Step-by-Step Analytical

Methods Validation and

Protocol in the Quality System
Compliance Industry
BY GHULAM A. SHABIR

Introduction peatability and intermediate precision), specificity, detec-
Methods Validation: Establishing documented evidence tion limit, quantitation limit, linearity, range, and robustness
that provides a high degree of assurance that a specific (Figure 1). In addition, methods validation information
method, and the ancillary instruments included in the should also include stability of analytical solutions and sys-
7
method, will consistently yield results that accurately reflect tem suitability.
the quality characteristics of the product tested.
Health Canada (HC) has also issued guidance on meth-
8
Method validation is an important requirement for any ods validation entitled Acceptable Methods Guidance. HC
package of information submitted to international regula- has been an observer of ICH, and has adopted ICH guide-
tory agencies in support of new product marketing or clini- lines subsequent to its reaching Step Four of the ICH
cal trials applications. Analytical methods should be vali- process. An acceptable method predates ICH, and HC
dated, including methods published in the relevant pharma- plans to revise this guidance to reflect current ICH termi-
copoeia or other recognized standard references. The suit- nology.
ability of all test methods used should always be verified Figure 2 shows the data required for different types of
under the actual conditions of use and should be well docu- analysis for method validation. Where areas of the Accept-
mented. able Methods Guidance are superseded by ICH Guidelines
1 2
Methods should be validated to include consideration of Q2A and Q2B, HC accepts the requirements of either the
characteristics included in the International Conference on ICH or Acceptable Methods Guidance; however, for
1, 2
Harmonization (ICH) guidlines addressing the validation method validation, ICH acceptance criteria are preferred.
of analytical methods. Analytical methods outside the scope HCs Acceptable Methods Guidance provides useful guid-
of the ICH guidance should always be validated. ance on methods not covered by the ICH guidelines (e.g.,
ICH is concerned with harmonization of technical re- dissolution, biological methods), and provides acceptance
quirements for the registration of products among the three criteria for validation parameters and system suitability tests
major geographical markets of the European Community for all methods.
(EC), Japan, and the United States (U.S.) of America. The HC has also issued templates recommended as an ap-
recent U.S. Food and Drug Administration (FDA) methods proach for summarizing analytical methods and validation
3-5
validation guidance document, as well as the United States data ICH terminology was used when developing these tem-
6
Pharmacopoeia (USP), both refer to ICH guidelines. plates.
The most widely applied typical validation characteris- This paper suggests one technique of validating meth-
tics for various types of tests are accuracy, precision (re- ods. There are numerous other ways to validate methods, all

4 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
 Ghulam A. Shabir

Figure 1
___________________________________________________________________________
ICH, USP, and FDA Methods Validation Characteristics Requirements for Various Types of Tests

Validation Assay Testing for Impurities Identification

Characteristics Quantitative Limit
Accuracy Yes Yes No No
Precision - Repeatability Yes Yes No No
Precision - Intermediate Yes1 Yes* No No
Precision
Specificity Yes Yes Yes Yes
Detection limit No No Yes No
Quantitation limit No Yes No No
Linearity Yes Yes No No
Range Yes Yes No No
Robustness Yes Yes No No
7
* In cases where reproducibility has been performed, intermediate precision is not needed.

Figure 2
____________________________________________________________________
Health Canada Methods Validation Parameter Requirements for Various Types of Tests

Validation Identity Active Ingredients Impurities / Degradation

Physico-Chemical
Parameters Tests Drug Drug Products
Tests
Substance Product Quantitative Limit Tests
Precision
(of the system) No Yes Yes Yes 1 Yes
Precision
(of the method) No 1 Yes Yes 1 Yes
Linearity No Yes Yes Yes No Yes
Accuracy No Yes Yes Yes 1 Yes
Range No 1 Yes Yes No Yes
Specificity Yes 1 Yes Yes Yes *
Detection Limit 1 No No Yes Yes *
Quantitation Limit No No No Yes No *
Ruggedness 1 Yes Yes Yes Yes Yes
* May be required depending upon the nature of the test.

