic substrate for amidolytic test record the chromatograms for a length of time to en-(see Reagents, Indicators, and Solutions—Reagent Specifi-sure complete elution, including salt and solvent peaks.cations) for factor Xa in water to obtain a concentration Calculate the total peak areas under each of the Stan-of about 3 mM. Dilute with pH 8.4 buffer to obtain a dard solution and Sample solution chromatograms, ex-solution having a concentration of 0.5 mM.cluding salt and solvent peaks at the end.Standard solutions: Reconstitute the entire contents of Calibration curve: Plot the retention times on the x-an ampul of USP Enoxaparin Sodium for Bioassays RS axis against the peak molecular weights on the y-axis with water, and dilute with pH 7.4 buffer to obtain four for the peaks from USP Enoxaparin Sodium Molecular dilutions in the concentration range between 0.025 and Weight Calibrant A RS and USP Enoxaparin Sodium Mo-0.2 Anti-Factor Xa IU/mL.lecular Weight Calibrant B RS, and fit the data to a Sample solutions: Proceed as directed for Standard so-third-order polynomial, using suitable gel permeation lutions to obtain concentrations of Enoxaparin Sodium chromatography (GPC) software.similar to those obtained for the Standard solutions.Calculations: Compute the data, using the same GPC Analysissoftware; determine the weight-average molecular Samples:Standard solutions, Sample solutions, Human weight, M w, for each of the duplicate chromatograms of antithrombin III solution, pH 7.4 buffer, Factor Xa solu-the Standard solution and the Sample solution; and take tion, Chromogenic substrate solution, and Acetic acid the average for each solution. Correct the mean value solutionof M w to the nearest 50. The Chromatographic system is Label 18 suitable tubes: B1 and B2 for blanks; T1, T2, suitable if M w for USP Enoxaparin Sodium RS is within T3, and T4 each in duplicate for the dilutions of the 150 Da of the labeled M w value. The M w for the Sample Sample solutions; and S1, S2, S3, and S4 each in dupli-solution is between 3800 and 5000 Da. Using the same cate for the dilutions of the Standard solutions. [NOTE—software, determine for each of the duplicate Sample Treat the tubes in the order B1, S1, S2, S3, S4, T1, T2,solution chromatograms the percentage of Enoxaparin T3, T4, T1, T2, T3, T4, S1, S2, S3, S4, B2.] To each Sodium chains with molecular weights lower than 2000tube add the same volume, V (20–50µL), of Human Da, M2000, the percentage of Enoxaparin Sodium chains antithrombin III solution and an equal volume, V, of with molecular weights in the range 2000–8000 Da,either the blank (pH 7.4 buffer) or an appropriate dilu-M2000–8000, and the percentage of Enoxaparin Sodium tion of the Sample solutions or the Standard solutions.chains with molecular weights greater than 8000 Da,Mix, but do not allow bubbles to form. Incubate at M8000. Average the duplicate values, and express to the37° for 1.0 min. Add to each tube 2V (40–100µL) of nearest 0.5%.Factor Xa solution, and incubate for 1.0 min. Add a 5V Analysis(100–250µL) volume of Chromogenic substrate solu-Samples:Standard solution A, Standard solution B, tion. Stop the reaction after 4.0 min with a 5V Standard solution C, Cesium chloride solution, and Sam-(100–250µL) volume of Acetic acid solution. Measure ple solutionthe absorbance of each solution at 405 nm, using a Concomitantly determine the absorbances of the Ce-suitable spectrophotometer (see •Ultraviolet-Visible sium chloride solution (blank), Sample solution, and Spectroscopy 〈857〉•(CN 1-May-2016)) against blank B1. The Standard solutions at 330.3 nm, using a sodiumreading of blank B2 relative to blank B1 is NMT ± 0.05hollow-cathode lamp and an air–acetylene flame. Us-absorbance unit.ing the absorbances of Standard solutions A–C, deter-Calculations: For each series, calculate the regression mine the sodium content in the Sample solution after of the absorbance against log concentrations of the an appropriate blank correction.Sample solutions and of the Standard solutions, and cal-Acceptance criteria: 11.3%–13.5% on the dried basis culate the potency of the Enoxaparin Sodium in IU ofIMPURITIESAnti-Factor Xa activity/mL, using statistical methods forparallel-line assays. The four independent log relativepotency estimates are then combined to obtain the Delete the following:final geometric mean. Its confidence limits are calcu-lated. Express the Anti-Factor Xa activity of Enoxaparin••H EAVY M ETALS, Method I〈231〉: NMT 30µg/g, using a Sodium/mg. 2.7% solution in water•(Official 1-Jan-2018) Acceptance criteria: The potency is NLT 90 and NMT125 Anti-Factor Xa IU/mg on the dried basis.SPECIFIC TESTS•P H 〈791〉: 6.2–7.7 for a 10.0% solution in water OTHER COMPONENTS•LOSS ON D RYING〈731〉: Dry 1g in a vacuum at 70° for 6•B ENZYL A LCOHOL C ONTENT h: it loses NMT 10.0% of its weight.Mobile phase: Acetonitrile, methanol, and water(3:1:16)Standard solution: 0.1mg/mL of USP Benzyl Alcohol Change to read:RS in waterSample solution: Weigh 0.5g of Enoxaparin Sodium•S PECIFIC A BSORBANCEinto a 10-mL volumetric flask, and dissolve in 5.0mL of(See •Ultraviolet-Visible Spectroscopy 〈857〉•(CN 1-May-2016).) 1N sodium hydroxide. Allow to stand at room temper-Sample solution: 0.5mg/mL of Enoxaparin Sodium in ature for about 1 h. Add 1.0mL of glacial acetic acid,0.01 N hydrochloric aciddilute with water to volume, and mix.Analysis: Obtain the UV spectra of the Standard solution Chromatographic system and the Sample solution between 200 nm and 300 nm (See Chromatography 〈621〉, System Suitability.)against a 0.01 N hydrochloric acid blank.Mode: LC Calculate the specific absorbance at the wavelength of Detector: UV 256 nm maximum absorbance at 231±2 nm, with reference Column: 4.6-mm × 15-cm stainless steel; packing L7to the dried substance:Flow rate: 1.0mL/min, maintained constant to ±10%Injection volume: 20µL Result = A× 100×1000/[M×l× (100 −E)] AnalysisA= absorbance at the wavelength of maximum Samples:Standard solution and Sample solutionabsorbanceCalculate the percentage of benzyl alcohol in the por-M= weight of Enoxaparin Sodium in the Sample tion of Enoxaparin Sodium taken:solution (mg)Result = (r U/r S) × (C S/C U) × 100l= path length (typically 1cm)E= loss on drying (%)r U= peak area of benzyl alcohol from the Sample Acceptance criteria: 14.0–20.0 on the dried basis solution•B ACTERIAL E NDOTOXINS T EST〈85〉: It contains NMT 0.01 r S= peak area of benzyl alcohol from the Standard USP Endotoxin Unit/IU of Anti-Factor Xa activity.solution•A NTI-F ACTOR II a A CTIVITYC S= concentration of benzyl alcohol in the Acetic acid solution, pH 7.4polyethylene glycol 6000Standard solution (mg/mL)buffer, pH 7.4 buffer, pH 8.4 buffer, and HumanC U= concentration of Enoxaparin Sodium in the antithrombin III solution: Proceed as directed in theSample solution (mg/mL)Assay for Anti-Factor Xa Activity, except that the concen-Acceptance criteria: NMT 0.1%tration of the Human antithrombin III solution is 0.5•N ITROGEN D ETERMINATION, Method II〈461〉: 1.8%–2.5%Antithrombin III Unit/mL.on the dried basis Thrombin human solution: Reconstitute thrombinhuman (see Reagents, Indicators and Solutions—ReagentSpecifications) in water, and dilute in pH 7.4polyethylene Change to read:glycol 6000 buffer to obtain a solution having a concen-tration of 5 Thrombin Units/mL.•S ODIUM C ONTENT Chromogenic substrate solution: Prepare a solution of (See •Atomic Absorption Spectroscopy 〈852〉•(CN 1-May-2016).) a suitable chromogenic substrate for an amidolytic test Cesium chloride solution: 1.27mg/mL of cesium chlo-(see Reagents, Indicators, and Solutions—Reagent Specifi-ride in 0.1 N hydrochloric acid cations) for thrombin in water to obtain a concentration Standard solution A: 0.0025% of sodium chloride in of about 3 mM. Immediately before use, dilute with pH Cesium chloride solution8.4 buffer to 0.5 mM.Standard solution B: 0.0050% of sodium chloride in Standard solutions: Reconstitute the entire contents of Cesium chloride solution an ampul of USP Enoxaparin Sodium for Bioassays RS Standard solution C: 0.0075% of sodium chloride in with water, and dilute with pH 7.4 buffer to obtain four Cesium chloride solution dilutions having concentrations in the range between Sample solution: Transfer 50.0mg of Enoxaparin So-0.015 and 0.075 IU of Anti-Factor IIa activity/mL.dium to a 100-mL volumetric flask, and dissolve in anddilute with Cesium chloride solution to volume.Sample solutions: Proceed as directed under Standard ADDITIONAL REQUIREMENTSsolutions to obtain concentrations of Enoxaparin Sodium•P ACKAGING AND S TORAGE: Preserve in tight containers, similar to those obtained for the Standard solutions.and store below 40°, preferably at room temperature.Analysis: Proceed as directed in the Assay for Anti-Factor•USP R EFERENCE S TANDARDS〈11〉Xa Activity, except to use Thrombin human solution in-USP Benzyl Alcohol RSstead of Factor Xa solution and to use Human antithrom-USP Endotoxin RSbin III solution as described P Enoxaparin Sodium RSCalculations: For each series, calculate the regression of USP Enoxaparin Sodium Molecular Weight Calibrant A RS the absorbance against log concentrations of the Sam-USP Enoxaparin Sodium Molecular Weight Calibrant B RS ple solutions and of the Standard solutions, and calculate USP Enoxaparin Sodium for Bioassays RSthe potency of the Enoxaparin Sodium in IU of Anti-Factor IIa activity/mg, using statistical methods for par-allel-line assays. The four independent dilution estimatesare then combined to obtain the final weighted mean.Then calculate the confidence limits. Express the Anti-Enoxaparin Sodium InjectionFactor IIa activity of Enoxaparin Sodium/mg.Acceptance criteria: It has a potency of NLT 20.0 and DEFINITIONNMT 35.0 Anti-Factor IIa IU/mg on the dried basis.Enoxaparin Sodium Injection is a sterile solution of Enox-•M OLAR R ATIO OF S ULFATE TO C ARBOXYLATE aparin Sodium in Water for Injection. Its appearance is Mobile phase: Carbon dioxide-free water analyzed for clarity and degree of color, using a validated Sample solution: 5mg/mL of Enoxaparin Sodium in method. Its potency value is NLT 90% and NMT 110% of carbon dioxide-free water the potency stated on the label in terms of International Chromatographic system Anti-Factor Xa Units (IU). It may contain, in multiple-dose (See Chromatography 〈621〉, System Suitability.)containers, a suitable antimicrobial preservative, such as Mode: LC benzyl alcohol.Detector: IonColumn: Two columns; one 1.5-cm × 2.5-cm column,IDENTIFICATIONpacked with an anion-exchange resin L64packing; and•A.one 1.5-cm × 7.5-cm column, packed with a cation-Analysis: Transfer the total contents of a single-dose exchange resin L65packing. The outlet of the anion-container or 0.4mL from a multiple-dose container to a exchange column is connected to the inlet of the cat-glass test tube, add 2mL of water and 1mL of 2% ion-exchange column.(w/v) protamine sulfate solution, and mix.Flow rate: 1mL/min Acceptance criteria: A creamy white precipitate is Analysis formed.Sample:Sample solution•B. U LTRAVIOLET A BSORPTION〈197U〉[N OTE—Regenerate the anion-exchange column and the Medium: 0.01 N hydrochloric acidcation-exchange column with 1N sodium hydroxide Standard solution: 500µg/mLand 1N hydrochloric acid, respectively, between two Sample solution: Transfer the total content of a single-injections.]dose container or 0.4mL from a multiple-dose con-Inject the Sample solution into the anion-exchange col-tainer to a 100-mL volumetric flask. Dilute with Medium umn, and collect the eluate from the cation-exchange to volume.column in a beaker at the outlet until the ion detector Acceptance criteria: The spectra exhibit maxima at reading returns to the baseline value. Quantitatively231±2 nm.transfer the eluate to a titration vessel containing a•C. I DENTIFICATION T ESTS—G ENERAL, Sodium〈191〉: Meets magnetic stirring bar, and dilute with carbon dioxide-the requirementsfree water to about 60mL. Position the titration vesselASSAYon a magnetic stirrer, and immerse the electrodes.Note the initial conductivity reading, and titrate withapproximately 0.1 N sodium hydroxide added in Change to read:100-µL portions. [N OTE—Prepare the sodium hydroxidesolution in carbon dioxide-free water.] Record the bu-•A NTI-F ACTOR X a A CTIVITYret reading and the conductivity meter reading after Acetic acid solution: Glacial acetic acid and water each addition of the sodium hydroxide solution.(42:58)Calculations: Plot the conductivity measurements on pH 7.4polyethylene glycol 6000 buffer: Dissolve the y-axis against the volumes of sodium hydroxide 6.08g of tris(hydroxymethyl)aminomethane and 8.77g added on the x-axis. The graph will have three linear of sodium chloride in 500mL of water. Add 1.0g of sections—an initial downward slope, a middle slight polyethylene glycol 6000, adjust with hydrochloric acid rise, and a final rise. For each of these sections draw the to a pH of 7.4, and dilute with water to 1000mL.best-fit straight lines, using linear regression analysis. At pH 7.4 buffer: Dissolve 6.08g of tris(hydroxymethyl)-the points where the first and second straight lines in-aminomethane and 8.77g of sodium chloride in tersect and where the second and third lines intersect,500mL of water. Adjust with hydrochloric acid to a pH draw perpendiculars to the x-axis to determine the of 7.4, and dilute with water to 1000mL.volumes of sodium hydroxide taken up by the sample pH 8.4 buffer: Dissolve 3.03g of tris(hydroxymethyl)-at those points. The point where the first and second aminomethane, 5.12g of sodium chloride, and 1.40g lines intersect corresponds to the volume of sodium hy-of edetate sodium in 250mL of water. Adjust with hy-droxide taken up by the sulfate groups (V S). The point drochloric acid to a pH of 8.4, and dilute with water to where the second and third lines intersect corresponds500mL.to the volume of sodium hydroxide consumed by the Human antithrombin III solution: Reconstitute a vial sulfate and the carboxylate groups together (V T).of antithrombin III (see Reagents, Indicators, and Solu-Calculate the molar ratio of sulfate to carboxylate:tions—Reagent Specifications) in water to obtain a solu-tion containing 5 Antithrombin III Units/mL. Dilute this Result = V S/(V T−V S)solution with pH 7.4polyethylene glycol 6000 buffer toobtain a solution having a concentration of 1.0 Anti-Acceptance criteria: The molar ratio of sulfate to car-thrombin III Unit/mL.boxylate is NLT 1.8.。

