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Document Number:

MAN-000037

Revision #: 03

Revised:

31 Oct 2023

Title: 

ORA Lab Manual Vol. II - Methods, Method Verification and Validation (ORALAB.5.4.5)

Page 1 of 34

For the most current and official copy, check QMiS.

Sections in This Document

Purpose ....................................................................................................................................2

Scope .......................................................................................................................................2

Responsibility............................................................................................................................2

Background...............................................................................................................................3

References ...............................................................................................................................4

Procedure .................................................................................................................................5

6.1. Method Selection ...........................................................................................................5

6.2. Method Evaluation & Record .........................................................................................5

6.3. Standard Method Verification.........................................................................................6

6.3.1. Chemistry.........................................................................................................6

6.3.2. Microbiology.....................................................................................................8

6.3.3. Other Scientific Disciplines ..............................................................................9

6.4. Validation of Method Performance.................................................................................9

6.5. Validation of Method Modifications: ...............................................................................9

6.6. Validation Tools............................................................................................................10

6.7. Validation Protocol Guidance.......................................................................................11

6.7.1. General ..........................................................................................................11

6.7.2. Chemistry.......................................................................................................11

6.7.3. Microbiology...................................................................................................12

6.7.4. Other Scientific Disciplines ............................................................................13

6.8. Documentation.............................................................................................................13

6.8.1. Method Verification ........................................................................................13

6.8.2. Method Validation ..........................................................................................14

Glossary/Definitions................................................................................................................15

Records ..................................................................................................................................17

Supporting Documents ...........................................................................................................17

Document History ...................................................................................................................18

Change History.......................................................................................................................19

Attachments............................................................................................................................19

Attachment A - Food and Feed Chemistry Modification Criteria.............................................20

Attachment B - Drug Chemistry Modification Criteria .............................................................24
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Attachment C - ORA Validation (non-compendial) and Verification Guidance for Human Drug 

Analytical Methods.......................................................................................................28

Purpose

This procedure provides basic requirements to Office of Regulatory Science 

(ORS) laboratories for development, validation, and verification of method 

performance specifications for new methods, modified methods or procedures 

previously validated externally.

Scope

ORS laboratories verify standard method performance and validate new or 

modified methods introduced into the laboratory. 

Refer to MAN-000043 ORA Lab Manual Vol. II - Ensuring the Quality of Test 

Results (ORA-LAB.5.9) for continuing verification of acceptable performance 

for each analytical batch (EAB) through a quality control program.

Responsibility

A. ORS Office of Research Coordination, Evaluation and Training 

(ORCET)

1. Directs, monitors, and coordinates ORS method development and 

validation activities designed to address regulatory gaps.

2. Issues call for proposals, assigns reviewers, approves proposals, 

and maintains approved and completed project list.

3. Tracks research progress, achievement of deliverables, and 

evaluation of impact.

B. Laboratory Management:

1. Provides resources and ensures implementation of method 

verification and validation procedures.

2. Develops and executes approved method development and 

validation program.

C. Analysts: 

1. Adhere to written protocol for method performance verification, 

validation, or modification.
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2. Submit method development and validation needs through local 

management in response to ORCET initiatives and/or programmatic 

needs.

3. Develop and submit proposals for method validation or verification in 

accordance with approved procedures.

4. Accomplish approved projects in timely manner.

5. Adhere to the highest standards of intellectual honesty and ethical 

standards in formulating, conducting, and presenting method 

development and validation work.

6. Follow Agency level and ORCET research and publication 

requirements and guidelines.

7. Submit new validation proposals into the Component Automated 

Research Tracking System (CARTS) to include projected timelines 

and milestones. 

D. Quality System Manager:

1. Ensures all processes are executed to ensure traceability and 

defensibility of data provided for method development, method 

validation, and method verification in accordance with ORS policy 

and maintaining accreditation.

2. Ensures an SOP exists or is written when use of an original Method 

does not provide guidance required in testing processes, for 

instance where the original method has been modified. 

3. Provides concurrence for approval with the Laboratory Management 

for requests and ensures that completed analytical studies have met 

all quality validation and/or verification criteria.

4. Maintains records of completed verified/validated studies.

5. Monitors progress/status of open method validation, verification, and 

modification.

Background

As a regulatory agency, it is imperative that the analytical methods ORS 

laboratories employ for surveillance, compliance and outbreak investigations 

continue to meet the highest standard of review and performance. 
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When a standard analytical method is verified, the laboratory is required to 

demonstrate with objective evidence that it can achieve specified performance 

characteristics/ parameters proven during the initial validation study. 

A validation study must establish pertinent performance characteristics (e.g., 

accuracy, precision, sensitivity, limit of detection, etc.) that demonstrate or 

confirm a method is suitable for its intended purpose. 

Any significant modification to existing procedures involving a major change in 

part of the original process that will affect the instruments and/or samples used 

to produce data must be validated.

References

A. Code of Federal Regulations. Title 40, Part 136, Appendix B, Definition 

and Procedure for the Determination of the Method Detection Limit

B. The Analytical Laboratory Accreditation Criteria Committee (ALACC) 

How to Meet ISO 17025 Requirements for Method Verification, AOAC 

International, 2007

C. ISO/IEC 17025:2017 General requirements for the competence of 

testing and calibration laboratories.

D. AOAC International Guidelines for Laboratories Performing 

Microbiological and Chemical Analysis of Food, Dietary Supplements, 

and Pharmaceuticals – An Aid to Interpretation of ISO/IEC 17025:2017; 

August 2018.

E. Guidelines for the Validation of Analytical Methods for the Detection of 

Microbial Pathogens in Foods and Feeds, 3rd Edition

F. Guidelines for the Validation of Chemical Methods for the FDA FVM 

Program, 3rd Edition

G. USFDA Center for Veterinary Medicine, ‘Mass Spectrometry for 

Confirmation of the Identity of Animal Drug Residues’, Guidance for 

Industry # 118, 2003

H. Food and Drug Administration, “Acceptance Criteria for Confirmation of 

Identity of Chemical Residues using Exact Mass Data within the Office 

of Foods and Veterinary Medicine”

I. USP <621> (Chromatography)

J. SANCO/12495/2011 ‘Method Validation and Quality Control Procedures 

for Pesticide Residue Analysis in Food and Feed’ 
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Document Number:

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Title: 

ORA Lab Manual Vol. II - Methods, Method Verification and Validation (ORALAB.5.4.5)

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Procedure

6.1. Method Selection

A. An integral part of the laboratory quality system is the use of standard 

methods. Standard methods are used, whenever possible, unless 

otherwise specified by the Compliance Program or the customer. 

B. Methods of analysis that are specified in law or regulations must be 

followed in accordance with those requirements.

C. Non-standard methods are used in cases where a standard method 

does not exist, and the customer has agreed to its use. These methods 

must be validated prior to use with a clear expression of quality 

objectives and testing parameters, or criteria included within the 

validation records. 

D. If a standard test method is not available for the requested analysis, a 

new method may be developed and validated. See MAN-000050 ORA 

Laboratory Manual, Volume III Section 6 -ORS Method Development 

and Validation Program. 

