A Practical Approach to

Validation of HPLC Methods

Under Current Good
Manufacturing Practices
GHULAM A. SHABIR

Introduction

Regulatory analytical procedures are of two types: comAnalytical methods validation is an important regulatory
pendial and noncompendial. The noncompendial analytical
requirement in pharmaceutical analysis. High-Performance
procedures in the USP are those legally recognized as reguLiquid Chromatography (HPLC) is commonly used as an
latory procedures under section 501(b) of the Federal Food,
analytical technique in developing and validating assay
Drug and Cosmetic Act. When using USP analytical methmethods for drug products and drug substances. Method valods, the guidance recommends that information be provided
idation provides documented evidence, and a high degree of
for the following characteristics: specificity of the method,
assurance, that an analytical method employed for a specific
stability of the analytical sample solution, and intermediate
test, is suitable for its intended use. Over recent years, reguprecision. Compendial analytical methods may not be stabillatory authorities have become increasingly aware of the neity indicating, and this concern must be addressed when decessity of ensuring that the data submitted to them in appliveloping a drug product specification, because formulation
cations for marketing authorizations have been acquired
based interference may not be considered in the monograph
using validated analytical methodology. The International
specifications. Additional analytical tests for impurities may
Conference on Harmonization (ICH) has introduced guidebe necessary to support the quality of the drug substance or
1,2
lines for analytical methods validation. The U.S. Food and
drug product. Noncompendial analytical methods must be
Drug Administration (FDA) methods validation draft guidfully validated. The most widely applied validation charac3-5
ance document, as well as United States Pharmacopoeia
teristics are accuracy, precision (repeatability and intermedi6
(USP) both refer to ICH guidelines.
ate precision), specificity, detection limit,
These draft guidances define regulatory
quantitation limit, linearity, range, and staand alternative analytical procedures and Figure 1
bility of analytical solutions.
____________________
stability-indicating assays. The FDA has
The parameters that require validation
The chemical structure of
proposed adding section CFR 211.222 on
and the approach adopted for each particethyl 4-hydroxybenzoate.
analytical methods validation to the curular case are dependent on the type and
rent Good Manufacturing Practice
applications of the method. Before under7
(cGMP) regulations. This would require
taking the task of method validation, it is
pharmaceutical manufacturers to estabnecessary that the analytical system itself
lish and document the accuracy, sensitivis adequately designed, maintained, cali8
ity, specificity, reproducibility, and any
brated, and validated. The first step in
other attribute (e.g., system suitability,
method validation is to prepare a protostability of solutions) necessary to valicol, preferably written with the instrucdate test methods.
tions in a clear step-by-step format. This

Equipment and Instrumentation Qualification

29

Ghulam A. Shabir

approach has been reported previously. In

this paper, it is intended to review and
demonstrate practical approaches to analytical method validation in detail with
reference to an HPLC assay of ethyl 4-hydroxybenzoate (Figure 1). Ethyl 4-hydroxybenzoate alone or in combination
with other esters of p-hydroxybenzoic
acid, or with other antimicrobial agents, is
used as a preservative in pharmaceutical
formulations.

Figure
2
______________________________________________
Graph measured peak area versus ethyl 4-hydroxybenzoate concentration demonstrating linearity.

Experimental
Chemicals and reagents.
All chemicals and reagents were of the
highest purity. HPLC-grade acetonitrile was
obtained from Merck (Darmstadt, Germany). Water was purified with a Millipore
Milli-Q system (Watford, UK). Ethyl 4-hydroxybenzoate (Batch #1005425) was supplied by Lancaster Synthesis (Morecambe, England).
HPLC instrumentation.
The HPLC system used for the validation studies consisted of a Waters Alliance 2690 Separations Module to a
996 photodiode-array (PDA) detector. The control of the
HPLC system and data collection was by a Compaq computer equipped with Waters Millennium32 software (version 3.20). The second HPLC system used for intermediate
precision studies consisted of Perkin Elmer: model series
200 UV visible detector, series 200 LC pump, series 200 autosampler, and a series 200 peltier LC column oven were
used to chromatograph the solutions. The data was acquired
via PE TotalChrom Workstation data acquisition software,
(Version 6.2.0) using PE Nelson series 600 LINK interfaces.
Both HPLC systems including software (Food and Drug Administration (FDA), 21 Code of Federal Regulations (CFR)
Part 11) were validated prior to use for the test method validation.
All chromatographic experiments were performed in the
isocratic mode. A C18 symmetry analytical column from
Waters (located in Milford, MA, United States) 3.9 x 150
mm, 5 mm particle size was used. The mobile phase consisted of a mixture of acetonitrile, water solution (65:35,
v/v). The flow rate was set to 1.0 ml/min, and the oven temperature to 25C. The injection volume was 20 l, and the
detection wavelength was set at 254nm.
30

