Analytical method- Content, Development, validation, Transfer & Life Cycle Management

ANALYTICAL METHOD (DEVELOPMENT,
VALIDATION, TRANSFER & LIFE CYCLE
MANAGEMENT)
Prepared by: MD. MIZANUR RAHMAN MIAJEE
1

ANALYTICAL METHOD VALIDATION
REGULATORY GUIDANCE:
• ICH Q2B- Validation of Analytical Procedures: Methodology
• USP <1225> Validation of Compendial Procedures
• USP <1226> Verification of Compendial Procedures
• USP <1220> The Analytical Procedure Lifecycle – Proposed
• USP <621> Chromatography
• FDA Guidance- Analytical Procedures and Methods Validation for Drugs
and Biologics
• CDER Reviewer Guidance- Validation of Chromatographic Methods
• Other Journal
2

CONTENT OF IDEAL ANALYTICAL METHOD (AS PER FDA):
• Principle/Scope: A description of the basic principles of the
analytical test/technology (i.e., separation, detection); target
analyte(s) and sample(s) type (e.g., drug substance, drug
product, impurities)
• Apparatus/ Equipment: All required qualified equipment and
components (e.g., instrument type, detector, column type,
dimensions, and alternative column, filter type).
3

• Operating Parameters: Qualified optimal settings and ranges
(include allowed adjustments supported by compendial
sources or development and/or validation studies) critical to
the analysis (e.g., flow rate, components temperatures, run
time, detector settings, gradient, head space sampler). A
drawing with experimental configuration and integration
parameters may be used, as applicable.
4

Reagents/Standards: The following should be listed where
applicable:
• Description of reagent or standard
• Grade of chemical (e.g., USP/NF, American Chemical Society,
High Performance or Pressure Liquid Chromatography, or
Gas Chromatography and preservative-free)
• Source (e.g., USP reference standard, qualified in-house
reference material, WHO International Standard/Reference
Material, CBER standard)
5

• Purity (for pure chemicals only), State (e.g., dried, undried),
and concentration
• Potencies (where required by CFR, USP)
• Storage conditions
• Directions for safe use (as per current Safety Data Sheet)
• Validated or documented shelf life
6

Sample Preparation:
Procedures (e.g., extraction method, dilution or
concentration, desalting procedures and mixing by sonication,
shaking or sonication time) for the preparations for individual
sample tests. A single preparation for qualitative and
replicate preparations for quantitative tests with appropriate
units of concentrations for working solutions (e.g., µg/ml or
mg/ml) and information on stability of solutions and storage
conditions.
7

• Standards Control Solution Preparation: Procedures for the
preparation and use of all standard and control solutions with
appropriate units of concentration and information on
stability of standards and storage conditions, including
calibration standards, internal standards, system suitability
standards, etc.
8

• Procedure: A step-by-step description of the method (e.g.,
equilibration times, and scan/injection sequence with blanks,
placeboes, samples, controls, sensitivity solution (for impurity
method) and standards to maintain validity of the system
suitability during the span of analysis) and allowable
operating ranges and adjustments if applicable.
9

• System Suitability: Confirmatory test(s) procedures and
parameters to ensure that the system (equipment,
electronics, and analytical operations and controls to be
analyzed) will function correctly as an integrated system at
the time of use. The system suitability acceptance criteria
applied to standards controls and samples, such as peak
tailing, precision and resolution acceptance criteria, may be
required as applicable.
10

• Calculations: The integration method and representative
calculation formulas for data analysis (standards, controls,
samples) for tests based on label claim and specification (e.g.,
assay, specified and unspecified impurities and relative
response factors). This includes a description of any
mathematical transformations or formulas used in data
analysis, along with a scientific justification for any correction
factors used.
11

