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

(also shown in Figure 1). pie and standard injections, etc.). The per-
, , , , , , 1 1 4 J
If an equivalent response was obtained cent recovery should then be calculated.
Figure 1 . Peak area ratio (circles)
at each concentration, the data points and response factor (squares) The fourth approach is the technique
would form a straight line with a zero versus concentration for mannitol. of standard additions, which can also be
slope. The response factors plotted in Fig- (Top) Concentration range is 5-80 mg/mL. For used to determine recovery of spiked ana-
ure 1 (top) vary greatly over the range and peak area ratio line, y = 0.09775 + 0.080569*
lyte. This approach is used if it is not pos-
and correlation coefficient = 0.99952. (Bottom)
fall only within 15% of the target concen- Concentration range is 12-28 mg/mL. For peak sible to prepare a blank sample matrix
tration. A second set of mannitol data, over area ratio line, y = 0.027 + 0.08625x and without the presence of the analyte. This
a narrower range of concentrations, is correlation coefficient = 0.99965. can occur, for example, with lyophilized
material, in which the speciation in the
Analytical Chemistry News & Features, May 1, 1996 3 0 7 A
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- Statistical equivalence is often used as a searchers have experienced the conse-
its of stability should be tested. Samples measure of acceptable interlaboratory re- quences of invalid methods and realized
and standards should be tested over at sults. An alternative, more practical ap- that the amount of time and resources
least a 48-h period, and quantitation of proach is the use of "analytical equiva- required to solve problems discovered
components should be determined by lence" in which a range of acceptable re- later exceeds what would have been ex-
comparison to freshly prepared standards. sults is chosen prior to the study and used pended initially if the validation studies
If the solutions are not stable over 48 h, to judge the acceptability of the results had been performed properly. We hope
storage conditions or additives should be obtained from the different laboratories. that we have provided a guide to help you
identified that can improve stability. An example of reproducibility criteria wend your way efficiently through the
An example of stability criteria for as- for an assay method could be that the assay method validation maze and eliminate
say methods is that sample and standard results obtained in multiple laboratories many of the problems common to inade-
solutions and the mobile phase will be sta- will be statistically equivalent or the mean quately validated analytical methods.
ble for 48 h under defined storage condi- results will be within 2% of the value ob-
tions. Acceptable stability is < 2% change in tained by the primary testing lab. For an I wish to thank Bruce Burgess, Joseph Glajch,
standard or sample response, relative to impurity method, results obtained in multi- and the DuPont Merck radiopharmaceuticals
freshly prepared standards. The mobile ple laboratories will be statistically equiva- methods quality team for their contributions
in formulating many of the concepts presented
phase is considered to have acceptable sta- lent or the mean results will be within 10% in this paper.
bility if aged mobile phase produces (relative) of the value obtained by the pri-
equivalent chromatography (capacity fac- mary testing lab for impurities > lwt%,
tors, resolution, or tailing factor) and as- within 25% for impurities from 0.1-1.0 wt%, References
say results are within 2% of the value ob- and within 50% for impurities <0.1wt%. (1) U.S. Pharmacopeia 23, pp. 1982-84,
United States Pharmacopeial Convention,
tained with fresh mobile phase. Inc., 1994.
For impurity methods, the sample and Is it robust? (2) International Conference on Harmonisa-
standard solutions and mobile phase will The robustness of a method is its ability to tion, Draft Guideline on Validalion ofAna-
lytical Procedures: Definitions and Termi-
be stable for 48 h under defined storage remain unaffected by small changes in pa- nology, Federal Register, Volume e0, pp.
conditions. Acceptable stability is < 20% rameters such as percent organic content 11260, March 1,1995.
change in standard or sample response at and pH of the mobile phase, buffer concen- (3) Reviewer Guidance, Validation of Chro-
matographii Methods, Center for Drug
the limit of quantitation, relative to tration, temperature, and injection volume. Evaluation and Research, Food and Drug
freshly prepared standards. The mobile These method parameters may be evalu- Administration, 1994.
phase is considered to have acceptable ated one factor at a time or simultaneously (4) Guideline for Submitting Samples and Ana-
as part of a factorial experiment (13). Ob- lytical Data for Methods Validalion, Food
stability if aged mobile phase produces and Drug Administration, 1987.
equivalent chromatography and if impu- taining data on the effects of these parame- (5) Clarke, G. S. /. Pharm. Biomed. Anal.
rity results at the limit of quantitation are ters may allow a range of acceptable values 1994,12,643.
to be included in the final method proce- (6) Inman, E. L.; Frischman, J. K.; ;imenezz
within 20% of the values obtained with P. J.; Winkel, G. D.; Persinger, M. L.;
fresh mobile phase. dure For example if column performance Rutherford, B. S. /. Chromatogr. Sci.
changes over time adjusting the mobile- 1987,25,252.
phase strength to compensate for changes (7) Wilson, T. D.J. Pharm. Biomed. Anal.
Establish precision, Round 2 1990,8,389.
The remaining precision studies comprise in the column may be allowed if such data (8) Adamovics, J. A.. Chromatographic Analy-
much of what historically has been called are incliidpH in the validation sis ofPharmaceuticals, M. Dekker, New
York, 1990.
ruggedness. Intermediate precision (2) is An example of robustness criteria is (9) Hokanson, G. C. Pharm. Technol. 1994,
the precision obtained when the assay is that the effects of the following changes in 18,118.
performed by multiple analysts, using chromatographic conditions will be de- (10) Carr. G. P.; Wahlich, J. C. /. Pharm.
Biomed. Anal. 1190,0,613.
multiple instruments, on multiple days, in termined: methanol content in mobile (11) Cassidy, R.; Janoski, M. LC-GC1992,10,
one laboratory. Different sources of re- phase adjusted by ± 2%, mobile-phase pH 692.
agents and multiple lots of columns should adjusted by + 0.1 pH units, and column (12) Dorschel, C. A.; Ekmanis, J. L.; Ober-
holtzer, J. E.; Warren, F. V.; Bidling-
also be included in this study. Intermedi- temperature adjusted by ± 5 "C. If these meyer, B. A. Anal. Chem. 1989, 61,
ate precision results are used to identify changes are within the limits that produce 951 A.
which of the above factors contribute sig- acceptable chromatography, they will be (13) Virlichie, J. L.; Ayache, A S.T.P. Pharma
incorporated in the method procedure. Pratiques s995,5,37.
nificant variability to the final result.
The last type of precision study is re-
/. Mark Green is a principal research scien-
producibility (2), which is determined by Doing it right the first time
tist at DuPont Merck Pharmaceutical Co.
testing homogeneous samples in multiple Performing a thorough method validation
whose research interests include chiral chro-
laboratories, often as part of interlabora- can be a tedious process, but the quality matography and radioanalytical tech-
tory crossover studies. The evaluation of of data generated with the method is di- niques. Address correspondence ebout this
reproducibility results often focuses more rectly linked to the quality of this pro- article to Green at DuPont Merck Pharma-
on measuring bias in results than on de- cess. Time constraints often do not allow ceutical Co., 331 Treble Cove Rd.. North
termining differences in precision alone. for sufficient method validations. Many re- Billerica, MA 01862.

Analytical Chemistry News & Features, May 1, 1996 3 0 9 A