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Analytical Method Validation Parameters An Updated Review

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Int. J. Pharm. Sci. Rev. Res., 61(2), March - April 2020; Article No. 01, Pages: 1-7 ISSN 0976 – 044X

Review Article

Analytical Method Validation Parameters: An Updated Review

Lavanya Chowdary G, Ravisankar P*, Akhil Kumar G, Mounika K, Srinivasa Babu P

Department of Pharmaceutical Analysis, Vignan Pharmacy College, Vadlamudi, Guntur, A.P, India.
*Corresponding author’s E-mail: [email protected]

Received: 08-02-2020; Revised: 22-03-2020; Accepted: 27-03-2020.

ABSTRACT
Analytical method development aids to understand the critical process parameters and to minimize their influence on accuracy and
precision. A validated systematic approach ensures that it provides consistent, reliable, and accurate data. The parameters depicted
here are according to ICH guidelines and include accuracy, precision, specificity and limit of detection, the limit of quantitation,
linearity, range and robustness. Method validation ensures that the selective method will give reproducible, reliable, and consistent
results adequate for the intended purpose. It is, therefore, necessary to define precisely both the conditions in which the procedure
is to be used and the purpose for which it is intended. Method validation is, therefore, a fundamental component of the measures
that a laboratory should establish to be able to create reliable analytical data.
Keywords: Validation, precision, specificity, accuracy, ICH guidelines.

INTRODUCTION The weight of constituents of the matrix is modified so as

to keep constant average weight.

A nalytical method validation is the process of

demonstrating that analytical procedures are
suitable for their intended use. More specifically,
analytical method validation is a matter of establishing
documented evidence that the specified method will
Look like formulation composition:
In these types of formulations, composition of the active
ingredient proportionally increases as the strength
increases but the average weight of dosage form remains
consistently provide accurate test results that evaluate a
constant by a minor change in weight of one of the
product against its defined specification and quality
excipient.
attributes. The method should be validatable,
transferable, robust, reliable, accurate and precise for day- Look alike formulation concept is applicable only for the
to-day activities in the Quality Control environment. The Drug Product having less content of active ingredient.
method should not enter the validation phase unless it is These current validation characteristics describe the
fully developed. Validation experiments must be properly validation parameters stated by the International
documented and performed on qualified and calibrated Conference on Harmonization [ICH] guidelines Q2 (R1)9-11.
instrumentation and equipment.1-8
Different Types of Validation characteristics:
There are different types of formulation compositions
➢ Precision.
available:
➢ Accuracy.
Dose proportional formulation composition.
➢ Specificity.
Pseudo dose proportional formulation composition.
➢ Linearity.
Look alike formulation composition.
➢ Range.
Dose proportional formulation composition:
➢ Detection Limit.
In these types of formulations, composition of active and
inactive ingredients proportionally increases as the ➢ Quantitation Limit.
strength increases. In this case, method validation can be
➢ Ruggedness.
performed on any of the strengths.
➢ Robustness.
Pseudo Dose proportional formulation composition:
System Suitability
In this type of formulations, composition of the active
ingredient proportionally increases as the strength System suitability is defined by ICH as "the checking of a
increases but the average weight of dosage form remains system, before or during the analysis of unknowns, to
constant. ensure system performance." System suitability criteria
may include such factors as plate count, tailing, retention,
and/or resolution. System suitability criteria should also
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include a determination of reproducibility (%RSD) when a N = Number of theoretical plates.

system suitability "sample" (a mixture of main components Ve = elution volume, retention time or retention
and expected by-products/interferences) is run. distance (mL, sec, or cm).
h = peak height.
System suitability testing is an integral part of analytical
wb = width of the peak at the base line (mL, sec, or cm).
procedures.
The plate number depends on column length. The
• If the % RSD specification is below 2.0 % five replicates
theoretical plate number is the measure of column
are used.
efficiency. As stated by plate theory, the analyte will be in
• If the % RSD specification above 2.0 %, six replicates instant equilibrium with the stationary phase and the
are used. column has to be divided into the number of hypothetical
plates and each plate consists of a fixed height and analyte
The parameters used in the system suitability tests (SST)
spends finite time in the plate. Height equivalent to a
report are as follows:
theoretical plate (HETP) is given by the following formula:
• The number of theoretical plates or Efficiency (N). HETP = L/N, Where, (1)
• Capacity factor (K).
L = length of column.
• Separation or Relative retention (α).
• Resolution (Rs). N = plate number.
• Tailing factor (T). Capacity ratio or Capacity factor (k′)
• Relative Standard Deviation (RSD).
t –t (2)
Number of theoretical plates/Efficiency (N) k′ = R M
t
M
In a specified column, efficiency is defined as the
measurement of the degree of peak dispersion and it The above said capacity factor sometimes is called as a
should have the column characteristics. The efficiency is retention factor which has no dimension and independent
conveyed in terms of the number of theoretical plates’. from the flow rate of mobile phase as well as column
The formula of calculation of N is illustrated bellow in the dimensions which is the measure of the extent of retention
following Figure 1. (Half height method). relating to an analyte relative to an un-retained peak.
Where tR implies the retention time of the sample peak
and retention time of an un-retained peak is tM.
k' = 0 means no compound is left in the column. Generally
the value of k' is > 2.

