CM 1224 02 - VVTP

Validation, Verification,
and Transfer of Analytical Procedures

Dr. Sharad D. Mankumare
Director, RSL and Verification Programs, USP India.
Updated: January 2018

Horacio Pappa
Course ID: CM-1224-02
Disclaimer
Because USP text and publications may have legal implications in the U.S. and elsewhere, their language
must stand on its own. The USP shall not provide an official ex post facto interpretation to one party, thereby
placing other parties without that interpretation at a possible disadvantage. The requirements shall be
uniformly and equally available to all parties
In addition, USP shall not provide an official opinion as to whether a particular article does or does not comply
with compendial requirements, except as part of an established USP verification or other conformity
assessment program that is conducted separately from and independent of USP's standard-setting activities.
Certain commercial equipment, instruments or materials may be identified in this presentation to specify
adequately the experimental procedure. Such identification does not imply approval, endorsement, or
certification by USP of a particular brand or product, nor does it imply that the equipment, instrument or
material is necessarily the best available for the purpose or that any other brand or product was judged to be
unsatisfactory or inadequate.
This course material is USP Property. Duplication or distribution without USP’s written permission is
prohibited.
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© 2018 USP
Dr. Sharad D. Mankumare
USP Affiliation: USP India Staff member

Title: Director, RSL and Verification Programs,
Dr. Sharad Mankumare is a Director of Reference Standards Laboratory and

Verification Programs at the USP facility in Hyderabad, India. He is responsible for the
collaborative testing of reference standards for the USP NF, medicinal compendia and
food chemical codex.
Dr. Sharad joined USP-India in June 2012. He has more than 21 years of experience in
generic pharmaceutical industry. Before joining USP, Dr. Sharad has worked with
several company includes Cipla, German Remedies, Glenmak and Sandoz (Novartis).
He is a FDA certified technical personnel for chemical and instrumental analysis. Dr.
Sharad is holding Ph.D. in chemistry and Master’s degree in Organic chemistry from
Mumbai University. He has published more than 06 scientific articles in international
and national journals.
.
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Topics
 USP Documentary Standards

 Definition and Validation Parameters
 Comparison of USP <1225> and ICH Q2 (R1)
 Specificity
 Precision
 Accuracy
 Linearity and Range
 Detection/Quantification Limit
 Robustness
 USP <621> Chromatography — System Suitability
 USP <1226> Verification of Compendial Procedures
 USP <1224> Transfer of Analytical Procedures
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USP Documentary Standards
Three critical components
1. Monographs
2. General Chapters
3. General Notices
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© 2018 USP
USP General Chapters
<1> to <999>: Required
 Standardize the use of common tests and procedures for compendial topics
 These chapters are often referenced in individual monographs
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USP General Chapters
<1000> to <1999>: Informational
 Provide guidance to assist compendial users on relevant topics
 These chapters are not normally referenced in individual monographs
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Why
Analytical method development and validation are the continuous and inter-
dependent task associated with the research and development, quality control and
quality assurance departments.
It helps in establishment of product-specific acceptance criteria and stability of

results.
An effective analytical method development and its validation can provide significant
improvements in precision and a reduction in bias errors. It can further help to avoid
costly and time consuming exercises.
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
 USP <1226> Verification of Compendial Procedures USP
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Validation in USP
The term “Validation” appears in:
USP <1010> Analytical Data-Interpretation and Treatment

USP <1043> Ancillary Materials Cell, Gene, and Tissue-Engineered Products
USP <1117> Microbiological Best Laboratory Practices
USP <1120> Raman Spectrophotometry
USP <1223> Validation of Alternative Microbiological Methods
USP <1092> The Dissolution Procedure: Development and Procedure
USP <1225> Validation of Compendial Procedure
and many others
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Definitions of Validation: USP
USP <1225>:
“ Validation of an analytical procedure is the process by which it is established by

laboratory studies, that the performance characteristics of the procedure meet
the requirements for the intended analytical applications.”
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© 2018 USP
Definitions of Validation: ICH
Q2(R1) VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND

METHODOLOGY
“The objective of validation of an analytical procedure is to demonstrate that it

is suitable for its intended purpose.”
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Definitions for Verification: USP
USP <1226> Verification of Compendial Procedures: “Verification consists of

assessing selected analytical performance characteristics, such as those that
are described in chapter <1225>, to generate appropriate, relevant data rather
than repeating the validation process.”
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Validation Characteristics
Validation characteristics to be selected according to type of method:

Accuracy
Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Robustness (it is not part of the formal validation process)
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
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Overall Purposes
Regulatory versus Procedures Suitable for Compendial

Requirements
ICH: “This document presents a discussion of the characteristics for

consideration during the validation of the analytical procedures included as part of
registration applications submitted within the EC, Japan and USA.”
USP: “According to Section 501 of the Federal Food, Drug, and Cosmetic Act,
assays and specifications in monographs of the United States Pharmacopeia and
the National Formulary constitute legal standards.
The Current Good Manufacturing Practice regulations [21 CFR 211.194(a)]
require that test methods, which are used for assessing compliance of
pharmaceutical articles with established specifications, must meet proper
standards of accuracy and reliability.”
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Submissions to the Compendia
Monograph Submission Requirements
 ICH: No such section included
 USP: Submissions should include:

 Statement of rationale (need, capability, advantages)
 Complete description of procedure
 Validation results based upon characteristics
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Types of Procedures
Finding and Recognizing the Tests
ICH:
1. Assay
2. Quantitative tests for impurities
3. Limit tests for the control of impurities
4. Identification
USP:
 Category 1: Like ICH 1
 Category 2: Like ICH 2,3
 Category 3: Performance tests like dissolution
 Category 4: Like ICH 4
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Definitions of Specificity
ICH:
“Specificity is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present. Typically these might include
impurities, degradants, matrix, etc. Lack of specificity of an individual analytical
procedure may be compensated by other supporting analytical procedure(s).”
USP:
<1225> references the ICH definition. It adds: “[NOTE—Other reputable
international authorities (IUPAC, AOAC-I) have preferred the term “selectivity,”
reserving “specificity” for those procedures that are completely selective.]
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Definitions of Specificity
Implications:
 Identification: to ensure the identity of an analyte.
 Purity Tests: to ensure that the analytical procedures performed allow an
accurate statement of the content of impurities in the sample, i.e., related
substances test, elemental impurities, residual solvents content, etc.
 Assay (content or potency): to provide an exact result which allows an accurate
statement on the content or potency of the analyte in the sample.
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Definitions of Precision
ICH:
“The precision of an analytical procedure expresses the closeness of agreement
(degree of scatter) between a series of measurements obtained from multiple
sampling of the same homogeneous sample under the prescribed conditions.
Precision may be considered at three levels: repeatability, intermediate precision and
reproducibility.”
USP:
“The precision of an analytical procedure is the degree of agreement among
individual test results when the procedure is applied repeatedly to multiple samplings
of a homogeneous sample.”
The definition also discusses repeatability, intermediate precision and reproducibility.
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ICH:
 “Repeatability expresses the precision under the same operating conditions over a
short interval of time. Repeatability is also termed intra-assay precision.”
 “Intermediate precision expresses within-laboratories variations: different days,
different analysts, different equipment, etc.”
 “Reproducibility expresses the precision between laboratories (collaborative studies,
usually applied to standardization of methodology).”
USP:
 “Repeatability refers to the use of the analytical procedure within a laboratory over a
short period of time using the same analyst with the same equipment.”
 “Intermediate precision (also known as ruggedness) expresses within-laboratory
variation, as on different days, or with different analysts or equipment within the same
laboratory.”
 “In this context, reproducibility refers to the use of the analytical procedure in different
laboratories, as in a collaborative study.”
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“The precision of an analytical procedure is usually expressed as the variance,

standard deviation or coefficient of variation of a series of measurements.”
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Definitions of Accuracy
ICH:
“The accuracy of an analytical procedure expresses the closeness of agreement
between the value which is accepted either as a conventional true value or an
accepted reference value and the value found. This is sometimes termed
trueness.”
USP:
“The accuracy of an analytical procedure is the closeness of test results obtained
by that procedure to the true value. The accuracy of an analytical procedure
should be established across its range. […In VIM and ISO documents, “accuracy”
has a different meaning. In ISO, accuracy combines the concepts of
unbiasedness (termed “trueness”) and precision.] ”
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VIM-International Vocabulary of Metrology; ISO-International Organisation for standardisation.
© 2018 USP
Precision vs... Accuracy
Precise but not Accurate but not Precise and

accurate precise Accurate
 Precisionis expressed as RSD

 Accuracy is expressed as % of recovery
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Definitions of Detection Limit
ICH:
“The detection limit of an individual analytical procedure is the lowest amount of
analyte in a sample which can be detected but not necessarily quantitated as an
exact value.”
USP:
“The detection limit is a characteristic of limit tests. It is the lowest amount of
analyte in a sample that can be detected, but not necessarily quantitated, under
the stated experimental conditions.
Thus, limit tests merely substantiate that the amount of analyte is above or below
a certain level. The detection limit is usually expressed as the concentration of
analyte (e.g., percentage, parts per billion) in the sample.”
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Definitions of Quantitation Limit
ICH:
“The quantitation limit of an individual analytical procedure is the lowest amount of
analyte in a sample which can be quantitatively determined with suitable precision and
accuracy. The quantitation limit is a parameter of quantitative assays for low levels of
compounds in sample matrices, and is used particularly for the determination of
impurities and/or degradation products.”
USP:
“The quantitation limit is a characteristic of quantitative assays for low levels of
compounds in sample matrices, such as impurities in bulk drug substances and
degradation products in finished pharmaceuticals. It is the lowest amount of analyte in a
sample that can be determined with acceptable precision and accuracy under the stated
experimental conditions. The quantitation limit is expressed as the concentration of
analyte (e.g., percentage, parts per billion) in the sample.”
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Definitions of Linearity
ICH:
“The linearity of an analytical procedure is its ability (within a given range) to
obtain test results which are directly proportional to the concentration (amount) of
analyte in the sample.”
USP:
“The linearity of an analytical procedure is its ability to elicit test results that are
directly, or by a well-defined mathematical transformation, proportional to the
concentration of analyte in samples within a given range.”
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Definitions of Range
ICH:
“The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations)
for which it has been demonstrated that the analytical procedure has a suitable
level of precision, accuracy and linearity.”
USP:
“The range of an analytical procedure is the interval between the upper and lower
levels of analyte (including these levels) that have been demonstrated to be
determined with a suitable level of precision, accuracy, and linearity using the
procedure as written. The range is normally expressed in the same units as test
results (e.g., percent, parts per million) obtained by the analytical procedure”
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The Choice of Performance
Characteristics
USP <1225> Validation of Compendial Procedures
Performance Category I Category II Category III Category IV

