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HPLC METHOD VALIDATION FOR PHARMACEUTICALS: A REVIEW

Article · July 2013

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 International Standard Serial Number (ISSN): 2319-8141
International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES
IMPACT FACTOR 1.89***
ICV 2.40***
Pharmaceutical Sciences Review Article……!!!

Received: 15-07-2013; Accepted: 25-07-2013

HPLC METHOD VALIDATION FOR PHARMACEUTICALS: A REVIEW
Harshad V. Paithankar*
MET’s Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra, India- 422003.

KEYWORDS:

ABSTRACT
Validation, HPLC,
Validation of an analytical procedure is to demonstrate that it is
Analytical, ICH, USFDA.
suitable for its intended purpose. Chromatographic methods play
For Correspondence:
significant role in the pharmaceutical industry from the drug
Harshad V. Paithankar*
discovery, development, formulations and quality control. A
Address: MET’s
validated analytical method ensures that it provides consistent,
Institute of Pharmacy,
reliable and accurate data. So these methods help pharmaceutical
Bhujbal Knowledge City,
analyst to ensure quality products are released for market. This
Adgaon, Nashik,
review describes general approach towards validation process and
Maharashtra, India-
validation parameters to be considered during validation of a
422003.
HPLC method. It also refers to various regulatory requirements.
Email:
The parameters described here are according to ICH guidelines
[email protected]
and include accuracy, precision, specificity and limit of detection,
limit of quantitation, linearity, range and robustness.

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 International Standard Serial Number (ISSN): 2319-8141
INTRODUCTION:
In pharmaceutical industry, Validation is an important part of quality control and quality
assurance. Various regulatory authorities give special emphasis on the validation of all the
processes used in the industry. Validation is a formal and systematic way to demonstrate
suitability of the method to provide useful data to ensure that the process or the method gives
satisfactory and consistent results within the scope of the process. The analytical methods refer to
the way of performing the analysis. “Validation is the process by which it is established, by
laboratory studies, that the performance characteristics of the method meet the requirements for
the intended application”[1-3]. All the analytical methods that are intended for analyzing any
sample will need to be validated. The current good manufacturing practices suggest that quality
should be built into the product, and testing alone cannot be relied on to ensure product quality.
Pharmaceutical products need to maintain high quality in order to provide safe and effective usage.
From the analytical point of view, analytical methods used to test these products should have
quality attributes built into them. Validation ensures these quality attributes are built into the
method. Validation of analytical methods is an essential but time consuming activity for most
analytical laboratories. But it results inexpensive, eliminates frustrating repetitions and leads to
better time management in the end. The analytical methods need to be validated or revalidated
before initial use of the method in routine analysis, when transferred from one laboratory to
another, whenever the conditions or method parameters for which the method has been validated
change and change is outside the original scope of the method [4].
Chromatography is defined as a procedure by which solutes are separated by dynamic differential
migration process in a system consisting of two or more mobile phases, one of which moves
continuously in a given direction and in which the individual substances exhibit different
mobilities by reason of differences in absorption, partition, solubility, vapor pressure, molecular
size or ionic charge density [5]. When mobile phase used is liquid the type of chromatography is
called liquid chromatography. High performance liquid chromatography (HPLC) is a modern form
of liquid chromatography that uses small particle columns through which the mobile phase is
pumped at high pressure. The separation of components depends on the extent of interaction
between the solute component and the stationary phase. The component that has lowest affinity for
the stationary phase will elute first. HPLC is becoming a preferred method of analysis among
various analytical methods for pharmaceuticals. HPLC methods provide rapid analysis, greater
sensitivity, high resolution, easy sample recovery, precise and reproducible results [6].
Method validation has received considerable attention from industrial committees and regulatory
agencies. The USFDA have developed guidelines for the analytical method validation.

