9
METHOD VALIDATION
Rosario LoBrutto and Tarun Patel
9.1 INTRODUCTION
The method validation process is to confirm that the method is suited for its
intended purpose. Although the requirements of validation have been clearly
documented by regulatory authorities [ICH, USP, and FDA], the approach to
validation is varied and open to interpretation. Validation requirements differ
during the development process of pharmaceuticals. The method validation
methodologies in this chapter will focus on the method requirements for
preliminary and full validation for both drug substance and drug product.
Preliminary method validation is generally performed in the earlier phases of
development up to Phase IIa because at this time ICH Q2A and Q2B [1] are
not yet binding. A more extensive validation (full validation) is performed
for methods used in later stages of drug development (after Phase IIa) and
for methods that will be used to evaluate marketed products. Specific require-
ments or methodologies for validation depending on the life cycle of the
potential drug candidate in each specific area in the drug development process
will be addressed in the corresponding chapter.
An analytical method is a laboratory procedure that measures an attribute
of a raw material, drug substance, or a drug product. Analytical method vali-
dation is the process of demonstrating that an analytical method is reliable
and adequate for its intended purpose. Any method that is utilized to deter-
mine results during drug substance and formulation development will have to
be validated. Reliable data for release of clinical supplies, stability, and setting
shelf life can only be generated with appropriate validated methods.
455
HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto
Copyright © 2007 by John Wiley & Sons, Inc.
Validation of high-performance liquid chromatography (HPLC) methods
focus mainly on the following:

tocols are not required. Sometimes an internal Standard Operating Procedure
(SOP) suffices and a generic validation protocol does not need to be used.
Usually, for Phase I, validation experiments may be carried out concurrently
with the analysis of the first batch of clinical supplies or the first delivery of
drug substance to be used for clinical supplies. However, depending on the
pharmaceutical organization method validation may need to be performed
prior to the analysis of material that will be used for clinical supplies.
For analytical method validation during full development (after final syn-
thesis has been set for drug substance and after final market formulation has
been set for drug product) corresponding to the definitive control procedure
for new drug application (NDA), a specific validation protocol has to be
written. Before start of the experimental work, the protocols must be written
456 METHOD VALIDATION
*Related substances described in this chapter encompass degradation products, and synthetic
by-products.
by an analytical chemist and approved by a quality assurance department.
Some of the items that are necessary to be specified in the validation proto-
col are listed below:
•
The analytical method for a given product or drug substance
•
The test to be validated
•
The test parameters for each test, including type and number of solutions
and number of injections
•
The acceptance criteria for each parameter based on an internal SOP
(product or method-specific adaptations may be necessary and are accept-
able, if justified)
•

approved with the validation report submission.
9.2 VALIDATION REPORT
A validation report is written during early and full development, and approval
by QA is required. Existing method validation data from earlier stages of
VALIDATION REPORT 457
development may be used for full development if the HPLC method has not
changed. Minor changes such as change in equilibration time may be accept-
able, and the preliminary validation performed for early phase may be used.
These data can be referred to in the validation report, and reference to the
original data must be given.
The validation report should contain reference to the analytical methods
(specific code number used as identifier within the pharmaceutical organiza-
tion) and the corresponding drug substance or product name. Note that for
early-phase method validation reports the results maybe filled in a predefined
table and compared against the acceptance criteria. However, for late-phase
validation, more explicit reports are generated explaining each and every
experiment, with detailed steps of sample and standard preparation.
The list of reference materials (reference standards with the appropriate
certificate of analysis) as well as the list of calibrated and qualified instruments
used in the validation experiments should be documented in the report.
For drug substances the list of the batches of drug substances, notebook
number/reference number for any individual impurities, or solutions or inter-
mediates used should be listed. For drug products the list of the batches of
drug substances, drug product, and the grade/quality of excipients should be
listed. The test parameters and acceptance criteria should be listed together
with the results for each test, and the results passed or failed should be indi-
cated. The validation report should also contain whether the method valida-
tion was successful and if any changes had to be applied to the analytical
method, and then the final analytical method must be resubmitted for QA
approval.