equally acceptable when scientifically justified. required accuracy, and required sensitivity. (Note: Most of
Prepare a Protocol the acceptance criteria come from the characterization
study.) Furthermore, some tests may be omitted, and the
The first step in method validation is to prepare a proto- number of replicates may be reduced or increased based on
col, preferably written, with the instructions in a clear step- scientifically sound judgment.
by-step format, and approved prior to their initiation. This A test method is considered validated when it meets the
approach is discussed in this paper. The suggested accep- acceptance criteria of a validation protocol. This paper is a
tance criteria may be modified depending on method used, step-by-step practical guide for preparing protocols and per-

A n a l y t i c a l M e t h o d s Va l i d a t i o n 5
Ghulam A. Shabir

forming test methods validation with reference to High Methods validation must have a written and approved
Performance Liquid Chromatography (HPLC) (use simi- protocol prior to its initiation. A project controller will se-
lar criteria for all other instrumental test method valida- lect a validation Cross-Functional Team (CFT) from var-
tion) in the quality system compliance industry. ious related departments and functional areas. The project
controller assigns responsibilities. The following tables il-
Analytical Methods Validation Protocol lustrate one suggested way of documenting and preserv-
Approval Cover Page ing a record of the approvals granted at the various phases

Summary Information

Summary Information
Organization name
Site location
Department performing validation
Protocol title
Validation number
Equipment
Revision number

Project Controller

Project Name Signature Date

Controller

Document Approval

Document Approval
Department /
Functional Area Name Signature Date
Technical Reviewer
End Lab Management
Health & Safety
Quality Assurance
Documentation Control
(reviewed and archived by)

Revision History

Revision History
Revision No. Date Description of change Author

6 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
 Ghulam A. Shabir

of the validation: 1.1.1. Test procedure

The specificity of the assay method will be investigated
Writing a Test Method Validation Protocol by injecting of the extracted placebo to demonstrate the
absence of interference with the elution of analyte.
Analytical method validations should contain the fol-
lowing information in detail: 1.1.2. Documentation
Purpose: This section provides a short description of Print chromatograms.
what is to be accomplished by the study.
Project scope: Identify the test methods and which prod- 1.1.3. Acceptance criteria
ucts are within the scope of the validation. The excipient compounds must not interfere with the
Overview: This section contains the following: a gen- analysis of the targeted analyte.
eral description of the test method, a summary of the char-
acterization studies, identification of method type and vali- 1.2. Linearity
dation approach, test method applications and validation
protocol, the intended use of each test method application, 1.2.1. Test procedure
and the analytical performance characteristics for each test Standard solutions will be prepared at six concentra-
method application. tions, typically 25, 50, 75, 100, 150, and 200% of target
Resources: This section identifies the following: end concentration. Three individually prepared replicates at
user laboratory where the method validation is to be per- each concentration will be analyzed. The method of
formed; equipment to be used in the method validation; standard preparation and the number of injections will
software to be used in the method validation; materials to be be same as used in the final procedure.
used in the method validation; special instructions on han-
dling, stability, and storage for each material. 1.2.2. Documentation
Appendices: This section contains references, signa- Record results on a datasheet. Calculate the mean, stan-
ture, and a review worksheet for all personnel, their specific dard deviation, and Relative Standard Deviation (RSD)
tasks, and the documentation of their training. Listings of all for each concentration. Plot concentration (x-axis) ver-
equipment and software necessary to perform the method sus mean response (y-axis) for each concentration. Cal-
validation should be found here along with document and culate the regression equation and coefficient of deter-
materials worksheets used in the method validation and in mination (r2). Record these calculations on the
the test method procedure(s). datasheet.

1. Analytical Performance Characteristics 1.2.3. Acceptance criteria

Procedure The correlation coefficient for six concentration levels
Before undertaking the task of methods validation, it is will be 0.999 for the range of 80 to 120% of the target
necessary that the analytical system itself be adequately concentration. The y-intercept must 2% of the target
designed, maintained, calibrated, and validated. All per- concentration response. A plot of response factor versus
sonnel who will perform the validation testing must be concentration must show all values within 2.5% of the
properly trained. Method validation protocol must be target level response factor, for concentrations between
9,10
agreed upon by the CFT and approved before execution. 80 and 120% of the target concentration. HC states
For each of the previously stated validation characteristics that the coefficient of determination for active ingredi-
(Figure 1), this document defines the test procedure, doc- ents should be 0.997, for impurities 0.98 and for bio-
umentation, and acceptance criteria. Specific values are logics 0.95.8
taken from the ICH, U.S. FDA, USP, HC, and pertinent
literature as references. (See the References section at the 1.3. Range
end of this article for further definitions and explanations.)
1.3.1. Test procedure
1.1. Specificity The data obtained during the linearity and accuracy
studies will be used to assess the range of the method.