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 International Standard Serial Number (ISSN): 2319-8141
The United State Pharmacopoeia had developed methodology for specific applications and general
chapters on different analytical aspects of FDA-regulated industry. In USP, chapter <1225> on
“validation of compendial methods” describes parameters to be used for the validation of
analytical methods [7]. Chapter <621> on “chromatography” has useful recommendation on how
GC and HPLC methods can be studied and modified [5]. ICH published two guidelines for method
validation. Q2A describes terminologies and definitions for the various parameters to be
considered for validation [8]. Q2B gives description of the methods to be used for estimating the
validation characteristics. It also provides flexibility in the procedures [9]. The EURACHEM has
published detailed guide as Fitness for Purpose of Analytical Methods. It describes how important
it is for the analytical performance and the analytical problem to be suited [10]. It describes the
importance of method validation and indicates when, how and who should perform validation[11].
1. Validation Process:
Successful validation requires cooperative efforts of several departments of organization including
regulatory affairs, quality control, quality assurance and analytical development. Therefore a well
planned process should be followed during validation. Possible steps for a complete method
validation are listed below:
Steps in method validation [12-13]:
1. Develop a validation protocol or operating procedure for the validation
2. Define the application, purpose and scope of the method
3. Define performance parameters and acceptance criteria
4. Define validation experiments
5. Verify relevant performance characteristics of equipment
6. Qualify materials, e.g. standards and reagents
7. Perform pre-validation experiments
8. Adjust method parameters and acceptance criteria if necessary
9. Perform full validation experiments
10. Develop SOPs for executing method in routine
11. Define criteria for revalidation
12. Define type and frequency of system suitability tests for routine
13. Document validation experiments and results of validation.
1.1. Validation protocol
Validation protocol prepared is a document that indicates the company’s approach for validation
[12. It ensures consistent and efficient execution of validation projects and also answers auditor
during audits. The validation protocol is an ideal tool for training all the employees working for

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 International Standard Serial Number (ISSN): 2319-8141
validation. The validation protocol should include:
1. Introduction: Firms validation policy, general description
2. Organizational structure: Description of all personal responsibilities for all validation
activities
3. Process and product description: Makes a brief description of the process and product or
reference to adequate documents.
4. Specific process considerations: describes critical characteristics of the process.
5. Key acceptance criteria: General statement on acceptance criteria for the process.
6. Documentation format: The format used for protocol and report is described.
7. Required SOPs: a list of relevant SOPs should be mentioned.
8. Planning and Scheduling: describes the resources, equipments and chemicals to be used,
including time plan of the project.
9. Change control: includes description or reference to the critical parameters variations in the
process or product.
For each individual validation project a project plan should be developed. It outlines what is to be
done in order to get a specific method or procedure validated. The plan should be include a time
table with specific tasks, deliverables and owners.
1.2. Revalidation
Revalidation is necessary whenever a method is changed and the new parameter is outside the
operating range. The operating parameters need to be specified with ranges clearly defined. In case
of methods for quantitation of impurities, if a new impurity is found that makes the method
deficient in its specificity, it needs modification and revalidation. Changes in equipment or
chemical quality may also have critical effects on method. So any such change needs revalidation.
2. Validation Parameters
The analytical methods which need to be validated are classified as per ICH are classified as
following [8]:
Identification tests: To ensure identity of an analyte.
Quantitative test for impurities: to accurately and quantitatively reflect the purity of a sample
Limit test for impurities: to reflect purity characteristics of the sample
Assay of drug substance and drug products: to measure accurately and quantitatively the analyte
present in the sample. These methods also include analysis for content uniformity and
measurement of analyte from dissolution samples.
The characteristics which need to be validated for the different types of method are also mentioned
in ICH guidelines [8].

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 International Standard Serial Number (ISSN): 2319-8141
These are tabulated below in table 1.
Table 1: ICH characteristics
Characteristics Test for impurities
Identification Assay
Quantitative Limit test

Accuracy X √ X √
Precision
Repeatability X √ X √
Intermediate precision X √ X √
Specificity √ √ √ √
Detection limit X X √ X
Quantitation limit X √ X X
Linearity X √ X √
Range X √ X √

