含量方法确认方案.doc

SOP No.: US-AD-PT-US002-01 METHOD VERIFICATION PROTOCOL FOR ASSAY OF CYTARABINE阿糖胞苷含量检查方法确认方案Area to be Distributed：AD, QA分发部门：分析部，质量保证部Print Name:姓名：Title:职位：Signature:签名：Date:日期： Author:起草人： Approver:批准人：Approver:批准人：Reviewer:审核人：Approver:批准人：Contents 目录一、Introduction3二、Scope3三、Responsibility3四、Definitions4五、Project5六、References18一、 Introduction简介# is in the process of developing Cytarabine Injection, a liquid product for parenteral administration, for #. Cytarabine is a chemotherapy agent with a molecular weight of 243.2. The API from Zhejiang Hisun Pharmaceutical Co. Ltd, which is approved by FDA, is used for Cytarabine Injection by Lummy. The assay method for Cytarabine is the method in the current USP34. The HPLC method for assay test of Cytarabine in USP is a quantitative method. The method will be verified according to USP, including system suitability, specificity, precision, linearity, stability of solution and mobile phase.阿糖胞苷注射液是一种临床用液体产品，#药业为赛金药业研发该品种。阿糖胞苷是一种化学药，分子量为243.2。#研发阿糖胞苷注射液所用原料药来源于浙江海正药业，是获得了FDA认证的原料药。阿糖胞苷含量检测方法来自现行USP34，HPLC含量检查方法，是定量检测方法。依据USP进行确认，本次确认内容包括系统适用性、专属性、精密度、线性、溶液稳定性和流动相稳定性。二、 Scope范围This protocol applies to the verification for assay of Cytarabine.该方案适用于阿糖胞苷含量测定的方法确认。三、 Responsibility职责Name姓 名Department所在部门Title职称/职务Responsibility职 责Group leader, responsible for approve of protocols, records and reports, and organize the implementation of the protocol组长，负责方案、记录和报告的审核并组织方案的实施Initiate and implement the protocol起草并执行方案Approve protocols, records and reports方案、记录和报告的审核批准Monitor the implementation of the protocol监督确认方案的实施四、 Definitions 定义1System suitability系统适用性System suitability tests are an integral part of gas and liquid chromatographic methods. They are used to verify that resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. The tests are based on the concept that the equipment, electronics, analytical operations, and sample to be analyzed constitute an integral system that can be evaluated as such.系统适用性试验是气相色谱和液相色谱方法的必要组成部。用以确认该分析方法色谱系统的分离度和重现性是适用的。该试验把分析设备、电子仪器、分析操作和分析样品作为一个整体来进行评估。2Specificity专属性Specificity is the ability to assess the analyte unequivocally in the presence of components which may be expected to be present. Typically these might include process impurities, degradates, matrix, etc.专属性是指在一些预期组分的存在下，被分析物被明确检测评估的能力。通常这些预期组分是指工艺杂质，降解产物，基质等。3Precision 精密度The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.分析过程的精密度是指同一样品在指定的条件下所做的一系列测试值的相近程度（离散度）。3.1Repeatability重复性Repeatability expresses the precision under the same operating conditions over a short interval of time.重复性是指同一个实验室相同操作条件下短期内测定实验结果的精密度。3.2Intermediate precision中间精密度Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipments, etc.中间精密度是指在同一实验室内改变其他条件，包括不同的日期，不同的人，不同的仪器等的精密度。4Linearity线性The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.一个分析方法的线性是指在给定范围内获得的实验结果与分析物的浓度（数量）成正比的能力。五、 Project方案内容1Acceptance criteria可接受标准Items确认参数Acceptance criteria可接收标准System suitability系统适用性Five replicate injections of standard solution, RSD2.0%.