I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y

Preparation of mobile phase.

The mobile phase was prepared by adding 650 ml of
HPLC-grade acetonitrile in 1000 ml of water (65:35, v/v).
The mobile phase was filtered under a vacuum through 0.45
mm nylon filters and degassed before use. Also, the mobile
phase continuously was degassed with an on-line degasser.
Preparation of standard and sample solutions.
Ethyl 4-hydroxybenzoate (100 mg) was weighed accurately and added to a 100 ml volumetric flask before being
dissolved in acetonitrile. A 2.0 ml aliquot of stock solution
was diluted to 100 ml in the mobile phase, yielding a final
concentration of 20 g/ml. Standard solutions for the evaluation of ethyl 4-hydroxybenzoate linearity were prepared
over a concentration range of 5.0-40 g/ml, to 25, 50, 75,
100, 150, and 200% in the mobile phase.

Results and Discussion

Validation of the chromatographic method:
Linearity and range
The linearity of the method should be tested in order to
demonstrate a proportional relationship of response versus
analyte concentration over the working range. The linearity
range for evaluation depends on the purpose of the analytical
test method. The ICH guidelines specified a minimum of
five concentration levels, along with certain minimum spec-

Ghulam A. Shabir

Figure
3
______________________________________________

n = 3) and the following regression equation

was found by plotting the peak area (y) verResults of assessment of the linearity of the HPLC method
sus the ethyl 4-hydroxybenzoate concentrafor the assay of ethyl 4-hydroxybenzoate employing the antion (x) expressed in g/ml: y = 29935x +
alytical working standard dissolved in mobile phase
51338 (r2 = 1.000). The demonstration coefficient (r2) obtained for the regression line
Concentration EP peak area Peak area
Concentrademonstrates the excellent relationship beRSD (%)
as percent of as mean of
tion (g/ml)
tween peak area and concentration of ethyl 43 injections
20 g/ml
hydroxybenzoate (Figure 2). The data obtained from linearity experiments are pre5
0.13
792862
25
sented in Figure 3. The range is derived from
10
0.16
1535889
50
linearity studies, and depends on the intended
15
0.21
2308902
75
application of the test method. It is estab20
0.13
3057149
100
lished by confirming that the assay procedure
30
0.10
4546415
150
provides an acceptable degree of linearity,
40
0.25
6027790
200
accuracy, and precision when applied to samCorrelation coefficient: r2 = 1.000; Equation for
ples containing amounts of analyte within, or
regression line: y = 29935x + 51338 (n = 3)
at the extremes of the specified range, of the
test method. The range is normally expressed
in the same units as the test results obtained
by the method. In this study, the data obtained
Figure
4
______________________________________________
during the linearity and accuracy studies was
Accuracy/recovery of ethyl 4-hydroxybenzoate from
used to assess the range of the assay method.
samples with known concentration
The precision data for this assessment was the
precision of the three replicate samples anaPercent of
Recovery (%)
RSD (%) of
Sample
lyzed at each level in the accuracy studies.
(n = 3)
area response
nominal
number
The valid analytical range of the method is
factor
that range of concentrations, which pass the
50
99.67
0.16
1
linearity and accuracy criteria, and yields an
99.78
0.21
75
2
RSD of < 2%. The linearity data described
100
99.85
0.13
3
earlier demonstrates acceptable linearity for
99.87
0.10
150
4
ethyl 4-hydroxybenzoate over the range of 80
Mean recovery: 99.8%; RSD 0.09%
to 120% of the target concentration. The RSD
values obtained for the recovery of ethyl 4-hyified ranges. For assay, the minimum specified range is from
droxybenzoate at 50, 75, 100, and 150% of target are 0.16,
80-120% of the target concentration. For an impurity test,
0.21, 0.13, and 0.10%, respectively. Each value was the result
the minimum range is from the reporting level of each imof three individual sample preparations and analysis. These
purity to 120% of the specification. Acceptability of lineardata support a method range of 80 to 120% of the target conity data is often judged by examining the correlation coefficentration.
cient and y-intercept of the linear regression line for the reAccuracy/recovery studies
sponse versus concentration plot. The regression coefficient
2
(r ) is > 0.998 is generally considered as evidence of acceptThe accuracy of an analytical method is the closeness of
6
able fit of the data to the regression line. The y-intercept
test results obtained by that method to the true value. Accushould be less than a few percent of the response obtained for
racy is usually determined in one of four ways. First, accuthe analyte at the target level. The Percent Relative Standard
racy can be assessed by analyzing a sample of known conDeviation (RSD), intercept, and slope should be calculated.
centration (reference materials), and comparing the meaIn the present study, linearity was studied in the concentrasured value to the true value. The second approach is to comtion range 5.0-40 g/ml (25-200% of nominal concentration,
pare test results from the new method with results from an