6.2. Method Evaluation & Record

A. Any method selected for use must be appropriate to the requirements of 

the regulatory function and must be within the capabilities of the 

laboratory. Analytical methods are to be evaluated based on attributes 

such as accuracy/trueness, precision, specificity/selectivity, sensitivity, 

ruggedness/robustness, matrix, and practicality. 

B. The Laboratory Method Validation and Verification Process Form in 

QMiS shall be used to provide a required record of evaluation and 

approval in the laboratory by designated official(s) to determine if a 

validation or verification is deemed necessary. The method plan 

proposal, evaluation, decisions, and justifications shall be included in 

the process form created for this purpose.

C. This form is initiated concurrently with submitting research projects 

through the CARTS system. 

D. For instructions in using this Process Form refer to WI-000487 

Instructions for Using the Method Validation and Verification Process 

Form in QMiS. 
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6.3. Standard Method Verification

A. Standard or FDA official methods require verification as confirmation 

that the laboratory can meet the test method performance 

specifications. Verification of a test method demonstrates that the 

laboratory has met the test method’s performance specifications and 

must be completed before the method is used for routine testing.

B. Verification ensures that the laboratory can obtain comparable results 

on the same matrix, using equivalent equipment and circumstances, as 

the standard method; in other words, the method is suitable under 

actual conditions of use in a laboratory.

C. For general guidance in submitting a method verification refer to SOP000107 Method Verification.

6.3.1. Chemistry

The requirements below outline general principles for quantitative and 

qualitative chemistry methods. 

For additional special considerations and requirements for drug chemistry 

work, please refer to Attachment C. Compendial drug methods should follow 

the compendial requirements. 

For additional special considerations and requirements for food chemistry 

work, please refer to the Guidelines for the Validation of Chemical Methods for 

the FDA FVM Program, 3rd Edition.

6.3.1.1. Quantitative Chemistry

A. Minimum Requirements

1. Meets System suitability requirements, where applicable

2. Accuracy, 

3. Precision, 

4. Working Range/Linearity

5. Method Detection Level/Limit of Detection (MDL/LOD)

6. Limit of Quantitation (LOQ)

B. Critical requirements for quantitative analysis are the accuracy and the 

precision (also known as repeatability and reproducibility) obtained from 

actual lab data which are reflected in the measurement uncertainty. 
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C. Analyze reference materials, standard, or spike samples at ≥ 2 

concentration levels in triplicate. Consider any relevant regulatory 

limits/action levels when selecting spike ranges (e.g., spiking at 0.5x 

any applicable limit). Run a matrix blank when available and a method 

blank with each spike level.

D. Determine working range or target level. As guidance for a calibration 

curve, the mid-point is set at the target level with the lowest calibrator at 

one-half this concentration and the highest calibrator at twice this 

concentration. For a one-point calibration, use the target level.

E. The R2 (coefficient of determination) for a calibration curve must be 

equal to or better than documented in the method. If the R2 is not 

specified in the method:

1. Single analyte / single matrix methods R2 should be ≥0.995

2. Multi residue, multi class mass spectrometer analysis may have a 

R2 value of ≥ 0.990 with a written justification included in the study 

package narrative(s).

F. Method Detection Limit (MDL)/Limit of Detection (LOD)

1. If the MDL is stated in the method, verify by running the method 

stated concentration. The result should be within ± 30% of the 

known value. 

2. If the MDL is not provided in the method, determine detection limit 

by either method detection limit (MDL) according to 40 CFR, Part 

136, Appendix B or limit of detection (LOD). LOD may be 

determined by analyzing sample blanks, calculating the standard 

deviation, and expressed as the mean plus 3 standard deviations.

G. Limit of Quantitation (LOQ) is the minimum amount/concentration that 

can be quantified with acceptable precision.

H. If available, prepare and analyze a Quality Control Sample/Certified

Reference Material (CRM).

1. The concentration must be within the range specified within the 

Certificate of Analysis for CRM or 80% - 120% of stated or known 

value for other control material used. 

2. This check is intended as an independent check of technique, 

methodology and standards.

I. For a standard curve, analyze calibration standards containing known 

amounts of analyte.
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J. Calculate percent recovery. The result should be within +20% of the 

known value for acceptable method performance.

6.3.1.2. Qualitative Chemistry Methods 

A. Meet system suitability requirements, if applicable.

B. Establish a detection limit (LOD). The detection limit is the lowest level 

of analyte that gives a positive response that can be reliably 

distinguished from zero (term is usually restricted to the response of the 

detection system. When applied to the complete analytical method it is 

referred to as the MDL)

C. Linearity

1. Analyze ≥3 matrices where available

2. Analyze a blank. The result must be negative.

3. Analyze a quality control sample or reference material, if available. 

The results must be positive. 

4. Analyze ≥ 3 matrix spikes for each matrix tested. The matrix or 

sample should be spiked at the relevant regulatory limits/action 

levels. The result must be positive.

6.3.2. Microbiology 

A. Meet method system suitability requirements, if applicable.

B. Prepare and analyze a positive culture control to assess and 

demonstrate accuracy.

1. Accuracy must be > 95%.

2. A positive culture control must exhibit positive growth or exhibit 

expected characteristics to assure the system is working. For 

example, turbidity in a tube filled with enrichment broth showing 

growth or a physical (phenotypic) change to the bacterial culture 

showing a positive test result.

C. Include all other controls as required by SOP-001064 Microbiological 

Controls for Food and Feed Sample Analysis and SOP-000288 

Microbiological Controls for Medical Product Sample Analysis.

D. Sample duplicates are not required when precision is calculated by the 

number of false negatives or false positives. Precision must be < 5%.
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6.3.3. Other Scientific Disciplines

Laboratories will address, at their local level, method verification of scientific 

disciplines not discussed in this procedure. At a minimum, scientific disciplines 

must meet the requirements to demonstrate lack of contamination, accuracy, 

precision, detection limits, quantitation limits, and linearity as applicable. 

6.4. Validation of Method Performance 

A. Non-standard and laboratory-developed methods must be validated. 

This activity is planned and assigned to qualified personnel. The 

method’s performance characteristics are based on the intended use of 

the method. For example, if the method will be used for qualitative 

analysis, there is no need to test and validate the method’s linearity 

over the full dynamic range of the equipment.

B. Typical validation characteristics which should be considered are:

1. accuracy,

2. precision,

3. specificity,

4. detection limit,

5. limit of quantitation, 

6. linearity,

7. range, and

8. ruggedness and robustness.

(See Section 7 for definitions of these characteristics)

6.5. Validation of Method Modifications: 

A. If there is any difference between the current samples (matrix or 

equipment type) and those for which the method was originally 

validated, the extent of the difference and its impact must be assessed. 

See Attachment A & B for general chemistry guidelines for allowable 

modifications to a method before a revalidation protocol is needed.

B. For protocol requirements for modification to Food and Feed chemistry 

methods, refer to Guidelines for the Validation of Chemical Methods for 

the FDA FVM Program, 3rd Edition. 

C. For protocol requirements for modification to food microbiology 

methods, refer to the Guidelines for the Validation of Analytical Methods 
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for the Detection of Microbial Pathogens in Foods and Feeds 3rd 

Edition.

