FDA剛重磅發布《BioanalyticalMethodValidationGuidanceforIndustry》

　　 I. INTRODUCTION

　　 引言

　　 This guidance helps sponsors of investigational new drug applications (INDs) or applicants of new drug applications (NDAs), abbreviated new drug applications (ANDAs), biologic license applications (BLAs), and supplements validate bioanalytical methods used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies that require pharmacokinetic, toxicokinetic, or biomarker concentration evaluation.2 This guidance can also inform the development of bioanalytical methods used for nonclinical studies that require toxicokinetic or biomarker concentration data. For studies related to the veterinary drug approval process such as investigational new animal drug applications (INADs), new animal drug applications (NADAs), and abbreviated new animal drug applications (ANADAs), this guidance may apply to blood and urine BA, BE, and pharmacokinetic studies.

　　 該指南有助于研究性新藥申請（IND）或新藥申請（NDAs）申請人，縮寫新藥申請（ANDA），生物學許可申請（BLA）和補充劑的申請人驗證用于人臨床藥理學的生物分析方法，生物利用度BA）以及需要進行藥代動力學，毒代動力學或生物標志物濃度評估的生物等效性（BE）研究。本指南還可以為需要毒代動力學或生物標志物濃度數據的非臨床研究開發生物分析方法。對于有關獸藥批準過程的研究，如研究性新動物藥物應用（INADs），新動物藥物應用（NADAs）和簡化新動物藥物應用（ANADAs），本指南可能適用于血液和尿液BA，BE，和藥代動力學研究。

　　 The information in this guidance applies to bioanalytical procedures such as chromatographic assays (CCs) and ligand binding assays (LBAs) that quantitatively determine the levels of drugs, their metabolites, therapeutic proteins, and biomarkers in biological matrices such as blood, serum, plasma, urine, and tissue such as skin.

　　 本指南中的信息適用于定量確定生物基質（如血液，血清，血漿和血漿）中藥物，其代謝物，治療性蛋白質和生物標志物水平的生物分析程序，如色譜分析（CC）和配體結合分析（LBA）尿液和皮膚等組織。

　　 This final guidance incorporates public comments to the revised draft published in 2013 and provides recommendations for the development, validation, and in-study use of bioanalytical methods. The recommendations can be modified with justification, depending on the specific type of bioanalytical method. This guidance reflects advances in science and technology related to validating bioanalytical methods.

　　 本最終指南將2013年發布的修訂草案的公眾意見納入其中，并為生物分析方法的開發，驗證和研究中的使用提供建議。根據生物分析方法的具體類型，可以根據理由修改建議。本指南反映了科學技術在驗證生物分析方法方面的進展。

　　 In general, FDA’s guidance documents do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.

　　 一般來說，FDA的指導文件不會建立法律上可執行的責任。相反，指導原則描述了FDA目前關于某一主題的想法，只應將其視為建議，除非引用具體的監管或法定要求。在機構指南中使用這個詞意味著建議或推薦某些東西，但不是必需的。

　　II. BACKGROUND

　　 The 2001 guidance for industry on Bioanalytical Method Validation was originally based on the deliberations of two workshops described in publications entitled:

　　 II。 背景

　　 2001年生物分析方法驗證行業指南最初是基于出版物中描述的兩個研討會的討論，這兩個研討會的題目是：

　　? Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies

　　 ?分析方法驗證：生物利用度，生物等效性和藥代動力學研究

　　? Bioanalytical Methods Validation: A Revisit With a Decade of Progress

　　 生物分析方法驗證：帶有十年進展的重訪

　　Additional workshops, summarized in the following publications, have informed subsequent revisions (e.g., the 2013 draft guidance for industry entitled Bioanalytical Method Validation):

　　 以下出版物中總結的其他研討會已通知后續修訂（例如，2013年生物分析方法驗證行業指南草案）：

　　? Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays

　　 ?定量生物分析方法驗證和實施：色譜和配體結合分析的最佳實踐

　　 ? The AAPS/FDA Workshop on Incurred Sample Reanalysis

　　 ?AAPS / FDA關于發生樣品再分析的研討會

　　? The AAPS Workshop on Crystal City V — Quantitative Bioanalytical Method Validation and Implementation: 2013 Revised FDA Guidance

