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Method development and validation: Skills and tricks

Article  in  Chronicles of Young Scientists · March 2012

DOI: 10.4103/2229-5186.94303

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Review Article

Method development and validation: Skills and tricks

Abstract

Because of the introduction of a lot of drugs in the market every year, it becomes necessary to develop newer
analytical methods for such drugs. Method development can take a number of forms. At one extreme, it involves
adapting an existing method or making minor changes so as to make it suitable for the new application, as
for developing a method for the estimation of drugs using a complex analytical technique like HPLC. After the
development, there is a need of method validation. Method validation is defined as the process of proving that
an analytical technique is acceptable for the intended use and this is an important requirement for analytical
purpose. Validation is done according to the guidelines of ICH and FDA. Here, in this review, we have discussed
method development and the various parameters used for method validation, namely accuracy, precision, limit
of detection, limit of quantification, specificity, robustness, ruggedness, and range.

Key words:
Analytical, FDA, high performance liquid chromatography, ICH

Introduction every aspect of society, and there are innumerable reasons

for making these measurements. Clearly, it is important to
In industries, new measurement technologies can determine the correct result and be able to show that it is
only be adopted if a sound scientific rationale for the correct. Therefore, a method validation is required.[2] For
application has been developed, proven, and justified e.g., the expanding use of innovative botanical ingredients
and the developed method has been approved by internal in dietary supplements and foods has resulted in a flurry
company procedures.[1] A number of drugs are being of research aimed at the development and validation of
introduced in the market every year, and these are either analytical methods for the accurate measurement of active
new drugs or the modification of the existing moieties. ingredients.[4]
Because of the possible uncertainties in the continuous
use of these drugs, appearance of some new toxicities and Method Development
patient resistance or introduction of some better drug
leads to a time lag from the date of introduction of drug in Method validation often evolves from method
the market to the date of its inclusion in pharmacopoeias. development. Method development can take a number
So there is a need to develop and validate newer analytical of forms.[5] At one extreme, it may involve adapting an
techniques for such drugs as it may not be available in existing method, making minor changes so that it is
pharmacopoeias.[2] suitable for a new application.[6] It requires a lot of effort,
and there is a degree of doubt initially to whether the
It is internationally recognized that a developed method method will be successful. It involves working on various
should necessarily be validated as these validation methods ideas simultaneously and then finally picking one of
also show the qualification and competency of the analytical those. Various steps involved in method development and
laboratory.[3] Analytical measurements are associated with validation are:[7]

Access this article online

Quick Response Code Ashok Kumar, Lalit Kishore, Navpreet Kaur, Anroop Nair
Website: M. M. College of Pharmacy, M. M. University,
http://www.cysonline.org Mullana, Ambala, Haryana, India
Address for correspondence:
Dr. Ashok Kumar,
DOI:
M. M. College of Pharmacy,
10.4103/2229-5186.94303 Mullana, Ambala, Haryana, India.
E-mail: [email protected]

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Kumar, et al.: Method development and validation

Method development plan definition monobasic: acetonitrile:40% tetrabutylammonium

