Cilnidipine PPT - Pavan Kalyan kalyanppt

Cilnidipine PPT - Pavan Kalyan.ppt

1. SUBMITTED BY
K. PAVAN KALYAN GOUD
M. Pharm
H.T.No: 353122885007
UNDER THE GUIDANCE OF
G. PRIYANKA
M. Pharm

DHANVANTHARI COLLEGE OF PHARMACEUTICAL SCIENCES
Tirumala Hills, Centre City, Appannapally, Mahabubnagar, 509001
Faculty of Pharmacy
PALAMURU UNIVERSITY
MAHABUBNAGAR – 509001

2. INTRODUCTION
DRUG PROFILE
REVIEW OF LITERATURE
AIM AND OBJECTIVES
PLAN OF WORK
EXPERIMENTAL METHODS
METHOD DEVELOPMENT
METHOD VALIDATION
STABILITY STUDIES
SUMMARY
CONCLUSION
BIBLIOGRAPHY
2

3. Chromatography may be defined as a method of separating mixture components in to individual components through
equilibrium distribution between two phases. Essentially, technique of chromatography is based on the differences in the
rate at which the components of a mixture move through a porous medium (called Stationary phase) under the influence
of some solvent or gas (called mobile phase).
TYPES OF CHROMATOGRAPHY:
High Performance Liquid Chromatography
Paper Chromatography
Thin Layer Chromatography
Column Chromatography
Gas Chromatography
Ion Exchange Chromatography
Gel Filtration Chromatography
High Performance Thin Layer Chromatography
3
INTRODUCTION

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC):
HPLC Is a powerful tool in analysis, it yields high performance and high speed.
It is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of a
mixture.
HPLC is a type of liquid chromatography with the main principle of separation is “ADSORPTION”. The components
in the mixture travel according the their relative affinities towards stationary phase.
High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid
chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures
of up to 400 atmospheres. That makes it much faster.
4
INTRODUCTION

5. 5
INSTRUMENTATION OF HPLC

6. As per ICH guidelines:
1. Linearity and Range
2. Precision
(a) Repeatability.
(b) Intermediate Precision.
(c) Reproducibility.
3. Accuracy
4. Robustness
5. Specificity
6. Quantitation Limit
7. Detection Limit
8. Stability
9. System Suitability
6

7. 7

8. 8
IUPAC NAME:

3-O-(2-methoxy ethyl) 5-O-[(E)-3-phenyl prop-2-enyl] 2, 6-dimethyl-4-(3-nitro phenyl)-1, 4-
dihydropyridine-3, 5-dicarboxylate
MOLECULAR FORMULA
:
C
27
H
28
N
2
O
7
MOLECULAR WEIGHT
:
492.528g/mol
SOLUBILITY:

Cilnidipine is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide
(DMF), sparingly soluble in aqueous buffers, insoluble in water, sparingly soluble in
methanol, soluble in chloroform, acetone.
CATEGORY :

Anti-Hypertensive Agent

9. 9
MECHANISM OF
ACTION:

Cilnidipine acts on the L-type calcium channels of blood vessels by blocking the incoming
calcium and suppressing the contraction of blood vessels, thereby reducing blood pressure.
Cilnidipine also works on the N-type calcium channel located at the end of the sympathetic
nerve, inhibiting the emission of norepinephrine and suppressing the increase in stress blood
pressure.
BIOAVAILABILITY:
Cilnidipine is reported to present very low bioavailability determined to be approximately 13%.

10. 10

11. 11
REVIEW OF LITERATURE
1. P. Ravi Sankar, et al. (2019): Good quality chromatographic separation of Cilnidipine was carried out by
using thermo scientific model C18 Column (4.6 mm i.d. × 250 mm, 5 µm particle size) (based on 99.999% ultra-
high purity silica) using mobile phase consisting acetonitrile: methanol (50:50% v/v) at flow rate of 1.0 mL/min.
The λmax of the Cilnidipine in methanol (as a solvent) was found to be 242 nm. The drug following linearity in
the concentration range of 2-10µg/mL with a correlation coefficient value of 0.9999 for both UV and HPLC
methods. The regression equation for Cilnidipine UV method was found to be Y = 0.081x + 0.0017 and for
HPLC method was Y = 25517x - 4755. LOD and LOQ were found as 0.15095µg/mL and 0.45742µg/mL for UV
method and for HPLC method it was found to be 0.003µg/mL and 0.0099µg/mL.
2. Tiwari Balakrishna, et al. (2020): The method was validated as per the ICH guidelines. The method was
validated for linearity, precision (repeatability, intermediate precision), accuracy, specificity, and robustness, limit
of detection and limit of quantification. Cosmosil (4.6 X 250mm, 5 μ) column was used for separation. The
selected wavelength for Cilnidipine was 241nm. The mobile phase consists Methanol: Potassium dihydrogen
phosphate buffer (50:50). Flow rate was delivered at 1.0 mL/min. Appropriate dilutions of standard stock
solutions were prepared as per the get desired concentrations in the range of 100-500 mcg/ml. The RT obtained
was 4.8165 minutes.

