Cilindipine Final Edited

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ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF CILNIDIPINE

BY UHPLC IN SOLID DOSAGE FORMS


A Dissertation submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR
ANDHRA PRADESH, INDIA

In partial fulfillment of the requirements


For the award of degree in
BACHELOR OF PHARMACY
Submitted By
A. INDUMATHI (16CP1R0002),
A .KOKILA(16CP1R0003) C. HEMAPRIYA(16CP1R0008)
M.WASIM AKRAM(16CP1R0027) S.AMJAD ALI(16CP1R0042)

Under the guidance of

G.Deepthi, M.Pharm,
Assistant Professor
Department of Pharmaceutical analysis

SESHACHALA COLLEGE OF PHARMACY


NAGARI ROAD, PUTTUR -517 583, A.P
AUGUST- 2020
Introduction:
• Cilnidipine is a dihydropyridine calcium antagonist.

• Cilnidipine is indicated for the management of hypertension for end-organ

protection.

•  It is reported to be useful in elderly patients and in those with diabetes and

albuminuria.

• Cilnidipine has been increasingly used in patients with chronic kidney disease.

• So far, several analytical methods using high-performance liquid chromatography (HPLC) combined

with ultraviolet/visible (UV) and have been reported for the quantitative determination of Cilnidipine.

• However, these methods have some limitations and need for improvements

• So in this study we aim to develop a reliable validated analytical method for estimation of Cilnidipine using UHPLC.
LITERATURE REVIEW:

• Kadam, A. & Hamrapurkar, P. & Patil, S. & Manoharan, M. & Suryagandha, A..
(2015). Development and validation of stability indicating RP-HPLC method for
the estimation of cilnidipine in bulk and pharmaceutical dosage form.
International Journal of Pharmaceutical Sciences Review and Research. 30. 177-
181.

• Parihar, Y. & Kotkar, T. & Mahajan, M.P. & Sawant, S.D.. (2014). Development
and validation of RP-HPLC method for simultaneous estimation of telmisartan
and cilnidipine in bulk and tablet dosage form. Pharmanest. 5. 2321-2325.

• Minase, A. S., M. N. Dole, and S. D. Sawant. “DEVELOPEMENT AND VALIDATION


OF ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF CILNIDIPINE AND
OLMESARTAN MEDOXOMIL IN BULK AND TABLET DOSAGE FORM BY RP-
HPLC”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 6,
no. 7, July 2014, pp. 508-11,
DRUG PROFILE
DRUG PROFILE: Cilnidipine:
Structure:

IUPAC Name: 3-O-(2-methoxyethyl) 5-O-[(E)-3-phenylprop-2-enyl]


2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-
dicarboxylate

Molecular formula: C27H28N2O7

Molecular weight 492.5 g/mol

Mechanism of action: Cilnidipine decreases blood pressure and is used


to treat hypertension and its comorbidities. Due to
its blocking action at the N-type and L-type
calcium channel, cilnidipine dilates both arterioles
and venules, reducing the pressure in the capillary
bed
Side effects: dizziness, fast heartbeat, and swelling of face, lips,
tongue, eyelids, hands and feet,stomach pain,
diarrhea and hypotension.
Marketed Formulations of Cilindipine

Dosage
Brand Name Dose Manufacturer
Form

Cilindipine Tablet 10mg Cipla company


AIM AND OBJECTIVE:
• Present work is aimed to develop a new, simple,
fast, rapid, accurate, efficient and reproducible
UHPLC method for the estimation of Cilnidipine.
The developed method will be validated according
to ICH guidelines.

Objective of the work :


• The analytical method for the estimation of
Cilnidipine will be developed by UHPLC method by
optimizing the chromatographic conditions. 
• The developed method is validated according to
ICH guidelines for various parameters specified in
ICH guidelines, Q2.
MATERIALS & INSTRUMENTS
Materials:

• Cilnidipinetablet prepared at In house ,

• Distilled water,

• Acetonitrile,

• Potassium dihydrogen phosphate,

 Ortho-phosphoric acid.