D. Modifications of methods that alter the fundamental nature of the 

method shall be validated to demonstrate that equivalent results are 

obtained; do not adversely affect the precision and accuracy of the data 

obtained; and that the method is suitable for its intended use.

E. Each major modification is verified against the original method. 

F. Additional statistics that may be employed to validate method 

modifications are:

1. The t test for significance of difference between the two data set 

means. If your t-stat value is less than or equal to the t-critical value 

(0.5 would provide 95% confidence level), there is no statistically 

significant difference between your methods.

2. The f test provides a ratio of the calculated data from the validated 

method and the modified method. A high f-statistic would indicate a 

significant difference.

6.6. Validation Tools

The following tools can be used to substantiate a method’s ability to meet 

satisfactory specifications of performance:

A. Blanks (matrix, reagent, system): Use of various types of blanks 

enables assessment of how much is attributable to the analyte and how 

much is attributable to other causes.

B. Reference materials and certified reference materials: Use of known 

materials can be used to assess the accuracy of the method, as well as 

obtaining information on interferences.

C. Fortified (spiked) materials and solutions: Recovery determinations can 

be made from fortification or spiking with a known amount of analyte.

D. Incurred materials: These are materials in which the analyte of interest 

has been introduced/added to the bulk at some point prior to the 

material being sampled.

E. Measurement standards: These are substances or stable materials 

used for calibration or identification purposes. When placed periodically 

in an analytical batch, checks can be made that the response of the 

analytical process to the analyte is stable.
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F. Replication: Replicate analysis provides a means of checking for 

changes in precision in an analytical process which could adversely 

affect the results.

G. Statistics: Statistical techniques are employed to evaluate accuracy, 

precision, linear range, limits of detection and quantification, and 

measurement uncertainty. 

6.7. Validation Protocol Guidance

6.7.1. General

Note: Refer to program validation guidance documents such as Guidelines for 

the Validation of Analytical Methods for the Detection of Microbial Pathogens 

in Foods and Feeds and Guidelines for the Validation of Chemical Methods for 

the FDA FVM Program for specific requirements pertaining to the program. 

A. Laboratories record their validation protocol, the performance 

characteristics measured, and acceptance limits for the validation of 

non-standard and laboratory developed methods. The validation record 

also includes a statement indicating the method is fit for the intended 

use.

B. Validation studies are planned and approved with local management 

and the Laboratory Quality System Manager using the Laboratory 

Method Validation and Verification Process Form in QMiS. Additional 

approval of the final protocol will usually be required by the ORS QSM 

and Program Managers or Coordinators. 

C. The extent of validation will depend on constraints imposed such as 

time, cost, amount of sample or standard, future use of method, or type 

of information (quantitative, qualitative, screening). Due to these 

constraints, not all characteristics may be applicable. 

D. Method validations performed to address emergency/limited use will 

undergo more rigid validation protocols if the method continues to be 

used for more wide-spread non-emergency regulatory use.

Basic guidelines for determining performance characteristics are outlined 

below.

6.7.2. Chemistry 

A. Perform system suitability requirements: i.e. injection repeatability, peak 

resolution, relative retention time for liquid chromatography analyses.

B. Quantitative measurements: Determine detection limit, either method 

detection limit (MDL) according to 40 CFR, Part 136, Appendix B or limit 
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of detection (LOD). LOD may be determined by analyzing sample 

blanks, calculating the standard deviation, and expressed as the mean 

plus 3 standard deviations. 

C. For qualitative measurements, determine the concentration threshold 

below which specificity becomes unreliable.

D. Quantitative measurements: Determine calibration range if a standard 

curve is to be used or determine the target calibration standard and 

linearity if only a one calibration point is to be used.

E. Quantitative measurements: Prepare and analyze spiked blanks, 

solvent or matrix samples of known concentration utilizing at least three 

different concentration levels: low, middle, and high. These samples are 

carried through the complete sample preparation procedure. Matrix 

effects can be assessed with these samples. Accuracy (percent 

recovery) and precision (relative standard deviation or relative percent 

recovery) are calculated from the results.

F. Analyze blanks (reagent, solvent, and matrix).

G. Evaluate interferences. i.e. spectral, peak symmetry, physical, chemical 

or memory by analyzing a sample containing various suspected 

interferences in the presence of the measure:

1. Spectral interference may be observed when an overlap of a 

spectral line from another element or background contribution 

occurs. 

2. Physical interference may occur from effects associated with sample 

transport processes on instruments.

3. Chemical interferences can be characterized by compound 

formation, ionization, or vaporization effects. 

4. Memory interference occurs from contribution of signal from 

previous sample to sample being tested. 

6.7.3. Microbiology

A. Meet method system suitability requirements, if applicable. The 

suitability of the method is checked and confirmed by comparing with 

requirements typical for the intended use of the method. For example, a 

filtration method for a non-filterable food, a five-day test where three 

days are needed, a 1-gram test where 100 grams are needed, surface 

tests for Colony Forming Units (CFU)/square area where CFU/gram is 

needed.
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B. Include un-inoculated medium control to assess contamination from the 

laboratory. This control is considered a blank and is to exhibit no 

growth.

C. Prepare and analyze positive and negative culture controls. A negative 

control is a typical, negative or no growth and the positive control is 

positive or shows microbial growth.

D. A spike positive culture control is prepared and analyzed. Unless 

otherwise specified, it is recommended that a 25-gram sample be 

spiked with an inoculum of 30 cells or less. This assesses the matrix 

effects as well as the sensitivity of the method.

E. Evaluate interferences. This assesses the selectivity and specificity of 

the method.

6.7.4. Other Scientific Disciplines

Laboratories will address, at their local level, method validation of scientific 

disciplines not discussed in this procedure. 

6.8. Documentation 

6.8.1. Method Verification

A. Verification studies are planned and approved with local management 

and the Quality System Manager. 

B. Objective evidence of standard method verification can be in method 

verification reports, analyst worksheets, memos of analyses, and 

method quality control records. These records can be attached to the 

Laboratory Method Validation and Verification Process Form or a 

separate Document InfoCard can be created in QMiS for these records 

which will be linked to the Process Form. 

C. Method verification reports include at a minimum:

1. A summary (e.g. standard method used, method criteria, how 

sample was prepared and analyzed, equipment used, measurement 

uncertainty, analysts demonstrating competence).

2. Analytical testing data generated during the Study.

3. Verification statistical evaluation (e.g. accuracy, precision, LOQ, 

MDL, linearity, etc.).
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6.8.2. Method Validation

Validation studies are initially planned and approved with local management 

and the Laboratory Quality System Manager and then submitted in CARTS 

concurrently with the Method Validation and Verification Process Form; refer to 

MAN-000050 ORA Lab Manual Vol. III Section 6 - ORS Method Development 

and Validation Program (III-06)

A. Submit method validation plans, results, and documentation to 

Laboratory Management and the QSM through the Process Form in 

QMiS for review and approval.

B. The validation information contains all relevant data to confirm that 

method performance specifications, as defined by the established 

acceptance criteria, are met. 