依诺肝素钠新版美国药典标准

Revision Bulletin1Official December 1, 2008Add the following:the dried basis. The ratio of anti-factor X a activity to anti-factor II a activity is between 3.3 and 5.3.Packaging and storage—Preserve in tight containers, and store be-low 40°, preferably at room temperature.sEnoxaparin SodiumUSP Reference standards 〈11〉—USP Benzyl Alcohol RS . USP En-dotoxin RS . USP Enoxaparin Sodium RS . USP Enoxaparin Sodium Solution for Bioassays RS . USP Enoxaparin Sodium MolecularWeight Calibrant A RS. USP Enoxaparin Sodium Molecular Weight Calibrant B RS.Identification—A: Ultraviolet Absorption 〈197U 〉—Solution:500µg per mL.Medium:0.01N hydrochloric acid. The spectra exhibit maxima at 231±2 nm.B:13C NMR spectrum (see Nuclear Magnetic Resonance 〈761〉)—Standard solution—Dissolve 200mg of USP Enoxaparin Sodium RS in a mixture of 0.2mL of deuterium oxide and 0.8mL of water.Add 0.05mL of deuterated methanol to serve as an internal reference.Test solution—Dissolve 200mg of Enoxaparin Sodium in a mix-ture of 0.2mL of deuterium oxide and 0.8mL of water. Add 0.05mL of deuterated methanol.[9041-08-1].Procedure—Transfer the Standard solution and the Test solution to NMR tubes of 5-mm diameter. Using a pulsed (Fourier trans-Change to read:form) NMR spectrometer operating at not less than 75MHz for 13C,record the 13C NMR spectra of the Standard solution and the Test » Enoxaparin Sodium is the sodium salt of a de-solution at 40°. The spectra are similar.polymerized heparin. It is obtained by alkaline de-C:The ratio of the numerical value of the anti-factor X a activ-polymerization of heparin benzyl ester. The starting ity, in Anti-Factor X a IU per mg, to the numerical value of the anti-factor II a activity, in Anti-Factor II a IU per mg, as determined by material, heparin, is obtained exclusively from porcine the Assay (anti-factor X a activity) and the Anti-factor II a activity,intestinal mucosa. •Heparin source material used in the respectively, is not less than 3.3 and not more than 5.3.manufacture of Enoxaparin Sodium complies with the D:Molecular weight distribution and weight-average molecu-compendial requirements stated in the Heparin Sodium lar weight—monograph.•(RB 01-Dec-2008) Enoxaparin Sodium consists Mobile phase—Prepare a 0.5M lithium nitrate solution. Pass of a complex set of oligosaccharides that have not yet through a membrane filter having a porosity of 0.45 µm or less, and degas with helium.been completely characterized. The majority of the Standard solution—Dissolve about 10mg of USP Enoxaparin components have a 4-enopyranose uronate structure at Sodium RS, accurately weighed, in 1mL of Mobile phase .the non-reducing end of their chain. About 20 percent Test solution—Dissolve about 10mg of Enoxaparin Sodium, ac-of the materials contain a 1,6-anhydro derivative on the curately weighed, in 1mL of Mobile phase.reducing end of the chain, the range being between 15Chromatographic system (see Chromatography 〈621〉)—The and 25 percent. The weight-average molecular weight high performance size exclusion chromatograph is equipped with a of Enoxaparin Sodium is 4,500 Da, the range being be-differential refractive index detector, a 6- × 40-mm guard column tween 3,800 and 5,000 Da; about 16 percent have a and two 7.8- × 300-mm analytical columns in series, both analytical and guard columns prepacked with packing L59, and used at room molecular weight of less than 2,000 Da, the range being temperature. The flow rate is about 0.6mL per minute maintained between 12.0 and 20.0 percent; about 74 percent have a constant to ±1.0%.molecular weight between 2,000 and 8,000 Da, the Procedure—Reconstitute one vial each of USP Enoxaparin So-range being between 68.0 and 82.0 percent. Not more dium Molecular Weight Calibrant A RS and USP Enoxaparin So-than 18.0 percent have a molecular weight higher than dium Molecular Weight Calibrant B RS in 1mL of Mobile phase.Separately inject 20 µL of USP Enoxaparin Sodium Molecular 8,000 Da. When prepared as a solution, the solution is Weight Calibrant A RS and USP Enoxaparin Sodium Molecular analyzed for clarity and degree of color using a vali-Weight Calibrant B RS, record the chromatograms, and measure the dated method. The degree of sulfation is not less than retention times. Inject in duplicate 20 µL of each of the Standard 1.8 per disaccharide unit. It has a potency of not less solution and the Test solution, and record the chromatograms for a than 90 and not more than 125 Anti-Factor X a Interna-length of time to ensure complete elution, including salt and solvent peaks. Calculate the total area under each of the Standard solution tional Units (IU) per mg, and not less than 20.0 and not and Test solution chromatograms, excluding salt and solvent peaks more than 35.0 Anti-Factor II a IU per mg, calculated onat the end.Calibration curve—Plot the retention times on the x -axis against the peak molecular weights on the y -axis for the peaks in the chro-matograms of USP Enoxaparin Sodium Molecular Weight Calibrant A RS and USP Enoxaparin Sodium Molecular WeightCopyright 2008 The United States Pharmacopeial Convention All Rights Reserved.2 Revision BulletinOfficial December 1, 2008Calibrant B RS, and fit the data to a third-order polynomial using a Mobile phase:carbon dioxide-free water.suitable gel permeation chromatography (GPC) software.Test solution—Dissolve an accurately weighed quantity of about Calculations—Compute the data, using the same GPC software 50mg of Enoxaparin Sodium in 10mL of carbon dioxide-free and determine the weight-average molecular weight, M W , for each water.of the duplicate chromatograms of the Standard solution and the Chromatographic system—The liquid chromatographic system Test solution, and take the average for each solution. Correct the consists of two peristaltic pumps, a six-port injection valve, an ion mean value of M W to the nearest 50. The Chromatographic system detector, and two columns—one 1.5- × 2.5-cm column packed with is suitable if M W of USP Enoxaparin Sodium RS is within 150 Da an anion-exchange resin L64 packing and one 1.5- × 7.5-cm column of the labeled M W value. The M W for the Test solution is between packed with a cation-exchange resin L65 packing. The outlet of the 3,800 and 5,000 Da. Using the same software, determine for each of anion-exchange column is connected to the inlet of the cation-ex-the duplicate Test solution chromatograms the percentage of Enox-change column. The flow rate is about 1mL per minute.aparin Sodium chains with molecular weights lower than 2000 Da,Procedure—[NOTE —Regenerate the anion-exchange column M 2000, the percentage of Enoxaparin Sodium chains with molecular and the cation-exchange column with 1N sodium hydroxide and weights in the range 2000 to 8000 Da, M 2000–8000, and the percentage 1N hydrochloric acid, respectively, between two injections.] Inject of Enoxaparin Sodium chains with molecular weights greater than the Test solution into the anion-exchange column, and collect the 8000 Da, M 8000. Average the duplicate values and express to the eluate from the cation-exchange column in a beaker at the outlet nearest 0.5%. M 2000 is between 12.0% and 20.0%, M 2000–8000 is be-until the ion detector reading returns to the baseline value. Quantita-tween 68.0% and 82.0%, and M 8000 is not more than 18.0%.tively transfer the eluate to a titration vessel containing a magnetic E:It responds to the test for Sodium 〈191〉.stirring bar, and dilute with carbon dioxide-free water to about Specific absorbance (see Spectrophotometry and Light-Scattering 60mL. Position the titration vessel on a magnetic stirrer and im-〈851〉)—merse the electrodes. Note the initial conductivity reading and ti-trate with approximately 0.1N sodium hydroxide added in 100-µL Test solution—Dissolve 50.0mg of Enoxaparin Sodium in portions. [NOTE —Prepare the sodium hydroxide solution in carbon 100mL of 0.01N hydrochloric acid.dioxide-free water.] Record the burette reading and the conductivity Procedure—Obtain the UV spectra of the Standard solution and meter reading after each addition of the sodium hydroxide solution.the Test solution between 200nm and 300nm against 0.01N hy-Calculations—Plot the conductivity measurements on the y -axis drochloric acid blank. Calculate the specific absorbance at the against the volumes of sodium hydroxide added on the x -axis. The wavelength of maximum absorbance at 231±2 nm, with reference graph will have three linear sections—an initial downwards slope, a to the dried substance, using the following formula:middle slight rise, and a final rise. For each of these sections draw the best-fit straight lines using linear regression analysis. At the A × 100×1000 / [M × l × (100 – E )]points where the first and second straight lines intersect and where in which A is the absorbance at the wavelength of maximum ab-the second and third lines intersect, draw perpendiculars to the x-sorbance; M is the weight, in mg, of Enoxaparin Sodium in the Test axis to determine the volumes of sodium hydroxide taken up by the solution; l is the pathlength (typically l = 1cm); and E is the loss on sample at those points. The point where the first and second lines drying, in percent. The specific absorbance is between 14.0 and intersect corresponds to the volume of sodium hydroxide taken up 20.0, calculated on the dried basis.by the sulfate groups (V S ). The point where the second and third Bacterial endotoxins 〈85〉—It contains not more than 0.01 USP lines intersect corresponds to the volume of sodium hydroxide con-Endotoxin Unit per IU of anti-factor X a activity.sumed by the sulfate and the carboxylate groups together (V T ). Cal-pH 〈791〉:between 6.2 and 7.7 of a 10.0% solution in water.culate the molar ratio of sulfate to carboxylate by the formula:Loss on drying 〈731〉—Dry 1g in a vacuum at 70° for 6 hours: it V S /(V T – V S )loses not more than 10.0% of its weight.The molar ratio of sulfate to carboxylate is not less than 1.8.Nitrogen content, Method II 〈461〉:between 1.8% and 2.5%, cal-culated on the dried basis.Benzyl alcohol content—Heavy metals, Method I 〈231〉—Prepare a 5% solution in water:Mobile phase—Prepare a filtered and degassed mixture of water,the limit is not more than 0.0030%.acetonitrile, and methanol (80:15:5 v/v).Sodium content (see Spectrophotometry and Light-Scattering Standard solution—Dissolve 100mg of USP Benzyl Alcohol RS 〈851〉)—in 200mL of water. Transfer 5mL of this solution to a 25-mL volu-metric flask, and dilute with water to volume.Cesium chloride solution—Prepare a solution of cesium chloride in 0.1N hydrochloric acid containing 1.27mg per mL.Test solution—Weigh 0.5g of Enoxaparin Sodium into a 10-mL volumetric flask, and dissolve in 5.0mL of 1N sodium hydroxide.Standard solutions—Dissolve an accurately weighed quantity of Allow to stand at room temperature for about 1 hour. Add 1.0mL sodium chloride in Cesium chloride solution to obtain a solution of glacial acetic acid, dilute with water to volume, and mix.having a known concentration of about 0.2% sodium. Dilute accu-rately measured volumes of this solution with Cesium chloride so-Chromatographic system (see Chromatography 〈621〉)—The liq-lution having known concentrations of 0.0025%, 0.0050%, and uid chromatograph is equipped with a 256-nm detector and a 4.6-0.0075% of sodium.mm × 15-cm stainless steel column that contains L7 packing. The flow rate is about 1.0mL per minute maintained constant to ±10%.Test solution—Transfer an accurately weighed quantity of about 50.0mg of Enoxaparin Sodium to a 100-mL volumetric flask, and Procedure—Separately inject equal volumes (about 20 µL) of the dissolve in and dilute with Cesium chloride solution to volume.Standard solution and the Test solution, record the chromatograms,and measure the peak responses.Procedure—Concomitantly determine the absorbances of the Ce-sium chloride solution (blank), Test solution, and Standard solu-Calculation—Calculate the percentage of benzyl alcohol in tions at 330.3nm using a sodium hollow-cathode lamp and an Enoxaparin Sodium taken by the formula,air–acetylene flame. Using the absorbances of Standard solutions,determine the sodium content in the Test solution after appropriate (A T × C S )/(A S × C T )blank correction. The sodium content, calculated on the dried basis,in which A T is the benzyl alcohol peak area in the Test solution; C S is between 11.3% and 13.5%.is the concentration, in mg per mL, of benzyl alcohol; A S is the area Molar ratio of sulfate to carboxylate (see Chromatography of the benzyl alcohol peak in the Standard solution; and C T is the〈621〉)—Copyright 2008 The United States Pharmacopeial Convention All Rights Reserved.Revision Bulletin3 Official December 1, 2008concentration, in mg per mL, of Enoxaparin Sodium. The percent- Human antithrombin III solution—Reconstitute a vial of an-age of benzyl alcohol is not more than 0.1%.tithrombin III (see Reagent Specifications in the section Reagents,Indicators, and Solutions) in water to obtain a solution containing 5 Anti-factor II a activity—Antithrombin III Units per mL. Dilute this solution with pH 7.4 Acetic acid solution, pH 7.4 Polyethylene glycol 6000 buffer, pHPolyethylene glycol 6000 buffer to obtain a solution having a con-7.4 Buffer, pH 8.4 Buffer, and Human antithrombin III solution—centration of 1.0 Antithrombin III Unit per mL.Proceed as directed under Assay (anti-factor X a activity), except thatFactor X a solution—Reconstitute an accurately weighed quantity the concentration of the Human antithrombin III solution is 0.5 An-of bovine factor X a (see Reagent Specifications in the section Re-tithrombin III Unit per mL.agents, Indicators, and Solutions) in pH 7.4 Polyethylene glycol Thrombin human solution—Reconstitute thrombin human (see6000 buffer to obtain a solution that gives an increase in absorbance Reagent Specifications in the section Reagents, Indicators and So-value at 405nm of not more than 0.20 absorbance units per minute lutions) in water, and dilute in pH 7.4 Polyethylene glycol 6000when assayed as described below but using as an appropriate vol-buffer to obtain a solution having a concentration of 5 Thrombinume (V, in µL) of pH 7.4 Buffer instead of VµL of the enoxaparin Units per mL.solution.Chromogenic substrate solution—Prepare a solution of a suitableChromogenic substrate solution—Prepare a solution of a suitable chromogenic substrate for an amidolytic test (see Reagent Specifi-chromogenic substrate for amidolytic test (see Reagent Specifica-cations in the section Reagents, Indicators, and Solutions) fortions in the section Reagents, Indicators, and Solutions) for factor thrombin in water to obtain a concentration of about 3 mM. Imme-X a in water to obtain a concentration of about 3 mM. Dilute with diately before use, dilute with pH 8.4 Buffer to 0.5 mM.pH 8.4 Buffer to obtain a solution having a concentration of 0.5 Standard solutions—Dilute USP Enoxaparin Sodium Solution for mM.Bioassays RS with pH 7.4 Buffer to obtain four dilutions havingStandard preparations—Dilute USP Enoxaparin Sodium Solu-concentrations in the range between 0.015 and 0.075 IU of anti-tion for Bioassays RS with pH 7.4 Buffer to obtain four dilutions in factor II a activity per mL.the concentration range between 0.025 and 0.2 USP Anti-Factor X a Test solutions—Proceed as directed under Standard solutions to IU per mL.obtain concentrations of Enoxaparin Sodium similar to those ob-Assay preparations—Proceed as directed for Standard prepara-tained for the Standard solutions.tions to obtain concentrations of Enoxaparin Sodium similar to Procedure—Proceed as directed under Assay (anti-factor X a ac-those obtained for the Standard preparations.tivity), except to use Thrombin human solution instead of Factor X aProcedure—Label 18 suitable tubes: B1 and B2 for blanks; T1, solution and to use the Human antithrombin III solution as de-T2, T3, and T4 each in duplicate for the dilutions of the Assay prep-scribed above.arations; and S1, S2, S3, and S4 each in duplicate for the dilutions Calculations—For each series, calculate the regression of the ab-of the Standard preparations. [NOTE—Treat the tubes in the order sorbance against log concentrations of the Test solutions and of the B1, S1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, S2, S3, S4, Standard solutions, and calculate the potency of the enoxaparin so-B2.] To each tube add the same volume, V, (20 to 50 µL) of Human dium in IU of anti-factor II a activity per mg using statistical meth-antithrombin III solution and an equal volume, V, of either the ods for parallel-line assays. The four independent dilution estimates blank, pH 7.4 Buffer, or an appropriate dilution of the Assay prepa-are then combined to obtain the final weighted mean. Then calcu-rations or the Standard preparations. Mix, but do not allow bubbles late the confidence limits. Express the anti-factor II a activity of to form. Incubate at 37° for 1.0 minute. Add to each tube volumeEnoxaparin Sodium per mg, calculated on the dried basis. It has a2V (40 to100 µL) of Factor Xa solution, and incubate for 1.0 min-potency of not less than 20.0 and not more than 35.0 anti-Factor II a ute. Add 5V (100 to 250 µL) volume of Chromogenic substrate so-IU per mg.lution. Stop the reaction after 4.0 minutes with 5V (100 to 250 µL) Assay (anti-factor X a activity)—volume of Acetic acid solution. Measure the absorbance of each so-lution at 405nm using a suitable spectrophotometer (see Spectro-Acetic acid solution—Transfer 42mL of glacial acetic acid to aphotometry and Light-Scattering 〈851〉) against blank B1. The read-100-mL volumetric flask, dilute with water to volume, and mix.ing of blank B2 relative to the blank B1 is not more than ±0.05 pH 7.4 Polyethylene glycol 6000 buffer—Dissolve 6.08g ofabsorbance units.tris(hydroxymethyl)aminomethane and 8.77g of sodium chloride inCalculations—For each series, calculate the regression of the ab-500mL of water. Add 1.0g of polyethylene glycol 6000, adjustsorbance against log concentrations of the Assay preparations and with hydrochloric acid to a pH of 7.4, and dilute with water toof the Standard preparations, and calculate the potency of the 1000mL.enoxaparin sodium in IU of anti-factor X a activity per mL using pH 7.4 Buffer—Dissolve 6.08g of tris(hydroxymethyl)ami-statistical methods for parallel-line assays. The four independent nomethane and 8.77g of sodium chloride in 500mL of water. Ad-log relative potency estimates are then combined to obtain the final just with hydrochloric acid to a pH of 7.4, and dilute with water togeometric mean. Its confidence limits are calculated. Express the 1000mL.anti-factor X a activity of Enoxaparin Sodium per mg, calculated on pH 8.4 Buffer—Dissolve 3.03g of tris(hydroxymethyl)ami-the dried basis. The potency is not less than 90 and not more thannomethane, 5.12g of sodium chloride and 1.40g of edetate sodium125 Anti-Factor Xa IU per mg.s2S(USP31)in 250mL of water. Adjust with hydrochloric acid to a pH of 8.4,and dilute with water to 500mL.Copyright 2008 The United States Pharmacopeial Convention All Rights Reserved.。

