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Assay Validation
Timothy L. Schofield
a
a
Merck Research Laboratories, West Point, Pennsylvania, U.S.A.
Online Publication Date: 23 April 2003
To cite this Section Schofield, Timothy L.(2003)'Assay Validation',Encyclopedia of Biopharmaceutical Statistics,1:1,63 71
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Assay Validation
Timothy L. Schofield
Merck Research Laboratories, West Point, Pennsylvania, U.S.A.
INTRODUCTION
Upon completion of the development of an assay, and
prior to implementation, a well-conceived assay valida-
tion is carried out to demonstrate that the procedure is fit
for use. Regulatory requirements specify the assay
characteristics that are subject to validation and suggest
experimental strategies to study these properties. These
requirements, when united with sound statistical experi-
mental design, furnish an effective and efficient valida-
tion plan. That plan should also address the form of
statistical processing of the experimental results and
thereby ensure reliable and meaningful measures of the
properties of the method.
This entry will introduce the validation parameters
used to describe the characteristics of an analytical
method. Performance metrics associated with those
parameters will be defined, with illustrations of their
utility in application. Realistic and relevant acceptance
criteria are proposed, as a basis for evaluating a pro-
cedure. An example of a validation protocol will il-
lustrate the design of a comprehensive exploration, in
conjunction with the statistical methods used to extract
the relevant information.
TERMINOLOGY
Most of the terminology related to assay validation can
be found in the entry on Assay Development. Some terms
that are unique to this topic are relative standard devia-
tion, spike recovery, and dilution effect.
The relative standard deviation (RSD) is a measure of
the proportional variability of an assay and is usually
expressed as a percentage of the measured value:
RSD 100
^ s assay
^x
%
Note that the RSD is the same as the coefficient of
variation in statistics. Other conventions for calculating
the RSD, which are appropriate for measurements that are
lognormally distributed, utilize s
log
x^
, the log standard
deviation or the fold variability:
RSD 100^ s
log^x
% or RSD 100FV 1%
100e
^ slog^x
1%
Note that the log standard deviation provides an
approximation to the RSD; it should be restricted to
levels below 20% RSD. An important feature of the RSD
is that it is calculated from the predicted measurements
for a sample, such as concentrations, and not from an
intermediate, such as the instrument readout. These are
the same only in the case of linear calibration.
Spike recovery is the process of measuring an analyte
that has been distributed as a known amount into the
sample matrix, with results usually reported as percent
recovery or percent bias. Dilution effect is a measure
of the proportional bias measured across a dilution series
of a validation sample; it is usually reported as percent
bias per dilution increment (usually percent bias per
twofold dilution).
REGULATORY REQUIREMENTS
The U.S. Pharmacopeia specifies that the Validation of
an analytical method is the process by which it is
established, in laboratory studies, that the performance
characteristics of the method meet the requirements for
the intended analytical applications.
[1]
Key to this
definition is the treatment of a validation study as an
experiment, with the goal of that experiment being to
demonstrate that the assay is fit for use. As an experiment,
a validation study should be suitably designed to meet its
goal, while the acceptance criteria should yield oppor-
tunities to judge the assay as unsuitable as well as suit-
able, possibly requiring further development. This does
not suggest that it is the role of the validation study to
optimize the assay; this should be completed during the
assay development. In fact, the properties of the assay are
probably well understood by the time the validation
experiment is undertaken. The validation experiment
should serve as a snapshot of the long-term routine
performance of the assay, thereby documenting its
characteristics so that we can make reliable decisions
during drug development and control.
The International Conference on Harmonization (ICH)
has devoted two topics of its guidelines to analytical
method validation. The first topic, entitled Guideline on
Validation of Analytical Procedures: Definitions and
Terminology,
[2]
offers a list of validation parameters
that should be considered when implementing a validation
experiment and guidance toward the selection of those
Encyclopedia of Biopharmaceutical Statistics 63
DOI: 10.1081/E-EBS 120007388
Copyright D 2003 by Marcel Dekker, Inc. All rights reserved.
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parameters. The second topic, entitled Validation of
Analytical Procedures: Methodology,
[3]
provides dir-
