Center for Drug Evaluation 
and Research  (CDER) 
Reviewer Guidance' 
Validation of 
Chromatographic Methods 
November  1994 
CMC  3  TABLE OF CONTENTS 
I.   INTRODUCTION ...............................................  1 
II.   TYPES OF CHROMATOGRAPHY .................................  2 
A .  High Performance Liquid Chromatography (HPLC) . . . . . . . . . . . . . . .   2 
1.   Chiral Chromatography  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   2 
2 .  Ion-exchange Chromatography  .........................  3 
3 .  Ion-pair1Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . .   3 
4 .  Normal Phase Chromatography .........................  3 
5 .  Reversed Phase Chromatography .......................  3 
6 .  Size Exclusion Chromatography  . . . . . . . . . . . . . . . . . . . . . . . .   4 
B.   Gas Chromatography (GC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   4 
C .  Thin-Layer Chromatography (TLC) ............................  5 
Ill.   REFERENCE STANDARDS ......................................  5 
IV.   PARAMETERS FOR VALIDATION OF HPL CHROMATOGRAPHIC 
METHODS FOR DRUG SUBSTANCE AND DRUG PRODUCT . . . . . . . . . .   7 
A .  Accuracy ................................................  8 
B.   Detection Limit and Quantitation Limit  . . . . . . . . . . . . . . . . . . . . . . . . .   8 
C .  Linearity  ................................................ 11 
D.   Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  13 
1.   Repeatability ........................................ 13 
a .  Injection Repeatability . . . . . . . . . . . . . . . . . . . . . . . . . . .  13 
b.   Analysis Repeatability ........................... 15  2 .  Intermediate Precision ................................ 15 
3 .  Reproducibility ...................................... 16 
E.   Range .................................................. 16 
F.   Recovery ................................................ 16 
G .  Robustness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16 
H .  Sample Solution Stability  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  17 
I.   Specificitylselectivity ....................................... 17 
J .  System Suitability Specifications and Tests . . . . . . . . . . . . . . . . . . . . .  21 
1.   Capacity factor ....................................... 22 
2 .  Precisionllnjection repeatability . . . . . . . . . . . . . . . . . . . . . . . . .  22 
3 .  Relative retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  22 
4 .  Resolution  .......................................... 22 
5 .  Tailing factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2.3 
6 .  Theoretical plate number  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  26 
K .  General Points to Consider ..................................  28 
V .  COMMENTS AND CONCLUSIONS ................................ 29 
VI .  ACKNOWLEDGEMENTS  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  29 
VII .  REFERENCES  ................................................ 29  REVIEWER GUIDANCE' 
VALIDATION OF CHROMATOGRAPHIC METHODS 
1.  INTRODUCTION 
The purpose of this technical review guide is to present the issues to consider when 
evaluating chromatographic test methods from a regulatory perspective. The 
document discusses the points to note and weaknesses of chromatography so that 
CDER reviewers can ensure that the method's performance claims are properly 
evaluated, and that sufficient information is available for the field chemist to assess the 
method.  Analytical terms, as defined by the International Conference of 
Harmonization (ICH), 1993, have been incorporated in this guide. 
Chromatographic methods are commonly used for the quantitative and qualitative 
analysis of raw materials, drug substances, drug products and compounds in biological 
fluids. The components monitored include chiral or achiral drug, process impurities, 
residual solvents, excipients such as preservatives, degradation products, extractables 
and leachables from container and closure or manufacturing process, pesticide in drug 
product from plant origin, and metabolites. 
The objective of a test method is to generate reliable and accurate data regardless of 
whether it is for acceptance, release, stability or pharmacokinetics study.  Data are 
generated for the qualitative and quantitative testing during development and post- 
approval of the drug products. The testing includes the acceptance of raw materials, 
release of the drug substances and products, in-process testing for quality assurance, 
and establishment of the expiration dating period. 
Validation of a method is the process by which a method is tested by the developer or 
user for reliability, accuracy and preciseness of its intended purpose. Data thus 
'This guidance has been prepared by the Analytical Methods Technical 
Committee of the Chemistry Manufacturing Controls Coordinating Committee (CMC 
CC) of the Center for Drug Evaluation and Research at the Food and Drug 
Administration. Although this guidance does not create or confer any rights for or on 
any person and does not operate to bind FDA or the industry, it does represent the 
agency's current thinking on the validation of chromatographic methods.  For additional 
copies of this guidance, contact the Division of Communications Management, HFD- 
210, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857 (Phone: 301-594-1012). 
