Method Validation in the

Pharmaceutical Industry
Kate Arnot

Analytical Development, PAR&D (AW)

6th November 2002

Overview
The drug development process
General recommendations
Examples
Assay
Dissolution test for an immediate release tablet
Identity
Identification test for a drug substance
Impurities
Quantitative test residual solvents in a
drug substance
Limit test residual solvents in a tablet

DRUG DEVELOPMENT PROCESS

Years

10

11 12 13

Start

14

15

16

Approval
Clinical Trials

Initial
research

Drug
discovery

Phase 1

Phase 2

Phase 3

50-100
people

100-200
people

500-5000
people

Phase 4

Full Scale
Manufacture

Clinical
Development

Toxicology and Pharmacokinetics

Chemical development
Pharmaceutical development
Sales and Marketing

Cost

2M

10,000 compounds

150M

300M

1 new medicine
3

A phased approach to analytical

validation
Phase I Limited validation, focussing on key
method attributes eg.specificity, limits of
quantitation and linearity
Phase II Starting to include accuracy and
precision data to support specifications
Phase III Full validation according to ICH
guidelines will be completed for all analytical
methods prior to submission of marketing
applications
4

General recommendations
Design your validation studies to reflect
the way that the method will be applied
in routine use as far as possible
Ensure that you have considered the
specification you want to achieve in
designing your studies
Know your method before you start to
validate!
5

Assay - Dissolution test for an

immediate release tablet
Dissolution testing is used to evaluate the
release of drug substance from tablets
Dissolution apparatus 2 (paddles), 50rpm,
37C, aqueous surfactant as dissolution
medium, UV analysis, sampling at a single 45
minute timepoint
Typical Specification: Q = 80 at 45 minutes (ie
each of 6 units should achieve 85%
dissolution)
6

Dissolution specification USP

acceptance table
S1, 6 units
Each unit is not less that Q+5%
S2, 6 units
Average of 12 units (S1+S2) is equal to or
greater than Q and no unit is less than Q-15%
S3, 12 units
Average of 24 units (S1+S2+S3) is equal to
or greater than Q, not more than 2 units are
less than Q-15%, and no unit is less than Q25%
7

Dissolution apparatus

Typical dissolution profile

%

100

90

80
70

60
% dissolved

D
i
s
s
o
l
u
t
i
o
n

50

40

30
20

10

0
0

15

30

45

60

75

time (mins)

Time (mins)

Validation parameters evaluated

Specificity
Linearity
Accuracy and range
Precision
Repeatability
Intermediate
precision

Robustness
10

Specificity
Demonstrate absence of interference from
excipients and reagents at the monitoring
wavelength
For example calculate the absorbance of a
placebo solution as a percentage of the
absorbance of a standard solution
Typically results of less that 2% would be
considered acceptable
Specificity may also be inferred from linearity
and accuracy data
11

Linearity
Range studied 0 to 150% of nominal (7 solutions,
including a blank solution)
Linearity experiments conducted in the presence and
absence of excipients
Determine equation of the line, correlation coefficient,
95% confidence interval of the slope and the
intercept. Verify that the 95% confidence interval of
the intercept includes zero, or that the result is
insignificant in the context of the experiment
Check that the response for both experiments is the
same calculate the slopes as a % of each other
Typically results of 98 to 102% would be considered
acceptable
12

Linearity in the presence of placebo

1.6

y = 42.983x + 0.004
r = 0.99989
95% CI of slope 0.0730
95% CI of intercept 0.016

1.5

A
b
s
o
r
b
a
n
c
e

1.4

1.3

1.2

1.1

Ab
0.9
sor
ban
0.8
ce
0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

0.02

0.022

0.024

0.026

0.028

0.03

0.032

0.034

0.036

0.038

concentration (g/100ml)

Concentration (g/100ml)

13

Linearity in the absence of placebo

1.6
1.5

y = 43.033x + 0.006
r = 0.99990
95% CI of slope 0.695
95% CI of intercept 0.009

1.4
1.3
1.2
1.1
1
0.9

Absorbance

A
b
s
o
r
b
a
n
c
e

0.8
0.7
0.6

Comparison of slopes:
(42.983/43.033) x 100
= 99.9%

0.5
0.4
0.3
0.2
0.1
0
0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

0.02

0.022

0.024

0.026

0.028

0.03

0.032

0.034

0.036

0.038

concentration (g/100ml)

Concentration (g/100ml)