↓ hydroxide (90:10:0.2).[9] Another example is reverse phase
Background information gathering HPLC for the estimation of ezitimibe in its tablet dosage
↓ form,[10] a method for this drug was developed and validated.
Laboratory method development Reverse phase HPLC has been used extensively for various
↓ drug combinations such as cefixime and dicloxacilin,
Generation of test procedure ropinirole hydrochloride, paracetamol and tramadol,
↓ ofloxacin and ornidazole, and so on.[11-14]
Methods validation protocol definition
↓ Problems in method development
Laboratory methods validation 1. Stored samples are initially accurate but slowly become
↓ inaccurate with low bias
Validated test method generation 2. Absorption issue: A serially diluted curve is concave. The
↓ response factors drop with decreasing concentration.
Validation report An increased exposure due to number of dilutions,
surface area contact, and time may cause this problem
A well-developed method is always to validate.[7] While 3. Homogeneity: the sample to be analysed gets partitioned.
using any analytical technique for the estimation of the
drug it needs a proper method to be developed. Let us take These problems can be overcome by adding a surfactant to
an example of a most complex analytical technique, i.e., the sample under test.
high performance liquid chromatography (HPLC) which
is complex in the sense that there are a wide variety of Method Validation
equipments, columns, eluents, and other parameters for
operation which makes it so. There are various aspects The word validation originated from the Latin word validus
which should be kept in mind while developing a method for meaning strong, and suggests that something has been
HPLC.[8] They are explained as follows: proved to be true, useful, and of an acceptable standard.[15]
• Selection of the HPLC method which includes choosing Method validation can be defined as the process of proving
either of the two, reverse phase or normal phase HPLC that a particular developed analytical method is acceptable
depending upon the nature of the sample, for example, for its intended use.[7,16-19] Validation is an important
for polar analytes we use reverse phase HPLC so that requirement in the practice of an analytical process. Method
we obtain better retention and resolution and for low validation can be interpreted as the process of defining an
or medium polarity samples we generally prefer normal analytical requirement, and confirming that the method
phase chromatography under consideration has performance capabilities consistent
• Then, choosing a proper mobile phase for the given with that the application requires.[5] In connection with
analyte is the most crucial stage in developing a method biotechnological synthesis of pharmaceutical drugs, validated
for HPLC. A mobile phase which has the capability of methods for quantification of both the product and the
pulling the analyte from the column is chosen. When substrate at different time intervals are essential for proper
dealing with weak acids and bases, we have to adjust the calculation of rate coefficients.[20] The current trend is in the
pH also as it affects the retention direction of phase-dependent methods development and
• A stationary phase is generally C18 bonded in the case validation. Analytical methods are progressively optimized,
of reverse phase HPLC and cyano-bonded in the normal and a preliminary validation package is furnished as part of
phase the IND application before Phase I safety trials are initiated.
• Then, the detectors are selected based on the nature All analytical methods should be fully optimized and
of the analyte. We observe that whether it has validation should be completed before the NDA is submitted
chromophores which will enable their detection in UV at the end of Phase III studies.[7,21] Method validation is a
while using UV-detectors. Fluorescence detectors are continuous process, and the final goal of validation of an
used in the case of trace analysis and in preparative analytical method is to ensure that every future measurement
HPLC refractive index detectors are used. in routine analysis will be close enough to the unknown
true value for the content of the analyte in the sample.[22,23]
Therefore, these criteria are kept in mind while developing a Because of the ongoing advances in analytical chemistry
method for HPLC and this developed method is further being technologies, analytical methods are updated over time; thus,
validated using various parameters which are defined later in validation and cross validation of methods become important
this review; for example, development and validation for the for accurate interpretation of data collected over years.[24-29]
determination of the level of EDTA in nonalcoholic drinks
using HPLC. In this a reverse phase C18 column was used Validation is performed with a formal, approved, and signed
and the column was eluted in 0.01 M ammonium phosphate methods validation protocol in quality assurance (QA) unit.

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Kumar, et al.: Method development and validation

Validation is complete when we: behavior, chemical reactivity, etc.) to that of a reference
1. Demonstrate that you have met all the acceptance standard. Testing for impurities can be either a quantitative
criteria test or a limit test for the impurity in a sample. The test is
2. Clearly document the results in a cGMP compliant intended to reflect the purity characteristics of the sample.
fashion Different validation characteristics are required for a
3. Show how you met the acceptance criteria in a final quantitative test than for a limit test. Assay procedures are
methods validation report, including references intended to measure the analyte present in a given sample.
to raw data, all of which have been reviewed and In the perspective of this document, the assay represents
approved by the appropriate personnel including peers, a quantitative measurement of the major component(s) in
management, and QA. the drug substance.[33]