• Data Reporting: A presentation of numeric data that is consistent with
instrumental capabilities and acceptance criteria. The method should
indicate what format to use to report results (e.g., percentage label claim,
weight/weight, and weight/volume) with the specific number of
significant figures needed. The American Society for Testing and Materials
(ASTM) E29 standard describes a standard practice for using significant
digits in test data to determine conformance with specifications. For
chromatographic methods, you should include retention times (RTs) for
identification with reference standard comparison basis, relative retention
times (RRTs) (known and unknown impurities) acceptable ranges and
sample results reporting criteria
12

GENERAL POINT MUST BE CONSEDERED FOR ANALYTICAL METHOD
(FDA Reviewer Guidance):
• The sample and standard should be dissolved in the mobile
phase. If that is not possible, then avoid using too high level
of the organic solvent as compared to the level in the mobile
phase.
• The sample and standard concentrations should be close if
not the same.
• The samples should be bracketed by standards during the
analytical procedure
13

GENERAL POINT CONSEDER FOR ANALYTICAL METHOD:
• Filtration of the samples before injection is occasionally
observed. Filtration will remove particulates (centrifugation
performs the same function) that may clog columns.
Adhesion of the analyte to the filter can also happen. This will
be of importance especially for low level impurities. Data to
validate this aspect should be submitted by the applicant.
14

15

• An analytical procedure is developed to test a defined characteristic
of the drug substance or drug product against established
acceptance criteria for that characteristic.
• Method development should begin with an initial risk assessment
and follow with multivariate experiments. Knowledge gained during
these studies on the sources of method variation can help you
assess the method performance.
ANALYTICAL METHOD DEVELOPMENT
16

What is QbD
A systematic approach to development that begins with
predefined objectives and emphasizes product and process
understanding and process control, based on sound science
and quality risk management.
Benefits of Application of QbD Approach to Analytical Methods
• Development of a robust method
• Applicable throughout the life cycle of the product
Regulatory flexibility −
• Movements within “Design Space” are not considered a change in
method.
17

18

• What Is An ATP?
• Analytical Target Profile is a part of QbD which is a predefined,
documented statement of the requirements for the quality of the
reportable value, which the analytical procedure must generate in
order for the procedure to be considered fit for purpose Using
Current Industry Acceptance Criteria. The procedure must be able
to accurately quantify [drug] in the [FPP] in the presence of
[Impurity/degradation product] with the following requirements for
the reportable values: Accuracy = 100.0% ± 3.0% and precision ≤
2.0% relative standard deviation (RSD).
19

• What Is in ATP?
In ATP Target profile for the analytical method to be predefined. Some
example are given below:
1. Sample sonication time: as per QbD different sonicatione time
should be considered to get optimum solubility (ATP)
2. Chromatographic Run Time: A target run time should be selected
and as per QbD, different run time to be performed to get optimum
one.
3. Sample & standard solution concentration in impurity analysis:
Concentration of sample is very important in impurity analysis to
cover Reporting threshold limit as per ICH Q2B. A target
concentration to be selected before development work.
20

Parameters that may be evaluated during method development:
During early stages of method development, the robustness of methods
should be evaluated because this characteristic can help you decide which
method you will submit for approval. Other characteristics that may be
considered during method development are:
• Specificity
• Linearity
• Accuracy
• Precision
• Range
• Quantitation limit
• Detection limit.
• Solution Stability
21

Describe the
sample
Establish
Goals
Consider sample
preparation
Choose
detector
Choose
chromatographic
mode
First Run
Optimize
separation
Predict
Problems
Validate and
Release
method
22

Collection of literature:
Analytical method development starts with literature search
where in various pharmacopeias like USP, EP, BP, JP etc and
chromatographic journals are referred to check the
availability of suitable analytical methods. If any suitable
method is found, it is still necessary to perform method
optimization and validation to prove that the method can be
successfully adapted for its intended use.
23

Sample information: The synthetic route from raw material to
finished dosage forms (structures) of the molecule should be
collected and the impurities originating from starting materials
of synthesis, degradation products, excipients, solvents etc.
need to be considered. The method should be designed
based on the closely related structures and get the best
resolution between the closely related compounds. The
structures of impurities, starting material, intermediates and
degradation products are compared with the structure of drug
substances and arrive at the polarity whether they are less
polar or more polar than the compound of interest.
24

