XXXHPLC分析方法的验证方案(7.5改后)
通用分析方法验证方案
xxxx含量分析方法验证方案起草签名日期审核签名日期批准签名日期变更历史1 验证目的 (3)2 验证范围 (3)3 验证时间 (3)4职责 (3)5 接受标准 (3)6 检测条件 (4)6.1 仪器、对照品、测试品 (4)6.2 检测方法 (4)7 验证内容 (5)7.1 专属性 (5)7.2 系统适应性 (6)7.3 线性及范围 (6)7.4 准确度 (8)7.5 精密度 (9)7.5.2 方法精密度 (10)7.5.3 中间精密度 (11)7.6 耐用性 (12)7.7 溶液稳定性 (14)7.8 三批样品含量测定 (15)7 偏差及异常处理 (16)8 验证结果结论与评价 (17)1 验证目的本分析方法验证方案的目的在于验证XXX含量分析方法，能有效的用于含量测定。
2 验证范围1)系统适应性2)专属性3)线性及范围4)精密度5)准确度6)耐用性7)溶液稳定性8)三批样品含量测定3 验证时间2013年1月4职责质量分析室分析员：起草验证方案并付诸实施，起草验证报告并做好相关的原始记录。
质量分析室副主任：审核验证方案及验证报告；准备相关仪器、试剂及人员安排，在验证实施过程中提供技术指导。
质量分析室主任：批准验证方案及验证报告。
6检测条件6.1仪器、对照品、测试品6.1.1 仪器6.1.2 对照6.1.3 测试品6.2检测方法6.2.1色谱条件色谱柱：Welch Ultimate RP 4.6*250mm 5um进样量：20ul柱温：25℃检测波长：298nm流动相A：20Mmol/L的磷酸二氢钾溶液（用氢氧化钾调节pH=5.70）流动相B:乙腈6.2.2溶液配制对照溶液: 称取约25.0mgXXXX对照品，置25ml容量瓶中，用流动相A溶解并稀释至刻度，作为对照品溶液。
供试液：称取约25.0mgXXXX供试品，置25ml容量瓶中，用流动相A溶解并稀释至刻度，作为供试品溶液。
7验证内容7.1专属性7.1.1 溶液配制B10：取25.0mgB10，精密称定，置25 ml容量瓶中，用流动相A并稀释至刻度，摇匀，即可。
XXX分析方法转移实施方案
XXX分析方法转移实施方案XXX分析方法转移方案————————————————————————————————作者：————————————————————————————————日期：XXXX分析方法转移方案XXXXXX有限公司起草人: ___________ ____ _ 日期: __________ _ 审核人: ___________ ____ _ 日期: __________ _ 批准人: ___________ ____ _ 日期: __________ _ XXXXXXXX有限公司审核人: ___________ ____ _ 日期: __________ _ 批准人: ___________ ____ _ 日期: __________ _目录1.目的 62.文件编号 63.样品 64.分析部门 65.时间 66.检测项目及规格要求77.分析方法描述87.1外观87.2溶解性87.3红外吸收光谱法(IR) 87.4紫外吸收光谱法(UV) 87.5液相质谱(LC-MS) 97.6高效液相色谱法(HPLC) 97.7含量107.7.1色谱条件107.7.2溶液配制107.7.3进样序列107.7.4系统适用性标准107.7.5含量计算107.8有关物质117.8.1色谱条件117.8.2流动相117.8.3溶液配制117.8.4进样序列127.8.5系统适用性标准127.8.6有关物质计算127.9水分137.9.1水分测试条件137.9.2系统适用性137.9.3样品测试137.10干燥失重137.11熔点147.12炽灼残渣147.13重金属157.14溶剂残留167.14.1色谱条件167.14.2样品及标准溶液制备16 7.14.3进样序列177.14.4计算177.15粒度177.16铜离子含量187.16.1仪器设备187.16.2溶液的配制187.16.3进样序列187.16.4系统适用性标准188.方法转移过程199.总结报告191.目的XXXX有限公司对XXXX分析方法进行确认：性状、鉴别、含量、有关物质、水分、干燥失重、熔点、炽灼残渣、重金属、残留溶剂、粒度以及铜离子含量的测定方法均参照《中国药典》2010年版二部凡例与附录方法进行测定。
HPLC分析方法的开发与验证
HPLC分析方法的开发与验证分析方法开发与验证在不同行业有不同的要求，医药化学行业对于质量的控制非常严格，高效液相分析是控制产品质量的重要手段，其开发与验证对其它行业有很好的借鉴意义。
一、分析方法开发（Development）分析方法的开发主要包括色谱柱的选择、流动相的选择、检测波长的选择和梯度的优化几个方面。
目前高效液相多做反相使用，所以本文主要以反相为例进行讲解。
1. 色谱柱的选择（Column）原料药生产对产品的纯度和杂质含量的要求非常苛刻，要求检测使用的色谱柱有较高的理论塔板数，能提供更好的分离度，从而对可能存在的杂质有更大的分离的可能性，所以5um填料的色谱柱长要250mm，3.5um填料的柱长要150mm，基本上都是各个粒径柱长最长的。
我比较喜欢近两年新出的亚二微米填料的色谱柱，50mm柱长就能提供很高的理论塔板数，而且柱长和粒径小了，流速增加很多，能节省很多的分析时间，极大的提高工作效率。
一般选用直径为4.6mm或3.0mm的柱子，太细了可能会增大柱外效应。
填料的孔径对于小分子合成药物不需要考虑，普通的分析柱都在100A左右，能满足分析检测的需要。
对于API分析方法开发，一般要求必须做色谱柱的筛选实验，最少使用三种不同类型的色谱柱，每种类型三只，要来自于不同厂家。
三种类型包括：▪1) 普通的C18或C8色谱柱，如Waters的Symmetry C18或C8，YMC的Pack Pro C18或C8，Agilent的Zorbax Eclipse C18等，其它公司如菲罗门和热电也有相应的色谱柱；▪2) 封端处理或者极性嵌入型色谱柱，如Waters的SymmetryShield RP18或RP8，XTerra RP18或RP8，YMC的ODS AQ，AgilentZorbax Bonus-RP，DIKMA SpursilC18等，其它公司如“菲罗门”（Luna）和“热电”（Hypersil 、Syncronis）也有相应的色谱柱；▪3) 填料用其它官能团修饰过的色谱柱，如苯基、氨基、氰基等色谱柱等，很多公司都有。
HPLC验证方案
设备/系统名称：高效液相色谱仪设备/系统型号：Waters515泵、2487检测器方案编号：VM-QC-001-01仪器编号：R211-JY-04方案制备姓名：日期：方案审核姓名：日期：姓名：日期：方案审核/批准姓名：日期：姓名：日期：目录1.目的 (4)2.范围 (4)3.组织与分工 (4)4.偏差解决 (5)5.变更控制 (5)6.确认内容 (5)6.1安装确认 (5)6.2运行确认 (7)6.3 性能确认 (8)7.验证结果汇总及评价 (12)8.验证周期 (13)9.设备确认总结报告 (14)10.附件 (14)10.1偏差记录 (15)10.2.偏差和解决报告 (16)10.3.附录文件清单 (17)11. 方案完成后审核/批准 (18)1.目的为确认waters515HPLC+waters UV2487高效液相色谱系统测试数据准确可靠，性能稳定，特制订本验证方案，对高效液相色谱仪进行验证。
验证过程应严格按照本方案规定的内容进行。
2.范围2.1 设备验证内容包括：安装确认、运行确认、性能确认。
2.2 该方案适用于本公司实验室高效液相色谱系统的验证。
3.组织与分工3.1成员组成：部门姓名职务3.2 分工（1）由QC负责制订本验证方案。
（2）由QA负责确认本验证方案。
（3）由QC负责进行本验证方案的具体执行。
（4）由QC负责编写验证报告。
3.3.职责划分：（1）验证小组负责本验证方案的设计、批准及实施。
（2）QC负责按本验证方案对受试仪器进行验证，并及时报告验证结果，提供验证原始数据。