12. 12
REVIEW OF LITERATURE
3. Ruhina Tanweer, et al. (2017): Separation was achieved on Symmetry C18 column (4.6 x 150mm), with
mobile phase consisting of ortho phosphoric acid buffer pH 4 and Acetonitrile in 60:40, V/V. The flow rate was
maintained at 1 ml/min and the analyte was monitored at 240nm wavelength. The retention time for Cilnidipine
was found to be 2.35 min. The linearity of the method was observed in the concentration range of 5-25ppm and
correlation coefficient was found to be 0.999. The percentage assay of Cilnidipine was found to be 98.733%.
The method was robust and rugged as observed from insignificant variation in the results of analysis by changes
in flow rate and analysis being performed on different days.
4. A. Kadam, et al. (2015): Efficient chromatographic separation was achieved on Kromasil C18 column (250mm X
4.6mm, 5μm) using isocratic elution and 240nm as a detection wavelength. The optimized mobile phase consists of
methanol, sodium di-hydrogen ortho phosphate buffer (PH=3) and acetonitrile [75:18:7] with a flow rate of 1 mL/min.
The reliability and analytical performance of the proposed HPLC method were validated for limit of detection, limit of
quantification, linearity, range, precision, accuracy, specificity and system suitability. The developed method is linear
in the range of 5-15 μg/mL with correlation coefficient being 0.9996. The relative standard deviation for intra and
inter day precision were below 1.2%.

13. 13
REVIEW OF LITERATURE
5. Prajapati Ketulkumar, et al. (2017): The separation was attained by Column C18 (25 cm × 0.46 cm) Hypersil BDS
and Buffer (pH 5.5): Acetonitrile (40:60 v/v) (Flow rate-1.0 ml/min) as mobile phase at a flow rate 1ml per minute,
Detection was carried out of wavelength of 240 nm retention time of irbesartan and Cilnidipine were found to be 3.730
and 6.040 min respectively. The method has been validated for linearity, accuracy and precision. Linearity observed for
range of 1-3μg/mL for Cilnidipine and 30-90μg/mL for irbesartan. The percentage recovery obtained for Cilnidipine and
irbesartan were found to be in range 100.61 ± 0.46, 100.68 ± 0.80 respectively. Development method was found to
be accurate, Precise and rapid for simultaneous estimation of Irbesartan and Cilnidipine in their combined dosage
form.
6. N. Sunitha, et al. (2015): Separation was performed on a ODS column (250 × 4.6mm ID, 5 µm) with buffer:
acetonitrile (42:58A), flow rate of 1.0 ml/ min and UV detection at wavelength 240 nm. Retention time of olmesartan
and Cilnidipine were found to be 2.317min and 3.763 min respectively. Linearity of olmesartan and Cilnidipine were in
the range of 50-300 µg/mL and 25-150 µg/mL respectively. %RSD of the olmesartan and Cilnidipine were and found
to be 0.6 and 0.36 respectively. % assay was obtained as 100.14 and 100.01 for olmesartan and Cilnidipine
respectively. LOD, LOQ values are obtained from regression equations of olmesartan and Cilnidipine were 0.43 mcg /
ml, 1.31 mcg / ml and 1.17 mcg / ml, 3.53 mcg / ml respectively. Regression equation of olmesartan is y = 19780x +
2601 and of Cilnidipine is y = 24481x + 8646 with regression co-efficient value was 0.999.

14. 14
REVIEW OF LITERATURE
7. Ramankit Jaiswal, et al. (2022): The chromatographic separation was performed using the C-18 reverse phase
column Oyster BDS Premium C18 column (4.6 × 150 mm, 5 µm particle size). The flow rate was set to 1.2 ml/min,
and the chromatogram was recorded at 240 nm. The proposed method was validated in compliance with the
International Conference on Harmonization (ICH) guidelines. Results: The calibration curves plotted were found to be
linear over the concentration range of 40-200 ng/ml in Wistar rat plasma, with a regression coefficient of R2>0.999,
suggesting that the linearity was within limits. The LOD and LOQ of the present method were found to be 2.184 and
4.518 ng/ml, respectively. Drug extraction recoveries from Wistar rat plasma were found to be >98%.
8. Farhana V Buchiya, et al. (2020): The separation was carried out using Luna C18 100A? (250mm × 4.6 mm i.d.) 5
mm reverse phase column (Phenomenex, Luna®) in gradient mode, with mobile phase containing Acetonitrile: Water
(85:15, v/v). The flow rate is 1.0 ml/min and effluents are monitored at 240 nm. Chromatogram showed peak at a
retention time of 2.083 min for Cilnidipine and 5.458 min for Valsartan. The method is validated for system
suitability, linearity, precision, accuracy specificity, ruggedness, robustness, LOD and LOQ. Recovery of Cilnidipine and
Valsartan is found to be 100.36% and 100.14% respectively. The LOD and LOQ for estimation of Cilnidipine and
Valsartan are found to be 0.037mg/ml, 0.31mg/ml and 0.206mg/ml, 0.62mg/ml respectively.

15. 15
REVIEW OF LITERATURE
9. Manoranjani Medikonda, et al. (2022): Separation was done by using X-bridge phenyl column (150x4.6 mm, 3.5m)
with mobile phase of buffer and Acetonitrile in the 30:70 v/v ratio was pumped through a column with a 1 ml/min flow
rate. The buffer used in this process was 0.1 percent tri ethyl amine in 1 lt of water and adjust its pH-2.5 with 0.1
percent ortho phosphoric acid. At ambient temperature, chromatography was isocratically performed and run time was
8 min. 240 nm was the optimized wave length. Results: Cilnidipine and Ramipril retention times were 2.79 min, 5.11
min. respectively. This approach offers strong linearity over a range of 2-30 µg/ml Cilnidipine 1-15 µg/ml Ramipril
concentrations. The regression coefficient R2>0.999 from the calibrated curve implies that the linearity of the system
was within the range.
10. Neelima Kudumula, et al. (2014): The separation of the two drugs was based on Inertsil ODS 3V (250 × 4.6 mm,
i.d., 5 μm) column in a Gradient mode. The mobile phase consisting of 0.025 M KH2PO4 buffer whose pH was adjusted
to 2.5 using dilute OPA (solvent A) & Acetonitrile (solvent B), set with gradient programming for 15 min at a flow rate of
1ml/min & the detection of the drugs at 240nm using a variable PDA detector. The retention times of Chlorthalidone
and Cilnidipine were found to be 3.872 min & 7.668 minutes respectively. Calibration curves were linear for CHL and
CIL at concentration ranges of range of 200-600 μg/ml and 160-480 μg/ml with the regression coefficient of 0.999 for
both the drugs and precise with (% RSD < 2).