• Selection of diluents: The diluent should be chosen based on
the solubility of impurities, degradation products, starting
materials, intermediates and the analyte. The diluent should
be compatible with the mobile phase to get a better peak
shape of analyte.
25

• Selection of stationary phase: The selection of bonded phase can
be based on the polarity of molecule and its by-products. For RP-LC,
a wide variety of columns are accessible covering a large range of
polarity by cross-linking Si-OH groups with linear alkyl chains like
C8, C18 and phenyl groups (-C6H6), nitrile groups (-CN), , different
embedded hybridized groups amino groups (-NH2) etc. C18
columns are the commonly used columns in HPLC method analysis.
C8 or Octyl bonded phases are also used occasionally. Like C18,
they are non-polar, but not as hydrophobic.
26

• Detectors:
Various detectors used in HPLC instrument include UV-Visible
detector, photodiode array detector, fluorescence detector,
conductivity detector, refractive index detector, electro-
chemical detector, mass spectrometer detector and
evaporative light scattering detector. UV-Visible detectors are
typical in many laboratories as they can detect a wide array of
compounds
27

• pH range:
Method development within the different pH ranges from 1 to
12 for better chromatographic resolution between two or
more peaks of an analyte depends upon three main factors,
column efficiency, selectivity and retention time. The
ionizable analytes are either bases or acids and they affect
the above three factors dramatically with change in pH.
Retention time can be improved by changing the pH that will
lead to easy separation of ionizable analytes from non-ionized
forms.
28

• pH range:
To achieve optimum resolution, it requires change in the pH of
mobile phase. Method development can proceed by
investigating parameters of chromatographic separations first
at low pH and then at higher pH until optimum results are
achievd.
29

• Mobile phase composition:
Mobile phase composition (or solvent strength) plays an
important role in RP-HPLC separation. Acetonitrile (ACN),
methanol (MeOH) and tetrahydrofuran (THF) are commonly
used solvents in RP -HPLC having low UV cut- off of 190, 205
and 212nm respectively. These solvents are miscible with
water. Mixture of acetonitrile and water is the best initial
choice for the mobile phase during method development.
30

• Column temperature:
Separation of many samples can be enhanced by selecting the
right column temperature. Higher column temperature
reduces system backpressure by decreasing mobile phase
viscosity, which in turn allows use of longer columns with
higher separation efficiency. The optimum temperature is
dependent upon the nature of the mixture components. The
overall separation can be improved by the simultaneous
changes in column temperature and mobile phase
composition
31

• Analytical Method Transfer:
What is analytical method transfer: It 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 transferred analytical procedure as intended.
32

• Transfer may be-
• One R & D development lab to another R & D development
lab
• R & D development lab to quality control lab inside same unit
• R & D development lab to quality control lab of other unit
• One facility to another intra-company facility
• One company to another company such as a contract
research organization
33

Procedure of Analytical Method Transfer: Analytical
Method Transfer (AMT) can be done by two ways:
1. Comparative Testing
2. Co-validation between Two or More Laboratories
34

1. Comparative Testing: analysis of a predetermined number of
samples of the same lot by both the sending and the receiving
units.
Prerequisite: A preapproved transfer protocol prepared by
sending unit that stipulates the details of the procedure, the
samples that will be used, and the predetermined acceptance
criteria, including acceptable variability
Criteria: Meeting the predetermined acceptance criteria is
necessary to assure that the receiving unit is qualified to run
the procedure.
35

2. Co-validation Between Two or More Laboratories: The
transferring unit can involve the receiving unit in an inter
laboratory co-validation, including them as a part of the
validation team at the transferring unit and thereby obtaining
data for the assessment of reproducibility
Prerequisite: a preapproved transfer or validation protocol
that provides the details of the procedure, the samples to be
used, and the predetermined acceptance criteria
Criteria: Meeting the predetermined acceptance criteria is
necessary to assure that the receiving unit is qualified to run
the procedure.
36

