ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF AZELNIDIPINE BY UV VISIBLE SPECTROSCOPY

1. ANALYTICAL METHOD DEVELOPMENT AND VALIDATION
OF
AZELNIDIPINE BY UV VISIBLE SPECTROSCOPY
GUIDED BY PRESENTED BY
Prof. Dr. P. Malairajan NAME: ANANT NAG
Head Of Department ID.No: 17PBH005
Department Of Pharmaceutical Sciences SUBJECT: ADVANCE INSTUMENTATIO TECHNIQUE
SIHAS, SHUATS, Prayagraj,U.P,india Subject Code: BP814PW
Date Of Submission: 14/06/2021

2. List of Content
1.Introduction
2.Litrature Review
3.Aim & Objective
4.Material & Method
5.Result and discussion
6.Conclusion
7.References
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3. List of tables
1.Table: UV RANGE IN SPECTROSCOPY
2.Table: LITERATURE REVIEW
3.Table: Reported methods for assessment of Azelnidipine.
4.Table: Reported methods for assessment of Azelnidipine.
5.Table: Linearity Calibration Data.
6.Table: Calibration Curve of Azelnidipine
7.Table: Repeatability Data
8.Table: Intraday Precision
9.Table: Interday data
10.Table:Accuracy Data
11.Table: ROBUSTNESS DATA
12.Table: Assay Data
13.Table: Summary Data
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4. analytical method development –
• it is a process whose main purpose is to prove any analytical method in pharmaceutical
industry suitable in api measurement, method & development is the process of proving that an
analytical method is acceptable for use to measure the concentration of an api in a specific
compounded dosage form which allow simplified procedures to be employed to verify that an
analysis procedure, accurately and consistently will deliver a reliable measurement of an
active ingredient in a compounded preparation.
• analytical method development allows to confirm that an accurate and reliable potency
measurement of a pharmaceutical preparation can be performed.
INTRODUCTION
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5. analytical method validation –
• it is process of establishing documentary evidence demonstrating that a particular, process or
activity carried out in testing and production maintains desired level of compliance at all
stages.
• the analytical method validation is essential for analytical method development and tested
extensively for specificity, linearity, accuracy, precision, range, detection limit, quantization
limit, and robustness.
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6. steps of method development and method validation:
• * method development plan definition
• *background information gathering
• * laboratory method development, it includes various stages namely sample preparation,
• specific analytical method, detection and data processing
• *generation of test procedure
• *a well-developed method should be easy to validate. a method should be developed
• with the goal to rapidly test preclinical samples, formulation prototypes, and
• commercial samples. there are five common types of analytical methods, each with its
• own set of validation requirements
• *identification tests
• *potency assays
• *quantitative tests for impurities
• *limit test for the control of impurities
• *specific tests
• it can be done for various instruments like uv visible spectroscopy.
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7. Azelnidipine
• azelnidipine (marketed under the brand name calblock ) is a dihydropyridine calcium channel blocker.
• it has a gradual onset and has a long-lasting hypotensive effect, with little increase in heart rate.
• azelnidipine is light yellow to yellow crystalline powder.
• it’s molecular formula of azelnidipine is c₃₃h₃₄n4o₆.
• molecular weight is 582.646 g/mol.
• it is insoluble in water, slightly soluble in methanol, soluble in ethyl acetate, freely soluble in acetone and
in acetic acid.
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8. UV-VISIBLE SPECTROSCOPY
• spectroscopy is the absorption, emission or scattering of electromagnetic radiation by atoms or molecules
or their ions used for qualitative or quantitative studies of sample.
• absorption spectrophotometric: when a molecule exposed to electromagnetic radiation, certain amount of
energy associated with particular radiation absorbed by molecule.
• there is transfer of energy from beam of radiant energy to molecule. the study of this is called absorption
spectrophotometric.
• origin of absorption spectra: absorption of light in ultraviolet and visible regions gives rise to the absorption
spectra.
• an absorption spectrum is observed, when radiation of range of frequencies is passed through substance. the
range of radiation in emr spectrum used in absorbance spectrum is as follows.
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Table: 1 ( UV RANGE IN SPECTROSCOPY)

9. • analytical method development by uv visible spectroscopy-
• analytical method development allows to confirm that an accurate and reliable
potency measurement of a pharmaceutical preparation can be performed. for this
azelnidipine, distilled water and methanol were used as solvent. it also involves
preparation of stock solution, preparation of working standard and preparation of
calibration curve.
• analytical method validation by uv visible spectroscopy-
• analytical method validation allows in establishing documentary evidence
demonstrating that a particular, process or activity carried out in testing and
production maintains desired level of compliance at all stages. various validation
parameters includes linearity, precision, accuracy, lod & loq and robustness
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10. Literature review
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Table:2 (LITERATURE REVIEW)