C. The method validation package includes:

1. A study summary (how sample was prepared and analyzed, 

equipment used, robustness, measurement uncertainty, analysts 

demonstrating competence)

2. Data generated in the study

3. Validation study statistical evaluation and acceptance criteria 

(accuracy, precision, LOQ, MDL, linearity, etc.)

4. Laboratory Method Procedure. 

Note: infrequently used or non-routine methods do not need a 

Laboratory Procedure until they become routine. In these instances 

a copy of the method itself will be included, if possible, or information 

providing where it is located.

D. A record of the person(s) authorizing adoption of the method and the 

date authorization was granted is included in the signature tracking of 

the QMiS Validation and Verification Process Form.

E. A statement on the validity of the method, detailing its fitness for the 

intended use is included on the Laboratory Method Validation and 

Verification Process Form as part of the final approval signature 

process for the Form

F. Management and the Quality System Manager review and approve 

validated methods for routine use. The validation package is retained 

by the Quality System Manager in QMiS.
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Glossary/Definitions

A. Accuracy – Accuracy is a measure of how closely the average value of 

the measurements, or the mean is to the true value. 

B. Analytical batch – An analytical batch consists of samples which are 

analyzed together with the same method sequence and the same lots of 

reagents and with the manipulations common to each sample within the 

same time period or in continuous sequential time periods.

C. Detection limit – A detection limit is the lowest amount of analyte in a 

sample which can be detected but not necessarily quantitated as an 

exact value. It is often called the limit of detection (LOD) which is the 

lowest concentration level that can be determined statistically different 

from a blank at a specified level of confidence. It is determined from the 

analysis of sample blanks. Method detection limit (MDL) is the minimum 

concentration of a substance than can be measured and reported with 

99% confidence that the analyte concentration is greater than zero. It is 

determined from analysis of a sample in a given matrix containing the 

analyte. 

D. Limit of quantitation (LOQ) – This is the level above which quantitative 

results may be determined with acceptable accuracy and precision. 

Limit of quantitation (or quantification) is variously defined but must be a 

value greater than the Method Detection Limit (MDL) and should apply 

to the complete analytical method. 

E. Linearity – Linearity is the ability of the method to elicit results that are 

directly proportional to analyte concentration within a given range.

F. Method Detection Limit (MDL) - The minimum amount or concentration 

of analyte in the test sample that can be reliably distinguished from 

zero. MDL is dependent on sensitivity, instrumental noise, blank 

variability, sample matrix variability, and dilution factor.

G. Non-standard method – This refers to a method that is not taken from 

authoritative and validated sources. This includes methods from 

scientific journals and unpublished laboratory-developed methods.

H. FDA "official" methods - This refers to methods found in compendia 

specified in the FD&C Act and prescribed in the CFR and methods in 

applications and petitions that have official status are included. These 

methods include those in the United States Pharmacopeia, National 

Formulary, Homeopathic Pharmacopeia of the United States, Official 

Methods of Analysis of the Association of Official Analytical Chemists 

(AOAC) International or any supplement of any of them, American 
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Public Health Association (APHA) Compendium of Methods for the 

Microbiological Examination of Foods, FDA compliance programs, the 

Pesticide Analytical Manual (PAM), the Food Additives Analytical 

Manual, the Food Chemicals Codex, FDA Bacteriological Analytical 

Manual (BAM), FDA Macroanalytical Procedures Manual (MPM), and 

ORA. Laboratory Information Bulletins (LIBs) that are included in 

compliance programs and special assignments. Standard methods are 

preferred for use and are verified for use in the laboratory. A standard 

method may be supplemented with additional details in the form of a 

laboratory procedure to ensure consistent application. Those methods 

specified by the manufacturer of the equipment are considered as 

standard methods. Standard methods are verified according to the 

procedures described above. 

I. Precision – Precision is the agreement between a set of replicate 

measurements without assumption of knowledge of the true value. 

Repeatability expresses the precision under the same operating 

conditions over a short period of time. The precision is described by 

statistical methods such as a standard deviation or confidence limit of 

test results. Intermediate precision expresses within-laboratory 

variations, such as different days, different analysts, and different 

equipment can be expressed with RSD. Reproducibility expresses the 

precision between laboratories and can be expressed with RPD.

J. Range – A range is the interval between the upper and lower 

concentration of analyte in sample for which it has been demonstrated 

that the analytical procedure has an acceptable level of accuracy, 

precision, and linearity.

K. Ruggedness or robustness – Ruggedness is a measure of an analytical 

procedure’s capacity to remain unaffected by small, but deliberate 

variations in method parameters and provides an indication of its 

reliability during normal usage.

L. Selectivity – The extent to which a method can determine specific 

analyte(s) in a mixture(s) or matrix(ces) without interferences from other 

components of similar behavior. Selectivity is generally preferred in 

analytical chemistry over the term Specificity.

M. Sensitivity – The change in instrument response which corresponds to a 

change in the measured quantity (e.g., analyte concentration). 

Sensitivity is commonly defined as the gradient of the response curve or 

slope of the calibration curve at a level near the LOQ.
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N. Specificity – In quantitative analysis, specificity is the ability of a method 

to measure analyte in the presence of components which may be 

expected to be present. The term Selectivity is generally preferred over 

Specificity.

O. Standard method – Standard methods are those published by 

international, regional, or national standards-writing bodies; by 

reputable technical organizations; in legal references; and FDA 

published methods. FDA “official” methods are considered as standard 

methods. 

P. Validation (method) – A method validation is the process of establishing 

the performance characteristics and limitations of a method and the 

identification of the influences which may change these characteristics 

and to what extent.

Q. Verification – A verification is the confirmation by examination and 

provision of objective evidence that specified requirements have been 

fulfilled.

Records

A. QMiS Laboratory Method Validation and Verification Process Form in 

QMiS

B. Validation and verification documentation and reports

Supporting Documents

A. SOP-001064 Microbiological Controls for Food and Feed Sample 

Analysis

B. SOP-000288 Microbiological Controls for Medical Product Sample 

Analysis

C. SOP-000107 Method Verification

D. WI-000487 Instructions for Using the Method Validation and Verification 

Process Form in QMiS

E. MAN-000050 ORA Lab Manual Vol. III Section 6 - ORS Method 

Development and Validation Program (III-06)
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Document History

Revision

#

Status*

(D, I, R) Date Author Name and Title Approving Official Name and Title

1.1 I 10/03/2003 LMEB LMEB

1.2 R 09/09/2005 LMEB LMEB

1.3 R 11/15/2007 LMEB LMEB

1.4 R 02/25/2009 LMEB LMEB

1.5 R 04/01/2009 LMEB LMEB

1.6 R 02/03/2012 LMEB LMEB

1.7 R 09/08/2014 LMEB LMEB

02 R 06/30/2020 LMEB LMEB

03 R SEE QMIS LMEB LMEB

* - D: Draft, I: Initial, R: Revision
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Change History

Revision 

# Change

02

• Revisions made as needed to align this procedure with new ISO/IEC 17025 and 

AOAC requirements. 