Send one self-addressed adhesive label to assist the offices in processing your 
request. An electror~icv ersion of this guidance is also ava~lablev ia Internet the World 
Wide Web (WWW) ( connect to the FDA Home Page at WWW.FDA.GOV/CDER and 
go to the "Regulatory Guidance" section).  generated become part of the methods validation package submitted to CDER. 
Methods validation should not be a one-time situation to fulfil Agency filing 
requirements, but the methods should be validated and also designed by the developer 
or user to ensure ruggedness or robustness. Methods should be reproducible when 
used by other analysts, on other equivalent equipment, on other days or locations, and 
throughout the life of the drug product. Data that are generated for acceptance, 
release, stability, or pharmacokinetics will only be trustworthy if the methods used to 
generate the data are reliable. The process of validation and method design also 
should be early in the development cycle before important data are generated. 
Validation should be on-going in the form of re-validation with method changes. 
II.  TYPES OF CHROMATOGRAPHY 
Chromatography is a technique by which the components in a sample, carried by the 
liquid or gaseous phase, are resolved by sorption-desorption steps on the stationary 
phase. 
A.  High Performance Liquid Chromatography (HPLC) 
HPL chromatographic separation is based on interaction and differential 
partition of the sample between the mobile liquid phase and the stationary 
phase. The commonly used chromatographic methods can be roughly 
divided into the following groups, not necessarily in order of importance: 
1.  Chiral 
2.  Ion--exchange 
3.  Ion--pair/affinity 
4.  Normal phase 
5.  Reversed phase 
6.  Size exclusion 
1.  Chiral Chromatography 
Separation of the enantiomers can be achieved on chiral stationary 
phases by formation of diastereomers via derivatizing agents or 
mobile phase additives on achiral stationary phases. When used 
as an impurity test method, the sensitivity is enhanced if the 
enantiomeric impurity elutes before the enantiomeric drug.  2.  Ion-exchange Chromatography 
Separation is based on the charge-bearing functional groups, 
anion exchange for sample negative ion (XI, or cation exchange 
for sample positive ion (X').  Gradient elution by pH is common. 
3.  Ion-pair1Affinity Chromatography 
Separation is based on a chemical interaction specific to the target 
species. The more popular reversed phase mode uses a buffer 
and an added counter-ion of opposite charge to the sample with 
separation being influenced by pH, ionic strength, temperature, 
concentration of and type of organic co-solvent(s). Affinity 
chromatography, common for macromolecules, employs a ligand 
(biologically active molecule bonded covalently to the solid matrix) 
which interacts with its homologous antigen (analyte) as a 
reversible complex that can be eluted by changing buffer 
conditions. 
4.  Normal Phase Chromatography 
Normal phase chromatography is a chromatographic technique 
that uses organic solvents for the mobile phase and a polar 
stationary phase. Here, the less polar components elute faster 
than the more polar components. 
5.  Reversed Phase Chromatography 
The test method most commor~lys ubmitted to CDER is the 
reversed phase HPLC method.  UV detection is the most common 
detection technique. 
Reversed phase chromatography, a bonded phase 
chromatographic technique, uses water as the base solvent. 
Separation based on solvent strength and selectivity also may be 
affected by column temperature and pH.  In general, the more 
polar components elute faster than the less polar components. 
UV detection can be used with all chromatographic techniques. 
The concern for this type of detector is the loss of sensitivity with 
lamp aging, and varying sensitivity at the low level depending on 
design and/or manufacturer. A point to note is that observations 
on the HPL chromatograms, by UV detection in combination with 
reversed-phase HPLC, may not be a true indication of the facts for  the following reasons: 
Compounds much more polar than the compound of interest 
may be masked (elute together) in the solvent FronVvoid 
volume. 
Compounds very less polar than the analyte may elute 
either late during the chromatographic run or are retained in 
the column. 
Compounds with lower UV extinction coefficients or different 
wavelength maxima may not be detectable at the low level 
relative to the visibility of the analyte since only one 
wavelength is normally monitored. 
6.  Size Exclusion Chromatography 
Also known as gel permeation or filtration, separation is based on 
the molecular size or hydrodynamic volume of the components. 
Molecules that are too large for the pores of the porous packing 
material on the column elute first, small molecules that enter the 
pores elute last, and the elution rates of the rest depend on their 
relative sizes. 
6.  Gas Chromatography (GC) 
Gas chromatography is based on the volatilized sample transported by 
the carrier gas as the moving phase through the stationary phase of the 
column where separation takes place by the sorption/desorption process. 
Samples for gas chromatographic analysis are normally low molecular 
weight compounds that are volatile and stable at high temperature.  In 
this respect, residual solvents in drug substances and drug products are 
suitable for gas chromatographic analysis. Chemical derivatives can also 
be formed to achieve volatility and thermal stability. 