Characteristic
Quant Limit
Test
Accuracy Yes Yes * * No

Precision Yes Yes No Yes No
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

* May be required, depending on the nature of the specific test
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ICH Q2 (R1)
Performance Assay Impurities Identification

Characteristic
Quant Limit Test
Accuracy Yes Yes No No
Precision
Repeatability Yes Yes No No
Interm. Precision Yes (1) Yes (1) No No
Specificity (2) Yes Yes Yes Yes
LOD No No (3) Yes No
LOQ No Yes No No
Linearity Yes Yes No No
Range Yes Yes No No

(1) in cases where reproducibility has been performed, intermediate precision is not needed
(2) lack of specificity of one analytical procedure could be compensated by other supporting procedure(s)
(3) may be needed in some cases
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Pre-validation Assessment
 Equipment- Suitable for expected accuracy?
 Reference Materials- Suitably characterized?
 Analytical method- Is procedure finalized?
 Validation Protocol- Management / QA approved?
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
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Specificity
 Identification Test
 Assay
 Impurity Test
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Specificity
“In the case of identification tests, the ability to select between compounds of
closely related structure that are likely to be present should be demonstrated. This
should be confirmed by obtaining positive results (perhaps by comparison to a
known reference material) from samples containing the analyte, coupled with
negative results from samples that do not contain the analyte and by confirming
that a positive response is not obtained from materials structurally similar to or
closely related to the analyte.” [<1225> USP]
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Specificity
May be applied to materials structurally similar to or closely related to the

analyte (which are likely to be present) to confirm that a positive response is not
obtained
The choice of such potentially interfering materials should be based on

sensible judgment considering the interferences that could occur
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Specificity
It is most often the case in USP monographs that more than one procedure is
used in the identification
Example: Hydralazine Hydrochloride

1. Infrared Absorption <197M>
2. The retention time of the major peak of the Sample solution corresponds to that
of the Standard solution, as obtained in the Assay.
3. Identification Tests—General, Chloride <191>
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Specificity (IR <197>)
“The IR absorption spectrum of the preparation of the test specimen, previously

dried under conditions specified for the corresponding Reference Standard unless
otherwise specified, or unless the Reference Standard is to be used without
drying, exhibits maxima only at the same wavelengths as that of a similar
preparation of the corresponding USP Reference Standard.”
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Specificity
It is important to remember (from USP <621>):
“Chromatographic retention times are characteristic of the compounds they

represent but are not unique.
Coincidence of retention times of a test and a reference substance can be used as

a feature in construction of an identity profile but is not insufficient on its own to
establish identity.”
“Absolute retention times of a given compound vary from one chromatogram to the
next.”
 “The deviations of RR, RF, or t values measured for the test substance from the
values obtained for the reference compound and mixture should not exceed the
reliability estimates determined statistically from replicate assays of the reference
compound.”
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Specificity
Identification Test
Assay
Impurity Test
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Specificity
From Q2(R1):
“…to provide an exact result which allows an accurate statement on the content
or potency of the analyte in a sample.”
“Specificity is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present. Typically these might include
impurities, degradants, matrix, etc.”
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Specificity
Identification Test
Assay
Impurity Test
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Specificity
Further elaboration from USP <1225>:
“Purity Tests: ensure that all the analytical procedures performed allow an
accurate statement of the content of impurities of an analyte (e.g., related
substances test, heavy metals limit, residual solvents).”
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Specificity
Representative chromatograms should be used to demonstrate specificity, and

individual components should be appropriately labeled
Critical
separations - can be demonstrated by the resolution of the two
components which elute closest to each other
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Specificity (Impurities Tests)
There are two basic approaches to establishing specificity for procedures

designed to measure impurities:
With impurity or degradation product standards
Without impurity or degradation product standards
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Specificity
When impurities are available
 Impurities Test
– Spiking drug substance or drug product with appropriate levels of impurities
– Demonstration of the separation of these impurities individually and from other
components in the sample matrix
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Specificity
When impurities are not available
 Demonstrate by comparing the test results of samples containing impurities or

degradation products to a second well-characterized procedure (pharmacopeial
method, validated independent procedure)
 Include samples stored under relevant stress conditions: light, heat, humidity,
acid/base hydrolysis, and oxidation
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Specificity (Stress Conditions)
Carried out in solution and/or solid state

Conditions more severe than accelerated stability testing
One batch of material
Thermolytic,hydrolytic, oxidative, and photolytic degradation mechanisms in the
drug substance and drug product
Analyte peaks are evaluated for peak purity in samples sufficiently stressed to
effect 10 - 15% degradation
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Forced Degradations
Guideline for submitting samples and analytical data for methods

validation, FDA, 1987.
 Drug substances:
– Heat (50°C)
– Light
– Hydrolysis: acid (HCl 0.1 N) / base (NaOH 0.1 N)
– Oxidation (H2O2 3%)
 Drug Products
– Heat
– Light
– Humidity (85%)
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Forced Degradations (cont)
 Other conditions may be necessary
 Exposure time, temperature and concentration can be adjusted to obtain an

appropriate degradation of the sample (10% to 15%)
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Specificity (Peak Purity Tests)
 To show that the analyte chromatographic peak is not attributable to more than
one component
 Subsequent analyses using other techniques: diode array, mass spectrometry,

different chromatographic system, etc.
 Software for the evaluation
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Basic Statistics
Definitions:
Alpha (α) :
α is the amount of uncertainty we're willing to live with when we estimate a parameter.
Default values as per IUPAC for α is 0.05 each i.e. acceptable probability of accepting
wrong values is 5%.
Confidence:
Confidence is the amount of certainty we have when we estimate parameter.
Confidence = 1- α
A frequently used level of confidence is 0.95, also referred to as 95% confidence.
Confidence limits are the lower and upper bounds for an estimate, based on confidence level.
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Basic Statistics
Confidence interval (CI): A range of values so defined that there is a

specified probability that the value of a parameter lies within it.
Upper and lower 95% confidence limits:
Imagine a sampling distribution of mean with x
at the centre
The standard error of the mean (x)=
s= standard deviation
N= Number of observations
Upper and lower 95% confidence limits for

estimating µ :
The two tailed critical value that cut of areas
adding up to 0.05 of the area under
distribution.
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Basic Statistics
Definitions:
Null hypothesis (H0): The null hypothesis states that a population parameter (such
as the mean, the standard deviation, and so on) is equal to a hypothesized value.
The null hypothesis is often an initial claim that is based on previous analyses or
specialized knowledge.
Alternative Hypothesis (H1) : The alternative hypothesis states that a population

parameter is smaller, greater, or different than the hypothesized value in the null
hypothesis. The alternative hypothesis is what you might believe to be true or hope
to prove true.
The ANOVA F-statistic (F): It is a ratio of the between Group Variation divided by the
within Group Variation.
A large F is evidence against H0, since it indicates that there is more difference between groups than
within groups.
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Example of specificity
Hypothesis:
Conc. of H0= All means are equal
Level sub Area
H1= Not all means are equal;
1 0 2150 Significance level; α=0.05
1 0 2200
1 0 2350
1 0 2210 Interpretation of data : 2 sample t-tests:
1 0 2167  Mean of level 1 =2196.2
1 0 2100  Mean of level 2 =2303.7
2 0.25 2350
 Stdev of level 1 =85.0
 Stdev of level 2 =68.6
2 0.25 2400
 t value = -2.41
2 0.25 2278
 p-Value = 0.039 hence H1 is valid.
2 0.25 2205
2 0.25 2322
Conclusion:
2 0.25 2267
 Since p-value is less than 0.05, there is difference in area
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Specificity
 Assessment of the analyte in the presence of other components (impurities,

degradation products, excipients)
Peak Purity (spectral homogeneity)
If not specific enough, combination of more than one analytical procedure
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
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Precision
Degreeof agreement among results from individual samplings of a

homogeneous sample
Repeatability, Intermediate Precision, Reproducibility
Precision should be evaluated across the specified quantitation range of the

method
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Precision
 Repeatability: is precision under the same operating conditions for a

short period of time. ICH recommends a minimum of 9 measurements
within the given range of the procedure (3 concentrations / 3 replications)
or a minimum of 6 replications at 100%
 Intermediate Precision: indicates intra-laboratory variations; different

days, different analysts, different equipment
 Reproducibility: indicates inter-laboratory variations
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Precision
Determination -
 Assay individual preparations from homogeneous samples
 Calculate Standard Deviation or Relative Standard Deviation
Method precision should include all sources of variation from sample preparation
to rounding the final test result
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Intermediate Precision
Intermediate Precision study variables
 Analyst A, B, C
 Day 1, 2, 3
 Equipment L, M, N
 Column x, y, z
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Degree of reproducibility of test results as a function of the

variables
Inte rme diate Pre cision Ev aluation
for
Bulk Substance Assay by HPLC
1.6
1.2
% RSD
0.8
0.4
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Hypothesis:
H0= All means are equal
H1= Not all means are
equal;
Significance level; α=0.05
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Precision
Summary
Reproducibility
FDA: At least 2 laboratories within the Company
– FDA (CDER), Reviewer Guidance: Validation of Chromatographic Methods, Nov. 1994
FDA: for Multi-laboratory follow ASTM 691

– “Standard practice for Conducting an Interlaboratory Study to Determine the Precision of a
Test Method”
AOAC protocol 8 samples - 8 laboratories.