16. 16
REVIEW OF LITERATURE
11. Leena Sawaikar, et al. (2020): The separation was achieved on waters C18 column, (100mm x 4.6mm, 2.5µm)
using a mobile phase consisting of phase A, (0.1 %Formic acid in methanol: Acetonitrile in the ratio 80:20 v/v) phase
B, (10 mM ammonium acetate) in gradient mode, at a flow rate of 1.0mL/min. The wavelength selected for detection
was 240 nm and a PDA detector was employed for the detection. The retention time of Chlorthalidone was
6.047±0.2 min and Cilnidipine was 12.642 ±0.2 min. The developed method was validated for specificity, linearity,
accuracy precision and robustness. There was no interference seen in the blank at the retention time of the drugs
hence the method was specific. The linear regression seen in calibration curve of both the drugs was 0.999.
12. Hetal Patel*, et al. (2019): The objective of the recent study was to develop a simple, accurate and precise
RPHPLC method with subsequently validate as per ICH guidelines for the determination of Cilnidipine and Bisoprolol
Fumarate using mobile phase (mixture of a Phosphate Buffer: Methanol 60:40) as the solvent. The proposed method
involves the measurement of Retention time at analytical wavelength 225 nm was selected. The Retention time of
Cilnidipine and Bisoprolol Fumarate was found to be 4.053 and 5.730 respectively. The linearity of the proposed
method was investigated in the range of 10-30 µg/ml for Cilnidipine and 5-15 µg/ml for Bisoprolol Fumarate
respectively. The method was validated for its linearity, accuracy and precision. Both inter-day and intra-day variation
was found to be showing less 2 % RSD.

17. 17
REVIEW OF LITERATURE
13. Pramod Kumar Goyal, et al. (2023): The method was carried out on Hypersil ODS C 5µ column (250 x 4.6 mm)
with a mobile phase consisting of Acetonitrile: 0.025 M Dibasic phosphate buffer pH 3.0 with phosphoric acid (70: 30
v/v) and flow rate of 1 mL/min. Detection was carried out at 245 nm. The retention time for Cilnidipine and Bisoprolol
fumarate was found to be 3.04 min and 15.114 min, respectively. The Cilnidipine and Bisoprolol fumarate followed
linearity in the concentration range of 5 - 25 µg/mL (r= 0.998) and 2.5 - 12.5 µg/mL (r= 0.9993). The developed
method was validated for linearity and range, accuracy, precision, and assay. Cilnidipine and Bisoprolol fumarate was
subjected to acid and alkali hydrolysis, oxidation and thermal degradation.
14. Shirleen Miriam Marques, et al. (2023): Design of Experiments was employed to optimize the RP-HPLC method.
The critical method parameters (CMPs) were screened using the Taguchi OA design. The selected parameters were
further optimized using the Box-Behnken design with the retention time (Rt), peak area, number of theoretical plates
(NTP) and tailing factor (Tf) as the dependent variables. The optimized chromatographic parameters for the RP-HPLC
method were: concentration of acetonitrile: 78% v/v, flow rate: 0.7 mL/min and oven temperature: 25 ˚C. The Rt,
peak area, NTP and Tf were 9.03 min, 1174428 mV-min, 11054 and 1.133 respectively. The optimized method
demonstrated excellent linearity (R2 = 0.9998) having a range of 50–4000 ng/mL and LOD and LOQ were 6.76 and
20.48 ng/mL, respectively.

18. 18
REVIEW OF LITERATURE
15. Vishal Singh Solanki, et al. (2018): The chromatographic separation was achieved by using Hypersil- keystone
C18 (4.6 x 250mm, 5μm) under isocratic conditions The mobile phase consisted of methanol and triple distilled
water (80/20, v/v) having pH 7 with a flow rate of 1.0 mL/min. The eluents were monitored at 225 nm for
simultaneous measurement. The selected chromatographic conditions were found to effectively separate CDP (Rt:
3.25 min), ATL (Rt: 5.366 min) and CTD (Rt: 9.025 min) having good resolution. The developed method was validated
for linearity, accuracy, precision, LOD, LOQ, robustness and for system suitability parameters as per ICH guidelines. In
this study, an excellent linearity was obtained with r2 = 0.999, r² = 0.999, r² = 1, for CDP, ATL and CTD respectively.
16. Biswajit Samantaray, et al. (2023): Cilnidipine was quantified using UV spectroscopy. Action separation of
Cilnidipine was achieved by using a C18 column. Mobile phase combination of methanol: water (90:10 v/v) was
tense at the flow of 1 ml/min. Detection At 241 nm, detection was carried out. In accordance with the International
Conference on Harmonization's (ICH) Q2R1 recommendations, validation parameters were assessed. With a
parametric value (r2) of 0.999, the standardization curve for Cilnidipine was linear over the range of 2–10 mg/ml.
The tactic was noticed to be accurate (101.66% recovery), precise (intraday, 1.65 and inter day, 1.38) and robust (%
RSD was calculated to be 0.66, 0.58 and 0.81 for variation in mobile phase composition, wave length and flow
velocity respectively) for the analysis of Cilnidipine.