Common detectors are flame ionization (FID) for carbon-containing 
compounds, electron capture (ECD) for halogenated compounds, flame 
photometric (FPD) for corrlpounds containing sulphur or phosphorous 
and nitrogen-phosphorous (NPD) for compounds containing nitrogen or 
phosphorous. Chiral separation also can be achieved by gas 
chromatography. Separation by the packed column is rapidly being 
replaced by the capillary colurrln that provides improved resolution and 
analysis speed. The location of the analyte on the gas chromatogram is  described by retention time (R,) which is similar to HPLC. 
C.  'Thin-Layer Chromatography ('TLC) 
Thin-layer chromatography is the simplest of the more common 
chromatographic techniques. Separation is based on migration of the 
sample spotted on a coated (stationary phase) plate with one edge 
dipped in a mixture of solvents (mobile phase). The whole system is 
contained in an enclosed tank. 
Detection techniques include fluorescence, UV and sprays (universal and 
specific) for compounds that are not naturally colored. The location of the 
analyte on the TLC plate is described by the R, value which is the ratio of 
the migration distance of the compound of interest to the mobile phase 
front. 
Of the three techniques, gas, liquid and thin-layer, TLC is the most universal test 
method as all components are present on the plate and with appropriate detection 
techniques, all components can be observed. However, it normally is not as accurate 
or sensitive as HPLC. TLC has a higher analytical variation than HPLC, although one 
sees the "whole picture" when appropriate detection schemes are selected. 
Ill.  REFERENCE STANDARDS 
A reference standard is a highly purified compound that is well characterized. 
Chromatographic methods rely heavily on a reference standard to provide accurate 
data. Therefore the quality and purity of the reference standard is very important. Two 
types of reference standards, chemical and nuclidic, exist. With the latter, the radio- 
label purity should also be considered as well as the chemical purity. 
As described in the Guideline for Submitting Samples and Analytical Data for Methods 
Validation, the two categories of chemical reference standards are as follows: 
USPINF reference standard that does not need characterization, and 
non-compendia1 standard that should be of the highest purity that can be 
obtained by reasonable effort and should be thoroughly characterized to 
assure its identity, strength, quality and purity. 
The points to note are: 
Most USPINF reference standards do not state the purity of the 
compound.  The purity correction factor for non-USP reference standards is 
recommended to be included in the calculation of the test method. 
In addition to structurally-related impurities from the synthesis process, 
other process impurities like heavy metals, residual solvents, moisture 
(bound and unbound), pesticides for products of plant origin, and 
degradation products can also contribute to the lack of purity in the 
reference standard. 
The drying of the reference standard before use, if stated in the method, 
will eliminate residual solvent(s), unbound moisture and sometimes 
bound moisture (depending on the drying conditions).  The drying step is 
always included for hygroscopic compounds.  On the other hand, drying 
can result in the loss of a hydrate or cause degradation in heat-sensitive 
compounds. 
Chromatographic test methods use either external or internal standards for 
quantitation. 
A.  An external standard method is used when the standard is analyzed on a 
separate chromatogram from the sample. Quantitation is based on a 
comparison of the peak arealheight (HPLC or GC) or spot intensity (TLC) 
of the sample to that of a reference standard of the analyte of interest. 
The external standard method is more appropriate for samples as follows: 
1.  Sample with a single target concentration and narrow 
concentration range, e.g., acceptance and release tests. 
2.  Simple sample preparation procedure. 
3.  Increased baseline time for detection of potential extraneous 
peaks, e.g., impurities test. 
B.  With an internal standard method, compound of known purity that does 
not cause interference in the analysis is added to the sample mixture. 
Quantitation is based on the response ratio of compound of interest to the 
internal standard vs the response ratio of a similar preparation of the 
reference standard (HPLC or GC). This technique is rarely used for TLC 
methods. 
The internal standard method is more appropriate for samples as follows:  1.  Complex sample preparation procedures, e.g., multiple 
extractions. 
2.  Low concentration sample (sensitivity being an issue), e.g., 
pharmacokinetics studies. 
3.  Wide range of concentrations expected in the sample for analysis, 
e.g., pharmacokinetics studies. 
Although CDER does not specify whether the method must use an internal or 
external standard for quantitation, it is commonly observed that HPLC methods 
for release and stability and TLC methods use external standards; and methods 
for biological fluids and GC methods use internal standards. 
The workina concentration is the target concentration of the compound of interest as 
described in the method.  Keeping the concentrations of the sample and the standard 
close to each other for the external standard method iniproves tlie accuracy of the 
method. 
Recommendations: 
1.  Include the purity correction factor, if knowti, of the reference standard in 
the calculation. 