　　?AAPS水晶城V研討會  -定量生物分析方法驗證和實施：2013年修訂的FDA指南

　　Validated analytical methods for the quantitative evaluation of analytes (i.e., drugs, including biologic products, and their metabolites) and biomarkers in a given biological matrix (e.g. blood, plasma, serum, or urine) are critical for the successful conduct of nonclinical, biopharmaceutics, and clinical pharmacology studies. These validated methods provide critical data to support the safety and effectiveness of drugs and biologic products. Validating the analytical method ensures that the data are reliable by addressing certain key questions, including:

　　 用于對特定生物基質（如血液，血漿，血清或尿液）中的分析物（即藥物，包括生物產品及其代謝物）和生物標志物進行定量評估的有效分析方法對于非臨床生物制藥的成功實施至關重要，和臨床藥理學研究。這些經過驗證的方法提供了關鍵數據來支持藥物和生物制品的安全性和有效性。通過解決某些關鍵問題，驗證分析方法可確保數據的可靠性，其中包括：

　　? Does the method measure the intended analyte? For example, does anything interfere with the measurement, and is the method specific or selective for the analyte?

　　? What is the variability associated with these measurements? For example, what are the accuracy and precision of the method?

　　? What is the range in measurements that provide reliable data? For example, what is the sensitivity of the method (e.g., what is the lower limit of quantitation (LLOQ) of the method, and what is the upper limit of quantitation the method (ULOQ)?)

　　? How do sample collection, handling, and storage affect the reliability of the data from the bioanalytical method? For example, what steps need to be followed while collecting samples? Do the samples need to be frozen during shipping? What temperatures are required to store the samples, and how long can the samples be stored?

　　?該方法是否測量預期的分析物？例如，是否有什么干擾測量，并且是分析物特有的還是選擇性的方法？

　　?這些測量有何變化？例如，該方法的準確性和精確度是什么？

　　?提供可靠數據的測量范圍是多少？例如，該方法的靈敏度是多少（例如，該方法的定量下限（LLOQ）是多少，該方法的定量上限是多（ULOQ）？）

　　?樣本采集，處理和存儲如何影響生物分析方法數據的可靠性？例如，收集樣本時需要遵循哪些步驟？運輸過程中是否需要凍結樣品？需要什么樣的溫度來存儲樣品，樣品可以存儲多長時間？

　　 When changes are made to a validated method, the sponsor should conduct additional validation (i.e., partial or cross validation).

　　The fit-for-purpose (FFP) concept states that the level of validation should be appropriate for the intended purpose of the study. The key questions listed above should be evaluated relative to the stage of drug development. Pivotal studies submitted in an NDA, BLA, or ANDA that require regulatory decision making for approval, safety or labeling, such as BE or pharmacokinetic studies, should include bioanalytical methods that are fully validated. Exploratory methods that would not be used to support regulatory decision making (e.g., candidate selection) may not require such stringent validation. This FFP concept applies to drugs, their metabolites, and biomarkers.

　　 The analytical laboratory conducting toxicology studies for regulatory submissions should adhere to 21 CFR 58, Good Laboratory Practices (GLPs).9 The bioanalytical method for human BA, BE, and pharmacokinetic studies must meet the criteria specified in 21 CFR 320 Bioequivalence and Bioavailability Requirements (i.e., 21 CFR 320.29).

　　 The following sections discuss the development, validation, and in-study use of bioanalytical methods and how best to document validation methods and results. Refer to the Glossary for the definitions of assay parameters and analytical terms used in this guidance.