ection on the design and analysis of results from a
validation experiment.
ASSAY VALIDATION PARAMETERS
AND VALIDATION DESIGN
It is convenient for the purposes of planning a validation
experiment to dichotomize assay validation parameters
into two categories: parameters related to the accuracy of
the analytical method and parameters related to variabil-
ity. In this way, the analyst will choose the test sample set
that will be carried forward into the validation, in accord
with accuracy parameters, and devise a replication plan
for the validation to satisfy the estimation of the
variability parameters.
The validation parameters related to the accuracy of an
analytical method are accuracy, linearity, and specificity.
The accuracy of an analytical method expresses the
closeness of agreement between the value which is
accepted either as a conventional value or an accepted
reference value, and the value found. Accuracy is usu-
ally established by spiking known quantities of analyte
(see the entry Assay Development for definitions of terms)
into the sample matrix and demonstrating that these can
be completely recovered. Conventional practice is to
spike using five levels of the analyte: 50%, 75%, 100%,
125%, and 150% of the declared content of analyte in the
drug (note that this is more stringent than the ICH, which
requires a minimum of nine determinations over a
minimum of three concentration levels). The experi-
menter is not restricted to these levels but should plan
to spike through a region that embraces the range of
expected measurements from samples that will be tested
during development and manufacture. Accuracy can also
be determined relative to a referee method; in the case
of complex mixtures, such as combination vaccines,
accuracy can be judged relative to a monovalent control.
The linearity of an analytical procedure is its ability
(within a given range) to obtain test results that are
directly proportional to the concentration (amount) of
analyte in the sample. This parameter is often confused
with the graphical linearity of the standard curve; in
fact, the guidelines encourage the experimenter to eval-
uate linearity by visual inspection of a plot of signals
as a function of concentration or content. The prin-
ciple of analytical linearity, however, should not be
confused with an abstract property, such as the
straightness of the standard curve, but rather should
be based upon the attribute that graded concentrations
of the analyte yield measurements that are in proper
proportion. Thus for example, if a sample is tested in
twofold dilution, the measured concentrations in those
samples should yield a twofold series. In this way, linearity
can be established from the spiking experiment outlined
earlier, while a dilution series is usually employed when
the assessment of linearity cannot be achieved through
spiking (i.e., when the purified analyte does not exist, such
as in vaccines).
Specificity is the ability to assess unequivocally the
analyte in the presence of components which may be
present. Thus specificity speaks to the variety of sample
matrices containing the analyte, including process inter-
mediates and stability samples. The ICH guidelines
provide a comprehensive description of the means to es-
tablish specificity in identity tests and in analytical
methods for impurities and content. When the samples
are available, specificity can be established through
testing of the analyte-free matrix.
The assay validation parameters related to variability
are precision, repeatability, ruggedness, limit of detection,
and limit of quantitation. The precision of an assay
expresses the closeness of agreement (degree of scatter)
between a series of measurements obtained from multiple
sampling of the same homogeneous sample under the
prescribed conditions. Precision is frequently called in-
terrun variability, where a run of an assay represents the
independent preparation of assay reagents, tests samples,
and a standard curve. The repeatability of an assay
expresses the precision under the same operating con-
ditions over a short interval of time, and is frequently
called intrarun variability. Repeatability and precision
can be studied simultaneously, using the levels of a
sample required to establish accuracy and linearity, in a
consolidated validation study design such as that de-
picted in Table 1. Here precision is associated with the
multiple experimental runs, while repeatability is asso-
ciated with the run-by-level interaction. In many cases,
the experimenter may wish to test true within-run
replicates rather than employ this subtle statistical artifact.
The runs in this design can be strategically allocated
to ruggedness parameters, such as laboratories, opera-
tors, and reagent lots, using a combination of nested and
factorial experimental design strategies. Thus operators
Table 1 Strategic validation design employing five levels of
the analyte tested in k independent runs of the assay
Level
Run 1 2 3 4 5
1 y
11