2.  State .the working concentrations of the standard and sample in the 
method. 
IV.  PARAMETERS FOR VALIDATION OF HPL CHROMATOGRAPHIC 
METHODS FOR DRUG SUBSTANCE AND DRUG PRODUCT 
Though many types of HPL chromatographic techniques are available; the most 
commonly submitted method, the reversed-phase HPLC with UV detection, is selected 
to illustrate the parameters for validation. The criteria for the validation of this 
technique can be extrapolated to other detection methods and chromatographic 
techniques.  For acceptance, release or stability testing, accuracy should be optimized 
since the need to show deviation from the actual or true value is of the greatest 
concern. 
A.  Accuracy 
Accuracy is the measure of how close the experimental value is to the 
true value.  Accuracy studies for drug substance and drug product are recommended 
to be performed at the 80, 100 and 120% levels of label claim as stated in 
the Guideline for Submitting Samples and Analytical Data for Methods 
Validation. 
For the drug product, this is performed frequently by the addition of 
known amounts of drug by weight or volume (dissolved in diluent) to the 
placebo formulation working in the linear range of detection of tlie 
analyte. This would be a true recovery for liquid formulations.  For 
formulations such as tablet, suppository, transdermal patch, this could 
mean evaluating potential interaction of the active drug with the 
excipients in the diluent.  From a practical standpoint, it is difficult to 
manufacture a single unit with known amount of active drug to evaluate 
recovery. This test evaluates the specificity of the method in the 
presence of the excipients under the chromatographic conditions used for 
the analysis of the drug product. It will pick up recovery problems that 
could be encountered during the sample preparation and the 
chromatographic procedures. However, it does not count the effect of the 
manufacturing process. 
At each recorr~mendedle vel studied, replicate samples are evaluated. 
The RSD of the replicates will provide the analysis variation or how 
precise the test method is. The mean of the replicates, expressed as % 
label claim, indicates how accurate the test method is. 
Recommendations: 
Recovery data, at least in triplicate, at each level (80, 100 and 120% of 
label claim) is recommended. The mean is an estimate of accuracy and 
the RSD is an estimate of sample analysis precision. 
B.  Detection Limit and Quantitation Limit 
These limits are normally applied to related substances in the drug 
substance or drug product. specifications on these limits are submitted 
with the regulatory impurities method relating to release and stability of 
both drug substance and drug product. 
Detection limit is the lowest concentration of analyte in a sample that can 
be detected, but not necessarily quantitated, under the stated  experimental conditions.  Quantitation limit is the lowest concentration of 
analyte in a sarr~pleth at can be determined with acceptable precision and 
accuracy under the stated experimental conditions. 
With UV detectors, it is difficult to assure the detection precision of low 
level compounds due to potential gradual loss of sensitivity of detector 
lamps with age, or noise level variation by detector manufacturer. At low 
levels, assurance is needed that the detection and quantitation limits are 
achievable with the test method each time. With no reference standard 
for a given impurity or means to assure detectability, extraneous peak(s) 
could "disappearlappear."  A crude method to evaluate the feasibility of 
the extraneous peak detection is to use the percentage claimed for 
detection limit from the area counts of the analyte.  For example, 
detection limit claim of 0.01% for the analyte integrated area count of 
50,000 will give an area count of 5 that is not detectable. 
Though USP expresses detection limit and quantitation limit in terms of 2 . 
or 3, and 10 times noise level respectively, this concept is not very 
practical.  Noise level on a detector during the method development 
phase may be different when samples are assayed on different detectors, 
etc. The use of standard(s) in the test method at the quantitation limit 
level (proposed by the applicant) is assurance that the impurity can be 
observed and quantitated. 
Detector sensitivity can vary with the model number andlor manufacturer 
as illustrated in Table 1 for the analysis of a compound by two 
commercial detectors. The data should not be taken as the expected 
ratio of sensitivity of the two detectors.  It is not known if other parameters 
which can also play a part, e.g., age of lamp, column, were considered 
when setting these limits.  Table I.  Comparison of Detector Sensitivity Limits in Two 
Commercial Detectors. 
II  I  I 
Detector 1  Detector 2 
I  I 
II  I  II 
Quantitatio  0.21%  0.07% 
n ~imit 
1  I  I 
Detection  0.16% 
I  I 
Limit 
One also should be cautious that baseline noise is not interpreted as 
extraneous peaks.  Undulations may be observed at the void volume if 
the diluent for the sample is different from the solvents (proportion and 
type) used in the mobile phase. 
If a reference standard for the compound of interest is available, a 
standard close to the quantitation limit or the specification could be used. 