14

Accuracy and Range

3 samples at each of 5 different concentration
levels were evaluated: 50%, 85%, 100%,
115% and 125% of nominal
Samples were analysed against standards as
per the method and % recoveries calculated
Typically results in the range 95 to 105%
would be considered acceptable
The overall standard deviation and relative
standard deviation may be used as an
estimate of precision
15

Example Accuracy Data

Experiment 1
50% 97.3
85% 96.8
100%96.5
115%96.0
125%95.8
Mean96.5
sd
0.7

Experiment 2
50% 99.3
85% 99.4
100%99.6
115%98.7
125%98.9
Mean99.2
sd
0.5
16

Example Accuracy Data continued

Experiment 1
Samples mixed, and allowed to stand overnight

Experiment 2
Samples stirred continuously
Time
1
Recovery
98.8%
(hours) 4
99.1%
21
99.2%
27
99.3%
17

Accuracy/Linearity how do
you prepare your samples?
Prepare samples containing drug substance
plus an appropriate quantity of ground
placebo tablet
Prepare samples containing drug substance
plus appropriate quantities of an excipient mix
Prepare samples by using different weights
from a ground active tablet preparation,
making a correction for nominal concentration
Make sure that you are confident in your
sample extraction technique
18

Precision - Repeatability
One analyst conducting multiple replicates of
the same sample, on the same day using the
same equipment, and applying the method as
written.
For dissolution experiments correcting for
tablet weight may be appropriate
Determine the mean, the range, the standard
deviation and the relative standard deviation.

19

Precision Intermediate precision

A second operator conducts the repeatability
experiment on a different day using different
equipment where possible.
Determine the individual data and overall
mean, range, standard deviation and relative
standard deviation.
Consider testing statistically that there is no
significant difference between operators
before combining the data set.
20

Robustness
A number of attributes were evaluated prior to
validation:
Standard and sample solution stability
Effect of pH and the requirement to buffer
and/or degass the dissolution medium
Reagents from different suppliers were
evaluated
Sampling tubes, filters and sampling and
filtration speeds were evaluated
Single point vs profile testing
21

Identification Infrared method

for a drug substance
Infrared spectroscopy is frequently used in
identification testing
Sample is dispersed in KBr, analysed using
the DRIFTS technique, and the spectrum
obtained visually compared with that of a fully
characterised reference standard analysed
under the same conditions.
Typical Specification: Result consistent with
reference standard
22

Validation parameters evaluated

Specificity

Identification testing should

optimally be able to discriminate
between compounds of closely
related structure which are likely
to be present (ICH Q6A)
The choice of such potentially
interfering materials should be
based on sound scientific
judgement with a consideration
of the interferences that could
occur (ICH Q2B)
23

Specificity
Demonstrate specificity between drug substance and
other potentially interfering compounds
eg. starting materials, isolated intermediates and
other materials manufactured or formulated in the
same plant
Consideration should also be given to
enantiomers, counter ions/salts, solvates and
polymorphs if necessary.

24

1.0

1.3
1.2
1.1

82968I01

Drug substance

0.9

0.8

1.0
0.7

0.9
0.6
Kubelka-Munk

0.8
Kubelka-Munk

Development
compound

0.7
0.6

0.5

0.4

0.5
0.3

0.4
0.3

0.2

0.2
0.1

0.1
0.0
4000

3500

3000

2500

2000

1500

1000

3500

500

3000

0.85

0.70
0.65
0.60
0.55

Isolated
intermediate

0.55
0.50
0.45
0.40

0.50

1500

1000

500

Compound formulated
in same plant

0.35

0.45

Kubelka-Munk

Kubelka-Munk

2000

0.60

0.80
0.75

2500

W avenumbers (cm-1)

Wavenumbers (cm-1)

0.40
0.35
0.30

0.30
0.25
0.20

0.25
0.15

0.20

0.10

0.15
0.10

0.05

0.05
-0.00

0.00
-0.05
4000

-0.05
3500

3000

2500

2000
Wavenumbers (cm-1)

1500

1000

500

4000

3500

3000

2500

2000

1500

1000

Wavenumbers (cm-1)

25

500

Impurities Quantitative test for residual

solvents in drug substance
A range of solvents are used in the
manufacture of a drug substance. It is
necessary to quantify these, and
demonstrate that they comply with ICH
Q3C requirements
Capillary GC method with flame
ionisation detection.
Typical Specification: 0.5% w/w
maximum
26

