有关物质方法确认方案.doc

.METHOD VERIFICATION PROTOCOL FOR CHROMATOGRAPHIC PURITY OF CYTARABINE阿糖胞苷有关物质检查方法确认方案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、References25一、 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 Chromatographic purity method for Cytarabine is the method in the current USP34. The HPLC method for Chromatographic purity test of Cytarabine is a quantitative method. The method will be verified according to USP, including system suitability, specificity, LOD, LOQ, precision, solution stability and mobile phase stability.阿糖胞苷注射液是一种临床用液体产品，#药业为#药业研发该品种。阿糖胞苷是一种化学药，分子量为243.2。莱美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。阿糖胞苷色谱纯度检测方法来自现行USP34，色谱纯度检查方法，是定量检测方法。依照USP，本次确认内容包括系统适用性，专属性，检测限,定量限、精密度、溶液稳定性和流动相稳定性。 二、 Scope范围This protocol applies to the verification for chromatographic purity of cytarabine.该方案适用于阿糖胞苷色谱纯度的方法确认。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、记录和报告的审查批准并组织方案的实施。 Initiate and implement the protocol起草并执行方案Approve protocols, records and reports方案、记录和报告的审查批准Monitor the implementation of the protocol监督确认方案的实施四、 Definitions 定义 1Limit of Detection (LOD)检测限（LOD）The limit of detection of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.分析规程中的检测限是指样品中的被分析物在该分析方法中能被检测，但不需要准确定量的最低浓度。The detection limit is determined by analyzing samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.检测限是通过分析样品已知浓度的被分析物，从而建立其可稳定检测的最低浓度水平。2Limit of Quantitation（LOQ）定量限（LOQ）The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. 分析方法中的定量限是指样品中被分析物能够被定量检测并保证一定精密度和准确度的最低浓度。The limit of quantitation is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.定量限是在样品中低浓度化合物被定量检测的参数，该参数经常被用于杂质和/或降解产物的测定。3system 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。4Specificity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。5Precision 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）。5.1Repeatability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。5.2Intermediate precision中间精密度Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, 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。五、 Project方案内容 1Acceptance criteria可接受标准Items 确认参数Acceptance Criteria可接受标准System suitability系统适用性Six replicate injections of Standard solution, RSD 3.0%.重复进样标准溶液6次，RSD 3.0%Resolution1.25 (between cytarabine and uridine)阿糖胞苷与尿苷分离度1.25The S/N ratio of the Cytarabine peak and the noise near the Cytarabine in the System sensitivity solution is not less than 10.系统灵敏度溶液中阿糖胞苷峰与其附近噪音的信噪比不小于10。Specificity专属性Diluent can not have interfering peak that can affect sample measured.空白不含干扰样品测定的峰。Resolution1.25 (between cytarabine and uridine)阿糖胞苷与尿苷分离度1.25Resolution 1.5 (between uracil and cytarabine，uridine and uracil arabinoside)尿嘧啶与阿糖胞苷、尿苷与阿糖尿苷分离度均1.5Limit of Detection (LOD) 检测限Cytarabine阿糖胞苷S/N3, LOD of each specimen0.015% (concentration : 0.75g/mL)S/N3，每个物质的LOD0.015%(浓度:0.75g/mL) Uracil尿嘧啶Uridine尿苷Uracil Arabinoside阿糖尿苷Limit of Quantitation (LOQ)定量限Cytarabine阿糖胞苷S/N10，LOQ of each specimen0.05% (concentration : 2.5g/mL)S/N10，每个物质的LOQ0.05%（浓度:2.5g/mL）Uracil尿嘧啶Uridine尿苷Uracil arabinoside阿糖尿苷Precision 精密度Repeatability 重复性RSD20% (for 6 results of individual impurity which is above 0.03% and the total impurities)RSD20%(对于6次试验测得的大于0.03%的每个杂质和总杂质)Intermediate precision 中间精密度RSD20% (for 6 results of individual impurity which is above 0.03% and the total impurities)RSD20%(对6次试验测得的大于0.03%每个杂质和总杂质)RSD30% (for 12 results of individual impurity which is above 0.03% and the total impurities)RSD30%(对12次试验测得的大于0.03%每个杂质和总杂质)Solution stability溶液稳定性System suitability solution: three peaks of Cytarabine, Uridine and Uracil arabinoside exist.