　　 當對經過驗證的方法進行更改時，申辦者應進行額外的驗證（即部分驗證或交叉驗證）。

　　 符合目的（FFP）的概念指出，驗證的水平應適合研究的預期目的。上面列出的關鍵問題應該相對于藥物開發階段進行評估。在NDA，BLA或ANDA中提交的需要作出批準，安全或標簽（如BE或藥代動力學研究）的監管決策的關鍵研究應包括經過充分驗證的生物分析方法。不用于支持監管決策（例如，候選人選擇）的探索性方法可能不需要這樣嚴格的驗證。該FFP概念適用于藥物，其代謝物和生物標志物。

　　 進行毒理學研究的分析實驗室應遵守21 CFR 58，良好實驗室規范（GLP）.9人體BA，BE和藥代動力學研究的生物分析方法必須符合21 CFR 320生物等效性和生物可用性要求（即21 CFR 320.29）。

　　 以下部分將討論生物分析方法的開發，驗證和研究中的使用以及如何最好地記錄驗證方法和結果。請參閱術語表以獲取本指南中使用的測定參數和分析術語的定義。

　　III. BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION

　　 A. Guiding Principles

　　 The purpose of bioanalytical method development is to define the design, operating conditions, limitations, and suitability of the method for its intended purpose and to ensure that the method is optimized for validation.

　　 Before the development of a bioanalytical method, the sponsor should understand the analyte of interest (e.g., determine the physicochemical properties of the drug, in vitro and in vivo metabolism, and protein binding) and consider aspects of any prior analytical methods that may be applicable.

　　 The elements and acceptance criteria of method development and validation are summarized in Table 1. Table 2 describes how the sponsor should document the development and validation of the bioanalytical assay and where it should be stored or submitted.

　　 Method development involves optimizing the procedures and conditions involved with extracting and detecting the analyte. Method development includes the optimization of the following bioanalytical parameters (which are discussed in greater detail in section III.B) to ensure that the method is suitable for validation:

　　? Reference standards ? Critical reagents ? Calibration curve ? Quality control samples (QCs) ? Selectivity and specificity ? Sensitivity ? Accuracy ? Precision ? Recovery ? Stability of the analyte in the matrix

　　 Bioanalytical method development does not require extensive record keeping or notation. However, the sponsor should record the changes to procedures as well as any issues and their resolutions during development of the bioanalytical method to provide a rationale for any changes during the development of the method.

　　 Bioanalytical method validation proves that the optimized method is suited to the analysis of the study samples. The sponsor should:

　　III。生物分析方法的開發和驗證

　　 A.指導原則

　　 生物分析方法開發的目的是確定方法的設計，操作條件，限制和適用性，以確保其方法達到最佳效果。

　　 在開發生物分析方法之前，申辦者應該了解感興趣的分析物（例如確定藥物的物理化學性質，體外和體內代謝以及蛋白質結合）并考慮可能適用的任何現有分析方法的方面。

　　 方法開發和驗證的要素和驗收標準總結在表1中。表2描述了申辦者如何記錄生物分析測定的開發和驗證以及應該如何存儲或提交。

　　 方法開發涉及優化與提取和檢測分析物有關的程序和條件。方法開發包括優化以下生物分析參數（將在第III.B節中詳細討論），以確保該方法適用于驗證：

　　?參比標樣?關鍵試劑?校準曲線?質控樣品（QC）?選擇性和特異性?靈敏度?準確度?精密度?回收率?基質中分析物的穩定性

　　 生物分析方法開發不需要大量的記錄保存或符號。但是，申辦者應該在開發生物分析方法的過程中記錄程序的變化以及任何問題和解決方案，以便為方法開發過程中的任何變化提供基本原理。

　　 生物分析方法驗證證明，優化的方法適合分析研究樣品。申辦者應該：

　　? Conduct a full validation of any new bioanalytical method for the analysis of a new drug entity, its metabolite(s), or biomarkers.

　　? Conduct a full validation for any revisions to an existing validated method that adds a metabolite or an additional analyte.

　　? Establish a detailed, written description (e.g., protocol, study plan, and/or standard operating procedure (SOP)) for the bioanalytical method before initiating validation. The description should identify procedures that control critical parameters in the method (e.g., environmental, matrix, procedural variables) from the time of collection of the samples to the time of analysis to minimize their effects on the measurement of the analyte in the matrix.

　　? Document and report (in the method validation report) all experiments used to make claims or draw conclusions about the validity of the method.

