有关物质方法确认方案

1、METHOD VERIFICATION PROTOCOL FORCHROMATOGRAPHIC 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：Sig nature: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、Definitio

2、ns3五、Project5六、References25Introduction简介# is in the process of developing Cytarabine Injection, a liquid product for parenteral adm ini strati on, for #. Cytarabi ne is a chemotherapy age nt with a molecular weight of 2432 The API from Zhejiang Hisun Pharmaceutical Co. Ltd, which is approved by FDA

3、, 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, specifi

4、city, 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。Scope范围This protocol applies to the verific

5、ation for chromatographic purity of cytarabine.该方案适用于阿糖胞苷色谱纯度的方法确认。Responsibility职责四、Definitions定义NameDepartme ntTitleResp on sibility姓名所在部门职称/职务职责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。Init

6、iate 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1 Limit of Detectio n (LOD)检测限(LOD)The limit of detecti on of an in dividual an alytical procedure is the lowest amount of an alyte in a sample which can be d

7、etected but not n ecessarily qua ntitated 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 detect ion limit is determ ined by an alyz ing samples with known concen trati ons 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

8、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。2 Limit of Qua ntitatio n (LOQ)定量限(LOQ)The qua ntitati on limit of an in dividual an alytical 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

9、limit of qua ntitati on is a parameter of qua ntitative assays for low levels of compo unds in sample matrices, and is used particularly for the determ in ati on of impurities an d/or degradati on 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。3 system suitability系统适用性System suitability tests

10、are an integral part of gas and liquid chromatographic methods. They are used to verify that resoluti on 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 t

11、o be analyzed constitute an in tegral 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。4 Specificity专属性Specificity is the ability to assess the analyte unequivocally in the presenee of components which may be expected to be

12、 prese nt. Typically these might in clude 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。5 Precisi on精密度The precisi on of an an alytical procedure expresses the close ness of agreeme nt (degree of scatter) betwee n a series of measureme nts obta

13、ined from multiple sampli ng of the same homoge neous sample un der the prescribed con diti ons.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)。5.1 Repeatability重复性Repeatability expresses the precision under the same operating conditions over a short in terval 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。5.2 I

14、n termediate precisi on中间精密度In termediate precisi on expresses withi n-laboratories variati ons: differe nt days, differe nt an alysts, differe nt equipme nt, 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1Accepta nee criteria可接受标准Items确认参数Accepta nee Criteria可接受标准System suitabilit

15、y 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 1.25 (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 theCytarabine 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Di

16、lue nt can not have in terferi ng peak that can affect sample measured.空白不含干扰样品测定的峰。Resolution 1.25 (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 arab ino side)尿嘧啶与阿糖胞苷、尿苷与阿糖尿苷分离度均1.5Limit of Detect ion (LOD) 检测限Cytarab ine 阿糖胞苷S/N 3,

17、 LOD of each specimenW 0.015% (concentration :0.75 口 g/mL)S/N 3，每个物质的 LOD W 0.015%(浓度:0.75 口 g/mL)Uracil尿嘧啶Uridi ne尿苷UracilArab ino side阿糖尿苷Limit of Quan titati on (LOQ) 定量限Cytarab ine 阿糖胞苷S/N 10, LOQ of each specimenW 0.05% (concentration :2.5 g/mL)S/N 10,每个物质的 LOQ W0.05% (浓度:2.5 口 g/n)Uracil尿嘧啶Uri

18、di ne尿苷Uracilarab ino side阿糖尿苷Precisio n精密度Repeatability 重复性RSDW 20% (for 6 results of in dividual impurity which is above 0.03% and the total impurities)RSDW 20%(对于6次试验测得的大于0.03%的每个杂质和总杂 质)In termediate precisi on 中间精密度RSDW 20% (for 6 results of in dividual impurity which is above 0.03% and the tot

19、al impurities)RSDW 20%(对6次试验测得的大于 0.03%每个杂质和总杂质)RSDW 30% (for 12 results of in dividual impurity which is above 0.03% and the total impurities)RSDW 30%(对12次试验测得的大于 0.03%每个杂质和总杂质)Soluti on stability溶液稳定性System suitability solution: three peaks of Cytarabine, Uridine and Uracil arab ino side exist.系统适

20、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 isbetween 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

21、 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, RSD 1.25 (between Cytarabine and Uridine) 阿糖胞苷与尿苷分离度1.25The S/N ratio of the Cytarab ine p