核磁共振内标法定量分析肝素钠中多硫酸软骨素

核磁共振内标法定量分析肝素钠中多硫酸软骨素高照明;张玉冰;于永良【摘要】@@ 1 引言rn肝素钠是一种由葡萄糖胺,L-艾杜糖醛苷、N-乙酰葡萄糖胺和D-葡萄糖醛酸交替组成的黏多糖硫酸脂钠盐,是临床大量应用的抗凝血药.研究证明,肝素钠还具有抗炎、抗过敏、抗病毒,抗癌等多种生物学功能.2008年初,欧美各国发生了肝索钠注射液事件,致使81人死亡.随后,美国FDA应用核磁共振方法在肝素钠原料药中定性查出杂质多硫酸软骨素(OSCS),并认为OSCS是引起用药异常的杂质.肝素与OSCS均属于黏多糖硫酸脂聚合物,分子量分布都有一定宽度,且结构及性质相似,因此,常规的化学和生物检测方法很难区分[2].%The quantitative analysis of Oversulfated Chondroitin Sulfate by 1 H-NMR internal standard method has been developed in this paper. The optimum experimental conditions have been described. The linear range of the method was found to be 0. 1％-5 ％. The regression equation derived was y＝2. 7929x-0.0317(r＝0. 9994). The limit of detection was 0. 1％. The average recovery rate was shown to be 98. 1％(n＝6), and the RSD was 0.35 ％(n＝6). The results showed that 1 H-NMR can be used for the quantitative determination of contaminants in heparin.【期刊名称】《分析化学》【年(卷),期】2011(039)004【总页数】2页(P601-602)【作者】高照明;张玉冰;于永良【作者单位】生态化工教育部重点实验室,青岛科技大学化工学院,青岛,266042;生态化工教育部重点实验室,青岛科技大学化工学院,青岛,266042;生态化工教育部重点实验室,青岛科技大学化工学院,青岛,266042【正文语种】中文肝素钠是一种由葡萄糖胺,L-艾杜糖醛苷、N-乙酰葡萄糖胺和D-葡萄糖醛酸交替组成的黏多糖硫酸脂钠盐,是临床大量应用的抗凝血药。

肝素钠事件

班级：组别：组学号姓名：“肝素钠”的严重药害事件一、肝素钠的简介肝素钠( heparin sodium) 是黏多糖硫酸酯类抗凝血药，用于预防和治疗血栓栓塞性疾病，在进行人工心肺、腹膜透析或血液透析时作为抗凝剂使用，即对患者进行多剂量静脉注射以便在很短的时间内溶掉血凝块。

我国是肝素钠原料药的主要生产国，也是全球最大的出口国。

二、“肝素钠”事件简介2007年10月－2008年3月，美国百特公司接到报告称有 81 例患者在使用其生产的肝素钠多剂量瓶装注射液后死亡，另有 300 多人出现了过敏反应和其他不良反应。

经初步调查，上述问题产品的活性成分都来自同一家供应商——美国SPL 公司位于中国常州的一个工厂。

美国 FDA 随即在国内外对不良事件的原因展开调查，并最终用非标方法在这些活性成分中检验出一种类肝素物质的污染物——多硫酸软骨素。

“多硫酸软骨素”为硫酸软骨素的磺化产品，属于硫酸化多糖。

由于临床不良反应与检测出的“多硫酸软骨素”的相关性尚未确定，国家药监局正在组织专家对此进行实验研究。

后经 FDA 承认，由于其混淆了常州 SPL 公司与中国另一家拼写相仿的厂家名称，误以为该公司已通过 GMP 检查，因此未对其进行现场检查，没有发现风险隐患，最终引发上述严重不良事件。

三、“肝素钠”事件对产业的影响2008年2月28日，美国百特医疗公司(以下简称“美国百特”)正式宣布召回大量肝素类产品，包括多剂量瓶装肝素、单剂量瓶装肝素和一种静脉给药留置针。

这是该公司首次宣布大量召回肝素类产品。

虽然本次美国百特产品不在中美双方共同签署的《药品医疗器械安全合作协议》合作框架，但其肝素钠不良事件调查可能已促使FDA考虑将合作检查全球化。

“百特事件”引起了不小的波澜，但未对国内肝素原料生产企业造成负面影响。

业内人士指出，作为一种存在了几十年的成熟技术，中国的肝素提取早就达到了标准化，但由于以前国产低分子肝素制剂受到产品质量、国际专利保护及国际药品市场准入等条件的限制，无法大规模进入国际市场。

