This document presents the analytical method development and validation of azelnidipine using UV-visible spectroscopy. It outlines the methodology, including the calibration curve and validation parameters like linearity, accuracy, and precision, ensuring compliance with ICH guidelines. The study aims to develop a rapid, sensitive, and economic method for estimating azelnidipine in drug formulations.

1. Analytical Method development and validation of azelnidipine by
UV-Visible Spectroscopy
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Presented to:
Prof. (Dr). P.Malairajan
(Hod, Department of Pharmaceutical
Sciences, SIHAS, SHUATS, Prayagraj,
U.P, India)
Presented By:
Name: Imdad H. Mukeri
Id No: 17BPH084
Subject: Advance Instrumentation
Technique
Course code: BP814PW
Date of Submission: 14/06/2021
1

2. Introduction
 Pharmaceutical analysis comprises the procedures necessary
to determine the identity, strength, quality and purity of
substances of therapeutic importance.
 Quality is important in every product or service, but it is vital
in medicines as it involves life.
 Analytical techniques that are generally used for drug analysis
are
• Chromatographic methods,
• Spectral methods,
• Biological and Microbiological methods
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3. Introduction to UV-Visible spectroscopy
• UV-Visible spectroscopy is type of absorption spectroscopy in which light
of ultra-violet and visible region (200-400nm and 400-800nm) is absorbed
by the molecule which results in the excitation of the electrons from the
ground state to higher energy state.
Instrumentation
Figure 1: Instrumentation of double Beam UV-Visible spectroscopy
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4. The fundamental law that governs the quantitative
spectrophotometric analysis
Beer -Lambert law.
• It states that the intensity of a beam of parallel monochromatic radiation
decreases exponentially with the number of absorbing molecules i.e
absorbance is proportional to the concentration and passes through a
medium of homogeneous thickness. A combination of these two laws
yields the Beer-Lambert law.
Mathematically, Beer Lambert law is expressed as
A=a b c (Where, A=absorbance or optical density, a= absorptivity or
extinction coefficient, b=path length of radiation through sample (cm),
c=concentration of solute in solution. Both b and a are constant so a is
directly proportional to the concentration c
• When c is in gm/100 ml, then the constant is called A (1%, 1 cm)
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5. Drug profile of azelnidipine
• Molecular weight: 582.646 g/mol
• Molecular formula: C33H34N4O
• Melting point and pka: 122-123c and 7.89
• Absorption: Orally absorbed
• Bioavailability: Less than 50%
• Half life: 16-24 hrs
• Plasma protein binding and Cmax: : ~90% and 3.0-13.1 ng/ml
• Metabolism: Metabolized by cytochrome P450 (CYP) 3A4 in the liver and
has no active metabolite(5).
• Storage: Stored In tightly closed container in cool, dry and well maintained
area.
• Solubility: Slightly soluble in methanol, freely soluble in acetone , soluble
in ethyl acetate, sparingly soluble in water
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Figure 2: Chemical structure of azelnidipine and their
IUPAC name
5

6. Objective
The Objective of this dissertation work is as follows
 Aim of the present work is to Develop some new analytical
methods for the estimation of azelnidipine and drug
formulations
 To Develop rapid, sensitive and selective method
 Economic and accurate method
 Method validation according to ICH guidelines.
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7. Methodology: UV-Visible spectroscopy
 Selection of analytical wavelength
 Preparation of stock solutions
 Calibration curve for the Azelnidipine
 Sample preparation for determination of azelnidipine from
dosage form
 Validation of Spectrophotometric methods
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8. Selection of wavelength (λ max)
• For the selection of analytical wavelength range for method 100µg/ml
azelnidipine was scanned in the spectrum mode from 200nm to 400nm
against distilled methanol as blank. Wavelength range was selected
around wavelength maxima (257nm).
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
200 220 240 260 280 300 320 340 360 380 400
UV-Visible spectroscopy
Wavelength nm
A
B
S
O
R
B
A
N
C
E
Figure 3: λ max of azelnidipine (100μg/ml)
8