重复进标准溶液5次，RSD2.0%Resolution2.5(between Cytarabine and Uracil arabinoside)阿糖胞苷与阿糖尿苷分离度2.5Specificity专属性Blank (water) can not have interfering peak that can affect sample measured.空白(水)不含干扰样品测定的峰。Resolution2.5,(between Cytarabine and Uracil arabinoside)阿糖胞苷与阿糖尿苷分离度2.5Precision精密度Repeatability重复性The RSD for 6 assay results of Cytarabine is 2.0%.6次结果中阿糖胞苷含量的RSD2.0%。Intermediate precision中间精密度The RSD for 6 assay results of Cytarabine is 2.0%.6次结果中阿糖胞苷含量的RSD2.0%。The RSD for 12 assay results of Cytarabine is 2.0%.12次结果中阿糖胞苷含量的RSD不得过2.0%。Linearity线性The correlation coefficient (R2) is NLT 0.999, y intercept is NMT 5% of standard response.相关系数（R2）不得低于0.999，y轴截距不得大于标准响应值的5%。Stability of solution 溶液稳定性Resolution solution: two peaks of Cytarabine and Uracil arabinoside exist分离度测试液：阿糖胞苷与阿糖尿苷峰存在Recovery from aged standard preparation is between 98.0%102.0% against that from initial results.与初始结果比较，放置后的标准溶液回收率在98.0%102.0%之间。The assay result from aged assay preparation is between 98.0%102.0% against that from initial results.与初始结果比较，放置后的含量应在98.0%102.0%之间。Stability of mobile phase流动相稳定性The appearance is is clear, without apparent change.溶液澄清无明显变化。 Five replicate injections of standard solution, RSD2.0%.重复进标准溶液5次，RSD2.0%Resolution2.5(between Cytarabine and Uracil arabinoside)阿糖胞苷与阿糖尿苷分离度2.52Reagents, sample, reference standards, and instruments试剂、样品、标准品、仪器2.1Reagents试剂Monobasic sodium phosphate (HPLC grade)磷酸二氢钠（HPLC grade）Mibasic sodium phosphate (HPLC grade)磷酸氢二钠（HPLC grade）Methanol (HPLC grade) 甲醇（HPLC grade）Water（purified, fresh daily ）水（纯化水，每天新制）2.2Sample样品Cytarabine (Zhejiang Hisun Pharmaceutical Co. Ltd, 1 batch)阿糖胞苷（浙江海正药业股份有限公司，1批）2.3Reference standards标准品USP Cytarabine RS(current lot)阿糖胞苷USP标准品（现行批）USP Uracil Arabinoside RS(current lot)阿糖尿苷USP标准品（现行批）2.4Instruments仪器HPLC instrument 液相色谱仪Waters e2695Electronic balance电子天平XS205 Dual rangTransparent degrees detector澄明度仪YB-3Test method 测试方法USP 34 Cytarabine (in the appendix)3.1Chromatographic condition色谱条件Column:色谱柱：Bondapak C18, 2504.6mm,10mMobile phase:流动相：Phasphate buffer : methanol = 95 : 5 (v:v)磷酸盐缓冲液:甲醇=95:5 (v:v)Flow rate:流速：1.0 ml / minDetection wavelength:检测波长：254nmColumn temperature:柱温：25Injection volume:进样体积：10lPhosphate buffer:磷酸盐缓冲液：Dissolve 0.73g of monobasic sodium phosphate and 1.4g of dibasic sodium phosphate in 1L of water, mix, and filter.将0.73g磷酸二氢钠和1.4g磷酸氢二钠溶于1L水中，混匀，过滤。Mobile phase:流动相Prepare a filtered and degassed mixture of Phosphate buffer and methanol (95:5). Make adjustments if necessary. 磷酸缓冲盐溶液：甲醇（95:5），过滤脱气即得。必要时调整比例。Standard preparation:标准液：Weigh accurately about 20 mg of USP Cytarabine RS, transfer into a 200-ml volumetric flask, dissolve and dilute with water to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称取约20mgUSP的阿糖胞苷标准品至200ml容量瓶中，用水溶解后稀释至刻度，混匀，即得。浓度约为0.1mg/ml。Resolution solution:分离度测试溶液：Weigh accurately about 10 mg of USP Uracil arabinoside RS, transfer into a 100-ml volumetric flask, dissolve and dilute with Standard preparation to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称取约10mg阿糖尿苷至100ml容量瓶中，用标准液溶解后稀释至刻度，混匀，即得。