There are numerous method validation examples in the literature [9–18].
Each company has their own approach and own set of acceptance criteria for
different analytical assays, but these approaches must be within the confines
of their line unit QA department and be in accordance with any regulatory
provisions. In the next section a description for each of the parameters to be
validated (figures of merit) are described in detail and examples are given
for each.
ASSIGNMENT OF VALIDATION PARAMETERS 459
TABLE 9-1. Early Development
Type of Tests to Be Validated
Weight Percent/Assay/Content Impurity Testing:
Validation Parameters Identity Uniformity/Dissolution Quantitative Test
a
Specificity Yes Yes Yes
Linearity No Yes
b
Yes
b
Accuracy No Yes
c
Yes
d
Precision (repeatability) No Yes Yes
e
Limit of detection No No Yes
f
Limit of quantitation No
g
No Yes
d

onal information concerning related substances and degradation products.
For example, one method would use reversed-phase (RP) separation mode on
a C18 column, and the second method would use a strong cation exchange
(SCX) column [20]. The orthogonal methods may show different selectivities
toward the degradation products, thereby demonstrating the orthogonal
nature of the two separation techniques. The accuracy would be demonstrated
460 METHOD VALIDATION
TABLE 9-2. Full Development
Type of Tests to Be Validated
Weight Percent/Assay/Content Impurity Testing:
Validation Parameters Identity Uniformity/Dissolution Quantitative Test
Specificity
a
Yes Yes Yes
Linearity No Yes Yes
Accuracy No Yes Yes
Precision (repeatability) No Yes Yes
Precision (intermediate No Yes Yes
precision)
b
Precision (reproducibility) No
cc
Range No Yes Yes
Limit of detection No No Yes
e
Limit of quantitation No
d
No Yes
Stability of the solutions No Yes Yes
Robustness

{only DP}
Dissolution Accuracy (mean)
(drug
•
Recovery 95–105%
product)
•
S
rel
for recovery ≤2.5%
Precision
•
Repeatability S
rel
≤ 2.0%, n ≥ 6 {at Q time}
•
Intermediate precision Project specific.
Linearity n ≥ 6
•
Correlation coefficient r ≥ 0.990
•
y-intercept (absolute value) ≤5%
•
Residual standard deviation ≤2.5%
Stability of solutions
•
Sample ≤2.0% change over specified time
•
Reference standard ≤2.0% change over specified time
Specificity

462 METHOD VALIDATION
TABLE 9-3. Continued
Quality
Characteristics Parameter to be Validated Acceptance Criteria
Weight Accuracy—DS
percent—
•
Comparison of methods % difference of the mean of two
drug (i.e., titration, DSC, PSA) methods ≤2.0%
substance Precision
•
Repeatability S
rel
≤ 2.0%, n ≥ 6, DP
S
rel
≤ 1.0%, n ≥ 6, DS
•
Intermediate precision S
rel
≤ 2.0%, n ≥ 4 [when combined from
two analysts]
Linearity n ≥ 6
•
Correlation coefficient r ≥ 0.998
•
y-intercept ≤2.0%
•
Residual standard deviation ≤2.0%
Stability of solutions

substances Level 0.1–<0.2%, S
rel
≤ 20%, n ≥ 6
(degradation Level 0.2–<0.5%, S
rel
≤ 10%, n ≥ 6
products) Level 0.5–<5%, S
rel
≤ 5%, n ≥ 6
Level ≥ 5%, S
rel
≤ 2.5%, n ≥ 6
Drug substance
•
Intermediate precision Level < 0.1%, S
rel
≤ 40%, n ≥ 4
(synthetic by- [all replicates combined Level 0.1–<0.2%, S
rel
≤ 30%, n ≥ 4
products and from two analysts] Level 0.2–<0.5%, S
rel
≤ 15%, n ≥ 4
degradation Level 0.5–<5%, S
rel
≤ 7.5%, n ≥ 4
products) Level ≥ 5%, S
rel
≤ 4.0%, n ≥ 4
Specificity Known peaks are separated.