y
12
y
13
y
14
y
15
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
n y
n1
y
n2
y
n3
y
n4
y
n5
64 Assay Validation
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might be nested within laboratory, while reagent lot can
be crossed with operator.
The robustness of an analytical method is a measure
of its capacity to remain unaffected by small, but
deliberate, variations in method parameters, and might
be more suitably established during the development
of the assay (see the entry on Assay Development).
Note that while ruggedness parameters represent uncon-
trollable factors affecting the analytical method, robust-
ness parameters can be varied and should be controlled
when it has been observed that they have an effect on
assay measurement.
The limit of detection (LOD) and limit of quantitation
(LOQ) of an assay can be obtained from replication of the
standard curve during the implementation of the con-
solidated validation experiment. The LOD of an analyt-
ical procedure is the lowest amount of analyte in a
sample that can be detected but not necessarily quanti-
tated as an exact value, while the LOQ is the lowest
amount of analyte in a sample that can be quantitatively
determined with suitable precision and accuracy. The
limit of quantitation need not be restricted to a lower
bound on the assay, but might be extended to an upper
bound of a standard curve, where the fit becomes flat (for
example, when using a four-parameter logistic regression
equation to fit the standard curve).
Finally, the composite of the ranges identified with
acceptable accuracy and precision is called the range of
the assay.
CHOICE OF VALIDATION PARAMETERS
The choice of parameters that will be explored during an
assay validation is determined by the intended use as well
as by the practical nature of the analytical method. For
example, a biochemical assay using a standard curve to
establish drug content might require the exploration of all
of these parameters if the assay is to be used to determine
low as well as high levels of the analyte (such as an assay
used to determine drug level in clinical samples or an
assay that will be used to measure the content of an
unstable analyte). If the assay is used, however, to de-
termine content in a stable preparation, the LOD and LOQ
need not be established. On the other hand, an assay for an
impurity requires adequate sensitivity to detect and/or
quantify the analyte; thus the LOD and LOQ become the
primary focus of the validation of an impurity assay.
Many of these assay validation parameters have lim-
ited meaning in the context of biological assay and
various other potency assays. There is no means to
explore the accuracy and linearity of assays in animals or
tissue culture, where the scale of the assay is defined by
the assay; thus validation experiments for this sort of
analytical method are usually restricted to a study of the
specificity and ruggedness of the procedure. As discussed
previously, the accuracy of a potency assay may be
limited to establishing linearity with dilution when the
purified analyte is unavailable to conduct a true spike-
recovery experiment.
VALIDATION METRICS AND
ACCEPTANCE CRITERIA
As previously defined, the goal of the validation ex-
periment is to establish that the analytical method is fit for
use. Thus for an impurity assay, for example, the goal of
the validation experiment should be to show that the
sensitivity of the analytical procedure (as measured by the
LOD for a limit assay and by the LOQ for a quantitative
assay) is adequate to detect a meaningful level of the im-
purity. Assays for content and potency are usually used to
establish that a drug conforms to specifications that have
been determined either through clinical trials with the
drug or from process/product performance. It is important
to point out that the process capability limits should
include the manufacturing distribution of the product
characteristic under study, the change in that character-
istic because of instability under recommended storage
conditions, as well as the effects on the measurement of
the characteristic because of the performance attributes of
the assay. In the end, the analytical method must be cap-
able of reliably discriminating satisfactory from unsat-
isfactory product against these limits. The portion of the
process range that is a result of measurement, including
measurement bias as well as measurement variability,
serves as the foundation for setting acceptance criteria for
assay validation parameters.
Validation parameters related to accuracy are rated
on the basis of recovery or bias (bias = 100 recovery,
usually expressed as a percentage), while validation para-
meters related to random variability are appraised on
the basis of variability (usually expressed as % RSD).
These can be combined with the process variability to
establish the process capability of the measured
characteristic:
[4]
CP
Specification range
6 Product variation
Specification range
6