For monitoring peak(s) with no reference standard for the impurity, a 
diluted reference standard of the drug substance is recommended. The 
method should then check that the high and low concentrations are 
operating in the linear range of detection of the drug substance. 
Otherwise the information that is expressed as % area or height of the 
drug substance peak from the same HPL chromatogram will be biased. It 
should also be noted that the extraneous peak using area count does not 
consider the detection response which depends on the UV extinction 
coefficient or absorptivity of the compound. 
Recommendations: 
1.  Analysis repeatability and injection repeatability data at the 
quantitation limit. 
2.  Use of an additional reference standard at the quantitation limit 
level in the test method.  C.  Linearity 
The linear range of detectability that obeys Beer's Law is dependent 
onzthe compo~~nadna  lyzed and detector used.  The working sample 
concentration and samples tested for accuracy should be in the linear 
range. 
Figures 1 and 2 illustrate the behavior of UV response vs. 
concentration of a (a) linear and (b) non-linear relationship.  A point to 
note is that when monitoring impurity peaks expressed as percent 
area of the parent drug substance, the impurity observed may not be 
a true reflection of the theoretical amount if the non-linear section of 
the concentration curve is employed.  In addition, the actual amount 
will be obtained only if the extinction coefficient or absorptivity values 
are the same for both impurity and parent compound,  Impurity 
reference standards are often needed. 
Figure 1.  Concentrations vs.  Peak Areas of Standards to Illustrate 
Linearity. 
regression coefficient = 0.999998 
intercept = 0.103 
slope = 0.000011 
Concentrat ion 
rwa&--.   .. , 
wwow- 
.  .. 
4owwo-- 
wwooo-- 
~wwo-- 
-  . . 
I-k--+.t  I-(-.-  1 - .--.  I  ,-4   I--  t;.kL 
70  Y)  ,lo  Figure 2.  Concentrations vs. Peak Areas of Standards Outside the Linear Range. 
Concentration ug/mL 
Recommendations: 
The linearity range for examination depends on -the purpose of the test 
method.  For example, the recorr~mendedr ange for an assay method 
* 
for content would be NLT  20% and the range for an 
assaylimpurities combination method based on area % (for impurities) 
would be +20% of target concentration down to the limit of 
quantitation of the drug substance or impurity.  Under most 
circumstances, regression coefficient (r) is 2 0.999.  Intercept and 
slope should be indicated.  D.  Precision 
Precision is the measure of how close the data values are to each other 
for a number of measurements under the same analytical conditions. 
ICH has defined precision to contain three components: repeatability, 
intermediate precision and reproducibility. Ruggedness as defined in 
USP XXll <I2 25>, 1990 incorporates the concepts described under the 
terms "intermediate precision", "reproducibility" and "robustness" of this 
guide. 
I.  Repeatability 
a.  Injection Repeatability 
Sensitivity is the ability to detect small changes in the 
concentration of the analyte in the sample.  Sensitivity can 
be partially controlled by monitoriug the specification for 
injection reproducibility (system suitability testing). 
The sensitivity or precision as measured by multiple 
injections of a homogeneous sample (prepared solution) 
indicates the performance of the HPLC instrument under the 
chromatographic conditions and day tested.  The 
information is provided as part of the validation data and as 
a systeni suitability test. The specification, as the 
coefficient of variation in % or relative standard deviation 
(RSD), set here will dete~mineth e variation limit of the 
analysis. The tighter the value, the more precise or 
sensitive to variation one can expect the results. This 
assumes that the chromatograph does not malfunction after 
the system suitability testing has been performed.  Keep in 
mind, however, that it does not consider variations due to 
the drug product manufacturing and laboratory sample 
preparation procedures. As an illustration for injection and 
R, variation, Table 2 provides representative data collected 
when a leak developed in the chromatographic system 
during sampling. The set of four duplicate samples were 
injected sequentially. Variations in peak area and drift of 
retention times are noted.  Sets of typical data from a well- 
behaved system for comparison are shown in Table 3.  Table 2.  Representative Injection Repeatability Data for an HPL 
Chromatographic System that Developed a Leak During 
Sampling. 
Sample  R,  Peak Area  A R,  A Peak Area 
A1  5.62  21 55699  0.04  35233 
A2  5.66  21 20466 
B1  5.87  2205659  0.26  82696 
82  6.13  2288355 
C1  6.21  2227066  0.27  38213 
C2  6.48  2265279 
Dl  6.73  25818 88  0.26  20128 
D2  6.99  2602016  
Table 3.  Representative Injection Repeata,bility Data for Select 
Formulations from a Normally Functional HPL 
Chromatographic System. 