Transfer Waiver:
• The new product’s composition is comparable to that of an
existing product and/or the concentration of active ingredient
is similar 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
• The analytical procedure transferred is the same as or very
similar to a procedure already in use.
37

Transfer Waiver:
• The personnel in charge of the development, validation, or
routine analysis of the product at the transferring unit are
moved to the receiving unit. If eligible for transfer waiver, the
receiving unit should document it with appropriate
justifications.
38

• ELEMENTS RECOMMENDED FOR THE TRANSFER OF
ANAYTICAL PROCEDURES:
• Transferring unit:
• provide training to the receiving unit, training should be
documented, providing the analytical procedure, the
reference standards, the validation reports, and any
necessary documents, as well as for providing the necessary
training and assistance to the receiving unit as needed during
the transfer.
39

• Receiving unit:
• Provides qualified staff or properly trains the staff before the
transfer, 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.
40

• Both unit: Compare and discuss data as well as any deviations
from the protocol. This discussion addresses any necessary
corrections or updates to the final report and the analytical
procedure as necessary to reproduce the procedure. A single
lot of the article
41

• Content of transfer protocol: Below mentioned parameters
must be included in transfer protocol
• Objective
• Scope
• Responsibilities of the transferring and receiving units,
• Materials and instruments that will be used,
• Analytical procedure
• Experimental design
• Acceptance criteria for all tests and/or methods included in
the transfer. 42

• Analytical Procedure for AMT: The procedure should be
written with sufficient detail and explicit instructions, so that
a trained analyst can perform it without difficulty. A pre-
transfer meeting between the transferring and receiving units
is helpful to clarify any issues and answer any questions
regarding the transfer process. If complete or partial
validation data exist, they should be available to the receiving
unit, along with any technical details required to perform the
test in question.
43

• What is analytical method validation
Validation is the process of demonstrating that an analytical
procedure is suitable for its intended purpose
44

• Importance of Analytical Method Validation
 Validation of a method involves using experimental design to prove that the
method can produce accurate and precise results within the scope of its
intended use
 Understanding the application and limitations of the test method will allow for
accurate assessment of sample information, ranging from process outputs to
commercial release testing and many steps in between.
 If the method validation has not been performed or has been performed in an
inadequate manner, the method is not proven to provide reliable data.
• A majority of audit findings fell into three main categories: 1. the use of a non-
validated method for critical decision making; 2. inadequate method
validation that did not provide the necessary information; 3. method
validation that lacked appropriate controls to maintain the integrity of the
validation 45

• Types of Analytical Method need to validate
 Identification tests
 Quantitative tests for impurities;
 Limit tests for the control of impurities; and
 Assay tests for the active component in a drug substance, drug product,
or other selected component(s) in the drug product.
46

• Noncompendial Analytical Procedures: Requires Complete Validation
• Compendial Analytical Procedures: Requires Verification
47

• Why verification:
 Drug substance's synthetic route,
 The method of manufacture for the drug product
 Drug substances from different suppliers may have different impurity
profiles that are not addressed by the compendial test procedure
 The excipients in a drug product can vary widely among manufacturers
and may have the potential to directly interfere with the procedure or
cause the formation of impurities that are not addressed by the
compendia procedure
 Drug products containing different excipients, antioxidants, buffers, or
container extractives may affect the recovery of the drug substance from
the matrix
(But as per current thinking of FDA, FDA prefer complete validation for
compendia method instead of verification)
48

• Following parameter to be considered for Analytical Method
Validation:
 Specificity
 Accuracy
 Precision (Repeatability, Intermediate Precision & Reproducibility)
 Linearity
 Range
 Quantitation limit (LOQ)
 Detection limit (LOD)
 Robustness
49