（3）验证小组组长全面协调各项验证工作，负责报告验证结果、审核验证的评价与建议以及相应的结论，并在验证过程中提供相关的技术支持。
4. 偏差解决偏差指的是任何的测试失败，偏离规格，缺失文件，或重大偏离已建立的程序而导致潜在的GMP符合性影响。
对于一个偏差事件，偏差必须被审核并评估确定导致偏差的根源。
另外，根据设备的状态对偏差的影响进行分析。
药品生产设备清洗分析方法验证方案（HPLC）
药品生产设备清洗分析方法验证方案（HPLC）方案名称：******共线生产设备清洗的分析方法验证方案方案登记号：MVa-P0011-00引用标准：******质量标准药业有限公司验证方案目录1.验证方案审批 (1)2.概述 (2)3.验证目的 (2)4.验证范围 (2)5.验证日期 (2)6.验证组织及职责分工 (2)7.验证内容 (3)7.1方法概述 (3)7.2样品信息及残留限度 (3)7.3仪器和仪器参数 (4)7.4溶液的制备 (4)7.5验证内容及过程记录 (5)7.5.1系统适用性试验 (5)7.5.2峰定位及专属性试验 (6)7.5.3线性试验 (6)7.5.4取样回收率及精密度试验 (7)7.5.5定量限与检测限试验 (10)7.5.6溶液稳定性 (10)7.6验证过程偏差分析 (11)7.7结论 (11)8.再验证 (11)8.1再验证条件 (11)8.2再验证周期 (11)1.验证方案审批起草：审核：批准：生效：修订历史：2.验证概述****、****、****等药品为我公司XX车间XX设备共线生产的品种。
为保证最大限度降低药品生产过程中污染及交叉污染等风险，我公司根据GMP（2010年版）的要求，制定XX车间XX设备清洗验证方案。
按照VC-P -0 ，清洁验证方案中所描述的，依据共线生产所有品种的毒性及溶解性，我们选择****药品作为清洗验证的目标化合物，作为清洁溶剂（后续试验所用溶剂应一致，如冲洗、棉签擦拭等），由矩阵法（制剂）或10ppm法（原料）确定残留药物的限度，根据设备的具体情况确定可操作的清洗方法（如淋洗法，擦拭法等），按照设备清洁草案进行设备的清洗验证，根据验证结果建立清洁规程，以确保设备在清洗后药物的残留量符合生产工艺和GMP的要求。
根据我公司自主开发的****药品质量标准，通过对XX设备清洗液分析方法的摸索、模拟，我们确定对****药品质量标准中的含量检测方法进行优化，以确保该方法具有较高的灵敏度，适合XX设备清洗液的检测，并对涉及的分析方法进行相应的验证，保证清洗验证检测结果的准确性和可信性。
HPLC的验证
HPLC的验证目录1. 验证目的V alidation purpose (2)2. 验证范围V alidation Scope (2)3. 验证周期V alidation Cycle (2)4. 规程Regulations (2)4.1.确认验证所需文件Qualification required documents (2)4.2.所用试剂与玻璃仪器的准备Reagent and glass instruments preparation (3)4.3.流量控制阀检测Flux control valve test (3)4.4.流量比例控制阀检测Proportion test (3)4.5.基线噪声和基线漂移的检定Baseline Noise & Baseline Drift (4)4.6.最小检测浓度Minimal Detectable Concentration (4)4.7.检测器性能检查Inspect apparatus test (5)4.8.定性、定量重复性Qualitative and Quantitative analysis (5)4.9.偏差Deviation (6)1. 验证目的Validation purpose本验证的计划任务是：确认仪器达到设计要求。
验证仪器的性能是否符合性能要求。
2. 验证范围Validation Scope3.1本验证主要是对试验室的高效液相色谱仪进行性能确认。
3.2验证高效液相色谱仪的流量控制器的性能、二元泵流量比例性能、进样体积控制阀与检测器的性能检查。
3.3相关验证：本确认方案应确保所使用的电子天平经过校验，所使用的容量仪器经过检定。
3. 验证周期Validation Cycle每年三个月对高效液相色谱仪的泵进行流量性能、二元泵流量比例性能的检查，并进行进样体积控制阀与检测器的性能检查。
4. 规程Regulations4.1.确认验证所需文件Qualification required documents目的核实在本次确认中所需的SOP、参考资料等文件均是可用的程序查找和本次确认相关的文件，确保公司文件均为经批准的现行版本，记录文件名称、文件编码、修订版次等内容。
HPLC方法验证及方案
SECTION XV SECTION 15 Analytical Methods(TYPICAL ANALYTICAL METHOD VALIDATION)1.PURPOSET he purpose of this Standard Analytical Procedure is to demonstrate the procedure required to validate in-house HPLC analytical methods and to show that the methods are stability-indicating. Methods based on the USP but modified for stability indicating test purposes require full in-house validation.This procedure ensures that the Product Development Process and Process Qualification Batch analysis is based on a foundation of Good Laboratory Practice using validated test procedures.2.RESPONSIBILITYThe Head of Analytical Development in coordination with the managers of QC and Regulatory Affairs at the proposed manufacturing site.3.FREQUENCYFor each non-compendial analytical method intended for ANDA (or OTC ANDA) manufactured products.For Stability-Indicating Assays and limit testing of impurities that may be based on compendial methods. Each Product strength will follow the full method validation procedure.4.PROCEDURE[a].Method ValidationNon-compendial methods validation will follow the USP direction for parameters needed for the validation of test methods.Typical parameters for validating assays and other non-compendial analytical methods designed for providing quantitative results shall include :• Accuracy• Recovery• Precision ( System reproducibility, Method reproducibility )• Specificity• Linearity• Range• Ruggedness (different analyst s / days /different equipment models / columns) [b].Placebo Analysis.A mixture of non-actives (placebo) shall be prepared and subjected to analysis.No interfering peaks shall be observed in the graph of the placebo chromatogram.