浓度约为0.1mg/ml。Assay preparation:供试液：Transfer about 20mg of Cytarabine, accurately weighed, to a 200-ml volumetric flask, dissolve and dilute with water to volume, and mix, the solution has a concentration of about 0.1mg/ml.精密称取阿糖胞苷约20mg，置200ml容量瓶中，用水溶解并稀释至刻度，混匀，浓度约为0.1mg/ml。3.2Procedure实验过程Separately inject equal volumes (about 10L) of the Resolution solution, the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peak. Calculate the assay of Cytarabine by the following formula:将相同体积（约10L）的分离度测试液、标准液和供试液分别注入色谱仪中，记录色谱图，计算峰面积。用下式计算阿糖胞苷的含量：Assay(as-is)=P(Cs / Cu) (ru / rs)in which,其中P is the content of USP Cytarabine RS;P为阿糖胞苷USP标准品的含量；Cs is the concentration of USP Cytarabine RS in the Standard preparation, mg /ml; Cs为标准液中阿糖胞苷USP标准品的浓度，mg/ml；Cu is the concentration of Cytarabine in the Assay preparation, mg / ml; Cu为供试液中阿糖胞苷的浓度，mg/ml；ru is the peak response of Cytarabine in the Assay preparation; ru为供试液中阿糖胞苷的峰响应值；rs is the peak response of USP Cytarabine RS in the Standard preparation: rs为标准液中阿糖胞苷USP标准品的峰响应值。Assay( dried basis)= Assayas-is%1-loss on drying4Verification确认4.1System suitability系统适用性4.1.1Preparation of solutions:溶液制备：Standard preparation:标准液：Weigh accurately about 20 mg of USP Cytarabine RS, transfer into a 200-ml volumetric flask, dissolve and dilute with water to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称取约20mgUSP的阿糖胞苷标准品至200ml容量瓶中，用水溶解后稀释至刻度，混匀，即得。浓度约0.1mg/ml。Resolution solution:分离度测试溶液：Weigh accurately about 10 mg of USP Uracil arabinoside RS, transfer into a 100-ml volumetric flask, dissolve and dilute with Standard preparation to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称取约10mg阿糖尿苷至100ml容量瓶中，用标准液溶解后稀释至刻度，混匀，即得。浓度约0.1mg/ml。4.1.2Procedure实验过程Inject equal volumes (about 10L) of the blank(water), the Resolution solution, the Standard preparation into the chromatograph, record the chromatograms. Calculate the relative standard deviation (RSD) of peak area for replicate injections. 取相同体积（大约10L）空白（水）、分离度测试溶液、标准溶液进样，记录色谱图。计算重复进样峰面积相对标准偏差(RSD)。Injections S.N.Description No. of injection(s)1Blank(water)12Resolution solution13Standard preparation54.1.3Acceptance criteria可接受标准Five replicate injections of Standard preparation, RSD 2.0%.标准溶液重复进5次，RSD 2.0%4.2Specificity专属性4.2.1Preparation of solutions溶液配制Standard preparation:标准液：Refer to 4.1.1。Selectivity preparation:选择性溶液Weigh accurately about 10 mg of USP Uracil arabinoside RS into a 100-ml volumetric flask, dissolve and dilute with Standard preparation to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称定约10mg阿糖尿苷USP标准品至100ml容量瓶中，用标准液溶解后稀释至刻度，混匀。浓度约0.1mg/ml。Unspiked test preparation:供试液Weigh accurately about 20 mg of Cytarabine sample, transfer into a 200-ml volumetric flask, dissolve and dilute with water to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称取约20mgUSP的阿糖胞苷供试品至200ml容量瓶中，用水溶解后稀释至刻度，混匀，即得。浓度约0.1mg/ml。4.2.2Procedure实验过程Inject equal volumes (about 10L) the blank(water), Selectivity solution and Unspiked test solution into the chromatograph and record the chromatograms. 