SPECIFICITY /SELECTIVITY
Ability to assess unequivocally the analyte in the presence of components
that may be expected to be present, such as impurities, degradation
products, and matrix components.
• Identification: Ensure the identity of the analyte.
• Impurity: Ensure that all of the analytical procedures performed allow an
accurate statement of the content of impurities of an analyte (e.g.,
related substances test, heavy metals limit, or organic volatile impurities).
• Assays: Provide an exact result, which allows an accurate statement on
the content or potency of the analyte in a sample
50

SPECIFICITY /SELECTIVITY
• Procedure:
When Impurities are available (Specified identified impurity): Done by
spiking pure substances (drug substance or drug product) with
appropriate levels of impurities and/or excipients and demonstrating that
the assay result is unaffected by the presence of these materials (by
comparison with the assay result obtained on unspiked samples). For the
impurity test, the discrimination may be established by spiking drug
substance or drug product with appropriate levels of impurities and
demonstrating the separation of these impurities individually and/or from
other components in the sample matrix.
51

Accuracy
The accuracy of an analytical procedure is the closeness of test results
obtained by that procedure to the true value.
Evaluate by analyzing synthetic mixtures: known quantity of API with
excipients/ Impurity
• Recommended Data: Accuracy should be assessed using a minimum of 9
determinations over a minimum of 3 concentration levels covering the
specified range (e.g., 3 concentrations /3 replicates each of the total
analytical procedure).
• Accuracy should be reported as percent recovery by the assay of known
added amount of analyte in the sample or as the difference between the
mean and the accepted true value together with the confidence intervals.
52

Precision
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 precision of an
analytical procedure is usually expressed as the standard deviation or
relative standard deviation (coefficient of variation) of a series of
measurements
A. Repeatability
• Repeatability should be assessed using:
(1) A minimum of 9 determinations covering the specified range for the
procedure (e.g., 3 concentrations/3 replicates each); or
(2) A minimum of 6 determinations at 100% of the test concentration.
53

Precision
B. Intermediate Precision
The extent to which intermediate precision should be established depends on
the circumstances under which the procedure is intended to be used. The
applicant should establish the effects of random events on the precision
of the analytical procedure. Typical variations to be studied include days,
analysts, equipment, etc. It is not necessary to study these effects
individually. The use of an experimental design (matrix) is encouraged.
54

Precision
C. Reproducibility
Reproducibility is assessed by means of an inter laboratory trial.
Reproducibility should be considered in case of the standardization of an
analytical procedure, for instance, for inclusion of procedures in
pharmacopoeias. These data are not part of the marketing authorization
dossier.
Recommended Data
The standard deviation, relative standard deviation (coefficient of variation),
and confidence interval should be reported for each type of precision
investigated
55

Linearity & Range
Linearity: 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
Parameter to check: The correlation coefficient, y-intercept, slope of the regression
line, and residual sum of squares should be submitted.
Number of sample/ Injection: For the establishment of linearity, a minimum of five
concentrations is recommended. Other approaches should be justified.
56

Linearity & Range
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 assay of a drug substance or a finished (drug) product:
Normally from 80% to 120% of the test concentration
For content uniformity: Covering a minimum of 70% to 130% 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
57

Linearity & Range
• For dissolution testing: +/-20% over the specified range; e.g., if the specifications
for a controlled released product cover a region from 20% after 1 hour: up to 90%
after 24 hours: the validated range would be (0-110)% of the label claim
• 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.
58

Detection limit
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 or parts per billion) in the
sample.
A. Based on Visual Evaluation:
• Visual evaluation may be used for non-instrumental methods but may
also be used with instrumental methods.
• The detection limit is 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 59

Detection limit
B. Based on Signal-to-Noise:
This approach can only be applied to analytical procedures which exhibit
baseline noise. Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing
the minimum concentration at which the analyte can be reliably detected.
Signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for
estimating the detection limit.
60

Detection limit
C. Based on the Standard Deviation of the Response and the Slope
The detection limit (DL) may be expressed as:
where
= the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The
estimate of may be carried out in a variety of ways, for example:
61