11. Table3: Reported methods for assessment of Azelnidipine.
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12. Table 4. Reported methods for assessment of Azelnidipine.
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13. AIM AND OBJECTIVE
• several works have been carried out of method development and validation
of azelnidipine by analytical methods like uv spectroscopy.
• it describes development of a new rapid, simple, sensitive and reproducible
methods for analysis of azelnidipine in its simplicity and sensitivity and
applicable in routine analysis.
• its also describes development of validation work as per ich guidelines.
• the objective was to develop uv method for azelnidipine method
development
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14. MATERIALS AND METHODS
• -analytical method development by uv spectrophotomete
• -chemicals and reagents:-
• pure drug sample of azelnidipine was provided. distilled grade water and methanol were used
as solvent for drugs.
• -apparatus and instrument:-
• instrument used were double beam uv-visible spectrophotometer having 2 matched quartz
cells with 1 cm light path, electronic balance, volumetric flasks & pipettes.
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15. • -spectrophotometric conditions:-
for the selection of analytical wavelength, standard solution of azelnidipine
was scanned in the spectrum mode from 400nm to 200nm. from the
spectrum, 𝜆 max of azelnidipine, 257 nm was selected for method.
• -preparation of calibration curve of azelnidipine:-
appropriate volume of aliquots 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1ml and 1.2 ml
from working azelnidipine stock solutions were transferred to different
volumetric flasks of 10 ml capacity. the volume was adjusted to the mark
with diluents to obtain concentration of 2-14 mg/ml.
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16. analytical method validation by uv spectroscopy
1. linearity:-
the linearity of response for azelnidipine was assessed by analysis of six independent
levels of concentration in range of 2-14 mg/ml in terms of slope, intercept and
correlation coefficient values of calibration curve.
2. precision:-
the precision of analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple sampling of homogenous
samples. this is usually expressed as the standard deviation or the relative standard
deviation (coefficient of variation)
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17. 3. accuracy:-
accuracy was determined by performing recovery studies by spiking different
concentration of pure drug in pre-analyzed sample solution of 4μg/ml. to
preanalyzed sample solution, a known amount of working standard solution
of azelnidipine (0.33, 0.42 and 0.48 ml of 100 μg/ml) was added in 10 ml
volumetric flask and made up to mark with diluents which was at different
level i.e. 80%, 100% and 120%. the solutions were analyzed by proposed
method. the mean % recovery was calculated from peak areas obtained.
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18. 4. limit of detection (lod) and limit of quantification (loq):-
a. lod
the lod was estimated from the set of five calibration curves used to determine method
linearity. calibration curve was repeated for 6 times and the standard deviation of
the intercept was calculated then lod was calculated as follow: from the formula
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.
b. loq
the loq was estimated from the set of five calibration curves used to determine method
linearity.
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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19. • robustness:- the robustness of an analytical method is a measure of its capacity to
remain unaffected by small by small but deliberate variation in method parameters
and provides an indication of its reliability during normal usage. the evaluation of
robustness should be considered during the development phase and depends on the
type of procedure under study. it should show there liability of an analysis with
respect to deliberate variations in method parameter.
• assay :- in assay of azelnidipine tablet formulation at first absorbance of standard
concentration measured three times (1%) at 1000 ug (10 ug/ml) found to be mean
value 325 and sample concentration at 10 ug (0.01ug/ml) found to be mean value
0.319 at 275nm. then percentage assay calculated by values of standard
concentration and sample concentration.
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20. RESULT AND DISCUSSION
• analytical method development by uv visible spectroscopy
• spectroscopy to optimize the uv parameters; several mobile phase compositions
were tried. distilled grade water and methanol were used as solvent for drugs.
• stock solution of 1000 μg/ml and working standard of 100 μg/ml were prepared. a
satisfactory separation and good peak symmetry for azel was obtained.
• methanol and water (80:20 v/v) used at a good flow rate of to get better
reproducibility. quantification was achieved with uv detection based on peak area.
• from the spectrum, 𝜆 max of azel, 257 nm was selected for method.
• complete resolution of the peaks with clear baseline was obtained.
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21. ANALYTICAL METHOD VALIDATION BY UV VISIBLE SPECTROSCOPY
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linearity:
the linearity of response for azelnidipine was assed by analysis of seven
independent levels of concentration in range of 2-14ug/ml in terms of slope,
intercept and concentration coefficient values of calibration
TABLE 5- Linearity Calibration Data.

22. The calibration curve was found to be linear over different concentration range in mobile phase.
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Table:6 (Calibration Curve of Azelnidipine)

23. • precision
• -repeatability
for repeatability middle concentration was selected i.e. 15μg/ml and repeatability carried out by
taking the absorbance of the solution six times.
• mean of the absorbance was calculated and standard deviation was founded out along with
percentage standard deviation.
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Repeatability was found to be 8.37% this was not within the normal range
Table: 7 Repeatability Data

24. Intraday Precision: ( WITHIN THE DAY)
• for intraday precision 3 concentrations from stock solution were prepared i.e. 5, 10&15 μg/ml.
• absorbance of each concentration was measured at time interval of two hours. then mean, standard
deviation & %rsd was calculated.
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Table.8 (Intraday Precision)