Equipment and Instrumentation Qualification

31

Ghulam A. Shabir

Method validation
provides documented
evidence, and a high
degree of assurance, that
an analytical method
employed for a specific
test, is suitable for its
intended use.

concentration. The ICH2 recommends collecting data from a

minimum of nine determinations over a minimum of three
concentration levels covering the specified range (e.g., three
concentrations, three replicates each).
In the present study, a number of different solutions
were prepared with known added amounts of ethyl 4-hydroxybenzoate and injected in triplicate. Percent recoveries of response factor (area/concentration) were calculated. The results of accuracy studies are shown in Figure
4, and it is evident that the method is accurate within the
desired recovery range.

existing alternate well-characterized procedure that is known

to be accurate. The third approach is based on the recovery
of known amounts of analyte. This is performed by spiking
analyte in blank matrices. For assay methods, spiked samples are prepared in triplicate at three levels over a range of
50-150% of the target concentration. The percent recovery
should then be calculated. The fourth approach is the technique of standard additions, which can also be used to determine recovery of spiked analyte. This approach is used if it
is not possible to prepare a blank sample matrix without the
presence of the analyte. Accuracy criteria for an assay
method (FDA) is that the mean recovery will be 100 2% at
each concentration over the range of 80-120% of the target

In order to design a chromatographic system for the

analysis of an active component of a pharmaceutical product, it is essential to have a good knowledge of; (1) susceptibility of the drug to degradation and its degradation
pathway; (2) assay interference by possible degradants or
synthesis precursors; and (3) assay interference by chemicals employed in sample preparation and excipients in the
formulation.
Degradation products may be formed by acid/base hydrolysis, oxidation, Ultraviolet (UV) irradiation, heat, light,
etc., however, it is not within the scope of this paper to discuss in detail the elucidation of degradation pathways.
In the present study, initially, a reference standard of

Specificity of the Assay and Degradation

of Active Constituent

Figure
5
________________________________________________________________
HPLC chromatogram of ethyl 4-hydroxybenzoate.

32

I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y

Ghulam A. Shabir

Figure
6
________________________________________________________________
HPLC chromatogram for placebo. The analyte peak was eluted at 1.58 minutes.

ethyl 4-hydroxybenzoate was chromatographed. Figure 5 clearly demonstrates

that ethyl 4-hydroxybenzoate is well separated from any potential interference. Assay
interference was investigated by injecting
placebo. No interfering peaks (Figure 6)
were observed. Therefore, this method was
specific for ethyl 4-hydroxybenzoate.