[c].The stability of the Standard solution is assessed by re-injection of the standard solution after24 x n hours (where n = number of days the Standard will be used).Standard Preparation for AssayComparison of standard solutions for Assay of Active material, injected after one month and freshly prepared demonstrate that the standard solutions are stable and does not lose its quality after one month if refrigerated.Standard Preparation for ImpurityComparison of standard solutions of Guanine, injected after one month and freshly prepared demonstrate that the standard solutions are stable and does not lose its quality after 1 month if refrigerated.Name of standards Storage conditions Difference. relativeto freshly preparedstandard[Active] 100%4°C<2%[Impurity] 100%4°C<2%Standard Solutions are stored at controlled temperatures and light conditions as per labeling.[d].Stability Indicating Procedures.For the Stability Indicating Method, the product sample shall include forced degradation by stressed analysis. Conditions of concentration and reaction time may vary depending on the active drug substance and drug product e.g. :• Oxidation-(H2O2 plus standing time).• Base Hydrolysis-(NaOH x N plus standing time).• Acid Hydrolysis-(HCl conc. plus standing time).• Sun light-(24 hours standing time).• Heat-(x degrees C).Summary of Stability Indicating ResultsStressed Conditions Temp.Time Raw Material;Tablets(°C)(hr)RemainingSubstance.(%)Peak Purity,(Figure)RemainingSubstance(%)PeakPurity,(Figure)Solution heating9012100.2pure98pure Solid heating160 2101.3pure92pure Sunlight 765 w/m24014101.1pure84.8pure 3,3N Sodium Hydroxide701099.8pure100.2pure 10%Hydrogen Peroxide37 377.5pure90.5pure 5% Hydrochloric Acid Room2079.7pure78.6pure[e]Specificity and Suitability (Resolution and Tailing Factors).When a satisfactory separations of all the degradation peaks have been achieved through the forced degradation reactions, a Resolution Factor (according to the USP requirements) between the main active peak and the nearest degradant peak is calculated using the USP formula.A Tailing Factor (according to the USP formula) is calculated for the main active peak.[f] System Suitability TestA mixture of [Active] AS. standard at the concentration about [0.1]mg/mL and of [Impurity] AS. standard at the concentration about [0.01]mg/mL according to Method SI-1000 was prepared and injected into the HPLC system.For chromatogram obtained the following values were calculated (according to USP):1. Relative Retention Time for [Impurity] peakRRT = RT [Impurity] = 2.65 =0.31RT [Active] 8.452. Tailing factor for [Active] peakT=W2=94.2= 1.1f 0.05 fThe values depict the specificity of the method for resolution between the main peak and impurity peak. (values shown for demonstrations purposes).Peak PurityThe photo diode-array is used for the evaluation of the stability indicating nature of the assay method number SI-1000 for [000]mg and [000]mg tablets using a Waters 996™ Unit, controlled by the chromatography manager Millennium 2010™.Peak purity and match results are reported as:Purity Angle is a measure of spectral non-homogeneity across a peak - i.e. the weighed average of all Spectral Contrast Angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum.Purity