取相同体积（约10L）空白（水）、选择性溶液以及供试液注入色谱仪，记录色谱图。Injections S.N.Description No. of injection(s)1Blank(water)12Unspiked test preparation13Selectivity preparation14.2.3Acceptance criteria可接受标准Blank (water) can not have interfering peak that can affect sample measured.空白(水)不含干扰样品测定的峰。Resolution2.5 (between Cytarabine and Uracil arabinoside)阿糖胞苷与阿糖尿苷分离度2.54.3Precision精密度4.3.1Repeatability重复性4.3.1.1Preparation of solutions:溶液配制Standard preparation:标准液：Refer to 4.1.1Assay preparation：供试液：Weigh accurately about 20 mg of Cytarabine, transfer into a 200-ml volumetric flask, dissolve and dilute with water to volume, mix well. The solution has a concentration of about 0.1 mg / ml.精密称取约20mg阿糖胞苷供试品至200ml容量瓶中，用水溶解后稀释至刻度，混匀，即得。浓度约0.1mg/ml。Prepare six Assay preparations.准备6份供试溶液。4.3.1.2Procedure实验过程Test 6 Assay preparations by one analyst, record the chromatograms. Calculate the assay on the mean peak area of the 5 duplicate standard injections and the relative standard deviation (RSD) of assay. 由1名分析员测试6份试验溶液，记录色谱图。以标准液重复5针平均峰面积计算含量及含量相对标准偏差(RSD)。Injections S.N.Description No. of injection(s)1Blank(water)12Resolution solution13Standard preparation 154Standard preparation 225Standard preparation 116Assay preparationEach in duplicate*7Standard preparation 1(bracketing)1*Up to six injections of Assay preparation can be made at once.4.3.1.3Acceptance criteria可接受标准The RSD for 6 assay results of Cytarabine 2.0%6次结果中阿糖胞苷含量的RSD不得过2.0%。4.3.2Intermediate precision中间精密度Prepare six Assay preparations using the same batch of Cytarabine and in the same manner with those in 4.3.1. Inject the solutions into the chromatograph using different HPLC and different columns by different analysts on different dates.采用与4.3.1中相同批次的阿糖胞苷样品，配制6份试验溶液，使用不同的仪器和色谱柱，由不同的分析员，在不同时间试验。4.3.3Acceptance criteria可接受标准The RSD for 6 assay results of Cytarabine 2.0%.6次结果中阿糖胞苷含量的RSD不得过2.0%。The RSD for the 12 assay results of Cytarabine 2.0%.12次结果中阿糖胞苷含量的RSD不得过2.0%。4.4Linearity线性4.4.1Preparation of solutions溶液制备Linearity stock preparation：线性储备液Weigh accurately about 50 mg of Cytarabine, transfer into a 50-ml volumetric flask, dissolve and dilute with water to volume, mix well. The solution has a concentration of about 1.0mg/ml.精密称取约50mg阿糖胞苷供试品至50ml容量瓶中，加水溶解并稀释至刻度，混匀。浓度约为1.0mg/ml。Linearity preparations：线性溶液80%:Transfer 4.0 ml of the Linearity stock preparation into a 50-ml volumetric flask, dilute with water to volume, mix well. The solution has a concentration of about 0.08mg/ml.80%：量取4.0 ml线性储备液至50-ml容量瓶中，用水稀释至刻度，混匀，即得。浓度约为0.08mg/ml。90%:Transfer 4.5 ml of the Linearity stock preparation into a 50-ml volumetric flask, dilute with water to volume, mix well. The solution has a concentration of about 0.09mg/ml.90%：量取4.5 ml线性储备液至50-ml容量瓶中，用水稀释至刻度，混匀，即得。浓度约为0.09mg/ml。100%:Transfer5.0 ml of the Linearity stock preparation into a 50-ml volumetric flask, dilute with water to volume, mix well. The solution has a concentration of about 0.10mg/ml.100%：量取5.0 ml线性储备液至50-ml容量瓶中，用水稀释至刻度，混匀，即得。