A n a l y t i c a l M e t h o d s Va l i d a t i o n 7
Ghulam A. Shabir

Linearity - Data Sheet Electronic file name:

Concentration Concentration as % Peak Area (mean of Peak Area
(mg/ml) of Analyte Target three Injections) RSD (%)

5 (e.g.) 25
10 50
15 75
20 100
30 150
40 200
Equation for regression line = Correlation coefficient (r2) =

Range - Data Sheet Electronic file name:

Record range:

Accuracy - Data Sheet Electronic file name:

Sample Percent Amount of Standard Recovery (%)
of Nominal (mean of (mg)
three injections) Spiked Found
1 75 (e.g.)
2 100
3 150
Mean
SD
RSD%

Repeatability - Data Sheet Electronic file name:

Injection No. Retention Time (min) Peak Area Peak Height

Replicate 1
Replicate 2
Replicate 3
Replicate 4
Replicate 5
Replicate 6
Replicate 7
Replicate 8
Replicate 9
Replicate 10
Mean
SD
RSD%

8 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
 Ghulam A. Shabir

The precision data used for this assessment is the preci- Record the retention time, peak area, and peak height on
sion of the three replicate samples analyzed at each level the datasheet. Calculate the mean, standard deviation,
in the accuracy studies. and RSD.
1.3.2. Documentation 1.5.3. Acceptance criteria
Record the range on the datasheet. The FDA states that the typical RSD should be 1% for
drug substances and drug products, 2% for bulk drugs
1.3.3. Acceptance criteria and finished products. HC states that the RSD should be
The acceptable range will be defined as the concentra- 1% for drug substances and 2% for drug products. For
tion interval over which linearity and accuracy are ob- minor components, it should be 5% but may reach
8
tained per the above criteria, and in addition, that yields 10% at the limit of quantitation.
9
a precision of 3% RSD.
1.6. Intermediate Precision
1.4. Accuracy
1.6.1. Test procedure
1.4.1. Test procedure Intermediate precision (within-laboratory variation) will
Spiked samples will be prepared at three concentrations be demonstrated by two analysts, using two HPLC sys-
over the range of 50 to 150% of the target concentration. tems on different days and evaluating the relative per-
Three individually prepared replicates at each concen- cent purity data across the two HPLC systems at three
tration will be analyzed. When it is impossible or diffi- concentration levels (50%, 100%, 150%) that cover the
cult to prepare known placebos, use a low concentration analyte assay method range 80 to 120%.
of a known standard.
1.6.2. Documentation
1.4.2. Documentation Record the relative % purity (% area) of each concentra-
For each sample, report the theoretical value, assay tion on the datasheet.
value, and percent recovery. Calculate the mean, stan-
dard deviation, RSD, and percent recovery for all sam- Calculate the mean, standard deviation, and RSD for the
ples. Record results on the datasheet. operators and instruments.

1.4.3. Acceptance criteria 1.6.3. Acceptance criteria

The mean recovery will be within 90 to 110% of the the- The assay results obtained by two operators using two
oretical value for non-regulated products. For the U.S. instruments on different days should have a statistical
9, 10
pharmaceutical industry, 100 2% is typical for an RSD 2%.
assay of an active ingredient in a drug product over the
9
range of 80 to 120% of the target concentration. Lower 1.7. Limit of Detection
percent recoveries may be acceptable based on the needs
of the methods. HC states that the required accuracy is a 1.7.1. Test procedure
bias of 2% for dosage forms and 1% for drug sub- The lowest concentration of the standard solution will be
8
stance. determined by sequentially diluting the sample. Six
replicates will be made from this sample solution.
1.5. Precision - Repeatability
1.7.2. Documentation
1.5.1. Test procedure Print the chromatogram and record the lowest detectable
One sample solution containing the target level of ana- concentration and RSD on the datasheet.
lyte will be prepared. Ten replicates will be made from
this sample solution according to the final method pro- 1.7.3. Acceptance criteria
2
cedure. The ICH references a signal-to-noise ratio of 3:1. HC rec-
ommends a signal-to-noise ratio of 3:1. Some analysts
1.5.2. Documentation calculate the standard deviation of the signal (or response)