– Statistical Manual of the AOAC W.J. Youden and E.H. Steiner
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
 USP <621> Chromatography - System Suitability
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Accuracy
Definitions:
USP- “The closeness of the test result obtained by the procedure to the true
value.”
ICH-“The closeness of the test result obtained by the method to a value that is
accepted as conventionally true value or as a reference value.”
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Accuracy
Determination with drug substance Q2(R1):
“…applicationof an analytical procedure to an analyte of known purity (e.g.;

reference material)”
“…comparison of the results of the proposed analytical procedure with those of

a second well-characterized procedure, the accuracy of which is stated and/or
defined”
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Accuracy
Determination with drug product Q2(R1):
“…applicationof the analytical procedure to synthetic mixtures of the drug

product components to which known quantities of the drug substance to be
analysed have been added.”
“…in cases where it is impossible to obtain samples of all drug product

components , it may be acceptable either to add known quantities of the analyte
to the drug product or to compare the results obtained from a second, well
characterized procedure, the accuracy of which is stated and/or defined”
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Accuracy
From USP <1225>:

“Accuracy is calculated as the percentage of recovery by the assay of the known
added amount of analyte in the sample, or as the difference between the mean
and the accepted true value, together with confidence intervals.”
“The ICH documents recommend that accuracy should be assessed using a
minimum of nine determinations over a minimum of three concentration levels,
covering the specified range (i.e., three concentrations and three replicates of
each concentration).”
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Case Study – Accuracy-API (Assay)
Standard at 100%
Standard Weight (mg) Standard Concentration(mg/mL) Injection Area Response
1 2413217
2 2416832
25.05 0.100 3 2422015
4 2426770
5 2427690
Average 2421305
Std Deviation 6256.24
%RSD 0.26
Accuracy at 80%
Preparation Weight of the sample (mg) Sample Concentration (mg/mL) Area Response Sample Concentration recovered (mg/mL) % Assay % Recovered
1 24.55 0.079 1914136 0.079 80.0 100.0
2 24.47 0.078 1907677 0.079 79.7 99.6
3 24.99 0.080 1945001 0.080 81.3 101.6
Average 80.3 100.4
Std Deviation 0.85 1.06
%RSD 1.06 1.06
Accuracy at 100%
1 25.01 0.100 2423648 0.100 100.0 100.0
2 24.90 0.100 2398279 0.099 99.0 99.0
3 25.06 0.100 2399911 0.099 99.0 99.0
Average 99.3 99.3
%RSD 0.58 0.58
Accuracy at 120%
1 24.59 0.118 2843002 0.117 119.3 99.4
2 24.49 0.118 2831390 0.117 118.8 99.0
3 24.51 0.118 2863135 0.118 120.2 100.2
Average 119.4 99.5
%RSD 0.59 0.59
Recovery (%)= ( % Assay / Theoretical Assay ) x100 % RSD (n=9) 0.84
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Case Study – Accuracy-API (Assay)
 Preparation of Assay standard solution (0.1mg/mL): Weighed and

transferred about 25 mg of USP RS into a 50 mL of volumetric flask dissolved
and made up to the mark with diluent. Pipetted 5.0 mL into a 25 mL volumetric
flask and dilute to volume with diluent.
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Case Study – Accuracy-Drug product (Assay)
Accuracy - Doxycycline Tablets, 100 mg,
API Conc.
API Conc.
% Level Prep Results ID Peak Area Found % Recovery2 Mean Recovery
Added1 (mg/mL)
(mg/mL)
1 2649 0.10871 1950409 0.10935 100.6
110 2 2650 0.10871 1930715 0.10824 99.6 99.7
3 2651 0.10871 1920557 0.10767 99.0
1 2652 0.11889 2113053 0.11847 99.6
120 2 2653 0.11889 2134804 0.11969 100.7 100.0
3 2654 0.11889 2112461 0.11843 99.6
1 2657 0.12908 2314031 0.12973 100.5
130 2 2658 0.12908 2308495 0.12942 100.3 100.4
3 2659 0.12908 2309676 0.12949 100.3

1 API Concentration Added = Spiked Standard Concentration + (Concentration of Stock Sample Solution*Mean Assay Value)
2 %Recovery = (API Conc. Found (mg/mL)/API Conc. Added (mg/mL))*100
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Accuracy
From USP <1225>:
“Assessment of accuracy can be accomplished in a variety of ways, including

evaluating the recovery of the analyte (percent recovery) across the range of
the assay, or evaluating the linearity of the relationship between estimated and
actual concentrations. The statistically preferred criterion is that the confidence
interval for the slope be contained in an interval around 1.0, or alternatively, that
the slope be close to 1.0. In either case, the interval or the definition of
closeness should be specified in the validation protocol.
The acceptance criterion will depend on the assay and its variability and on the
product. Setting an acceptance criterion based on the lack of statistical
significance of the test of the null hypothesis that the slope is 1.0 is not an
acceptable approach.”
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Case Study
Recovery:
Amount added (mg) Amound observed (mg) Recovered 40
y = 0.9989x + 0.3272
24.35 24.70 101.4% R² = 0.9974
25.15 25.41 101.0%
25.15 25.17 100.1% 35
30.04 30.79 102.5%
observed
29.74 30.18 101.5%
30.14 30.38 100.8%
30
35.33 35.70 101.0% SUMMARY OUTPUT
34.63 34.56 99.8%
36.73 37.01 100.8% Regression Statistics
Mean: 101.0% Multiple R 0.99870599 25
SD: 0.7945 R Square 0.997413654
Adjusted R Square 0.997044177
Ecuacion de la recta
Standard Error 0.253568809
Typical acceptance criteria: Observations 9
20
Encontrado = a + ( b x Añadido)
20 25 30 35 40
b=
added 0.9989
 Mean Recovery ANOVA a= 0.3272 Valor
df SS MS F Significance F
 Individual recovery Regression 1 173.57152 173.5715 2699.521582 2.56314E-10
 Regression analysis of known vs estimated Residual 7 0.450079988 0.064297
Total 8 174.0216
 Slope within 0.9-1.1
Coefficients Standard Error t Stat P-value Lower 95% Upper 95%
 95% confidence interval of slope includes 1 Intercept 0.327238102 0.585575609 0.558832 0.593694893 -1.057428184 1.711904388
X Variable 1 (slope) 0.998888347 0.019225319 51.95692 2.56314E-10 0.953427693 1.044349002
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Accuracy
Impurities
 If impurities are available
– Spike sample with impurities
 If impurities are unavailable
– Comparison with a reference method
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Case Study – Accuracy-API (Organic Impurities)
Impurity Standard at 100 %
Area of Impurity
Preparation Weight taken (mg) Average Area
Injection 1 Injection 2
1 47149 46736 46943
2 2.048 46927 47239 47083
3 47299 47352 47326
Average 47117
Std Deviation 193.750
%RSD 0.41
Impurity Content in in Test Sample

Area of Impurity
Preparation Weight taken (mg) Average Area Impurity % (w/w)
1 50.02 4547 4688 4618 0.050
2 50.27 4556 4607 4582 0.050
3 50.73 4493 4651 4572 0.049
Average 0.050
Std Deviation 0.001
%RSD 1.201
Impurity Content in Spiked Sample

Spiked Sample (Sample + Lower Limit Impurity )
Area of
Impurity A % Obtained Impurity
Preparation Weight taken (mg) Average Area % Impurity added % Recovery
%(w/w)
1 50.20 9434 9495 9465 0.102 0.051 102.85
2 50.81 9427 9454 9441 0.100 0.050 100.91
3 50.26 9463 9444 9454 0.101 0.051 100.89
Average 0.10 0.05 101.55
Std Deviation 0.001 0.001 1.126
%RSD 0.99 1.14 1.11
Impurity (%) = (Impurity area in Test Sample x Concentration of Impurity Standard x100)/ (Impurity standard area x Concentration of Test Sample)
% Obtained Impurity = Impurity area in Spike Sample x Concentration of Impurity Standard x100)/ (Impurity standard area x Concentration of Spiked Sample)
% Impurity added = Concentration of impurity/ Concentration of sample x 100
Recovery (%) = (% impurity obtained - Impurity % in Test sample)/ (% Impurity added) x 100
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(continued)
Spiked Sample (Sample + 50% Impurity )
Area of Impurity % Obtained Impurity