• Revision to formatting and other changes were made in all the document sections 

to provide policy clarification and enhance areas like documentation 

requirements, method modification requirements, etc. 

• Addition of requirement to submit validation requests in CARTS

• Significant revision was also made to the documents in the Attachments section.

03

• Transferred to new ORA TEMPLATE-000054.

• Added reference to MAN-000043 for continuing verification of acceptable performance for 

each analytical batch (EAB) through a quality control program to scope to point out this 

document does not address continuing verification (QC).

• Added requirement for validation of non-standard methods prior to use (Section 6.1).

• Added instruction for use of Laboratory Method Validation and Verification Process Form 

throughout document.

• Added requirement to initiation of form concurrently with submitting research projects 

through CARTS.

• Added reference to SOP-000107 Method Verification

• Added requirement for rigid validation protocols for methods validated to address 

emergency/limited use if method will be continually used for widespread non-emergency 

regulatory 

• Addition to Appendix B (Highlighted)

Attachments

List of Attachments

Attachment A - Food and Feed Chemistry Modification Criteria ................................. 20

Attachment B - Drug Chemistry Modification Criteria..................................................24

Attachment C - ORA Validation (non-compendial) and Verification Guidance for 

Human Drug Analytical Methods .........................................................28
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Attachment A - Food and Feed Chemistry Modification Criteria

If adjustments to method conditions are needed, the following documents may be 

consulted for specifications to sample preparation or operation parameter 

conditions: USP <621> (Chromatography), SANCO/12495/2011, or an FDA LIB 

that qualifies as a standard method for the set of target analytes in a given matrix.

A. Sample Inlet

1. LC

a. Mobile phase pH: the pH of the aqueous buffer used in the 

preparation of the mobile phase can be adjusted to within ± 0.2 pH 

units of the value or range specified. Applies to both gradient and 

isocratic separations.

b. Concentration of salts in buffer: The concentration of the salts used 

in the preparation of the aqueous buffer employed in the mobile phase 

can be adjusted to within ± 10%, provided the permitted pH variation is 

met. Applies to both gradient and isocratic separations.

c. Mobile Phase Composition: The following adjustment limits apply to 

minor components of the mobile phase (specified at ≤ 50%). The 

amount(s) of these components can be adjusted by ± 30% relative. 

However, the change in any component cannot exceed ± 10% 

absolute, nor can the final concentration of any component be reduced 

to zero. Examples of adjustments for binary and ternary mixtures are 

given below:

i. Binary Mixtures: Specified Ratio of 50:50 – Thirty percent of 

50 is 15% absolute, but this exceeds the maximum permitted 

change of ± 10% absolute in either component. Therefore, the 

mobile phase ratio may be adjusted only within the range 40:60 

to 60:40.

Specified Ratio of 95:5 – Thirty percent of 5 is 1.5% absolute. 

However, because adjustments of up to ± 2% are allowed, the 

ratio may be adjusted within the range of 93:7 to 97:3.

Specified Ratio of 2:98 – Thirty percent of 2 is 0.6% absolute. 

In this case an absolute adjustment of -2% is not allowed 

because it would reduce the amount of the first component to 

zero. Therefore, the maximum allowed adjustment is within the 

range of 1.4:98.6 to 2.6 to 97.4.

ii. Ternary Mixtures: Specified Ratio of 60:35:5 – For the second 

component, thirty percent of 35 is 10.5% absolute, which 

exceeds the maximum permitted change of ± 10% absolute in 

any component. Therefore, the second component may be 

adjusted only within the range of 25 to 45% absolute. For the 
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third component, thirty percent of 5 is 1.5% absolute. Since ± 

2% absolute is permitted and provides more flexibility, the third 

component may be adjusted within the range of 3 to 7% 

absolute. In all cases, enough of the first component is used to 

give a total of 100%.

d. Flow rate: May be adjusted by as much as ± 50%. Consult the column 

configuration table in USP <621> (Table 2) when particle size is 

changed, because smaller particle columns will require higher linear 

velocities for the same performance (as measured by reduced plate 

height).

e. Injection volume: The injection volume can be adjusted as far as it is 

consistent with accepted precision, linearity, and detection limits. Note 

that excessive injection volume can lead to unacceptable band 

broadening, causing a reduction in the number of theoretical plates (N, 

which is a measure of column efficiency) and resolution, which applies 

to both gradient and isocratic separations.

2. GC

a. Carrier gas. Hydrogen for FID use, Helium for MS use. Hydrogen can 

be used with MS detection if sensitivity does not vary more than 10% in 

the working range for every target analyte in the sample.

b. Flow rate: The flow rate can be adjusted by as much as ± 50%, 

provided that the carrier gas system can be maintained under control 

at the desired set points.

c. Injection volume: The injection volume and split volume (which is 

controlled by the split ratio) can be varied as much as possible, if the 

detection and repeatability are satisfactory for the entire range 

concentrations of the calibration curve.

B. Sample Analysis

1. LC

a. Stationary phase type: The column stationary phase type can be 

changed according to the USP L-rating number (consult the USP LC 

column equivalence chart).

b. Column dimensions: For isocratic separations, the particle size 

and/or length of the column may be modified provided that the ratio of 

the column length (L) to the particle size (dp) remains constant or into 

the range between -25% and 50% of the prescribed L/dp ratio. 

Alternatively (as for the application of particle-size adjustment to 

superficially porous particles), other combinations of L and dp can be 

used provided that N is within -25% to 50%, relative to the prescribed 

column. For gradient elution separations, changes in length, column 
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inner diameter, and particle size are not recommended. If UHPLC is 

used, then the columns dimensions should be chosen such that the 

resolution of all target analytes is conserved.

c. Column temperature: The column temperature can be adjusted by as 

much as ± 10°C. Column thermostating is recommended to improve 

control and reproducibility of retention time, which applies to both 

gradient and isocratic separations.

2. GC

a. Stationary phase type: The column stationary phase type can be 

changed according to the USP G-rating number (consult the USP GC 

column equivalence chart). Stationary film thickness can be adjusted 

by as much as -50% to 100%. A capillary column can be used in 

substitution of a packed column if performance (resolution of target 

analytes and analysis time) is demonstrated to be equivalent or 

superior.

b. Column dimensions: Column length can be adjusted by as much as ± 

70%. Column inner diameter can be adjusted by as much as ± 50%.

c. Temperature program: Oven temperature can be to any rate that the 

instrument can reproducibly deliver, and up to a temperature that will 

not produce excessive column bleed (typically 20°C below the 

stationary phase’s prescribed maximum allowable operating 

temperature). The retention index of target analytes should not vary by 

more than 10 index units in the modified method, and peak shapes 

(peak asymmetry) should be consistent (or better) than in the method 

that is being modified.