肝素钠事件

我国是肝素钠原料药的主要生产国，也是全球最大的出口国。

经初步调查，上述问题产品的活性成分都来自同一家供应商——美国SPL 公司位于中国常州的一个工厂。

美国 FDA 随即在国内外对不良事件的原因展开调查，并最终用非标方法在这些活性成分中检验出一种类肝素物质的污染物——多硫酸软骨素。

“多硫酸软骨素”为硫酸软骨素的磺化产品，属于硫酸化多糖。

由于临床不良反应与检测出的“多硫酸软骨素”的相关性尚未确定，国家药监局正在组织专家对此进行实验研究。

这是该公司首次宣布大量召回肝素类产品。

“百特事件”引起了不小的波澜，但未对国内肝素原料生产企业造成负面影响。

肝素钠分析报告

肝素行业分析报告一、肝素行业和主要产品的简介 (3)1、肝素简介及临床应用 (3)2、肝素类产品 (4)（1）肝素粗品 (6)（2）肝素原料药中间体 (6)（3）精制肝素原料药 (6)（4）标准肝素制剂 (7)（5）低分子量肝素 (7)（6）低分子量肝素制剂 (8)（7）肝素类产品在抗肿瘤方面的具体应用 (9)二、行业技术水平及发展趋势 (10)1、肝素行业技术水平及发展趋势 (10)（1）行业技术水平 (10)（2）发展趋势 (11)2、我国肝素行业发展历程 (12)（1）粗品出口阶段 (12)（2）整体产业提升阶段 (12)（3）肝素原料药高速发展与肝素制剂突破阶段 (12)（4）未来发展趋势 (13)三、行业监管 (13)1、行业主管部门及监管体制 (13)2、行业主要法律法规 (14)（1）药品生产许可证制度 (14)（2）新药证书和药品批准文号制度 (15)（3）药品生产质量管理规范（GMP）和药品经营质量管理规范（GSP） (15)（4）药品的知识产权保护制度 (15)（5）药品定价制度 (16)（6）处方药和非处方药分类管理制度 (16)（7）国家药品标准制度和药品召回制度 (17)3、行业相关产业政策 (17)（1）产业政策 (17)（2）国家基本药物制度 (18)四、医药行业发展概况 (19)五、肝素类产品市场需求情况 (20)1、肝素类药品市场需求概况 (20)（1）肝素类药品市场需求增长的驱动因素 (20)（2）肝素类药品市场需求情况 (23)（3）肝素类产品市场供求情况 (23)2、肝素原料药市场需求 (24)3、肝素制剂市场需求情况 (25)（1）我国标准肝素制剂市场需求情况 (26)（2）我国低分子量肝素制剂市场需求状况 (27)六、行业的关联性、上下游行业发展状况对本行业的影响 (28)1、上游行业的发展及对肝素行业的影响 (29)2、下游行业发展对行业的影响 (30)七、我国肝素行业出口情况 (31)1、我国对肝素钠原料药的出口政策 (33)2、国际药典标准、国际认证证书及主要国家对肝素原料药进口政策 (33)3、主要进口国同类产品的竞争情况 (33)4、贸易摩擦对产品出口的影响 (34)八、进入本行业的主要障碍 (34)1、技术壁垒 (34)2、行政许可准入壁垒 (35)3、药品出口认证壁垒 (35)4、人才壁垒 (35)5、资金实力壁垒 (36)6、产品质量壁垒与品牌壁垒 (36)7、成本及规模经济壁垒 (36)8、市场渠道壁垒 (37)九、行业利润水平的变动趋势及变动原因 (37)1、完整的产业链与利润水平 (37)2、肝素原料药的利润水平变动趋势 (38)3、标准肝素制剂的利润水平变动趋势 (38)4、低分子量肝素钙制剂的利润水平变动趋势 (39)十、行业的周期性、区域性及季节性特征 (39)十一、影响行业发展的因素 (40)1、有利因素 (40)（1）国家产业政策扶持 (40)（2）丰富的肝素粗品供应 (41)（3）行业集中度提高 (41)（4）肝素制剂进入《国家基本药物目录》和《国家医保目录》 (41)（5）产业基础与运行环境持续改善 (42)（6）全球抗凝血和抗血栓药品市场增长迅速，肝素类药品临床适用范围日益拓宽 (42)（7）对肝素类药品认识程度日益加深 (42)（8）随着国内经济和科技的发展，国内药企的研发能力提高 (43)2、不利因素 (43)（1）肝素类药品的政府定价政策 (43)（2）肝素粗品价格上涨 (44)（3）融资渠道不畅 (44)（4）人民币的升值 (44)十二、肝素原料药行业竞争格局 (45)1、国际市场的竞争格局与主要厂商情况 (45)2、国内市场的竞争格局与主要厂商情况 (46)十三、标准肝素制剂行业竞争格局 (48)十四、低分子量肝素制剂行业竞争格局 (49)一、肝素行业和主要产品的简介1、肝素简介及临床应用肝素（Heparin）是一种由葡萄糖胺、L-艾杜糖醛酸、N-乙酰葡萄糖胺和D-葡萄糖醛酸以及他们的硫酸化衍生物组成的糖胺聚糖。