系统适用性溶液：阿糖胞苷、尿苷与阿糖尿苷3个峰存在。Standard solution: recovery from aged standard solution is between 90.0%110.0% against that from initial results.标准溶液：与初始结果比较，放置后的标准溶液回收率在90.0%110.0%之间。Test solution: the RSD of % impurity results which is above 0.03% and the total impurity between aged test solution and the initial results is NMT 30%.供试品溶液：放置后的供试液中与初始结果中大于0.03%的杂质及总杂质含量RSD不大于30%。Mobile phase stability流动相稳定性The appearance is clear, without apparent change.外观应澄清无明显变化。Six replicate injections of standard solution, RSD3.0%.重复进标准溶液6次，RSD3.0%Resolution1.25 (between Cytarabine and Uridine)阿糖胞苷与尿苷分离度1.25The S/N ratio of the Cytarabine peak and the noise near the Cytarabine in the System sensitivity solution is not less than 10.系统灵敏度溶液中阿糖胞苷峰与其附近噪音的信噪比不小于10。2Reagents, sample, reference standards, and instruments试剂、样品、标准品、仪器2.1Reagents试剂Monobasic sodium phosphate (HPLC)磷酸二氢钠（HPLC）Dibasic sodium phosphate (HPLC)磷酸氢二钠（HPLC）Sodium hydroxide (AR) 氢氧化钠（AR）Phosphoric acid (AR) 磷酸（AR）Methanol (HPLC) 甲醇（HPLC）Uridine (AR) 尿苷（AR）Uracil (AR) 尿嘧啶（AR）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 rangpH meterpH计Metrohm 8273Test method 测试方法USP 34 Cytarabine（in the appendix）3.1Chromatographic condition色谱条件Column:色谱柱：Supelcosil LC-18-DB, 2504.6mm，5mMobile phase:流动相：See gradient见梯度Flow Rate:流速：1.0 mL/minDetection wavelength:检测波长：254 nmColumn temperature:柱温：20Injection volume:进样体积：20lPhosphate buffer:磷酸盐缓冲液：Prepare a solution containing 0.01M monobasic sodium phosphate and 0.01M dibasic sodium phosphate in a suitable container. Adjust with 0.1M sodium hydroxide or 0.1M phosphoric acid to a pH of 7.0.配制浓度为0.01M的磷酸二氢钠和0.01M的磷酸氢二钠溶液，用0.1M氢氧化钠或0.1M磷酸调pH至7.0。Solution A:溶液APrepare a filtered and degassed mixture of Phosphate buffer and methanol (49:1).Make adjustments if necessary. Prepare this solution fresh daily.磷酸缓冲盐溶液：甲醇（49:1），过滤脱气即得。必要时调整比例，每天新配。Solution B:溶液BPrepare a filtered and degassed mixture of Phosphate buffer and methanol (7:3).Make adjustments if necessary. Prepare this solution fresh daily.磷酸缓冲盐溶液：甲醇（7:3），过滤脱气即得。必要时调整比例，每天新配。Gradient：梯度Time (minute)时间（分钟）Solution A溶液ASolution B溶液B01000101000200100250100301000501000System suitability solution:系统适用性溶液：Weigh accurately about 20 mg of Uridine, transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uridine Stock Solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg尿苷至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为尿苷储备液，溶液浓度大约是0.1mg/mL。Weigh accurately about 20 mg of USP Uracil arabinoside RS , transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uracil arabinoside Stock Solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg阿糖尿苷USP标准品至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为阿糖尿苷储备液，溶液浓度大约是0.1mg/mL。Weigh accurately about 25 mg of USP Cytarabine RS, transfer into a 5-mL volumetric flask, transfer 1.0 mL of Uridine Stock Solution and 1.0 mL of Uracil Arabinoside Stock Solution into the volumetric flask, dissolve and dilute to volume with water, mix well to obtain System suitability solution, the solution contain about 0.02, 0.02, 5.0 mg/mL, respectively.精密称定25mg阿糖胞苷USP标准品至5mL容量瓶中，分别加入1.0mL尿苷储备液和1.0mL阿糖尿苷储备液，用水溶解后稀释至刻度，混匀，即得系统适用性溶液，浓度分别为0.02、0.02、5.0mg/ml。Standard solution: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. Transfer 2.0mL of obtained solution to a 50-mL volumetric flask, dilute with water to volume and mix well, the solution has concentration of about 4g /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。