Dosage Form  n  Mean &  SD  RSD 
Inhalation  10  1993162 +  0.25% 
Solution  5029 
Solution for  10  1722253  0.37% 
Inhalation  6288 
Capsule  10  1744320 +  0.18% 
3133  Recommendations: 
As part of methods validation, a minimum of 10 injections 
with an RSD of 11% is recommended. With the methods 
for release and stability studies, an RSD of I1 % RSD for 
precision of the system suitability tests for at least five 
injections (n  5) for the active drug either in drug substance 
2 
or drug product is desirable.  For low level impurities, higher 
variations may be acceptable. 
b.  Analysis Repeatability 
Determination, expressed as the RSD, consists of multiple 
measurements of a sample by the same analyst under the 
same analytical conditions. For practical purpose, it is often 
combined with accuracy and carried out as a single study. 
See section 1V.A under Accuracy. 
lntermediate Precision 
lntermediate precision was previously known as part of 
ruggedness. The attribute evaluates the reliability of the method in 
a different environment other than that used during development of 
the method. The objective is to ensure that the method will provide 
the same results when similar samples are analyzed once the 
method development phase is over. 
Depending on time and resources, the method can be tested on 
multiple days, analysts, instruments, etc. 
lntermediate precision in the test method can be' partly assured by 
good system suitability specifications. 'Thus, it is important to set 
tight, but realistic, system suitability specifications. 
Recommendations: 
As a minimum, data generated as described under section 1V.A 
Accuracy, for two separate occasions, is recommended to indicate 
the intermediate precision of the test method.  3.  Reproducibility 
As defined by ICH, reproducibility expresses the precision 
between laboratories as in collaborative studies.  Multiple 
laboratories are desirable but not always attainable because of the 
size of the firm. 
Recommendations: 
It is not normally expected if intermediate precision is 
accomplished. 
E.  Range 
Range is the interval between the high and low levels of analyte studied. 
See also sections 1V.A and C under Accuracy and Linearity respectively. 
The ranges recommended in sections 1V.A and C under Accuracy and 
Linearity can be applied to other analytes, e.g., preservatives. 
F.  Recovery 
Recovery is expressed as the amountJweight of the compound of interest 
analyzed as a percentage to the theoretical amount present in the 
medium. 
Full recovery should be obtained for the compound(s) of interest.  During 
the sample preparation procedure, the compound of interest is recovered 
from excipients in the formulation matrix ranging from a simple aqueous 
solution to complex cream formulation, and from potential adhesion to 
container/closure components, e.g., glass vial, metered valve.  In 
general, a simpler sample preparation procedure will result in a lower 
variation of recovery. Data collection for recovery are discussed in 
section 1V.A under Accuracy. 
G.  Robustness 
ICH defines robustness as a measure of the method's capability to 
remain unaffected by small, but deliberate variations in method 
parameters.  Robustness car1 be partly assured by good system 
suitability specifications. Thus, it is important to set tight, but realistic, 
system suitability specifications.  Testing varying some or all conditions, e.g., age of columns, colurr~nty pe, 
column temperature, pH of buffer in mobile phase, reagents, is normally 
performed. 
Recommendations: 
Data obtained from studies for robustness, though not usually submitted, 
are recommended to be included as part of method validation. 
H.  Sample Solution Stability 
Solution stability of the drug substance or drug product after preparation 
according to the test method should be evaluated according to the test 
method. Most laboratories utilize autosamplers with overnight runs and 
the sample will be in solution for hours in the laboratory environment 
before the test procedure is completed. This is of concern especially for 
drugs that can undergo degradation by hydrolysis, photolysis or adhesion 
to glassware. 
Recommendations: 
Data to support the sample solution stability under normal laboratory 
conditions for the duration of the test procedure, e.g., twenty-four hours, 
shouldbe generated.  In exceptional cases where multiple days are 
needed for sample preparation or solution storage, an appropriate 
stability time should be selected. 
I.  Specificitylselectivity 
The analyte should have no interference from other extraneous 
components and be well resolved from them. A representative HPL 
chromatogram or profile should be generated and submitted to show that 
the extraneous peaks either by addition of known compounds or samples 
from stress testing are baseline resolved from the parent analyte. 
Examples of the extraneous peaks are as follows: 
For the drug substance or raw material, the related substances to 
consider are process impurities (which include isomeric impurities) 
from the synthesis process, residual pesticides, solvents, and 
other extraneous components from extracts of natural origin.  For the drug product, the related substances may be impurities 
present in the active drug, degradation products, interaction of the 
active drug with excipients, extraneous components, e.g., residual 
solvents from the excipients or manufacturing process, leachables 
. 
or extractables from the container and closure system or from the 
manufacturing process. 