Validation parameters evaluated

Specificity
Linearity
Accuracy
Precision
Reproducibility
Intermediate precision

Range
Limits of detection and
quantitation
Robustness
27

Specificity
Demonstrate method is specific for all
the solvents used in the registered
synthetic process
Can use linearity data for solvents in the
presence and absence of the drug
substance to show that the drug
substance does not interfere
28

22000

0.0
1.0

28000

2.0
Time (min)

N -M e th y l-2 -p y rro lid o n e

D im e th y ls u lp h o x id e

A c e to n itrile
Tolu e n e

uV
26000

T rie th y la m in e
E th y l A c e ta te
P ro p a n -2 -o l

24000

Specificity chromatogram

3.0

29

Linearity
Range studied 0 to 150% of nominal (9 solutions,
including a blank solution and a solution at the LOQ)
Drug substance required without solvent, or low
levels of solvent and then make a correction
Determine equation of the line, correlation coefficient,
95% confidence interval of the slope and the
intercept. Verify that the 95% confidence interval of
the intercept includes zero, or that the result is
insignificant in the context of the experiment

30

Accuracy
Calculate recoveries from the linearity
experiments by analysing the solutions
against an external standard as per the
method.
Typically results in the range 80 to 120%
would be considered acceptable
The overall standard deviation and relative
standard deviation may be used as an
estimate of precision
31

Table 1

Residual solvents by GC - accuracy of ethyl acetate in the presence of drug

substance

Concentration ethyl
acetate % nominal
(with respect to
standard
concentration)

Ethyl acetate
added
theoretical
(l/100 ml)

Ethyl acetate
theoretical
(mg/100 ml)a

Ethyl acetate
determined
(mg/100 ml)

Recovery
(% w/w)

0b

NA

10

1.0

0.902

0.844

93.5

15

1.5

1.353

1.289

95.3

25

2.5

2.255

2.017

89.4

50

5.0

4.510

4.495

99.7

75

7.5

6.765

6.874

101.6

100

10.0

9.020

8.991

99.7

125

12.5

11.275

11.435

101.4

150

15.0

13.530

13.616

100.6

Mean

97.7

Standard deviation

4.4

% RSD

4.5

This figure is corrected for the density of ethyl acetate (0.902 g/ml)
The 0 level solution represents a test solution of drug substance to which no ethyl acetate was
added. A correction for the level of ethyl acetate found in this solution was made for all other test
solutions prepared.
NA Not applicable
b

32

Intermediate precision - Japanese

style
Expt No
Operator
GC
Column

1
1
1
1

2
1
2
2

3
1
2
1

4
1
1
2

5
2
1
1

6
2
2
2

7
2
2
1

8
2
1
2

Experiments are conducted on different days with each analyst

using each instrument/column combination in turn
Report results, overall mean, overall standard and relative
standard deviation, and 95% CI of sd
33

Range
Determined from the linearity, accuracy and
precision data

34

Limit of Quantitation and

Detection
Limit of quantitation established as 0.05%
w/w
Samples at this concentration included in
linearity and accuracy experiments
Limit of detection estimated as 0.02% w/w by
visual inspection of chromatograms

35

18000

0.0
2.762 DMSO

0.809 EtOAc

20000

22000

1.086 ACN

uV

3.061 NMP

24000

LOQ chromatogram

1.0
2.0
Time (min)
3.0

36

Robustness
A number of attributes were evaluated prior to
validation using an experimental design
approach:
Injector temperature
Detector temperature
Initial temperature
Ramp rate
Hold time
Split flow
Column head pressure
37

Impurities Limit test for residual

solvent in a tablet
An alcohol is used as a preservative in
a purchased film coating concentrate. It
is removed during the manufacturing
process, but this needs to be
demonstrated
Fast GC method with flame ionisation
detection.
Need to demonstrate that tablets
comply with requirements for Class 3
solvents ie less than 0.5% w/w
38

Validation parameters evaluated

Specificity

Limit of quantitation
Linearity

For solvents arising

from the drug
substance and
tablet manufacturing
process
0.05% w/w
0.05% to 0.5% w/w

39

References
ICH www.ICH.org
International Pharmaceutical Product Registration
Aspects of Quality, Safety and Efficacy, eds
Cartwright and Matthews, Ellis Horwood, 1994,
Chapter 8, Analytical validation
The validation of analytical methods for drug
substances and drug products in UK pharmaceutical
laboratories, G S Clarke, J. Pharm. Biomed. Anal.,
12. 643 652 (1994)
FDA www.FDA.gov
Technical review guide: Validation of chromatographic
methods
40