(w/w)
1 50.20 28230 28056 28143 0.306 0.255 100.57
2 50.81 28062 28077 28070 0.301 0.252 99.78
3 50.26 28263 28157 28210 0.306 0.255 100.57
Average 0.30 0.25 100.31
Std Deviation 0.003 0.002 0.456
%RSD 0.95 0.68 0.45
Spiked Sample (Sample + 100% Impurity )

(w/w)
1 50.20 51495 51380 51438 0.557 0.510 99.50
2 50.81 51522 51400 51461 0.550 0.504 99.30
3 50.26 51173 51747 51460 0.556 0.509 99.50
Average 0.55 0.51 99.43
Std Deviation 0.004 0.003 0.115
%RSD 0.68 0.63 0.12
Spiked Sample (Sample +150% Impurity )

(w/w)
1 50.20 99248 99504 99376 1.075 1.020 100.53
2 50.81 99104 99296 99200 1.060 1.008 100.24
3 50.26 99280 99514 99397 1.074 1.019 100.54
Average 1.07 1.02 100.44
Std Deviation 0.008 0.007 0.170
%RSD 0.78 0.66 0.17
Impurity (%) = (Impurity area in Test Sample x Concentration of Impurity Standard x100)/ (Impurity standard area x Concentration of Test Sample)
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Case Study – Accuracy-API (Organic impurities)
 Preparation of Impurity Standard stock solution (0.01-mg/mL): Weighed

and transferred about 2.0 mg of Impurity into a 200 mL volumetric flask and
sonicate to dissolve in about 120 mL of diluent. Diluted to volume and mixed
well by inversion.
 Preparation of Impurity Standard solution (0.001-mg/mL): Taken 2.0 mL of
Impurity Standard stock solution into 20 mL of volumetric flask and made up to
the mark with diluent.
 Sample solution (0.2-mg/mL): Weighed and transferred about 50 mg of API
into a 50 mL of volumetric flask and sonicated to dissolve in about 30 mL of
diluent. Diluted to volume and mixed well by inversion. Taken 4.0 mL of above
solution into 20 mL of volumetric flask and made up to the mark with diluent.
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 Spiked sample solution (solution containing 0.2-mg/mL of API and 0.001-

mg/mL Impurity) : Weighed and transferred about 50 mg of API into a 50mL of
volumetric flask, and sonicate to dissolve in about 30 mL of diluent. Diluted to
volume and mixed well by inversion. Taken 4.0 mL of above solution and 2.0 mL
of Impurity Standard stock solution into 20 mL of volumetric flask and made up
to the mark with diluent.
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System suitability chromatogram
Impurity Standard chromatogram
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Unspiked sample chromatogram
Spiked sample chromatogram
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Case Study – Accuracy-Drug Product (Organic Impurities)
Impurity
Tablet Spiked Sample (Sample + Lower Limit Impurity )
Average weight of tablet
Preparation Label claim (mg) Weight taken (mg) Equivalent weight (mg) Area of Impurity Obtained Impurity (%w/w) Impurity added (%w/w) % of Recovery
(mg)
1 804.55 20.01 103110 0.1028 0.1024 100.4
2 201.03 5 804.73 20.02 104118 0.1038 0.1024 101.4
3 804.33 20.01 103886 0.1036 0.1024 101.2
Average 101.0
Std Deviation 0.52
%RSD 0.51
Tablet Spiked Sample (Sample + 100% Impurity )

(mg)
1 804.87 20.02 508512 0.5199 0.5125 101.4
2 201.03 5 804.14 20.00 502462 0.5142 0.5125 100.3
3 804.65 20.01 506059 0.5177 0.5125 101.0
Average 100.9
Std Deviation 0.56
%RSD 0.55
Tablet Spiked Sample (Sample + 150% Impurity )

(mg)
1 804.51 20.01 1014121 1.0408 1.025 101.5
2 201.03 5 804.35 20.01 1003882 1.0302 1.025 100.5
3 804.62 20.01 1000471 1.0267 1.025 100.2
Average 100.7
Std Deviation 0.71
Impurity (%) = (Impurity area in Test Sample x Concentration of Impurity Standard x100)/ (Impurity standard area x Concentration of Test Sample) %RSD 0.71
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 Preparation of Impurity Stock solution-I (0.1mg/mL): Weighed and

transferred about 2 mg of Impurity into a 20 mL of volumetric flask, dissolved in
15 mL of Acetonitrile and made up to the mark with diluent.
 Preparation of Impurity Stock solution-2 (0.010 mg/mL): Taken 2 mL of
Impurity Stock solution-I in to 20 mL of volumetric flask and made up to the mark
with diluent.
 Preparation of Standard solution (0.001 mg/mL): Taken 2.0 mL of Impurity
stock solution-2 into 20 mL of volumetric flask and mad up to the mark with
diluent.
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 Preparation of Tablets sample solution (1.0 mg/mL): Weighed and

transferred Tablet powder equivalent to 20 mg of API into a 20 mL of volumetric
flask. To this solution 15 mL of diluent was added and sonicated for 30 min. Now
this solution was made up to the mark with diluent and centrifuged the solution
for 10 min at 3000 rpm and collected supernatant liquid.
 Preparation of Tablets Spiked sample solution (1.0mg/mL of Sample +
0.10% Impurity): Weighed and transferred Tablet powder equivalent to 20 mg of
into a 20 mL of volumetric flask. To this solution 2 mL of Impurity Stock solution-
2, 15 mL of diluent was added and sonicated for 30 min. Now this solution was
made up to the mark with diluent and centrifuged the solution for 10 min at 3000
rpm and collected supernatant liquid.
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Un spiked sample chromatogram
Spiked sample chromatogram
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
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Linearity and Range – Impurities
USP <1225>:
 “The linearity of an analytical procedure is its ability to elicit test results that are
directly, or by a well-defined mathematical transformation, proportional to the
concentration of analyte in samples within a given range.”
 “The range of an analytical procedure is the interval between the upper and
lower levels of analyte (including these levels) that have been demonstrated to
be determined with a suitable level of precision, accuracy, and linearity using the
procedure as written.”
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Q2(R1): Recommended Ranges to Validate
For the assay of a drug substance or a finished (drug) product: normally from 80
to 120 percent of the test concentration
For content uniformity, covering a minimum of 70 to 130 percent of the test
concentration, unless a wider more appropriate range, based on the nature of
the dosage form (e.g., metered dose inhalers), is justified
For dissolution testing: +/-20 % over the specified range
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Linearity and Range Considerations for Validation Protocol
Acceptance criteria should balance scientific rigor with practical needs
Linearity -
 Minimum r2 value-  0.99 up to  0.9999
 Y intercept- statistically insignificant, within n% of the response of the standard

solution
 Suitable for assay by a single point standard
Range - necessary for accurate and precise results
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Regression Analysis
r2 and r are a evaluation of the degree of fit of the data points to the regression line
The coefficient of determination (r2) measures the proportion of variation that is

explained by the model. Ideally, r2 should be equal to one, which would indicate zero error.
The correlation coefficient (r) is the correlation between the predicted and observed
values. This will have a value between 0 and 1; the closer the value is to 1, the better the
correlation.
Additional statistical analysis is recommended to provide estimates of systematic errors,

not just the correlation or results.
For instance, in method comparison studies, if one method gives consistently higher
results than the other method, the results would show linear correlation and have a high
correlation coefficient, despite a difference between the two methods.
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Linearity
Calibration model Requirements

Single-point calibration  Linear response function
 Negligible systematic error
 Homoscedasticity
Multiple-point calibration (linear, unweighted)  Linear response function

 Homoscedasticity
Multiple point calibration (linear, weighted)  Linear response function
Multiple point calibration (non-linear)  Continuous response function

Area normalization  For main peak
‒ Linear response function
‒ Negligible systematic error
‒ Homoscedasticity
 For impurities
‒ Linear response function
‒ Negligible systematic error
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ICH Guideline
Performed directly on the drug substance by dilution of a standard stock solution
AND / OR:
Separate weighing of synthetic mixtures of the drug product components
ICH recommends minimum of five concentrations
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Linearity
Amount(ppm) Response Log amount Response/amount

1 1350 0.0000 1350.000
3 3600 0.4771 1200.000 Plotting the sensitivity (response/amount) gives a clear indication
10 12200 1.0000 1220.000 of the linear range.
20 24200 1.3010 1210.000
30 36800 1.4771 1226.667 Plotting the amount on a logarithmic scale has a significant
40 49280 1.6021 1232.000 advantage for wide linear ranges. Rc designates the line of
50 59836 1.6990 1196.720
60 66500 1.7782 1108.333
constant response.
80000 1500.000
y = 1148.1x + 1008.9
R² = 0.995 R
70000 e
1400.000
R s
60000 p
e
s o 1300.000 1.05 Rc
50000
p n
o 40000
s Rc
1200.000
n e
0.95 Rc
s 30000 /
e A 1100.000
20000 m
o
10000 u 1000.000 Calibration range
n
0 t 900.000
0 10 20 30 40 50 60 70
0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 1.2000 1.4000 1.6000 1.8000 2.0000
Amount (ppm)
Log Amount
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Regression Analysis
Y- intercept, slope, and residuals
y
Response
x
y/x = slope
D y = residual
Y-intercept
0 0.05 0.1 0.15 0.2