新版的肝素钠欧洲药典标准

NOTE ON THE MONOGRAPHThis monograph has been thoroughly revised further to the contamination events in 2008to update and strengthen the quality standards for unfractionated heparin.The style and presentation have also been updated in line with the latest version of the Style guide.Definition:the minimum potency limit has been raised after an enquiry among European manufacturers in line with the quality of currently marketed heparin batches; only1grade of heparin has been kept as the present2-tiered specification no longer reflects the situation in Europe.Production:the tests for nuclear magnetic resonance spectrometry(NMR)and capillary electrophoresis previously introduced in the1st-step revision applicable from1August 2008have been deleted,as detailed tests are now provided under Identification and Tests;statements have been added to emphasise the need for a reliable quality assurance system throughout production and,based on current practice among European manufacturers,for confirming the identity of the source species as well as the absence of any material issued from other species likely to contaminate the drug substance.This monograph is also revised to harmonise the information related to the source species for substances of human and animal origin and its presentation in monographs.The statement relative to the origin of the substance is moved under Definition accordingly and a paragraph is added regarding the health of the animals used for the preparation of heparin sodium.Identification:the tests for specific optical rotation(shown not to be discriminative enough)and zone electrophoresis have been replaced by the highly specific1H-NMR and strong anion-exchange liquid chromatography(SAX-HPLC)tests;1H-NMR has been selected for its ability not only to allow identification of heparin,but also to alert users to possible contaminations,whatever the nature of the contaminant;identification of the counterion is now based on the test for sodium by atomic absorption spectrometry described under Tests.Nucleotidic impurities:the limit has been tightened,based on current batch data. Protein:the Lowry test method has been introduced to replace the non-specific absorbance test;it is foreseen that this method be replaced by method2of general chapter2.5.33,in line with Ph.Eur.general policy,and users are therefore kindly requested to carry out the Ph.Eur.method and to submit their results during the public enquiry.Related substances:a SAX-HPLC-based test has been introduced,allowing the differentiation of natural contaminants linked to the production process(such as dermatan sulfate and chondroitin sulfate)from chemically synthesised contaminants;a limit for the sum of dermatan sulfate and chondroitin sulfate,which co-elute in this method,is proposed,further to consideration of current batch data.Nitrogen:a lower limit has been added,based on current batch data.Heavy metals:method C has been replaced by method F,in line with the general policy for heavy metals tests.Sulfated ash:in view of the highly specific tests introduced into the monograph,this test has become redundant and has therefore been deleted.XXXX:0333HEPARIN SODIUMHeparinum natricumDEFINITIONPreparation containing the sodium salt of a sulfated glycosaminoglycan present in mammalian tissues.It is prepared either from the lungs of oxen or from the intestinal mucosae of pigs,oxen or sheep.On complete hydrolysis,it liberates D-glucosamine,D-glucuronic acid,L-iduronic acid,acetic acid and sulfuric acid.It has the characteristic property of delaying the clotting of freshly shed blood.The potency of heparin sodium intended for parenteral administration is not less than150IU/mg,calculated with reference to the dried substance.The potency of heparin sodium not intended for parenteral administration is not less than120IU/mg,calculated with reference to the dried substance.Potency:minimum180IU/mg(dried substance).PRODUCTIONIt is prepared either from the lungs of oxen or from the intestinal mucosae of pigs,oxen or sheep.The animals from which heparin sodium is derived must fulfil the requirements for the health of animals suitable for human consumption.All stages of production and sourcing are subjected to a suitable quality assurance system.The identity of the source species and the absence of material from the other species is verified by appropriate testing during production.It is produced by methods of manufacturing designed to minimise or eliminate substances lowering blood pressure and to ensure freedom from contamination by over-sulfated glycosaminoglycans.It complies with the following additional requirements.Nuclear magnetic resonance spectrometry(2.2.33).The1H NMR spectrum obtained with a frequency of at least300MHz complies with the specifications approved by the competent authority.Capillary electrophoresis(2.2.47).The electropherogram obtained complies with the specifications approved by the competent authority.CHARACTERSAppearance:white or almost white,hygroscopic powder.Solubility:freely soluble in water.IDENTIFICATIONA.It delays the clotting of recalcified citrated sheep plasma(see Assay).B.Dissolve0.40g in water R and dilute to10.0ml with the same solvent.The specific optical rotation(2.2.7)is not less than+35.C.Examine by zone electrophoresis(2.2.31)using agarose for electrophoresis R asthe supporting medium.To equilibrate the agarose and as electrolyte solution use a mixture of50ml of glacial acetic acid R and800ml of water R adjusted to pH3by addition of lithium hydroxide R and diluted to1000.0ml with water R.Test solution.Dissolve25mg of the substance to be examined in water R and dilute to10ml with the same solvent.Reference solution.Dilute heparin sodium BRP with an equal volume of water R. Apply separately to the strip2µl to3µl of each solution.Pass a current of1mAto2mA per centimetre of strip width at a potential difference of300V for about10min.Stain the strips using a1g/l solution of toluidine blue R and remove the excess by washing.The ratio of the mobility of the principal band or bands in the electropherogram obtained with the test solution to the mobility of the band in the electropherogram obtained with the reference solution is0.9to1.1.B.Nuclear magnetic resonance spectrometry(2.2.33).Preparation:dissolve40mg of the substance to be examined in0.7ml of a solution containing20µg/ml of deuterated sodium trimethylsilylpropionate R and12µg/ml of sodium edetate R in deuterium oxide R.Comparison:dissolve40mg of heparin sodium for NMR identification CRS in0.7ml of a solution containing20µg/ml of deuterated sodium trimethylsilylpropionate R and12µg/ml of sodium edetate R in deuterium oxide R.If stored,the sodium edetate and deuterated sodium trimethylsilylpropionate solutions must be kept in high-density,natural polyethylene bottles. Apparatus:spectrometer operating at minimum300MHz.Acquisition of1H-NMR spectra:—number of transients:minimum16;it is adjusted until the signal-to-noise ratio is at least2000:1for the heparin methyl signal;—temperature:about25°C;test sample and reference spectra have to be obtained at the same temperature;—acquisition time:2s;—repetition time(acquisition time plus delay):minimum4s;—spectral width:10-12ppm,centred at around4.5ppm;—pulse width:to give a flip angle between30°and90°.Processing:—trimethylsilylpropionate reference signal set at0.00ppm;—exponential line-broadening window function(LB):0.3Hz;—phasing(manually if needed);—baseline correction;—zero filling:equal to data points used in acquisition.Results:—the large heparin sodium signals must be present:2.04ppm,3.27ppm(doublet),4.34ppm,5.22ppm and5.42ppm,all within±0.03ppm;—the1H-NMR spectrum obtained has to be superimposed on the1H-NMR spectrum obtained with heparin sodium for NMR identification CRS;the2spectra arenormalised so as to have a similar intensity;dermatan sulfate with a methyl signal at2.08±0.03ppm may be observed;no unidentified signals larger than4percent compared to the height of the heparin signal at5.42ppm are present in theranges0.10-2.00ppm,2.12-3.20ppm and5.70-8.00ppm;signals from the solvent or process-related substances may be present and have to be identified to be accepted;variations in the intensity of some signal regions of the spectrum of heparin mayoccur:the intensity-variable regions are between3.35ppm and4.55ppm,where the signal pattern is approximately kept but intensity varies.Note:a signal occurring at2.10ppm may be observed and is not to be confused with that of dermatan sulfate.C.Liquid chromatography(2.2.29)as described in the test for related substances with the following modifications.Injection:test solution(a)and reference solution(a).Retention time:dermatan sulfate and chondroitin sulfate=about22min; heparin=about26min;over-sulfated chondroitin sulfate=about33min.System suitability:reference solution(a):—peak-to-valley ratio:minimum1.5,where H p=height above the baseline of the peak due to dermatan sulfate+chondroitin sulfate and H v=height above the baseline of the lowest point of the curve separating this peak from the peak due to heparin. The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.2.heparin3.over-sulfated chondroitin sulfate1.dermatan sulfate+chondroitinsulfateFigure0333.-1.–Chromatogram for identification test C for heparin sodium:referencesolution(a)Results:the principal peak in the chromatogram obtained with test solution(a)is similar in retention time and shape to the principal peak in the chromatogram obtained with reference solution(a).D.The residue obtained in the test for sulfated ash(see Tests)gives reaction(a)of sodium(2.3.1).It complies with the test for sodium(see Tests).TESTSAppearance of solution.The solution is clear(2.2.1)and not more intensely coloured than intensity5of the range of reference solutions of the most appropriate colour(2.2.2, Method II).Dissolve a quantity equivalent to50000IU in water R and dilute to10ml with the same solvent.pH(2.2.3):5.5to8.0.Dissolve0.1g in carbon dioxide-free water R and dilute to10ml with the same solvent. Protein and Nucleotidic impurities.Dissolve40mg in10ml of water R.The absorbance (2.2.25)measured at260nm is not greater than0.150.20and that measured at280nm is not greater than0.15.Protein:maximum0.5per cent(dried substance).Solution A.Mix2volumes of a10g/l solution of sodium hydroxide R and2volumes of a 50g/l solution of sodium carbonate R and dilute to5volumes with water R. Solution B.Mix2volumes of a12.5g/l solution of copper sulfate R and2volumes of a 29.8g/l solution of sodium tartrate R and dilute to5volumes with water R.Solution C.Mix1volume of solution B and50volumes of solution A.Solution D.Dilute a phosphomolybdotungstic reagent(1)2-to4-fold in water R.Suitable dilutions produce solutions of pH10.25±0.25after addition of solutions C and D to the test and reference solutions.Test solution.Dissolve the substance to be examined in water R to obtain a concentration of5mg/ml.Reference solutions.Dissolve bovine albumin R in water R to obtain a concentrationof100mg/ml.Prepare dilutions of the solution in water R to obtain not fewer than5reference solutions having protein concentrations evenly spaced over a suitable range situated between5µg/ml and100µg/ml.Blank:water R.Procedure.To1ml of each reference solution,of the test solution and of the blank,add 5ml of solution C.Allow to stand for10min.Add0.5ml of solution D,mix and allowto stand at room temperature for30min.Determine the absorbances(2.2.25)of the solutions at750nm,using the solution prepared from the blank as compensation liquid. e a2nd-order polynomial regression curve of absorption against concentration.Related substances.Liquid chromatography(2.2.29).Test solution(a).Dissolve an accurately weighed quantity of about50mg of the substance to be examined in5.0ml of water R.Mix using a vortex mixer until dissolution is complete.(1)Folin-Ciocalteu’s phenol reagent from Merck(reference1.09001.0500)is suitable.Test solution(b).Dissolve an accurately weighed quantity of about0.1g of the substance to be examined in1.0ml of water R.Mix using a vortex mixer until dissolution is complete.Mix500µl of the solution and250µl of1M hydrochloric acid,then add50µl of a250mg/ml solution of sodium nitrite R.Mix gently and allow to stand at room temperature for40min before adding200µl of1M sodium hydroxide to stop the reaction. Reference solution(a).Dissolve the contents of a vial of heparin for system suitability CRS in water R to obtain concentrations of10mg/ml of heparin sodium, 0.2mg/ml of dermatan sulfate and0.05mg/ml of over-sulfated chondroitin sulfate.Mix using a vortex mixer to homogenise.Reference solution(b).Mix400µl of a125mg/ml solution of heparin sodium CRS with100µl of water R and mix using a vortex mixer.Add250µl of1M hydrochloric acid,then add50µl of a250mg/ml solution of sodium nitrite R.Mix gently and allow to stand at room temperature for40min before adding200µl of1M sodium hydroxide to stop the reaction.Reference solution(c).Dissolve the contents of a vial of heparin for system suitability CRS in water R to obtain concentrations of50mg/ml of heparin sodium,1mg/ml of dermatan sulfate and0.25mg/ml of over-sulfated chondroitin sulfate.To 500µl of the solution add250µl of1M hydrochloric acid,then add50µl of a250mg/ml solution of sodium nitrite R.Mix gently and allow to stand at room temperature for40min before adding200µl of1M sodium hydroxide to stop the reaction.Reference solutions are stable at room temperature for24h.Precolumn:—size:l=0.05m,Ø=2mm;—stationary phase:anion exchange resin R(13µm)(2).Column:—size:l=0.05m,Ø=2mm;—stationary phase:anion exchange resin R(9µm)(3);—temperature:40°C.Mobile phase:—mobile phase A:dissolve0.80g of sodium dihydrogen phosphate R in2litres of water R and adjust to pH3.0with phosphoric acid R;—mobile phase B:dissolve0.80g of sodium dihydrogen phosphate R in2litres of water R,add280g of sodium perchlorate R(4)and adjust to pH3.0with phosphoric acid R;filter and degas;Time (min)Mobile phase A(per cent V/V)Mobile phase B(per cent V/V)0-10752510-3575→025→10035-400100 Flow rate:0.22ml/min.Detection:spectrophotometer at202nm.(2)AG11-HC from Dionex(reference052963)is suitable.(3)AS11-HC from Dionex(reference052961)is suitable.(4)Normapur from VWR/Prolabo(reference27988.232)is suitable.Equilibration:15min.Injection:20µl of test solution(b)and reference solutions(b)and(c).System suitability:—the chromatogram obtained with reference solution(b)shows no peak at the retention time of heparin;—resolution:minimum3.0between the peaks due to dermatan sulfate+chondroitin sulfate and over-sulfated chondroitin sulfate in the chromatogram obtained with reference solution(c).The following chromatogram is shown for information but will not be published in the European Pharmacopoeia.1.dermatan sulfate+chondroitin sulfate2.over-sulfated chondroitin sulfate Figure0333.-2.–Chromatogram for the test for related substances of heparin sodium:reference solution(c)Results:—sum of dermatan sulfate and chondroitin sulfate:not more than the area of the corresponding peak in the chromatogram obtained with reference solution(c)(2.0per cent);—any other impurity:absence.Nitrogen(2.5.9):not more than1.5per cent to2.5per cent(dried substance),determined on0.100g.Sodium:9.5per cent to12.5per cent(dried substance).Atomic absorption spectrometry(2.2.23,Method I).Test solution.Dissolve50mg of the substance to be examined in a1.27mg/ml solution of caesium chloride R in0.1M hydrochloric acid and dilute to100.0ml with the same solvent.Reference solutions.Prepare reference solutions containing25ppm,50ppm and75ppm of Na,using sodium standard solution(200ppm Na)R diluted with a1.27mg/ml solution of caesium chloride R in0.1M hydrochloric acid.Source:sodium hollow-cathode lamp.Wavelength:330.3nm.Atomisation device:flame of suitable composition(for example11litres of air and2litres of acetylene per minute).Heavy metals(2.4.8):maximum30ppm.0.5g complies with test CF.Prepare the reference solution using1.5ml of lead standard solution(10ppm Pb)R.Loss on drying(2.2.32):maximum8.0per cent,determined on1.000g by drying at60°C over diphosphorus pentoxide R for3h at a pressure not exceeding670Pa.Sulfated ash(2.4.14):30per cent to43per cent,determined on0.20g and calculated with reference to the dried substance.Bacterial endotoxins(2.6.14):less than0.01IU per International Unit of heparin,if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins.ASSAYCarry out the assay of heparin(2.7.5).The estimated potency is not less than90per cent and not more than111per cent of the stated potency.The confidence limits of the estimated potency(P=0.95)are not less than80per cent and not more than125per cent of the stated potency.STORAGEIn an airtight container.If the substance is sterile,store in a sterile,airtight,tamper-proof container.LABELLINGThe label states:—the number of International Units per milligram;—the animal species of origin;—where applicable,that the substance is suitable for use in the manufacture of parenteral preparations.ReagentsDeuterated sodium trimethylsilylpropionate.C8H92H4NaO2Si.(M r172.3).XXXXXXX. [24493-21-8].Sodium3-(trimethylsilyl)(2,2,3,3-2H4)propionate.TSP-d4.Degree of deuteration:minimum98per cent.White or almost white powder.。

低分子肝素质量标准研究_李京

肝素是临床广泛应用的一类抗凝血药物，属于高度硫酸化的糖胺聚糖。2008 年以来，国内外对肝素类产品的质量及质量标准问题十分关注［1-2］，欧洲和美国药典几次修订了肝素钠的药典标准，《中国药典》2010 年版也对肝素标准进行了大幅提高［3］。2009 年和 2010 年连续 2 年的全国药品评价性抽验结果表明，目前我国市场上的肝素钠注射液质量是可靠的［4］。低分子肝素［5］（ low-molecular-weight heparin，LMWH）是由肝素分级或降解得到具有较低相对分子质量的组分或片段，其抗血栓作用优于肝素，生物利用度高、出血倾向小，是肝素的第二代产品。2011 年 LMWH 注射剂评价性抽验暴露出我国 LMWH 产品质量及质量标准都存在一些问题，多批产品相对分子质量与活性不合格，质量标准与国外原研厂家的差距较大［6］。抗凝药市场销售额逐年上升，国内有多家企业同时进行新药申报，市场竞争激烈，对产品质量要求高。LMWH 已于 2012 年进入我国基本药物目录，是《中国药典》2015 年版备选品种，质量标准提高工作迫在眉睫。
ABSTＲACT： OBJECTIVE To revise the national specification low molecular weight heparin for improving the quality and quality control level its domestic products as candidate new varieties Chinese Pharmacopoeia 2015 edition. METHODS Domestic and imported original products and related information were collected，including 27 batches raw material from 11 manufacturers and 49 batches injections from 13 manufacturers. Domestic low molecular weight heparin products were classified according to the production process. The analysis focused on the verification structure，molecular weight and activity，process impurities，and degradation impurities. Various physical，chemical and biological methods were used，such as ion chromatography，size exclusion chromatography，reversed phase chromatography，gas chromatography，NMＲ，atomic absorption spectrometry，micro-chromogenic substrate methods，and so on. ＲESULTS The domestic low molecular heparin products were divided into three categories： dalteparin sodium，enoxaparin sodium，and nadroparin calcium. Eight draft specifications the raw material and preparations have been made. In the draft specifications，eight items have been added： structure type，production，1，6-anhydro derivatives for enoxaparin sodium，free sulfate，nitrite，benzyl alcohol，ultraviolet absorption maximum specific absorption enoxaparin sodium，and residual solvent； 15 items have been revised： Chinese name，English name，definition，characters，pH value，molecular weight and molecular weight distribution，anti-FⅩa activity，the ratio between anti-FⅩa and anti-FⅡa activity，the color the solution，sodium，sulfate and carboxylate ratio，loss on drying，volume injection，storage， preparation； one item has been deleted： light absorption at 260 and 280 nm. CONCLUSION The current draft specifications low molecular weight heparin have been greatly improved than the national specifications established in 2005. The products are divided into three categories. A lot items have been added and the limits are more reasonable. The draft specifications are roughly equal with the Europe

达肝素钠注射液

达肝素钠注射液达肝素钠注射液（Dalteprin Sodium Injection）（法安明）主要成分：达肝素钠组成：1支单剂量注射器达肝素钠2500 IU（抗-Xa）5000 IU（抗-Xa）7500 IU（抗-Xa）氯化钠适量注射用水加至0.2ml 加至0.2ml 加至0.3ml效价以低分子量肝素第一国际标准中所描述的国际抗-Xa单位（IU）表示。

【性状】本品为无色或淡黄色的澄明液体。

【药理毒理】达肝素钠是一种含有达肝素钠（低分子量肝素钠）的抗血栓剂。

达肝素钠是从猪肠粘膜提取的低分子肝素钠，其平均分子量为5000。

达肝素钠主要通过抗凝血酶（AT）而增强其对凝血因子X a和凝血酶的抑制，从而发挥其抗血栓形成的作用。

达肝素钠增强抑制凝血因子Xa的能力，相对高于其延长血浆凝血时间（APTT）的能力。

达肝素钠对血小板功能和血小板粘附性的影响比肝素小，因而对初级阶段止血只有很小的影响。

尽管如此，达肝素钠的某些抗血栓特性仍被认为是通过对血管壁或纤维蛋白溶解系统的影响而形成的。

达肝素钠比肝素的急性毒性低。

在毒理研究中重复出现的最明显的反应为大剂量注射后注射部位的局部出血。

这种反应的发生率及程度均为剂量相关的，而且不累加。

局部出血反应与抗凝作用中剂量相关的改变有关，用APTT和抗-Xa的活性来测量。

等剂量研究表明，达肝素钠与肝素相比，没有更强的骨质稀少作用。

不论是何种给药途径、剂量和治疗周期，均未发现器官毒性。

也未发现致突变反应。

未发现孕期的胚胎毒性和致畸作用以及发殖力、围产期或产后的影响。

【药代动力学】皮下注射的生物利用度约为90%。

静脉注射后消除半衰期约为2小时，皮下注射后为3-4小时。

尿毒症病人的半衰期将延长。

治疗剂量范围（30-120IU/kg）外，半衰期与剂量略相关。

药物主要通过肾脏消除。

【适应症】治疗急性深静脉血栓。

急性肾功能衰竭或慢性肾功能不全者进行血液透析和血液过滤期间防止在体外循环系统中发生凝血。

达肝素钠标准(EP 8.0)

