METHOD VERIFICATION PROTOCOL FOR CHROMATOGRAPHIC PURITY OF CYTARABINE 阿糖胞苷有关物质检查方法确认方案Contents目录一、In troduct ion (3)二、Scope (3)三、Resp on sibility (3)四、Defin iti ons (4)五、Project (6)六、Refere nces (27)In troduct ion简介## is in the process of developing Cytarabine Injection, a liquid product forpare nteral adm ini strati on, for ##. Cytarab ine is a chemotherapy age nt with a molecular weight of 243.2. The API from Zhejia ng Hisun Pharmaceutical Co. Ltd, which is approvedby FDA, is used for Cytarab ineInjection by Lummy.The Chromatographic purity method for Cytarab ine isthe method in the current USP34. The HPLC method for Chromatographicpurity test of Cytarabine is a quantitative method. The method will be verified according to USP<1226>, including system suitability, specificity,LOD, LOQ, precisi on, soluti on 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<1226>，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 verification for chromatographic purity of cytarab ine.该方案适用于阿糖胞苷色谱纯度的方法确认。
Resp on sibility职责四、Defin iti ons定义1 Limit of Detectio n (LOD)检测限(LOD )The limit of detecti on of an in dividual an alytical procedure is the lowestamount of an alyte in a sample which can be detected but not n ecessarily qua ntitated asan 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 knownconcentrations of analyte and by establishing the minimum level at which the an alyte canbe 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。
2 Limit of Quantitation (LOQ)定量限(LOQ)The qua ntitati on limit of an in dividual an alytical procedure is the lowestamount of analyte in a sample which can be quantitatively determinedwith suitable precisi on 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 levelsof compounds in sample matrices, and is used particularly for the determ in ati on ofimpurities 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 are an in tegral part of gas and liquidchromatographic methods. They are used to verify that resolution andreproducibility of the chromatographic system are adequatefor thean alysis to be done. The tests are based on the con cept that theequipme nt, electr oni cs, an alytical operati ons, and sample to be an alyzed con stitute 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。
Specificity 专属性Specificity is the ability to assess the an alyte un equivocally in the prese nee of componentswhich may be expected to be present. Typically thesemight 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。
Precisi on 精密度The precision of an analyticalprocedureexpresses the closeness ofagreeme nt (degree of scatter) betwee na seriesof measureme ntsobta ined from multiple sampli ng of the same homoge neous sample un der the prescribedcon 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)。
Repeatability 重复性Repeatability expresses the precisi on un der the same operati ng con diti ons 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。
In termediate precisi on 中间精密度 In termediateprecisi onexpresses withi n-laboratoriesvariati ons:differe ntdays, 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5.15.2等的精密度。
五、Project方案内容1 Accepta nee criteria2 Reage 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)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Cytarab ine (Zhejia ng His un 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 Cytarabi ne RS(curre 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Waters e2695 XS205 Dual rang Metrohm 827pH 计3 Test method 测试方法USP 34 Cytarabi ne(in the appe ndix)3.1 Chromatographic con diti on 色谱条件Supelcosil LC-18-DB, 250 x 4.6mm , 5 m See gradie nt 见梯度 1.0 mL/min254 nm Column 20 °C temperature:柱温：Injection volume: 20 pl进样体积：Phosphate buffer: 磷酸盐缓冲液：Prepare a soluti on containing 0.01M mono basic sodium phosphate and0.01M dibasic sodium phosphate in a suitable contain er. 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。