Precision
Precision is the measure of the degree
of repeatability of an analytical method
under normal operation, and is normally
expressed as the percent relative standard
deviation for a statistically significant
number of samples. Precision may be performed at three different levels: repeatability, intermediate precision, and reproducibility.

Figure
7
______________________________________________
Demonstration of the repeatability of the HPLC assay for
ethyl 4-hydroxybenzoate as shown by the results of 10
replicate injections of one solution at 100 percent of the
test (20 mg/ml) concentration
Injection
number

RT (min)

Peak height
(V)

Peak area
(V s)

1
2
3
4
5
6
7
8
9
10
Mean
RSD (%)

1.57
1.58
1.58
1.58
1.57
1.57
1.57
1.58
1.58
1.58
1.58
0.18

855847
858249
854532
856705
857058
854755
855098
854078
856416
849916
855265
0.25

3109735
3100787
3099540
3103544
3101464
3099731
3102575
3103159
3104217
3091891
3101665
0.14

Repeatability
Repeatability (intra-day assay precision) is the results of
the method operating over a short time interval under the
same conditions (intra-assay precision). It should be determined from a minimum of nine determinations covering the
specified range of the procedure (for example, three levels,

three repetitions each), or from a minimum of six determinations at 100% of the test or target concentration. A precision criterion for an assay method is that the instrument precision (RSD) will be 1%, and for the impurity assay, at the
limit of quantitation, the instrument precision (repeatability)
will be 5%. Documentation in support of precision studies
should include the standard deviation, relative standard devi-

Equipment and Instrumentation Qualification

33

Ghulam A. Shabir

Figure
8
_____________________________________________________________
Demonstration of the intermediate precision of the HPLC assay for ethyl
4-hydroxybenzoate results in relative percent purity area
HPLC system 1
Sample

S1
(50%)

S2
(100%)

S3
(150%)

S1
(50%)

S2
(100%)

S3
(150%)

Operator 1, day 1
Operator 1, day 2
Operator 2, day 1
Operator 2, day 2
Mean
(HPLC systems)
Mean (Operators)
RSD (criteria 2%)
HPLC systems
Operators

99.83
99.76
99.71
99.53
99.71

99.79
99.74
99.76
99.62
99.73

99.76
99.74
99.77
99.57
99.71

99.76
99.82
99.75
99.79
99.78

99.83
99.80
99.76
99.81
99.80

99.83
99.78
99.69
99.82
99.78

99.79
S1+S1
0.05
0.06

99.79
S2+S2
0.05
0.04

99.78
S3+S3
0.05
0.05

99.70

99.74

99.71

ation, coefficient of variation, and confidence interval. In this

study, precision of the method was evaluated through the repeatability of the method (intra-assay precision) by assaying
ten replicate injections of ethyl 4-hydroxybenzoate at the
same concentration (20 g/ml), during the same day, under
the same experimental conditions. The RSD values of the retention time, area, and height of ethyl 4-hydroxybenzoate
peak were found to be < 0.3%, as presented in Figure 7.

Intermediate Precision
Intermediate precision (inter-day variation) is the results
from within lab variations, due to random events, such as different days, analysts, equipment, etc. In determining intermediate precision, experimental design should be employed, so
that the effects (if any) of the individual variables can be monitored. Precision criteria for an assay method is that the intraassay precision will be 2%, and for impurity assay, at the
limit of quantitation, the instrument precision will be 5%,
and the intra-assay precision will be 10%. In this study, intermediate precision (within-laboratory variation) was
demonstrated by two operators, using two HPLC systems,
and evaluating the relative percent purity data across the two
HPLC systems at three concentration levels (50%, 100%,
150%) that cover the ethyl 4-hydroxybenzoate assay method
range (5.0-40 g/ml). The mean and RSD across the systems
and analysts were calculated from the individual relative per34

HPLC system 2

I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y

cent purity mean values at 50, 100, and 150% of the test concentration. The RSD values presented in Figure 8 were less
than 1% for both systems and operators, and illustrated the
good precision of the analytical method.