Submission of data from stress testing of the drug substance using acid 
and base hydrolysis, temperature, photolysis and oxidation according to 
the Guideline for Submitting Samples and Analytical Data for Methods 
Validation is recommended. Representative HPL chromatograms are 
recommended for stressed and non-stressed samples that include test 
methods for impurities, preservatives, etc. and placebo sample. With the 
impurities test method, the HPL chromatogram should indicate the 
presence of impurities at the level of detectionlquantitation claimed. The 
chromatograms should be legible, labeled, and the time or time scale and 
attenuation should be indicated. 
Points to note are as follows: 
1.  The parent peak may be expanded, e.g., by increasing the 
concentration, attenuation change, so that extraneous 
peaks can be observed at a reasonable size to evaluate 
stability-indicating capability. See comments in section 1V.B 
under Limits of DetectionIQuantitation. 
2.  The baseline should be on-scale as off-scale baseline 
(observed as a flat straight line) can hide minor peaks. 
Peak purity can be determined by the photo-diode array detector.  Low 
level extraneous components present under the compound of interest, 
however, may not interfere or influence the UV spectrum of the analyte. 
Figures 3 and 4 illustrate the combination of UV spectroscopy and HPL 
chromatography by photo-diode array detection using (a) 3-dimensional 
plot and (b) conventional HPL chromatogram. The analyte elutes at 4.7 
minutes. It should be noted that the quality of the UV spectra for the low 
level components is poor.  Figure 3.  A Representative 3-Dimensional Plot of the HPL Chromatogram 
with the UV Spectra. 
Tim i-. --r nnnr 
I1  14. 1  mirr  Figure 4.  A Representative Conventional HPL Chromatogram with UV 
Spectra. 
When stressed samples are used, an appropriate detectortintegrator 
setting should be selected.  For example, to be able to detect low 
levels, e.g.,  0.1%   degradation products, the parent peak should be of 
a size that at least a 0.1 % detectability or area count is feasible. 
Recommendations: 
Representative HPL chromatograms should be submitted fo;  stressed 
and non-stressed samples that include impurities test method, 
preservative(s), etc, with the related placebo sample.  Representative 
HPL chromatogram(s) to show selectivity by the addition of known 
extraneous compounds also should be submitted.  J.  System Suitability Specifications and Tests 
The accuracy and precision of HPLC data collected begin with a well- 
behaved chromatographic system.  The system suitability 
specifications and tests are parameters that provide assistance in. 
achieving this purpose.  This section explains the terms as indicated in 
Figure 5, and provides recommendations and illustrations. 
Figure 5.  Definition of Terms for the System Suitability Parameters. 
Where 
W,  =  width of the peak determined at either 5% (0.05) or 10% (0.10) from the 
baseline of the peak height 
f  =  distance between peak maximum and peak front at W, 
to =  elution time of the void volume or non-retained components 
t,  =  retention time of the analyte 
 ,t =  peak width measured at baseline of the extrapolated straight sides to baseline  t,  =  retention time of the analyte 
 ,t =  peak width measured at baseline of the extrapolated straight sides to baseline 
1.  Capacity factor (k') 
k' = (t,  - to) Ito  
The capacity factor is a measure of where the peak of interest is 
located with respect to the void volume, i.e., elution time of the 
non-retained components. 
Recommendations: 
The peak should be well-resolved from other peaks and the void 
volume.  Generally the value of k' is > 2. 
2.  Precisionllnjection repeatability (RSD) 
Injection precision expressed as RSD (relative standard deviation) 
indicates the performance of the HPL chromatograph which 
includes the plumbing, column, and environmental conditions, 
the time the samples are analyzed. It should be noted that sample 
preparation and manufacturing variations are not considered. 
Recommendations: 
RSD of s 1% for n 2 5 is desirable. 
3.  Relative retention (a) 
Relative retention is a measure of the relative location of two 
peaks. This is not an essential parameter as long as the resolution 
(R,)  is stated. 
4.  Resolution (R,) 
R,  is a measure of how well two peaks are separated.  For reliable 
quantitation, well-separated peaks are essential for quantitation. 
This is a very useful parameter if potential interference peak(s) 
may be of concern. The closest potential eluting peak to the  R,  is minimally influenced by the ratio of the two compounds 
being measured.  The resolution of peaks as indicated by the R, 
values is shown in Figure 6. 
Figure 6.  Separation of Peaks as Indicated by R, Values. 
Recommendations: 
R,  of > 2 between the peak of interest and the closest 
potential interfering peak (impurity, excipient, degradation 
product, internal standard, etc.) is desirable. 