　　? Validate the measurement of each analyte in the biological matrix. The specific requirements and acceptance criteria for each bioanalytical parameter are listed in Table 1.

　　B. Bioanalytical Parameters of CCs and LBAs

　　 The bioanalytical parameters applicable to CCs and LBAs are discussed below. Issues unique to either CCs or LBAs are specifically identified.

　　1. Reference Standards and Critical Reagents

　　 The sponsor should appropriately characterize and document (e.g. determine the identity, purity, and stability) all reference standards and critical reagents, such as antibodies, labeled analytes, and matrices and store them under defined conditions.

　　 a. Reference standards

　　The purity of reference standards used to prepare calibrators and QCs can affect the study data. Therefore, the sponsor should use authenticated analytical reference standards with known identities and purities to prepare solutions of known concentrations. The reference standard should be identical to the analyte； however, when this scenario is not possible, the sponsor can use an established chemical form (e.g., free base, free acid, or salt) of known purity.

　　 The sponsor should provide the certificates of analyses (CoA), including the source, lot number, and expiration date (with the exception of United States Pharmacopeia (USP) standards) for commercially available reference standards. For internally or externally generated reference standards that do not have a CoA, the sponsor should provide evidence of the standard’s identity and purity in addition to the source and the lot number. When using expired reference standards, the sponsor should provide an updated CoA or re-establish the identity and purity of the standard. If the reference standard expires, the sponsor should not make stock solutions with this lot of standard unless the standard’s purity is re-established. For internal standards (ISs), the sponsor does not have to provide a CoA or evidence of purity if it demonstrates that the IS is suitable for the specific use (e.g., lack of interference with an analyte).

　　?對任何新的生物分析方法進行全面驗證，以分析新藥物實體，其代謝物或生物標記物。

　　?對添加代謝物或額外分析物的現有驗證方法的任何修訂進行全面驗證。

　　?在開始驗證之前，為生物分析方法建立詳細的書面描述（例如，方案，研究計劃和/或標準操作程序（SOP））。描述應該確定控制方法中的關鍵參數（例如，環境，基體，程序變量）從采樣到分析時間的程序，以使其對矩陣中分析物測量的影響最小化。

　　?記錄和報告（在方法驗證報告中）用于提出索賠的所有實驗，或者得出關于該方法有效性的結論。

　　?驗證生物基質中每種分析物的測量結果。表1列出了每個生物分析參數的具體要求和驗收標準。

　　B. CC和LBA的生物分析參數

　　 下面討論適用于CC和LBA的生物分析參數。具體確定CC或LBA獨有的問題。

　　1.參考標準和關鍵試劑

　　 申辦者應對所有參考標準品和關鍵試劑（如抗體，標記分析物和基質）進行適當表征和記錄（如確定其身份，純度和穩定性），并將其存儲在特定條件下。

　　 一個。參考標準

　　用于制備校準品和QC的參考標準的純度會影響研究數據。因此，申辦者應使用具有已知身份和純度的認證分析參考標準來制備已知濃度的溶液。參考標準應與分析物相同；然而，當這種情況不可能時，申辦者可以使用已知純度的確定的化學形式（例如游離堿，游離酸或鹽）。

　　 申辦者應提供分析證明（CoA），包括來源，批號和截止日期（美國藥典（USP）標準除外）。對于沒有CoA的內部或外部生成的參考標準，除了來源和批號外，申辦者還應提供標準身份和純度的證據。使用過期參考標準時，申辦者應提供最新的CoA或重新確定標準的身份和純度。如果參考標準到期，除非標準的純度重新建立，否則發起人不應該使用這個標準的庫存解決方案。對于內部標準（ISs），如果申辦者證明信息系統適合于特定用途（例如缺乏干擾w），則申辦者不必提供CoA或純度證據。

　　b. Critical reagents

　　 The sponsor should appropriately characterize and document (i.e., determine the identity, purity and stability) the critical reagents, including – but not limited to – any reference standards, antibodies, labeled analytes, and matrices.