Threshold is the sum of Noise Angle and Solvent Angle. It is the limit of detection of shape differences between two spectra.Match Angle is a comparison of the spectrum at the peak apex against a library spectrum.Match Threshold is the sum of the Match Noise Angle and Match Solvent Angle. Noise Angle is a measure of spectral non-homogeneity caused by system noise.Peak Purity (Cont.)Solvent Angle is a measure of spectral non-homogeneity caused by solvent composition.It the purity angle is smaller than the purity threshold and the match angle is smaller than the match threshold, this indicates that no significant differences between spectra are detected. There is no spectroscopic evidence for co-elution and the peak is considered pure.[f]Relative Retention Time of Main and Additional peaks.Each stressed analysis shall indicate the percentage by which the Main peak is decreased as well as the RRT for any other Additional peaks.If the RRT of an Additional peak corresponds to a known degradant/impurity etc. it shall be stated.The peak purity of the main peak shall be given for each stressed analysis (where possible).[g].Validation of limit testing for impurity methods shall include :*Specificity*Detection Limit(DL)*Quantitation Limit(QL)Detection Limit (DL)The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detested but not necessary quantitated as an exact value.Quantitation Limit(QL)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. Used in the determination of impurities and or degradation products.[h].Contents of a typical HPLC Analytical Validation Protocolrefer Method No. A-0340-01-1299Validation of HPLC Analytical MethodMethod No: A-0340-01-1299[1]Introduction - A brief description is given of the following parameters :*Method and Edition # used*Batch # of samples tested (test the lowest and the highest label strength)*Type of detector used to analyze stressed samples*Stress testing of Standard solution to determine origin of Additional peaks.[2]System Reproducibility - PrecisionTen replicate (single) injections of the standard solution at the nominal concentration described in the method is performed and the RSD calculated. The Results (sample # and peak areas) are tabulated. The Average Peak Area, SD and RSD are shown in the table. Target values for RSD = 0.5 to 1.0(Keep this standard solution for the stability of Standard Solutions - Point 9)SYSTEM REPRODUCIBILITYSAMPLE No.PEAK AREAS1.2.3.4.5.6.7.8.9.10.Average Peak Area Standard Deviation Relative Standard Deviation === 0.5 - 1.0[3]Method Reproducibility - PrecisionThe full analytical method # is carried out and repeated Ten times on the finished product (batch #) and the RSD is calculated. Two HPLC injections are performed per method assay and the peak areas are averaged. The Results (assay %) are tabulated. The Average Assay %, SD and RSD are calculated and shown in the tabulations. Target values for RSD = 1.5 to 3.0.METHOD REPRODUCIBILITYSAMPLE NoBatch No:ASSAY %12345678910Average Assay % Standard Deviation Relative Standard Deviation.=== 1.5 - 3.0[4]AccuracyThe Accuracy of an analytical procedure expresses the closeness of agreement between the true value and the value found.Ten replicate (single) injections of the standard solution at the nominal concentration of x mg/100 mL as described in the Analytical Method / Ed # [00] is made and the percent deviation from the true values as determined from the linear regression line is calculated.The Results (Peak areas and % accuracy) are tabulated.The Mean, SD and C.of.V are shown in the tabulations[4]Accuracy (continued).A C C U R A C YINJECTIONNo PEAKAREACALCULATEDCONC.