√ indicate this need to be evaluated

x indicate this need not to be evaluated
The united state pharmacopoeia (USP) has classified these methods into four categories and also
specifies which parameters to be considered for validation of different types of methods [7].
Category I: Analytical methods for quantitation of measurement of bulk drug substances or active
ingredients including preservatives in finished pharmaceutical products.
Category II: Analytical methods for determination of impurities in bulk drugs or for the
determination of degradation compounds in finished pharmaceutical products.
Category III: Analytical methods for the determination of performance characteristics (e.g.
dissolution, drug release).
Category IV: identification tests.
Table 2: characteristics required for the validation as per USP
Analytical performance Category I Category II Category Category
characteristics Quantitative Limit test III IV
Accuracy √ √ * *
Precision √ √ √
Specificity √ √ √ * √
Limit of detection √ *
Limit of quantitation √ *
Linearity √ √ *
Range √ √ * *
Ruggedness √ √ √

√ indicates the parameter need to be considered

* indicates parameter may be considered depending on the nature of the test.
The parameters for validation need to be selected as per the regulatory requirements. The
parameters considered in chromatographic method validation are discussed below.
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 International Standard Serial Number (ISSN): 2319-8141
2.1. Selectivity and Specificity
Selectivity of the analytical method is defined as the degree to which a method can quantify the
analyte in the presence of inerferents [14]. The other components which may be present include
impurities, degradants, matrix, etc. The term specificity and selectivity is often used
interchangeably. The term specific generally refers to a method that produces a response for a
single analyte only, while the term selective refers to a method that provides responses for a
number of chemical entities that may or may not be distinguished from each other. The
International Union of Pure and Applied Chemistry (IUPAC) have expressed the view that
“Specificity is the ultimate of Selectivity’. The IUPAC discourages use of the term specificity and
instead encourages the use of the term selectivity [15].
Specificity study of the chromatographic method is performed by the separation of the analyte
from the other potential components such as impurities, degradants or excipients etc. In addition
forced degradation studies are carried out to challenge the method. The forced degradation studies
are of particular importance when the impurities are not available. During forced degradation
studies, the sample is subjected to the stressed conditions of light, heat, humidity, acid/base
hydrolysis and oxidation. The scheme which is generally used for forced degradation studies for
drug substances and drug products are summarized in table 3 below [15]. The selectivity of
chromatographic methods may be assessed by examination of peak homogeneity or peak purity
test. Peak purity test shows that there is no co-elution of any sample component. For this, peak
purity assessment is done by using PDA or MS detectors. Representative chromatograms with
peaks labeled should be included with resolution, plate count and tailing factor reported in the
validation report.
Table 3: Table showing different forced degradation conditions to be used for drug substances and
drug products
Sample Forced degradation study
Drug substances
Solid Photolytic, thermal, humidity
Solution/suspension Acid/Base hydrolysis, oxidative
Drug products
Solid Photolytic, oxidative, thermal, humidity
Semisolid Photolytic, oxidative, thermal, humidity
Solution/suspension Photolytic, thermal, oxidative, hydrolysis
2.2. Linearity
Linearity of a method is its ability to obtain test results that are directly proportional to the sample
concentration over a given range. For HPLC methods, the linear relationship between detector

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response (peak area and height) and sample concentration is determined. The relationship can be
demonstrated directly on drug substance by dilution of standard stock or by separate weighing of
the sample components, using the proposed procedures.
Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte
concentration or content. If there is linear relationship, test results should be evaluated by
appropriate statistical methods, for example, by regression analysis. Data from the regression line
is helpful to provide mathematical estimates of the degree of linearity. It is generally expressed in
terms of variance around the slope of regression line. In some cases, the analytical responses
should be described by the appropriate function of the analyte concentration. The widely used
linearity ranges and acceptance criteria for various pharmaceutical methods are listed in the table 4
[16].
Table 4: Linearity ranges and Acceptance criteria for various pharmaceutical methods
Test Linearity levels and ranges Acceptance criteria
Five levels, Correlation coefficient,
Assay
50-150% of label claim R ≥0.999
Five to eight levels, % y intercept NMT 2.0%
Dissolution
10-150% of label claim R ≥0.99
Five levels, % y intercept NMT 5.0%
Related substances
LOQ to acceptance criteria R ≥0.99

2.3. Precision
Precision of an analytical method expresses the closeness of agreement between a series of
measurements obtained from multiple sampling of the same homogeneous sample under the
prescribed conditions. Precision may be considered at three levels: repeatability, intermediate
precision and reproducibility.
Repeatability is the precision under the same operating conditions over a short interval of time. It
is also termed as intra-assay precision. It is assessed by making six sample determinations at 100%
concentration or by preparing three samples at three concentrations in triplicates covering the
specified range for the procedure. It involves repeated determination of same sample.
Intermediate precision expresses within laboratories variation: different days, different analyst,
different equipments, etc. It is the term synonymous with the term ‘ruggedness’, defined by USP.
The extent to which intermediate precision should be established depends on the circumstances
under which the procedure is intended to be used. To study intermediate precision, use of an
experimental design is encouraged. The intermediate precision is generally studied by multiple
preparations of sample and standard solution.