9. Method development by UV-Visible
spectroscopy
• It is the process of formulating the material, condition, and
protocol for measuring the analyte
• To develop a suitable UV-Visible method for determination of
azelnidipine with the compositions of methanol was used as
diluents
• Finally good % RSD was found with diluents methanol.
• On scanning the standard solution against diluents in entire
UV-Visible range of 200-800nm, good response was found at
257nm for 100µg/ml solution.
• There is no interference from diluents methanol through the
entire UV-Visible range 200-800nm.
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10. Method life cycle
optimization
Validation
Development
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Figure 4 : method development life cycle
10

11. Steps involved in method development
Analyte standard characterization
Method requirements
Literature search and prior methodology
Choosing a method
Instrumental setup and initial studies
Optimization
Documentation of analytical figures of merit
Evaluation of method development with actual samples
Determination of percent RSD of actual sample and
demonstration of quantitative sample analysis.
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12. Analytical method validation
• Validation of analytical procedures is the process of
determining the suitability of a given methodology for
providing useful analytical data.
• Validation is the formal and systematic proof that a method
compiles with the requirements for testing a product when
observing a defined procedures.
Purpose of Method Validation
• Identification of Sources and Quantitation of Potential errors
• Determination if Method is Acceptable for Intended Use
• Establish Proof that a Method Can be Used for Decision
Making
• Satisfy FDA Requirements
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13. Various validation parameters
Figure 5: Eight Steps of Analytical Method Validation
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14. 1. Linearity
• Ability of an assay to elicit a
direct and proportional response
to changes in analyte
concentration.
• By Visual Inspection of plot of
signals vs. analyte concentration
• By Appropriate statistical
methods
 Linear Regression (y = mx + c)
 Correlation Coefficient, y-intercept
(b), slope (m)
• Acceptance criteria: Linear
regression r2 > 0.95
• Requires a minimum of 5
concentration levels
S.N Concentration (μg/ml) Absorbance
1 2 0.187
2 4 0.326
3 6 0.424
4 8 0.534
5 10 0.722
6 12 0.951
7 14 1.019
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y = 0.0722x + 0.017
R² = 0.9826
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14 16
Calibration curve of azeldipine
Concentraion
A
b
s
o
r
b
a
n
Figure 6: Calibration Curve of Azelnidipine at 257 nm
14
Table 1: Calibration Curve of linearity

15. 2.Range
• The specified range is normally derived from linearity studies
and depends on the intended application of the procedure
e.g.. 2-14µg/ml.
• It is established by confirming that the analytical procedure
provides an acceptable degree of linearity, accuracy and
precision when applied to samples containing amounts of
analyte within or at the extremes of the specified range of the
analytical procedure.
• For Drug Substance & Drug product Assay 80 to 120% of test
Concentration i.e 2-14µg/ml.
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16. 3.Accuracy
• Closeness of the test results
obtained by the method to the
true value.
• Should be established across
specified range of analytical
procedure.
• Should be assessed using a
minimum of 3 concentration
levels, each in triplicate (total
of 9 determinations)
• Should be reported as %RSD of
known amount added
• Acceptance criteria : % RSD ≤
2%
S.N SPIK
E %
TOTAL
CONCE
NTRATI
ON
ABSO
RBAN
CE
MEAN STD
DEVI
ATIO
N
%
RSD
1
80%
27µg/ml
27µg/ml
27µg/ml
1.001
1.010
1.023
1.011 0.011 1.09%
2
100%
30µg/ml
30µg/ml
30µg/ml
1.023
1.041
1.047
1.037 0.0124 1.20%
4
120
%
33µg/ml
33µg/ml
33µg/ml
1.073
1.081
1.091
1.081 0.0090 0.83%
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Table 2: accuracy data