A n a l y t i c a l M e t h o d s Va l i d a t i o n 9
Ghulam A. Shabir

Intermediate Precision - Datasheet Electronic file name:

Relative % Purity (% area)
Instrument 1 Instrument 2
Sample S1 S2 S3 S1 S2 S3
(50%) (100%) (150%) (50%) (100%) (150%)
Operator 1, day 1
Operator 1, day 2
Operator 2, day 1
Operator 2, day 2
Mean (Instrument)
Mean (Operators)
RSD% S1 + S1 S2 + S2 S3 + S3
Instruments
Operators

Limit of Detection - Data Sheet Electronic file name:

Record sample data results: (e.g., concentration, S/N ratio, RSD%)

Limit of Quantitation - Data Sheet Electronic file name:

Record sample data results: (e.g., concentration, S/N ratio, RSD%)

of a number of blank samples and then multiply this num- centration that gives an RSD of approximately 10% for
8
ber by two to estimate the signal at the limit of detection. a minimum of six replicate determinations.

1.8. Limit of Quantitation

1.9. System Suitability
1.8.1. Test procedure
Establish the lowest concentration at which an analyte in 1.9.1. Test procedure
the sample matrix can be determined with the accuracy System suitability tests will be performed on both HPLC
and precision required for the method in question. This systems to determine the accuracy and precision of the
value may be the lowest concentration in the standard system by injecting six injections of a solution contain-
curve. Make six replicates from this solution. ing analyte at 100% of test concentration. The following
parameters will be determined: plate count, tailing fac-
1.8.2. Documentation tors, resolution, and reproducibility (percent RSD of re-
Print the chromatogram and record the lowest quantified tention time, peak area, and height for six injections).
concentration and RSD on the datasheet. Provide data
that demonstrates the accuracy and precision required in 1.9.2. Documentation
the acceptance criteria. Print the chromatogram and record the data on the
datasheet
1.8.3. Acceptance criteria
The limit of quantitation for chromatographic methods 1.9.3. Acceptance criteria
has been described as the concentration that gives a sig- Retention factor (k): the peak of interest should be well
nal-to-noise ratio (a peak with height at least ten times as resolved from other peaks and the void volume; gener-
2
high as the baseline noise level) of 10:1. HC states that ally k should be 2.0. Resolution (Rs): Rs should be 2
the quantitation limit is the best estimate of a low con- between the peak of interest and the closest eluted peak,

10 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
 Ghulam A. Shabir

System Suitability Data Sheet Electronic file name:

System Suitability Acceptance Results Criteria Met/
Parameter Criteria Not Met
HPLC 1 HPLC 2
Injection precision for
retention time (min) RSD 1%
Injection precision for
peak area (n = 6) RSD 1%
Injection precision for
peak height RSD 1%
Resolution (Rs) Rs = 2.0
USP tailing factor (T) T = 2.0
Capacity factor (k) K = 2.0
Theoretical plates (N) N = 2000

Robustness - Data Sheet Electronic file name:

Explain / record sample data:

which is potentially interfering (impurity, excipient, and temperature adjusted by 5C. If these changes are within
degradation product). Reproducibility: RSD for peak the limits that produce acceptable chromatography, they
9, 10
area, height, and retention time will be 1% for six injec- will be incorporated in the method procedure.
tions. Tailing factor (T): T should be 2. Theoretical
3
plates (N): 2000.

1.10. Robustness 2. Appendices

As defined by the USP, robustness measures the capac- List all appendices associated with this protocol. Each
ity of an analytical method to remain unaffected by appendix needs to be labeled and paginated separately
small but deliberate variations in method parameters.
Robustness provides some indication of the reliability of
an analytical method during normal usage. Article Acronym Listing
Parameters, which will be investigated, are percent or-
ganic content in the mobile phase or gradient ramp, pH CFT: Cross-Functional Team
of the mobile phase, buffer concentration, temperature, EC: European Community
and injection volume. These parameters may be evalu- FDA: Food and Drug Administration
ated one factor at a time or simultaneously as part of a HC: Health Canada
factorial experiment. HPLC: High Performance Liquid
Chromatography
The chromatography obtained for a sample containing ICH: International Conference on
representative impurities, when using modified parame- Harmonization
ter(s), will be compared to the chromatography obtained RSD: Relative Standard Deviation
using the target parameters. The effects of the following U.S.: United States
changes in chromatographic conditions will be deter- USP: United States Pharmacopoeia
mined: methanol content in mobile phase adjusted by
2%, mobile phase pH adjusted by 0.1 pH units, column

A n a l y t i c a l M e t h o d s Va l i d a t i o n 11
Ghulam A. Shabir

List of Appendices
Appendix Document Title Total Pages
No.