%ACCURACY12345678910Mean (% Accuracy) =Standard Deviation =% Coef. of Variation =[5]Recovery (Extraction time)The extraction efficiency is demonstrated by varying the extraction time of prepared sample solutions as described in the analytical method #. Two HPLC injections are performed per method assay and the peak areas are averaged. The extraction time suitable to ensure complete extraction is highlighted.Not less than three different extraction times are used namely 0.5 T, T and 1.5 T (where T is the extraction time of the method).[5]Recovery (Extraction time - tabulations continued).The Results (Extraction time and Assay %) are tabulated as shown.RECOVERY - EXTRACTION% ASSAYTIME IN MINUTESBatch No:0.5 TT1.5 T[6]Recovery (spiked placebo samples).Five spiked admixtures of the active substance and the non-active vehicle (placebo) at concentrations of about 50 % to 150 % of the stated concentration required by the assay procedure is prepared and analyzed to show the percentage active recovery. Two HPLC injections are performed per method assay and the peak areas are averaged.The Results (Theoretical conc. Actual conc. and % recovery ) are tabulated.The Average Recovery, SD and the % Coefficient of Variation are given.[6]Recovery (spiked placebo samples tables - continued).T he recovery results are shown graphically (peak area Vs conc. (mg/100 mL). These results also show extraction method and detector linearity.RECOVERYStandard solution mg/100mL Peak Area =CONC. Theoretical (mg/100ml)PEAK AREAFOUNDCONC.FOUND(mg/100ml)PERCENTAGERECOVERY5075100125150Mean (% Recovery) =Standard Deviation =% Coef of Variation =The Linear Regression value, Slope and Y-Intercept are shown in the GRAPH. The placebo chromatogram (vehicle only) is shown to highlight the absence of Additional Peaks[7]Linearity and range.T he linearity on an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of the analyte in the test sample.F ive Standard solutions in a concentration range of (about) 50 % to 150 % of the stated concentration required by the assay procedure are prepared and analyzed by the stated method.T wo HPLC injections are performed per method assay and the peak areas are averaged.[7]Linearity and range - (continued).T he Area count and concentration range is plotted. Linear regression analysis willdemonstrate the acceptability of the method for quantitative analysis over the full spectrum of the concentration range. Detector linearity is demonstrated.The Results (Range conc. and peak areas ) are tabulated.LINEARITY AND R A N G ECONC. Batch No:PEAK AREAS50 %75 %100 %125 %150 %Linear RegressionY-Intercept Slope ===The results are shown graphically (peak area Vs range conc. (mg/100 mL).GRAPH OF LINEARITYConc. mg/100mLPe akAre a200004000060000800001000001200000255075100125150[8]RUGGEDNESS&Robustness.Ruggedness measures the lack of ex ternal influence on the test results whereas robustness measures the lack of in ternal influences on the test results.The Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and thus providing an indication of its reliability normal usage.The method may be evaluated for specificity using two different columns. No differences in specificity, selectivity or column performance should be observed. RobustnessRobustness