Reproducibility
Reproducibility1 is determined by testing homogeneous
samples in multiple laboratories, often as part of inter-laboratory crossover studies. An example of reproducibility criteria
for an assay method could be that the assay results obtained
in multiple laboratories will be statistically equivalent, or the
mean results will be within 2% of the value obtained by the
primary testing lab. For an impurity method, results obtained
in multiple laboratories will be statistically equivalent, or the
mean results will be within 10% (relative) of the value obtained by the primary testing lab for impurities. Reproducibility is not normally expected if intermediate precision
is performed.

Limit of Detection and Quantitation

The Limit of Detection (LOD) and Limit of Quantitation
(LOQ) tests for the procedure are performed on samples
containing very low concentrations of analyte. LOD is defined as the lowest amount of analyte that can be detected
above baseline noise; typically, three times the noise level.

Ghulam A. Shabir

Figure
9
________________________________________________________________
HPLC chromatogram for limit of detection (2 ng/ml)

Figure
10
________________________________________________________________
HPLC chromatogram for limit of quantitation (5 ng/ml).

Figure
11
________________________________________________________________________
Stability of ethyl 4-hydroxybenzoate in solution (n = 6)
Time
(hour)

RT (min)

Peak area
RSD (%)

Peak Height
RSD (%)

Percent
recovery

0
24
48

0.14
0.18
0.29

0.70
0.15
0.30

0.80
0.27
0.51

99.88
99.82
99.78

Percent of initial

99.35

Equipment and Instrumentation Qualification

35

Ghulam A. Shabir

Figure
12
_______________________________________________________
Demonstration of the system suitability of the HPLC assay for ethyl
4-hydroxybenzoate
Results

System Suitability
Parameter

Acceptance
Criteria

Injection precision for

area (n = 10)

RSD 1%

0.15

0.11

RSD 1%

0.18

0.11

USP tailing (T) for ethyl

4-hydroxybenzoate peak

T2

1.05

1.03

Theoretical plates (N) for

ethyl 4-hydroxybenzoate
peak

N = > 2000

5276

6628

Injection precision for

retention time (min)

HPLC system 1 HPLC system 2

LOQ is defined as the lowest amount of analyte which can

be reproducibly quantitated above the baseline noise, that
gives S/N = 10.
In this study, LOD for a 20 l injection of ethyl 4-hydroxybenzoate standard (signal to noise = 3) was 2.0 g/ml
(Figure 9), and the LOQ (signal to noise = 10) was 5 g/ml
(Figure 10) and RSD < 2% (n = 6).

mal lighting conditions for 48 hours, and were shown to be

stable with no significant change in ethyl 4-hydroxybenzoate
concentrations over this period (Figure 11). This is indicated
by <1% changes in area between T = 0 hours and T = 48
hours. Based on these data that show quantitative recovery
through 48 hours, ethyl 4-hydroxybenzoate solutions can be
assayed within 48 hours of preparation.

Stability of Analytical Solutions

System Suitability

Samples and standards should be tested over at least a 48

hour period (depends on intended use), and quantitation of
components should be determined by comparison to freshly
prepared standards. A stability criterion for assay methods is
that sample and standard solutions and the mobile phase will
be stable for 48 hours under defined storage conditions. Stability is considered to be acceptable when the change in the
standard or sample response is within 2% relative to freshly
prepared standards. In this study, the stability of ethyl 4-hydroxybenzoate solutions was investigated. Therefore, test solutions of ethyl 4-hydroxybenzoate were prepared using the
conditions cited in Section 2.4. They were chromatographed
at the beginning, and after 24 and 48 hours. The stability of
ethyl 4-hydroxybenzoate and the mobile phase were calculated by comparing area response and area percent of two
standards at 20 g/ml over time. Standard solutions stored in
a capped volumetric flask on a laboratory bench under nor-

System suitability tests are an integral part of HPLC

methods, and are used to verify that the accuracy and precision of the system are adequate for the analysis to be performed.
Parameters, such as plate count, tailing factor, resolution,
and repeatability (RSD of retention time and area for six repetitions) are determined and compared against the specifications set for the method. The parameter to be measured and
their recommended limits4,9 obtained from the analysis of the
system suitability sample are shown in Figure 8. In the present study, the system suitability test was performed on both
HPLC systems to determine the accuracy and precision of
the system, by injecting ten injections of a solution containing 20 g/ml of ethyl 4-hydroxybenzoate. RSD for peak area
and retention time < 1%, tailing factor (T) < 2 and theoretical plate (N) were > 5000 for both HPLC systems, as can be
seen in Figure 12.