Figure 1: Half height method relating to determination of

N.
N = Efficiency / Number of theoretical plates.
Ve = Retention time of analyte.
h = Height of the peak.
w 1/2 = Gaussian function of the peak width at the half-
height.
4-Sigma/tangential method (USP method)
Figure 3: Determination of capacity factor/ capacity ratio.
With the help of signa/tangential method N is calculated
Relative retention or separation factor (α)
which is shown in the following figure 2 duly noting the
formula for calculation of N.
𝛼 = 𝑡2 − 𝑡𝑎 ⁄𝑡1 − 𝑡𝑎 (3)

𝛼 = Relative retention.
𝑡2 = Retention time calculated from point of injection.
𝑡𝑎 = Unretained peak time (Retention time (tR) of an inert
component not retained by the column).
𝑡1 = the retention time from the point of injection of
Figure 2: Sigma/tangential method relating to the reference peak defined. (Suppose no reference peak is
determination of N. found, the value would be zero).
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Resolution (Rs) Where:

As = peak asymmetry factor.
Resolution is the capability of the column to separate 2
B = distance from the point at peak midpoint to the
drugs in 2 individual peaks or chromatographic zones and
trailing edge. (measured at 10 % of peak height).
it is improved by enhancing column length, reduction of
A = distance from the leading edge of peak to the
particle size and rising temperature, altering the eluent or
midpoint. (measured at 10 % of peak height).
stationary phase. It can be told in terms of the ratio of
separation of the apex of two peaks by the tangential Ideally, peaks should be Gaussian in shape or totally
width average of the peaks. By using the following formula, symmetrical. Determination of tailing and asymmetric
the resolution is calculated. factors is shown in Figure 5.

Figure 4: Determination of resolution between two peaks.

tR1 and tR2 are the retention times for the two peaks of
components.
tw1 and tw2 = At the baseline lies between tangents drawn
to the sides of the peaks. (Tangents are drawn at 0.6 times
the peak height). If the peaks are correctly symmetric,
provided the valley between the two peaks should touch Figure 5: Determination of tailing and asymmetric factor.
the baseline Rs is 1.5. Generally good value of resolution is Acceptance criteria (limits) of system suitability
Rs ≥2 should be adequate and preferred normally. parameters are shown in the following Table 1.
Resolution factor (R) Table 1: Acceptance criteria for system suitability
Resolution is a function of capacity factor, the function of parameters.
selectivity and a function of efficiency (or) number of S.No Parameter name Acceptance
theoretical plates (N). In order to separate any two peaks, criteria
you must have the right capacity factor ideally between 2
1 Number of theoretical plates or > 2000
and 10, but appropriate selectivity is required i.e., ideally Efficiency (N)
1.2 and enough efficiency i.e., a number of theoretical
plates (more than 2000 theoretical plates). The resolution 2 Capacity factor (K) <1
should be ≥ 1.5. 1.5 defines baseline resolution. 3 Separation or Relative retention (α) >1
4 Resolution (Rs) > 1.5
k′ α−1 N
R= × × √ - (5) 5 Tailing factor or Asymmetry(T) <2
1+k′ α 4

Tailing factor or Asymmetry factor 6 Relative Standard Deviation (RSD) <2

Chromatographic peak assumed to have a Gaussian shape Specificity

under ideal conditions. However in practical conditions,
One of the significant features of HPLC is its ability to
there is always a deviation from the normal distribution
generate signals free from interference. Specificity refers
which indicates non-uniform migration and non-uniform
to the strength of the analytical method to differentiate
distribution process. Hence the regulatory organizations
and quantify the analyte in complex mixtures. An
like USP and EP have recommended this as one of the
investigation of specificity is to be conducted during the
system suitability parameters. The asymmetry factor and
determination of impurities and validation of identification
tailing factor are roughly the same and rarely accurate and
tests.
equal in most cases. Values should normally between 1.0-
1.5 and values greater than 2 are unacceptable. The peak An ICH guideline defines specificity as the ability to assess
asymmetry is computed by utilizing the following formula. unequivocally the analyte in the presence of other
compounds that may be likely to be present. Typically
As = B/A (6)
these might be impurities, degradants, matrix, etc.

