Verification of applicability

of the validated/compendial
API analytical method
for the final formulation
Assay, dissolution test and
degradants
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Guidelines
ICH Q2A
Validation of Analytical Methods: Definitions and
Terminology (CPMP/ICH/381/95)

ICH Q2B
Validation of Analytical Procedures: Methodology
(CPMP/ICH/281/95)

ICH Q6A
Specifications: Test Procedures and Acceptance
Criteria for New Drug Substances and New Drug
Products: Chemical Substances
(CPMP/ICH/367/96 corr)
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

API
Assay
Validation with respect to:
Specificity, linearity/range, accuracy, precision,
robustness

Impurities
Validation with respect to:
Specificity, linearity/range, accuracy, precision,
limit of detection (LOD), limit of quantitation (LOQ) ,
robustness
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

FPP
Formulation of the drug product
Presence of further APIs
Presence of excipients (individual formulation)
Presence of known impurities/degradants of
all APIs and potential new degradants or
incompatibility products

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Requirements
Capability of the analytical method(s):
Assay of each API in the presence of the
other APIs and all impurities/degradants
Assay of each degradant in the presence of
all APIs and all other degradants/impurities
Influence of formulation components should
be excluded/controlled

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Revalidation I
Revalidation of analytical methods with respect to:
Specificity

presence of new API(s) and impurities/degradants/formulation

components

Range

test concentrations of API(s) versus FPP

Accuracy

influence of formulation components

Precision

influence of formulation and sample preparation

LOD/LOQ

test concentrations of API(s) versus FPP)

Robustness

change of column material, column parameters, solvents)

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Revalidation II
Revalidation reflected by ICH Q2A:
Revalidation may be necessary in the
following circumstances:
Changes in the synthesis of the drug substance
Changes in the composition of the finished
product
Changes in the analytical procedure
(e.g. robustness)

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity
Identification
Discrimination between compounds of closely
related structures
positive results (from samples containing the
analyte)
negative results (from samples that do not contain
the analyte)
components structurally similar to the analyte do
not give positive results

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity II
Assay and impurities
Chromatographic procedures
Representative chromatograms with appropriate
labelling of individual components
Investigation at an appropriate level

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity III

Chromatogram with
retention times and
chemical structures
of:
(1) arteannuin B
(2) artemisitene
(3) artemisinin
(4) artemisinic acid
(5) artemether (IS)
Analytical standard
containing 1.2g/ml
of each analyte and
0.4 g/ml IS
From: F.C.W. Van Nieuverburgh et al., J Chromatogr. A 1118 (2006) 180-187
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity IV
Assay and impurities/degradants
Discrimination of analytes where
impurities/degradants are available
Assay
Demonstration of discrimination of the analyte in the
presence of all impurities/degradants and/or excipients
f. ex. assay result unaffected by presence of spiked
impurities/degradants:
- Injection of pure API
- Injection of API plus impurities/degradants

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity V
Assay and impurities/degradants
Discrimination of analytes where
impurities/degradants are available
Impurities/Degradants
Demonstration of separation of impurities/degradants
individually and/or from excipients
f. ex. spiking of API with appropriate levels of
impurities/degradants and/or excipients:
Chromatographic profiles of API with and
without impurities/degradants/excipients

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity VI
Assay and impurities/degradants
Discrimination of analytes where
impurities/degradants are not available
Comparison of the test procedure to a second
well-characterized (independent) procedure
Samples
Test samples containing impurities/degradants
Test samples stored under relevant stress conditions
(potential degradants arising during shelf life)

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity VII
Assay and impurities/degradants
Discrimination of analytes where
impurities/degradants are not available
Assay
Comparison of test results by the two independent
procedures

Impurities/Degradants
Comparison of impurity profiles

Peak purity assessment

Demonstration that the analyte peak is attributable to
only one component
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity VIII
Peak purity
Overlapping
peaks in HPLC
(simulation)

From: Prof. Siegfried Ebel, University of Wuerzburg, in: Stavros Kromidas, Validierung
in der Analytik, Wiley-VCH