36

I n s t i t u t e o f Va l i d a t i o n Te c h n o l o g y

Ghulam A. Shabir

Conclusion
It is clear from the various guidelines issued by regulatory authorities that analytical methodology should be thoroughly validated under Current Good Manufacturing Practice (cGMP). HPLC assay of active ingredients in pharmaceutical products, and subsequent method validation, can be
complex and time-consuming. However, a well-defined protocol and documented validation plan simplifies and shortens the process, while also providing regulatory agencies
with evidence that the analytical system and method is suitable for its intended use. This paper is intended to provide
guidance on how to perform method validation for HPLC
that generates both useful and meaningful data that meets all
FDA, USP, and ICH validation requirements for pharmaceutical analysis.

Article Acronym Listing

CFR:
cGMP:
FDA:
HPLC:
ICH:
LOD:
LOQ:
PDA:
RSD:
USP:
UV:

Code of Federal Regulations

current Good Manufacturing Practice
Food and Drug Administration
High Performance Liquid Chromatography
International Conference on Harmonization
Limit of Detection
Limit of Quantitation
Photodiode Array
Relative Standard Deviation
United States Pharmacopoeia
Ultra Violet

Acknowledgements

References

I thank Abbott Laboratories and MediSense for permission to publish this article. I would also thank to Dr Nigel
Forrow for his comments on the text.

1. Text on Validation of Analytical Procedures. ICH, Q2A, FDA,

Federal Register, Vol. 60, (March), 1995. p. 11260.
2. Validation of Analytical Procedures: Methodology. ICH, Q2b.
FDA, Federal Register, Vol. 62, (May), 1997. p. 27463.
3. Analytical Procedures and Methods Validation: Chemistry,
Manufacturing and Controls Documentation, FDA, Federal
Register (Notices) 65 (169), August, 2000, p. 52776.
4. Validation of Chromatographic Methods, Reviewer Guidance, Centre for Drug Evaluation and Research, FDA, 1994.
5. Guideline for Submitting Samples and Analytical Data for
Methods Validation. FDA, February 1987.
6. Validation of Compendial Methods. USP 25-NF 20, (1225),
United States Pharmacopeal Convention, Rockville, MD,
2002, p. 2256.
7. Current Good Manufacturing Practice for Finished Pharmaceuticals, Proposed Rules, FDA, HHS, 21 CFR Part 211,
Federal Register, Vol. 61, 87 (May), 1996. p. 20106.
8. Shabir, G.A., Validation of HPLC Methods for Pharmaceutical Analysis: Understanding the Differences and Similarities
between Validation Requirements of the U.S. FDA, USP and
ICH. Journal of Chromatography A. 987 (2003). pp. 57-66.
9. Chromatography. USP 23 (621), United States Pharmacopeal Convention, Rockville, MD, 1994, p. 1776.

About the Author

Ghulam Shabir, BSc (Hons), MSc, FIQA, CIM, is a
Principal Scientist in Analytical R&D at Abbott Laboratories, MediSense Products UK, where he is responsible for analytical methods development and
validation and instrument qualification. He has over
17 years of experience in the pharmaceutical industries, and has received the Technical Excellence
and the Spot Award from Abbott Laboratories. He
has also won the Education Award from the Institute of Manufacturing UK. Mr. Shabir has written for
over 26 industry publications, and his work has
been presented at the worlds premier international
conferences. He is a Fellow of the Institute of Quality Assurance, Companion of the Institute of Manufacturing, and Committee member of the Society of
Chemical Industry Separation Science & Technology UK.
Mr. Shabir can be reached by phone at +44(0) 1993
863099 or by email at [email protected]

Originally published in the May, 2004 issue of the Journal of Validation Technology

Equipment and Instrumentation Qualification

37