19. 19
REVIEW OF LITERATURE
17. G. Sai Sri Lakshmi, et al. (2022): A simple reverse phase isocratic elution has been developed to separate the all
four active ingredients using water and acetonitrile was used as mobile phase in ratio of 60:40 v/v. A isocratic elution
has been developed and validated, on a reverse phase X-Bride Phenyl column (150 X4.6 mm, 3.5μ) with a flow rate of
1mL/min by monitoring at 240nm of wavelength. Results: The mean retention times of Lamivudine, Ramipril and
Cilnidipine were found to be 3.156and 6.889min respectively. Linearity of Ramipril and Cilnidipine was found to be 2.5-
15μg/mL and 2.5-15μg/mL respectively. The accuracy of the proposed method was determined by performing
recovery studies and was found to be between 98-102%. The repeatability testing for both sample and standard
solutions was found as %RSD<2.0% which is within the acceptable limits showing that the method is precise as well.
18. Rameshwar Gholve, et al. (2021): The chromatographic analysis was accomplished at ambient temperature on
Xttera RP18 (100 x 4.6 mm, 3.5 µm) column and 1 mL/min flow rate by using Eluent composed of 10 mM phosphate
buffer pH 2.6 with Acetonitrile (300:700, v/v). The UV detection at the wavelength of 240 nm was carried out using 20
µL injection volume. The Cilnidipine retention time was found to be 3.029 min. The method in the range of 40.0573 –
120.1719 µg/mL was found to be linear (R2 = 0.999) with a detection limit and quantitation limit of 1.2038 and
3.6478 μg/mL, respectively. The mean recovery % over the three tested levels of 50, 100, and 150% were found to be
98.74, 99.60, and 98.23%, respectively. The mean % assay of 99.29 for method repeatability and 98.82 for
intermediate precision were found with % RSD of 0.68 and 0.31, respectively.

20. 20
REVIEW OF LITERATURE
19. Hitendra S. Joshi, et al. (2013): Isocratic RP-HPLC method was developed on Waters C18 250 mm x 4.6mm, 5
 
μm column using mobile phase as acetonitrile (ACN): buffer pH 3.0 with orthophosphoric acid (68:
 32) at a flow rate
of 1.0
 mL/min and the detection was carried out at 245 nm using photodiode array detector. Forced degradation
study was carried out by oxidation, hydrolysis, photolysis, and heating the drug. The method was validated for
specificity, linearity, precision, accuracy, robustness, and solution stability. The method was found to be linear in the
concentration range of 40–160
 μg/mL with correlation coefficient of 0.9990 for Telmisartan and 10–40 μg/mL with
correlation coefficient of 0.9989 for Cilnidipine.
20. Pravin Y. Khandagale, et al. (2017): A specific, accurate, precise and reproducible RP-HPLC method development
validation for simultaneous estimation of Cilnidipine and Telmisartan in combined pharmaceutical dosage form. The
proposed HPLC method utilizes Youglin C18 column (250 mm x 4.6 mm) 5 µm particle sizes and mobile phase
consisting of Acetonitrile: 0.05% ortho phosphoric acid (60: 40) at a flow rate of 0.7 ml/min with UV detection 236
nm. The retention time of Cilnidipine and Telmisartan were found to be 4.650 min and 8.050 min respectively. The
method was validated in terms of accuracy, precision, Linearity and robustness and was successfully applied for the
determination of investigated drugs in combined dosage form.

21. 21
REVIEW OF LITERATURE
21. Ms. Priyal Shah, et al. (2021): The developed method consisted of mobile phase Methanol: water (50:50 v/v).
The flow rate was 0.8 mL/min with a run time of 10 min and detection at 270 nm. The assay was performed on
marketed formulation that showed 99.52% labelled claim. During the forced degradation studies, Efonidipine showed
maximum degradation (10 %) under oxidative stress followed by 8 % photo degradation. The drug showed lower
degradation under acid, base and thermal stress conditions, to the extent of 4 %, 3 % and 6 % respectively. It was
also observed that the retention time of the degradants under photolytic and oxidative degradation were the same
probably due to the formation of the same product.
22. Kumar A, et al. (2017): A Reversed Phase High Performance Liquid Chromatographic (RP-HPLC) method using
symmetry C18, 5.0 mm column was developed for the determination of Efonidipine Hydrochloride Ethanolate (EFD).
The mobile phase acetonitrile and water ratio was selected 85: 15 via flow rate were 0.8 mL/min and elution was
monitored at 254 nm. Response was a linear function of concentration over the range 20-140 μg/ml (R2=0.9994)
and the limits of detection was 681.83 ng/ml. The limit of quantification was 2.06μg/ml. The coefficient of variation
for intra-assay and inter-assay precision was less than or equal to 1.5% and the accuracy was 104.0-105.0% and
method was validated accordance with International Conference on Harmonization (ICH) guidelines to check content
uniformity.