22、eak and the no ise n ear 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。2Reage nts, sample, refere nee sta ndards, and in strume nts试剂、样品、标准品、仪器2.1 Reage nts试剂Mono basic sodium phosphate (HPLC) 磷酸二氢钠(HPLC)Dibasic sodium phosphate (HPLC) 磷酸氢二钠(HPLC

23、)Sodium hydroxide (AR)氢氧化钠(AR)Phosphoric acid (AR) 磷酸(AR)Metha nol (HPLC)甲醇(HPLC)Uridi ne (AR)尿苷(AR)Uracil (AR)尿嘧啶(AR)Water(purified, fresh daily)水(纯化水，每天新制)2.2 Sample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)2.3 Refere nee sta ndards标准品USP Cytarabine RS(curre

24、nt lot) 阿糖胞苷USP标准品(现行批)USP Uracil arabi noside RS(curre nt lot)阿糖尿苷USP标准品(现行批)2.4 In strume nts仪器HPLC in strume nt液相色谱仪Electr onic bala nee电子天平pH meterpH计Waters e2695XS205 Dual rangMetrohm 8273Test method测试方法USP 34 Cytarabine( in the appendix)3.1 Chromatographic con diti on色谱条件Column:Supelcosil LC-1

25、8-DB, 250 4.6mm, 5(im色谱柱：See gradie nt见梯度1.0 mL/min254 nmMobile phase:流动相：Flow Rate:流速：Detecti on wavele ngth:检测波长：Column temperature: 20 C柱温：Injection volume:20 口1进样体积：Phosphate buffer:磷酸盐缓冲液：Prepare a soluti on containing 0.01M mono basic sodium phosphate and 0.01M dibasic sodium phosphate in a su

26、itable 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 d

27、aily.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 (mi nute)时间(分钟)Solution A溶液ASolution B溶液B0100010

28、1000200100250100301000501000System suitability soluti on: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

29、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 arabi no side RS , tra nsfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well to obta in Uracil arabinoside Stock Solution , the solution has a concentration of about 0.1mg/mL.精密

30、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/mLWeigh accurately about 25 mg of USP Cytarab ine RS, tran sfer into a 5-mL volumetric flask, tran sfer 1.0 mL of Uridi ne Stock Solution and 1.0 mL of Uracil Arabi no side Stock Soluti on in to the volumetric flask, dissolve and dilut

31、e to volume with water, mix well to obtai n System suitability soluti on, the soluti on con tai n 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。Stan dard soluti on:标准液：Weigh accurately about 20 mg o

32、f USP Cytarabi ne RS, tran sfer into a 200-mL volumetric flask, dissolve and dilute with water to volume, mix well. Tran sfer 2.0mL of obta ined soluti on to a 50-mL volumetric flask, dilute with water to volume and mix well, the solution has concentration of about 4 g /mL 。 卩精密称取约20mgUSP的阿糖胞苷标准品至20

33、0mL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4由/mL。Test soluti on:供试液：Tran sfer about 25mg of cytarab ine, accurately weighed, to a 5.0-mL volumetric flask, dissolve in and dilute with water to volume, and mix, the soluti on has a concen trati on of about 5mg/mL. NOTE-prepare this s

34、oluti on 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。)3.2 Procedure实验过程Inject equal volumes (about 20口 LSystem suitability solution, the Standard solutionand the Test soluti on into the chromatograph, record the chromatograms, and measure the peak resp on ses. Calculate t

35、he perce ntage of uracil arab ino side in the porti on of Cytarab ine take n by the formula:将相同体积(约20 口 L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 conten t of USP Cytarabi ne RS in the Sta ndard solutio n;P为标准液中阿糖胞苷 USP标准品的含量Cs is the concen t

36、rati onof USP Cytarabi ne RS in the Stan dard soluti on, mg / mL;Cs为标准液中阿糖胞苷 USP标准品的浓度，mg/mL ;Ci is the concen trati on of Cytarab ine in the Test soluti on, mg / mL;Ci为(阿糖胞苷)样品浓度， mg/mL ;1.34 is the relative resp onse factor for uracil arab ino side;1.34为阿糖尿苷相对响应因子；ri is the peak resp onse of uraci

37、l arab ino side in the Test soluti on;ri为供试液中阿糖尿苷的峰响应值；rs is the peak resp onse of USP Cytarab ine RS in theSta ndard soluti on: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 perce ntage of all other impurities in the porti on of Cytarab ine take n by the formula:

38、其它杂质含量按下式计算：P(Cs / Ci )(ri/Frs)in which,式中P is the conten t of USP Cytarabi ne RS in the Sta ndard solutio n;P为标准液中阿糖胞苷 USP标准品的含量Cs is the concen trati onof USP Cytarabi ne RS in the Stan dard soluti on, mg / mL;Cs为标准液中阿糖胞苷 USP标准品的浓度，mg/mL ；Ci is the concen trati on of Cytarab ine in the Test soluti

39、 on, mg/mL;Ci为(阿糖胞苷)样品浓度， mg/mL ；ri is the peak resp onse of each impurity in the Test soluti on;ri为供试液中各单个杂质的峰面积；rs is the peak resp onse of USP Cytarab ine RS in theSta ndard soluti on;rs为标准液中阿糖胞苷 USP标准品的峰面积；F is the relative resp onse factor, equals 2.5 for the Uracil peak, with a relative rete n

40、ti ontime time of 0.55, 1.5 for peaks with relative rete nti on 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 tha n 0.10% of any in dividual impurity is found, and not more tha n 0.30% of total impur

41、ities is found (in cludi ng uracil arab ino side).单个杂质不得过0.10%，总杂质不得过0.30% (包括阿糖尿苷)。Verificati on确认4.14.1.1System suitability系统适用性Preparati on of soluti ons:溶液制备：Impurity stock solution :杂质储备液：Weigh accurately about 20 mg of Uridi ne, tran sfer into a 200-mL volumetric flask, dissolve and dilute wit

42、h water to volume, mix well to obtain Uridine Stock Solution , the soluti on has a concen trati on 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 Arabi no side RS , tran sfer into a 200-mL volumetric flask, dissolve and dilute w

43、ith water to volume, mix well to obta in Uracil Arab ino side Stock Soluti on, the soluti on has a concen trati on 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 soluti on:系统适用性测试液：Weigh accurately about 25 mg of USP Cytarab ine RS, tran sfer

44、 into a 5-mL volumetric flask, transfer accurately 1.0 mL of Uridine Stock Solution and 1.0 mL of Uracil Arab ino side Stock Soluti on into the volumetric flask, dissolve and dilute to volume with water, mix well. (0.02mg/mL for Uridi ne, 0.02 mg/mL for Uracil Arab ino side, 5.0 mg/mL for Cytarab in

45、 e).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 )。Stan dard soluti on:标准液：Weigh accurately about 20 mg of USP Cytarabi ne RS, tran sfer into a 200-mL volumetric flask, dissolve and dilute to volume with water, mix well. Transfer

46、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 4口 g/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4 口 g/mL。System sen sitivity soluti on:系统灵敏度溶液：Weigh accurately about 20 mg of US

47、P Cytarabi ne RS, tran sfer 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 2口 g/mL.精密称定约20mg阿糖胞苷USP标准品至200mL容量瓶中，用水

48、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2 口 g/mL。4.1.2Procedure实验过程Separately inject the System suitability solution , the Standard solution and the System sen sitivity soluti on into the chromatograph, record the chromatograms. Calculate the relative sta ndard deviati on (RSD) of the Stan dard s

49、oluti on, the resoluti on 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。Injectio nsS.N.Descripti onNo. of injectio n(s)1Bla nk(water)12System sen sitivity soluti on13System suitability solu

50、ti on14Bla nk(water)3 at in itial gradie nt for 12m in*5Stan dard soluti on6To elim in ate carry over of the in jector4.1.3Accepta nee 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

51、10。Six replicate injections of Standard solution, RSD 3.0%.标准溶液重复进6次，RSD 1.25. 系统适用性测试液中阿糖胞苷与尿苷分离度1.25。4.2 Specificity专属性4.2.1 Preparati on of soluti ons: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 wate

52、r 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 Arabi no side RS , tran sfer into a 200-mL volumetric flask, dissolve and dilute with water

53、 to volume, mix well to obta in 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 , tran sfer into a 200-mL volumetric flask, dissolve and dilute with water

54、to volume, mix well to obtain Uracil stock solution , the solution has a concen trati on 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 Uridin

55、e 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 forUracil Arab ino side, 5.0 mg/mL for Cytarab ine)精密称定约25mg阿糖胞苷U

56、SP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 )。Un spiked test soluti on: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