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Validation methods 1
Bioanalytical methods validation for
pharmacokinetic studies
P.L. Toutain
Toulouse Feb. 2008
ECOLE
NATIONALE
VETERINAIRE
T O U L O U S E
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Validation methods 2
Validation methods
Selective and sensitive analytical
methods for the quantitative
determination of drugs and their
metabolites (analytes) are critical
for successful performance of PK
and bioequivalence studies
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Validation methods
Validation of analytical methods
includes all the procedures
recommended to demonstrate that a
particular method, for a given
matrix, is reliable and reproducible
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Validation methods
1. A priori validation:
Pre-study validation for analytical
method development and method
establishment
2. In-life validation
(Routine validation)
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Regulatory requirements
G.L.P.
(e.g.; bioequivalence, Toxicokinetics)
S.O.P. (standard operating procedure)
(from sample collection to reporting)
Record keeping
Chain of sample custody (chane des garanties)
Sample preparation
Analytical tools
Procedures for quality control and verification of
results
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A priori validation
makes sure the method
is suitable for its
intended use
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A priori validation:
criteria to be validated
1. Calibration curve
2. Accuracy
3. Precision (repeatability, reproducibility)
4. Limit of quantification (LOQ)
5. Limit of detection (LOD)
6. Sensitivity
7. Specificity/selectivity
8. Stability of the analyte in the matrix under study
9. Others (ruggedness, agreement,)
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1. Calibration curve
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Definition
It is the relationship between known
concentrations and experimental response
values

Goal
Determine the unknown concentration of
a sample
Calibration curve
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Response: dependent variable
(peak,area ..)
Y

(
o
b
s
e
r
v
e
d
)

Y
X
y = ax + b
Independent variable:
exactly known
concentrations
Calibration curve
x1
y1
xn
Yn
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Response: dependent variable
Y

(
o
b
s
e
r
v
e
d
)

Y
X
y = ax + b
Independent variable:
X
estimated concentration
^
Calibration curve
x1
y1
xn
Yn
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x
Response
x
Response
GOOD BAD
^ ^
Calibration curve
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Calibration curve
Construction
5 to 8 points over the analytical domain
replicates are required to test linearity
3 to 5 replicates per levels
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Standard calibration curve

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Calibration curve
The calibration curve should be prepared
in the same biological matrix (e.g. plasma )
as the sample in the intended study by
spiking with known concentration of the
analyte (or by serial dilution).
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Reference Standard
Calibration standards and quality control samples
(QC)
Authenticated analytical reference standard
should be used to prepare (separately) solution
of known concentration
certified reference standards
Never from a marketed drug formulation
commercially supplied reference standards
other material of documented purity
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Building the calibration curve:
a regression problem
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Building the calibration curve:
a regression problem
In statistics, regression analysis is a statistical
technique which examines the relation of a
dependent variable (response variable or dependent
variable i.e. Y) that is for us the response of the
analytical apparatus (peak, area..) to specified
independent variables (explanatory variables or
independent variable i.e. X) that is for us the
concentration of calibrators .
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Linear regression : see Wikipedia
Linear regression - Wikipedia, the free
encyclopedia
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Linear regression : Wikipedia
In statistics, linear regression is a regression method
that models the relationship between a dependent
variable Y, independent variables Xi, i = 1, ..., p, and a
random term . The model can be written as:



where 0 is the intercept ("constant" term), the is are
the respective parameters of independent variables, and
p is the number of parameters to be estimated in the
linear regression.

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Linear regression : Wikipedia
This method is called "linear"
because the relation of the
response (the dependent variable Y)
to the independent variables is
assumed to be a linear function of
the parameters.

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Linear regression : Wikipedia
It is often erroneously thought that the reason the technique
is called "linear regression" is that the graph of Y = 0 + x
is a straight line or that Y is a linear function of the X
variables. But if the model is (for example)




the problem is still one of linear regression, that is, linear in
x and x2 respectively, even though the graph on x by itself is
not a straight line. In other words, Y can be considered a
linear function of the parameters (, , and ), even though it
is not a linear function of x.
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Statistical requirements to
build a calibration curve
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Statistical requirements to build a
calibration curve
1. Standard concentration (X) are known without error
2. Variance of response (Y) should be constant over the
analytical domain (homoscedasticity hypothesis); this
equivalent to say that the random errors i are
homoscedastic i.e., they all have the same variance.
3. The random errors i have expected value 0.
4. The random errors i should be independent from Y and
are uncorrelated.

