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 postapproval 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, HFD210, 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~ic version of this guidance is also ava~lable via 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~ly submitted 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 radiolabel 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~mended level 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~ple that 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. 

Quantitatio 0.21% I 0.07% II n ~imit II II Detector 1 I Detector 2 

I I 

1 Detection I 0.16% I 

I 

I Limit I 

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~~nd analyzed 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-- 70 Y) - ,- ,lo t;.kL 
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~mended range 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 <I 225>, 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~mine the 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 wellbehaved 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. 

Table 3. Representative Injection Repeata,bility Data for Select 

Formulations from a Normally Functional HPL 

Chromatographic System. 

Sample 

A1 

A2 

B1 

82 

C1 

C2 

Dl 

D2 

R, 

5.62 

5.66 

5.87 

6.13 

6.21 

6.48 

6.73 

6.99 

Dosage Form 

Inhalation 

Solution 

Solution for 

Inhalation 

Capsule 

Peak Area 

21 55699 

21 20466 

2205659 

2288355 

2227066 

2265279 

2581 888 

260201 6 

n 

10 

10 

10 

A R, 

0.04 

0.26 

0.27 

0.26 

Mean & SD 

1993162 + 

5029 

1722253 

6288 

1744320 + 

3133 

A Peak Area 

35233 

82696 

38213 

20128 

RSD 

0.25% 

0.37% 

0.18% 
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 I 1% RSD for 

precision of the system suitability tests for at least five 

injections (n 2 5) for the active drug either in drug substance 

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~n type, 

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. 

Ti mi. rnnnr 

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 wellbehaved 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) I to 

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 52 
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~portance especially 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. <I 225>. 

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.