22. 22
REVIEW OF LITERATURE
23. Ch. Gopinath, et al (2023): The chromatographic system consisted of Inertsil ODS (4.6*250mm, 5µ) column and
the separation was achieved by using ambient temperature with a mobile phase containing mobile Phase 70% buffer
30% CAN, Phosphate Buffer pH 3.0. The samples were monitored at 225 nm for detection at a flow rate of 1.2
mL/min and the retention time was about 8 mins for Efonidipine Hydrochloride Fig.1 Ethanolate and Telmisartan
respectively. The calibration curve was linear over the concentration range 50-250 and 100-500 for Efonidipine
Hydrochloride Ethanolate and Telmisartan respectively. The proposed method is accurate in the range of 99.89% -
100.23% recovery and precise (%RSD of intraday variation and % RSD of inter day variation were found to be within
the acceptance criteria).
24. Swati Bharati, et al. (2023): The biological samples were prepared using the methanolic extraction procedure to
eliminate solvent effects. Using a C18 column, a pH-adjusted mobile phase of methanol and water (90:10, v/v) at a
flow rate of 1 mL/min, and UV detection at 252 nm was utilized for chromatographic separation. The retention time of
EFE was found to be 5.2 min. The developed bioanalytical method was validated with different validation parameters.
The validation parameter results are within the acceptable range of the International Council for Harmonization
guidelines. The method was successfully employed to evaluate the pharmacokinetic properties in Wistar albino rats
following a single oral administration of 10 mg/kg of EFE.

23. 23
REVIEW OF LITERATURE
25. Dr. Sushil D. Patil, et al. (2021): A Stability Indicating RP HPLC method was develop and validated for the
determination of Azelnidipine using Phenomenex C18 column (25 cm × 4.6 mm, 5μm) with mobile phase consisting
of Methanol: Water (75: 25% v/v). The flow rate was kept constant 1.0 mL/min and eluent was detected at 256 nm.
In calibration curve experiments, Linearity was found to be in concentration range 2-14 μg/ml (R2=0.9985) with
regression equation y = 160134x + 3313.7 Azelnidipine was subject to stress condition including alkaline, acidic,
oxidation, wet heat, thermal degradation and photolysis. Azelnidipine is more sensitive towards acid degradation. Also
there was no interference of excipient and degradation product at retention time of Azelnidipine, indicating specificity
of the method.

24. OBJECTIVES:
To develop RAPID, simple, precise, accurate and ECONOMIC analytical method for the determination of Cilnidipine
in bulk form and Marketed pharmaceutical dosage form.
To develop simple method which focuses on improving a better method when compared to previously reported
analytical methods
To develop Simple, Cost effective RP HPLC method for Estimation of Cilnidipine in bulk & pharmaceutical dosage
form.
To Validate the developed method as per ICH guidelines.
24
AIM :
The main aim of the present study is to determine the method development and validation for the estimation of
Cilnidipine in bulk form and marketed tablets dosage forms by RP-HPLC.

25. 25

26. 26
Collection of literature for selected drug
Extensive literature survey for selection of appropriate solvents to dissolve respective selected drug and
preparation of stock solution
Study of drug profile
Selection of chromatographic conditions
Selection of mobile phase
Method trails on HPLC by using different solvents and columns
Development of RP-HPLC method which is different from the finished articles
Setting up of beginning UV and chromatographic conditions for the strategy improvement in unadulterated and
pharmaceutical dose shapes.
Optimization of initial chromatographic and spectrophotometric conditions.
Analytical method validation of the developed RP- HPLC method.
Carryout stability studies to find out stability related impurities.
Quantitative determination of Cilnidipine in pharmaceutical dosage form using the method developed and
validated.

27. 27

28. 28
LIST OF INSTRUMENTS USED
S. No.Instruments/Equipments/Apparatus
1. HPLC with Empower2 Software with Isocratic with UV-Visible Detector (Waters).
2. T60-LAB INDIA UV – Vis spectrophotometer
3. Electronic Balance (SHIMADZU ATY224)
4. Ultra Sonicator (Wensar wuc-2L)
5. Thermal Oven
6. Symmetry ODS RP C
18,5m, 15mm x 4.6mm i.d.
7. P
H
Analyzer (ELICO)
8. Vacuum filtration kit (BOROSIL)

29. 29
LIST OF CHEMICALS USED
 
S.No.
 
Name
Specifications  
Manufacturer/SupplierPurity Grade
1. Doubled distilled water 99.9% HPLC Sd fine-Chem ltd; Mumbai
2. Methanol 99.9% HPLC Loba Chem; Mumbai.
3. Dipotassium hydrogen
orthophosphate
96% A.R. Sd fine-Chem ltd; Mumbai
4. Acetonitrile 99.9% HPLC Loba Chem; Mumbai.
5. Potassium dihydrogen
orthophosphate
99.9% A.R. Sd fine-Chem ltd; Mumbai
6. Sodium hydroxide 99.9% A.R. Sd fine-Chem ltd; Mumbai
7. Hydrochloric acid 99.9% A.R. Loba Chem; Mumbai.
8. Hydrogen Peroxide 99.9% A.R. Loba Chem; Mumbai.

30. 30

31. 31
Selection of Wavelength:
The Standard & Sample Stock Solutions were prepared separately by dissolving standard & sample in a solvent in
mobile phase diluting with the same solvent. (After optimization of all conditions) for UV analysis. It scanned in the
UV spectrum in the range of 200 to 400nm. This has been performed to know the maxima of Cilnidipine, so that
the same wave number can be utilized in HPLC UV detector for estimating the Cilnidipine. The scanned UV
spectrum is attached in the following page,
Sample & Standard Preparation for the UV-Spectrophotometer Analysis:
25 mg of Cilnidipine standard was transferred into 25 ml volumetric flask, dissolved & make up to volume with
mobile phase.
Further dilution was done by transferring 0.5 ml of the above solution into a 10ml volumetric flask and make up to
volume with mobile phase.

32. 32
Selection of Wavelength:
Observation: While scanning the Cilnidipine solution we observed the maxima at 235nm. The UV spectrum
has been recorded on T60-LAB INDIA make UV – Vis spectrophotometer model UV-2450.