Concentration
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Regression Analysis
Each residual :( y  y
ˆ)
Which brings us to the concept of residual variance. So the regression line minimizes the sum of the square
deviations of the y-direction from the scatter plot points to the line.
 ( y  yˆ )
2
S 
i i
i
Where n= number of points in the scatter plot
n2
y x
This is also called residual variance, because it's the variance, it's also called the mean square. In this case, the
mean square residual, or MS residual.
Analogous to the variance in its square root, the standard deviation, you can find the square root of residual
variance and this is called the standard error of estimate.
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Confidence Intervals
The random error in the y-direction:
 i ˆi
( y  y ) 2
Sy x  i Where is the exp value against x concentration

n2 and is the value calculated from regression line.
Standard deviation for the slope (b):

Sy Confidence limit of the slope of line:
Sb  x
 i
( x
i
 x ) 2 b ± t(n-2) Sb
where the t-value is taken at the desired

Standard deviation for the intercept (a): confidence level and (n-2) degrees
 i
x 2 of freedom
Sa  S y i Confidence limit for the intercept
n ( xi  x )
x 2
a ± t(n-2) Sa
i
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Residue Analysis
Análisis de residuos
20
15
10
Residuals
5
0
-5
-10
-15
-20
0 5 10 15 20 25 30 35
C (m g/m l)
- Check that the graph has a random pattern.

- A trend indicates a failure in the chosen model.
- An increase or decrease in residue dispersion indicates that the data is not homoscedastic.
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Residue Analysis
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Example of Linearity
Concentration Area
0.050 1250
0.050 1260
0.050 1155
0.105 2625
0.105 2550
0.105 2700
0.120 3000
0.120 3150
0.120 3090
0.150 3750
0.150 3800
0.150 3755
0.175 4375
0.175 4402
0.175 4450
0.250 6250
0.250 6311
0.250 6288
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Hypothesis:
H0= Conc. has no effect on

Response
H1= Conc. has effect on response
In this case, P is less than 0.05

hence
H1 is valid .
R square means 99.88% values
are explaining the linear
relationship, while 0.12% is
because of residual error.
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SUMMARY OUTPUT
Regression Statistics Equation (y=mx+c) : 25247x -12.23

Multiple R 0.999419737
Intercept (c) = -12.23 (The value of y when x=0)
R Square 0.998839811
Adjusted R Square 0.998767299 Standard Error, (SE intercept) = 33.27
Standard Error 56.67013031 95% CI of SE (intercept)= -82.76 to 58.31
Observations 18
Slope (m) = 25247
ANOVA Standard error (SE of slope) = 215.11
df SS MS F Significance F 95% CI of SE (slope) = 24791 to 25703
Regression 1 44238000.44 44238000.44 13774.85595 6.4499E-25
Coefficient of determination (r2)=0.9988
Residual 16 51384.05872 3211.50367
Total 17 44289384.5
Correlation coefficient (r)= 0.9994
(This will be between 0 to 1, the closure the value 1, the better the
correlation)
Coefficients Standard Error t Stat P-value Lower 95% Upper 95%
Intercept -12.22610471 33.2736504 -0.36744104 0.718105085 -82.76309252 58.31088311
Regression SS = 44238000
X Variable 1 25247.47839 215.1168729 117.3663323 6.450E-25 24791.45099 25703.50579 (Regression sum of squares is the amount of variability in the response)
Residual SS = 51384
Typical acceptance criteria: (Residual sum of squares is the variability about regression line, the
amount of uncertainty remains)
 Valid calibration model e.g. r and r2 Total SS = 44289384

(The total sum of squares is the total amount of variability in the response )
 Residual plots shows random scatter and no systematic trends
 95% confidence interval of intercept includes zero
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Q2(R1): Recommended Ranges to Validate
 For the determination of an impurity: from the reporting level of an impurity to

120% of the specification
– For impurities known to be unusually potent or to produce toxic or unexpected
pharmacological effects, the detection/quantitation limit should be commensurate with the level
at which the impurities must be controlled
 If assay and purity are performed together as one test and only a 100%
standard is used, linearity should cover the range from the reporting level of the
impurities to 120% of the assay specification
105
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
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Detection/Quantification Limits
 Detection Limit – The minimum concentration of analyte that can be detected

under the established experimental conditions
 Quantification Limit – The lower concentration of analyte that can be

determined with acceptable precision and accuracy
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Threshold Limits
Q3A(R2): Impurities in New Drug Substances
Maximum Daily Reporting Identification Qualification

Dose Threshold Threshold Threshold
</= 2g/daily 0.05% 0.1% o 1.0 mg per 0.15% o 1.0 mg per
day (whichever is day (whichever is
lower) lower)
> 2g/daily 0.03% 0.05% 0.05%
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Q3B(R2): Impurities in New Drug Products :
ICH Threshold for impurities in Drug Products

Maximum Daily Dose Threshold
Reporting thresholds
≤ 1 g/day 0.1%
>1g 0.05%
Identification thresholds
<1 mg 1.0% or 5 µg TDI, whichever is lower
1 mg -10 mg 0.5% or 20 µg TDI, whichever is lower
>10 mg - 2 g 0.2% or 2 mg TDI, whichever is lower
>2 g 0.10%
Quantification thresholds
<10 mg 1.0% or 50 µg TDI, whichever is lower
10 mg - 100 mg 0.5% or 200 µg TDI, whichever is lower
> 100 mg - 2 g 0.2% or 3 mg TDI, whichever is lower
>2g 0.15%
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Basic Approaches to Determination
USP <1225> (and ICH Q2) lists the following

 Visual
 Signal-to-Noise ratio
 Blank standard deviation and regression line
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Visual
 I can see it!!

 Determined by the analysis of samples with known concentrations of analyte
and by establishing the minimum level at which the analyte can be reliably
detected
 Seems more appropriate for LOD
 Subject to analyst interpretation
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Signal-to-Noise Ratio
 Q2 and USP <1225> -- Compare

measured signals from samples with 10:1
known low concentrations of analyte with
those of blank samples
 Requirements
S/N > 3 (or 2) for LOD
3:1
S/N > 10 for LOQ
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Signal-to-Noise Ratio
h: range of the background noise in a

chromatogram obtained after injection
or application of a blank, observed
over a distance equal to 5 times the
width at half-height of the peak in the
chromatogram obtained with the
prescribed reference solution and, if
possible, situated equally around the
place where this peak would be found
S/N = 2H/h
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Based on the standard deviation of the response:
3.3  S 10  S
LOD  LOQ 
b b
S = Standard deviation of the response based on either

 The standard deviation of the blank
 Residual standard deviation of the regression line (standard error)
b = Slope of the calibration curve
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Options for S:
SD of blank responses
– Analyze various blanks and calculate the SD
Residual SD from regression line
 ( y  yˆ )
i i
2
Sy x  i
n2
 The residual standard deviation is also called standard error or random error in the y direction.
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Basis for this Approach
5%
Blank
 The 3.3 and 10 SD values
5%
+3.3 Sigma
+10 Sigma
are intended to separate the
distribution of responses at
LOD and LOQ from the
distribution of blank
samples
+10 SD
 Dividing by the slope, b,
converts differences in y
(response) axis to
+3.3 SD
differences in x
(concentration) axis
blank response
LOD LOQ 116

© 2018 USP
Topics

 Specificity
 Precision
 Accuracy
 Robustness
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Robustness
Definition - The ability to remain unaffected by small but deliberate variations in

method parameters
Ruggedness vs. Robustness

– They are different
– Ruggedness: Performance under normal variability
– Robustness: Small but deliberate changes
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Robustness
Experimental design
 One factor at a time
 Simple to Analyze
 Time Consuming
 Interactions between factors are not detectable
 Factorial Designs
– Full Factorial (Useful for 2 to 4 factors)
• Can determine main effects of the factors manipulated on response variables
• Can determine effects of factor interactions on response variables
• Can estimate levels at which to set factors for best result
• Time consuming
– Fractional Factorial (Useful 5 or more than 5 factors)
– Plackett-Burman –
 Very efficient screening designs where only main effects are of interest
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Full Factorial Design (2k Runs)
% Organic
TEA TEA
Level=2 (high and low)

Flow Flow
Factors= 5
pH pH 25 = 32 experiments
Particle size
TEA TEA
Flow Flow
pH pH
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Fractional Factorial Design – 5 Factors
% Organic
TEA TEA
"A factorial experiment in which

only an adequately chosen fraction
Flow of the treatment combinations
Flow
pH required for the complete factorial
pH
Particle size experiment is selected to be run."
16 experiments
TEA TEA
Flow Flow
pH pH
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Placket Burman
Plackett-Burman (PB) designs are used for screening experiments because, in a PB design, main
effects are, in general, heavily confounded with two-factor interactions.
7 factors (A-G) 2 levels
Exp. Factors Result
A B C D E F G
1 + + + + + + + y1
2 + + - + - - - y2
3 + - + - + - - y3
4 + - - - - + + y4
5 - + + - - + - y5
6 - + - - + - + y6
7 - - + + - - + y7
8 - - - + + + - y8
Eff 
 y    y 
𝑇=
2
𝐸𝑓𝑓
4 𝑆𝑅 122
© 2018 USP
Summary
Design type Description No. of factors

