Report

A Practical Guide to
Analytical Method Validation
Doing a thorough
method validation
can be tedious, but the
consequences of not
doing it right are
wasted time, money,
and resources

T he ability to provide timely, accu-

rate, and reliable data is central
to the role of analytical chemists
and is especially true in the discovery, de-
velopment, and manufacture of pharma-
ceuticals. Analytical data are used to
screen potential drug candidates, aid in
the development of drug syntheses, sup-
port formulation studies, monitor the sta-
bility of bulk pharmaceuticals and formu-
lated products and test final products for
release The quality of analytical data is a
key factor in the of a drug devel-
opment program The of method
development and validation has a direct
impact on the quality of these data
Although a thorough validation cannot
rule out all potential problems, the pro-
cess of method development and valida-
tion should address the most common
ones. Examples of typical problems that
can be minimized or avoided are synthe-
sis impurities that coelute with the analyte
peak in an HPLC assay; a particular type
J. Mark Green
DuPont Merck Pharmaceutical Co.
of column that no longer produces the sep-
aration needed because the supplier of the
column has changed the manufacturing
process; an assay method that is trans-
ferred to a second laboratory where they
are unable to achieve the same detection
limit; and a quality assurance audit of a val-
idation report that finds no documenta-
tion on how the method was performed
during the validation.
Problems increase as additional people,
laboratories, and equipment are used to
perform the method. When the method is
used in the developer's laboratory, a
small adjustment can usually be made to
make the method work, but the flexibility
to change it is lost once the method is
transferred to other laboratories or used
for official product testing. This is espe-
cially true in the pharmaceutical indus-
try, where methods are submitted to regu-
latory agencies and changes may require
formal approval before they can be imple-
mented for official testing. The best way
to minimize method problems is to per-
form adequate validation experiments dur-
ing development.
What is method validation?
Method validation is the process of proving
that an analytical method is acceptable for
its intended purpose. For pharmaceutical
methods, guidelines from the United
States Pharmacopeia (USP) (1), Interna-
tional Conference on Harmonisation (ICH)
(2), and the Food and Drug Administration
(FDA) (3, 4) provide a framework for per-
forming such validations. In general, meth-
ods for regulatory submission must include
studies on specificity, linearity, accuracy,

0003-2700/96/0368-305A/$12.00/0 Analytical Chemistry News & Features, May 1, 1996 305 A

© 1996 American Chemical Society
 Report
precision, range, detection limit, quantita-
tion limit, and robustness.
Although there is general agreement
about what type of studies should be done,
there is great diversity in how they are per-
formed (5). The literature contains diverse
approaches to performing validations (as in
References 6-10). This Report presents an
approach to performing validation studies
that encompasses much of the current lit-
erature and provides practical guidance.
This approach should be viewed with the
understanding that validation requirements
are continually changing and vary widely
depending on the type of drug being tested
the stage of drug development and the
regulatory group that will review the drug
aDDlication For our purposes we will dis-
cuss validation studies as they apply to
chromatncrraphic methridQ althmitrh the
same Drincioles aDDly to other analytical
techninues
In the early stages of drug develop-
ment, it is usually not necessary to per-
form all of the various validation studies.
Many researchers focus on specificity, lin-
earity, accuracy, and precision studies
for drugs in the preclinical through Phase
II (preliminary efficacy) stages. The re-
maining studies are performed when the
drug reaches the Phase III (efficacy) stage
of development and has a higher proba-
bility of becoming a marketed product.