•
Recovery Level 0.2–<0.5%: 80–120%
Level 0.5–<5%: 90–110%
Level ≥ 5%: 95–105%
•
S
rel
for recovery Level < 0.5%: ≤10%,
Level 0.5–<5%: ≤5%
Level ≥ 5%: ≤2.5%
For all, n = 9 (at least three
concentrations), a weighted average
maybe used based on the level and
the S
rel
.
Stability of solutions [report
two decimal places]
•
Reference standard Level < 5% of
theoretical 100% Change ≤10%
concentration over specified
time
Level ≥ 5% of
theoretical 100% Change ≤2.0%
concentration over specified
time
•
Sample Related substances (impurities)
Level < 0.5% Change ≤20% over

overlap in this case) and results may be biased, leading to a higher weight
percent of the material.
However, these titration methods can be used in early development when
a reference standard is not available. Also, the spectrometric-based assay
methods such as ultraviolet (UV) may be nonspecific because most of the drug
substance impurities contain a similar chromophore as the parent molecule. If
UV is used, UV absorption is measured at one or more wavelengths and the
absorbance value is recorded for a particular concentration. Sandor Gorog
has critically evaluated the difference between specific and nonspecific assay
methods in the European and US Pharmacopoeias [23]. The difference
between the mean and the accepted true value with a defined confidence inter-
val should be reported in the acceptance criteria.
The accuracy can also be demonstrated by recovery of drug substance
spiked into a placebo for a drug product. The accuracy can also be demon-
strated by recovery of the impurity spiked to a drug substance or into a
placebo with drug substance. The percentage recovery with the certain accep-
tance criteria at each defined level is reported.
Accuracy should be assessed using a minimum of nine determinations at a
minimum of three concentration levels covering the specified range (e.g., 3
concentrations/3 replicate preparations of each in the total analytical proce-
dure) within the ranges shown in Table 9-4.
Accuracy is performed to determine recovery of an active or degradation
products from a drug product or recovery of related substances from a drug
substance. The experiment is designed to recover the total amount of active
or degradation product from a drug product or a specific impurity or impuri-
ties from a drug substance. For recovery of the active for assay and CU, a
known amount of drug substance in solution is spiked into the placebo blend.
The influence of sample preparation steps for tablets must be taken into con-
464 METHOD VALIDATION
sideration such as grinding, sonication, and extraction. For assay determina-

except the amount should be from reporting level to at least 120% of the
ASSIGNMENT OF VALIDATION PARAMETERS 465
TABLE 9-4. Minimal Concentration Ranges for Accuracy Test (Wider Range May
Be Used)
Type of Analytical Procedure Range, at Minimum, to Be Covered
Assay (content) 80–120% of declared content
Assay (CU) 70–130% of declared content
Assay (dissolution) ±30% of specified range (for immediate release
dosage form).
If the specification for a controlled release product
(modified release or sustained release) covers
a region from 20% (after 1 hr) to 90% (after
24 hr), the validated range would cover 50% of
1-hr limit (20% × 50% = 10%) to 130% of the
label claim (label claim × 1.3).
Degradation products/impurities Reporting level to at least 120% of specification
limit.
466 METHOD VALIDATION
TABLE 9-5. Range for Recovery Experiment for CU and Assay Method to Be
Defined in Method Validation Protocol
Target Concentration Number of
of Solutions Target Concentration Amount Preparations/Number
(%) of Solutions (mg/mL) Injected (µg) of Injections
130 1.30 13.0 3/1
115 1.15 11.5 3/1
100 1.00 10.0 3/1
85 0.85 8.5 3/1
70 0.70 7.0 3/1
TABLE 9-6. Example of Actual Sample Preparation in Method Validation Protocol
Milliliters of Stock Actual Final