33. 33
Preparation of Standard Solution:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark
with the same Methanol.
Further pipette 0.1ml of the above Cilnidipine stock solutions into a 10ml volumetric flask and dilute up to the
mark with Methanol.
Procedure:
Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions
of proper peak elution for performing validation parameters as per ICH guidelines.
Mobile Phase Optimization:
Initially the mobile phase tried was Methanol and Methanol: Water with varying proportions. Finally, the mobile
phase was optimized to Acetonitrile: Methanol: 0.1% OPA in proportion 60:30:10% v/v/v.

34. 34
Optimization of Column:
The method was performed with various C18 columns like, X- bridge column, Xterra, and C18 column. Symmetry
ODS (C18) RP Column, 250 mm x 4.6 mm, 5µm was found to be ideal as it gave good peak shape and resolution at
1.0ml/min flow.
Preparation of 0.1% OPA Solution:
0.1% OPA was prepared by taking 1 ml of OPA in 1000 ml HPLC grade water. Filter and sonicate the solution
by vacuum filtration and ultra sonication.
Preparation of Mobile Phase:
600ml of HPLC Grade Acetonitrile, 300ml of HPLC Grade Methanol and 100ml 0.1% OPA were mixed well and
degassed in ultrasonic water bath for 15 minutes. The solution was filtered through 0.45 µm filter under vacuum
filtration.
Diluent Preparation:
The Mobile phase was used as the diluent.

35. 35
Trails for the Method Development:
Trail 1:
Chromatographic Conditions:
Column: Develosil C
18, ODS, Reverse Phase, 250
mm x 4.6 mm, 5µm, Column
Column temperature: 38°C
Wavelength: 235nm
Mobile phase ratio: Methanol : Acetonitrile = 40 : 60
Flow rate: 1.0ml/min
Injection volume: 20µl
Run time: 10.0min
Observation: In this trial it shows improper separation of peak in the chromatogram. So it
requires more trials to obtain good peaks.
Drug Name Rt Peak Area Tailing FactorPlate Count
Cilnidipine 7.840 6785463 0.54 2012

36. 36
Trails for the Method Development:
Trail 2:
Chromatographic Conditions:
Column: Inertsil C18, ODS, Reverse Phase, 250
mm x 4.6 mm, 5µm, Column
Column temperature: 35°C
Wavelength: 235nm
Mobile phase ratio: Methanol : Acetonitrile
= 55 : 45% v/v
Flow rate: 1.0ml/min
Injection volume: 15µl
Run time: 10min
Observation: In this trial it shows improper separation of peak in the chromatogram and
peak splitting was observed. So it requires more trials to obtain good peaks.
Drug Name Rt Peak Area Tailing Factor Plate Count
Cilnidipine 4.964 6458752 0.85 2415

37. 37
Trails for the Method Development:
Trail 3:
Chromatographic Conditions:
Column: Phenomenex Luna C18, ODS, Reverse
Phase, 250 mm x 4.6 mm, 5µm, Column
Column temperature: 30°C
Wavelength: 235nm
Mobile phase ratio: Acetonitrile : Water = 50:50%
v/v
Flow rate: 1.0ml/min
Injection volume: 15µl
Run time: 10.0min Observation: In this trial it shows improper separation of peak in the chromatogram and
stabilization was good. So it requires more trials to obtain good peaks.
Drug Name Rt Peak Area Tailing FactorPlate Count
Cilnidipine 3.343 552545 0.79 2931

38. 38
Trails for the Method Development:
Trail 4:
Chromatographic Conditions:
Column: Symmetry C18, ODS, Reverse Phase, 250
mm x 4.6 mm, 5µm, Column
Column temperature: Ambient
Wavelength: 235nm
Mobile phase ratio: Methanol : Water = 70:30% v/v
Flow rate: 1.0ml/min
Injection volume: 10µl
Run time: 7.0min
Observation: In this trial it shows broad peak and tailing factor is there in the
chromatogram. So it requires more trials to obtain good peaks.
Drug Name Rt Peak Area Tailing Factor Plate Count
Cilnidipine 2.614 3265845 1.04 2852

39. 39
Trails for the Method Development:
Trail 5:
Chromatographic Conditions:
Column: Symmetry C18, ODS, Reverse Phase, 250
mm x 4.6 mm, 5µm, Column
Column temperature: Ambient
Wavelength: 235nm
Mobile phase ratio: ACN : Methanol: 0.1% OPA =
70:25:5% v/v/v
Flow rate: 1.0ml/min
Injection volume: 10µl
Run time: 7.0min Observation: In this trial it shows broad peak and tailing factor is there in the
chromatogram. So it requires more trials to obtain good peaks.
Drug Name Rt Peak Area Tailing FactorPlate Count
Cilnidipine 2.685 7254681 1.08 3124

40. 40
Trails for the Method Development:
OPTIMIZED CHROMATOGRAPHIC CONDITION:
Chromatographic Conditions:
Column: Symmetry ODS (C18) RP Column, 250 mm x
4.6 mm, 5µm
Column temperature: Ambient
Wavelength: 235nm
Mobile phase: ACN : Methanol: 0.1% OPA = 60:30:10%
v/v/v
Flow rate: 1.0ml/min
Injection volume: 10µl
Run time: 6.0min
Observation: In this trial it shows proper separation of peak and more plate count in the
chromatogram and the tailing factor is within the limit. So it is an optimized chromatogram.
Drug Name Rt Peak Area Tailing Factor Plate Count
Cilnidipine 2.631 203654 1.18 3152

41. 41

42. 42
SYSTEM SUITABILITY:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks
add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution)
Further pipette 0.1ml of the above Cilnidipine stock solution into a 10ml volumetric flask and dilute up to the
mark with diluents.
Procedure:
The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD
for the area of five replicate injections was found to be within the specified limits.

