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Birgit Schmauser

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April 2008 1
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Pharmaceutical Development with Focus
on Paediatric formulations
Pharmaceutical Development with Focus
on Paediatric formulations
WHO/FIP Training Workshop
Hyatt Regency Hotel
Sahar Airport Road
Andheri East, Mumbai, India
28 April 2008 2 May 2008
Birgit Schmauser
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April 2008 2
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Analytical Method Development Analytical Method Development
Presented by: Presented by:
Birgit Schmauser, PhD Birgit Schmauser, PhD
Federal Institute for Drugs Federal Institute for Drugs
and Medical Devices (BfArM) and Medical Devices (BfArM)
b.schmauser@bfarm.de b.schmauser@bfarm.de
Birgit Schmauser
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April 2008 3
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Analytical Method Development Analytical Method Development
In this presentation: In this presentation:
Standards in developing analytical methods for Standards in developing analytical methods for
Originator and multisource generic FPPs Originator and multisource generic FPPs
Specifications Specifications
Stability Stability
Parallel development of analytical methods for Parallel development of analytical methods for
cleaning validation cleaning validation
Birgit Schmauser
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April 2008 4
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Analytical Method Development Analytical Method Development
Originator Originator, , First First- -time time Generic Generic and Multisource Generic and Multisource Generic
Multisource Generic Multisource Generic First First- -time time Generic Generic Originator Originator
Pharmacopoeias Pharmacopoeias Information from Information from
regulatory agencies regulatory agencies
(publicly available) & (publicly available) &
literature data literature data
Originator Originator s s
specifications specifications
API quality API quality
standards standards
Pharmacopoeias Pharmacopoeias Information from Information from
regulatory agencies regulatory agencies
(publicly available) & (publicly available) &
literature data literature data
Originator Originator s s
specifications specifications
FPP quality FPP quality
standards standards
Verify Verify identity, potency, identity, potency,
purity of API and FPP by purity of API and FPP by
pharmacopoeial methods pharmacopoeial methods
and in and in- -house methods house methods
Derive Derive identity, potency, identity, potency,
purity of API and FPP purity of API and FPP
by in house methods by in house methods
Establish Establish identity, identity,
potency, purity of potency, purity of
API and FPP by API and FPP by
in in- -house methods house methods
Analytical Analytical
methods methods
Birgit Schmauser
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April 2008 5
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Analytical Method Development Analytical Method Development
HPLC HPLC- -method to assay potency and purity method to assay potency and purity risk assessment risk assessment
Multisource Generic Multisource Generic First First- -time time Generic Generic Originator Originator
Selectively Selectively screen screen/detect /detect
any impurity or degradant any impurity or degradant
Establish Establish potency potency
Identify Identify
impurities/degradants impurities/degradants
Verify impurities from Verify impurities from
Pharmacopoeia Pharmacopoeia
Characterise in Characterise in- -house house
impurities/degradants impurities/degradants
(Response factors) (Response factors)
Derive Derive impurities/degradants impurities/degradants
from Originator from Originator
Characterize in Characterize in- -house house
impurities/degradants impurities/degradants
Calculate response factors Calculate response factors
Characterise Characterise all all
impurities/degradants impurities/degradants
Calculate Response factors Calculate Response factors
(qualification by clinical (qualification by clinical
use) use)
Use pharmacopoeial Use pharmacopoeial
reference materials reference materials
Extract Extract (& (& reproduce reproduce) reference ) reference
materials materials
Establish Establish reference reference
materials materials
Implement for routine use Implement for routine use Adapt Adapt/modify to/for routine use /modify to/for routine use Adapt Adapt to routine use to routine use
Birgit Schmauser
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April 2008 6
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Analytical Method Development Analytical Method Development
Interchangeability (IC) of Interchangeability (IC) of multisource generic FPPs multisource generic FPPs
( (Essential similarity Essential similarity with Innovator FPP) with Innovator FPP)
Pharmaceutical Pharmaceutical + + Bioequivalence Bioequivalence
Equivalence Equivalence
IC = IC = PE PE + + BE BE
Birgit Schmauser
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April 2008 7
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Analytical Method Development Analytical Method Development
Pharmaceutical equivalence Pharmaceutical equivalence
FPPs meet the FPPs meet the same same or or comparable comparable standards standards by by
use of equivalent analytical methods use of equivalent analytical methods