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Reproducibility is the precision obtained by analysis between laboratories. It is generally assessed
during collaborative studies at the time of technology or method transfer. It is assessed by means
of an inter-laboratory trial.
The precision data is generally expressed in the form of standard deviation, relative standard
deviation and confidence intervals. To ensure precision of method for major analytes, RSD should
be ≤ 2 %. For low level impurities, RSD of 5-10 % is usually acceptable [17].
2.4. Range
Range of an analytical method is the interval between the upper and lower concentration of analyte
in the sample (including these concentrations) for which it has been demonstrated that the
analytical procedure has a suitable level of precision, accuracy and linearity. The range is normally
derived from the linearity studies and depends on the intended application of the procedure. The
following minimum specified ranges should be considered [10]:
• For the assay method, normally covering from 80 to 120 percent of the test
concentration.
• For content uniformity, covering minimum of 70 to 130 percent of the test
concentration, based on the nature of the dosage form.
• For dissolution testing, ±20 % over the specified range.
• For impurity determination, from reporting level of impurity to 120 % of the
specification.
The range of a method is confirmed when linearity, accuracy and precision criteria are fulfilled
[2].
2.5. Accuracy
The accuracy of an analytical method expresses the closeness of agreement between the value
accepted either as a conventional true value or an accepted reference value and the value found.
Practically no measurement process is ideal, therefore, the true or actual value cannot be exactly
known in any particular measurement. The accepted true value for accuracy assessment can be
assessed by analyzing a sample with known concentration. The accuracy studies are usually
carried out by determining the recovery of the spiked sample of analyte into the matrix of the
sample (a placebo) or by comparing the result to the results of a certified reference material of
known purity. If the placebo of the sample is not available, the technique of standard addition is
used. In case of methods for quantitation of impurities, the sample with known amount of
impurities is assessed. Accuracy should be assessed using minimum of nine determinations over a
minimum of three concentration levels covering the specified range (for e.g., three concentrations/
three replicates each of the total analytical procedure).

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Accuracy should be reported as percent recovery by the assay of known added amount of analyte
in the sample or as the difference between the means and the accepted true value together with the
confidence intervals. The concentration should cover the range of concern. The expected recovery
depends on the sample matrix, the sample processing procedure, and the analyte concentration.
The reported limits for accuracy for drug substances and products are 98.0 – 102.0 % and 97.0 –
103.0 % respectively. For the impurity determination, range from 50 - 150 % of average recovery
may be accepted [2].
2.6. Limit of Detection
The limit of detection of an individual analytical procedure is the lowest amount of analyte in the
sample which can be detected but not necessarily quantified as an exact value. The detection limit
can be determined in different ways.
The simplest approach is based on the signal to noise ratio. The signal to noise ratio is determined
by comparing measured signals from samples with known low concentration of analyte with those
of blank samples. The concentration showing signal to noise ratio between 3:1 or 2:1 is generally
considered as acceptable detection limit.
The other approach is based on the standard deviation of the response and the slope. The detection
limit may be expressed as:
LOD =

Where, σ = the standard deviation of the response

S = the slope of the calibration curve
The slope may be estimated from the calibration curve of the analyte. The σ can be estimated as
the standard deviation of the blank. The value of σ can also be estimated based on the calibration
curve. For this the specific calibration curve should be studied using sample containing analyte in
the range of detection limit. The residual standard deviation of a regression line or the standard
deviation of the y-intercept of regression lines may be used as standard deviation.
Another approach for the estimation of the detection limit is base on visual evaluation. This
method is applicable to non-instrumental methods but may be applied to the instrumental methods.
The LOD is determined by the analysis of samples with known concentrations of analyte and by
establishing the minimum level at which the analyte can be reliably detected. The relevant
chromatograms are sufficient for the justification of the detection limit.
2.7. Limit of Quantitation
The Quantitation limit of an individual analytical procedure is the lowest amount of analyte in the
sample which can be quantitatively determined with suitable precision and accuracy.