130%-Recovery–3 1281.6 129.0509
% Average
recovery = 99.31
SD 0.18
%RSD 0.18
specification limit. Note that the reporting level can never be lower than the
limit of quantitation (LOQ) of the method. However, during early-phase val-
idation for drug products, if authentic degradation products are not available,
then low amounts of API are added (LOQ to 120% specification limit of
largest impurity) to the placebo and the recovery experiment is performed.
Once degradation products of known purity become available (isolated or
synthesized), a spiked recovery experiment should be performed. For drug
substances these may be directly spiked into the drug substance (DS), and for
the drug product (DP) these may be spiked into the DS + placebo.This spiked
experiment is conducted to determine whether a sample preparation proce-
dure is able to completely extract active and degradation products from the
sample matrix. For drug substances, a known amount of spiked impurities
(authentic samples) is added to the active pharmaceutical ingredient and the
recovery experiment is performed.The purity (A% − water − residual solvents
− inorganic impurities) of the impurity that will be spiked must be known as
well in order to calculate the actual amount added to the respective DS so that
the theoretical amount of the impurity that would be in the solution can be
determined. This is because the API may have some amounts of the same
known degradation products may already present in the API drug substance.
This must be accounted for in the calculation. Therefore, the amount of the
impurity that is present in the matrix (DS) must be known. The total of
the spiked amount of impurity and the amount of impurity that is present in
the drug substance must be used to determine the overall amount of impurity.
Also, for drug products when authentic degradation products are added to
placebo in presence of API, the purity factor of the isolated degradation

dure (recovery procedure) requires filtering the sample solution prior to the
solution being injected into an HPLC system, then a check for adsorption of
the components onto the filter membrane must be performed.The experiment
should be set up to conduct the filter step and centrifuge on the same solu-
tion. So, for the same solution (reference standard solution as well as a sample
solution), an aliquot of solution is passed through a membrane filter and col-
lected after 2, 4, 6, 8, and 10 mL. In addition, the same solution (not filtered)
is centrifuged and supernatant is collected. All solutions are then injected on
a HPLC system. Since the identical solution has gone through different paths,
the peak areas from the chromatogram should be identical (with some accept-
able variability due to injection precision of the analytical instrumentation).
If there is no change in peak areas between centrifuged and filtered solutions,
then it can be stated that the membrane for that particular filter does not cause
adsorption of the analyte(s). If the peak areas between centrifuged and fil-
tered solution are different (filtered solution shows smaller peak areas than
the centrifuged solution), then it can be stated that the membrane in that par-
ticular filter is adsorbing the analyte(s). However, sometimes an increase in
peak areas is observed as greater volumes are passed through the filter (e.g.,
2–6 mL). Therefore, the minimum volume that needs to be passed through the
filter to get constant peak areas that are comparable to the centrifuged peak
areas must be determined. If those areas are within (2.0%), then the desig-
nated amount of volume that is needed to pass through the filter before the
468 METHOD VALIDATION
TABLE 9-9. Spiked Recovery Results
Impurity A Impurity A Theoretical Actual Recovery
% in Matrix % Spiked Overall Overall Overall
0.2 0.1 0.3 0.31 103.3%
0.2 0.2 0.4 0.42 105.0%
0.2 0.4 0.6 0.59 98.3%
solution can be collected for HPLC analysis must be noted. These types of

longer extraction times t
1
,t
2
and t
3
are used and higher extraction volumes V
1
,
V
2
, and V
3
are used. The extraction volumes employed should use the same
extraction time specified in the method (t
0
).
ASSIGNMENT OF VALIDATION PARAMETERS 469
TABLE 9-10. Generalized Procedure to Evaluate Both
Kinetic and Thermodynamic Factors
Experiment
Number Extraction Time Extraction Volume
1 t
0
V
0
2 t
1
V
0

Lastly, to really prove that the sample preparation procedure or the recov-
ery procedure is completely extracting the API and degradation products, uti-
lization of a homogenizer must be considered.
In general, homogenizers are utilized for automated sample preparation
procedures in workstations such as TPWII (Caliper Life Sciences, 68 Elm
Street, Hopkinton, MA 01748 or www.caliperls.com). Homogenizers are made
up of a stainless steel blade that rotates up to 20,000 rpm. The following
example illustrates why homogenizers play a vital role in sample preparation.
A modified release (MR) product under development gives a drug release
profile for at least 8 hours. This corresponds to the release rate of drug sub-
stance or the API from the dosage form within 8 hours at 37°C in the disso-
lution media (apparatus I as described in the United States Pharmacopoeia
(USP) 〈711〉 at 100 rpm). For example, when a sample preparation procedure
was developed for assay and degradation products for a modified release drug
product, it was determined that a sonication time of about 30 minutes with
about 4 hours of mechanical shaking provided adequate extraction efficiency
of the drug from the dosage form. In contrast, if a stand-alone homogenizer
was utilized for the same procedure for this dosage form, then the total sample
extraction time was found to be about 5 minutes with intermittent stops
required by the system software (e.g., TPWII takes about 2 to 5 seconds from
end of one pulse to the start of another pulse). The homogenizer provides the
energy needed to break the dosage form and to extract the API very efficiently
when compared to conventional sonication and mechanical shaking. The final
sample preparation procedure was finalized as follows:
Two pulses at 8000 rpm for 10 seconds each
Six pulses at 15,000 rpm for 15 seconds each
Soak/settle time for 2 minutes (to allow all particles to settle to the bottom
of the vessel, which allowed for a facile filtration step)
9.4.2 Precision
Precision provides an indication of random errors and can be broken down