Full Factorial - Measures responses at all combinations of factor level 2-15
- Run factor at 2 level
Fractional - Measures responses for subset of original full design 2-15
factorial - Run factor at 2 level
Plackett-Burman - Identifies significant main Effect 2-47
- Run factor at 2 level
Different available software's:

 ACD
 Minitab
 Design experts
 Others
123 123
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Topics

 Specificity
 Precision
 Accuracy
 Robustness
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USP <621> Chromatography
System Suitability
System Suitability
Definition - System suitability tests are an integral part of gas and liquid
chromatographic methods. They are used to verify that the resolution and
reproducibility of the chromatographic system are adequate for the analysis to
be done. The tests are based on the concept that the equipment, electronics,
analytical operations, and samples to be analyzed constitute an integral system
that can be evaluated as such.
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System Suitability
Provides assurances that the system is working properly at the time of analysis
Ensures that both methodology and instrumentation are performing within

expectations prior to the analysis of the test samples
Should be monitored during run time to verify that the criteria remain realistic
and achievable
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System Suitability
Parameters for a chromatographic method

 Resolution - specificity
 Column efficiency - specificity
 Relative Standard Deviation - precision
 Tailing Factor - accuracy and precision
 QL sensitivity solution
 Capacity factor - specificity
 Reference Standard Check - analyst
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System Suitability
Resolution (R)
 Function of column efficiency (N)
 Separation factor (α)
 Capacity factor (k)
 Measure of the resolving power of the system
 Generally, not less than 2.0
 Most closely eluting pair
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System Suitability – Resolution
2(t 2  t1 ) 1 (  1) k
R R N x x
W2  W1 4  1 k
>1.5 for the critical pair
R = 1,0 R = 1,5 R = 2,0
FDA (CDER), Reviewer Guidance: Validation of Chromatographic Methods, Nov. 1994  Efficiency , separation factor and Retention factor
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System Suitability – Resolution
Peak-to-Valley Ratio
p / v  H p Hv
Pemetrexed sodium: p/v NLT 1.5
Somatropin : Resolution, NMT 0.4

for the ratio of the valley height,
between the dimer and the monomer,
and the dimer peak height
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System Suitability
Column Efficiency (N)

 Only one peak of interest
 Measure of peak sharpness
 Detection of trace components
 Generally not less than 2000 (HPLC)
 Isocratic/isothermal system
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System Suitability – Column Efficiency
N= 16 (tR/w)2 (Manual calculation)
N= 5.54 (tR/wh/2)2 (Electronic calculation)
– tR = Retention time of the substance

– W = peak width at baseline
– Wh/2 =peak width at half-height
Note: In the case of dispute, only equations based on peak width at baseline are
to be used
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System Suitability
Relative Standard Deviation (RSD)
 Replicate injections of a Standard preparation

 Assessment of repeatability of the system
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System Suitability
Repeatability Requirements
Data from five replicate injections of the analyte to calculate the %RSD if the
requirement is 2.0% or less
Data from six replicate injections are used if the relative standard deviation
requirement is more than 2.0%
For the Assay of drug substances, when no maximum relative standard
deviation is stated
K: constant (0.349)
K .B n B: Upper limit – 100
% RSD 
t 90%, n  1 n: number of injections
t90%,n-1: Student’s t at the 90% probability level with n-1 degrees
of freedom
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System Suitability
Repeatability Requirements
Number of individual injections

3 4 5 6
K .B n
B (per cent) Maximum permitted relative standard deviation % RSD 
2.0 0.41 0.59 0.73 0.85 t 90%, n  1
2.5 0.52 0.74 0.92 1.06
3.0 0.62 0.89 1.10 1.27
Example 1
K=0.349
B=102-100=2
n = 3 ; √3=1.7321
t 90%, 3-1 =2.920 (two tailed)
0.349 x 2 x 1.7321 = 0.41

2.920
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System Suitability
Tailing Factor (T)

 Measure of peak symmetry
 Equals one for perfectly symmetrical peaks
 Value increases as tailing becomes more pronounced
 Peak asymmetry increases, accuracy and precision becomes less reliable
137
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System Suitability
Tailing factor (Symmetry factor)
W0.05
As 
2f
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System Suitability
Signal to noise ratio
S/N = 2H/h
where H is the height of the peak measured from the peak apex to a
baseline extrapolated over a distance 5 times the peak width at its half-
height; and h is the difference between the largest and smallest noise values
observed over a distance 5 times the width at the half-height of the peak
and, if possible, situated equally around the peak of interest
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System Suitability – Allowed Variations

USP <621> includes adjustments that can be made to the chromatographic
system in order to meet system suitability requirements
“The user should verify the suitability of the method under the new conditions by
assessing the relevant analytical performance characteristics potentially affected
by the change.”
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System Suitability – Allowed Variations for HPLC
The amount of minor component can be adjusted by ±30% relative. However,

the change in any component cannot exceed ±10% absolute (i.e., in relation to
the total mobile phase).
The pH of the aqueous buffer used in the preparation of the mobile phase can
be adjusted to within ±0.2 units of the value or range specified.
Wavelength of UV-Visible Detector (HPLC): Deviations from the wavelengths

specified in the procedure are not permitted.
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Column inner diameter (HPLC): Can be adjusted if the linear velocity is kept
constant.
Injection volume: can be reduced as far as is consistent with accepted
precision and detection limits; no increase is permitted.
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Chromatography <621>
“Adjustments to the specified chromatographic system may be necessary in order

to meet system suitability requirements. In other circumstances, it may be
desirable to use an HPLC column with different dimensions to those prescribed in
the official procedure (different length, internal diameter, and/or particle size). In
either case, changes in the chemical characteristics (L designation) of the
stationary phase will be considered a modification to the method and will require
full validation.”
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 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% to +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.
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Flow rate (HPLC): When column dimensions have been modified, the flow rate
can be adjusted using the following formula:
dc22  dp1
F2  F1
dc12  dp2
– dc1 and dc2: respective column diameters
– dp1 and dp2: respective particle sizes
– Additionally the flow rate can be adjusted ± 50%
Column Temperature (HPLC): The column temperature can be adjusted by as

much as ±10°. Column thermostating is recommended to improve control and
reproducibility of retention time.
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dc22  dp1
F2  F1
dc1  dp2
2
Relative Values
L, mm dc, mm dp, µm L/dp F N Pressure 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
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Adjustments: Isocratic vs. Gradient
Adjustment Isocratic Gradient
pH of Mobile Phase Allowed Allowed

Concentration of Salts in Buffer Allowed Allowed
Allowed Not allowed
Ratio of Components in Mobile Phase
Column dimensions (length, diameter) Allowed Not allowed

Particle size Allowed Not allowed
Flow rate Allowed Not allowed
Injection volume Allowed Allowed
Column temperature Allowed Allowed
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Harmonization - Pharmacopoeial Discussion Group (PDG)
USP has participated since PDG’s inception in 1989

Informal body of representatives from
– European Pharmacopoeia (EDQM)
– Japanese Pharmacopoeia (MHLW)
– United States Pharmacopeia (non-governmental)
– WHO (observer since 2001)
Linked to ICH (until 2011)
Focused on harmonizing general chapters and excipient monographs
Steady but limited progress (Retrospective Harmonization)
– 29 out of 36 General Chapters
– 46 out of 62 Excipients
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Liquid chromatography: isocratic elution
Column parameters and flow rate
 Stationary phase: no change of the physico-chemical characteristics of the

stationary phase permitted
 Column dimensions:
– 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 in the range between -25 per cent to +50 per
cent of the prescribed L/dp ratio.. Further adjustments in method conditions (mobile phase,
temperature, pH, etc.) may be required, within the permitted ranges described under System
Suitability and Adjustment of chromatographic conditions in this chapter.
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When the particle size is changed, the flow rate may require adjustment,
because smaller-particle columns will require higher linear velocities for
the same performance (as measured by reduced plate height). Flow rate
changes for both the change in column diameter and particle size can
be made by:
F2 = F1 (dc22 dp1) / (dc12 dp2)
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Injection volume
When the column dimensions are changed, injection volume adjustment may be
guided by,
Vinj,2 = Vinj,1 (L2 dc,22) / (L1 dc,12)
Vinj,1 = original injection volume
Vinj,2 = new injection volume
L1 = original column length
L2 = new column length
dc,1 = original column internal diameter
dc,2 = new column internal diameter
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Liquid chromatography: isocratic elution (cont)
Independent of changing column dimensions, Injection volume may be varied provided System
Suitability criteria remain within their established acceptability limits. When Injection volume
is decreased, special attention should be given to (limit of) detection and repeatability of the
peak(s) to be determined. An increase is permitted provided in particular linearity and separation
of the peak(s) to be determined remain satisfactory
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Liquid chromatography: gradient elution
A change in column dimensions, and thus in column volume, impacts the gradient volume
which controls selectivity. Gradients are adjusted to the column volume by changing the
gradient volume in proportion to the column volume. This applies to every gradient segment
volume. Because the gradient volume is the gradient time, tG, multiplied by the flow rate, F,
the gradient time for each gradient segment needs to be adjusted to maintain a constant ratio
of the gradient volume to the column volume (expressed as L x dc2). Thus, the new gradient
time, tG2 can be calculated from the original gradient time, tG1, the flow rate(s), and the column
dimensions as follows:
tG2 = tG1 x (F1 / F2) [(L2 x dc22) / (L1 x dc12)]
Thus, the change in conditions for gradient elution requires three steps:
– (1) adjust the column length and particle size according to L/dp,
– (2) adjust the flow rate for changes in particle size and column diameter, and
– (3) adjust the gradient time of each segment for changes in column length, diameter and flow rate. The
example below illustrates this process.
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Variable Original Adjusted Conditions Comment