C. Sample Detection

1. LC

a. Non-MS Detectors: Reference samples should provide adequate S/N 

ratios (>10) over the working range.

b. MS Detectors

Table 1 MS 

MS detector/Characteristics 

Acquisition 

Requirements for Identification

Resolution Typical 

Systems 

(Examples)

Minimum 

number of 

Ions

Other

Unit Mass 

Resolution

Single MS

Quadrupole 

Ion trap 

Full scan,

Limited m/z range 

Selected ion 

monitoring (SIM) 

3 ions 

S/N ≥ 3

Analyte peaks from 

both product ions in the 

extracted ion 
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Table 1 MS 

MS detector/Characteristics 

Acquisition 

Requirements for Identification

Resolution Typical 

Systems 

(Examples)

Minimum 

number of 

Ions

Other

Time of Flight 

(TOF)

chromatogram must 

overlap 

Ion ratio from sample 

extracts should be 

within ± 30% of 

average of calibration 

standards from same 

sequence. 

Please consult 

CVM118 reference for 

additional information. 

MS/MS

Triple Quad 

Ion trap 

Q-trap

Q-TOF

Q-Orbitrap 

Selected or 

multiple reaction 

monitoring (SRM, 

MRM)

Mass resolution 

for precursor-ion 

Isolation equal to 

or better than unit 

mass resolution 

2 product 

ions 

Accurate 

Mass 

Measurement

High 

Resolution MS 

Q-TOF

Q-Orbitrap 

FT-ICR-MS

Magnetic 

Sector MS 

Full scan

Limited m/z range 

SIM 

Fragmentation 

with or without 

precursor-ion 

selection 

2 ions with 

mass 

accuracy ≤

5 ppm 

S/N ≥ 3

Analyte peaks from 

precursor and/or 

product ion(s) in the 

extracted ion 

chromatograms must 

fully overlap 

Please consult CVM 

118 for additional 

information. 

2. GC

a. Non-MS Detectors: Reference samples should provide adequate S/N 

ratios (>10) over the working range.

b. MS-MS and HRMS: refer to Table 1 MS.

 
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Attachment B - Drug Chemistry Modification Criteria

If adjustments to operating conditions are needed, each of the following is the maximum 

specification (USP General Chapter <621> Chromatography) that can be considered. All 

adjustments falling outside the maximum specifications will be considered as method 

modifications and will be subject to the method modification protocol. If a firm method is 

required to be used, no modification should be made to the method unless it is cleared 

with the customer or requester through immediate Office (IO) before testing. In general, if 

there is need to change certain parameters in the method, it is recommended that the 

analyst discuss the changes with supervisor to ensure the acceptability and compliance.

pH of Mobile Phase (HPLC): The pH of the aqueous buffer used in the preparation of the 

mobile phase can be adjusted to within ±0.2 pH units of the value or range specified. 

Applies to both gradient and isocratic separations.

Concentration of Salts in Buffer (HPLC): The concentration of the salts used in the 

preparation of the aqueous buffer used in the mobile phase can be adjusted to within 

±10%, provided the permitted pH variation is met. Applies to both gradient and isocratic 

separations.

Ratio of Components in Mobile Phase (HPLC): The following adjustment limits apply to 

minor components of the mobile phase (specified at ≤50%). The amount(s) of these 

component(s) can be adjusted by ±30% relative. However, the change in any component 

cannot exceed ±10% absolute (i.e., in relation to the total binary phase), Adjustment can 

be made to one minor component in a ternary mixture. Examples of adjustments for binary 

and ternary mixtures are given below.

Binary Mixtures:

• Specified Ratio of 50:50 – Thirty percent of 50 is 15% absolute, but this exceeds the 

maximum permitted change of ±10% absolute in either component. Therefore, the 

mobile phase ratio may be adjusted only within the range of 40:60 to 60:40. 

• Specified Ratio of 2:98 – Thirty percent of 2 is 0.6% absolute. Therefore, the 

maximum allowed adjustment is within the range of 1.4:98.6 to 2.6:97.4.

Ternary Mixtures: 

• Specified Ratio of 60:35:5 – For the second component, thirty percent of 35 is 

10.5% absolute, which exceeds the maximum permitted change of ±10% absolute 

in any component. Therefore, the second component may be adjusted only within 

the range of 25 to 45% absolute. 

• For the third component, thirty percent of 5 is 1.5% absolute. In all cases, quantity 

of the first component is used to give a total of 100%. 
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• Therefore, mixture ranges of 50:45:5–70:25:5 or 58.5:35:6.5-61.5:35:3.5 would 

meet the requirement. 

Wavelength of UV-Visible Detector (HPLC): Deviations from the wavelengths specified 

in the method are not permitted. The procedure specified by the detector manufacturer, or 

another validated procedure, is to be used to verify that error in the detector wavelength is, 

at most, ±3 nm.

Column Length (GC): May be adjusted by as much as ±70%.

Column Length (HPLC): See Particle Size (HPLC) below

Column Inner Diameter (HPLC): Can be adjusted if the linear velocity is kept constant. 

See Flow Rate (HPLC) below.

Column Inner Diameter (GC): Can be adjusted by as much as ±50%.

Film Thickness (Capillary GC): May be adjusted by as much as –50 to +100%.

Particle size (HPLC): For isocratic separations, the particle size and/or the length of the 

column may be modified provided that the ratio of the column length (L) to the particle size 

(dp) remains constant or into the range between −25% and 50% of the prescribed L/dp 

ratio. 

Alternatively (as for the application of particle-size adjustment to superficially porous 

particles), other combinations of L and dp can be used provided that the number of 

theoretical plates (N) is within −25% to 50%, relative to the prescribed column. 

Caution should be used when the adjustment results in a higher number of theoretical 

plates that generate smaller peak volumes, which may require adjustments to minimize 

extra-column band broadening by factors such as instrument plumbing, detector cell 

volume and sampling rate, and injection volume. 

For gradient separations, changes in length, column inner diameter, and particle size are 

not allowed.

Particle size (GC): Changing from a larger to a smaller or from a smaller to a larger 

particle size GC mesh support is acceptable if the chromatography meets the 

requirements of system suitability and the same particle size range ratio is maintained. 

The particle size range ratio is defined as the diameter of the largest particle divided by the 

diameter of the smallest particle

Flow Rate (GC): The flow rate can be adjusted by as much as ±50%. [Note—When the 

monograph specifies a linear velocity parameter, the allowed velocity adjustment is 

between +50% and −25%, provided the carrier gas system can be maintained under 

control at the desired set points.]

Flow Rate (HPLC): When the particle size is changed, the flow rate may require 

adjustment, because smaller-particle columns will require higher linear velocities for the 
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same performance (as measured by reduced plate height). Flow rate changes for both a 

change in column diameter and particle size can be made by: 

F2 = F1 × (dc22 × dp1)

(dc12 × dp2)

where F1 and F2 are the flow rates for the original and modified conditions, 

respectively, dc12 and dc22 are the respective column diameters, and dp1 and 

dp2 are the particle sizes.

When a change is made from ≥3-μm to <3-μm particles in isocratic separations, an 

additional increase in linear velocity (by adjusting flow rate) may be justified, provided that 

the column efficiency does not drop by >20%. Similarly, a change from <3-μm to ≥3-μm 

particles may require additional reduction of linear velocity (flow rate) to avoid reduction in 

column efficiency by >20%. Changes in F, dc, and dp are not allowed for gradient 

separations. 

Additionally, the flow rate can be adjusted by ±50% (isocratic only).