The process of validating a method
cannot be separated from the actual devel-
opment of the method conditions, be-
cause the developer will not know whether
the method conditions are acceptable un-
til validation studies are performed. The
development and validation of a new ana-
lytical method may therefore be an itera-
tive process. Results of validation studies
may indicate that a change in the proce-
dure is necessary, which may then re-
quire revalidation. During each validation
study key method parameters are deter-
mined and then used for all subsequent
validation steps To minimize repeti-
tious studies and ensure that the valida-
tion data are generated under conditions
ertuivalent to t h e final p r o c e d u r e WP
recommend the following sequence of
chirlipc
Establish minimum criteria
The first step in the method development
and validation cycle should be to set
minimum requirements, which are essen-
tially acceptance specifications for the
method. A complete list of criteria should
be agreed on by the developer and the
end users before the method is developed
so that expectations are clear.
For example, is it critical that method
precision (RSD) be < 2%? Doee she meehod
need to be accurate to within 2% of the
target concentration? Is it acceptable to
have only one supplier of the HPLC col-
umn used in the analysis? During the
actual studies and in thefinalvalidation re-
port, these criteria will allow clear judg-
ment about the acceptability of the analyti-
cal method.
Examples of minimum criteria are pro-
vided throughout this article that indicate
practical ways to evaluate the acceptabil-
ity of data from each validation study. The
statistics generated for making compari-
The development
and validation of
a new analytical
method may be an
iterative process.
sons are similar to what analysts will gen-
erate later in the routine use of the method
and therefore can serve as a tool for eval-
uating later questionable data. More rigor-
ous statistical evaluation techniques are
available and should be used in some in-
stances, but these may not allow as direct
a comparison for method troubleshoot-
ing during routine use.
Demonstrate specificity
For chromatographic methods, develop-
ing a separation involves demonstrating
specificity, which is the ability of the
method to accurately measure the analyte
response in the presence of all potential
sample components. The response of the
analyte in test mixtures containing the ana-
lyte and all potential sample components
(placebo formulation, synthesis intermedi-
ates, excipients, degradation products,
process impurities, etc.) is compared with
the response of a solution containing
only the analyte. Other potential sample
components are generated by exposing
the analyte to stress conditions sufficient
to degrade it to 80-90% purity. For bulk
pharmaceuticals, stress conditions such as
heat (50 *C), light (600 FC)) acid (0.1 N
HC1), base (0.1 N NaOH)) and oxidant
(3% H202) are typical. For formulated
products, heat, light, and humidity (85%)
are often used.
The resulting mixtures are then ana-
lyzed, and the analyte peak is evaluated for
peak purity and resolution from the near-
est eluting peak. If an alternate chromato-
graphic column is to be allowed in the fi-
nal method procedure, it should be identi-
fied during these studies. Once acceptable
resolution is obtained for the analyte and po-
tential sample components, the chromato-
graphic parameters, such as column type,
mobile-phase composition, flow rate, and
detection mode are considered set.
An example of specificity criteria for an
assay method is that the analyte peak will
have baseline chromatographic resolution
of at least 1.5 from all other sample compo-
nents. If this cannot be achieved, the unre-
solved components at their maximum ex-
pected levels will not affect the final assay
result by more than 0.5%. An example of
specificity criteria for an impurity method
is that all impurity peaks that are > 0.1% bb
area will have baseline chromatographic
resolution from the main component
peak(s) and, where practical, will have reso-
lution from all other impurities.
Demonstrate linearity
A linearity study verifies that the sample
solutions are in a concentration range
where analyte response is linearly pro-
portional to concentration. For assay meth-
ods, this study is generally performed by
preparing standard solutions atfivecon-
centration levels, from 50 to 150% of the
target analyte concentration. Five levels
are required to allow detection of curva-
ture in the plotted data. The standards are
evaluated using the chromatographic
conditions determined during the specific-
ity studies
Standards should be prepared and ana-
lyzed a minimum of three times. The 50 to
150% range for this study is wider than
what is required by the FDA guidelines. In
the final method procedure, a tighter
range of three standards is generally used,
such as 80,100, and 120% of target; and in
some instances, a single standard con-
centration is used.