Appendix 1
______________________________________________________________________________
Method Validation Personnel Signature and Review Worksheet

Analyst Name Dept. Validation Protocol Analyst Date

Activity Reference Signature

Comments:

Completed By: Signature: Date:

Appendix 2
______________________________________________________________________________
Equipment and Software Used in Method Validation Worksheet

Equipment Last Next Software Validation

Name/Module # Calibration Date Calibration Date Name and Version Reference

Comments:

Completed By: Signature: Date:

12 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y
 Ghulam A. Shabir

Appendix 3
______________________________________________________________________________
Document and Materials Used in Method Validation Worksheet

Complete Pre-protocol Execution

Document Edition/Version Material Name Supplier/ Expiration

Name/Ref. No. Number Lot Number Date

Comments:

Completed By: Signature: Date:

Appendix 4
_____________________________________________________________________________
Analytical Test Method Procedure

This procedure should include the entire testing method and all procedures associated with it.
This appendix can appear in any format, but it should always be included in the documentation

quality assurance, in-process control, R&D, and val-

from the body of the document. The following informa- idation in the pharmaceutical industry. Ghulam has
tion must be found on every page of each appendix: val- received many technical excellence industrial
idation protocol number; validation protocol title; ap- awards as well as academic awards for authoring
pendix number (e.g., 1, 2, 3, or A, B, C, ); and page best scientific papers. Ghulams work has ap-
X of Y. peared in many publications. He has given several
Acknowledgements presentations at international conferences as well
as having organized and moderated at sympo-
I thank Abbott Laboratories and MediSense for permis- siums. He holds a Masters Degree in Chemistry
sion to publish this article. I also thank Dr. Alison Ingham and Pharmaceutical Sciences. He can be reached
(Health Canada) for his comments on the text. by phone at 44-1993-863099, by fax at 44-1235-
467737, or by e-mail at [email protected].

About the Author

References
Ghulam Shabir is a Principal Scientist at Abbott
Laboratories, MediSense UK. His group is respon- 1. International Conference on Harmonization (ICH), Q2A:
sible for materials characterization, analytical meth- Text on Validation of Analytical Procedures, March 1995.
ods development, and validation and equipment 2. International Conference on Harmonization (ICH), Q2B: Val-
qualification. Ghulam is a Fellow of the Institute of idation of Analytical Procedures: Methodology, May 1997.
Quality Assurance and a Companion of the Institute 3. U.S. Center for Drug Evaluation and Research, Reviewer
of Manufacturing with 17 years of broad-based ex- Guidance: Validation of Chromatographic Methods, Novem-
perience in the areas of production, quality control, ber 1994.

A n a l y t i c a l M e t h o d s Va l i d a t i o n 13
Ghulam A. Shabir

4. U.S. FDA, Guidance for Submitting Samples and Analytical

Data for Methods Validation, Rockville, Md., USA, Center
for Drugs and Biologics, Department of Health and Human
Services, February 1987.
5. U.S. FDA DHHS, 21 CFR Parts 210 and 211, Current
Good Manufacturing Practice of Certain Requirements for
Finished Pharmaceuticals, Proposed Rule, May 1996.
6. Validation of Compendial Methods, <1225>, U.S. Pharma-
copoeia 26-National Formulary 21, United States Pharma-
copeial Convention, Rockville MD, 2003.
7. U.S. FDA, Guidance for Industry: Analytical Procedures and
Methods Validation: Chemistry, Manufacturing and Controls
Documentation, August 2000.
8. Drugs Directorate Guidelines, Acceptable Methods, Na-
tional Health and Welfare, Health Protection Branch,
Canada, July 1994. (This guidance is available from HC as
a print copy, but is soon to be released on the website
http://www.hc-sc.gc.ca/hpfb-dgpsa/tpd-dpt/).
9. M.J. Green, Anal. Chem., Vol. 68, 1996. p. 305A.
10. G.A. Shabir, J. Chromatogra. A, Vol. 987, 2003. p. 57.

14 I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y