25. Interday precision:
• for intraday precision 3 concentrations from stock solution were
prepared i.e. 5, 10&15 μg/ml.
• absorbance of each concentration was measured at time interval of three
days. then mean, standard deviation & %rsd were calculated.
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Table.9 ( Interday data)

26. accuracy:
• from the stock solution of azelnidipine 1ml of solution was pipette out in 3
volumetric flasks.
• further 80, 100 and 120% solution was added from the standard stock solution to
each volumetric flasks respectively. similarly two other sets were prepared.
• the absorbance of each set i.e. total 3 concentrations of 3 sets. mean, standard
deviation & %rsd were calculated.
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Table.10 ( Accuracy Data)

27. LIMIT OF DETECTION (LOD) and LIMIT OF QUANTIFICATION (LOQ)
a. lod
from the formula 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.
therefore,
lod = 3.3x (0.017/0.072) = 0.77
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b. loq
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.
therefore, loq = 10 x (0.017/0.072) = 2.36
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28. 28
robustness:
•robustness (15ug/ml) of the method.
•robustness study was carried out by considering different parameter the absorbance
was determined.
•mean of absorbance was calculated. after than standard deviation was determined
along with percentage relative standard deviation
In case of robustness average of %RSD was found to be 1.54%. It was found
to be within the normal range i.E. Less than 2 percent
Table.11 (ROBUSTNESS DATA)

29. ASSAY OF AZELNDIPINE TABLET FORMULATION
in assay of azelnidipine tablet formulation at first absorbance of standard concentration was
measured three ti es (1%) at 100ug/ml) was found to be mean value 325 and sample
concentration at 10ug (0.01ug/ml) was found to be mean value 0.319 at 257nm
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It was found to be 98.87%
Table.12(Assay Data)

30. 30
-Summary Of Validation Parameters
Table.13 ( Summary Data)

31. DISCUSSION
• the calibration curve was found to be linear over different
concentration range in mobile phase.
• linearity was found to be 0.9826. accuracy was found to be 2.42%.
repeatability, intra-day and inter-day precision were found to be
8.37%, 1.03-1.48% and1.92-5.58% respectively.
• these were within the normal range i.e. ≤2%. lod and loq were 0.77
and 2.36 respectively. it was less than 2 i.e. within the normal
range. finally robustness was found to be 1.54%. as it was less than
2% hence it was also within the normal range.
• the above method was found to be specific, linear, precise, and
accurate. this confirmed that the uv visible spectroscopy is suitable
method for estimation of azelnidipine
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32. CONCLUSION
• by uv- visible spectroscopic method azelnidipine was successfully
developed and validated
• for its intended purpose. uv visible spectroscopic method was found to be
simple and rapid.
• the % assay was found to be 91.69%. further the precision of the methods
was confirmed by the intra-day and interday analysis and the accuracy of
the methods was confirmed by recovery study.
• the %rsd was found to be < 2.0%.the method was shown to be specific,
linear, precise, and accurate. so this method can be applied for routine
analysis of azel in pure form and in its formulations without interference.
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33. REFERENCES
• 1. koike h, kimura t, kawasaki t, et al. azelnidipine, a long-acting calcium channel blocker with slow onset and high vascular affinity [j].
annu rep sankyo res lab, 2002; 54:1-64
• 2. an, huamin, wang chencai., determination of content and related substance of azelnidipine by hplc, huaxi yaoxue zazhi, 2006; 21(6):
581-582.
• 3. ding, li, li, ma pengcheng, determination of azelnidipine in muman plasma by liquid chromatography-electron ionization- mass
spectroscopy, journal of pharmaceutical and biomedical analysis, 2007; 43(2): 575-579.
• 4. rele r.v., patil s.p... ultra-violet spectrophotometric method for estimation of azelnidipine from bulk drug and pharmaceutical
formulation. asian j. research chem. 3(4): oct. - dec. 2010; page 1077-1079.
• 5. kunti d. raskapur*, mrunali m. patel, anandkumari d. captain quality assurance, a. r. college of pharmacy, vallabh vidhyanagar,
gujarat, india,department of pharmaceutical chemistry, a. r. college of pharmacy, vallabh vidhyanagar, gujarat, india. email:
kunti_9@yahoo.com (received: 20 aug 2011, revised and accepted: 28 sep 2011)
• 6. spectrophotometric estimation of azelnidipine in bulk and pharmaceutical dosage form by second order derivative methods rele
rajan v.central research laboratory, d. g. ruparel college, matunga, mumbai available online journal of chemical and pharmaceutical
research, 2014, 6(8):198-202
• 7.m.s. charde, a.s. welankiwar, j. kumar, method development by liquid chromatography with validation, international journal of
pharmaceutical chemistry, 4 (1) (2014) 57-61.
• 8. n. n. godswill, n. e. g. frank, m. y. j. edson, y. emmanue, b. j. martin, n.b. hermine, t. m. kingsley, l. l.n. b. constant, n. m. armand,
gc- fid method development and validation parameters for analysis of palm oil (elaeis guineensis jacq.) fatty acids composition, research
in plant sciences, 2 (3) (2014), 53
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34. THANK YOU
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