Validating over a wider range provides
confidence that the routine standard lev-
els are well removed from nonlinear re-
sponse concentrations, that the method
covers a wide enough range to incorpo-
rate the limits of content uniformity test-
ing, and that it allows quantitation of crude
samples in support of process develop-
ment. For impurity methods, linearity is
determined by preparing standard solu-
tions atfiveconcentration levels over a
range such as 0.05-2.5 wt%.
Acceptability of linearity data is often
judged by examining the correlation coeffi-
cient and ^intercept of the linear regres-
sion line for the response versus concen-
tration plot. A correlation coefficient of
> 0.999 is generally considered as evi-
dence of acceptable fit of the data to the re-
gression line. The ^intercept should be
less than a few percent of the response ob-
tained for the analyte at the target level.
Althoughtfieseare very practical ways
of evaluating linearity data, they are not
true measures of linearity (11,12). These
parameters, by themselves, can be mis-
leading and should not be used without a
visual examination of the response versus
concentration plot. An example of how
the use of correlation coefficients can be
misleading can be seen in data from an
HPLC method for quantitation of manni-
tol. This method uses an internal stan-
dard, so the data are recorded as peak
area ratios (mannitol area/internal stan-
dard area). Figure 1 is a plot of mannitol
peak area ratio versus mannitol concentra-
tion for standards analyzed by the method.
Although the correlation coefficient of the
linear regression is > 0.999 (top), the plot
indicates small deviations from linearity at
low and high concentrations. An alternate
way of evaluating the data is to plot re-
sponse factor [ (peak area ratio - y inter-
cept/concentration)] versus concentration
(also shown in Figure 1).
If an equivalent response was obtained
at each concentration, the data points
would form a straight line with a zero
slope. The response factors plotted in Fig-
ure 1 (top) vary greatly over the range and
fall only within 15% of the target concen-
tration. A second set of mannitol data, over
a narrower range of concentrations, is
shown in Figure 1 (bottom). The response
factors for all concentrations in this
range are within 1.5% of the target concen-
tration response. The near-zero slope of
the response factor plot indicates that a lin-
ear response is obtained over this con-
centration range.
At the completion of linearity studies,
the appropriate concentration range for
the standards and the injection volume
should be set for all subsequent studies.
An example of a linearity criteria for an
assay method is that the correlation coeffi-
cient for each of three curves (five con-
centration levels each) will be > 0.99 for
the range 80-120% of the target concentra-
tion. The ^-intercept will be < 2% of the
target concentration response. An alter-
nate criteria is that a plot of response fac-
tor versus concentration will show all
values within 2.5% of the target-level re-
sponse factor for concentrations between 80
and 120% of the target concentration. For
an impurity method, the correlation coeffi-
cient for each of three curves (five concen-
tration levels each) will be > 0.98 for the
range 0.1- 2.5% of the main component
concentration. The ^intercept will be < 10%
of the response produced for a 2.5 wt%
Figure 1 . Peak area ratio (circles)
and response factor (squares)
versus concentration for mannitol.
(Top) Concentration range is 5-80 mg/mL. For
peak area ratio line, y = 0.09775 + 0.080569*
and correlation coefficient = 0.99952. (Bottom)
Concentration range is 12-28 mg/mL. For peak
area ratio line, y = 0.027 + 0.08625x and
correlation coefficient = 0.99965.
Analytical Chemistry News & Features, May 1, 1996 3 0 7 A
impurity. An alternate criteria is that a plot
of response factor versus concentration will
show all values within 5% oo the mean re-
sponse factor for concentrations > 0.5 wt%
and within 10% of the mean response factor
for concentrations < 0.5 wt%.