Dalteparin sodium EUROPEAN PHARMACOPOEIA8.0Reference solution (a).Dilute 2.5mL of dimethylaminesolution R (impurity D)to 100.0mL with water R (solution A).Firmly attach the septum and cap to a 20mL ing a 10μL syringe,inject 10μL of solution A into the vial.Reference solution (b).Firmly attach the septum and cap to a 20mL ing a 10μL syringe,inject 10μL of solution A and 10μL of a 10g/L solution of triethylamine R into the vial.Column :–material :fused silica;–size :l =30.0m,Ø=0.53mm;–stationary phase :base-deactivated polyethyleneglycol R (ﬁlm thickness 1.0μm).Carrier gas :helium for chromatography R .Flow rate :13mL/min.Split ratio :1:1.Static head-space conditions that may be used :–equilibration temperature :60°C;–equilibration time :10min;–transfer-line temperature :90°C;–pressurisation time :30s.Temperature :Time (min)Temperature(°C)Column0-3353-1135→165Injection port 180Detector220Detection :ﬂame ionisation.Injection :1mL.System suitability :reference solution (b):–resolution :minimum 2.5between the peaks due to impurity D and triethylamine.Limit :–impurity D :not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a)(0.05per cent).Water (2.5.12):maximum 0.5per cent,determined on 1.00g.Sulfated ash (2.4.14):maximum 0.1per cent,determined on 1.0g.ASSAYDissolve 0.150g in 30mL of anhydrous acetic acid R .Titrate with 0.1M perchloric acid ,determining the end-point potentiometrically (2.2.20).1mL of 0.1M perchloric acid is equivalent to 18.22mg of C 6H 10N 6O.STORAGEAt a temperature of 2°C to 8°C,protected from light.IMPURITIESSpecified impurities :A,B,D .Other detectable impurities (the following substances would,if present at a sufﬁcient level,be detected by one or other of the tests in the monograph.They are limited by the general acceptance criterion for other/unspeciﬁed impurities and/or by the general monograph Substances for pharmaceutical use (2034).It is therefore not necessary to identify these impurities for demonstration of compliance.See also 5.10.Control of impurities in substances for pharmaceutical use ):C. A.3,7-dihydro-4H -imidazo[4,5-d ]-1,2,3-triazin-4-one(2-azahypoxanthine),B.5-amino-1H-imidazole-4-carboxamide,C.5-diazenyl-1H-imidazole-4-carboxamide,D.N -methylmethanamine.01/2008:1195DALTEPARIN SODIUM DalteparinumnatricumDEFINITIONDalteparin sodium is the sodium salt of a low-molecular-mass heparin that is obtained by nitrous acid depolymerisation of heparin from porcine intestinal mucosa.The majority of the components have a 2-O -sulfo-α-L -idopyranosuronic acid structure at the non-reducing end and a6-O -sulfo-2,5-anhydro-D -mannitol structure at the reducing end of their chain.Dalteparin sodium complies with the monographLow-molecular-mass heparins (0828)with the modifications and additional requirements below.The mass-average relative molecular mass ranges between 5600and 6400,with a characteristic value of about 6000.The degree of sulfatation is 2.0to 2.5per disaccharide unit.The potency is not less than 110IU and not more than 210IU of anti-factor Xa activity per milligram,calculated with reference to the dried substance.The anti-factor IIa activity is not less than 35IU/mg and not more than 100IU/mg,calculated with reference to the dried substance.The ratio of anti-factor Xa activity to anti-factor IIa activity is between 1.9and 3.2.PRODUCTIONDalteparin sodium is produced by a validated manufacturing and puriﬁcation procedure under conditions designed to minimise the presence of N-NO groups.The manufacturing procedure must have been shown toreduce any contamination by N-NO groups to approved limits using an appropriate,validated quantiﬁcation method.1988See the information section on general monographs (cover pages)EUROPEAN PHARMACOPOEIA 8.0DalteparinsodiumIDENTIFICATIONCarry out identiﬁcation test A as described in the monograph Low-molecular-mass heparins (0828)using dalteparin sodium CRS .Carry out identiﬁcation test C as described in the monographLow-molecular-mass heparins (0828).The followingrequirements apply.The mass-average relative molecular mass ranges between 5600and 6400.The mass percentage of chains lower than 3000isnot more than 13.0per cent.The mass percentage of chainshigher than 8000ranges between 15.0per cent and 25.0percent.TESTSAppearance of solution .Dissolve 1g in 10mL of water R .The solution is clear (2.2.1)and not more intensely coloured than intensity 5of the range of reference solutions of the most appropriate colour (2.2.2,Method II ).Nitrite .Not more than 5ppm.Examine by liquid chromatography (2.2.29).Rinse all volumetric flasks at least three times with water R before the preparation of the solutions.Test solution.Dissolve 80.0mg of the substance to be examined in water R and dilute to 10.0mL with the same solvent.Allow to stand for at least 30min.Reference solution (a).Dissolve 60.0mg of sodium nitrite R in water R and dilute to 1000.0mL with the same solvent.For the preparation of reference solution (b),use a pipette previously rinsed with reference solution (a).Reference solution (b).Dilute 1.00mL of reference solution (a)to 50.0mL with water R .Before preparing reference solutions (c),(d)and (e),rinse all pipettes with reference solution (b).Reference solution (c).Dilute 1.00mL of reference solution (b)to 100.0mL with water R (corresponding to 1ppm of nitrite in the test sample).Reference solution (d).Dilute 3.00mL of reference solution (b)to 100.0mL with water R (corresponding to 3ppm of nitritein the test sample).Reference solution (e).Dilute 5.00mL of reference solution (b)to 100.0mL with water R (corresponding to 5ppm of nitritein the test sample).The chromatographic procedure may be carried out using:–a column 0.125m long and 4.3mm in internal diameterpacked with a strong anion-exchange resin;–as mobile phase at a ﬂow rate of 1.0mL/min a solution consisting of 13.61g of sodium acetate R dissolved in water R ,adjusted to pH 4.3with phosphoric acid R and diluted to 1000mL with water R ;–as detector an appropriate electrochemical device with the following characteristics and settings:a suitable working electrode,a detector potential of +1.00V versus Ag/AgCl reference electrode and a detector sensitivity of 0.1μA full scale.Inject 100μL of reference solution (d).When thechromatograms are recorded in the prescribed conditions,the retention time for nitrite is 3.3to 4.0min.The test is not valid unless:–the number of theoretical plates calculated for the nitrite peak is at least 7000per metre per column (dalteparin sodium will block the binding sites of the stationary phase,which will cause shorter retention times and lower separation efﬁciency for the analyte;the initialperformance of the column may be partially restored using a 58g/L solution of sodium chloride R at a ﬂow rate of 1.0mL/min for 1h;after regeneration the column is rinsed with 200mL to 400mL of water R );–the symmetry factor for the nitrite peak is less than 3;–the relative standard deviation of the peak area for nitriteobtained from 6injections is less than 3.0per cent.Inject 100μL each of reference solutions (c)and (e).The test is not valid unless:–the correlation factor for a linear relationship between concentration and response for reference solutions (c),(d)and (e)is at least 0.995;–the signal-to-noise ratio for reference solution (c)is not less than 5(if the noise level is too high,electrode recalibration is recommended);–a blank injection of water R does not give rise to spurious peaks.Inject 100μL of the test solution.Calculate the content of nitrite from the peak areas in the chromatogram obtained with reference solutions (c),(d)and (e).Boron .Not more than 1ppm,determined by inductively coupled plasma atomic emission spectroscopy.Boron is determined by measurement of the emission from an inductively coupled plasma (ICP)at a wavelength speciﬁc to boron.The emission line at 249.733nm is e anappropriate apparatus,whose settings have been optimised as directed by the manufacturer.Test solution.Dissolve 0.2500g of the substance to beexamined in about 2mL of water for chromatography R ,add 100μL of nitric acid R and dilute to 10.00mL with the samesolvent.Reference solution (a).Prepare a 1per cent V/V solution of nitric acid R in water for chromatography R (blank).Reference solution (b).Prepare a 11.4μg/mL solution of boricacid R in a 1per cent V/V solution of nitric acid R in water for chromatography R (STD cal ).Reference solution (c).Dissolve 0.2500g of a referencedalteparin sodium with no detectable boron in about 2mL ofwater for chromatography R ,add 100μL of nitric acid R and dilute to 10.00mL with the same solvent (STD 0).Reference solution (d).Dissolve 0.2500g of a reference dalteparin sodium with no boron detected in about 2mL of a 1per cent V/V solution of nitric acid R in water forchromatography R ,add 10μL of a 5.7mg/mL solution of boric acid R and dilute to 10.00mL with the same solvent (STD 1).This solution contains 1μg/mL of boron.Calculate the content of boron in the substance to be examined,using the following correction factor:Loss on drying (2.2.32).Not more than 5.0per cent,determined on 1.000g by drying in an oven at 60°C over diphosphorus pentoxide R at a pressure not exceeding 670Pa for 3h.General Notices (1)apply to all monographs and other texts1989。

肝素钠_中国药典2015版公示标准

肝素钠GansunaHeparin Sodium■本品系自猪或牛的肠黏膜中提取的硫酸氨基葡聚糖的钠盐，属黏多糖类物质，是由不同分子量的糖链组成的混合物，由α-D-氨基葡萄糖（N-硫酸化，O-硫酸化，或N-乙酰化）和O-硫酸化糖醛酸（α-L-艾杜糖醛酸或β-D葡萄糖醛酸）交替连接形成聚合物，具有延长血凝时间的作用。

按干燥品计算，每1mg中抗Ⅱa因子效价不得少于180 IU，抗Xa因子效价与抗IIa因子效价比为0.9～1.1。

■[修订]■核酸取本品，加水溶解并稀释制成每1ml中含4mg的溶液，照紫外-可见分光光度法（附录ⅣA）测定，在260nm的波长处，吸光度不得大于0.10。

■[增订]■蛋白质取本品适量，精密称定，加水溶解并稀释制成每1ml中约含30mg的溶液，作为供试品溶液；另取牛血清白蛋白对照品适量，分别加水制成每1ml中各含0、10μg、20μg、30μg、40μg与50μg的溶液，作为对照品溶液，照蛋白质含量测定法（附录ⅦM 第二法）测定。

按干燥品计，含蛋白质不得过0.5%。

肝素钠说明书

肝素钠注射液说明书请仔细阅读说明书并在医师指导下使用【药品名称】通用名称：肝素钠注射液英文名称：heparin sodium lnjection 汉语拼音：gansuna zhusheye 【成份】本品主要成份为肝素钠。

肝素钠系自猪或牛的肠黏膜中提取的硫酸氨基葡聚糖的钠盐，属黏多糖类物质。

辅料为：苯酚、注射用水。

【性状】本品为无色至淡黄色的澄明液体。

【适应症】用于防治血栓形成或栓塞性疾病（如心肌梗塞、血栓性静脉炎、肺栓塞等）；各种原因引起的弥漫性血管内凝血（dic）；也用于血液透析、体外循环、导管术、微血管手术等操作中及某些血液标本或器械的抗凝处理。

【规格】 2ml:12500单位【用法用量】（1）深部皮下注射：首次5000～10000单位，以后每8小时8000～10000单位或每12小时15000～20000单位；每24小时总量约30000～40000单位，一般均能达到满意的效果。