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It is mainly affected by the detector sensitivity and accuracy of sample preparation. The
Quantitation limit can be determined in the similar way as that of the detection limit. It is the
concentration showing signal to noise ratio of 10:1. Based on the standard deviation of the
response and the slope it is calculated by the formula:

LOQ =

Where, σ = the standard deviation of the response

S = the slope of the calibration curve
The value of S and σ are estimated as for the detection limit.
The LOQ can also be established from the visual evaluation as the LOD. The analyte
concentration should be quantifiable with acceptable accuracy and precision at LOQ level. Typical
acceptance criteria for LOQ are mean recovery at this level between 50 – 150 % with % RSD of ≤
25 %.
2.8. Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters and provides an indication of its reliability
during normal usage. It is partially evaluated during method development stages. The aim of the
robustness study is to identify the critical operating parameters for the successful implementation
of the method. These parameters should be adequately controlled and a precautionary statement
included in the method documentation. In case of an HPLC method, robustness study involves
method parameters like pH, flow rate, column temperature and mobile phase composition which
are varied within a reasonable range. The system suitability parameters obtained for each condition
are studied to check the parameter which significantly affects the method.
Stability of the analytical solution and extraction time are other parameters which are also
evaluated as additional parameters during robustness study. Stability of analytical solution is
determined by assessing the results obtained by subjecting the analytical solution to the method
parameters for longer period of time e.g. 4 hrs, 12 hrs, 24 hrs, 48 hrs, etc. The acceptance criteria
are based on relative difference between initial value and the value at specified solution stability
time. For drug substances and products difference should be ≤ 2.0 % and for impurity
determination, it should be ≤ 10 %.2
When filtration is done during sample preparation filter paper study can be carried out. It involve
analysis by filtering sample solution through different types of filter paper.

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 International Standard Serial Number (ISSN): 2319-8141
2.9. System suitability
System suitability testing (SST) is an integral part of many analytical procedures. The tests are
based on the concept that the equipment, analytical operations and samples are the integral part of
the system that can be evaluated as such. System suitability test provide the added assurance that
on a specific occasion the method is giving, accurate and precise results. System suitability test are
run every time a method is used either before or during analysis. The results of each system
suitability test are compared with defined acceptance criteria and if they pass, the method is
deemed satisfactory on that occasion. In case of HPLC methods, system suitability tests ensure the
adequacy for performing the intended application on daily basis. The primary SST parameters
considered are resolution (Rs), repeatability (% RSD of peak response and retention time), column
efficiency (N), and tailing factor (Tf). The other SST parameters include retention factor (k) and
separation factor (α). The limits which are considered for the SST parameters are listed table 5
[17].
Table 5: Limits for system suitability tests

SST Limits
Resolution (Rs) >2.0

Repeatability (RSD) <1.0% for five replicates

Plate count (N) >2000

Tailing factor (Tf) ≤2.0

Separation factor (α) >1.0

3. Conclusion
The growing pharmaceutical industry demands various analytical methods for various
pharmaceutical products. To ensure quality of the product, it is necessary that the analytical
method used for assuring quality should give accurate and predictable results. For this the method
need to be validated. The HPLC methods are the preferred methods of analysis due to their
responsiveness. This review guides analysts to validate chromatographic methods in order to
comply with regulatory requirements.
4. REFERENCES
1. Chan C. C. (2008), Analytical Method Validation: Principles and Practices, in
Pharmaceutical Manufacturing Handbook: Regulations and Quality. Edited by Shyne Cox
Gad. John Wily and Sons.
2. Jimidar M. I., Heylen P. and Smet M. D. (2007), Method Validation in HPLC Method
Development for pharmaceuticals. Edited by Satinder Ahuja and Henrik Rasmussen.
Academic Press. London.
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3. USFDA. Draft Guidance for Industry: Analytical Procedures and Method Validation.
Center for Drug Evaluation and Research. Rockville, MD.
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