These assumptions imply that least-squares estimates of
the parameters are optimal in a certain sense

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Regression can be used
for prediction
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Regression can be used for prediction
These uses of regression (calibration curve) rely
heavily on the model assumptions being satisfied.
Calibration curve is misused for these purposes
where the appropriate assumptions cannot be
verified to hold

The misuse of regression is due to the fact that it
take considerably more knowledge and experience
to critique a model than to fit a model with a
software.

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Assessing the calibration curve
the calibration curve (here a statistical
model ) should be checked for two
different things:
1. Whether the assumptions of least-
squares are fulfilled
Analysis (inspection) of residuals
2. Whether the model is valid and useful
Test of linearity
Back calculations

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Validation of the calibration curve
Homogeneity of variance
Linearity
Back calculations
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Checking model assumptions
The model assumptions are
checked by calculating the
residuals and plotting them.
The residuals are calculated as
follows :

fitted observed Y Y siduals = Re
fittted observed Y Y siduals = Re
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Inspection of residuals
The following plots can be constructed to test the
validity of the assumptions:

1. A normal probability plot of the residuals to test normality. The
points should lie along a straight line.
2. Residuals against the explanatory variables, X.
3. Residuals against the fitted values, Y .
4. Residuals against the preceding residual.

There should not be any noticeable pattern to
the data in all but the first plot
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Validation of the calibration curve
Homogeneity of variance

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Problem of the homogeneity of variance
Cochran's test
Homogeneous Non homogeneous
"cone shaped"
Calibration curve: homogeneity of variance
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Calibration curve: linearity & homogeneity of variance
Inspection of a residuals plot
If the linear model and the assumption of homoscedasticity are valid, the
residual should be normally distributed and no trends should be apparent
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Calibration curve: linearity & homogeneity of variance
Inspection of a residuals plot
The fact that the
weighted residuals show
a fan-like pattern, getting
larger as X increase
suggest
heteroscedasticity and
the use of a weighting
procedure to reduce
variance heterogeneity
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Heterogeneity of variance
Commonly observed
Y has often a constant coefficient of variation
Weighted regression
weighing factor proportional to the inverse of
variance (1/X, 1/X)
After weighing, the coefficient of correlation
(r) can be lower but accuracy and precision
of prediction are better
Calibration curve: homogeneity of variance
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Calibration curve:
homogeneity of variance
Weighing factor=1/x
2
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Inspection of the residual plot
Weighted residues
Unweighted residues
Misfit evidenced by visual inspection of residuals despite the use of
weighted regression: does the simple linear model holds???
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Calibration curve : Linearity

- Specific tests of linearity should be used

- The coefficient of correlation (r) cannot
assess linearity except for r = 1

e.g.: r = 0.999 can be associated with a calibration
curve which is not a straight line
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R
e
s
p
o
n
s
e


Y
X
Calibration curve: linearity
Concentration
Test of linearity : Coefficient of correlation
r = 0.99
does not prove linearity
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Calibration curve: linearity
Test of lack of fit
Requires replicates
Should be carried out after weighing
ANOVA
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Y
Response
Test of lack-of-fit
It is a comparison of 2 variances
Variance 1
Mean estimated from
each set of data
Variance 2
Mean estimated
from the curve
?
=
X
Concentration
The case of very precise technique
Calibration curve: linearity
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Calibration curve: linearity
If no replicate
Y = ax + b vs Y= ax + cx + b
Y
X
Y
X
Test the significance of C
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Calibration curve: linearity
If non linearity
use the 2
nd
degree polynom
reduce the domain of the calibration curve
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Calibration curve:
Weight=1/X
2
& quadratic component

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Calibration curve:
Weight=1/X
2
& quadratic
component