Examples:

Adjustments in column length, internal diameter, particle size, and flow rate can be used in 

combination to give equivalent conditions (same N), but with differences in pressure and 

run time. Table 2 lists some of the more popular column configurations to give equivalent 

efficiency (N), by adjusting these variables. 

Table 2. Column Configurations

Length (L, 

mm)

Column Diameter (dc, 

mm)

Particle 

Size (dp, 

μm)

Relative Values

L/dp F N Pressure Run 

Time

250 4.6 10 25,000 0.5 0.8 0.2 3.3

150 4.6 5 30,000 1.0 1.0 1.0 1.0

150 2.1 5 30,000 0.2 1.0 1.0 1.0

100 4.6 3.5 28,600 1.4 1.0 1.9 0.5

100 2.1 3.5 28,600 0.3 1.0 1.9 0.5

75 4.6 2.5 30,000 2.0 1.0 4.0 0.3

75 2.1 2.5 30,000 0.4 1.0 4.0 0.3

50 4.6 1.7 29,400 2.9 1.0 8.5 0.1

50 2.1 1.7 29,400 0.6 1.0 8.5 0.1

For example, if a monograph specifies a 150-mm × 4.6-mm; 5-μm column operated at 1.5 

mL/min, the same separation may be expected with a 75-mm × 2.1-mm; 2.5-μm column 
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operated at 1.5 mL/min × 0.4 = 0.6 mL/min, along with a pressure increase of about four 

times and a reduction in run time to about 30% of the original.

Injection Volume (HPLC): The injection volume can be adjusted as far as it is consistent 

with accepted precision, linearity, and detection limits. Note that excessive injection 

volume can lead to unacceptable band broadening, causing a reduction in N and 

resolution, which applies to both gradient and isocratic separations.

Injection volume and split volume (GC):

The injection volume and split volume may be adjusted if detection and repeatability are 

satisfactory.

Column Temperature (HPLC): May be adjusted by as much as ±10°. Applies to both 

gradient and isocratic separations.

Oven Temperature (GC): May be adjusted by as much as ±10%.

Oven Temperature Program (GC): Adjustment of temperatures is permitted as stated 

above. For the times specified for the temperature to be held or for the temperature to be 

changed from one to another, an adjustment of up to ±20% is permitted.
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Attachment C - ORA Validation (non-compendial) and Verification Guidance for 

Human Drug Analytical Methods

A. Purpose: Products that are non-compendial, Over the counter (OTC), or 

pharmacy-compounded do not require a New Drug Application (NDA) or 

Abbreviated New Drug Application (ANDA) to be marketed. Nevertheless, 

ORS labs can be called upon to test these products. Instances also exist 

where “Standard Methods” (Compendial or NDA/ANDA methods) are not 

applicable to a certain product, require equipment not available in the 

laboratory, are outdated or not readily available, or are not the most efficient 

use of a laboratory’s resources. This attachment provides uniform guidance to 

ORS laboratories on minimum requirements for validation and verification of 

drug analytical methods developed for this purpose.

Validation, in simplest terms, is defined as the demonstration that an analytical 

method is suitable for its intended purpose. It is important to recognize that, 

especially in drug analysis, different types of methods exist for different 

purposes. These methods can be grouped into categories, each category 

requiring a different set of validation parameters. Categories of methods are 

discussed below.

The ORA Laboratory Manual directs that validation is required when a new 

method is developed, when an existing validated method is significantly 

modified, or when an existing validated method is applied to a sample matrix 

significantly different from that for which the method was developed.

Verification (sometimes also referred to as “method transfer”) is defined as an 

assurance that a laboratory other than the originator of a Standard Method or 

other previously validated method can obtain comparable results, using the 

same or equivalent equipment, as the originator of the method; in other words, 

that the method is suitable under actual conditions of use in a particular 

laboratory. 

In general, presented here are the minimum requirements for validation and 

verification of drug methods within ORS. This is primarily designed to address 

methods for single-occurrence or internal use: for a single sample or a small 

group of similar samples. Validation of methods intended for use by multiple 

labs, for publication in a scientific journal, or for establishment as a future 

“Standard Method” require additional validation; this is addressed in Notes (c) 

and (d), below. In any case, labs may in certain circumstances justifiably find 

the need to perform additional validation steps. However, the value of 

additional information gained by such work must be weighed against the 

resources expended in the process. 
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Also presented are acceptance criteria for each validation parameter. These 

must be considered carefully. These acceptance criteria apply to 

“conventional” dosage forms (tablets, capsules, solutions, aqueous injections, 

etc.) where matrix interference is usually minimal. For more complex matrices 

(creams, suppositories, suspensions, etc.), meeting these criteria may be 

impossible. Other considerations, such as reduction of spiking levels due to 

limited standard availability, may also cause difficulties in meeting the criteria. 

Such situations must be evaluated, approved, and documented on a case-bycase basis (see note a. below). 

B. References: 

1. International Conference on Harmonisation (ICH) Harmonised Tripartite 

Guideline: Validation of Analytical Procedures: Text and Methodology 

Q2(R1)

2. United States Pharmacopeia (USP) section <1225>: Validation of 

Compendial Procedures

3. United States Pharmacopeia (USP) section <1226>: Verification of 

Compendial Procedures.

C. Validation Parameters: The following validation parameters (referred to as 

“Analytical Performance Characteristics” in USP and ICH documents), are to 

be applied based on the category of method being validated, according to the 

chart below. Definitions are as commonly accepted by the scientific 

community and expressed in the USP and ICH documents referenced above.

1. Accuracy: Should be assessed by using a minimum 9 determinations over 

a minimum of three concentration levels, covering the specified range (i.e., 

three concentrations and three replicates of each concentration). Prepare 3 

sample preparations of composited sample, containing a known quantity of 

added analyte (“matrix spike”), so that the expected concentrations are as 

follows:

a. Assay: range at least 80%-120% of expected content

b. Content Uniformity: range at least 70%-130% of expected content 

(Note: if Assay and Content Uniformity methods are the same, 

accuracy determination ranging 70%-130% of expected content will 

satisfy requirements for both methods.) 

Based on the nature of the dosage form (e.g., metered dosed 

inhalers) a wider range maybe used with appropriate justification.
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c. Dissolution/release rate determinative step: range at least 20% less 

than lower dissolution limit to 20% greater than higher dissolution 

limit

Acceptance Criteria: 97.0% - 103.0% recovery for each spike level 

for APIs; 95.0% - 105.0% for finished dosage forms. (See note a. 

below)

2. Precision (repeatability): Repeatability should be assessed using a 

minimum of nine determinations covering the specified range for the 

procedure (i.e., three concentrations and three replicates of each 

concentration) or using a minimum of six determinations at 100% of the test 

concentration (e.g., prepare 6 sample solutions at 100% of the test 

concentration and inject each), unless otherwise specified.