17. 4.Precision
• The closeness of agreement (degree of scatter) between a
series of measurements obtained from multiple samplings of
the same homogeneous sample.
• Should be investigated using homogeneous, authentic
samples.
Precision Considered at 3 Levels
A. Repeatability
B. Intraday Precision
C. Interday precision
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18. A. Repeatability
• Express the precision under
the same operating conditions
over a short interval of time.
• Also referred to as Intra-assay
precision
• Should be assessed using
minimum of 9
determinations(3
concentrations/ 3 replicates)
• Acceptance criteria : % RSD ≤
2%
S.N Concent
ration
(µg/ml)
Absorba
nce
Mean Std.
deviatio
n
% RSD
1 15µg/ml 0.771
0.805 0.067 8.37%
2 15µg/ml 0.784
3 15µg/ml 0.816
4 15µg/ml 0.804
5 15µg/ml 0.730
6 15µg/ml 0.929
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Table 3 : Repeatability data
18

19. B. Intraday precision
• Determined by repeating the
above methods at different time
intervals (morning, afternoon and
evening) on the same day
(Intraday precision) and on three
consecutive days (interday
precision).
• The intraday variation for the
estimation of Azelnidipine was
carried out at three different
concentration levels of 5, 15and
25 µg/ml as shown in table .
• Acceptance criteria : % RSD ≤ 2%
C. Interday precision
• Determined by repeating the
above methods on three
consecutive days (interday
precision).
• The interday variation for the
estimation of Azelnidipine was
carried out at three different
concentration levels of 5, 15and
25 µg/ml as shown in table.
• Acceptance criteria : % RSD ≤ 2%
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20. S.N Concentration(µg/ml ) Absorbance Mean Std
deviation
%
RSD
1 2 3
1 5 µg/ml 0.436 0.441 0.432 0.436 0.0045 1.03%
2 15 µg/ml 1.267 1.311 1.289 1.289 0.022 1.71%
3 25 µg/ml 1.344 1.389 1.357 1.363 0.023 1.70%
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S.N Concentration (µg/ml
)
Absorbance Mean Std deviation % RSD
1 2 3
1 5 µg/ml 0.436 0.452 0.439 0.442 0.0085 1.92%
2 15 µg/ml 1.267 1.319 1.309 1.298 0.027 2.13%
3 25 µg/ml 1.344 1.382 1.446 1.390 0.0515 3.71%
Table 5: Interday Precision data
Table 4: Intraday Precision data
20

21. 5. Detection Limit (LOD)/ Quantitation Limit (LOQ)
Detection Limit (LOD)
• Lowest amount of analyte in a
sample that can be detected but
not necessarily quantitated.
• Acceptance criteria: Less than 2
• Based on Standard Deviation of
the Response and the Slope.
• From the formula, we calculate
LOD= (3.3*SD)/slope.
Where, SD= the standard deviation
of
y-intercept of 5 calibration curves.
Slope= the mean slope of the 5
calibration curves.
Quantitation Limit (LOQ)
• Lowest amount of analyte in a
sample that can be quantified
with suitable accuracy and
precision.
• Estimated by Signal to Noise Ratio
of 10:1
• Based on Standard Deviation of
the Response and the Slope
• The LOQ may be calculated as
LOQ = 10 × (σ/S).
Where, σ = Standard deviation of the
Y- intercepts of the five calibration
curves.
S = Mean slope of the five calibration
curves.
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22. 6.Robustness
• It 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 carryout by doing deliberate variation in method
parameter is done (i.e. change in wavelength, analysis by
person to person and changing room temperature).
Absorbance of any one concentration 15 µg/ml is measured at
three different wavelengths i.e. 256,257,258 nm and calculate
% RSD.
• Acceptance criteria : % RSD ≤ 2%
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23. S.
N
Parameter Absorbance Mean Std.
Deviation
% RSD
1
Wavelength
(256,257,258 nm)
256
257
258
0.494
0.4963 0.0068 1.37%
0.491
0.504
2
Analysis by person
to person
1st
2nd
3rd
0.476
0.484 0.0065 1.34%
0.481
0.491
3
By changing
Temperature
38c
43c
40c
0.436
0.442 0.0052 1.92%
0.452
0.439
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Table 6: Measure Robustness (15µg/ml) of the method
23