Same Same API API ((chemical chemical and and physical physical equivalence) equivalence)
Same Same dosage form dosage form and and route of administration route of administration
Same Same strength strength
Comparable Comparable labeling labeling
Equivalence in Equivalence in pharmaceutical development pharmaceutical development
Equivalence in Equivalence in stability stability
Equivalence in Equivalence in manufacture (WHO manufacture (WHO- -GMP) GMP)
Birgit Schmauser
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April 2008 8
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Analytical Method Development Analytical Method Development
Prequalification requirements Prequalification requirements
Validation of analytical methods is a Validation of analytical methods is a prerequisite prerequisite for for
prequalification of product dossiers prequalification of product dossiers
Non Non--compendial APIs and FPPs compendial APIs and FPPs are tested with are tested with methods developed by the methods developed by the
manufacturer manufacturer
For For compendial APIs and FPPs the applicability of pharmacopoeial methods compendial APIs and FPPs the applicability of pharmacopoeial methods to to
particular products particular products must be demonstrated (verification) must be demonstrated (verification)
Analytical methods must be developed and validated Analytical methods must be developed and validated
according to TRS according to TRS 823 823, , Annex Annex 5 5, , Validation of analytical Validation of analytical
procedures used in the examination of pharmaceutical procedures used in the examination of pharmaceutical
materials materials ; ICH Q ; ICH Q2 2 (R (R1 1) )
To be used within GLP and GMP environments To be used within GLP and GMP environments
Birgit Schmauser
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April 2008 9
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Analytical Method Development Analytical Method Development
METHODS METHODS PHARMACEUTICAL PHARMACEUTICAL CLINICAL CLINICAL
At At initial initial phase of pharmaceutical development phase of pharmaceutical development
To understand the To understand the profile profile of related of related
substances and to study substances and to study stability stability
To start To start measuring measuring the impact of the impact of
key product key product and and manufacturing manufacturing
process parameters process parameters on on consistent consistent
FPP quality FPP quality
To develop a stable and To develop a stable and
reproducible formulation for reproducible formulation for
the manufacture of the manufacture of
bioequivalence, dissolution, bioequivalence, dissolution,
stability and pilot stability and pilot- -scale scale
validation batches validation batches
To To determine determine
bioavailability bioavailability in in
healthy volunteers healthy volunteers
At At advanced advanced phase of pharmaceutical development phase of pharmaceutical development
To be To be robust, transferable, accurate robust, transferable, accurate
and and precise precise for specification for specification
setting, stability assessment and setting, stability assessment and
QC release of prequalified product QC release of prequalified product
batches batches
To To optimise optimise, , scale scale- -up up and and
transfer transfer a a stable stable and and
controlled controlled manufacturing manufacturing
process for the process for the
prequalification product prequalification product
To prove To prove
bioequivalence bioequivalence after after
critical variations critical variations to to
the prequalified the prequalified
dossier dossier
Use of analytical methods Use of analytical methods - - generics generics
Birgit Schmauser
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April 2008 10
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Analytical Method Development Analytical Method Development
Prerequisites Prerequisites for analytical method validation for analytical method validation
Six Ms
Quality of the Quality of the
analytical method analytical method
M Man an M Machine achine
qualified qualified
calibrated calibrated
robust robust
qualified qualified
M Methods ethods
suitable suitable
characterised characterised
documented documented
M Milieu ilieu M Material aterial M Management anagement
Quality Quality
Reference Reference
standards standards
Tempe Tempe- -
rature rature
Analysts Analysts
support support
skilled skilled
Humidity Humidity
Vibrations Vibrations
Time Time
Supplies Supplies
Irradi Irradi- -
ations ations
Birgit Schmauser
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April 2008 11
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Analytical Method Development Analytical Method Development
Method development life cycle Method development life cycle
Planning
Development and Validation Policy
Objectives/Requirements of Method
Information Gathering
Resource Gathering
Method development
Initital Method Development
Pre-Validation Evaluation
Method Optimization
Robustness
System Suitability
Development
Plan
Project
Customer Evaluation
Testing
Validation Experiments
Method Transfer
Experiments
Filed Method in Use
Periodically
Monitoring/Review
of Methods
in Control Labs
From: From: Analytical Chemistry in a GMP Environment Analytical Chemistry in a GMP Environment. Edited by J.M. Miller and J.B. Crowther, ISBN . Edited by J.M. Miller and J.B. Crowther, ISBN 0 0- -471 471- -31431 31431- -5 5, Wiley & Sons Inc. , Wiley & Sons Inc.