s
2
product
s
2
assay
Bias
2
_
Process capability, in turn, is related to the percentage of
measurements that are likely to fall outside of the spe-
cifications. Thus acceptance criteria on the amount of
bias and analytical variability can be established based
Assay Validation 65
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upon the knowledge of the product variability and the
desired process capability.
When the limits have not been established for a
particular product characteristic, such as during the early
development of a drug or biologic, acceptance criteria for
an assay attribute might be specified on the basis of a
typical expectation for the analytical method as well as on
the nature of the measurement in the particular sample.
Thus for example, high-performance liquid chromato-
graphy for the active compound in the final formulation of
a drug might be typically capable of yielding measure-
ments equal to or less than 10% RSD, while an immu-
noassay used to measure antigen content in a vaccine
might only be capable of achieving up to 20% RSD.
Measurement of a residual or an impurity by either me-
thod, on the other hand, may only achieve up to 50%
RSD, owing to the variable nature of measurement at low
analyte concentrations.
VALIDATION DATA ANALYSIS
Analysis of Parameters Related to Accuracy
Analytical accuracy and linearity can be parameterized as
a simple linear model, assuming either absolute or relative
bias. In one model, if we let m represent the known analyte
content and x is the measured amount, then the accuracy
can be expressed as x=m at a single concentration, or as
x=a+bm, where a=0 and b=1, across a series of
concentrations. This linear equation can be rewritten as
x m a b 1m
In this parameterization, a is related to the accuracy of the
analytical method and represents the constant bias in an
assay measurement (usually reported in the units of
measurement of the assay), while (b1) is related to the
linearity of the analytical method and represents the
proportional bias in an assay measurement (usually
reported in the units of measurement in the assay per
unit increase in that measurement, e.g., 0.02 mg per
microgram increase in content). Data from a spiking
experiment can be utilized to estimate a and b1, and
these (with statistical confidence limits if the validation
experiment has been designed to establish statistical con-
formance) can be compared with the acceptance cri-
teria established for these parameters.
To illustrate, consider the example in Table 2. A vali-
dation study has been performed in which samples have
been prepared with five levels of an analyte ([C] in mg),
and their content is determined in six runs of an assay.
These data are depicted graphically, along with the results
from their analysis, in Fig. 1. A linear fit to the data yields
the following estimates of intercept (note that the data
have been centered in order to obtain a centered
intercept) and slope:
^a
0
0:04 0:08; 0:01;
^
b 0:81 0:70; 0:85
The slope can be utilized to estimate the bias per unit
increase in drug concentration:
^
b 1 0:19 0:30; 0:15
This proportional bias is significant, owing to the fact that
the confidence interval excludes 0. It is more desirable,
however, to react to the magnitude of the bias and to
judge that there is an unacceptable nonlinearity when the
bias is in excess of a prespecified acceptance limit. Thus
there is a 0.2-mg decrease per unit increase in concen-
tration (as much as 0.3 mg per unit decrease in
concentration based on the confidence interval). If the
range in the concentration of the drug is typically 46 mg
(i.e., a 2-mg range), this would predict that the bias as a
result of nonlinearity is 0.4 mg (0.6 mg in the
confidence interval). This can be judged to be of con-
sequence or not in testing product; for example, if the
specification limits on the analyte are 51 mg/dose,
much of this range will be consumed by the proportional
Table 2 Example of results from a validation experiment
in which five levels of an analyte were tested in six runs of
the assay
[C] 1 2 3 4 5 6 Avg.
3 3.3 3.4 3.2 3.1 3.4 3.3 3.3
4 4.2 4.2 4.2 4.4 4.0 4.3 4.2
5 5.0 4.9 4.9 5.0 4.8 4.9 4.9
6 5.8 5.8 5.6 5.9 5.9 5.7 5.8
7 6.4 6.7 6.7 6.8 6.6 6.4 6.6
Fig. 1 Validation results from an assay demonstrating
proportional bias, with low-concentration samples yielding
high measurements and high-concentration samples yielding
low measurements.
66 Assay Validation
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bias, potentially resulting in an undesirable failure rate in
the measurement of that analyte.
When the purified analyte is unavailable for spiking,
and the levels of the analyte in the validation have been
attained through dilution, then the data generated from
that series can be alternatively evaluated using the model
x = am
b
(note that m can represent either the expected
concentration upon dilution or the actual dilution). Taking
the logs, the data from the dilution series is used to fit the
linear model,
logx loga b logm
The intercept of this model has no practical meaning,
while the proportional bias (sometimes called dilution
effect) can be estimated as 2
b 1
1 if m is in units of
concentration and as 2
b + 1
1 if m is in units of dilution
(note that the units of dilution effect are percent bias
per dilution increment; the dilution increment used here
is twofold).
To illustrate, consider the example in Table 3. A
dilution series of a sample is performed in each of the five
runs of an assay. These data are depicted graphically,
along with the results from their analysis, in Fig. 2. A
linear fit of log-titer to log-dilution yields an estimated
slope equal to
^
b 1:01 1:04; 0:98. The corres-
ponding dilution effect is equal to 2
1.01 + 1
1= 0.007
(i.e., a 0.7% decrease per twofold dilution).
The dilution effect can also be calculated as a function
of the estimated slopes for the standard (slope as a
function of dilution rather than concentration) and the
test sample:
Dilution effect 2
1
^
b
T
=
^
b
S
with the corresponding standard error a function of the
standard error of a ratio:
SE
^
b
T
=
^
b
S