（2）静脉注射：首次5000～10000单位，之后，或按体重每4小时100单位/kg，用氯化钠注射液稀释后应用。

（3）静脉滴注：每日20000～40000单位，加至氯化钠注射液1000mi中持续滴注。

滴注前可先静脉注射5000单位作为初始剂量。

（4）预防性治疗：高危血栓形成病人，大多是用于腹部手术之后，以防止深部静脉血栓。

在外科手术前2小时先给5000单位肝素皮下注射，但麻醉方式应避免硬膜外麻醉，然后每隔8～12小时5000单位，共约7日。

【不良反应】毒性较低，主要不良反应是用药过多可致自发性出血，故每次注射前应测定凝血时间。

如注射后引起严重出血，可静注硫酸鱼精蛋白进行急救。

偶可引起过敏反应及血小板减少，常发生在用药初5～9天，故开始治疗1个月内应定期监测血小板计数。

偶见一次性脱发和腹泻。

尚可引起骨质疏松和自发性骨折。

肝功能不良者长期使用可引起抗凝血酶-iii耗竭而血栓形成倾向。

【禁忌】对肝素过敏、有自发出血倾向者、血液凝固迟缓者（如血友病、紫癜、血小板减少）、溃疡病、创伤、产后出血者及严重肝功能不全者禁用。

肝素检验细则

肝素钠检验项目附细则一、效价测定操作步骤：1. 标准品制备：精密称量肝素钠标准品数量(mg)×197u/mg=总单位数×0.93=美国羊血浆法总单位数（uspu ）÷8 uspu/ml=需要实际加入的蒸馏水毫升数。

精确加入蒸馏水。

分装封口备用。

2. 效价测定：2.1 样品的制备：精确称量待测肝素粗品40~50mg 放入容量瓶中，再向其中加入1mg:1ml 比例的生理盐水，至样品全部溶解。

吸取0.5ml 上述溶液至西林瓶中，再按照公式：估效价÷16-0.5计算出生理盐水量，加入西林瓶中。

2.2 样品的测定：依据估计血浆标准凝固点±10ul 、±20ul 分别向试管中加入标准品和待测样品，再向各试管中加入1ml 血浆和0.8ml 的氯化钙生理盐水。

充分混匀后置37℃水浴锅中孵育1小时。

结果按照公式计算：实际效价=凝固点样品21V 凝固点21V 标准品 ×估效价结果要求：所需主要仪器及价格：电子天平单价：1000（物理）~2000（电子）rmb 水浴锅单价：800rmb二、比旋度测定当平面偏振光通过含有某些光学活性物质（如具有不对称碳原子的化合物）的液体或溶液时，能引起旋光现象，使偏振光的振动平面向左或向右旋转。