Validation guidelines Steps in the methods validation process

1. ICH Q2A text on validation of analytical procedures: Method evaluation and further method development
definitions and terminology (March 1995)[30] ↓
2. ICH Q2B validation of analytical procedures: Final method development and trial method
methodology (June 1997) validation
3. FDA (Draft) guidance for industry: analytical procedures ↓
and methods validation Formal method validation and report generation
4. Pharmacopoeias USP and European Pharmacopoeia. ↓
Formal data review and report issuance
Why is analytical method validation required?
Method validation is required for the following reasons:[19] Total time for method evaluation, validation, data/
1. For assuring the quality of the product documentation review, and reporting is approximately 6–10
2. For achieving the acceptance of the products by the months.[43]
international agencies
3. It is a mandatory requirement for accreditation as per Criteria that must be satisfied by a validation process
ISO 17025 guidelines 1. The whole method must be validated. It is quite usual
4. A mandatory requirement for registration of any to focus on the detection technique or the instrumental
pharmaceutical product or pesticide formulation. measurement, which often means that just this stage
is validated. However, the previous steps of sample
Validated methods are only acceptable for undertaking pretreatment, extraction, or preconcentration also
proficiency testing. belong to the method of analysis and are of utmost
importance. Therefore, they must all be validated
Validation not only improves the processes, but also confirms 2. The whole range of concentrations must be validated.
that the process is properly developed. For the manufacturer It is difficult to comply with this condition because
method validation is important in the following aspects: a method may work very well in one particular
• It deepens the understanding of processes and decreases concentration range but not in others
the risk of preventing problems 3. The whole range of matrices must be validated. It
• It decreases the risk of defect costs is well known that the matrix can have a decisive
• It decreases the risk of regulatory noncompliance effect on the analysis. Therefore, and for the sake of
• A fully validated process may require less in-process representativeness, several matrices must be submitted
controls and end product testing.[31,32] to method validation.[35]

Types of analytical procedures to be validated Performance characteristics for method validation

Discussion of the validation of analytical procedures is • Accuracy
directed to the four most common types of analytical • Precision
procedures: • Specificity
1. Identification tests • Limit of detection
2. Quantitative tests for impurities content • Limit of quantification
3. Limit tests for the control of impurities • Linearity
4. Quantitative tests of the active moiety in samples of a • Range
drug substance. • Robustness
• Ruggedness
Identification tests are intended to ensure the identity of
an analyte in a sample. This is achieved by comparison of Accuracy
a property of the sample (e.g., spectrum, chromatographic It is the closeness to the true value, measured by % recovery

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Kumar, et al.: Method development and validation

of sample spikes or % error in the analysis of a reference three categories: repeatability, intermediate precision,
sample.[36] Accuracy is normally studied as two components: and reproducibility.[42] Repeatability is obtained when one
‘trueness’ and ‘precision’.[6,37-41] The ‘trueness’ (of a method) operator using one piece of equipment over a relatively
is an expression of how close the mean of a set of results short time-span carries out the analysis in one laboratory.
(produced by the method) is to the true value. Trueness is At least five or six determinations at two or three different
normally expressed in terms of bias. concentrations should be done and the RSD calculated. [19]
Precision is usually stated in terms of standard deviation
Trueness or RSD. Both repeatability and reproducibility are generally
Two techniques are available and if possible both should be dependent on analyte concentration, and so should
performed. be determined at a number of concentrations and if
a. Method comparison: If you are introducing a new relevant, the relationship between precision and analyte
method into the department, which has already been concentration should be established.[5]
validated, compare the results from the two methods for • Repeatability: Repeatability expresses the precision
the same samples. It is recommended that a minimum under the same operating conditions over a short
of 10 samples are compared; however, more the results interval of time.[42] From the repeatability standard
better the comparison. If an analyser is being used deviation or sr it is useful to calculate the ‘repeatability
then this number should be at least 30 samples. Use a limit ‘r’’, which enables the analyst to decide whether
statistical package to compare the two methods using the difference between duplicate analyses of a
linear regression, thereby calculating any bias[5] sample, determined under repeatability conditions, is
b. Reference comparison: Obtain reference material from significant[6]
the relevant External Quality Assessment scheme along • Reproducibility: Reproducibility expresses the precision
with the statistical results showing all method mean, or between laboratories.[42] From the reproducibility
if available a certified reference material. Run the EQA standard deviation or sr it is useful to calculate the
samples as many times as possible (depending on available ‘reproducibility limit R’, which enables the analyst
sample) up to a maximum of 10 and determine the mean to decide whether the difference between duplicate
and standard deviation of these replicate tests. Compare analyses of a sample, determined under reproducibility
the results with the method mean obtained nationally.[5] conditions, is significant. These calculations can be
performed directly with the built-in statistics function
Accuracy should be established across a specified range of of the instrument, if available, or by using a pocket
analytical procedure. It should be assessed using a minimum calculator or a Personal Computer (PC) with a suitable
of three concentration levels, each in triplicate (total of nine software package (e.g. spreadsheet program).[6]
determinations). Results of the accuracy study should be
reported as: Specificity
• Percent recovery of the known amount added or It is the ability to measure the desired analyte in a complex
• The difference between the mean assay result and the mixture.[7] It is the degree of bias (or lack thereof) caused by
accepted value. expected sample components and common interferences,
determined by measuring the analyte with and without
A set of data showing the accuracy study is given in Table 1. anticipated interferences.[36] For example, it has been
Data give the information about the amount of analyte recommended that when developing an analysis for a drug
added and the percentage recovery of the same. in blood or plasma, that at least six independent sources of
blank matrix be tested for interferences.[36,43] It is not always
Precision possible to demonstrate that an analytical procedure is
The degree of agreement between replicate analyses of specific for a particular analyte (complete discrimination).
a homogenous sample, usually measured as the relative In this case, a combination of two or more analytical
standard deviation (RSD) of a set of replicates.[36] The procedures is recommended to achieve the necessary level
measured standard deviation can be subdivided into of discrimination.[42]