1
^
b
2
S
VAR
^
b
T

^
b
T
=
^
b
S

2
VAR
^
b
S

_ _

This has the advantage over the earlier calculation of

being a more reliable estimate of the underlying vari-
ability in the measurements, coming from the con-
tribution to the estimate of variability from the data for
the standard.
A special case of accuracy comes from a comparison
of an experimental analytical method with a validated
referee method. Paired measurements in the two
assays can be made on a panel of samples, and these
can be utilized to establish the linearity and the
accuracy of the experimental method relative to the
referee method. The paired measurements are a sample
from a multivariate population, and the linearity of
the experimental method to the referee method can
be estimated using principal component analysis.
[5]
A
concordance slope can be estimated from the elements
of the first characteristic vector of the sample covariance
matrix (a
21
, a
11
):
^
b
C

a
21
a
11
The standard error of the concordance slope is obtained
from these along with the other elements of the
characteristic value evaluation (a
i1
is the ith element of
the first characteristic vector and l
i
is the corresponding
characteristic roots):
SE
^
b
C

Va
21

a
2
21

Va
11

a
2
11
2
cova
21
; a
11

a
21
a
11
_ _
^
b
2
C

where
Va
i1

l
1
l
2
n l
2
l
1

2
_ _
a
2
i2
and
cova
21
; a
11

l
1
l
2
n l
2
l
1

2
_ _
a
12
a
22
Table 3 Example of results from a validation experiment in
which five twofold dilutions of an analyte were tested in five
runs of the assay
Dilution 1 2 3 4 5 Titer
1 75 90 93 79 72 81
2 43 45 46 35 40 83
4 20 23 22 18 21 83
8 11 10 12 10 10 85
16 4 5 6 4 5 78
Fig. 2 Results from a validation series demonstrating satisfact-
ory linearity, i.e., approximately a twofold decrease in response
with twofold dilution.
Assay Validation 67
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The concordance slope can be used, along with the
standard error of that estimate, to measure the discordance
of the experimental analytical method relative to the
referee method and its corresponding confidence
interval:
Discordance 2
b
C
1
This is similar to the dilution effect discussed earlier
and represents the percentage difference in reported
potency per twofold increase in result as measured in
the referee method.
When the two assays are scaled alike, the accuracy
of experimental method relative to the referee me-
thod can be assessed through a simple paired variate
analysis. When the percent difference is expected to be
constant across the two methods, the data are analyzed
on a log scale. An estimate of the simple paired dif-
ference in the logs can be used, in conjunction with the
standard error of the paired difference, to estimate the
percentage difference in measurements obtained from
the experimental procedure relative to the referee
method:
[6]