偏振光旋转的度数称为旋光度。

旋光度有右旋、左旋之分，偏振光向右旋转（顺时针方向）称为“右旋”，用符号“+”表示；偏振光向左旋转（逆时针方向）称为“左旋”，用符号“-”表示。

偏振光透过长1dm ，且每1ml 中含有旋光性物质1g 的溶液，在一定波长与温度下，测得的旋光度称为比旋度。

比旋度是旋光物质的重要物理常数，可以用来区别药物或检查药物的纯杂程度，也可用来测定含量。

物质的旋光度不仅与其化学结构有关，而且还和测定时溶液的浓度、光路长度以及测定时的温度和偏振光的波长有关。

旋光度测定法的测定方法主要包括以下几个方面：1．仪器旋光计：《中国药典》规定，应使用读数至0.01。

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Ratio: A240/A445, 1.30–1.50for Labeling 〈7〉, Labels and Labeling for Injectable Prod-•B. The retention time of the major peak of the Sample ucts•(CN 1-May-2016).solution corresponds to that of the Standard solution, asADDITIONAL REQUIREMENTS obtained in the Assay.ASSAYChange to read:•P ROCEDURE[N OTE—Use freshly prepared Standard solution and Sam-•P ACKAGING AND S TORAGE:•Preserve as described in Pack-ple solution, protected from light.]aging and Storage Requirements 〈659〉, Injection Packaging, Mobile phase: Acetonitrile and water (3:2)Sterile solids packaging; protect from light.•(CN 1-May-2016).Standard solution: 250µg/mL of USP Dactinomycin RS•L ABELING: Label it to include the statement “Protect from in Mobile phaselight.”Sample solution: 250µg/mL of dactinomycin from•USP R EFERENCE S TANDARDS〈11〉Dactinomycin for Injection diluted with Mobile phase.USP Dactinomycin RSFilter, if necessary, to obtain a clear solution. [N OTE—USP Endotoxin RSPrepare the solution by adding a suitable aliquot of Mo-bile phase to one container of Dactinomycin forInjection.]Chromatographic system(See Chromatography 〈621〉, System Suitability.)Mode: LC Add the following:Detector: UV 254 nmColumn: 3.9-mm × 30-cm; packing L1Flow rate: 2.5mL/minv Dalteparin SodiumInjection size: 10µLSystem suitability [9041-08-1].Sample:Standard solution[N OTE—The retention time for dactinomycin is 6 min.]Suitability requirementsColumn efficiency: NLT 1200 theoretical platesTailing factor: NMT 2Relative standard deviation: NMT 3.0%AnalysisSamples:Standard solution and Sample solutionCalculate the percentage of C62H86N12O16 in the por-tion of Dactinomycin for Injection taken:Result = (r U/r S) × (C S/C U) × 100r U= peak response from the Sample solutionr S= peak response from the Standard solutionC S= concentration of USP Dactinomycin RS in theStandard solution (µg/mL)DEFINITIONC U= nominal concentration of dactinomycin in the Dalteparin Sodium is the sodium salt of a low molecularSample solution (µg/mL)weight heparin obtained by nitrous acid depolymerization Acceptance criteria: 90.0%–120.0%of heparin from porcine intestine or intestinal mucosa.Heparin source material used in the manufacture of SPECIFIC TESTSDalteparin Sodium complies with the compendial require-•P H 〈791〉: 5.5–7.5, in the solution constituted as directedments stated in the Heparin Sodium monograph.in the labelingDalteparin Sodium is produced by a validated manufac-•L OSS ON D RYING〈731〉: Dry a portion in vacuum at aturing and purification procedure under conditions shown pressure not exceeding 5mm of mercury at 60° for 3 h:to minimize the presence of species containing the N–NO it loses NMT 4.0% of its weight.group. The majority of the components have a 2-O-sulfo-α-L-idopyranosuronic acid structure at the non-reducing Change to read:end and a 6-O-sulfo-2,5-anhydro-D-mannitol structure atthe reducing end of their chains. The weight-average mo-•O THER R EQUIREMENTS: It meets the requirements under lecular weight (M w) ranges between 5600 Da and 6400•Injections and Implanted Drug Products 〈1〉•(CN 1-May-2016).Da, with a characteristic value of about 6000 Da. The •B ACTERIAL E NDOTOXINS T EST〈85〉: NMT 100.0 USP Endo-percentage of chains lower than molecular weight 3000 toxin Units/mg of dactinomycin.Da is NMT 13.0%, and the percentage of chains higher •S TERILITY T ESTS〈71〉: Meets the requirements when than molecular weight 8000 Da ranges between 15.0% tested as directed for Test for Sterility of the Product to be and 25.0%. The degree of sulfation is NLT 1.8/disaccha-Examined, Membrane Filtration, each container being con-ride unit. The potency is NLT 110 and NMT 210 Anti-stituted aseptically by injecting Sterile Water for Injection Factor X a International Units (IU)/mg of activity, calculated through the stopper, and the entire contents of all the on the dried basis. The anti-factor IIa activity is NLT 35 containers being collected aseptically with the aid of IU/mg and NMT 100 IU/mg, calculated on the dried ba-200mL of Fluid A before filtering.sis. The ratio of anti-factor Xa activity to anti-factor IIaactivity is between 1.9 and 3.2.Change to read:IDENTIFICATION•A.1H NMR S PECTRUM•C ONSTITUTED S OLUTION: At the time of use, it meets the Standard solution: Dissolve 15mg of USP Dalteparin requirements for •Injections and Implanted Drug Products Sodium RS in 0.7mL of deuterium oxide with deuter-〈1〉, Specific Tests, Completeness and clarity of solutions and ated trimethylsilylpropionic (TSP) acid sodium salt. Thesample is freeze-dried to remove exchangeable P MonographsRedissolve the sample and repeat the freeze-drying step centage of chains lower than the molecular weight twice more before transferring the sample into an NMR3000 Da (M3000) is NMT 13.0%, and the percentage of tube.chains higher than the molecular weight 8000 Da Sample solution: Dissolve 15mg of Dalteparin Sodium(M8000) ranges between 15.0% and 25.0%.in 0.7mL of deuterium oxide (99.9%) with deuterated•C. A NTI-F ACTOR X A TO A NTI-F ACTOR II A R ATIOTSP. The sample is freeze-dried to remove exchangea-(See Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac-ble protons. Redissolve the sample and repeat the tionated and Low Molecular Weight Heparins 〈208〉, Anti-freeze-drying step twice more before transferring the Factor Xa and Anti-Factor IIa Assays for Low Molecular sample into an NMR tube.Weight Heparins.)Instrumental conditions Acceptance criteria: The ratio of the numerical value of (See Nuclear Magnetic Resonance Spectroscopy 〈761〉.)the anti-factor Xa activity, in Anti-Factor X a IU/mg, to Mode: NMR, pulsed (Fourier transform)the numerical value of the anti-factor IIa activity, in Frequency: NLT 500MHz for 1H Anti-Factor II a IU/mg, as determined by the Anti-Factor Temperature: 30°Xa Activity and Anti-Factor IIa Activity assays, is NLT 1.9 System suitability and NMT 3.2, respectively.Samples:Standard solution and Sample solution•D. I DENTIFICATION T ESTS—G ENERAL: Meets the require-Transfer the Standard solution and the Sample solution to ments for Sodium ContentNMR tubes of 5mm in diameter. Using a pulsedASSAY(Fourier transform) NMR spectrometer operating at•A NTI-F ACTOR X A A CTIVITYNLT 500MHz for 1H, acquire a free induction decay(See Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac-(FID) with NLT 32 scans using a 90° pulse, an acquisi-tionated and Low Molecular Weight Heparins 〈208〉, Anti-tion time of NLT 2 s, and at least a 10-s delay. ForFactor Xa and Anti-Factor IIa Assays for Low Molecular each sample, an initial short spectrum is collected (1Weight Heparins, Anti-Factor Xa Activity for Low Molecular scan), and the water resonance is then suppressed byWeight Heparin.)selective irradiation during the relaxation delay. FinalAnalysis: Proceed as directed in the chapter.spectra are recorded over 32 scans. For all samples,Acceptance criteria: The potency is NLT 110 and NMT the TSP methyl signal should be set to 0.00ppm. Re-210 Anti-Factor X a IU/mg on the dried basis.cord the 1H NMR spectrum of the Standard solution.Collect the 1H NMR spectrum with a spectral window OTHER COMPONENTSof at least 10 to −2ppm and without spinning. The•N ITROGEN D ETERMINATION, Method II 〈461〉: 1.5%–2.5% Standard solution shall be run at least daily when the on the dried basisSample solution is being run. All spectra are phased,•S ODIUM C ONTENTand linear baseline correction is applied to all spectra Cesium chloride solution: 1.27mg/mL of cesium chlo-before peak identification.ride in 0.1 M hydrochloric acidSuitability requirements Standard solution A: 0.0025% of sodium chloride in Chemical shift: The TSP methyl signal should be set Cesium chloride solutionto 0.00ppm for all samples.Standard solution B: 0.0050% of sodium chloride in Chemical shifts for system suitability: The ppm val-Cesium chloride solutionues for the methyl group of N-acetyl, the H-2 of N-Standard solution C: 0.0075% of sodium chloride in sulfo glucosamine, the H-2 of glucuronic acid plus Cesium chloride solution3-O-sulfo glucosamine, the H-1 of iduronic acid, and Sample solution: Transfer 50.0mg of Dalteparin So-the H-1 of 3-O sulfo glucosamine of dalteparin in the dium to a 100-mL volumetric flask, and dissolve in and Standard solution are present at 2.05, 3.28, 3.39,dilute with Cesium chloride solution to volume.5.01, and 5.51, respectively. Two additional signals,Analysiscorresponding to the H-1 of the 2-O-sulfo iduronic Samples:Cesium chloride solution, Standard solution A, acid linked to the terminal 2, 5-anhydromannitol and Standard solution B, Standard solution C, and Sample the H-1 of 2-O-sulfo iduronic acid are located at solution5.18–5.22ppm. The ppm values of these signals do Concomitantly determine the absorbances of the Ce-not differ by more than ±0.03ppm, Standard solution.sium chloride solution (blank), the Sample solution, and [N OTE—Depending on specific sample makeup and the Standard solutions at 330.3 nm, using a sodium instrument parameters, including the field strength hollow-cathode lamp and an air–acetylene flame. Us-of the NMR instrument, the two signals associated ing the absorbances of Standard solutions A, B, and C, with the H-1 of 2-O-sulfo iduronic acid at determine the sodium content in the Sample solution5.18–5.22ppm may appear well separated or as a after an appropriate blank correction.main signal with a shoulder.]Acceptance criteria: 10.5%–13.5% on the dried basis AnalysisSample:Sample solution IMPURITIESRecord the 1H NMR spectra of the Sample solution.•L IMIT OF N ITRITESAcceptance criteria: The ppm values for the methyl Mobile phase: Dissolve 13.6g of sodium acetate trihy-group of N-acetyl, the H-2 of N-sulfo glucosamine, the drate in 900mL of water in a 1000-mL volumetric flask.H-2 of glucuronic acid plus 3-O-sulfo glucosamine, the Adjust with orthophosphoric acid to a pH of 4.3, and H-1 of iduronic acid and the H-1 of the 2-O-sulfo dilute with water to 1000mL. Filter through a 0.45-µm iduronic acid linked to the terminal anhydromannitol,membrane.the H-1 of 2-O-sulfo iduronic acid and the H-1 of 3-O Nitrite stock standard solution: Dissolve 0.075g of so-sulfo glucosamine of dalteparin in the Sample solution dium nitrite in a 1000-mL volumetric flask with carbon are present at 2.05, 3.28, 3.39, 5.01, 5.18–5.22, and dioxide-free water (0.05g/L of nitrite).5.51, respectively. The ppm values of these signals do Nitrite standard solution: Dilute 1mL of Nitrite stocknot differ by more than ±0.03ppm.standard solution in a 100-mL volumetric flask with car-•B. M OLECULAR W EIGHT D ISTRIBUTION AND W EIGHT-A VERAGE bon dioxide-free water (500 ng/mL of nitrite).M OLECULAR W EIGHT Calibration standard solutions: Dilute Nitrite standard (See Low Molecular Weight Heparin Molecular Weight De-solution in carbon dioxide-free water to prepare four so-terminations 〈209〉.)lutions with the final nitrite concentrations of 2.5, 5, Acceptance criteria: The weight-average molecular15, and 25 ng/mL.weight (M w) ranges between 5600 Da and 6400 Da,with a characteristic value of about 6000 Da. The per-Sample solution: Weigh 80.0mg of Dalteparin Sodium r SA= response of boron from Standard solution A into a 20-mL volumetric flask, and dissolve in carbon r C= response of boron from the Calibration solution dioxide-free water.r B= response of boron from the BlankChromatographic system Acceptance criteria: NMT 1ppm(See Chromatography 〈621〉, System Suitability.)SPECIFIC TESTSMode: LC•A NTI-F ACTOR II A A CTIVITYDetector: Electrochemical detector containing a work-(See Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac-ing electrode (glassy carbon type) with the potentialtionated and Low Molecular Weight Heparins, 〈208〉, Anti-of +1.00 V against a silver–silver chloride referenceFactor Xa and Anti-Factor IIa Assays for Low Molecular electrodeWeight Heparins, Anti-Factor IIa Activity for Low Molecular Column: 3-mm × 15-cm; 5-µm packing L92Weight Heparin.)Column temperature: 30±5°Acceptance criteria: NLT 35 and NMT 100 Anti-Factor Column regeneration: 1M sodium chloride (NaCl) atII a IU/mg on the dried basis0.5mL/min for about 1 h. After regeneration, wash•M OLAR R ATIO OF S ULFATE TO C ARBOXYLATE the column with water and re-equilibrate with MobileMobile phase: Carbon dioxide-free water phase.Sample solution: 50mg of Dalteparin Sodium in 10mL Flow rate: 0.5mL/minof carbon dioxide-free waterInjection volume: 25µLChromatographic systemRun time: 10 min(See Chromatography 〈621〉, System Suitability.) System suitabilityMode: LCSamples:Calibration standard solutions and SampleDetector: IonsolutionColumn: Two columns: one 1.5-cm × 2.5-cm column, Suitability requirementspacked with an anion-exchange resin L64packing, and Column efficiency: NLT 4000 theoretical plates forone 1.5-cm × 7.5-cm column, packed with a cation-the nitrite peak for all Calibration solutions and Sampleexchange resin L65packing.1 The outlet of the anion-solution runsexchange column is connected to the inlet of the cat-Tailing factor: Between 0.8 and 1.2 for all Calibrationion-exchange column.solutions and Sample solution runsFlow rate: 1mL/minRelative standard deviation: Inject Calibration stan-Analysisdard solutions with 25 ng/mL concentration at leastSample:Sample solutionsix times. Calculate the relative standard deviation %[N OTE—Regenerate the anion-exchange column and the (%RSD) of the nitrite peak areas of the last six injec-cation-exchange column with 1N sodium hydroxide tions. The %RSD is NMT 2%.and 1N hydrochloric acid, respectively, between two Analysisinjections.]Samples:Calibration standard solutions and SampleWith the valve in the inject position, inject the Sample solutionsolution into the anion-exchange column, and collect Plot the areas of the nitrite peaks from the chromato-the eluate from the cation-exchange column in a grams of the Calibration standard solutions against re-beaker at the outlet until the ion detector reading re-spective concentrations of nitrite. Draw a best-fit re-turns to the baseline value. Quantitatively transfer the gression line through the points. The correlationeluate to a titration vessel containing a magnetic stir-coefficient is NLT 0.995. Calculate the concentrationring bar, and dilute with carbon dioxide-free water to of nitrite from the areas of the nitrite peak in theabout 60mL. Position the titration vessel on a mag-chromatogram of the Sample solution.netic stirrer, and immerse the electrodes. Note the ini-Acceptance criteria: NMT 5ppmtial conductivity reading, and titrate with approxi-•B ORONmately 0.1 N sodium hydroxide added in 100-µL [N OTE—Use only plastic labware, avoid glass.]portions. [N OTE—Prepare the sodium hydroxide solu-Blank: 1% (v/v) solution of nitric acid in watertion in carbon dioxide-free water.] Record the buret Calibration solution: Prepare a 11.4-µg/mL solution ofreading and the conductivity meter reading after each USP Boric Acid RS in the Blank.addition of the sodium hydroxide solution.Standard solution A: Dissolve 0.2500g of USP LowPlot the conductivity measurements on the y-axis Molecular Weight Heparin for Boron Analysis RS inagainst the volumes of sodium hydroxide added on about 2mL of water, add 100µL of nitric acid, andthe x-axis. The graph will have three linear sections—dilute with the Blank to 10.00mL.an initial downward slope, a middle slight rise, and a Standard solution B: Dissolve 0.2500g of USP Lowfinal rise. For each of these sections, draw the best-fit Molecular Weight Heparin for Boron Analysis RS instraight lines using linear regression analysis. At the about 2mL of Blank, add 10µL of a 5.7-mg/mL solu-points where the first and second straight lines inter-tion of USP Boric Acid RS, and dilute with the Blank tosect and where the second and third lines intersect,10.00mL. This solution contains 1µg/mL of boron.draw perpendiculars to the x-axis to determine the Sample solution: Dissolve 0.2500g of Dalteparin So-volumes of sodium hydroxide taken up by the sample dium in about 2mL of water, add 100µL of nitric acid,at those points. The point where the first and second and dilute with the Blank to 10.00mL.lines intersect corresponds to the volume of sodium Analysishydroxide taken up by the sulfate groups (V S). The Samples:Blank, Calibration solution, Standard solutionpoint where the second and third lines intersect corre-A, Standard solution B, and Sample solutionsponds to the volume of sodium hydroxide consumed Boron is determined by measurement of the emissionby the sulfate and the carboxylate groups together from inductively coupled plasma (ICP) at 249.733 nm(V T).or a suitable wavelength. Use an appropriate appara-Calculate the molar ratio of sulfate to carboxylate: tus with settings that have been optimized as directedby the manufacturer.Result = V S/(V T−V S) Calculate the content of boron in Dalteparin Sodiumusing the following correction factor:1The procedure is based on analyses performed with two columns: one 1.5-cm × 2.5-cm packed with anion-exchange resin Dowex 1X8 (200–400 mesh)and the other 1.5-cm × 7.5-cm packed with cation-exchange resin DowexF = (r SB – r SA) × 2/(r C – r B)50WX2 (100–200 mesh).r SB= response of boron from Standard solution BAcceptance criteria: The molar ratio of sulfate to car-Percentageboxylate is NLT 1.8.Concentra-(%,•P H 〈791〉: 5.5–8.0 for a 1.0% solution in water tion for comparison•L OSS ON D RYING 〈731〉Standard (µg RS perwith test Sample: 1gsolutionDilution mL)specimen)Analysis: Dry the Sample under vacuum at 70° for 6 h.A (1 in 2)500 1.0Acceptance criteria: NMT 10%B (1 in 4)2500.5•B ACTERIAL E NDOTOXINS T EST 〈85〉: It contains NMT 0.01C (1 in 10)1000.2USP Endotoxin Unit/IU of anti-factor Xa activity.D(1 in 20)500.1ADDITIONAL REQUIREMENTS•P ACKAGING AND S TORAGE : Preserve in tight, light-resistant Test solution—Dissolve an accurately weighed quantity of containers, and store below 40°, preferably at room Danazol in Solvent to obtain a solution containing 50mg temperature.per mL.•L ABELING : Label to state the number of Anti-factor X a In-Procedure—Apply separately 5µL of the Test solution and ternational Units of activity per mg.5µL of each Standard solution to a suitable thin-layer chro-•USP R EFERENCE S TANDARDS 〈11〉matographic plate (see Chromatography 〈621〉) coated with USP Boric Acid RSa 0.25-mm layer of chromatographic silica gel mixture. Posi-USP Dalteparin Sodium RS tion the plate in a chromatographic chamber and develop USP Endotoxin RSthe chromatograms in a solvent system consisting of a mix-USP Low Molecular Weight Heparin for Bioassays RSture of cyclohexane and ethyl acetate (7:3) until the solvent USP Low Molecular Weight Heparin for Boron Analysis RS front has moved about three-fourths of the length of the USP Low Molecular Weight Heparin Molecular Weight plate. Remove the plate from the developing chamber, mark Calibrant RSthe solvent front, and allow the solvent to evaporate in v USP39warm, circulating air. Examine the plate under short-wave-length UV light. Expose the plate to iodine vapors for 5min-utes. Compare the intensities of any secondary spots ob-served in the chromatogram of the Test solution with those of the principal spots in the chromatograms of the Standard solutions: the sum of the intensities of secondary spots ob-Danazoltained from the Test solution corresponds to not more than 1.0% of related compounds, with no single impurity corre-sponding to more than 0.5%.Assay—Dissolve about 100mg of Danazol, accuratelyweighed and previously dried, in about 50mL of alcohol ina 100-mL volumetric flask, swirl until dissolved, dilute with alcohol to volume, and mix. Transfer 2.0mL of this solution C 22H 27NO 2337.46to a 100-mL volumetric flask, dilute with alcohol to volume,Pregna-2,4-dien-20-yno[2,3-d ]isoxazol-17-ol, (17α)-.and mix. Similarly, dissolve an accurately weighed quantity 17α-Pregna-2,4-dien-20-yno[2,3-d ]isoxazol-17-ol of USP Danazol RS in alcohol to obtain a Standard solution [17230-88-5].having a known concentration of about 20µg per mL. Con-comitantly determine the absorbances of both solutions in » Danazol contains not less than 97.0percent 1-cm cells at the wavelength of maximum absorbance at and not more than 102.0percent of C 22H 27NO 2,about 285 nm, using alcohol as the blank. Calculate the quantity, in mg, of C 22H 27NO 2 in the portion of Danazol calculated on the dried basis.taken by the formula:Packaging and storage—Preserve in tight, light-resistant containers.5C (A U /A S )USP Reference standards 〈11〉—in which C is the concentration, in µg per mL, of USPUSP Danazol RS Danazol RS in the Standard solution; and A U and A S are the Identification—absorbances of the solution of Danazol and the Standard A: Infrared Absorption 〈197K 〉.solution, respectively.B: Ultraviolet Absorption 〈197U 〉—Solution: prepared as directed in the Assay .Specific rotation 〈781S 〉: between +21° and +27°.Test solution: 10mg per mL, in chloroform.Danazol CapsulesLoss on drying 〈731〉—Dry it at a pressure not exceeding 5mm of mercury at 60° to constant weight: it loses not » Danazol Capsules contain not less thanmore than 2.0% of its weight.90.0percent and not more than 110.0percent of Chromatographic purity—the labeled amount of C 22H 27NO 2.Solvent—Prepare a mixture of chloroform and methanol (9:1).Packaging and storage—Preserve in well-closed contain-Standard solutions—Dissolve an accurately weighed quan-ers.tity of USP Danazol RS in Solvent to obtain a solution having USP Reference standards 〈11〉—a known concentration of 1mg per mL. Dilute quantita-USP Danazol RStively with Solvent to obtain Standard solutions having the Identification—Shake the contents of a sufficient number following compositions:of Capsules, equivalent to about 50mg of Danazol, with 50mL of chloroform, and filter. Evaporate the filtrate on a steam bath with the aid of a stream of nitrogen to dryness:the IR absorption spectrum of a potassium bromide disper-sion of the residue, previously dried, exhibits maxima at the。