Birgit Schmauser
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April 2008 12
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Analytical Method Development Analytical Method Development
Validation should verify the suitability of an Validation should verify the suitability of an
analytical method analytical method for its intended purpose for its intended purpose
Validation should be founded on Validation should be founded on method method
development performed beforehand development performed beforehand that that
suggest the suggest the suitability and robustness suitability and robustness of the of the
method method
Validation may be performed in different ways Validation may be performed in different ways
(individual purpose) according to (individual purpose) according to common common
standards standards
Birgit Schmauser
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April 2008 13
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Validation protocol Validation protocol
Method principle / objective Method principle / objective
Listing of responsibilities Listing of responsibilities
Laboratories involved and their role in the validation Laboratories involved and their role in the validation
Method categorization Method categorization
List of reagents (including test lots) and standards List of reagents (including test lots) and standards
Test procedures to evaluate each validation parameter and proposed Test procedures to evaluate each validation parameter and proposed
acceptance criteria acceptance criteria
Plan or procedure when acceptance criteria are not met Plan or procedure when acceptance criteria are not met
Requirements for the final report Requirements for the final report
The validation process cannot proceed until the protocol and The validation process cannot proceed until the protocol and
all parties involved approve the acceptance criteria all parties involved approve the acceptance criteria
Birgit Schmauser
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April 2008 14
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Analytical Method Development Analytical Method Development
Innovator versus Generics Innovator versus Generics
Generics Generics Innovator Innovator
- - ++ R & D on API R & D on API
- - ++ Preclinical trials Preclinical trials
- - Method validation Method validation
summary summary
Clinical trials phase I and II Clinical trials phase I and II
- - Method validation Method validation
completed completed
Clinical trials phase III Clinical trials phase III
- - Validated methods Validated methods Post marketing phase IV Post marketing phase IV
Validated methods: Validated methods:
GMP and GLP GMP and GLP
Validated methods Validated methods Entering of Generics; Entering of Generics;
Pharmaceutical development, Pharmaceutical development,
Comparability with Innovator Comparability with Innovator
Birgit Schmauser
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April 2008 15
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Analytical Method Development Analytical Method Development
Validation Characteristics Validation Characteristics
Assay Assay Impurities Impurities Identification Identification
limit limit quantitative quantitative
++ - - ++ - - Accuracy Accuracy
++ - - ++ - - Precision Precision
++ ++ ++ ++ Specificity Specificity
- - ++ - - - - Detection Limit Detection Limit
- - - - ++ - - Quantitation Limit Quantitation Limit
++ - - ++ - - Linearity Linearity
++ - - ++ - - Range Range
++ ++ ++ ++ Robustness Robustness
Birgit Schmauser
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April 2008 16
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Analytical Method Development Analytical Method Development
Accuracy and precision Accuracy and precision
Accurate & Accurate &
precise precise
Accurate & Accurate &
imprecise imprecise
Inaccurate Inaccurate & &
precise precise
Inaccurate & imprecise Inaccurate & imprecise
Birgit Schmauser
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April 2008 17
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Analytical Method Development Analytical Method Development
Precision Precision
Expresses the Expresses the closeness of agreement closeness of agreement between a series of between a series of
measurements obtained from multiple sampling of the same measurements obtained from multiple sampling of the same
homogenous sample homogenous sample
Is usually expressed as the Is usually expressed as the standard deviation (S), standard deviation (S), variance variance
(S (S
2 2
) ) or or coefficient of variation coefficient of variation (RSD) (RSD) of a series of of a series of
measurements measurements
Precision may be considered at three levels Precision may be considered at three levels
Repeatability Repeatability (intra (intra--assay precision) assay precision)
Intermediate Precision Intermediate Precision (variability within a laboratory) (variability within a laboratory)
Reproducibility Reproducibility (precision between laboratories) (precision between laboratories)
Birgit Schmauser
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April 2008 18
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Analytical Method Development Analytical Method Development
Normal distribution, Normal distribution, probability function [P(x)] probability function [P(x)]
and confidence interval [CI] and confidence interval [CI]
Probability (P) Probability (P), that measurements from a normal distribution fall within [ , that measurements from a normal distribution fall within [- -xx
n n
, +x , +x
n n
] ]
for x for x
n n
= n = no o is described by the is described by the erf erf- -function function ! ! mean) mean)
An interval of An interval of 3 3 o o
covers covers 99 99..73 73% of values % of values
N
u
m
b
e
r

o
f

t
i
m
e
s

e
a
c
h

v
a
l
u
e

o
c
c
u
r
s
PP x x
nn
0.6826895 0.6826895 o o
0.9544997 0.9544997 2o 2o
0.9973002 0.9973002 o o
0.9999366 0.9999366 o o
0.9999994 0.9999994 o o
Values
o
2o
o
o
2o
o
Birgit Schmauser
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April 2008 19
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Analytical Method Development Analytical Method Development
Normal distribution, probability function [P(x)] and Normal distribution, probability function [P(x)] and confidence confidence
interval [CI] interval [CI]
Probability Probability- -P P Confidence interval [CI] Confidence interval [CI]
centered around the mean [] centered around the mean []
in units of sigma [ in units of sigma [o o] described by ] described by
inverse inverse erf erf- -function: function:
A CI of A CI of 95 95% includes values % includes values