determinations are essential when transferring analytical methods from the development laboratory to the commercial plant quality control laboratory. There may usually be a difference in columns or HPLC machine models used. Deliberate variations according to the following table were made to the critical parameters of the method such as column, flow rate and concentration of [organic acid] in the mobile phase. Using the System Suitability solution and LOQ solution as the Test Solutions the performance of the method was evaluated. Column 1: Phenomenex Bondclone 10µ, C-18, 300 x 3.9mm (OOH-2117-CD) Column 2: Waters µ-Bondapak 10µ, C-18, 300 x 3.9mm (27324)C O ND I T I O N RE S U L T SConditionNo.Column Flow RatemL/minBufferConc. (%)RRT T f RSDbet. LOQ of[Active]RSDbet. LOQ of[Impurity]11 2.50.10.3 1.1<10<1021 2.20.10.3 1.1<10<1031 2.80.10.3 1.1<10<1041 2.50.150.3 1.1<10<1052 2.50.10.3 1.1<10<10Notes on different terms frequently used:INTERMEDIATE PRECISIONT he analytical variation expressed between laboratories on different days; with different equipment; or different analysts is known as - intermediate precision. REPRODUCIBILITY (INTRA-LAB)T his intra-laboratory precision or the precision between laboratories is known as reproducibility or more specifically - intra-laboratory reproducibility. Both the above are ruggedness - and a USP requirement.[8]RUGGEDNESS&Robustness- (Tabulations - continued).The Results (Average assay % for Analyst 1 and 2 ) are tabulated.RUGGEDNESSANALYSTNo 1%ASSAYColumn IANALYSTNo 2%ASSAYColumn 212345678910Mean (% Accuracy) =Standard Deviation =% Coef of Variation =R obustness.The evaluation of robustness should be finalized at the end of the development phase - around the time of the process qualification lot manufacture. The robustness evaluation should be developed with the commercial laboratory equipment in mind. It should show the reliability of an analysis with respect to deliberate variations in the method parameters A consequence of robustness evaluation is that a series of system suitability parameters are established to ensure that the validity of the analytical procedure is maintained whenever used.Robustness is defined by both the USP and the ICH Tripartite guidelines as "a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal use " Robustness is defined both in the USP and ICH, but is not required.[9]Stability of Standard solutionsRe-chromatography of ten replicate single injections of the same standard solution (which have been allowed to stand for x hours ) against freshly prepared Standards showed no significant differences from the original results.STABILITY OF STANDARD SOLUTIONSmg/100mL Initial Analysis(Date)mg/100mL Repeat Analysis 2nd (Date)1 injection2 injection3 injection4 injection5 injection6 injection7 injection8 injection9 injection10 injection1 injection2 injection3 injection4 injection5 injection6 injection7 injection8 injection9 injection10 injectionMeanStandard Deviation Relative Standard Dev.=== NMT 2.0 %[10]Typical Chromatograms.Representative chromatograms of the following traces are routinely provided:-♦ System Suitability♦ Standard Solution♦ Drug Product♦ placeboTypical ChromatogramsWhen R epresentative Chromatograms are displayed - all peaks are LABELED with the peak name and RRT.R epresentative chromatogramDrug Product[11]Conclusion .