　　 Assay validation is important when there are changes to the critical reagents, such as lot-to-lot changes or switches to another reagent. For example, if there are changes to the labeled analytes, detector reagents, or antibodies, the sponsor should:

　　? Evaluate binding and re-optimize assays

　　? Verify performance with a standard curve and QCs

　　? Evaluate cross-reactivities

　　 2. Calibration Curve

　　During method development, the sponsor should choose the quantitation range of the assay and the concentrations of the calibration standards on the basis of the concentration range expected in a particular study. For LBAs, in addition to the calibration standards, anchor points outside the range of quantification can facilitate the fitting of the curve. Anchor points should not be used as part of the acceptance criteria for the run. For most LBAs, calibration (standard) curves are inherently nonlinear, and in general, more calibration standards are needed to define the fit over the calibration curve range for LBAs than for CCs. In addition, the response-error relationship for LBA standard curves is a variable function of the mean response (i.e., heteroscadisticity).

　　The sponsor should use the simplest model that adequately describes the concentration-response relationship, as well as an appropriate weighting scheme and regression equation. For LBAs, the concentration-response relationship is most often fitted to a four -or five-parameter logistic model, although other models can be assessed.

　　 When the method is validated, the calibration curve should be continuous and reproducible. The sponsor should prepare the calibration standards in the same biological matrix as the samples in the intended study. Study samples may contain more than one analyte. The sponsor should generate a calibration curve for each analyte in the sample. When surrogate matrices are necessary, the sponsor should justify and validate the calibration curves.

　　The requirements for the calibration curve, including the LLOQ, ULOQ, as well as the acceptance criteria are listed in Table 1.

　　 關鍵試劑

　　 申辦者應適當表征和記錄關鍵試劑（包括但不限于）任何參考標準品，抗體，標記分析物和基質，并記錄（即確定其身份，純度和穩定性）。

　　 當關鍵試劑發生變化時，分析驗證非常重要，如批次間變化或切換到其他試劑。例如，如果標記的分析物，檢測試劑或抗體發生變化，申辦者應該：

　　?評估結合并重新優化檢測

　　?使用標準曲線和QC驗證性能

　　?評估交叉反應性

　　 2.校準曲線

　　在方法開發過程中，申辦者應根據特定研究中預期的濃度范圍選擇測定的定量范圍和校準標準品的濃度。對于LBA，除了校準標準外，量化范圍之外的錨點可以幫助擬合曲線。不應將錨點用作運行驗收標準的一部分。對于大多數LBA，校準（標準）曲線本質上是非線性的，并且通常需要更多的校準標準來定義LBA的校準曲線范圍與CC的擬合。另外，LBA標準曲線的響應  -誤差關系是平均響應的可變函數（即異質性）。

　　申辦者應使用充分描述濃度  -反應關系的最簡單模型，以及適當的加權方案和回歸方程。對于LBA，濃度  -反應關系通常適用于四參數或五參數邏輯模型，但也可以評估其他模型。

　　 當該方法被驗證時，校準曲線應該是連續的和可重現的。申辦者應該在預期研究中的樣品制備相同的生物基質中的校準標準。研究樣品可能含有多種分析物。申辦者應該為樣品中的每種分析物產生校準曲線。當需要替代矩陣時，申辦者應該證明和驗證校準曲線。

　　表1列出了校準曲線的要求，包括LLOQ，ULOQ和驗收標準。

　　3. Quality Control Samples

　　 Quality controls are used to assess the precision and accuracy of an assay and the stability of the samples. Sponsors should prepare QCs in the same matrix as the study samples to be assayed with the validated method. Freshly prepared QCs are recommended for precision and accuracy analyses during method development, as stability data are generally not available at this time.

　　During method validation, QCs evaluate the performance of a method and the stability of an analyte. Performance QCs are included in validation runs to determine the precision and accuracy of the method (see section III.B). Stability QCs evaluate the stability of an analyte under various stress conditions (Refer to section III.B for the selection of QC concentrations).

　　The sponsor should prepare any calibration standards and QCs from separate stock solutions. However, if the sponsor can demonstrate the precision and accuracy in one validation run using calibrators and QCs prepared from separate stock solutions, then the sponsor can use calibrators and QCs prepared from the same stock solution in subsequent runs. The sponsor should make up calibrators and QCs in lots of blank matrix that is free of interference or matrix effects.