Table 1: Recovery studies for the determination of an analyte in tablet dosage form by initial rate method
Formulations Initial rate method
Amount taken Amount added Amount found % Recovery % RSD
(μg/ml) (μg/ml) (μg/ml)±SD
Analyte 5 4 9.01±0.018 100.46 0.200
5 9.98±0.057 99.68 0.571
6 11.08±0.016 101.38 0.144
Values are mean±SD for 3 determinations

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Kumar, et al.: Method development and validation

Specificity relates to the ability of the test to identify can be determined with acceptable precision [usually relative
negative results. Consider the example of the medical test standard deviation (RSD) < 10–25%] and accuracy.[6,36,42,44,45]
used to identify a disease. The specificity of a test is defined Several approaches for determining the quantitation limit
as the proportion of patients who do not have the disease (QL) are possible, depending on whether the procedure is
who will test negative for it. This can also be written as: a noninstrumental or instrumental. Approaches other than
those listed below may be acceptable.[42]
number of truenegatives • Limit of quantification is evaluated based upon the
specificity =
number of truenegatives + visual evaluation which is a type of noninstrumental
number off alse positives method. Using this method, QL is determined by the
analysis of the samples with known concentrations of
If a test has high specificity, a positive result from the test analyte and by establishing the minimum level at which
means a high probability of the presence of disease. the analyte can be quantified with acceptable accuracy
and precision
Limit of detection • Based on the signal-to-noise approach, this approach
The limit of detection (LOD) is the lowest concentration can only be applied to analytical procedures that exhibit
of the analyte in a sample that can be detected but baseline noise. Determination of the signal-to-noise
not necessarily quantified.[6,19,42] Several approaches ratio is performed by comparing measured signals from
for determining the detection limit (DL) are possible, samples with known low concentrations of the analyte
depending on whether the procedure is a noninstrumental with those of blank samples and by establishing the
or instrumental. Approaches other than those listed below minimum concentration at which the analyte can be
may be acceptable.[42] reliably quantified. A typical signal-to-noise ratio is 10:1
• Noninstrumental methods are based on the visual • The QL may be expressed as: QL = 10σ/S, where σ is the
evaluation. The DL is determined by the analysis of standard deviation of the response, S is the slope of the
samples with known concentrations of the analyte and calibration curve, the slope S may be estimated from the
by establishing the minimum level at which the analyte calibration curve of the analyte. The estimate of σ may
can be reliably detected be carried out in a variety of ways including:
• Based on signal-to-noise, this approach can only be (i)  Measurement of the magnitude of analytical
applied to analytical procedures which exhibit baseline background response is performed by analysing
noise. Determination of the signal-to-noise ratio is an appropriate number of blank samples and
performed by comparing measured signals from samples calculating the standard deviation of these
with known low concentrations of the analyte with responses
those of blank samples and establishing the minimum (ii)  Based on the calibration curve, a specific calibration
concentration at which the analyte can be reliably curve should be studied using samples, containing
detected. A signal-to-noise ratio between 3 or 2:1 is an analyte in the range of QL. The residual standard
generally considered acceptable for estimating the DL deviation of a regression line or the standard
• The DL may be expressed as: DL = 3.3σ/S, where σσ is deviation of y-intercepts of regression lines may be
the standard deviation of the response, S is the slope of used as the standard deviation.[42]
the calibration curve, the slope S may be estimated from
the calibration curve of the analyte. The estimation of σ Linearity
may be carried out in a variety of ways, for example: The linearity of an analytical method is its ability to
(i)  Based on the standard deviation of the response, elicit  test results that are (directly or by means of well-
the blank measurement of the magnitude of defined mathematical transformations) proportional to the
analytical background response is performed by concentration of analytes in samples within a given range
analysing an appropriate number of blank samples or proportional by means of well-defined mathematical
and calculating the standard deviation of these transformations.[19,46,47] Linearity may be demonstrated
responses directly on the test substance by preparing a series of dilution
(ii)  When determining the data from a calibration of a standard stock solution or by using separate weighing of
curve, a specific calibration curve should be studied synthetic mixtures of the test product components, using the
using samples containing an analyte in the range of proposed procedure.[19,42,48] Test results should be evaluated by
DL. The residual standard deviation of a regression appropriate statistical methods, for example, by calculation
line or the standard deviation of y-intercepts of a regression line by the method of least squares.[42]
of regression lines may be used as the standard
deviation.[42] Acceptability of linearity data is often judged by examining
the correlation coefficient and y-intercept of the linear
Limit of quantification regression line for the response versus concentration plot.
It is the concentration level above which the concentration A correlation coefficient of >0.999 is generally considered