Conditions
Column length (L) in mm 150 100 user choice
Column diameter (dc) in mm 4.6 2.1 user choice
Particle size (dp) in µm 5 3 user choice
L/dp 30.0 33.3 (1)
Flow rate in mL/min 2.0 0.7 (2)
Gradient adjustment factor 0.4 (3)
Gradient conditions
%B Time (min) Time (min)
30 0 0
30 3 (3x0.4)=1.2
70 13 [1.2+(10x0.4)]=5.2
30 16 [5.2+(3x0.4)]=6.4
(1) 11% increase within allowed L/dp change of -25% to +50%
(2) calculated using F2= F1 [(dc22 x dp1) / (dc12 x dp2)]
(3) calculated using tG2 = tG1 x (F1 / F2) [(L2 x dc22) / (L1 x dc12)]
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Injection volume:
When the column dimensions are changed, injection volume adjustment may be
guided by,
Vinj,2 = Vinj,1 (L2 dc,22) / (L1 dc,12)
Independent of changing column dimensions, injection volume may be varied

provided System Suitability criteria remain within their established acceptability
limits. When Injection volume is decreased, special attention should be given to
(limit of) detection and repeatability of the peak(s) to be determined.
An increase is permitted provided in particular linearity and separation of the

peak(s) to be determined remain satisfactory
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Case study 1
Column length Column diameter Particle size L/dp ratio Allowable L/dp
L, mm dc, mm dp, µm (-25% to +50%)
250 4.6 5(0.005 mm) 50,000 37,500 – 75,000
100 4.6 2.6(0.0026 mm) 38462
Can we use this new column - Yes
156 156
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Case study 2
Column Column Particle size L/dp Allowable L/dp Efficiency

length diameter dp, µm ratio (-25% to +50%) (N)
L, mm dc, mm
150 4.6 3(0.003 mm) 50,000 37,500 – 75,000 12560
150 4.6 2.6(0.0026 mm) 57692
100 4.6 2.6(0.0026 mm) 38462
75 4.6 2.6(0.0026 mm) 28846 16100

(15700-18840)
Alternatively for particle size adjustment of superficially porous particle; Efficiency N is within -25% to +50%
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System Suitability – Allowed Variations for GC
Column length (GC): can be adjusted by as much as ±70%.
Oven Temperature (GC): The oven temperature can be adjusted by as much

as ±10%.
Oven Temperature Program (GC): When the specified temperature must be

maintained or when the temperature must be changed from one value to
another, an adjustment of up to ±20% is permitted.
Column inner diameter (GC)— Can be adjusted by as much as ±50%
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System Suitability – Allowed Variations for GC
Film thickness (GC): can be adjusted by as much as -50% to 100%.
Particle Size (GC): Same particle size range ratio
Injection
Volume and Split Volume (GC):The injection volume and split
volume may be adjusted if detection and repeatability are satisfactory.
Flow rate (GC): can be adjusted by as much as ±50%.
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Topics

 Specificity
 Precision
 Accuracy
 Method Development - Robustness
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Verification of Compendial
Procedures
Verification in USP
Verification of microbiological procedures:

– USP <51> Antimicrobial Effectiveness
– USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests
– USP <71> Sterility Tests
– USP <1227> Validation of Microbial Recovery From Pharmacopeial Articles
Verification of biological procedures:

– USP <111> Design and Analysis of Biological Assays
– USP <1033> Validation of Biological Assays (in development)
– USP <1226> Verification of Compendial Procedures
– And others
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Definitions for Verification: USP
USP <1225> Validation of Compendial Procedures:

“users of analytical methods described in the USP-NF are not required to validate
accuracy and reliability of these methods, but merely verify their suitability under
actual conditions of use.” [21 CFR 211.194(a)(2)]
USP <1226> Verification of Compendial Procedures: “Verification consists of

assessing selected analytical performance characteristics, such as those that
are described in chapter <1225>, to generate appropriate, relevant data rather
than repeating the validation process.”
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Definitions for Verification: FDA
 Guide to Inspections of Pharmaceutical Quality Control Laboratories:
 “Methods appearing in the USP are considered validated and they are
considered validated if part of an approved ANDA”
 “For compendial methods firms must demonstrate that the method works under
the actual conditions of use.”
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A Risk-Based Approach
 “Verification requirements should be based on an assessment of the complexity

of both the procedure and the material to which the procedure is applied.”
 “The degree and extent of the verification process may depend on the level of
training and experience of the user, on the type of procedure and its associated
equipment or instrumentation, on the specific procedural steps, and on which
article(s) are being tested.”
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Basic Concepts
 Validation: Challenges the analytical method using a well defined sample

 Verification: Challenges the analytical environment using a well defined method
– Analyst (education, training, experience)
– Instrument
– Reagents
– Matrix
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Verification Process
There are no general verification protocols
Verification
DEGREE OF VERIFICATION
Perform the test

Full validation
as written
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Why is USP <1226> Needed?
21 CFR 211.194(a)(2): “users of analytical methods described in USP–NF are

not required to validate the accuracy and reliability of these methods, but merely
verify their suitability under actual conditions of use.
Response to industry inquiries
Official USP 30 Second Supplement
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USP <1226> Scope
 Applies to drug substances, drug products, and excipients.
 Application: titrations, chromatographic procedures, spectroscopic tests, etc.
 Verification is not required for basic compendial test procedures that are
routinely performed unless there is an indication that the compendial procedure
is not appropriate for the article under test.
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Verification
Typical industry practices for verification of compendial

procedures
 Specificity –
– Peak purity
– Matrix
 Accuracy: Recovery in the specification range
 Precision: Repeatability in the specification range
 LOD: Verification of detection at 50% of the specification
 LOQ: Verification of quantitation at 50% of the specification
 Usually not needed: Linearity, Range, and Robustness
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Verification
Common sources of failure
 Chromatographic column definition
 Dwell Volume
 Mobile phase preparation
 Sample preparation
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Case Study – API
Identification
– IR, UV Performance Validation Verification
Characteristic
– HPLC
Accuracy Yes Yes
Purity test
Precision Yes Yes
– Loss on drying, residue on ignition
– Heavy metals Specificity Yes Maybe
– pH LOD No No
– Related substances
LOQ Yes Yes
Assay
Linearity Yes No
– Titrimetric
– HPLC Range Yes No
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Case Study – API
Performance Validation Verification

Identification Characteristic
– IR, UV Accuracy Yes Yes
– HPLC
Precision Yes Yes
Purity test
Specificity Yes Maybe
– Loss on drying, residue on ignition,
– Heavy metals LOD No No
– pH LOQ No No
Linearity Yes No
Assay
Range Yes No
– GC/HPLC
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Case Study – Dosage Form
Identification Performance Validation Verification

Characteristic
– HPLC
Accuracy Yes Yes
Specific tests
Precision Yes Yes
– Particle in injections
– Uniformity Specificity Yes Yes
– Dissolution LOD No No
Impurities:
LOQ Yes Maybe
– Residual solvents Linearity Yes No
Assay Range Yes No
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Case Study – Dosage Form
Identification Performance Validation Verification

– HPLC Characteristic
Accuracy Yes Yes
Specific tests
– Particle in injections Precision Yes Yes
– Uniformity
Specificity Yes Yes
– Dissolution
Impurities: LOD No No
– Related substances LOQ No No

– Residual solvents
Linearity Yes No
Assay
– HPLC Range Yes No
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USP <1226> Summary
The intent of this chapter is to provide general information to laboratories on the

verification of compendial procedures that are being performed for the first time
to yield acceptable results utilizing the laboratories’ personnel, equipment, and
reagents
Not intended for retroactive application to already successfully established

laboratory procedures
Verificationconsists of assessing selected Analytical Performance

Characteristics, such as those which are described in USP <1225>, to generate
appropriate, relevant data rather than repeating the validation process
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Decision Tree
Verification
Protocol
YES SUITABLE NO
Adjustment
Implementation
(<621>)
Contact
YES SUITABLE NO
USP
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Topics

 Specificity
 Precision
 Accuracy
 Method Development - Robustness
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Transfer of Analytical
Procedures
Initial Considerations
 To date, there have been no published regulatory guidelines for transfer of

analytical procedures
 Usually not applicable to Pharmacopeial procedures, but may help
 21 CFR Part 211.194 (a) (2): “The suitability of all testing methods used shall be
verified under actual condition of use.
 Warning Letters
02 documents: ISPE: International society for pharmaceutical engineering and FDA guidance for Vet medicines. 180
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Method Transfer – 483’s Observations
Documentation Issues:
 Failure to adequately address all validation characteristics for type of procedure.
 Transfer of methods to QC lab does not contain predetermined acceptance
criteria.
Training Issues:
 Prior to transfer, the QC analyst is not always sufficiently trained.
 The analytical method states the transfer was completed on (DATE), but the
official transfer documents were not completed as of (DATE-6 month later).
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Definition from USP <1224>
“The transfer of analytical procedures (TAP), is the documented process that

qualifies a laboratory (the receiving unit) to use an analytical test procedure
that originated in another laboratory (the transferring unit), thus ensuring that
the receiving unit has the procedural knowledge and ability to perform the
analytical procedure as intended.”
182
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Scope of the Chapter
The purpose of this general informational chapter is to summarize the types of

transfers that may occur, including the possibility of waiver, and to outline the
potential components of a transfer protocol.
Does not provide, at this time, any statistical approach for the evaluation of the
results.
Does not encompass the transfer of microbiological or biological procedures.
As a General information chapter, USP <1224> does not contain any standard
nor other mandatory specifications
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When Transfer is Needed?
R&D to QC labs
Site A to Site B (same company or not)
QC to Contract lab
Instrument A to Instrument B
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Risk Based Approach
The extent of the transfer activities, and the implementation strategy should be
based on risk analysis that considers
 Experience and knowledge
 The specification of the product, and
 The complexity of the analytical procedure
ICH Q9-Quality Risk management 185