5.  Tailing factor (T) 
The accuracy of quantitation decreases with increase in peak 
tailing because of the difficulties encountered by3the integrator 
in determining wheretwhen the peal< ends and hence the 
calculation of the area under the peal<. Integrator variables are 
preset by the analyst for optimum calculation of the area for the  peak of interest.  Figures 7 and 8 illustrate the tailing factors 
and the effect on quantitation.  If the integrator is unable to 
determine exactly when an upslope or downslope occurs, 
accuracy drops. 
' 
Figure 7.  HPLC Peak with Various Tailing Factors. 
Tailing factor =  1.3  I\ 
Tailing factor = 3.7  I \  Figure 8.  Effect of Peak Tailing on Ouantitation. 
Recommendations: 
Tof 5 2   6.  Theoretical plate number (N) 
Theoretical plate number is a measure of column efficiency, that is, 
how many peaks can be located per unit run-time of the 
chromatogram. 
N is fairly constant for each peak on a chromatogram with a fixed 
set of operating conditions. H, or HETP, the height equivalent of a 
theoretical plate, measures the column efficiency per unit length 
(L) of the column.  Parameters which can affect N or H include 
peak position, particle size in column, flow-rate of mobile phase, 
column temperature, viscosity of mobile phase, and molecular 
weight of the analyte.  Figure 9 shows one set of compounds A, B, 
and C under two different chromatographic conditions resulting in, 
e.g., R,s for B to be 3 and 8.5 minutes, respectively. An 
examination of peak B  indicates that the theoretical plate values 
are different even though the peaks appear similar visually.  Figure 9.  Effect of Retention Times on Theoretical Plates.  Recommendations: 
The theoretical plate number depends on elution time but in 
general.should be > 2000. 
General Recommendation: 
System suitability testing is essential for the assurance of the quality 
performance of the chromatographic system. The amount of testing 
required will depend on the purpose of the test method.  For dissolution or 
release profile test methods using an external standard method, k', T and 
RSD are minimum recommended system suitability tests.  For 
acceptance, release, stability, or impuritiesldegradation methods using 
external or internal standards, kt, T, R,  and RSD are recommended as 
minimum system suitability testing parameters.  In practice, each method 
submitted for validation should include an appropriate number of system 
suitability tests defining the necessary characteristics of that system. 
Additional tests may be selected at the discretion of the applicant or the 
reviewer. 
K.  General Points to Consider 
Some basic points to note in the test method are: 
1.  The sample and standard should be dissolved in the mobile phase. 
If that is not possible, then avoid using too high a level of the 
organic solvent as compared to the level in the mobile phase. 
2.  The sample and standard concentrations should be close if not the 
same. 
3.  The samples should be bracketed by standards during the 
analytical procedure. 
4.  Filtration of the samples before injection is occasionally observed. 
Filtration will remove particulates (centrifugation performs the 
same function) that may clog columns. Adhesion of the analyte to 
the filter can also happen. This will be of irr~portancee specially for 
low level impurities. Data to validate this aspect should be 
submitted by the applicant.  V.  COMMENTS AND CONCLUSIONS 
HPL Chromatoaraphic Methods for Drua Substance and Drua Product. 
Methods should not be validated as a one-time situation, but methods should be 
validated and designed by the developer or user to ensure ruggedness or 
robustness throughout the life of the method. 
The variations due to the drug product manufacturing process, the laboratory 
sample preparation procedure and the instrument performance contribute to the 
accuracy of the data obtained 'from the analysis. With proper validation and tight 
chromatographic performance (system suitability) criteria, an improvement in the 
reliability of the data can be obtained. Variations, except from the drug product 
manufacturing process, will be minimized. Only with good reliable validated 
methods, can data that are generated for release, stability, pharmacokinetics be 
trust-worthy. 
VI.  ACKNOWLEDGEMENTS 
Comments froni Drs. Hoiberg, Poochikian, Blumenstein, Schroeder, Look, 
Tolgyesi (HFD-150); Dr. Layoff (HFH-300); Drs. Zimmerman and Piechocki 
(HFD-110); and the Analytical Methods Technical Committee: Dr. Sheinin, Mr. 
Shostak, Ms. Cunningham, Ms. Jongedyk, Mr. Leutzinger, Dr. Seggel, Ms. 
Sharkey and Mr. Smela are appreciated. 
VII.  REFERENCES 
1.  Guideline for Submitting Samples and Analytical Data for Methods 
Validation, February 1987. 
2.  United States Pharmacopeia, XXII, 1990. <I2 25>. 
3.  Text on Validation of Analytical Procedures, International Conference on 
Harmonization, September 1993.  Submitted by: 
Linda L. Ng, Ph.D. 
Approved by CMC CC: 
Charles Kumkumian, Ph.D.  Roger L. Williams, M.D.