精密移取该溶液2.0mL置50mL量瓶中，加水稀释至刻度，摇匀，即得, 浓度约为4g/mL。Test solution:供试液：Transfer about 25mg of cytarabine, accurately weighed, to a 5.0-mL volumetric flask, dissolve in and dilute with water to volume, and mix, the solution has a concentration of about 5mg/mL. NOTE-prepare this solution fresh daily.精密称取阿糖胞苷约25mg，置5mL容量瓶中，用水溶解并稀释至刻度，混匀，浓度约为5mg/mL。（注：此溶液每天新配。）3.2Procedure实验过程Inject equal volumes (about 20L) of the System suitability solution, the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of uracil arabinoside in the portion of Cytarabine taken by the formula:将相同体积（约20L）的系统适用性溶液、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中阿糖尿苷的含量：P(Cs / Ci )(ri/1.34rs)in which,其中，P is the content of USP Cytarabine RS in the Standard solution; P为标准液中阿糖胞苷USP标准品的含量Cs is the concentrationof USP Cytarabine RS in the Standard solution, mg / mL; Cs为标准液中阿糖胞苷USP标准品的浓度，mg/mL；Ci is the concentration of Cytarabine in the Test solution, mg / mL;Ci为（阿糖胞苷）样品浓度，mg/mL；1.34 is the relative response factor for uracil arabinoside; 1.34为阿糖尿苷相对响应因子；ri is the peak response of uracil arabinoside in the Test solution; ri为供试液中阿糖尿苷的峰响应值；rs is the peak response of USP Cytarabine RS in the Standard solution: rs为标准液中阿糖胞苷USP标准品的峰响应值。Not more than 0.20% is found.阿糖尿苷含量不得过0.20%。Calculate the percentage of all other impurities in the portion of Cytarabine taken by the formula: 其它杂质含量按下式计算：P(CS / Ci )(ri/Frs)in which, 式中P is the content of USP Cytarabine RS in the Standard solution; P为标准液中阿糖胞苷USP标准品的含量Cs is the concentrationof USP Cytarabine RS in the Standard solution, mg / mL; Cs为标准液中阿糖胞苷USP标准品的浓度，mg/mL；Ci is the concentration of Cytarabine in the Test solution, mg/mL;Ci为（阿糖胞苷）样品浓度，mg/mL；ri is the peak response of each impurity in the Test solution; ri为供试液中各单个杂质的峰面积；rs is the peak response of USP Cytarabine RS in the Standard solution;rs为标准液中阿糖胞苷USP标准品的峰面积；F is the relative response factor, equals 2.5 for the Uracil peak, with a relative retention time time of 0.55, 1.5 for peaks with relative retention times of 0.38, 0.43 and 1.14, and 1.0 for all other peaks.F为相对响应因子，尿嘧啶相对保留时间为0.55，相对响应因子为2.5，相对保留时间为0.38、0.43、1.14的杂质，相对响应因子为1.5，其它峰为1.0。Not more than 0.10% of any individual impurity is found, and not more than 0.30% of total impurities is found (including uracil arabinoside).单个杂质不得过0.10%，总杂质不得过0.30%（包括阿糖尿苷）。 4Verification确认4.1System suitability系统适用性4.1.1Preparation of solutions:溶液制备：Impurity stock solution:杂质储备液：Weigh accurately about 20 mg of Uridine, transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uridine Stock Solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg尿苷至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为尿苷储备液，浓度大约0.1mg/mL。Weigh accurately about 20 mg of USP Uracil Arabinoside RS , transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uracil Arabinoside Stock Solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg阿糖尿苷USP标准品至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为阿糖尿苷储备液，浓度大约0.1mg/mL。System suitability solution:系统适用性测试液：Weigh accurately about 25 mg of USP Cytarabine RS, transfer into a 5-mL volumetric flask, transfer accurately 1.0 mL of Uridine Stock Solution and 1.0 mL of Uracil Arabinoside Stock Solution into the volumetric flask, dissolve and dilute to volume with water, mix well. (0.02mg/mL for Uridine, 0.02 mg/mL for Uracil Arabinoside, 5.0 mg/mL for Cytarabine).精密称定25mg阿糖胞苷USP标准品至5mL容量瓶中，分别加入1.0mL尿苷储备液和1.0mL阿糖尿苷储备液，用水溶解后稀释至刻度，混匀（浓度分别为尿苷0.02mg/mL、阿糖尿苷0.02 mg/mL、阿糖胞苷5.0mg/mL）。 