(Closing Statement)A n appropriate conclusion should be given stating clearly that:“The method # IAG00-005 Ed. No [00] is shown to be accurate and precise for carrying out assay analysis as part of the Assay and Stability Studies for the Drug Product conforming to the formula as shown in Appendix 1”[12]References and Appendixes.A cknowledgment to references as well as attachments such as the drug productformula are attached at the end of the validation protocol.I t is important to emphasize that analytical validation applies to a drug formula anda set manufacturing procedure. Extraneous peaks and processing stresses are specific to a manufacturing procedure, equipment used and the nature of the excipients.References:1. "Validation of compendial methods" USP 23 <1225> USPC Rockville Maryland USA 1994.2. USP/NF XXIII USPC Rockville Maryland USA 1994.3. Scale up and Post approval Changes Manufacturing and Controls In vitro Dissolution and In Vivo Bioequivalence Documentation CEDER 1995 (SUPAC)4. International Conference on Harmonization "Guidelines on validation of Analytical Procedures:Definitions and Terminology; Federal Register (March 1, 1995.)5. ASTM Standard Guide For Conducting Ruggedness Tests E1169 American Society for testing Materials Philadelphia 1989.6. G. Kateman and L. Buydens, T he Ruggedness Test Quality Control in the Analytical chemistryJohn Wiley and Sons NY 2nd Edition 1993, pp118 125.Label the peakclearlyName and Retention time (8.78 min)。
某一种含量及溶出度检测的HPLC方法验证方案
55555品种含量及溶出度检测的HPLC方法验证批准签字目录55555公司............................................................................................... 错误！未定义书签。
目录31.简介 (6)2.研究日期 (6)3.HPLC方法描述 (6)3.1仪器 (6)3.2色谱条件 (6)3.3溶剂和试剂 (6)3.4流动相 (7)3.5试验溶液的制备 (7)4.用于55555品种含量及溶出度测定的HPLC法的验证 (7)4.1线性 (7)4.2含量检测准确度 (9)4.4.1样品溶液的配制 (9)4.3含量精密度 (11)4.4系统适应性 (12)5.偏差记录 (13)研究大纲表1：含量验证参数和可接受标准：含量测定（测试浓度: 0.50mg/mL）1.简介本试验方法是采用的《中国药典》2010版方法，由于药典方法是成熟的方法，故主要是对其系统适应性等参数进行验证。
2.研究日期计划开始日期：计划实验工作完成日期：3.HPLC方法描述3.1仪器容量瓶精确到0.01mg的天平PH计HPLC系统由以下设备组成：- 色谱系统： Waters 1525- 进样系统：Waters 717- 紫外检测器：Waters 2487- 数据处理系统：Waters EMPOWER- 色谱柱：Waters Symmetry® C18 3.5μm 4.6×150mm3.2色谱条件流速: 1.0mL/min.进样体积: 20 L洗脱模式: 等度运行时间柱温: 30°C检测波长: 263 nm3.3溶剂和试剂水超纯水甲醇色谱纯乙腈色谱纯醋酸钠分析纯冰醋酸分析纯3.4流动相醋酸钠缓冲液：取醋酸钠6.13g，加水750ml使溶解，用冰醋酸调节PH值至2.5。
流动相：醋酸钠缓冲液-乙腈=40:603.5试验溶液的制备样品溶液：精密称取55555品种内容物适量（约相当于品种50mg），置100ml 量瓶中，加甲醇适量，振摇使品种溶解，用甲醇稀释至刻度，摇匀，滤过。
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XXXHPLC分析方法的验证方案(7.5改后)XXX产品HPLC分析方法确认方案XXX制药有限公司确认方案审批表确认方案名称: XXX产品HPLC分析方法确认方案确认方案编号：KY-ZL-YZ-FF-00400目录1 概述2 确认目的3 确认范围4 确认小组成员与职责5 确认方案的审核与批准6 进度计划7 确认内容8 变更与偏差9 确认结果与评价报告10 确认合格证书1 概述我公司产品XXX产品采自《中国药典》2010年版二部，含量及有关物质、A、B、C的检验均使用高效液相色谱仪进行检验，两个检验项目检验参数等条件完全相同，且我公司未对《中国药典》2010年版二部中的方法进行过任何改变，因此按照2010新版GMP要求，需要进行分析方法确认。
本确认方案使用LC-2010AH型高效液相色谱仪对XXX产品采用《中国药典》2010年版二部“含量”、“有关物质”及“A、B、C”的检验方法进行确认，证明此方法在本公司实验室的适用性。
根据《药品GMP指南（质量控制实验室与物料系统）》分册要求，“含量”、“有关物质”及“A、B、C”项需确认项目如下：“是”代表该项内容需确认，“否”代表该项内容不需要确认2确认目的通过对XXX产品含量及有关物质A、B、C的检验方法的确认，证明此方法在本公司实验室的适用性。
3确认范围本确认方案适用于XXX产品含量及有关物质A、B、C、的检验方法进行确认。
4确认小组成员与职责5确认方案的起草与审批5.1 确认方案的起草与审批：确认方案由检验室起草，确认小组会审，质量负责人批准实施。
5.2 确认方案的培训：确认方案经批准后，实施前由确认方案的起草部门组织确认方案的参与者进行培训。
5.3 确认方案的修改：确认方案如需修改，由质量负责人批准实施，在确认报告中体现。
6 进度计划整个确认活动实施时间：确认时间：从_____年___月__日至_____年__ 月__ 日；起草报告：从_____年___月__日至_____年__ 月__ 日；7 确认内容7.1确认前仪器及材料检查7.1.1确认前，对下列试验用仪器和材料进行检查7.1.1.1岛津高效液相色谱仪7.1.1.2 TG-332A型分析天平7.1.1.3色谱柱：C18（4.6*250㎜）7.1.1.4对照品和试剂：产品X二醇物对照品、对硝基苯甲醛对照品、产品X对照品、羟苯甲酯对照品、羟苯乙酯对照品、羟苯乙酯对照品、庚烷磺酸钠，乙腈（色谱级），甲醇（色谱级），磷酸二氢钾、三乙胺、磷酸、蒸馏水7.1.1.5其他一些辅助的玻璃仪器（如量瓶、移液管等）7.1.2可接受标准：仪器和用具经校验且在效期内；对照品、试液、试剂与试药符合验证要求且在效期内；色谱柱使用状态正常。