43. 43
SYSTEM SUITABILITY: Results of System Suitability for Cilnidipine
S.No. Parameter Limit Result
1 Resolution Rs  2 8.47
2 Asymmetry T  2 Cilnidipine=0.23
3 Theoretical plateN  2000 Cilnidipine=2987
4 Tailing Factor T<2 Cilnidipine=1.17

44. 44
SPECIFICITY:
Preparation of Standard Solution:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7ml of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution). Further pipette 0.1ml of the above Cilnidipine stock solutions into a 10ml volumetric flask
and dilute up to the mark with diluents.
Preparation of Sample Solution:
Weight 10 mg equivalent weight of Cilnidipine sample into a 10mL clean dry volumetric flask and add about 7mL of
Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent.
Further pipette 0.1ml of Cilnidipine above stock solution into a 10ml volumetric flask and dilute up to the mark with
diluent.
Procedure:
Inject the blank and standard injections of Cilnidipine and check the any interference of impurities:

45. 45
SPECIFICITY:
Observation: In this test method blank, standard solutions were analyzed individually to examine the
interference. The above chromatograms show that the active ingredient was well separated from blank and
their excipients and there was no interference of blank with the principal peak. Hence the method is specific.
Chromatogram of Blank Solution Chromatogram of Cilnidipine Standard Solution

46. 46
LINEARITY AND RANGE:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7ml of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution)
Preparation of Level – I (6ppm of Cilnidipine):
Take 0.6ml of stock solution in to 10ml of volumetric flask and make up the volume up to mark with diluents and
sonicate the solution for bubble entrapment using ultrasonicator.
Preparation of Level – II (8ppm of Cilnidipine):
Take 0.8ml of stock solution in to 10ml of volumetric flask and make up the volume up to mark with diluents and
sonicate the solution for bubble entrapment using ultrasonicator.
Preparation of Level – III (10ppm of Cilnidipine):
Take 0.1ml of stock solution in to 10ml of volumetric flask and make up the volume up to mark with diluents and
sonicate the solution for bubble entrapment using ultrasonicator.

47. 47
LINEARITY AND RANGE:
Preparation of Level – IV (12ppm of Cilnidipine):
Take 0.12ml of stock solution in to 10ml of volumetric flask and make up the volume up to mark with diluents and
sonicate the solution for bubble entrapment using ultrasonicator.
Preparation of Level – V (14ppm of Cilnidipine):
Take 0.14ml of stock solution in to 10ml of volumetric flask and make up the volume up to mark with diluents and
sonicate the solution for bubble entrapment using ultrasonicator.
Procedure:
Inject each level into the chromatographic system and measure the peak area.
Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the
correlation coefficient.

48. 48
LINEARITY AND RANGE: Linearity Data of Cilnidipine
CONC.(µg/ml)MEAN AUC (n=6)
0ppm 0
6ppm 129013
8ppm 166523
10ppm 198315
12ppm 234151
14ppm 275819

49. 49
LINEARITY AND RANGE:
Calibration Curve of Cilnidipine

50. 50
PRECISION
REPEATABILITY
Preparation of Cilnidipine Product Solution for Precision:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7ml of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution)
Further pipette 0.1ml of the above Cilnidipine stock solutions into a 10ml volumetric flask and dilute up to the mark
with diluents.
Procedure:
The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for
the area of six replicate injections was found to be within the specified limits.

51. 51
PRECISION
REPEATABILITY
Results of Repeatability for Cilnidipine
HPLC Injection
Replicates of Cilnidipine
Retention Time
(Minutes)
Peak Area
(AUC)
Replicate – 1
2.572 197236
Replicate – 2
2.570 197762
Replicate – 3
2.573 195969
Replicate – 4
2.570 194724
Replicate – 5
2.574 198327
Replicate – 6
2.573 198711
Average
  197121.5
Standard Deviation
  1515.213
% RSD
  0.768667

52. 52
PRECISION
INTERMEDIATE PRECISION:
To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on
different days by maintaining same conditions.
Procedure:
Analyst 1:
The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for
the area of six replicate injections was found to be within the specified limits.
Analyst 2:
The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for
the area of six replicate injections was found to be within the specified limits.

53. 53
PRECISION
INTERMEDIATE PRECISION:
Results of Intra-Assay & Inter-Assay
Conc. of
Cilnidipine(API)
(µg/ml)
Observed Conc. of Cilnidipine (µg/ml) by the proposed method
Intra-Day Inter-Day
Mean (n=6) % RSD Mean (n=6) % RSD
8 7.46 0.62 8.05 0.96
10 10.87 0.85 9.43 0.71
12 11.81 0.92 12.04 0.65

54. 54
ACCURACY:
For preparation of 80% Standard stock solution:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution)
Further pipette 0.08ml of the above Cilnidipine stock solution into a 10ml volumetric flask and dilute up to the
mark with diluents.
For preparation of 100% Standard stock solution:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution)
Further pipette 0.1ml of the above Cilnidipine stock solution into a 10ml volumetric flask and dilute up to the mark
with diluents.

55. 55
ACCURACY:
For preparation of 120% Standard stock solution:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stock solution)
Further pipette 0.12ml of the above Cilnidipine stock solution into a 10ml volumetric flask and dilute up to the
mark with diluents.
Procedure:
Inject the Three replicate injections of individual concentrations (80%, 100%, 120%) were made under the
optimized conditions. Recorded the chromatograms and measured the peak responses. Calculate the Amount
found and Amount added for Cilnidipine and calculate the individual recovery and mean recovery values.