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Kumar, et al.: Method development and validation

as evidence of acceptable fit of the data to the regression 2

line. The y-intercept should be less than a few percent of the y = 0.0916x - 0.0057
response obtained for the analyte at the target level. Figure 1 R² = 0.9996
shows the linearity of an analyte over a concentration range 1.5
of 2–20 mg/ml with a correlation coefficient of 0.999.[49]
1
Aspects
• Test across the range (at least five concentrations)[30]
• Evaluate linearity by visual inspection of the plot and by 0.5
statistical techniques Series 1
• Calculate corr. coefficient, y-intercept, slope, and res.
sum of squares. 0
0 5 10 15 20 25
Range
-0.5
It is the concentration, upper and lower levels, which
meets the linearity, precision, and accuracy performance Figure 1: Figure shows the linearity of an analyte over a
characteristics.[36,42] The range is normally expressed in the concentration range of 2-20 µg/ml with a correlation coefficient
same units as the test results (e.g., percentage, parts per of 0.999
million) obtained by the analytical method.[19]
of a method’s robustness, a number of method parameters, for
The following minimum specified ranges should be example, pH, flow rate, column temperature, injection volume,
considered: [42] detection wavelength, or mobile phase composition, are varied
• for the assay of an active substance or a finished product: within a realistic range, and the quantitative influence of the
normally from 80 to 120% of the test concentration; variables is determined. If the influence of the parameter is
• for content uniformity, covering a minimum of 70– within a previously specified tolerance, the parameter is said
130% of the test; to be within the method’s robustness range.[19,51]
• concentration, unless a wider more appropriate range,
based on the nature of the dosage form (e.g., metered Ruggedness
dose inhalers), is justified; Ruggedness is normally evaluated during method
• for dissolution testing: ±20% over the specified range; development, typically by the originating laboratory, before
e.g., if the specifications for a controlled released product collaborating with other laboratories and is a measure how
cover a region from 20%, after 1 h, up to 90%, after 24 h, well a method stands up to less than perfect implementation.
the validated range would be 0–110% of the label claim. In any method there will be certain stages, which, if not
• for the determination of an impurity: from the reporting carried out with sufficient care, will have a severe effect
level of an impurity 1–120% of the specification; for on method performance, and may even result in complete
impurities known to be unusually potent or to produce loss of the activity of the method. These stages should be
toxic or unexpected pharmacological effects, the identified, usually as a part of method development, and if
detection/quantitation limit should be commensurate possible, their influence on method performance evaluated
with the level at which the impurities must be controlled. using ‘ruggedness tests’.[6] It is the variance in the analysis
of homogenous samples between analysts and laboratories.
To demonstrate an acceptable linear range, it is generally Ruggedness is a measure of reproducibility of test results
suggested to prepare five different standard solutions from under normal, expected operational conditions from
50% to 150% of the target analytical concentration.[15,47] laboratory to laboratory and from analyst to analyst.[19,51,52]