and is directly proportional over the relevant concentration range for the
target concentration of the analyte. It is recommended to perform the linear-
ity of the API and related substances independently; and once linearity has
been demonstrated, another linearity could be performed containing both API
and specific related substance if necessary. For this reason, a stock solution of
each substance (API, degradation products, synthetic by-product) must be pre-
pared separately (one per solution), and a serial dilution from this stock solu-
tion must be injected into an HPLC system (constant injection volume).There
are two major reasons to perform a linearity test on each solution indepen-
dently. First, each substance may not be pure and the linearity test for each
component may become confounded. This is especially true if the active drug
substance contains the impurity that linearity is being performed on and/or if
the impurity contains the active drug substance as an impurity. Second, when
each substance is studied independently, the calculation of relative response
factor (RRF) is much easier to determine. The ranges that should be covered
for the linearity test are described in Table 9-11.
At least five concentrations within the range specified above for the
linearity test should be used. When a linearity test is needed for an assay and
ASSIGNMENT OF VALIDATION PARAMETERS 471
degradation products test, it is generally recommended that five or more con-
centrations should be utilized to cover the entire range. The focus should be
on both (a) the lower limit (LOQ to 1.0%) for the degradation products and
(b) the higher limit (80–120%) for the assay of the active. If the assay method
is also used for content uniformity, the range 80–120% should be expanded
accordingly to 70–130%.
As stated in the recovery section, if authentic degradation products or
impurities are not available, then an API may be utilized to perform the lin-
earity test at the lower concentration range (reporting level to 1.0%). In addi-
tion for drug products, if assay and degradation products are calculated from
a single 100% reference standard solution (mass percent) or area percent nor-

content [only if 100% reference standard is
utilized to calculate low level of
degradation products]
and the proposed criteria are shown in Table 9-3.Although these are very prac-
tical ways of evaluating linearity data, they are not true measures of linearity
[25, 26]. The coefficient of correlation can be subject to misinterpretation and
may give a misrepresentation of linearity, since different datasets can yield
identical regression statistics [27, 28]. The parameters, correlation coefficients,
y-intercept, and %RSD by themselves can be misleading and should not be
used without a visual examination of the response versus concentration plot
[29]. A more statistically sound approach to examine linearity would include
examining the residuals from a linear regression. The residuals are the dis-
tances of the experimental points from the fitted regression line, measured in
a direction parallel to the response axis. Analysis of the residuals provides
further support that the calibration curve would be deemed linear if the resid-
ual response shows a normal distribution with a zero mean [30]. Although
correlation coefficients of the linear regression can be >0.99, the plots of the
response factor versus the concentration can shed light if there are any appar-
ent deviations from linearity.A slope close to zero (response factor versus con-
centration) would indicate that a linear response is obtained over the specified
concentration range. An additional acceptance criterion that could be consid-
ered is that the response factor will show %RSD of ≤2.0% across all concen-
tration levels between 80% and 120% of the target concentration (assay).
Also, this %RSD acceptance criterion could be applied to the low-level lin-
earity regions such that the response factor will show %RSD of ≤10% across
all concentration levels between LOQ to 120% of impurity specification level.
Additionally, this comparison of the response factor can be used to help justify
the LOQ above and beyond the typical S/N >10, injection precision require-
ments, and low-level linearity requirements.A simple test would be to compare
the response factor difference between the proposed LOQ and the 5× LOQ