　　4. Selectivity and Specificity

　　During method development, the sponsor should verify that the substance being measured is the intended analyte to minimize or avoid interference. Selectivity of the method is routinely demonstrated by analyzing blank samples of the appropriate biological matrix (e.g., plasma) from multiple sources. Depending on the intended use of the assay, the impact of hemolyzed samples, lipemic samples, or samples from special populations can be included in the selectivity assessment. When using liquid chromatography/mass spectrometry (LC/MS) methods, the sponsor or applicant should determine the effects of the matrix on ion suppression, ion enhancement, or extraction efficiency. Internal standards should be assessed to avoid interference with the analyte. Potential interfering substances in a biological matrix include endogenous matrix components such as metabolites, decomposition products – and from the actual study – concomitant medications and other xenobiotics. If a stabilizer or enzyme inhibitor is used during sample collection, the sponsor should evaluate the potential for interference on the quantitation of the analyte. Sponsors should make a scientific judgment about the need to assess these (and any other) potential interferences during method development.

　　During validation, the sponsor should confirm that the assay is free of potential interfering substances including endogenous matrix components, metabolites, anticipated concomitant medications, etc. If the study sample contains more than one analyte and the analytes are intended to be quantified by different methods, the sponsor should test each method for interference from the other analyte.

　　 The sponsor should analyze blank samples of the appropriate biological matrix (e.g. plasma) from at least six (for CCs) or ten (for LBAs) individual sources. The sponsor should ensure that there are no matrix effects throughout the application of the method. Refer to Table 1 for details of selectivity and specificity requirements and acceptance criteria.

　　 3.質量控制樣品

　　 質量控制用于評估測定的精確度和準確度以及樣品的穩定性。申辦者應該使用經過驗證的方法與待分析的研究樣品在相同的基質中制備QC。在方法開發過程中，建議使用新制備的QC進行精密度和準確度分析，因為此時通常不提供穩定性數據。

　　在方法驗證期間，QC評估方法的性能和分析物的穩定性。性能QCs包含在驗證運行中以確定方法的精確度和準確性（請參閱第III.B節）。穩定性QC評估各種應力條件下分析物的穩定性（關于選擇QC濃度，請參閱第III.B部分）。

　　申辦者應該從不同的庫存解決方案中準備任何校準標準和QC。但是，如果贊助商可以證明使用由不同儲備溶液制備的校準品和QC進行的一次驗證運行中的精確度和準確性，那么贊助商可以在隨后的運行中使用由相同儲備液制備的校準品和QC。贊助商應該在沒有干擾或基質影響的大量空白基質中制作校準品和QC。

　　4.選擇性和特異性

　　在方法開發過程中，申辦者應確認被測物質是預期分析物，以盡量減少或避免干擾。該方法的選擇性通過分析來自多個來源的合適生物基質（例如血漿）的空白樣品而被常規證明。根據測定的預期用途，溶血樣品，脂血樣品或來自特定人群的樣品的影響可以包括在選擇性評估中。當使用液相色譜/質譜（LC / MS）方法時，申辦者或申請人應確定基質對離子抑制，離子增強或提取效率的影響。應對內部標準進行評估以避免干擾分析物。生物基質中潛在的干擾物質包括內源性基質成分，如代謝物，分解產物  -以及實際研究  -伴隨藥物和其他異生素。如果在樣品采集期間使用穩定劑或酶抑制劑，申辦者應評估對定量分析物產生干擾的可能性。申辦者應該對方法開發過程中評估這些（以及其他任何潛在干擾）的需求做出科學判斷。

　　在驗證過程中，申辦者應確認測定不含潛在的干擾物質，包括內源性基質成分，代謝物，預期的伴隨藥物等。如果研究樣品含有多種分析物，并且分析物旨在通過不同的方法進行定量，申辦者應該測試每種方法是否受到來自其他分析物的干擾。