Note: for validation of impurity test procedures carried out As we discussed method development of HPLC, after
during development, it may be necessary to consider the developing a method it needs proper validation and all
range around a suggested (probable) limit; if assay and purity the above-mentioned parameters are studied for these
are performed together as one test and only a 100% standard developed methods.
is used, linearity should cover the range from the reporting
level of the impurities 1–120% of the assay specification.[42] Validation characteristics and requirements given by
WHO
Robustness There are a number of analytical methods used for the
The robustness of an analytical procedure is a measure of examination of pharmaceutical materials. Not all the
its capacity to remain unaffected by small, but deliberate characteristics referred above will need to be considered in
variations in method parameters and provides an indication of all cases. Analytical methods may be broadly classified as per
its reliability during normal usage.[36,42,50] For the determination WHO as follows:

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Kumar, et al.: Method development and validation

• Class A: Tests designed to establish identity, whether of Characteristics required to be validated according to USP are
bulk drug substances or of a particular ingredient in a given in Table 3.
finished dosage form
• Class B: Methods designed to detect and quantitative Revalidation
impurities in a bulk drug substance or finished dosage Revalidation means the repetition of the validation process.
form A validation status once reached is not static, but is subject
• Class C: Methods used to determine quantitatively the to everyday dynamics. A revalidation is necessary whenever
concentration of a bulk drug substance or of a major a method is changed and the new parameter is outside the
ingredient in a finished dosage form operating range. Processes and procedures should undergo
• Class D: Methods used to assess the characteristic of periodic critical re-evaluation to ensure that they remain
finished dosage forms, such as dissolution profiles and capable of achieving the intended results. As in industrial
content uniformity. manufacturing, process validation is carried out on the
basis of approved manufacturing instructions. If at a later
For all these classes given by WHO, characteristics stage, these manufacturing instructions are intended to be
that should be considered for them are given in changed, it is necessary to check, document, and possibly
Table 2.[53] show through revalidation that this change has no effect on
the product quality. For example, if ingredients such as APIs
Classification of analytical methods as per USP or critical excipients are changed or even their percentage
• Category I: Analytical methods for quantitation of quantity is changed, the original validation results are no
of major components of bulk drug substances or longer relevant.[54] Another example is that if the operating
active ingredients including preservatives in finished range of column temperature is changed from the specified
pharmaceutical products one, i.e., 35–40°C to 42°C then the method is required to be
• Category II: Analytical methods for determination revalidated.
of impurities in bulk drugs or for determination of
degradation compounds in finished pharmaceutical Conclusion
products
• Category III: Analytical methods for determination Method development and validation play an important role
of performance characteristics (e.g., dissolution, drug in the pharmaceutical industry. Methods resulted are used
release) in the QA of the drug entity. Validating a developed method
• Category IV: Identification tests.[51] is important as it is meaningless if the method cannot be

Table 2: Characteristic that should be considered for different types of analytical procedure
Parameters Class A Class B Class C Class D
Quantitative tests Limit tests
Accuracy – Yes – Yes Yes
Precision Yes Yes – Yes Yes
Robustness Yes Yes Yes Yes Yes
Linearity Yes Yes – Yes Yes
Range – – – – –
Selectivity Yes Yes Yes Yes Yes
Limit of detection – Yes Yes – –
Limit of quantification – Yes – – –

Table 3: Characteristics required for assay validation as per USP

Analytical performance Category I Category II Category III Category IV
characterisitics Quantitative tests Limit tests
Accuracy Yes Yes * *
Precision Yes Yes Yes
Specificity Yes Yes Yes * Yes
Limit of detection Yes *
Limit of quantification Yes *
Linearity Yes Yes *
Range Yes Yes * *
*Indicates that may be required depending upon the nature of the specific test

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Kumar, et al.: Method development and validation

reproduced. Validation is always a balance between costs, including measurement uncertainty and accuracy profiles. Trends Anal
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Source of Support: Nil, Conflict of Interest: None declared
51. United States Pharmacopoeial Convention. General Chapter 1225,

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