Detection limit
Based on the standard deviation of the blank
Measurement of the magnitude of analytical background response is
performed by analyzing an appropriate number of blank samples and
calculating the standard deviation of these responses.
Based on the calibration curve
A specific calibration curve should be studied using samples containing an
analyte in the range of DL. The residual standard deviation of a regression
line or the standard deviation of y-intercepts of regression lines may be
used as the standard deviation.
62

Detection limit
Recommended Data
The detection limit and the method used for determining the detection limit
should be presented. If DL is determined based on visual evaluation or
based on signal-to-noise ratio, the presentation of the relevant
chromatograms is considered acceptable for justification.
In cases where an estimated value for the detection limit is obtained by
calculation or extrapolation, this estimate may subsequently be validated
by the independent analysis of a suitable number of samples known to be
near or prepared at the detection limit.
63

Quantitation Limit
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 or parts per billion) in the sample.
QL to be determined as like Detection limit except signal-to-noise ratio is
10:1. and the calculation would be 64

Quantitation Limit
Recommended Data
The quantitation limit and the method used for determining the quantitation
limit should be presented.
The limit should be subsequently validated by the analysis of a suitable
number of samples known to be near or prepared at the quantitation limit
65

Robustness
The evaluation of robustness should be considered during the development
phase and depends on the type of procedure under study. It should show
the reliability of an analysis with respect to deliberate variations in
method parameters.
If measurements are susceptible to variations in analytical conditions, the
analytical conditions should be suitably controlled or a precautionary
statement should be included in the procedure.
One consequence of the evaluation of robustness should be that a series of
system suitability parameters (e.g., resolution test) is established to
ensure that the validity of the analytical procedure is maintained
whenever used.
66

Robustness
• Examples of typical variations are:
o Stability of analytical solutions
o Extraction time
• In the case of liquid chromatography, examples of typical variations are:
o Influence of variations of pH in a mobile phase
o Influence of variations in mobile phase composition
o Different columns (different lots and/or suppliers)
o Temperature
o Flow rate
67

Robustness
System suitability testing: These tests are used to verify that the
chromatographic system is adequate for the intended analysis. system
suitability tests are performed by collecting data from replicate injections
of standard or other solutions as specified in the individual monograph.
• Factors that may affect chromatographic behavior include the following:
Composition, ionic strength, temperature, and apparent pH of the mobile
phase•
o Flow rate, column dimensions, column temperature, and pressure
o Stationary phase characteristics, including type of chromatographic support
(particle-based or monolithic), particle or macropore size, porosity, and
specific surface area
o Reverse-phase and other surface modification of the stationary phases, the
extent of chemical modification (as expressed by end-capping, carbon loading,
and others) 68

Robustness
System Suitability: Unless otherwise specified in the individual monograph,
data from five replicate injections of the analyte are used to calculate the
relative standard deviation (RSD), if the requirement is ≤ 2.0%; data from
six replicate injections are used if the RSD requirement is >2.0%.
If adjustments of operating conditions are necessary in order to meet system
suitability requirements, each of the items in the following list is the
maximum variation that can be considered:
69

Robustness
pH of mobile phase (HPLC): ±0.2 units of the value or range specified.
Concentration of salts in buffer (HPLC): ±10% if the permitted pH variation
Ratio of components in mobile phase (HPLC): ±30% relative. However, the
change in any component cannot exceed ±10% absolute (i.e., in relation
to the total mobile phase)
Wavelength of UV-visible detector (HPLC): ±3 nm
Stationary phase: Length- between −25% and 50% of the prescribed L/dp
Flow rate (HPLC): ±50% (Based on other factors)
Injection volume (HPLC): Can be adjusted as far as it is consistent with
accepted precision, linearity, and detection limits
Column temperature (HPLC): As much as ±10°.
70

Life Cycle Management of Analytical Procedure
Once an analytical procedure (including compendial methods) is successfully
validated or verified and implemented, the procedure should be followed
during the life cycle of the product to continually assure that it remains fit
for its intended purpose.
71