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Type of Transfers
 Comparative testing
 Co-validation
 Revalidation
 Transfer waiver
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Comparative Testing
 Comparative testing is the most common method for TAP
 It is performed with validated procedures
 Typically does not challenge all validation parameters; it is a comparability study
 Requires the analysis of a predetermined number of samples of the same lot by

both the transferring and receiving units.
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Evaluation of the Results
 Comparing averages and precisions in both laboratories
 Equivalency test (general or statistical)
 USP <1010> Interpretation and treatment of analytical data
188
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Covalidation
 The transferring unit involves the receiving unit in an interlaboratory

covalidation, including them as a part of the validation team.
 Used when the procedure is not yet fully validated
 USP General Chapter Validation of Compendial Procedures <1225> provides
guidance about which characteristics are appropriate for testing.
 Statistical evaluation of the results may be challenging
189
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Evaluation of the Results
 Precision: Repeatability and intermediate precision. Reproducibility only if the

number of lab involved is appropriate.
 Accuracy
 Limit of quantitation
190
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Revalidation
 The receiving lab repeats some or all of the validation experiments to challenge
the applicable validation performance characteristics
 This approach is more time consuming and may be more difficult to observe
differences between different sites, operators, and instruments.
 Does not ensure equivalency of test methods between sites.
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Transfer Waiver
 The new product’s composition is comparable to that of an existing product and

is analyzed by procedures with which the receiving unit already has experience.
 The analytical procedure being transferred is described in the USP–NF and is
unchanged. Verification should apply in this case (see <1226>).
 The analytical procedure transferred is the same or very similar to a procedure
already in use.
 The personnel in charge of the development, validation, or routine analysis of
the product at the sending unit are moved to the receiving unit.
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Elements
 Training
 Protocol
 The analytical procedure
 The validation report
 Facilities and Instrumentation
 Samples and reference standards
 Transfer report
193
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Training
The transferring unit should provide training to the receiving unit, or the receiving
unit should run the procedure and identify any issues that may need to be
resolved before the transfer protocol is signed.
194
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Training
 Training depends on development stage, complexity of the method, and

experience with the instruments involved.
 Perform, if possible, all laboratory techniques needed to run the procedure

including any safety procedures or method precautions.
 Work out final details of the procedure
 Document results of training activities and analyst qualification
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Protocol
 A well-designed protocol should be discussed, agreed upon, and documented

before the implementation of TAP.
 The protocol should contain at least the following topics:

̶ Scope
̶ Roles and responsibilities of the transferring and receiving units
̶ Analytical procedure
̶ Experimental design and acceptance criteria
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Protocol
 All the details of the protocol should be discussed among the involved parts
– How many runs will be performed
– How many analysts / instruments will be involved
– The acceptance criteria
– If a transfer waiver can be used
– How to handle investigations of transfer failures
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The Analytical Procedure
 The procedure should be written with sufficient detail and explicit instructions so
that a trained analyst can perform it without difficulty.
 The number of replicates, and injection sequences in the case of
chromatography should be clearly expressed.
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 A pre-transfer meeting between the transferring and receiving units is helpful to

clarify any issues and answer any questions regarding the transfer process.
 Reviewed in advance during training phase

̶ Technology is well established in the receiving lab
̶ The receiving lab has not experience in the technique or some aspect of the procedure to be
transferred
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 If complete or partial validation data exist, they should be made available to the
receiving unit, along with any technical details required to perform the test in
question.
200
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Facilities and Instrumentation
 The receiving unit should ensures that the facilities and instrumentation are
properly calibrated and qualified as needed, and verifies that the laboratory
systems are in compliance with applicable regulations and in-house general
laboratory procedures.
 Laboratory infrastructure
 Compatibility of instruments
 Adjustments for dwell volume in HPLC gradients
 Chromatographic columns and consumables
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Facilities and Instrumentation
 A well designed system suitability test will help to detect instrument issues
 Precision
 Impurities detection (S/N ratio)
 Resolution
 Tailing
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Materials
Reference standards
 Use official RS if available
 Critical when the involved labs are in different geographical regions
Samples
 One lot, three lots, etc
 What lot should be used in the transfer
 Number of samples required
 Shipping requirements
 Spiked samples
Reagents
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Transfer Report
 When the TAP is completed, the receiving unit should prepare a Transfer Report
that describes the results obtained in relation to the acceptance criteria, along
with conclusions that confirm that the receiving unit is now qualified to run the
procedure.
 Data from both sites should be reported consistently.
 Both set of data are compared to the acceptance criteria in the protocol
 Include any amendment or deviation to the protocol, reason for these changes and the impact.
 Should be signed by both Transferring and Receiving labs
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Transfer Report
 If acceptance criteria are not met, an investigation should be initiated to

determine the cause of failure.
 An investigation may provide guidance about the nature and extent of the
remedial steps, which may vary from further analyst training and clarification to
more complex approaches depending on the particular procedure.
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Summary of Transfer
Transfer
YES
Validated? Can transfer be YES Transfer waiver
waived? (Option IV)
NO
NO
Can
Is transferring site YES Comparative
comparative testing to be
available? done? testing (Option I)
YES NO
Co-validation Revalidation
(Option II) (Option III)
206
© 2018 USP
Allowance for Alternative Procedures in USP
USP General Notices:
6.30 Alternatives and Harmonized Methods and Procedures
Alternative methods and/or procedures may be used if they provide advantages in

terms of accuracy, sensitivity, precision, selectivity, or adaptability to automation or
computerized data reduction, or in other special circumstances. Such alternative
procedures and methods shall be validated as described in the general chapter
Validation of Compendial Procedures <1225 > and must be shown to give equivalent
or better results.
207
© 2018 USP
Summary USP <1225>, <1226>, <1224>
 USP <1225> Validation of Compendial Procedures

– Validation will be required when
• An analytical procedure is used to test a non-official article.
• An official article is tested using an alternative procedure (see USP General Notices 6.30)

– Verification will be required the first time an official article is tested using a USP procedure

– Transfer will applies when a non-compendial procedure is moved from one lab to another
208
© 2018 USP
Summary – Validation / Verification / Transfer
Implementation of a new
procedure
NO
Validated? USP <1225>
YES
NO
Compendial? USP <1224>
YES
USP <1226> 209

© 2018 USP

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Validation, Verification,

and Transfer of Analytical Procedures

Updated: January 2018

USP Affiliation: USP India Staff member

Dr. Sharad Mankumare is a Director of Reference Standards Laboratory and

 USP Documentary Standards

Three critical components

<1> to <999>: Required

 These chapters are often referenced in individual monographs

<1000> to <1999>: Informational

 Provide guidance to assist compendial users on relevant topics

 These chapters are not normally referenced in individual monographs

It helps in establishment of product-specific acceptance criteria and stability of

 USP Documentary Standards

The term “Validation” appears in:

USP <1010> Analytical Data-Interpretation and Treatment

“ Validation of an analytical procedure is the process by which it is established by

Q2(R1) VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND

“The objective of validation of an analytical procedure is to demonstrate that it

USP <1226> Verification of Compendial Procedures: “Verification consists of

Validation characteristics to be selected according to type of method:

 USP Documentary Standards

Regulatory versus Procedures Suitable for Compendial

ICH: “This document presents a discussion of the characteristics for

Monograph Submission Requirements

 ICH: No such section included

 USP: Submissions should include:

Finding and Recognizing the Tests

“The precision of an analytical procedure is usually expressed as the variance,

Precise but not Accurate but not Precise and

 Precisionis expressed as RSD

Performance Category I Category II Category III Category IV

Accuracy Yes Yes * * No

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

Performance Assay Impurities Identification

Accuracy Yes Yes No No

Specificity (2) Yes Yes Yes Yes

LOD No No (3) Yes No

Linearity Yes Yes No No

Range Yes Yes No No

 Equipment- Suitable for expected accuracy?

 Reference Materials- Suitably characterized?

 Analytical method- Is procedure finalized?

 Validation Protocol- Management / QA approved?

 USP Documentary Standards

May be applied to materials structurally similar to or closely related to the

The choice of such potentially interfering materials should be based on

Example: Hydralazine Hydrochloride

“The IR absorption spectrum of the preparation of the test specimen, previously

It is important to remember (from USP <621>):

“Chromatographic retention times are characteristic of the compounds they

Coincidence of retention times of a test and a reference substance can be used as

Further elaboration from USP <1225>:

Representative chromatograms should be used to demonstrate specificity, and

There are two basic approaches to establishing specificity for procedures

With impurity or degradation product standards

Without impurity or degradation product standards

When impurities are available

When impurities are not available