Standard solution:标准液：Weigh accurately about 20 mg of USP Cytarabine RS, transfer into a 200-mL volumetric flask, dissolve and dilute to volume with water, mix well. Transfer 2.0mL of obtained solution into a 50-mL volumetric flask, dilute with water to volume and mix, the solution has a concentration of about 4g/mL.精密称定约20mg阿糖胞苷USP标准品至200mL容量瓶中，用水溶解并稀释至刻度。移取2.0mL至50mL容量瓶中，用水稀释至刻度并混匀，浓度大约4g/mL。System sensitivity solution:系统灵敏度溶液：Weigh accurately about 20 mg of USP Cytarabine RS, transfer into a 200-mL volumetric flask, dissolve and dilute to volume with water, mix well. Transfer 2.0 mL of obtained solution into a 100-mL volumetric flask, dilute with water to volume and mix, the solution has a concentration of about 2g/mL.精密称定约20mg阿糖胞苷USP标准品至200mL容量瓶中，用水溶解并稀释至刻度。移取2.0mL至100mL容量瓶中，用水稀释至刻度并混匀，浓度大约2g/mL。4.1.2Procedure实验过程Separately inject the System suitability solution, the Standard solution and the System sensitivity solution into the chromatograph, record the chromatograms. Calculate the relative standard deviation (RSD) of the Standard solution, the resolution of the System suitability solution and S/N of the System sensitivity solution. 分别将系统适用性测试液、标准液和系统灵敏度溶液注入色谱，记录色谱图。计算标准溶液的相对标准偏差(RSD)，系统适用性溶液的分离度和系统灵敏度溶液的信噪比。InjectionsS.N.Description No. of injection(s)1Blank(water)12System sensitivity solution13System suitability solution14Blank(water)3 at initial gradient for 12min*5Standard solution6* To eliminate carry over of the injector4.1.3Acceptance criteria可接受标准The S/N ratio of the Cytarabine peak and the noise near the Cytarabine in the System sensitivity solution is not less than 10.系统灵敏度溶液中阿糖胞苷峰与其附近噪音的信噪比不小于10。Six replicate injections of Standard solution, RSD 3.0%.标准溶液重复进6次，RSD 3.0%。Resolution between Cytarabine and Uridine in the System suitability solution is 1.25.系统适用性测试液中阿糖胞苷与尿苷分离度1.25。4.2Specificity 专属性4.2.1Preparation of solutions:溶液制备Impurity stock solution:杂质储备液：Weigh accurately about 20 mg of Uridine, transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uridine stock solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg尿苷至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为尿苷储备液，浓度大约0.1mg/mL。Weigh accurately about 20 mg of USP Uracil Arabinoside RS , transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uracil Arabinoside stock solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg阿糖尿苷USP标准品至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为阿糖尿苷储备液，浓度大约0.1mg/mL。Weigh accurately about 20 mg of Uracil , transfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obtain Uracil stock solution, the solution has a concentration of about 0.1mg/mL.精密称取约20mg尿嘧啶至200mL容量瓶中，用水溶解后稀释至刻度，混匀，作为尿嘧啶储备液，浓度大约0.1mg/mL。Selectivity solution:选择性溶液Weigh accurately about 25 mg of USP Cytarabine RS into a 5-mL volumetric flask, transfer 1.0 mL of Uridine stock solution, 1.0 mL of Uracil stock solution and 1.0 mL of Uracil Arabinoside stock solution into the volumetric flask. dissolve and dilute with water to volume, mix well. (0.02mg/mL for Uridine, 0.02mg/mL for Uracil, 0.02 mg/mL for Uracil Arabinoside, 5.0 mg/mL for Cytarabine)精密称定约25mg阿糖胞苷USP标准品至5mL容量瓶中，分别加入1.0mL尿苷储备液，1.0mL尿嘧啶储备液和1.0mL阿糖尿苷储备液，用水溶解后稀释至刻度，混匀（浓度分别为尿苷0.02mg/mL、尿嘧啶0.02 mg/mL、阿糖尿苷0.02 mg/mL、阿糖胞苷5.0mg/mL）。Unspiked test solution:供试液Weigh accurately about 25mg of Cytarabine sample into a 5-mL volumetric flask，dissolve and dilute to volume with water, the solution has a concentration of about 5.0mg/mL.精密称定约25mg阿糖胞苷样品至5mL容量瓶中，用水溶解后稀释至刻度，溶液浓度约5.0mg/mL。4.2.2Procedure实验过程Inject the Impu