浓度约为0.10mg/ml。110%:Transfer 5.5 ml of the Linearity stock preparation into a 50-ml volumetric flask, dilute with water to volume, mix well. The solution has a concentration of about 0.11mg/ml.100%：量取5.5 ml供试储备液至50-ml容量瓶中，用水稀释至刻度，混匀，即得。浓度约为0.11mg/ml。120%:Transfer 6.0 ml of the Linearity stock preparation into a 50-ml volumetric flask, dilute with water to volume, mix well. The solution has a concentration of about 0.12mg/ml.120%：量取6.0 ml线性储备液至50-ml容量瓶中，用水稀释至刻度，混匀，即得。浓度约为0.12mg/ml。4.4.2Injections S.N.Description No. of injection(s)1Blank(water)12Resolution solution13Linearity preparations：80%，90%，100%，110%，120%Each in duplicate4.4.3Acceptance criteria可接收标准Calculate the function of Cytarabine concentration VS the peak area. Record the correlation coefficient, y-intercept, slope of the regression line. 计算阿糖胞苷浓度与峰面积的方程。记录相关系数，y截距，斜率。The correlation coefficient (R2) is NLT 0.999.相关系数不得低于0.999。y intercept is NMT 5% of standard response.Y轴截距不得大于标准响应值的5%。4.5Stability of solutions溶液稳定性4.5.1Stability of standard preparation 标准液稳定性4.5.1.1Preparation of solutions溶液配制Standard preparation:标准液：Refer to 4.1.1Resolution solution:分离度测试溶液：Refer to 4.1.14.5.1.2Procedure实验过程One standard preparation is prepared and stored at ambient condition. The standard preparation will be analyzed on initial, day1, day 2, day 3 after preparation, and report recovery results at each time point .配制一份标准液并放置在试验环境中，分别于初始（0天）、1天、2天、3天检测标准液回收率，并报告每个考察点的结果。InjectionsS.N.Description No. of injection(s)1Blank(water)12Resolution solution13Standard preparation 154Standard preparation 225Standard preparation 116Aged standard preparation24.5.1.3Acceptance criteria 可接收标准Recovery from aged standard preparation is between 98.0%102.0% against that from initial results.与初始结果比较，放置后的标准液回收率在98.0%102.0%之间。4.5.2Stability of assay preparation供试液稳定性4.5.2.1Preparation of solutions溶液配制Standard preparation:标准液：Refer to 4.1.1Resolution solution:分离度测试溶液：Refer to 4.1.1Assay preparation:供试液：Weigh accurately about 20 mg of Cytarabine, transfer into a 200-ml volumetric flask, dissolve and dilute with water to volume, mix well. The solution has a concentration of about 0.1 mg/ml/精密称取约20mgUSP的阿糖胞苷至200ml容量瓶中，用水溶解后稀释至刻度，混匀，即得。浓度约为0.1mg/ml。4.5.2.2Procedure实验过程One Assay preparation is prepared and stored at ambient condition. The Assay preparation will be analyzed on initial, day1, day 2, day 3 after preparation, and report assay results at each time point.配制一份供试液并放置在试验环境中，分别检测供试液初始（0天）、1天、2天、3天的含量，并报告每个考察点的含量结果。InjectionsS.N.Description No. of injection(s)1Blank(water)12Resolution solution13Standard preparation 154Standard preparation 225Standard preparation 116Aged Assay preparation24.5.2.3Acceptance criteria 可接收标准The assay result from aged assay preparation is between 98.0%102.0% against that from initial results.与初始结果比较，放置后的含量应在98.0%102.0%之间。4.5.3Stability of resolution solution分离度测试液稳定性4.5.3.1Preparation of solutions溶液制备Resolution solution:分离度测试溶液：Refer to 4.1.14.5.3.2Procedure实验过程One resolution solu