1 1..95 95 o o around the mean around the mean
x x
pp
PP
1.28155 1.28155o o 0?00 0?00
1.64485 1.64485o o 0700 0700
1.95996 1.95996o o 070 070
2.57583 2.57583o o 077 077
3.29053 3.29053o o 0777 0777
Birgit Schmauser
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April 2008 20
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Analytical Method Development Analytical Method Development
Relationship of variability, probability and reliability of data Relationship of variability, probability and reliability of data
High variability of data (large High variability of data (large o o) generate large confidence intervals and ) generate large confidence intervals and
thus lower the reliability of the mean thus lower the reliability of the mean
Low variability of data (small Low variability of data (small o) o) generate small confidence intervals and generate small confidence intervals and
thus increase the reliability of the mean thus increase the reliability of the mean
Birgit Schmauser
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April 2008 21
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Analytical Method Development Analytical Method Development
Repeatability Repeatability
Six replicate sample preparation steps from a homogenously prepared tablet Six replicate sample preparation steps from a homogenously prepared tablet
mixture (nominal value of API mixture (nominal value of API 150 150 mg) mg)
Assay Assay Peak area Peak area Injection Injection
147.10 mg/98.06% 147.10 mg/98.06% 173865 173865 1 1
148.00 mg/98.66% 148.00 mg/98.66% 174926 174926 2 2
146.32 mg/97.54% 146.32 mg/97.54% 172933 172933 3 3
148.08 mg/98.72% 148.08 mg/98.72% 175011 175011 4 4
151.95 mg/101.30% 151.95 mg/101.30% 179557 179557 5 5
149.28 mg/99.52% 149.28 mg/99.52% 176425 176425 6 6
148.45 mg/98.96% 148.45 mg/98.96% 175453 175453 Mean Mean
1.98 mg/1.32% 1.98 mg/1.32% 2329 2329 SD ( SD (o o) )
1.32% 1.32% 1.32% 1.32% RSD RSD
Mean Mean 3 3 SD = SD =
Confidence interval of Confidence interval of 99 99..73 73% %
98 98..96 96 3 3x x1 1..32 32% = % = 95 95% %- - 102 102..92 92% %
Birgit Schmauser
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April 2008 22
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Analytical Method Development Analytical Method Development
Intermediate precision Intermediate precision
Expresses within Expresses within- -laboratories variations (different days, different analysts, laboratories variations (different days, different analysts,
different equipment etc.) different equipment etc.)
Peak area Peak area
analyst analyst 3 3
Peak area Peak area
analyst 2 analyst 2
Peak area Peak area
analyst 1 analyst 1
Injection Injection
177965 177965 175656 175656 173865 173865 1 1
178556 178556 175878 175878 174926 174926 2 2
177342 177342 176004 176004 172933 172933 3 3
178011 178011 176344 176344 175011 175011 4 4
179466 179466 175332 175332 179557 179557 5 5
179688 179688 174959 174959 176425 176425 6 6
178504 178504 175695 175695 175453 175453 Mean Mean
918 918 495 495 2329 2329 SD ( SD (o o) )
0.51% 0.51% 0.28% 0.28% 1.32% 1.32% RSD RSD
Analyst Analyst 1 1: : 98 98..96 96% % 3 3 x x 1 1..32 32% %
Analyst Analyst 2 2: : 99 99..12 12% % 3 3 x x 0 0..28 28
Analyst Analyst 3 3: : 100 100..70 70% % 3 3 x x 0 0..51 51
Average of Average of 3 3 analysts analysts 3 3SD SD: :
95 95% % - - 102 102..23 23% %
Mean Mean 3 3 SD: ( SD: (177252 177252 $$ 100 100%) %)
Birgit Schmauser
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April 2008 23
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Analytical Method Development Analytical Method Development
Reproducibility Reproducibility
Expresses the precision between laboratories Expresses the precision between laboratories
Collaborative studies, usually applied to standardisation of Collaborative studies, usually applied to standardisation of
methodology methodology
Transfer of technology Transfer of technology
Compendial methods Compendial methods
Birgit Schmauser
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April 2008 24
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Analytical Method Development Analytical Method Development
Accuracy Accuracy
Expresses the Expresses the closeness of agreement closeness of agreement between between the value which the value which
is accepted either as a conventional is accepted either as a conventional true value true value or or an accepted an accepted
reference value reference value and the and the value found value found
Sometimes referred to as Sometimes referred to as TRUENESS TRUENESS
true true mean mean
Birgit Schmauser
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April 2008 25
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Analytical Method Development Analytical Method Development
To find out whether a method is accurate: To find out whether a method is accurate:
Drug substance (assay) Drug substance (assay)
Application of the method to an analyte of known purity (e.g. reference Application of the method to an analyte of known purity (e.g. reference
substance) substance)
Comparison of the results of one method with those of a second well Comparison of the results of one method with those of a second well- -
characterised method (accuracy known) characterised method (accuracy known)
Drug product (assay) Drug product (assay)
Application of the method to synthetic mixtures of the drug product component Application of the method to synthetic mixtures of the drug product component
to which to which known quantities known quantities of the analyte have been added of the analyte have been added
Drug product may exceptionally be used as matrix Drug product may exceptionally be used as matrix
Drug substance/Drug product (Impurities) Drug substance/Drug product (Impurities)
Application of the method to samples spiked with Application of the method to samples spiked with known amounts known amounts of impurities of impurities
Birgit Schmauser
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April 2008 26
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Analytical Method Development Analytical Method Development
Accuracy Accuracy: : Application of the method to synthetic mixtures of the Application of the method to synthetic mixtures of the
drug product components drug product components
to which known quantities to which known quantities
of the analyte of the analyte
have been added have been added
Recovery Recovery reduced reduced
by by ~~10 10 15 15% %
From From: : Analytical Method Validation and Instrument Performance Verification Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. , Edited by Chung Chow Chan,Herman Lam, Y.C.