Demonstrate accuracy
The accuracy of a method is the closeness
of the measured value to the true value
for the sample. Accuracy is usually deter-
mined in one of four ways. First, accuracy
can be assessed by analyzing a sample of
known concentration and comparing the
measured value to the true value. Na-
tional Institute of Standards and Technol-
ogy (NIST) reference standards are often
used; however, such a well-characterized
sample is usually not available for new
drug-related analytes. The second ap-
proach is to compare test results from the
method with results from an exist-
ing alternate method that is known to be
accurate Again for pharmaceutical stud-
ies such an alternate
not
The third and fourth approaches are
based on the recovery of known amounts
of analyte spiked into sample matrix. The
third approach, which is the most widely
used recovery study, is performed by
spiking analyte in blank matrices. For as-
say methods, spiked samples are pre-
pared in triplicate at three levels over a
range of 50-150% of the target concentra-
tion. If potential impurities have been iso-
lated they should be added to the matrix
to mimic impure samples For impurity
methods spiked samples are prepared in
triplicate at three levels over a range that
covers the expected impurity content of
the sample such as 0 1-2 5 wt% The ana-
lyte levels in the spiked samples
determined using the same quantitation
p r o c e d u r e as will be u s e d in t h e final
method procedure (i p same number and
i I t 4. J J ' u e
levels of standards, same number of sam-
1 J 4 J J • • f 4- \ TT.
pie and standard injections, etc.). The per-
, , , , , , 1 1 4 J
cent recovery should then be calculated.
The fourth approach is the technique
of standard additions, which can also be
used to determine recovery of spiked ana-
lyte. This approach is used if it is not pos-
sible to prepare a blank sample matrix
without the presence of the analyte. This
can occur, for example, with lyophilized
material, in which the speciation in the
 Report

lyophilized material is significantly differ- criteria, which are required prior to rou-
ent when the analyte is absent. tine use of the method to ensure that it is
An example of an accuracy criteria for performing appropriately. Typically, the
an assay method is that the mean recov- process involves makingfiveinjections of
ery will be 100 ± 2% at each honcentration a standard solution and evaluating several
over the range of 80-120% of the target chromatographic parameters (1) such as
concentration. For an impurity method, resolution, area % reproducibility, number
the mean recovery will be within 0.1% ab- of theoretical plates, and tailing factor.
solute of the theoretical concentration or
10% relative, whichever is greater, for im- Establish the detection limit
purities in the range of 0.1-2.5 wt%. Figure 2. %RSD versus concentra- The detection limit of a method is the low-
tion for a GC headspace analysis of est analyte concentration that produces a
ethanol.
Determine the range response detectable above the noise level
The range of an analytical method is the of the system, typically, three times the
concentration interval over which accept- Thefirsttype of precision study is in- noise level. The detection limit needs to be
able accuracy, linearity, and precision are strument precision or injection repeatabil- determined only for impurity methods in
obtained. In practice, the range is deter- ity (3). A minimum of 10 injections of which chromatographic peaks near the de-
mined using data from the linearity and ac- one sample solution is made to test the tection limit will be observed. The detec-
curacy studies. Assuming that acceptable performance of the chromatographic in- tion limit should be estimated early in the
linearity and accuracy (recovery) results strument. The second type is repeatability method development-validation process
were obtained as described earlier, the or intra-assay precision (2). Intra-assay and should be repeated using the specific
only remaining factor to be evaluated is precision data are obtained by repeatedly wording of the final procedure if any
precision. This precision data should be analyzing, in one laboratory on one day, al- changes have been made. It is important to
available from the triplicate analyses of iquots of a homogeneous sample, each of test the method detection limit on differ-
spiked samples in the accuracy study. which has been independently prepared ent instruments, such as those used in the
Figure 2 illustrates how precision may according to the method procedure. different laboratories to which the method
change as a function of analyte level. The From these precision studies, the sample will be transferred. An example of a
%RSD values for ethanol quantitation by preparation procedure, the number of rep- detection limit criteria is that, at the 0.05%
GC increased significantly as the concen- licate samples to be prepared, and the level, an impurity will have S/N > 3.