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The definition has the following implications: Standard deviation

% RSD = -------------------------- x 100
• Identification test: Identification tests should be able
Mean
to differentiate compounds of closely related structure
which are expected to be present i.e., to assure the Accuracy
identity of an analyte.
Definition: It is the closeness of agreement between the
• Purity test: To ensure that the analytical procedure actual value and measured value.
performed allows an accurate statement of the content Accuracy is calculated as the percentage of recovery by the
of the impurity of an analyte i.e. related substances, assay of the known added amount of the analyte in the
residual solvents content, heavy metals, etc. sample or the difference between the mean and accepted
• Assay: To arrive at an accurate result, this permits a true value together with confidence intervals.
correct report on the potency or content of analyte in a The ICH guidance recommended to take a minimum of 3
sample. concentration levels covering the specified range and 3
Precision replicates of each concentration are analyzed (totally 3
* 3 = 9 determination)
Definition: The closeness of agreement between a series of
measurements multiple samplings of the same Specificity
homogeneous sample under prescribed condition. The Definition: The ability to assess unequivocally the analyte
precision of test method is usually expressed as the in the presence of components that may be expected to
standard deviation or relative standard deviation of a present, such as impurities, degradation products and
series of measurements. matrix components, etc.
Precision may be considered at three levels: Repeatability, Methodology:
Intermediate Precision and reproducibility.
Specificity shall be demonstrated by performing Placebo /
Method precision (Repeatability): blank interference and forced degradation studies.
Repeatability expresses the precision under the same 1. Blank interference:
operating conditions over a short interval of time.
repeatability is also termed intra-assay precision. Prepare blank solutions as per the test method and analyze
them as per the test method.
Intermediate Precision:
2. Placebo interference (In case of Drug products):
It expresses with in laboratory variations; different days,
different analysts, different equipment, etc. Prepare the placebo solution equivalent to the test
concentration (Subtract the weight of active ingredient)
Reproducibility: and analyze it as per the test method.
Precision between laboratories (mostly performed during 3. Force Degradation studies12:
analytical method transfer).
Degrade the sample forcefully under the various stress
Relative standard deviation: conditions like Light, heat, humidity, acid/base/water
This serves as a daily evaluation of the repeatability of the hydrolysis, and oxidation and ensure the degradation and
system. Often, the relative standard deviation calculated for peak purity.
as % RSD for five or six replicate injections of a reference Note: Based on the physio-chemical properties and
standard or working standard is measured at the beginning literature stress conditions can be decided.
of each set of analyses. Standard deviation is calculated
using the formula Linearity and Range
Linearity
The linearity of an analytical procedure is its ability (within
a given range) to obtain test results which are directly
proportional to the concentration (amount) of analyte in
the sample.

Where Range

s = standard deviation The range of analytical procedure is the interval between

the upper and lower concentrations of analyte in the
x = each value in the sample analytical procedure has a suitable level of precision,
accuracy, and linearity.
= mean of the values
N = the no. of values ( sample size)
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Methodology: Based on the standard Deviation of the Response and the

Slope:
At least 6 replicates per concentrations to be studied. Plot
a graph of concentration (on the x-axis) Vs mean response The detection limit (DL) may be expressed as:
(on Y-axis). Calculate the regression equation, Y –
The formula for calculating LOD is
intercept and correlation coefficient. Linearity shall be
established across the range.
LOD = 3.3 δ/S (7)
If linearity is not meeting the acceptance criteria, establish
the range of concentration in which it is linear. Where δ = standard deviation of intercepts of calibration
curves.
Correlation Co-efficient
S = the slope of the linearity plot.
A measure of the strength of linear association between
two variables. The correlation will always between -1.0 and The slope shall be estimated from the calibration curve of
+1.0. If the correlation is positive, we have a positive the analyte.
relationship. If it is negative, the relationship is negative.
Based on the Standard Deviation of the Blank
Measurement of the magnitude of analytical background
response is performed by analyzing an appropriate
number of blank samples and calculating the standard
deviation of these responses.
Where, Based on the Calibration Curve
N = Number of values or elements A specific calibration curve should be studied using
X = First Score samples containing an analyte in the range of DL. The
Y = Second Score residual standard deviation of a regression line or the
Σxy = Sum of the product of first and Second standard deviation of y-intercepts of regression lines may
Scores be used as the standard deviation.
Σx = Sum of First Scores
Quantitation Limit
Σy = Sum of Second Scores
Σx2 = Sum of square First Scores Definition: It is lowest amount of analyte in a sample,
Σy2 = Sum of square Second Scores which can be quantitatively determined with acceptable
accuracy and precision.
Detection Limit
Methodology:
Definition: It is the lowest amount of analyte in a sample
that can be detected but not necessarily quantitated under Following are different approaches:
the stated experimental conditions.
Visual evaluation method:
Methodology:
The visual evaluation may be used for non-instrumental
Following are different approaches: methods but may also be used with instrumental methods.
The quantitation limit is generally determined by the
Visual Evaluation Method:
analysis of samples with known concentrations of analyte
The visual evaluation may be used for non-instrumental and by establishing the minimum level at which the analyte
methods but may also be used with instrumental methods. can be quantified with acceptable accuracy and precision.
The detection limit is determined by the analysis of
Based on signal to noise ratio method:
samples with known concentrations of analyte and by
establishing the minimum level at which the analyte can be This approach can only be applied to analytical procedures
reliably detected. that exhibit baseline noise.
Based on Signal to Noise Ratio Method: Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known low
This approach can only be applied to analytical procedures
concentrations of analyte with those of blank samples and
which exhibit baseline noise. Determination of the signal-
by establishing the minimum concentration at which the
to-noise ratio is performed by comparing measured signals
analyte can be reliably quantified. A typical signal-to-noise
from samples with known low concentrations of analyte
ratio is 10:1.
with those of blank samples and establishing the minimum
concentration at which the analyte can be reliably
detected. A signal-to-noise ratio between 3 or 2:1 is
generally considered acceptable for estimating the
detection limit.