What to be done:
 Trend analysis on method performance at regular intervals
 New information and risk assessments (e.g., a better understanding of
product CQAs or awareness of a new impurity) may warrant the
development and validation of a new or alternative analytical method
 An appropriate number of retention samples should be maintained to
allow for comparative studies.
 The retention samples used in comparative studies should include
samples that represent marketed product and, when possible, pivotal
clinical trial material.
72

Re-Validation:
 When a change is made to an analytical procedure, such as a change in
equipment or reagent, then re-validation of all or part of the analytical
procedure needs to be considered.
 Method re-validation should focus on the critical performance
characteristics of the method, such as specificity, precision, and accuracy.
 The scope of re-validation should be risk-based.
73

• Analytical Method Comparability Studies:
Alternative Analytical Procedures
An alternative analytical procedure is an analytical procedure that is used in
place of the FDA approved analytical procedure. After approval, for an
NDA or ANDA, or for a procedure approved in a BLA but not included in an
FDA regulation, the addition, revision, or deletion of an alternative
analytical procedure that provides the same or increased assurance of the
identity, strength, quality, purity, or potency of the material being tested
as the analytical procedure described in the approved application, must
be documented in the next annual report
74

Stability Indicating Method (SIM)
If a procedure is a validated quantitative analytical procedure that can detect
changes in a quality attribute(s) of the drug substance and drug product
during storage, it is considered a stability- indicating test. To demonstrate
specificity of a stability-indicating test, a combination of challenges should
be performed. Some challenges include the use of samples spiked with
target analytes and all known interferences; samples that have undergone
various laboratory stress conditions; and actual product samples
(produced by the final manufacturing process) that are either aged or
have been stored under accelerated temperature and humidity
conditions.
75

Objective
The main objective of a stability indicating method is to monitor results
during stability studies in order to guarantee safety, efficacy and quality. It
represents also a powerful tool when investigating out-of-trend (OOT) or
out-of-specification (OOS) results in quality control processes.
Important factor to consider: The method should be sensitive to the
reportable impurity level, LOQ (Limit of Quantitation), which is typically
0.05% of label claim, should be established in the method, and the
method should be linear from LOQ to typically up to 150% of the nominal
standard (STD) concentration
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• How develop a SIM:
Generation of degraded samples for testing selectivity of the method
Determination of Limit of Quantification (LoQ) In close relation to the
determination of the amount of degradation is the evaluation of Limit of
Detection (LoD) and Limit of Quantification (LoQ) of the method. These
limits should be closely related to the Reporting, Identification and
Qualification of degradation products, as stated in ICH Q3B (R2). These
thresholds are determined either as percentage of drug substance or total
daily intake (TDI) of degradation product.
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• Force Degradation:
Objective of forced degradation studies:
 To establish degradation pathways of drug substances and drug products.
 To differentiate degradation products that are related to drug products from those
that are generated from non-drug product in a formulation.
 To elucidate the structure of degradation products.
 To determine the intrinsic stability of a drug substance in formulation.
 To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis
or photolysis of the drug substance and drug product
 To establish stability indicating nature of a developed method.
 To understand the chemical properties of drug molecules.
 To generate more stable formulations.
 To produce a degradation profile similar to that of what would be observed in a
formal stability study under ICH conditions.
• To solve stability-related problems 79