d t
n1
s
d
=

n
p
Percent difference 100e

d
1%
Note that as usual, the significance of the percentage
difference should be judged primarily on the basis of
practical considerations rather than statistical significance.
The specificity of an analytical procedure can be
assessed through an evaluation of the results obtained
from a placebo sample (i.e., a sample containing all
constituents except the analyte of interest) relative to
background. A statistical evaluation might be composed
of either a comparison of instrument measurements
obtained for this placebo, with background measure-
ments (i.e., through background-corrected placebo
measurements), or from measurements made on the
placebo alone. In either case, an increase over the
detection level of the assay would indicate that some
constituent of the sample, in addition to analyte, is con-
tributing to the measurement in the sample. This should
be assessed by noting that the upper bound on a con-
fidence interval falls below some prespecified level in
the assay.
Analysis of Parameters Related to Variability
Estimates of repeatability (intrarun, or within-run, vari-
ability) and precision (interrun, or between-run, variabil-
ity) can be established by performing a variance com-
ponent analysis on the results obtained from the data
generated to assess accuracy and linearity.
[7]
Other
sources of variability (e.g., laboratories, operators, and
reagent lots) can be incorporated into the pattern of runs
performed during the validation to estimate the effects of
these sources of variability. The expected mean squares
are shown in Table 4. From this, ^ s
2
withinrun
mean square
error (MSE) and ^ s
2
betweenrun
mean square for runs
(MSR)MSE=5 (note that the MSE is a composite of
pure error and the interaction of run and level; pure error
could be separated from the interaction by performing
replicates at each level; however, the within-run variabil-
ity will still be reported as the composite of pure error
and the level-by-run interaction). The total variability
associated with testing a sample in the assay can be
calculated from these variance component estimates as:
^ s
2
Total

^ s
2
B
r

^ s
2
W
nr
Note that this is the variance of y, where r represents the
number of independent runs performed on the sample
and n represents the number of replicates within a run.
A confidence interval can be established on the total
variability as follows:
[8]
^ s
2
Total

^ s
2
B
r

^ s
2
W
nr

1
Jr
_ _
MSR
n J
Jnr
_ _
MSE
c
1
MSR c
2
MSE

q
c
q
S
q
where J represents the number of replicates of a sample
tested in each run of the assay validation experiment (here
J=5 for the number of levels of the sample). The 1 2a
confidence interval on ^ s
2
Total
becomes:
^ s
2
Total

q
G
2
q
c
2
q
S
4
q

; ^ s
2
Total

q
H
2
q
c
2
q
S
4
q

_
_
_
_
Table 4 Analysis of variance table showing expected mean
squares as functions of intrarun and interrun components
of variability
Effect df MS
Estimated
mean square F
Level 4 Mean square
for levels
(MSL)
^ s
2
W
QL
Run 5 MSR ^ s
2
W
5^ s
2
B
MSR/MSE
Error 20 MSE ^ s
2
W
68 Assay Validation
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where
G
q
1
1
F
a;df
q
;1
; H
q

1
F
1a;df
q
;1
1;
F
a;df
q
;1
; F
1a;df
q
;1
represent the percentiles of the F-distribution.
The LOD and the LOQ are calculated using the
standard deviation of the responses (^ s) and the slope
of the calibration curve (
^
b) as LOD 3:3^ s=
^
b (note,
the 3.3 is derived from the 95th percentile of the stand-
ard normal distribution, 1.64; here, 2 1.64 = 3.3,
thereby limiting both the false-positive and false-
negative error rates to be equal to or less than 5%) and
LOQ 10^ s=
^
b (note, the 10 corresponds to the restriction
of no more than 10% RSD in the measurement variability;
RSD=100 ^ s=x < 0:10 gives x; thus if the restriction
was changed to not more than 20% RSD, then the
LOQ could be calculated as LOQ 5^ s=
^
b).
These estimates do not account for the calibration
curve, and they can be improved upon by acknowledging
the variability in prediction from the curve.
[9]
For a linear
fit, the LOD is depicted graphically in Fig. 3. The LOQ
can be calculated as prescribed in the ICH guideline,
solving for x
Q
in the following equation:
LOQ 10 ^ s