Lee and Xue Lee and Xue--Ming Zhang, ISBN Ming Zhang, ISBN 0 0--471 471--25953 25953--5 5, Wiley & Sons , Wiley & Sons
Birgit Schmauser
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April 2008 27
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Analytical Method Development Analytical Method Development
When to expect Accuracy problems When to expect Accuracy problems
Insufficient selectivity Insufficient selectivity of the method of the method
Impurity peaks are not resolved and account for assay value Impurity peaks are not resolved and account for assay value
Recovery Recovery is < is < 100 100% %
Irreversible adsorption of analyte to surfaces of the system Irreversible adsorption of analyte to surfaces of the system
Incorrect Incorrect assay value of a assay value of a reference standard reference standard
Due to decomposition of reference standard Due to decomposition of reference standard
Incorrect assay value due to Incorrect assay value due to change in matrix change in matrix
Analytical laboratory still uses the preceding matrix as standard Analytical laboratory still uses the preceding matrix as standard
Birgit Schmauser
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April 2008 28
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Analytical Method Development Analytical Method Development
Specificity Specificity
Is the ability to assess unequivocally the analyte in the presence of components Is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present (impurities, degradants, matrix) which may be expected to be present (impurities, degradants, matrix)
Identity testing Identity testing
To ensure the identity of an analyte To ensure the identity of an analyte
Purity testing Purity testing
To ensure accurate statement on the content of impurities of an analyte To ensure accurate statement on the content of impurities of an analyte
Assay Assay
To allow an accurate statement on the content of an analyte in a sample To allow an accurate statement on the content of an analyte in a sample
Birgit Schmauser
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April 2008 29
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Analytical Method Development Analytical Method Development
Specificity: Specificity: Overlay chromatogram of an impurity solution with a Overlay chromatogram of an impurity solution with a
sample solution sample solution
From From: : Analytical Method Validation and Instrument Performance Verification Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. , Edited by Chung Chow Chan,Herman Lam, Y.C.
Lee and Xue Lee and Xue--Ming Zhang, ISBN Ming Zhang, ISBN 0 0--471 471--25953 25953--5 5, Wiley & Sons , Wiley & Sons
Birgit Schmauser
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April 2008 30
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Analytical Method Development Analytical Method Development
Specificity and Specificity and stability stability
Stress stability testing to ensure the Stress stability testing to ensure the stability indicating potential stability indicating potential of an of an
analytical method analytical method
Apply diverse stress factors to the API Apply diverse stress factors to the API
Apply diverse stress factors to the FPP Apply diverse stress factors to the FPP
Stress conditions: e.g. Supplement Stress conditions: e.g. Supplement 2 2 of Generic Guideline; TRS of Generic Guideline; TRS 929 929, Annex , Annex 5 5
Assure that the API can be assessed specifically in the presence of Assure that the API can be assessed specifically in the presence of
known and unknown (generated by stress) impurities known and unknown (generated by stress) impurities
Assure that known impurities/degradants can be specifically assessed Assure that known impurities/degradants can be specifically assessed
in the presence of further degradants in the presence of further degradants
By By peak purity assessment peak purity assessment and (overlay of) and (overlay of) chromatograms chromatograms
Birgit Schmauser
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April 2008 31
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Analytical Method Development Analytical Method Development
Stress Stress stability studies versus stability studies versus forced degradation forced degradation studies studies
Stress stability Stress stability
(5 (5 15% decomposition) 15% decomposition)
Forced degradation Forced degradation Stress Stress
parameter parameter
pH pH 2 (2 weeks) 2 (2 weeks) 0.2 ml 0.2 ml 1N HCl 1N HCl / 5 ml API / 5 ml API- -solution / solution /
3h, 6h 3h, 6h, 12h, 24h7d (RT & 60 , 12h, 24h7d (RT & 60C) C)
Acid Acid
pH pH 10 (2 weeks) 10 (2 weeks) 0.2 ml 0.2 ml 1N NaOH 1N NaOH / 5 ml API / 5 ml API- -solution / solution /
3h, 6h 3h, 6h, 12h, 24h7d (RT & 60 , 12h, 24h7d (RT & 60C) C)
Base Base
1 g/ml 1 g/ml oxygen oxygen bubbled through (8 hours) bubbled through (8 hours)
0.1 0.1 2% 2% H H
2 2
OO
2 2
(24 hours) (24 hours)
0.2 ml 0.2 ml 5% 5% or or 35% 35% H H
2 2
OO
2 2
/ 5 ml API / 5 ml API- -
solution (RT, to 7d & 60 solution (RT, to 7d & 60C, C, 3h 3h) )
H H
2 2
OO
2 2
/ /
Oxygen Oxygen
- - 60 60C C / 5 ml solution ( / 5 ml solution (3h, 3h, 6h 6h7d) 7d) Heat Heat
60 60C (4 weeks) C (4 weeks) 105 105 C C / solid API ( / solid API (1d 1d and 7d) and 7d) Heat Heat