tration decreased from 1000 ppm to number of injections required for each
10 ppm. Higher variability is expected as sample in the final method procedure will Establish the quantitation limit
the analyte levels approach the detection be set. Two additional types of precision The quantitation limit is the lowest level of
limit for the method. The developer must studies are described later in Round 2. analyte that can be accurately and pre-
judge at what concentration the impreci- An example of precision criteria for cisely measured. This limit is required
sion becomes too great for the intended an assay method is that the instrument only for impurity methods and is deter-
use of the method. precision (RSD) will be < 1% and the intra- mined by reducing the analyte concentra-
An example of range criteria for an as- assay precision will be < 2%. For an impu- tion until a level is reached where the
say method is that the acceptable range will rity method, at the limit of quantitation, the precision of the method is unacceptable. If
be defined as the concentration interval instrument precision will be < 5% and the not determined experimentally, the quan-
over which linearity and accuracy are ob- intra-assay precision will be < 10%. titation limit is often calculated as the ana-
tained per previously discussed criteria and lyte concentration that gives S/N = 10. An
that yields a precision of < 3% RSD. For an Widen the scope example of quantitation limit criteria is
impurity method, the acceptable range will Once these validation studies are com- that the limit will be defined as the lowest
be defined as the concentration interval plete, the method developers should be concentration level for which an RSD
over which linearity and accuracy are ob- confident in the ability of the method to < 20% is obtained when an intra-assay pre-
tained per the above criteria, and that, in provide good quantitation in their own lab- cision study is performed.
addition, yields a precision of < 10% RSD. oratories. This result may be sufficient
for many methods, especially in the early Establish stability
Determine precision, Round 1 phases of drug development. The remain- During the earlier validation studies, the
The precision of an analytical method is ing studies should provide greater assur- method developer gained some informa-
the amount of scatter in the results ob- ance that the method will work well in tion on the stability of reagents, mobile
tained from multiple analyses of a homoge- other laboratories, where different opera- phases, standards, and sample solutions.
neous sample. To be meaningful, the pre- tors, instruments, and reagents are in- For routine testing in which many sam-
cision study must be performed using the volved and where it will be used over ples are prepared and analyzed each day,
exact sample and standard preparation much longer periods of time. it is often essential that solutions be stable
procedures that will be used in the final This is a good time to begin accumulat- enough to allow for delays such as instru-
method. ing data for two or more system suitability ment breakdowns or overnight analyses

308 A Analytical Chemistry News & Features, May 1, 1996

using autosamplers. At this point, the lim-
its of stability should be tested. Samples
and standards should be tested over at
least a 48-h period, and quantitation of
components should be determined by
comparison to freshly prepared standards.
If the solutions are not stable over 48 h,
storage conditions or additives should be
identified that can improve stability.
An example of stability criteria for as-
say methods is that sample and standard
solutions and the mobile phase will be sta-
ble for 48 h under defined storage condi-
tions. Acceptable stability is < 2% change in
standard or sample response, relative to
freshly prepared standards. The mobile
phase is considered to have acceptable sta-
bility if aged mobile phase produces
equivalent chromatography (capacity fac-
tors, resolution, or tailing factor) and as-
say results are within 2% of the value ob-
tained with fresh mobile phase.
For impurity methods, the sample and
standard solutions and mobile phase will
be stable for 48 h under defined storage
conditions. Acceptable stability is < 20%
change in standard or sample response at
the limit of quantitation, relative to
freshly prepared standards. The mobile
phase is considered to have acceptable
stability if aged mobile phase produces
equivalent chromatography and if impu-
rity results at the limit of quantitation are
within 20% of the values obtained with
fresh mobile phase.
Establish precision, Round 2
The remaining precision studies comprise
much of what historically has been called
ruggedness. Intermediate precision (2) is
the precision obtained when the assay is
performed by multiple analysts, using
multiple instruments, on multiple days, in
one laboratory. Different sources of re-
agents and multiple lots of columns should
also be included in this study. Intermedi-
ate precision results are used to identify
which of the above factors contribute sig-
nificant variability to the final result.