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity IX
Peak purity

Fatty acids were reacted with

ethylene oxide and separated
by HPLC (Fractions 1-6)

Fraction 5 was analysed

by MALDI

From: Dr. Michael Schmitt, Henkel KGaA, Dsseldorf, in: Stavros Kromidas, Validierung
in der Analytik, Wiley-VCH

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Specificity with FDCs

FDC (e.g. artesunate and amodiaquine)
One analytical method for both APIs
Capability of one method to quantify both APIs and
to separate/discriminate one API and its
impurities/degradants and potential incompatibility
products from the other API and its
impurities/degradants/incompatibility products
Some reference material for impurities/degradants will be
available (spiking experiments applicable)
Other degradants are not available as reference material
(stress testing necessary to generate in situ degradants)
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Range
Minimum specified ranges
Assay

80 120% of the test concentration

Content uniformity
70 130% of the test concentration
Dissolution
Q-20% - 120%

Impurities/Degradants

Reporting level to 120% of specification limit

Revalidation is necessary, if the ranges covered

during validation of the API-methods are
different from those of the FPP-methods
(different test concentrations)
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Accuracy
Assay
Application of the analytical procedure to synthetic
mixtures of the product components (placebo
mixture) to which known quantities of the analyte
have been added
In case certain product components are
unavailable:
Application of the analytical procedure to the product
to which known quantities of the analyte have been
added
Comparison of results obtained by a second
(independent) procedure with defined accuracy
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Accuracy II
Impurities/Degradants
Assessment of samples spiked with known
amounts of impurities/degradants
In case certain impurities/degradation
products are unavailable
Comparison of results obtained by a second
(independent) procedure with defined accuracy

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Precision
Assay and impurities/degradants
Repeatability
9 determinations (3 x 3) covering the specified range
or
6 determinations at 100% of the test concentration

Intermediate precision
Effects of random events on the precision of the procedure,
e.g.
Days
Analysts
Equipment

To be performed with a test solution prepared

from the drug product
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Detection Limit
Determination based on
Visual evaluation (non-instrumental and instrumental
methods)
Signal to Noise (baseline noise)
Standard deviation of response () and
slope (S)
DL=3.3/S
Estimation of S
from the calibration curve of the analyte
Estimation of
from the standard deviation of the blank
from the standard deviation (regression line or y-intercept)
of a calibration curve in the range of the DL
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Quantitation Limit
Determination based on
Visual evaluation (non-instrumental and instrumental
methods)
Signal to Noise (baseline noise)
Standard deviation of response () and
slope (S)
QL=10/S
Estimation of S
from the calibration curve of the analyte
Estimation of
from the standard deviation of the blank
from the standard deviation (regression line or y-intercept)
of a calibration curve in the range of the QL
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Robustness I
Reliability of an analysis with respect to
deliberate variations in method
parameters
Susceptibility to variations in analytical
conditions?
control of analytical conditions
or
precautionary statement
establishment of system suitability parameters
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Robustness II
Examples of variations
Stability of analytical solutions
Extraction time

In the case of liquid chromatography

Influence of variations of pH in a mobile phase

Influence of variations in mobile phase composition
Influence of columns (different lots and/or suppliers)
Influence of temperature
Influence of flow rate

In the case of gas chromatography

Influence of columns (different lots and/or suppliers)

Influence of temperature
Influence of flow rate

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Robustness III
Influence of pH of elution on separation of amino
acids by RP-HPLC

From: Waters, in: Stavros Kromidas, Validierung in der Analytik, Wiley-VCH

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Robustness
Electropherograms under identical conditions by
different analytical equipment

From: Dr. Michael Krmer, NOVARTIS, Basel, in: Stavros Kromidas, Validierung in der
Analytik, Wiley-VCH

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Dissolution
Applicability of the analytical method used for
assay and impurities/degradants
Sample preparation
Range

Applicability of the dissolution method

Appropriateness of drug release acceptance criteria
Solubility criteria of the APIs

Appropriateness of test conditions and acceptance

criteria
Dissolution affecting bioavailability
Changes in formulation or manufacturing variables affecting
dissolution
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Dissolution II
Applicability of the analytical method used
for assay and impurities/degradants
Potential parameters for revalidation
Sample preparation
Stability of analytes in the dissolution medium?
Preparation of an injectable sample volume according to
the analytical method?
Precision of analysis of the prepared dissolution sample?