365 365 nm or white fluorescent light / nm or white fluorescent light /
solid API ( solid API (1 1dd and and 7 7d) d)
UV or Light UV or Light
50 50C / 80% RH (4 weeks) C / 80% RH (4 weeks) - - Humidity Humidity
Birgit Schmauser
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April 2008 32
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Analytical Method Development Analytical Method Development
Limit of Detection (LOD, DL) Limit of Detection (LOD, DL)
The LOD of an analytical procedure is the lowest amount of analyte in sample The LOD of an analytical procedure is the lowest amount of analyte in sample
which can be which can be detected but not necessarily quantitated as an exact value detected but not necessarily quantitated as an exact value
Determination is usually based on Determination is usually based on
Signal to noise ratio (~ Signal to noise ratio (~3 3::1 1) ( ) (baseline noise baseline noise) )
or or
Standard deviation of response ( Standard deviation of response (o o) and Slope (S) ) and Slope (S)
3 3..3 3 o o/S /S
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April 2008 33
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Analytical Method Development Analytical Method Development
Limit of Quantitation (LOQ, QL) Limit of Quantitation (LOQ, QL)
The LOQ is the lowest amount of analyte in a sample which can The LOQ is the lowest amount of analyte in a sample which can
be quantitatively be quantitatively determined with suitable precision and accuracy determined with suitable precision and accuracy
The quantitation limit is used particularly for the The quantitation limit is used particularly for the determination of determination of
impurities and/or degradation products impurities and/or degradation products
Determination is usually based on Determination is usually based on
Signal to noise ratio (~ Signal to noise ratio (~10 10::1 1) ( ) (baseline noise baseline noise) )
or or
Standard deviation of response ( Standard deviation of response (o o) and Slope (S) ) and Slope (S)
10 10 o/ o/SS
Birgit Schmauser
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April 2008 34
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Analytical Method Development Analytical Method Development
Noise Noise
LOD LOD
Signal to Signal to Noise Noise = = 3 3: :1 1
LOQ LOQ
Signal to Signal to Noise Noise = = 10 10: :1 1
LOD, LOQ and Signal to Noise Ratio (SNR) LOD, LOQ and Signal to Noise Ratio (SNR)
Birgit Schmauser
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April 2008 35
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Analytical Method Development Analytical Method Development
LOQ LOQ
Quantitation by SNR is accepted Quantitation by SNR is accepted
Quantitation by Standard deviation of response ( Quantitation by Standard deviation of response (o o) and Slope (S) ) and Slope (S)
( (10 10 o/ o/S) is more adequate as it involves the S) is more adequate as it involves the response response of the actual of the actual
analyte analyte
Best to calculate in the region close to y Best to calculate in the region close to y- -intercept intercept
Birgit Schmauser
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April 2008 36
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Analytical Method Development Analytical Method Development
LOQ and impurities LOQ and impurities
In determination of impurities in APIs and FPPs the LOQ should In determination of impurities in APIs and FPPs the LOQ should
be determined be determined in the presence of API in the presence of API
LOQ should be LOQ should be NMT reporting level NMT reporting level
LOQ should be given LOQ should be given relative relative to the test concentration of API to the test concentration of API
Specificity of impurity determination should always be Specificity of impurity determination should always be
demonstrated in the presence of API demonstrated in the presence of API at API specification levels at API specification levels
Spiking of test concentration (API/FPP) with impurities at levels of their Spiking of test concentration (API/FPP) with impurities at levels of their
specification range specification range
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April 2008 37
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Analytical Method Development Analytical Method Development
Spiking Spiking
API test concentration (normalised) API test concentration (normalised)
0 0. .1 1 mg/ml ( mg/ml (100 100%) %)
Impurity spiking concentrations Impurity spiking concentrations
0 0. .001 001 mg/ml ( mg/ml (1 1%) %) specification limit specification limit
0 0. .0001 0001 mg/ml ( mg/ml (0 0. .1 1%) %) limit of quantitation (minimum requirement) limit of quantitation (minimum requirement)
API at test concentrations API at test concentrations
API below test concentrations API below test concentrations
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Linearity Linearity
of an analytical procedure is its ability ( of an analytical procedure is its ability (within a given within a given range range) )
to obtain test to obtain test results results which are directly which are directly proportional to the proportional to the
concentration concentration (amount) of analyte in the sample (amount) of analyte in the sample