The last type of precision study is re-
producibility (2), which is determined by
testing homogeneous samples in multiple
laboratories, often as part of interlabora-
tory crossover studies. The evaluation of
reproducibility results often focuses more
on measuring bias in results than on de-
termining differences in precision alone.
Statistical equivalence is often used as a
measure of acceptable interlaboratory re-
sults. An alternative, more practical ap-
proach is the use of "analytical equiva-
lence" in which a range of acceptable re-
sults is chosen prior to the study and used
to judge the acceptability of the results
obtained from the different laboratories.
An example of reproducibility criteria
for an assay method could be that the assay
results obtained in multiple laboratories
will be statistically equivalent or the mean
results will be within 2% of the value ob-
tained by the primary testing lab. For an
impurity method, results obtained in multi-
ple laboratories will be statistically equiva-
lent or the mean results will be within 10%
(relative) of the value obtained by the pri-
mary testing lab for impurities > lwt%,
within 25% for impurities from 0.1-1.0 wt%,
and within 50% for impurities <0.1wt%.
Is it robust?
The robustness of a method is its ability to
remain unaffected by small changes in pa-
rameters such as percent organic content
and pH of the mobile phase, buffer concen-
tration, temperature, and injection volume.
These method parameters may be evalu-
ated one factor at a time or simultaneously
as part of a factorial experiment (13). Ob-
taining data on the effects of these parame-
ters may allow a range of acceptable values
to be included in the final method proce-
dure For example if column performance
changes over time adjusting the mobile-
phase strength to compensate for changes
in the column may be allowed if such data
are incliidpH in the validation
An example of robustness criteria is
that the effects of the following changes in
chromatographic conditions will be de-
termined: methanol content in mobile
phase adjusted by ± 2%, mobile-phase pH
adjusted by + 0.1 pH units, and column
temperature adjusted by ± 5 "C. If these
changes are within the limits that produce
acceptable chromatography, they will be
incorporated in the method procedure.
Doing it right the first time
Performing a thorough method validation
can be a tedious process, but the quality
of data generated with the method is di-
rectly linked to the quality of this pro-
cess. Time constraints often do not allow
for sufficient method validations. Many re-
Analytical Chemistry News & Features, May 1, 1996 3 0 9 A
searchers have experienced the conse-
quences of invalid methods and realized
that the amount of time and resources
required to solve problems discovered
later exceeds what would have been ex-
pended initially if the validation studies
had been performed properly. We hope
that we have provided a guide to help you
wend your way efficiently through the
method validation maze and eliminate
many of the problems common to inade-
quately validated analytical methods.
I wish to thank Bruce Burgess, Joseph Glajch,
and the DuPont Merck radiopharmaceuticals
methods quality team for their contributions
in formulating many of the concepts presented
in this paper.
References
(1) U.S. Pharmacopeia 23, pp. 1982-84,
United States Pharmacopeial Convention,
Inc., 1994.
(2) International Conference on Harmonisa-
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lytical Procedures: Definitions and Termi-
nology, Federal Register, Volume e0, pp.
11260, March 1,1995.
(3) Reviewer Guidance, Validation of Chro-
matographii Methods, Center for Drug
Evaluation and Research, Food and Drug
Administration, 1994.
(4) Guideline for Submitting Samples and Ana-
lytical Data for Methods Validalion, Food
and Drug Administration, 1987.
(5) Clarke, G. S. /. Pharm. Biomed. Anal.
1994,12,643.
(6) Inman, E. L.; Frischman, J. K.; ;imenezz
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/. Mark Green is a principal research scien-
tist at DuPont Merck Pharmaceutical Co.
whose research interests include chiral chro-
matography and radioanalytical tech-
niques. Address correspondence ebout this
article to Green at DuPont Merck Pharma-
ceutical Co., 331 Treble Cove Rd.. North
Billerica, MA 01862.