1
1
n

x
Q
x
2
SXX
_
_
_
_
_
_
The limit of quantitation is depicted graphically in Fig. 4.
The LOQ is more complicated to calculate from a
nonlinear calibration function, such as the four-parameter
logistic regression equation, and involves the first-or-
der Taylor series expansion of the solution equation for
x
Q
[10]
(see the entry on Assay Development). The calcu-
lation of the LOQ for this equation is illustrated in Fig. 5.
FOLLOW-UP
The information from the analytical method validation
can be utilized to establish a testing format for the
samples in the assay. In particular, variance component
estimates of the within-run and between-run variabilities
Fig. 3 Determination of the LOD using the linear fit of the
standard curve plus restrictions on the proportions of false
positives and false negatives.
Fig. 4 Determination of the LOQ using the linear fit of the
standard curve and a restriction on the variability (% RSD) in
the assay.
Fig. 5 Quantifiable range determined as limits within which
assay variability (% RSD) is less than or equal to 20%.
Table 5 Predicted variability (% RSD) for a given number of
runs (r) and a given number of replicates within each run (n)
r/n 1 2 3 1111
1 6% 5% 5% 5.0%
2 4% 4% 4% 3.5%
3 3% 3% 3% 2.9%
Assay Validation 69
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can be used to predict the total variability associated
with a variety of testing formats. For example, consider
the case where component estimates have been deter-
mined to be equal to ^ s
B
0:05 (5%) and ^ s
W
0:03
(3%). Letting r represent the number of independent runs
that will be performed on a sample, and n be the number
of replicates within a run, the variabilities are listed in
Table 5. Suppose that, because of the process capability,
the analytical variability must be restricted to RSD3%.
As can be observed from the table, this cannot be ac-
complished using a single run of the assay; it must
therefore be achieved through a combination of multiple
runs with multiple replicates within each run.
ANALYTICAL METHOD QUALITY CONTROL
Assay validation does not end with the formal validation
experiment but is continuously assessed through assay
quality control. Control measures of variability and bias
are utilized to bridge to the assay validation and to
warrant the continued reliability of the procedure.
Extra variability in replicates can be detected utilizing
standard statistical process control metrics;
[4]
detection
and prespecified action upon detection can help maintain
the variability characteristics of an assay. For example,
when the measurement of an analyte is obtained from
multiple runs of an assay, those results can be examined
by noting the range (either log(max) log(min) or
max min, depending upon whether the measure-
ments are scaled proportionally or absolutely). A bound
on the range can be derived:
Range ^ s
dimension
d
2
kd
3

