What is validation?
Validation is the process
used to confirm that the
employed for a specific
test is suitable for its
Analytical methods need to be
validated or revalidated:
-before their introduction into
-whenever the analytical
-whenever the method is changed.
Center for Drug
Evaluation and Research,
A reference standard is a highly purified
compound that is well characterized.
Chromatographic methods rely heavily
on a reference standard to provide
accurate data. Therefore the quality of
the reference standard is critical.
Accuracy is the measure of how close the
experimental value is to the true value.
Recovery data, at least in triplicate, at
each level (80, 100 and 120% of label
The mean is an estimate of accuracy and
the RSD is an estimate of sample
LOD / LOQ
Limit of Detection
The lowest concentration of analyte in a
sample that can be detected, but not
necessarily quantitated. Usually s/n 2:1 or
Limit of Quantitation
The lowest concentration of analyte in a
sample that can be determined with
acceptable precision and accuracy under
the stated conditions. Usually s/n 10:1
That range of analyte concentrations over
which the detector yields a linear response.
The working sample concentration and samples
tested for accuracy should be in the linear
The linearity range depends on the purpose of the test
method. For example, the range for drug content
uniformity would be NLT ± 20%.
Regression coefficient (R2) NLT 0.999. Intercept and slope
should be indicated.
Measure of how close the data
values are to each other for a
number of measurements under
the same analytical conditions.
Precision is defined by three
Injection repeatability: Multiple
injections of the same sample in the
Analysis repeatability: Multiple
measurements of a sample by the same
analyst under the same analytical
Recommendation: A minimum of 10
injections with an RSD of 1%.
Evaluates the reliability of the method in a different environment other
than that used during development of the method.
The objective is to ensure that the method will provide the same
results when similar samples are analyzed once the method
development phase is over.
Depending on time and resources, the method can be tested on
multiple days, analysts, instruments, etc.
The precision between
laboratories as in
It is not normally expected if
intermediate precision is
Range and Recovery
Range: The interval between the high and low levels of analyte studied.
Recommendation is usually +/- 20%.
Recovery: The amount/weight of the compound of interest analyzed as a
percentage to the theoretical amount present in the medium.
Full recovery should be obtained for the compound(s) of interest.
Simpler sample preparation procedure will result in a lower variation of recovery.
Measure of the method's ability to
remain unaffected by small, but
deliberate variations in method
e.g., column type, column temperature, pH of
mobile phase, reagents, flow rate, etc.
Sample Solution Stability
Solution stability of the drug substance should be evaluated.
Most laboratories use autosamplers with overnight runs and the sample will be in
solution for hours before the test procedure is completed. This is of concern
especially for drugs that can undergo degradation by hydrolysis, photolysis or
adhesion to glassware.
Recommendations: Data to support the sample solution stability under normal
laboratory conditions for the duration of the test procedure should be generated.
Specificity and Selectivity
The analyte should have no
interference from other
extraneous components and be
well resolved from them.
System Suitability Tests.
The accuracy and precision of data begin
with a well-behaved chromatographic system.
The system suitability specifications and tests
are parameters that help achieve this
System Suitability Parameters - USP
Plate count > 2000 plates/meter
Tailing factor < 2
Resolution > 2
Partition ratio > 2
Relative retention > 1.5
Precision / repeatability RSD </= 1% for n >/= 5
The sample and standard should be dissolved in the mobile phase. If
that is not possible, then avoid using too much organic solvent as
compared to the mobile phase.
The sample and standard concentrations should be close if not the
The samples should be bracketed by standards during the analytical
If the sample is filtered, adhesion of the analyte to the filter can
happen. This will be of importance especially for low level impurities.
Data to validate this aspect should be submitted.
Hardware validation – IQ/OQ/PQ
Was the instrument installed as per vendor’s guidelines?
Is the system performing as per claimed specifications?
Is the analysis compliant for each sample? (System suitability
What is 21CFR-11?
CFR = Code of Federal Regulations
Codification of the general rules and
regulations of the US Federal
Divided into 50 books (titles).
Each title has several chapters.
21 CFR Part 11
Title 21 deals with the Food and Drug
Administration (FDA) rules and
Title 21-Part 11 defines the criteria
under which electronic records and
electronic signatures are considered to be
trustworthy, reliable and equivalent to
21 CFR Part 11 Regulations
Three Subparts :
1) Subpart A : General Provision
2) Subpart B : Electronic Records
3) Subpart C : Electronic Signatures
Subpart A : General Provision
•Regulations establish the criteria the FDA considers for electronic records and electronic
signature to be trustworthy, reliable, and generally equivalent to paper.
•Applies to all records in electronic form under any records requirement within any FDA
•Electronic records are considered equivalent to full handwritten signatures, initials, and
other general signings.
•Electronic records may be used in accordance with Part 11 unless paper records are
•Computer system (hardware and software), controls, and relevant documentation must
be available for review during FDA inspections.
Subpart B : Electronic Record
“Any combination of text, graphics, data, audio, pictorial, or other information
representation in digital form that is created, modified, maintained, archived, retrieved,
or distributed by a computer system.”
Must develop procedures and controls to ensure authenticity, integrity and
confidentiality, and that signer cannot repudiate the signed record.
The controls must:
Maintain accurate and complete records
Limit the system to authorized persons
Protect records through retention period
Contain audit trails that are secure, operator independent, computer-generated, time-stamped, cover the
creation , modification and deletion of records and do not obscure previous information
•Allow for the performance of operational system checks, authority checks, and device
checks to ensure system, record, and data integrity
•Ensure appropriate personnel qualifications
•Policies written and followed to hold personnel accountable for actions and to deter
•Control over system documentation including distribution, access, use, revision and change
•Must develop procedures and controls that ensure authenticity, integrity, and
confidentiality of electronic records and comply with all other parts of Section 11.10
•Must use additional measures (e.g. document encryption, digital signature standards) to
ensure authenticity, integrity, and confidentiality
•Signed electronic records must include the printed name of the signer, date and time of
signature, and the purpose of the signature (e.g. review, approval etc.) Each of these must
be readable by display or printout.
•Electronic signature and handwritten signatures must be linked to ensure signatures
cannot be excised, copied, transferred or falsified.
Subpart C : Electronic Signature
“A computer data compilation of any symbol or series of symbols executed, adopted, or
authorized by an individual to be the legally binding equivalent of the individual’s
•Must be unique to an individual and not reassigned
•Identity of individual must be verified by organization
•Must certify electronic signature system to the agency prior to or at the time of use
of the system
•Certification must be submitted in paper form and, upon agency request, provide
certification that signature is legally binding
•Non-Biometric signatures must:
Contain at least two different identification components (e.g. User ID and Password)
Single sign-on with multiple tasks: Use all identification components at first, with partial
identification for each task thereafter
Multiple sign-on without continuous access requires all identification components to be used
Be used only by the owner
Ensure use by other individuals is precluded and does not occur without collaboration by at
least two other individuals
•Biometric signatures must ensure use by the owner
Persons using electronic signatures must use controls to ensure security and integrity and
Assuring that no two individuals have the same combination of identification code and
Periodic check, recall, or revision of identification code and password
Loss management and replacement procedures
Testing of devices (i.e. tokens or cards) that produce or maintain identification codes or
passwords to ensure proper function and unaltered state.
•Unauthorized use safeguards
•Report attempts in to:
Management, as appropriate
• Information management
related to manufacturing
• Clinical data managementGCP
• Data Acquisition
• Laboratory InformationGLP
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