24. 7.Assay of Azelnidipine Tablet
• In assay of the azelnidipine tablet formulation. At first
Measure the absorbance of standard concentration three
times (1%) at 1000µg (10 µg/ml) was found to be mean value
325 and sample concentration at 10µg (0.01 µg/ml) was found
to be mean value 0.319 at 257 nm. Calculate the % assay of
the sample of the drug.
% assay = sample abs × Std. Conc. / Std. Abs × test Conc. × 100%
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S.N Std.Conc Abs (1000µg) Sample Conc.(10µg) % Assay
1 325 0.319 98.87%
Table 7: Assay of azelnidipine tablet
24

25. 8.Result and Discussion
S.N PARAMETER NORMAL RANGE RESULT
1 Linearity (R2) 0.999 0.9826
Slope (m) - 0.072
Y-intercept - 0.017
2 Accuracy % RSD ≤ 2% 2.42 %
3
Precision
Repeatability
% RSD ≤ 2%
8.37 %
1.03-1.48 %
1.92-5.58 %
Intraday
Interday
4 Limit of detection (LOD) Less than 2 0.77
5 Limit of Quantification
(LOQ)
Less than 2 2.36
6 Robustness % RSD ≤ 2% 1.37-1.92%
7 Assay of azelnidipine % Assay ±99.98% 98.87%
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Table 8: Summary of Validation Parameter
25

26. • The method was tested and validated for various parameters
according to ICH guidelines.
• In validation of UV-Visible Spectroscopy, it can be concluded
that spectroscopic method was found to be simple and rapid
method. Correlation coefficient of Linearity and The value of
% RSD for intra-day were within normal range and the
accuracy was found to be nearby normal range but in the
interday precision was found to be more than 2. This value
was out of normal range. The value of % assay was found to
be more than 98.87% for this method. Shows that the method
is almost accurate and free from the in interference used in
formulation.
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27. • The limit of detection and limit of quantification of APL were
found to be 0.77 and 2.36.
• The excipients in the commercial tablet preparation did not
interfere with the % assay .
• The developed UV-Visible Spectrophotometric method was
found to be least accurate, sensitive, precise, so this method
was almost accurate to apply in pharmaceutical tablet
formulation for quantitative estimation of Azelnidipine.
• The developed UV method was subjected to stability
indicating studies for Azelnidipine. It was found that the
interfering peak from solvent did not interfere with estimation
of drug and the developed method was found to be specific
for estimation of Azelnidipine.
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28. 9. Conclusion
• The proposed method development of UV-Visible
SPECTROSCOPY was found to be least rapid, precise, accurate
and sensitive in comparison to other.
• Many samples cannot be suitably analyzed by this method.
Hence developed method cannot be used for routine analysis
of AZP in various formulations.
• It was concluded that developed method is simple, almost
accurate, precise and reliable.
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29. 10.References
1. Guy RC. International Conference on Harmonisation. Encycl Toxicol Third
Ed. 2014;2:1070–1072.
2. Thakare L, Ahmad S, Shastry VM. Development and Validation of Uv-
Visible Spectrophotometric Method for Estimation of Cilnidipine and
Telmisartan in Bulk and Dosage Form. Indo Am J Pharm Res.
2017;7(04):8552–8559.
3. Jenisha Modi, Shivangi K. Patel, Namrata Parikh, Shreya R. Shah* PKP and
UMU. World Journal of Pharmaceutical Research. Infection. 2014;5(2):831-
847.
4. Rane AS, Mahajan SK. Validation and Forced Stability-Indicating Hptlc
Method for Determination of Azelnidipine. Rane al World J Pharm Res
World J Pharm Res SJIF Impact Factor 6 [Internet]. 2016;5(9):1053–1062.
Available from: www.wjpr.net
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30. Thank you!
Any question?
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