where ^ s
dimension
is the variability in the dimension of rep-
lication (here, run to run), d
2
and d
3
are the factors
used to scale the standard deviation to a range, and k is
a factor representing the desired degree of control (e.g.,
k = 2 represents approximately 95% confidence). Hav-
ing detected extra variability, additional measurements
can be obtained, and a result for the sample can be
determined from the remainder of the measurements
made after the maximum and minimum results have been
eliminated from the calculation. When extra variabil-
ity has been identified in instrument replicates of a
standard concentration or a dilution of a test sample, that
concentration of the standard or dilution of the test sample
might be eliminated from subsequent calculations.
Measurements on control sample(s) help identify shifts
in scale and therefore bias in a run of an assay. In-
formation in multiple control samples can typically be
reduced to one or two meaningful scores, such as an
average and/or a slope. These can help isolate problems
and provide information regarding the cause of a
deviation in a run of an assay. Suppose, for example,
that three controls of various levels are used to monitor
shifts in sensitivity in the assay. The average can be used
to detect absolute shifts, because of some underlying
source of constant variability, such as inappropriate
preparation of standards or the introduction of significant
background. The slope can be used to detect proportional
shifts as a result of some underlying source of pro-
portional variability.
Suitability criteria on the performance of column
peaks can detect degeneration of a column, while cha-
racteristics of the standard curve, such as the slope or
EC
50
(EC
50
corresponds to C in a four-parameter logistic
regression) help predict an assay with atypical or
substandard performance.
Care must be taken, however, to avoid either mean-
ingless or redundant controls. The increased false-positive
rate inherent in the multiplicity of a control strategy must
be considered, and a carefully contrived quality control
scheme should acknowledge this.
CONCLUSION
A carefully conceived assay validation should follow
assay development, and precede routine implementation
of the method for product characterization. The purpose
of the validation is to demonstrate that the assay is
fundamentally reliable, and possesses operating charac-
teristics that make it suitable for use. The statistical
estimates collected during the validation can also be used
to design a test plan, that provides the maximum relia-
bility for the minimum effort in the laboratory. Most
importantly, validation of the assay does not end with the
documentation of the validation experiment, but should
continue with assay quality control. The proper choice of
control samples and control parameters helps assure the
continued validity of the procedure during routine use in
the laboratory.
REFERENCES
1. USP XXII. General Chapter 1225, Validation of Compen-
dial Methods; 19821984.
2. Federal Register. International Conference on Harmoniza-
tion; Guideline on Validation of Analytical Procedures:
Definitions and Terminology; March 1, 1995; 11259
11262.
70 Assay Validation
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3. International Conference on Harmonization; Guideline on
Validation of Analytical Procedures: Methodology;
November 6, 1996.
4. Montgomery, D. Introduction to Statistical Quality Con-
trol, 2nd Ed.; Wiley: New York, 1991; pp. 203206, 208
210.
5. Morrison, D. Multivariate Statistical Methods; McGraw-
Hill: New York, 1967; 221258.
6. Snedecor, G.; Cochran, W. Statistical Methods, 6th Ed.;
Iowa State Univ. Press: Ames, IA, 1967; 91100.
7. Winer, B. Statistical Principles in Experimental Design,
2nd Ed.; McGraw-Hill: New York, 1971; 244251.
8. Burdick, R.; Graybill, F. Confidence Intervals on Variance
Components; Marcel Dekker: New York, 1992; 2839.
9. Oppenheimer, L.; Capizzi, T.; Weppelman, R.; Mehta, H.
Determining the lowest limit of reliable assay measure-
ment. Anal. Chem. 1983, 55, 638643.
10. Morgan, B. Analysis of Quantal Response Data; Chapman
& Hall: New York, 1992; 370, 371.
11. Massart, D.L.; Dijkstra, A.; Kaufman, L. Evaluation and
Optimization of Laboratory Methods and Analytical
Procedures; Elsevier: New York, 1978.
12. Currie, L.A. Limits for qualitative detection and quanti-
tative determination: Application to radiochemistry. Anal.
Chem. 1968, 40, 586593.
13. Cardone, M.J. Detection and determination of error in
analytical methodology. Part I. The method verification
program. J. Assoc. Off. Anal. Chem. 1983, 66, 1257
1281.
14. Cardone, M.J. Detection and determination of error in
analytical methodology. Part II. Correction for cor-
rigible systematic error in the course of real sample
analysis. J. Assoc. Off. Anal. Chem. 1983, 66, 1283
1294.
15. Miller, J.C.; Miller, J.N. Statistics for Analytical Chem-
istry, 2nd Ed.; Wiley: New York, 1988.
16. Caulcutt, R.; Boddy, R. Statistics for Analytical Chemists;
Chapman & Hall: New York, 1983.
Assay Validation 71
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