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What to include: The testing should include the effect of temperatures (in
10°C increments (ie, 50°C, 60°C) above that for accelerated testing),
humidity (ie, 75% relative humidity or greater) where appropriate,
oxidation, and photolysis on the drug substance. The testing should also
evaluate the susceptibility of the drug substance to hydrolysis across a
wide range of pH values when in solution or suspension.”
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How much is enough
Values anywhere between 5% to 20% degradation of the drug substance
have been considered as reasonable and acceptable for validation of
chromatographic assays. However, for small pharmaceutical molecules for
which acceptable stability limits of 90% of label claim is common,
pharmaceutical scientists have agreed that approximately 10%
degradation is optimal for use in analytical validation.
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Selection of drug concentration
Which concentration of drug should be used for degradation study has not
been specified in regulatory guidance. It is recommended that the studies
should be initiated at a concentration of 1 mg/mL . By using drug
concentration of 1 mg/mL, it is usually possible to get even minor
decomposition products in the range of detection
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What is peak purity: Peak purity is an analysis of spectral differences,
assuming the impurities are spectrally different from the analyte. The
spectral analysis is achieved using vector analysis algorithms The more
similar the spectra are, the closer the value is to 0.0 degrees; the more
spectrally different, the larger the value (90 degrees is the maximum).
When no significant spectral differences are found within a peak, one can
conclude that the peak is spectrally homogeneous and probably contains
a single compound.
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 Peak Purity algorithm analyzes all spectra within a peak
 The Apex Spectrum is the reference spectrum that it compares to.
 If Purity Angle < Purity Threshold, Within the noise of the system the peak
is spectrally homogeneous or spectrally pure.
 If Purity Angle > Purity Threshold , there is something within the peak that
cannot be explained by noise. The peak is not spectrally pure, the Purity
Flag field is checked.
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Purity Angle > Purity Threshold = Impure Peak
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Purity threshold > Purity Angle = Pure Peak
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Mass balance
• Mass balance correlates the measured loss of a parent drug to the
measured increase in the amount of degradation products. It is a good
quality control check on analytical methods to show that all degradation
products are adequately detected and do not interfere with quantitation
of the parent drug (i.e.,stability-indicating methods).
89

System Suitability
General Recommendation:
System suitability testing is essential for the assurance of the quality
performance of the chromatographic system. The amount of testing
required will depend on the purpose of the test method. For dissolution
or release profile test methods using an external standard method, k', T
and RSD are minimum recommended system suitability tests. For
acceptance, release, stability, or impurities / degradation methods using
external or internal standards, k', T, R, and RSD are recommended as
minimum system suitability testing parameters.
90

System Suitability
Capacity factor (k'): k' = (tR - to) /to
The capacity factor is a measure of where the peak of interest is located with
respect to the void volume, i.e., elution time of the non-retained
components.
Recommendations: The peak should be well-resolved from other peaks and
the void volume. Generally the value of k' is > 2.
Void volume: amount of dead volume in the column that is not taken up by
the particle size of the stationary phase
91

System Suitability
Resolution (R,):
R, is a measure of how well two peaks are separated. For reliable
quantitation, well-separated peaks are essential for quantitation. This is a
very useful parameter if potential interference peak(s) may be of concern.
The closest potential eluting peak to the R, is minimally influenced by the
ratio of the two compounds being measured. The resolution of peaks as
indicated by the R, values is shown in below Figure:
Recommendations: R, of > 2 between the peak of interest and the closest
potential interfering peak (impurity, excipient, degradation product,
internal standard, etc.) is desirable. 92

System Suitability
Tailing factor (T):
The accuracy of quantitation decreases with increase in peak tailing because
of the difficulties encountered by3the integrator in determining
wheretwhen the peal< ends and hence the calculation of the area under
the peal peak of interest. Below Figures illustrate the tailing factors and
the effect on quantitation. If the integrator is unable to determine exactly
when an upslope or downslope occurs, ' accuracy drops.
Recommendations: T of less than 2
93

System Suitability
Theoretical plate number (N):
Theoretical plate number is a measure of column efficiency, that is, how
many peaks can be located per unit run-time of the chromatogram. N is
fairly constant for each peak on a chromatogram with a fixed set of
operating conditions.
Recommendations: The theoretical plate number depends on elution time
but in general. should be > 2000.
94

System Suitability
Precision /injection repeatability (RSD): Injection precision expressed as RSD
(relative standard deviation) indicates the performance of the HPL
chromatograph which includes the plumbing, column, and environmental
conditions, the time the samples are analyzed. It should be noted that
sample preparation and manufacturing variations are not considered.
Recommendations: RSD of s 1% is desirable.
95

Example of FDA Warning letter
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