　　 申辦者應該分析appr的空白樣本

　　For LBAs, it is important to investigate any interference originating from structurally or physiologically similar analytes (i.e., exogenous interference) or matrix effects (i.e., endogenous interference). Investigating exogenous interference involves determining the cross-reactivity of molecules that could potentially interfere with the binding interaction, including molecules structurally related to the drug, any metabolites, concomitant medications (and their significant metabolites), or endogenous matrix components. The sponsor should evaluate each factor individually and in combination with the analyte of interest to determine its ability to cause interference. Matrix effects evaluation involves comparing calibration curves in multiple sources of the biological matrix against a calibration curve in the matrix for parallelism (serial dilution of incurred samples) and nonspecific binding. The sponsor should eliminate or minimize any significant interference. If such attempts are unsuccessful, the sponsor could consider the development of an orthogonal method to eliminate or minimize the interference.

　　 Carryover between samples can occur in analytical methods. The sponsor should eliminate any carryover during method development. If carryover cannot be eliminated, the sponsor should assess the impact of any carryover during method validation on the accuracy of the study sample concentrations.

　　5. Sensitivity

　　The LLOQ defines the method sensitivity and should be determined during method development. The method should be developed and validated such that it will be able to meet the requirements necessary for the intended study samples. The LLOQ evaluation can be done separately or as part of the precision and accuracy assessment for the calibration range. The specific requirements to validate sensitivity are listed in Table 1.

　　 6. Accuracy, Precision, and Recovery

　　Evaluating the accuracy and precision across the quantitation range during method development is essential to determine whether the method is ready for validation and involves analyzing replicate QCs at multiple concentrations across the assay range. Specifically, the sponsor should evaluate the performance at the LLOQ, low, mid and high QCs (and the ULOQ for LBAs) to determine if the method is suitable to analyze study samples.

　　 Method validation experiments for estimating accuracy and precision should include a minimum of three (for CCs) and six (for LBAs) independent runs (i.e., accuracy and precision (A & P) runs； see Table 1) conducted over several days. Each A & P run should include a calibration curve and multiple QC concentrations that are analyzed in replicates. The sponsor should determine the accuracy and precision of the method based on the performance of the QC in the A & P runs. The specific validation requirements for accuracy and precision and A & P runs are listed in Table 1. The sponsor should use freshly prepared calibrators and QCs in all A & P runs. Use of freshly prepared QCs in all A & P runs is preferred； however, if this is not possible, the sponsor should use freshly prepared QCs in one or more A & P runs.

　　The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent, but the extent of the recovery of an analyte and of the ISs should be consistent and reproducible. The sponsor should perform recovery experiments by comparing the analytical results of extracted samples with corresponding extracts of blanks spiked with the analyte post-extraction (i.e., to represent 100 percent recovery). Recovery evaluation is not necessary for LBAs unless sample extraction is involved. Recovery experiments should be performed as described in Table 1.

　　 對于LBA，研究來源于結構或生理學相似分析物（即外源干擾）或基質效應（即內源性干擾）的干擾很重要。研究外源性干擾涉及確定可能干擾結合相互作用的分子的交叉反應性，包括與藥物結構相關的分子，任何代謝物，伴隨藥物（及其重要代謝物）或內源性基質組分。申辦者應分別評估每個因素，并與感興趣的分析物結合，以確定其造成干擾的能力。基質效應評估涉及將生物基質的多個來源中的校準曲線與基質中的校準曲線進行比較（并行度（發生樣品的連續稀釋））和非特異性結合。申辦者應該消除或減少任何重大干擾。如果這種嘗試不成功，發起人可以考慮開發正交方法以消除或最小化干擾。

　　 樣品之間的結轉可能發生在分析方法中。發起人應該在方法開發過程中消除任何遺留物。如果無法消除遺留物，申辦者應評估方法驗證期間任何遺留物對研究樣本濃度準確性的影響。

　　5.靈敏度

　　LLOQ定義了方法靈敏度，應在方法開發過程中確定。應開發和驗證該方法，使其能夠滿足預期研究樣本所需的要求。 LLOQ評估可以單獨完成，也可以作為校準范圍精度和準確度評估的一部分。表1列出了驗證靈敏度的具體要求。