Linear &
Weighted residues
Linear &
Unweighted residues
Quadratic &
Weighted residues
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Coefficient of correlation
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Coefficient of correlation
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Coefficient of correlation
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Validation of the calibration curve:
Back calculations
back calculation of the concentrations of
calibration samples using the fitted curve
coefficients
The ULOQ calibrator must back-calculate to
within 15% of the nominal concentration.
At least four out of six non-zero standards
should meet the back-calculation criteria,
including the LLOQ and ULOQ standards.
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Calibration curve: Parallelism
If samples should be diluted with
blank plasma, parallelism should be
investigated with QC samples
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Freeze/thaw stability
Avoid freeze and thaw cycles
Enough aliquot samples should be
to be prepared
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Calibration curve: sensitivity
The sensitivity of an analytical method is its
ability to give response to small changes in
the absolute amount of analyte present
1
2
3
High sensitivity
Concentration (X)
added quantity
Response (Y)
measured
quantity
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Long term freezer stability
Required for some analytes and for
retrospective investigations
Re-assay QC after the study is
completed
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1
2
A1 A2 A2 A1 x x
Performance : The slope factor
Y
X
^ ^
Calibration curve: sensitivity
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Accuracy and precision
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Origin of the error :
Accuracy and precision
Systematic (not random)
bias
impossible to be corrected
accuracy
Random
can be evaluated by statistics
precision
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Good Precision
Good Accuracy
Poor Precision
Good Accuracy
Good Precision
Poor Accuracy
Poor Precision
Poor Accuracy
Gold
Standard
Silver
Standard
Off-Base
Model
Hit or
Miss Model
Bias and precision
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Accuracy

Closeness of determined value to the
true value.

The acceptance criteria is mean value
s 15% deviation from true value.

At LOQ, 20% deviation is acceptable.
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Accuracy
The accuracy is calculated using the following
equation :
Accuracy (%) = 100 x
Found value - Theoretical value
Theoretical value
The accuracy at each concentration level must
be lower than 15% except a LOQ (20%)
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Accuracy
Determination
by replicate analysis of the sample
containing known amount of analyte
5 samples for at least 3 levels
The mean value should be within 15%
of the actual value except at LOQ where
it should not deviate by more than 20%
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Precision

The closeness of replicate determinations
of a sample by an assay.

The acceptance criteria is s 15% CV.

At LOQ, 20% deviation is acceptable.

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Repeatability (r)
Agreement between successive measurements
on the same sample under the same conditions


Reproducibility (R)
The closeness of agreement between results
obtained with the same method under different
conditions
Precision
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Precision Considered at 3 Levels
Repeatability
Intermediate Precision
Reproducibility
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Repeatability
Express the precision under the same
operating conditions over a short
interval of time.
Also referred to as Intra-assay precision
(within day)
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Intermediate Precision
Express within-laboratory variations.
Between days variability
Known as part of Ruggedness in USP
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Reproducibility
Definition: Ability reproduce data
within the predefined precision
Repeatability test at two different labs
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Precision: measurement
Should be measured using a minimum
of 5 determinations per concentration
A minimum of 3 concentrations in the
range of expected concentrations
The precision at each concentration
should not exceed 15% except for the
LOQ (20%)
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Precision: measurement
for a single measurement : CV(%)
for intra-day and inter-day precision
ANOVA
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Precision: data analysis
Single level of concentration with repetition
e.g. 12, 13, 12, 14, 13, 14 g/mL
mean : 13.0 g/mL
SD: 0.8944 g/mL
CV% = SD/mean * 100 = 6.88%
CV% is also known as the relative standard deviation
or RSD
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Precision: data analysis
Several levels of concentration and several days
day 1 levels (g/mL) 0.5 5 20
Repetitions 0.4 5.2 20.5
0.5 5.1 21.0
0.4 4.9 19.8
0.6 5.2 18.8
day 2 and 3 : same protocol
ANOVA
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Precision: the statistical model
The statistical model (for each
concentration level)
Y = + day + c
: general mean
day: an effect (day, technician, or any
factor = inter )
c: error-random = intra
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ANOVA
Allows an estimation of the 2 variance terms
inter-day mean square (BMS)
intra-day mean square (WMS)
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Repeatability and reproducibility
SD for repeatability
o
r
= Var
(e)