If there is a If there is a linear linear relationship relationship test results should be test results should be
evaluated evaluated by appropriate by appropriate statistical methods statistical methods
Correlation coefficient (r) Correlation coefficient (r)
YY--intercept intercept
Slope of regression line Slope of regression line
Residual sum of squares Residual sum of squares
PLOT OF THE DATA PLOT OF THE DATA
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Usual acceptance criteria for a linear calibration Usual acceptance criteria for a linear calibration
curve curve
r > r > 0 0..999 999; ; y y- -intercept a < intercept a < 0 0 to to 5 5% of target concentration % of target concentration
RSD (wrt calibration curve) < RSD (wrt calibration curve) < 1 1..5 5- -2 2% %
r > 0.997 r < 0.997
From From: : Analytical Method Validation and Instrument Performance Verification Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and , Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and
Xue Xue--Ming Zhang, ISBN Ming Zhang, ISBN 0 0--471 471--25953 25953--5 5, Wiley & Sons , Wiley & Sons
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Range Range
The The range range of an analytical procedure is the interval of an analytical procedure is the interval
between the upper and lower concentration (amounts) of between the upper and lower concentration (amounts) of
analyte in the sample for which it has been demonstrated analyte in the sample for which it has been demonstrated
that the analytical procedure has a that the analytical procedure has a suitable level of suitable level of
precision, accuracy and linearity precision, accuracy and linearity
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Range Range
Assay Assay
80 80 to to 120 120% % of test concentration of test concentration
Content uniformity Content uniformity
70 70 to to 130 130% % of test concentration of test concentration
Dissolution Dissolution
QQ- -20 20% to % to 120 120% %
Impurities Impurities
Reporting level Reporting level 120 120% of specification limit (with respect % of specification limit (with respect
to test concentration of API) to test concentration of API)
Assay & Impurities Assay & Impurities
Reporting level to Reporting level to 120 120% of assay specification % of assay specification
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Linearity is limited to Linearity is limited to 150 150%of shelf life specification of impurities %of shelf life specification of impurities
Test concentration can be Test concentration can be
used to determine impurities used to determine impurities
To determine drug substance To determine drug substance
(assay) the test concentration (assay) the test concentration
must be diluted must be diluted
The range is The range is 0 0 ~ ~ 150 150% of % of
impurity specification impurity specification
From From: : Analytical Method Validation and Instrument Performance Verification Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. , Edited by Chung Chow Chan,Herman Lam, Y.C.
Lee and Xue Lee and Xue--Ming Zhang, ISBN Ming Zhang, ISBN 0 0--471 471--25953 25953--5 5, Wiley & Sons , Wiley & Sons
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Robustness Robustness
Robustness of an analytical procedure should show Robustness of an analytical procedure should show
the the reliability reliability of an analysis of an analysis with respect to with respect to deliberate deliberate
variations in method parameters variations in method parameters
The evaluation of robustness should be considered The evaluation of robustness should be considered
during the during the development phase development phase
If measurements are If measurements are susceptible susceptible to variations in to variations in
analytical conditions the analytical conditions the analytical conditions should analytical conditions should
be suitably controlled be suitably controlled or a or a precautionary statement precautionary statement
should be included in the procedure should be included in the procedure
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Influence of buffer pH and buffer concentration in mobile phase Influence of buffer pH and buffer concentration in mobile phase
on retention times of API and impurities on retention times of API and impurities
Conclusion: The buffer composition should be maintained in a range of Conclusion: The buffer composition should be maintained in a range of
85 85 0 0..5 5% %
Missing: Missing: Acceptance criterion for maximal deviation of retention time should be Acceptance criterion for maximal deviation of retention time should be
defined unless justified defined unless justified
Impurity C Impurity C Impurity B Impurity B Impurity A Impurity A API API
8 8..26 26 7.43 7.43 3.86 3.86 10.46 10.46 As is As is
8.38 8.38 7.51 7.51 3.94 3.94 10.45 10.45 buffer pH 5.9 buffer pH 5.9
8.34 8.34 7.49 7.49 3.94 3.94 10.46 10.46 buffer pH 6.9 buffer pH 6.9
6.66 6.66 6.16 6.16 3.43 3.43 7.84 7.84 Buffer conc. 83% Buffer conc. 83%
11.18 11.18 9.61 9.61 4.77 4.77 15.26 15.26 Buffer conc. 87% Buffer conc. 87%
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System suitability testing System suitability testing
Based on the concept that equipment, electronics, Based on the concept that equipment, electronics,