　　6.準確性，精確度和恢復

　　在方法開發過程中評估整個定量范圍內的準確度和精密度對于確定該方法是否可以進行驗證并涉及分析整個測定范圍內多個濃度的重復QC是至關重要的。具體而言，申辦者應評估LLOQ，低，中，高QC（以及LBA的ULOQ）的表現，以確定該方法是否適合分析研究樣本。

　　 用于估計準確性和精密度的方法驗證實驗應包括在數天內進行的最少三次（對于CC）和六次（對于LBA）獨立運行（即，精度和精度（A＆P）運行；參見表1）。每次A＆P運行應包括校準曲線和重復分析的多個QC濃度。申辦者應根據A＆P運行中QC的性能來確定方法的準確性和精確度。表1列出了準確度和精密度以及A＆P運行的具體驗證要求。贊助商應在所有A＆P運行中使用新制備的校準品和QC。在所有A＆P運行中使用新鮮制備的QCs是首選；然而，如果這不可能，贊助商應該在一次或多次A＆P運行中使用新配備的QC。

　　申辦者應優化分析物的回收率以確保萃取效率和可重復性。回收率不必為100％，但分析物和IS的回收率應該一致且可重現。申辦者應通過將提取樣品的分析結果與萃取后萃取分析物的相應提取物（即代表100％回收）進行比較來進行回收實驗。除非涉及樣本提取，否則恢復評估對于LBA不是必需的。恢復實驗應按照表1中的描述進行。

　　7. Stability

　　During method development, the sponsor should determine the chemical stability of the analyte in a given matrix, including the effects of sample collection, handling, and storage of the analyte. The sponsor should assess autosampler, benchtop, processed or extracted samples, freeze-thaw, stock solution, and long-term stability of the analyte. The sponsor should assess the stability in the same matrix as that intended for in-study samples； however, when the matrix is rare, the sponsor can explore the use of suitable surrogate matrices.

　　For drugs administered as fixed combinations, or part of a specific drug regimen, the stability of the analyte should be assessed in the presence of the other drug. The sponsor should also consider the stability of the analyte in the presence of other co-medications that are known to be regularly administered to patients for the indication of the drug under development.

　　Depending on the analyte as well as the sample collection and assay conditions, evaluating the stability of the analyte in whole blood during method development can be useful. For example, a drug can be unstable in whole blood or adsorb to cellular components during collection.

　　During validation, stability evaluations should cover the expected sample conditions before receipt at the analytical site (e.g., at the clinical site, during shipment, and at all other secondary sites) as well as during receipt and analysis at the analytical site. Validation of drug stability in a biological fluid is a function of the storage conditions, the physicochemical properties of the drug, the matrix, and the container system. The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems.

　　 If the storage conditions changed or the sample analysis occurred outside of the validated storage condition , the stability should be re-established under these new conditions. Stability testing of the analyte in whole blood should be revalidated if necessary (e.g., if the analytes are unstable during blood collection). The specific requirements and acceptance criteria for stability are listed in Table 1.

　　 Matrix-related stability experiments should compare stability QCs against freshly prepared calibration curves and freshly prepared QCs. Although the use of freshly prepared calibrators and QCs is the preferred approach, in some cases, (e.g., for macromolecules), it may be necessary to freeze them overnight. In such cases, the sponsor should provide valid justification and demonstrate the freeze-thaw stability.

　　 7.穩定性

　　在方法開發過程中，申辦者應確定給定基質中分析物的化學穩定性，包括樣品收集，處理和分析物儲存的影響。贊助商應評估自動進樣器，臺式，加工或提取的樣品，凍融，儲備液和分析物的長期穩定性。申辦者應評估與研究樣本相同基質的穩定性；然而，當矩陣很少時，申辦者可以探索使用合適的替代矩陣。

　　對于以固定組合或部分特定藥物方案給藥的藥物，應在其他藥物存在下評估分析物的穩定性。申辦者還應該考慮在已知定期向患者指示正在開發的藥物的其他聯合藥物的情況下分析物的穩定性。

　　根據分析物以及樣品采集