SD for reproducibility
o
R
= o
(day) +
o
(r)

variance for reproducibility is the sum of
the variance for repeatability and the
inter-day variance
Inter-day intra-day
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Precision: ANOVA
CV intra : 5%
CV inter : 8%
CV inter > CV intra
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The limit of quantification (LOQ)
LOQ is the lowest amount of
analytes in a sample which can be
determined with defined precision
and accuracy
LOQ : 20%
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Limit of quantification (LOQ)
The lowest standard on the
calibration curve is the LOQ if:
no interference is present in the blanks
at retention time of the analyte for this
concentration
the response (analyte peak) has a
precision of 20% and accuracy 80-120%
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Estimation of chromatographic baseline noise
N
p-p
N
p
W : Peak width
1
Baseline
noise
Largest variation
of the baseline
noise
(N )
p-p
Most important
deviation (N )
p
Sample chromatogram
Blanc chromatogram
(a)
(b)
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Three analytical areas
1 2 3
Xb
not detected
Area of
detection
Area of
quantification
or CV<20%
LOD LOQ
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The recovery of an analyte in an assay is the
detector response obtained from an amount of the
analyte added to and extracted from the biological
matrix, compared to the detector response obtained
for the true concentration of the pure authentic
standard

The recovery allows to determine the percent of lost
drug during sample preparation

Minimal extraction ratio required to ensure a good
repeatability


Recovery: definition
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Absolute recovery is evaluated using low,
medium, and high QC samples and at least three
times for each level

The extraction recovery of the analyte (s) and
internal standard(s) should be higher than 70%,
precise, and reproducible.

Recovery: Determination
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Recommended to be a close
analog of the analyte of interest

Advantages and limits


Recovery: Internal standard
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Recovery
) _ tan , / _ _ _( _
) _ , / _ _ _( _
cov Re
solution dard s mL ng x of area Peak
extract plasma mL ng x of area Peak
ery =100
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Specificity / Selectivity (1)
Specificity : for an analyte
ability of the method to produce a
response for a single analyte
metabolites
enantiomers
Selectivity: for a matrix
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Specificity / Selectivity (2)
Analyses of blank samples from different subjects
(n=6)
Blanks should be tested for interference using the
proposed extraction procedure and other
chromatographic conditions
Results should be compared with those obtained
with aqueous solution of the analyte at a
concentration near the LOQ
Blank plasma and pre-dose samples should be
without interference
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Specificity / Selectivity (3)
If more than 10% of the blank
samples exhibit significant
interference, the method should be
changed to eliminate interference
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Definition
The drug must keep all its properties during the
investigations

Stability at room temperature
An experiment should cover 6 to 24h

Stability in frozen biological samples : (-20C or -80C)
Stability sample should allow assay from day 0 to
day 20

Stability during a freeze / thaw cycle
Samples should be frozen and submitted to three
freeze / thaw cycles
Aliquotage is better than repeated freeze / thaw cycles
Stability
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In life validation
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In life validation
should be generated for each run
no replicate
should be validated
back calculation
quality control (QC)
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In life validation
Validation performed in each batch
(day) of study samples to be analyzed
Validation of the routine calibration
curve
QC samples
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In life validation:
validation of the calibration curve
Prepare routine calibration in the matrix
of interest
calibration samples, n>6 including blank
Validation of the routine calibration curve
QC samples
3 concentration levels
3 QC per level
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In life validation: calibration curve
separately prepared QC samples should
be analyzed with test samples
QC in duplicate at 3 different
concentrations (one <=3X LOQ, one in
midrange and one close to the high end
of range) should be incorporated in each
run
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In life validation: calibration curve
Decision rule
at least 4 of 6 QC should be within 20%
of their respective nominal value
2 out of 6 QC may be outside the 20%
of their respective nominal value but not
at the same level
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Y
X
Significant : Origin ?
NS : Keep the intercept
as an empirical
parameter
Intercept : Test hypothesis that the line goes
through the origin
Calibration curve
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In life validation:
Robustness/Stability assay of a drug
1.00
0.80
1.20
1.40
1.60
1.80
0.60
0 4 8 12 16 20
Time (days)
+ 2 SD
- 2 SD
Mean
Calculated concentration
(mg/ml)
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In life validation: the QC
to evaluate accuracy
to evaluate precision
to confirm LOQ
to evaluate robustness of the method
to confirm sample stability
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References
See Guidance for Industry (main guidances in the world)
Bioanalytical Method Validation
FDA May 2001: Bioanalytical Method Validation
ICH 1995
EMEA: no specific document
Published Workshop Reports
Shah, V.P. et al, Pharmaceutical Research: 1992; 9:588-592
Shah, V.P. et al, Pharmaceutical Research: 2000; 17: 1551-
1557

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To see this guidance
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To see this guidance