Acceptance Criteria: Drug products RSD less than or equal to 3.0%, unless 

otherwise specified; Drug active pharmaceutical ingredients - RSD less 

than or equal to 2.0%,

3. Linearity: Prepare and analyze a set with a minimum of 5 concentrations 

of analyte standard, across a minimum range as recommended by ICH 

based on the type of analysis described below: defined for Content 

Uniformity solutions under “accuracy,” above.

a. Assay of a drug substance (or a finished product): From 80% to 

120% of the test concentration

b. For content uniformity: A minimum of 70%–130% of the test 

concentration, unless a wider or more appropriate range based 

on the nature of the dosage form (e.g., metered-dose inhalers) is 

justified

c. For dissolution testing: ±20% over the specified range (e.g., if the 

acceptance criteria for a controlled-release product cover a 

region from 30% after 1 h, and up to 90% after 24 h, the 

validated range would be 10%–110% of the label claim).

d. Determination of an impurity: From 50% to 120% of the 

acceptance criterion

Perform the determination and generate a standard curve. The 

correlation coefficient, y-intercept, slope of the regression line, and 

residual sum of squares should be submitted. Acceptance Criteria: 

Linear Regression Coefficient of Determination r (2) greater than or 

equal to 0.995 (see note a. and e. below)
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4. Specificity: Assessment of specificity depends on the technique being 

used. Certain techniques (i.e., titrations) are non-specific by nature; a 

combination of two or more analytical procedures is necessary to achieve 

the required level of discrimination. Techniques such as HPLC-UV or UV 

spectrophotometry are somewhat more specific in nature: visual 

comparison of standard and sample spectra or chromatograms should be 

performed; no interferences should be apparent. Peak-purity technology 

should be used, when possible, to assist in this evaluation. Techniques 

such as IR spectrophotometry or mass spectrometry are highly specific: 

sample and standard maxima or bands should occur at the same 

wavelengths or masses.

When excipients, known impurity or degradant standards, are available, 

specificity can be additionally assessed by addition of these compounds to 

the primary analyte, to assure that interferences do not occur. 

5. Detection Limit (DL)/Limit of Detection (LOD): For chromatographic or 

spectrophotometric methods, determine the minimum level at which a 

compound can be detected, using analyte solutions of decreasing 

concentration. LOD generally defined as 3 times the noise level (signal to 

noise ratio (S/N) ≥ 3). Some applications may depend on standard 

deviation or slope of the calibration curve. Other scientifically-sound 

approaches may also be used.

For other types of methods, estimate through visual evaluation the 

minimum level at which a compound can be detected, using analyte 

solutions of decreasing concentration.

6. Quantitation Limit (QL)/Limit of Quantitation (LOQ): For 

chromatographic or spectrophotometric methods, determine the minimum 

level at which a compound can be quantitated, using analyte solutions of 

decreasing concentration. LOQ is generally defined as 10 times the noise 

level (S/N ≥ 10). The standard deviation should also be considered along 

with the S/N ratio. Other scientifically sound approaches may also be 

used.

For other types of methods, estimate through visual evaluation the 

minimum level at which a compound can be quantitated, using analyte 

solutions of decreasing concentration.

Notes: 

a. If acceptance criteria are not met, due to situations described in this 

paragraph, the occurrence should be evaluated in the form of a discussion 

between analyst(s), lab managers, and QA managers, with the purpose of 
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the analysis and the requirements of the customer considered. The 

specified acceptance criteria can then be modified, if sufficiently justified.

b. For most purposes, the quantitative or qualitative range of a method will be 

appropriately established through determination of linearity, accuracy, and 

LOD/LOQ, as described above.

c. The validation parameter “Robustness” is frequently discussed. When the 

method being validated is for single occurrence use or internal use, this 

determination may not be necessary. For a more complete validation, as 

in cases where the method is intended for publication or establishment as a 

future “Standard Method”, robustness should be assessed through 

variations in the analytical procedure (e.g., change in flow rate, use of 

differing equipment, different column lots, etc.) Consult with the lab 

manager for specifics on test requirements.

d. ICH and other guidelines recommend, for Accuracy determination, an 

assessment using a minimum of 3 replicates at each of 3 concentrations, 

thereby equating to a minimum of 9 determinations. This should be done 

when the method is intended for publication or establishment as a future 

“Standard Method.” For routine regulatory analytical purposes, 

ISO17025:2017 requirements will be considered met if each of the three 

single preparations evaluated under “Accuracy” meet the Acceptance 

Criteria. If one or more preparations fails to meet these criteria, the 

laboratory should conduct a failure investigation, to include an examination 

of possible causes for this failure.

e. For certain types of methods, e.g., Atomic Emission Spectroscopy, a nonlinear standard curve may be expected, and can be used. Linear 

Regression analysis would not apply to such situations. 

D. Categories of Methods; Validation Parameter Requirements

1. Category I: Quantitative Assessment of Major Components: (i.e. 

Assay, Content Uniformity, determinative step for Dissolution/Release 

Rate). Required parameters: Accuracy, Precision, Linearity, Specificity

2. Category IIa: Quantitative Assessment of Minor Components: (i.e. 

Impurity and Degradant quantitative determinations). Required 

parameters: Accuracy, Precision, Linearity, Specificity, Limit of 

Quantitation

3. Category IIb: Qualitative Assessment of Minor Components: (i.e. 

Impurity and Degradant Limit Tests). Required parameters: Specificity, 

Limit of Detection
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4. Category III: Performance Tests Components: (e.g. dissolution, drug 

release, particle size). Required parameter: Precision, all other 

parameters are considered based on nature of the specified test)

5. Category IV: Identification tests: Required parameter: Specificity

E. Verification of Methods: As is mentioned above, a laboratory must verify 

that any validated method (including USP or other “Standard Methods”) can be 

performed acceptably under actual conditions of use. Method Verification 

should be performed upon the first use of a method by a particular analyst on a 

particular instrument to document that the method performance criteria can 

be met. After this, instrument performance criteria (for example, system 

suitability parameters, criteria specified in the method, etc.) should be met as 

directed by the method or per batch of similar samples.

Verification should include, at a minimum:

1. Full system suitability testing, as defined in the compendial method, with 

acceptance criteria as defined in the compendium. If this is not applicable, 

system precision for chromatographic procedures should be assessed 

using five (RSD requirement ≤2.0%) or six (RSD requirement >2.0%) 

replicate injections or as specified in the method; specificity should be 

assessed using either a chromatographic resolution factor (>1.3) or a visual 

examination of chromatograms or spectra for freedom from interference.

2. Accuracy determination through analysis of a matrix spike (acceptance 

criteria:

97.0%103.0% recovery for APIs, 95.0%-105.0% for finished dosage forms; 

see note a. above).

F. Ad Hoc Sample Analyses (example Consumer Complaint Samples)

For ad hoc samples, such as consumer complaint samples, the analysis is 

intended for a particular sample without consideration for wider application. In 

addition, the amount of sample is usually limited, and the analytical results 

need to be expedited. In such cases, it may not be practical to perform full 

method validation or verification. However, efforts should be made to 

demonstrate accuracy, precision, and linearity. Precision can be assessed 

from the RPD (relative percent difference) of duplicate sample analyses. 

Accuracy may be assessed from a single or duplicate spike recovery. Method 

assessment results are documented in a memo of analysis that is included 

with the analytical report.
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Document Number:

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Revised:

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Title: 

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For the most current and official copy, check QMiS.