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Based on the standard Deviation of the Response and the Flow rate: (+/- 0.2ml/minutes).
Slope:
Mobile phase composition: (+/- 10% of organic phase).
The formula for calculating LOQ is
Column oven temperature: (+/- 5°C).
LOQ = 10 PH of buffer in mobile phase: (+/- 0.2 units).
(8)
δ/S
Filter suitability: (At least two filters).
Where δ = standard deviation of response. Methodology:
S = Mean of slopes of the calibration curves. a) Mobile phase variation: Prepare the mobile phases
by changing organic phase to +/-10 % of the mobile
Based on the Standard Deviation of the Blank
phase composition.
Measurement of the magnitude of analytical background
b) Flow rate: Change the flow rate by +/- 0.2 ml/minutes
response is performed by analyzing an appropriate
of the target flow rate mentioned in test method.
number of blank samples and calculating the standard
deviation of these responses. c) Temperature of the Column: Change the temperature
of the column by +/- 5.0°C of the target temperature
Based on the Calibration Curve
mentioned in Test method.
A specific calibration curve should be studied using
d) PH of the buffer of mobile phase: Prepare the mobile
samples, containing an analyte in the range of QL. The
phases by changing the pH of the buffer by +/- 0.2
residual standard deviation of a regression line or the
units of the pH mentioned in the test method.
standard deviation of y-intercepts of regression lines may
be used as the standard deviation. e) Filter Suitability: Prepare the test solution as per the
test method and filter through two different types of
Ruggedness
filters. Analyse the sample as per the test method and
Definition: Ruggedness is the degree of reproducibility of compare the results against the unfiltered /
test results obtained by the analysis of the same samples centrifuged sample.
under a variety of test conditions such as different
CONCLUSION
laboratories, analysis, instruments, reagent lots, elapsed
assay times, temperature, days, etc. It can be HPLC is probably the most important analytical technique
expressed as a lack influence of the operation and used in pharmaceutical analysis13,14. A skilled operator is
environmental variable on the test results of the analytical required to perform HPLC analysis. Method validation is an
method. significant requirement for any package of information
submitted to international regulatory agencies in support
Robustness
of novel product marketing or clinical trial applications.
Definition: It is a measure of the method's ability to remain Analytical methods should be validated, including methods
unaffected by small but deliberate variations in method published in the relevant pharmacopoeia or other
parameters and provides an indication of its reliability recognized standard references. The suitability of all test
during normal usage. If measurements are susceptible to methods used should always be verified under the real
variations in analytical conditions, the analytical conditions conditions of use and should be well documented.
should be suitably controlled or a precautionary statement Methods should be validated to include consideration of
should be included in the procedure. characteristics included in the International Conference on
Harmonization (ICH) guidelines14 addressing the validation
Examples of typical variations are:
of analytical methods. Generally to evaluate and interpret
• Stability of analytical solutions bioequivalence, bioavailability, toxicokinetic study and
pharmacokinetic data bioanalytical method validation 15,16
• Extraction time
plays an important role. In this, infact the quantitative
In the case of liquid chromatography, examples of typical determination of drug and its metabolites in the biological
variations are fluid can be performed.
• Influence of variations of ph in a mobile phase REFERENCES
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change deliberately and verify during method validation:

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Source of Support: Nil, Conflict of Interest: None.

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