56. 56
ACCURACY:
Accuracy Results for Cilnidipine
Conc. In ppm Conc. Found Peak Area % Recovery
8 8.035 161523 100.437
8 8.153 163815 101.912
8 8.061 162023 100.762
Avg. 101.037
S.D 0.775
%RSD 0.767046
Conc. In ppm Conc. Found Peak Area % Recovery
10 9.930 198315 99.30
10 10.033 200320 100.33
10 10.044 200540 100.44
Avg. 100.0233
S.D 0.628835
%RSD 0.628688
Conc. In ppm Conc. Found Peak Area % Recovery
12 11.981 238151 99.841
12 12.066 239819 100.55
12 12.215 242712 101.791
Avg. 100.7273
S.D 0.987021
%RSD 0.979894

57. 57
LIMIT OF DETECTION:
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which
can be detected but not necessarily quantitated as an exact value.
LOD = 3.3 × σ / s
Where
σ = Standard deviation of the response
S = Slope of the calibration curve
Result:
= 0.08µg/ml

58. 58
LIMIT OF QUANTITATION:
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample
which can be quantitatively determined.
LOQ = 10×σ/S
Where
σ = Standard deviation of the response
S = Slope of the calibration curve
Result:
= 0.24µg/ml

59. 59
ROBUSTNESS:
The analysis was performed in different conditions to find the variability of test results. The following conditions are
checked for variation of results. .
For Preparation of Standard Solution:
Accurately weigh and transfer 10 mg of Cilnidipine working standard into a 10ml of clean dry volumetric flasks add
about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent.
(Stock solution). Further pipette 0.1ml of the above Cilnidipine stock solution into a 10ml volumetric flask and dilute
up to the mark with diluents.
Procedure:
Influence of small changes in chromatographic conditions such as change in flow rate ( 0.1ml/min), Wavelength of
detection (2nm) & organic phase in mobile phase (5%) studied to determine the robustness of the method are
also in favour of (Table-6.25, % RSD < 2%) the developed RP-HPLC method for the analysis of Cilnidipine (API).

60. 60
ROBUSTNESS: Results for Robustness for Cilnidipine
Change in Parameter % RSD
Flow (1.1 ml/min) 0.68
Flow (0.9 ml/min) 0.39
More Organic 0.54
Less Organic 0.63
Wavelength of Detection (237 nm) 0.91
Wavelength of detection (233 nm) 0.93

61. 61
Estimation of Cilnidipine in Pharmaceutical Dosage Form (Assay):
Assay Results for Cilnidipine
Brand Name of
Cilnidipine
Labelled amount of
Drug (mg)
 
Mean ( SD) amount
(mg) found by the
proposed method (n=6)
Assay % ( SD)
Cilaheart-10 Tablet
(Mankind)
10mg 9.524 ( 0.635) 99.574 ( 0.258)

62. 62

63. 63
The results of the stress studies indicated the specificity of the method that has been developed. Cilnidipine was
stable in thermal and photolytic stress conditions. The result of forced degradation studies are given in the
following table.
Results of Forced Degradation Studies for Cilnidipine
Stress Condition Time Assay of active
substance
Assay of degraded
products
Mass Balance
(%)
Acid Hydrolysis (0.1 M
HCl)
24Hrs. 81.36 18.64 100.0
Basic Hydrolysis (0.I M
NaOH)
24Hrs. 83.37 16.63 100.0
Thermal Degradation (50
0
C)
24Hrs. 98.92 1.08 100.0
UV (254nm) 24Hrs. 96.33 3.67 100.0
3 % Hydrogen peroxide24Hrs. 89.41 10.59 100.0

64. 64

65. 65
To develop a precise, linear, specific & suitable stability indicating RP-HPLC method for analysis of Cilnidipine,
different chromatographic conditions were applied & the results observed are presented in previous chapters.
Isocratic elution is simple, requires only one pump & flat baseline separation for easy and reproducible results.
So, it was preferred for the current study over gradient elution.
In case of RP-HPLC various columns are available, but here Symmetry ODS RP C18,5µm, 15mmx4.6mm i.d.
Column was preferred because using this column peak shape, resolution and absorbance were good.
Mobile phase & diluent for preparation of various samples were finalized after studying the solubility of API in
different solvents of our disposal (methanol, Acetonitrile, dichloromethane, water, 0.1N NaOH, 0.1NHCl).
The drug was found to be Soluble in DMSO, ethanol, DMF, water, methanol and acetonitrile and slightly
soluble in dichloro methane. Utilizing these solvents with suitable arrangement more current techniques
can be created and approved.

66. 66
Discovery wavelength was chosen in the wake of examining the standard arrangement of medication more than
200 to 400nm. From the U.V range of Cilnidipine it is apparent that a large portion of the HPLC works can be
proficient in the wavelength scope of 210-300 nm helpfully. Further, a stream rate of 1 ml/min and an infusion
volume of 10µl were observed to be the best investigation.
The outcome demonstrates the created technique is amazingly, one more reasonable strategy for measure and
dependability related debasement examines which can help in the investigation of Cilnidipine in various details.

67. 67

68. 68
A sensitive& selective RP-HPLC method has been developed & validated for the analysis of Cilnidipine in Bulk
form and Marketed Pharmaceutical Dosage form by using RP-HPLC.
Encourage the proposed RP-HPLC technique has astounding affectability, accuracy and reproducibility.
The outcome demonstrates the created technique is amazingly, one more appropriate strategy for test,
immaculateness and solidness which can help in the examination of Cilnidipine in various definitions.

69. 69

70. 70
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