analytical operations and samples to be analysed analytical operations and samples to be analysed
constitute an integral system constitute an integral system that can be evaluated as that can be evaluated as
such such
Suitability parameters are established for each analytical Suitability parameters are established for each analytical
procedure procedure individually individually
Depend on the Depend on the type of analytical procedure type of analytical procedure
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Method Method stability stability
System suitability System suitability over time over time
Sample solution stability Sample solution stability
A solution of stavudine is stable for ~ A solution of stavudine is stable for ~ 2 2 h, then it starts to degrade h, then it starts to degrade
to thymine to thymine
Impurity Impurity--spiked sample solution stability spiked sample solution stability
A solution containing stavudine spiked with its impurity thymine A solution containing stavudine spiked with its impurity thymine does not allow to does not allow to
clearly distinguish between degradation and spike clearly distinguish between degradation and spike
A solution containing stavudine of a FPP A solution containing stavudine of a FPP--stability sample solution does not allow stability sample solution does not allow
to clearly distinguish between FPP to clearly distinguish between FPP--stability degradation and sample solution stability degradation and sample solution
degradation degradation
Should be analysed immediately Should be analysed immediately
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When to be surprised about validation data: When to be surprised about validation data:
Precision of Precision of
impurity determination impurity determination
Precision of Precision of
API determination API determination
Method precision of Method precision of
released API (dissolution) released API (dissolution)
% RSD 0.33 % RSD 0.33 2.25 2.25 System precision System precision
% RSD 0.0 % RSD 0.0 Method precision Method precision
% RSD 0.08 % RSD 0.08 Average peak area Average peak area
% RSD 2.0 % RSD 2.0 Acceptance criterion Acceptance criterion
% RSD 0.4 % RSD 0.4 Average peak area Average peak area
% RSD 10.0 % RSD 10.0 Acceptance criterion Acceptance criterion
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Specification range (USL Specification range (USL- -LSL) LSL)
Process variability (usually Process variability (usually 2 SD) 2 SD)
Analytical variability ( Analytical variability ( 3 3o o) )
~ NMT 30% of total specification range ~ NMT 30% of total specification range
Analytical variability Analytical variability Process variability Process variability
Reliability of evaluation of major process variables Reliability of evaluation of major process variables by analytical by analytical
procedures procedures depends on depends on analytical variability analytical variability
Impurities Impurities
LOQ LOQ and specification limit (e.g. qualification limits NMT 0.15%) and specification limit (e.g. qualification limits NMT 0.15%)
Response factors Response factors (LOQ modified by response factor) (LOQ modified by response factor)
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Methods for cleaning validation Methods for cleaning validation
Method for assay and related substances used in stability studies of API Method for assay and related substances used in stability studies of API
and FPP and FPP
Specificity Specificity (in (in samples taken from a cleaning assessment samples taken from a cleaning assessment))
L Linearity of response inearity of response (from (from 50% of the cleaning limit to 50% of the cleaning limit to 1 10x this concentration 0x this concentration; R ; R
2 2
0.9900) 0.9900)
Precision Precision
Repeatability Repeatability (RSD (RSD 5%) 5%)
i inter ntermediate precision [ruggedness (USP)] mediate precision [ruggedness (USP)]
Reproducibility Reproducibility
L Limits of detection and quantitation imits of detection and quantitation
AAccuracy or recovery from rinsate ccuracy or recovery from rinsate (( 80%) 80%), swabs , swabs (( 90%) 90%), and process surface , and process surface (( 70%) 70%)
RRange ange ((lowest level is at least 2x higher than lowest level is at least 2x higher than LOQ LOQ))
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Summary Summary
Analytical procedures play a critical role in pharmaceutical Analytical procedures play a critical role in pharmaceutical
equivalence and risk assessment/management equivalence and risk assessment/management
Establishment of Establishment of product product- -specific specific acceptance criteria acceptance criteria
Assessment of Assessment of stability stability of APIs and FPPs of APIs and FPPs
Validation of analytical procedures should demonstrate that Validation of analytical procedures should demonstrate that
they are they are suitable for their intended use suitable for their intended use
Validation of analytical procedures deserves Validation of analytical procedures deserves special attention special attention
during assessment during assessment of dossiers of dossiers for prequalification for prequalification
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THANK YOU THANK YOU