7.1.3结果记录：将结果记录在附件1《确认前仪器及材料检查表》中。
7.2确认涉及文件检查7.2.1确认前，对与此次确认相关的文件进行检查7.2.1.1XXX产品成品检验操作规程（KY-ZL-SOP-CP-00400）7.2.1.2 型高效液相色谱系统标准操作及维护保养规程（KY-SB-SOP-JY-03700）7.2.1.3 型高效液相色谱系统维护保养及清洁操作规程（KY-WS-SOP-JY-03700）7.2.1.4TG-332A型分析天平标准操作规程（KY-SB-SOP-JY-02300）7.2.1.5TG-332A型分析天平维护保养及清洁标准操作规程（KY-WS-SOP-JY-02300 7.2.1.6高效液相色谱法标准操作规程（KY-ZL-SOP-ZK-03300）7.2.1.7XXX产品HPLC分析方法的验证方案（KY-YZ-SOP-JY-00400）7.2.1.8XXX产品生产工艺规程（KY-SC-JB-GY-00400）7.2.1.9检验方法验证（确认）管理程序（KY-ZL-SMP-05800）7.2.2可接受标准：所有文件都应为现行版本，且经过批准。
7.2.3结果记录：将结果记录在附件2《确认前文件检查表》中。
7.3人员培训确认7.3.1检查并确认所有参与执行确认的人员是否已得到培训，并在培训记录上签字。
7.3.2可接受标准：验证小组成员均已经过培训，并在培训记录上签字。
7.3.3确认结果：将确认结果填入附件3《人员培训、参与情况确认记录》中。
7.4 确认项目7.4.1 “含量”项确认7.4.1.1重复性/准确度7.4.1.1.1空白基质的制备：精密称定硼酸：13.125g硼砂：0.6g玻璃酸钠：1g羟苯乙酯：0.5g按照XXX产品生产工艺规程（KY-SC-JB-GY-00400）配制加注射用水至1000ml。
7.4.1.1.2对照液制备：称取产品X对照品用注射用水配制成1ml含2.5mg的溶液，取本品用流动相定量稀释制成每1ml中含0.1mg的溶液摇匀，作为对照液备用。
对照液平行制备2份，标识为S-1、S-2（浓度为标示量的100%）。
7.4.1.1.3样品制备：称取产品X对照品用空白基质水溶解后定容，摇匀，备用。
平行制备浓度梯度为标示量80%、100%、120%的样品各3份，标识为A-1、A-2、A-3、A-4、A-5、A-6、A-7、A-8、A-9。
7.4.1.1.4照《高效液相色谱仪标准操作规程》测定。
色谱条件与系统适用性试验用十八烷基硅烷键合硅胶为填充剂；流动相A为0.01mol/L庚烷磺酸钠缓冲溶液（取磷酸二氢钾6.8g，用0.01mol/L庚烷磺酸钠溶液溶解并稀释至1000ml，再加三乙胺5ml，混匀，用磷酸调节PH值至2.5），流动相B 为甲醇；检测波长为277nm；按下表进行线性梯度洗脱。
另取产品X对照品、产品X 二醇物对照品、对硝基苯甲醛对照品、羟苯甲酯、羟苯乙酯与羟苯丙酯对照品各适量，加甲醇适量（产品X每10mg加甲醇1ml）使溶解，用流动相定量稀释成每1ml中含产品X0.5mg、产品X二醇物40ug与对硝基苯甲醛3ug、羟苯甲酯40ug、羟苯乙酯50ug 和羟苯丙酯20ug的混合溶液。
精密吸取该溶液10µl注入色谱仪，作为系统适用性溶液。
记录色谱图。
各相邻峰间的分离度均应符合要求）（应大于1.5）。
7.4.1.1.5测定：精密量取上述A-1、A-2、A-3、A-4、A-5、A-6、A-7、A-8、A-9溶液用流动相定量稀释制成每1ml中含0.1mg的溶液，摇匀，精密量取10ul，注入液相色谱仪，记录色谱图。
1）取S-1精密量取10ul，注入液相色谱仪记录色谱图，该溶液连续进样2针。
2）取S-2精密量取10ul，注入液相色谱仪，记录色谱图，该溶液连续进样1针。
3）分别精密量取稀释后的A-1、A-2、A-3、A-4、A-5、A-6、A-7、A-8、A-9溶液各10ul，注入液相色谱仪，记录色谱图。
4）计算S-1和S-2的F值，相对标准偏差应≤2.0%，以外标法计算稀释后的A-1、A-2、A-3、A-4、A-5、A-6、A-7、A-8、A-9溶液的含量。
结果计算含量（cx）=CR×Ax/ARcx-------- 为供试品的浓度；CR---------为对照品的浓度；Ax-------- 为供试品的峰面积；AR---------为对照品的峰面积。
7.4.1.1.6可接受标准：稀释后的A-1、A-2、A-3、A-4、A-5、A-6、A-7、A-8、A-9各浓度的含量均应在标示量的±3%，且各浓度样品含量的RSD%值应≤2.0%。
7.4.1.1.7确认记录和数据确认数据和结果记录于附件4《含量精密度/准确度确认数据和结果》中。
7.4.1.2专属性7.4.1.2.1 测试方法：7.4.1.2.1.1取空白基质进样1针，记录色谱图。
7.4.1.2.1.2 精密量取样品（用成品即可）用流动相A-B（68：32）定量稀释制成每1ml中含产品X0. 1mg的溶液，作为供试品溶液，精密量取10ul，注入液相色谱仪，进样1针，记录色谱图，获得产品X的保留时间。
7.4.1.2.1.3取产品X对照品用流动相定量稀释制成每1ml中含产品X0. 1mg的溶液，作为对照品溶液，注入液相色谱仪，进样1针，记录色谱图，获得产品X的保留时间。
7.4.1.2.1.4可接受标准：溶剂空白相应的峰对样品测定有无明显的干扰；供试品图谱中产品X主峰保留时间与对照品图谱中产品X主峰保留时间一致。
7.4.1.2.1.5 确认记录和数据确认数据和结果记录于附件5《专属性确认数据和结果》中。
7.4.2“有关物质”及“A、B、C”项确认7.4.2.1专属性7.4.2.1.1精密量取样品（用成品即可）用流动相A-B（68：32）定量稀释制成每1ml 中含产品X0.5mg的溶液，作为供试品溶液。
7.4.2.1.2对照溶液；取产品X对照品、产品X二醇物对照品、对硝基苯甲醛对照品、羟苯甲酯、羟苯乙酯与羟苯丙酯对照品各适量，加甲醇适量（产品X每10mg加甲醇1ml）使溶解，用流动相定量稀释成每1ml中含产品X0.5mg、产品X二醇物40ug与对硝基苯甲醛3ug、羟苯甲酯40ug、羟苯乙酯50ug和羟苯丙酯20ug的混合溶液。
作为对照溶液；照含量项下的色谱条件，取对照品溶液10ul，注入液相色谱仪，调节检测灵敏度，使产品X二醇物的峰高约为满量程的64%。
7.4.2.1.3精密量取供试品溶液与对照品溶液各10ul，分别注入液相色谱仪，进样1针，记录色谱图。
7.4.2.1.4可接受标准：供试品图谱中产品X、产品X二醇物、对硝基苯甲醛、羟苯甲酯、羟苯乙酯和羟苯丙酯各峰的保留时间与系统适用性图谱中相对峰的保留时间一致。
溶剂空白相应位置无峰的干扰。
7.4.2.1.5确认记录和数据确认数据和结果记录于附件5《专属性确认数据和结果》中。
7.4.2.2精密度7.4.2.2.1测试程序7.4.2.2.1.1取含量项下系统适用性溶液作为对照溶液D-1；另按同法制备一份对照品作为对照溶液D-2。
7.4.2.2.1.2取样品用含量测定项下的流动相A-B（68：32）定量稀释制成每1ml中含产品X0.5mg的溶液，配制6分平行样品作为供试品溶液分别为B-1～B-6。
7.4.2.2.1.3 D-1进样2针，D-2进样1针，然后B-1～B-6分别进针，记录色谱图。
7.4.2.2.1.4各相邻峰之间的分离度应≥1.57.4.2.2.1.5计算D-1和D-2的F值，相对标准偏差应≤2.0%，以外标法计算B-1、B-2、B-3、B-4、B-5、B-6溶液中各有关物质的含量。
计算：产品X二醇物（%）=S二醇物×C二醇物对照/ S二醇物对照×C供试品×100%对硝基苯甲醛（%）=S对硝基苯甲醛×C对硝基苯甲醛对照/ S对硝基苯甲醛对照×C供试品×100%羟苯甲酯（%）=S羟苯甲酯×C羟苯甲酯醛对照/ S羟苯甲酯对照×C供试品×100%羟苯乙酯（%）=S羟苯乙酯×C羟苯乙酯对照/ S羟苯乙酯对照×C供试品×100%羟苯丙酯（%）=S羟苯丙酯×C羟苯丙酯对照/ S羟苯丙酯对照×C供试品×100%7.4.2.2.1.2可接受标准：6个样品B-1、B-2、B-3、B-4、B-5、B-6含量的RSD%值应≤2.0%。