Analytical Methods Validation

M. Hatta Prabowo
Department of Pharmaceuticals Chemistry
Universitas Islam Indonesia

Chemical analysis
The bioanalytical part of bioequivalence trials
should be conducted according to the applicable
principle of Good Laboratory Practice (GLP)

Good Laboratory Practice

(GLP)

Test plan (Analytical protocol)

Sample traceability
Documentation, possible to reconstruct the
study
Analytical method validation report
Analytical report signed by responsible
investigator

Pre-study phase

The method used must be well characterised

Stability
Specificity
Accuracy

Precision
Limit

of quantitation
Response function

Validation objective
To demonstrate that the analytical procedure is
suitable for its intended purpose

Analytical method validation

Analytical
Procedure

Stability

Selectivity
Robustness

Validation

Accuracy

Calibration curve
Precision
Limit of Quantitation
LOQ

- within-run
- between-run

Recovery

Analytical procedure

Sample
preparation

Separation

Detection

Specificity (selectivity)

Ability of an analytical method to measure only

what it is intended to measure

Blank samples from six different subjects

Will other drugs, metabolites or endogenous
components interfere in the measurements?

Accuracy
The closeness of mean test results obtained by
the analytical method to the true value
(concentration) of the analyte.

Accuracy

Accuracy should be measured at minimum 3

levels
At least 5 determinations per concentration
The mean value should be within 15% of the
actual value
At the lower limit of quantitation level within
20% is accepted
x

x
x

Precision
The closeness of individual measurements of an
analyte when the procedure is applied
repeatedly to multiple aliquotes of a single
homogenous volume of biological matrix

Precision

Precision should be measured at minimum 3

levels
At least 5 determinations per concentration
The calculated CV should not exceed 15%
At the lower limit of quantitation level, CV
should not exceed 20%
Subdivided into within-run and between-run

Precision and Accuracy

Conc.
nmol/l

Accuracy
(%)

Precision

Within-run Between-run

0.76

0.6

%
5.1

23
122

3.6
3.9

1.7
1.3

%
6.1

18

1.8
1.3

18
18

Recovery

The extraction efficiency of an analytical

method

Recovery of an analyte need not be 100%

Lower limit of quantitation

The lowest standard on the calibration curve
should be accepted as the lower limit of
quantitation (LLOQ)

if

Lower limit of quantification

The analyte responce at LLOQ is at least 5

times the blank response
The peak should be identifiable and discrete
Precision within 20% CV
Accuracy of 80-120%

LLOQ (1.50 nmol/l) for

morphine
Sample No.
LLOQ 1
LLOQ 2
LLOQ 3
LLOQ 4
LLOQ 5
LLOQ 6
Mean:
SD:
CV%:

nmol/l
1.58
1.61
1.46
1.44
1.50
1.49
1.51
0.067
4.5

Accuracy
5.3%
7.3%
-2.6%
-4.0%
0.0%
-0.7%
0.9%

Calibration/Standard curve

A calibration curve is the relation between instrument response and

known concentrations of the analyte

Should be prepared in the same biological matrix as the samples

Should consist of 6-8 samples covering the expected range

Should include LLOQ and a blank sample

Should include a zero sample (with internal standard)

Same curve fitting, weighting in prestudy and study

Any changes should be documented

Calibration curve

Sample dilution

Any required sample dilutions should use like

matrix
Dilution QC sample should be used

Robustness
How many samples can be analysed in one run?

Robustness
115

Found concentration %

110
105
100
0

10

20

30

40

50

60

95
90
85
Sample No.

70

80

90

100

110

Stability of your substance

In Freeze/Thaw tests

In room temperature (4 h)

In the automatic injector

In stock solutions
In plasma during storage

Analytical method validation

Analytical
Procedure

Stability

Selectivity
Robustness

Validation

Accuracy

Calibration curve
Precision
Limit of Quantitation
LOQ

- within-run
- between-run

Recovery

References
1.

2.

3.

Guidance for Industry

Bioanalytical Method Validation FDA, May
2001
Workshop Report: Shah, V.P. Et al.,
Pharmaceutical Research: 1992; 9:588-592.
Workshop Report: Shah, V.P. et al.,
Pharmaceutical Research: 2000; 17:15511557

Costs

Validation = 130-180.000 SEK

Stability = 15-20.000 SEK for each time point
QA = 11.000 SEK/study

The study phase (1)

...in which the validated bioanalytical method

is applied to the actual analysis of samples
from the biostudy mainly in order to confirm
the stability, accuracy and precision.

The study phase (2)

Calibration curve in each run

Six Quality Control samples in each run
Pre-stablished SOPs for procedures (method)
Acceptance criteria for a run
- accuracy and precision of the calibration
curve
- accuracy and precision of the QC samples
- repeat analysis
It is preferable to analyse all study samples
from a subject in a single run

The study phase (3)

The QC samples should be used to accept or

reject the run (2 samples at 3 levels)
Four QC samples out of six should be within
15% of their nominal value
Two QC samples can be outside 15% but not
both at the same concentration

System suitability test

The lowest calibration sample is injected before
each run.
The system is accepted if:

Signal to noise ratio is above 5 for the substance.

The peak shape is acceptable after visual

inspection of the chromatogram

The retention times are within 10% of the previous

run.

The analytical report should include

Results for all calibration curves

Results for all quality control samples
Representative number of chromatograms
Should include data from subjects who
eventually dropped-out
Reanalysed samples and the reason for
reanalyses
The analytical validation report
The responsible investigator(s) should sign for
their respective section of the report

Chiral active substances

The bio-analytical method should be

enantiomeric
Unless
Both

products contain the same stable singel

enantiomer
Both products contain the racemate and both
enantiomers show linear pharmacokinetics

Also guidance for

Reference and test product

Data analysis
In vitro dissolution comlementary to a
bioequivalence study
Reporting of results
Application for products containing new active
substances
Application for products containing approved
active substances

is given