Range of test concentrations of API / impurities /

degradants according to the validated ranges?
Test concentration of prepared dissolution sample versus
test concentration of FPP sample
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Dissolution III
Applicability of the dissolution method
Appropriateness of drug release acceptance criteria
Solubility of the APIs (ICH Q6A Definitions)
Rapidly dissolving products
Not less than 80% of the label amount of the drug
substance dissolves within 15 minutes in each of the
following media: pH 1.2, pH 4.0, pH 6.8
Highly water soluble drugs
Drugs with a dose/solubility volume of less than or equal to
250 ml over a pH range of 1.2 to 6.8
Low solubility drugs
Drugs with a dose/solubility volume of more than
250 ml
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Dissolution IV
Appropriateness of drug release acceptance
criteria
Solubility of the APIs
Problem with low solubility drugs:
Solution of the drugs may become a time-limiting step
Dissolution also dependent on the strength of the drug
product
Dissolution test cannot reflect batch to batch consistency

Possible solution of the problem

Extending the dissolution volume
and
Validation of the dissolution procedure with extended volume
(applicability of the pharmacopoeial procedure)
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Dissolution V
Sink conditions
Ph. Eur 2.9.3: ..the material already in solution does
not exert a significant modifying effect on the rate of
dissolution of the remainder
Sink conditions normally occur in a volume of
dissolution medium that is at least
3 to 10 times the saturation medium
Consequently: the amount of API contained in the
dosage form should be soluble in NMT 300 ml of
dissolution medium

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Dissolution V
Applicability of the dissolution method

Appropriateness of test conditions and acceptance

criteria (ICH Q6A)
Dissolution significantly affecting bioavailability

Have relevant developmental batches exhibited unacceptable

bioavailability?
Development of test conditions and acceptance criteria
which can distinguish batches with unacceptable
bioavailability

Changes in formulation or manufacturing variables affecting

dissolution

Control of these changes by another procedure and acceptance

criterion
or
Development of test conditions and acceptance criteria which
can distinguish these changes

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Major problems
Solubility of Artemisinins
Sink condition cannot be established
(+) Addition of solubilizers could help establish a (dis)solution
test
(-) The test would disconnect dissolution and bioavalability
and could only serve as parameter for batch to batch
consistency
Disintegration could be considered as additional parameter

Stability of Artemisinins
Artesunate decomposes (to DHA) in buffers required
for dissolution testing (e.g. pH 1.2, pH 4.5)
Dissolution could only be performed at a neutral pH (~ 7.0)
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Deficiencies from PQ
Validation of precision
Precision of the drug substance solution lower than
precision of the drug product solution
Acceptance criteria for precision of the drug
substance solution wider than for precision of the
drug product solution
Acceptance criteria much wider than real values
assessed
Acceptance criteria of assay specifications and
precision do not match
(3 x RSD outside the specification range)
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Deficiencies from PQ II
Assay of API and impurities/degradants
No acceptable mass balance found between
assay of API and impurities/degradants
Quantitation limit of impurities too high
ICH requirement on threshold for identification and
qualification of unknown impurities cannot be
fulfilled

Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

Deficiencies from PQ III

Dissolution
Necessary information on development of
dissolution test not presented
Dissolution method (pharmacopoeial) not
presented along with development of
dissolution test and/or validation of
applicability of analytical methods
Test conditions and acceptance criteria of the
dissolution test not justified
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,

THANK
YOU
FOR
YOUR
ATTENTION
Dar es Salaam, August,

Dr. Birgit Schmauser, BfArM,