( All the above chemicals and solvents are from Rankem)


Instruments:

•Ultrasonicator-BVK enterprises

•Electronics Balance-Denver

•pH meter -BVK enterprises, India

•UHPLC: Agilent technologies 1220 inifinity series


( Serial no:DECAG00308 )

 Software:
CHEM STATION_open lab
Chromatographic condition
Chromatographic condition:

• Column: Supelcosil LC18( 3.6 cm X


4.6 mm; 3.0 microns)
• Wave length: 245nm
• Flow rate: 0.6ml/min
• Injection volume: 2µl
• Mobile phase: Buffer:acetonitrile(30:70)
preparation of Solutions
Preparation of Standard solution:

• Weighed accurately and transferred about 49.96mg of Cilnidipine standard into a 50 ml


volumetric standard flask.
• Dissolved and madeup to the volume with mobile phase.
• Diluted 5 ml of this solution to 50 ml with mobile phase.

Preparation of Sample solution:

• Weighed accurately and transferred powdered sample equivalent to about 5mg of


Cilnidipine into a 50 ml volumetric standard flask.
• Dissolved and madeup to the volume with mobile phase.

Preparation of buffer:

• Buffer: Dissolved 0.68 gms of Potassium dihydrogen phosphate in 100 of water;


adjusted the pH 3.0 with phosphoric acid.
METHOD DEVELOPMENT
METHOD DEVELOPMENT :
• Numerous trails were conducted for optimized method with alteration in mobile
phase concentration and flow rate.

Validation:
The validation of the optimized method was carried out using the validation parameters

1. System suitability
2. Specificity
3. Linearity
4. Precision
5. Accuracy
6. Robustness
7. Assay
RESULTS AND DISCUSSION: METHOD DEVELOPMENT :
Method development was done by changing various, mobile phase ratios, buffers etc.
Trials:
Chromatograph Trail-1 Trail-2 Trail-3 Trail-4
ic conditions (optimized)

Mobile phase Buffer :Acetonitrile Buffer :Acetonitrile Buffer :Acetonitrile Buffer :Acetonitrile
(40:60) (50:50) (40:60) (30:70)
Flow rate 1.0ml/min 0.6ml/min 0.6ml/min 0.6ml/min

Column Supelcosil LC18; 3.6 cm Supelcosil LC18; 3.6 cm Supelcosil LC18; 3.6 cm Supelcosil LC18; 3.6 cm
X 4.6 mm; 3.0 microns X 4.6 mm; 3.0 microns X 4.6 mm; 3.0 microns X 4.6 mm; 3.0 microns

Detector wave length 245nm 245nm 245nm 245nm

Injection volume 2L 2L 2L 2L

Run time 3 min 3 min 3 min 3 min

Diluent Mobile phase Mobile phase Mobile phase Mobile phase

Results Peak was eluted but In this Trail peak In this Trail peak Retention time and
peak shape was not was eluted but more was eluted but more peak shape is good
good and more void tailing factor and retention time and
volume. so, Retention time. So Less plate count so,
further trial is further trail was further trail was
carried out carried out. Carried out.
Trial chromatogram 1 Trial chromatogram 2

Trial chromatogram 3 Optimized chromatogram


Optimized conditions:

Chromatographic conditions:

• Mobile phase: Buffer :Acetonitrile (30:70)


• Flow rate : 0.6ml/min
• Column : Supelcosil LC18; 3.6 cm X
4.6 mm; 3.0 microns
• Detector wave length: 245nm
• Injection volume: 2L
• Run time : 3 min
• Diluent : Mobile phase
• Results : Retention time and peak
shape is good.
Optimized chromatogram

Observation: Cilnidipinewas eluted at 0.81 min with good

resolution. Plate count and tailing factor was very

satisfactory, so this method was optimized and to be validated


Validation
Validation:

The validation of the optimized method was carried out using the
validation parameters

1. System suitability
2. Specificity
3. Linearity
4. Precision
5. Accuracy
6. Robustness
7. Assay
1.System suitability:
• All the system suitability parameters were within the range
and satisfactory as per ICH guidelines
Cilnidipine
Sample ID
RT AREA

Injection -01 0.81 592.916

Injection -02 0.81 596.256

Injection -03 0.81 597.856

Injection -04 0.81 597.957

Injection -05 0.81 603.035

Average : 0.81 597.604


SD : 0.00 3.66
System Suitability Chromatogram % RSD : 0.00 0.61

Discussion: As per IP 2018plate count should be more than


2000, tailing factor should be less than 2 and RSD Not more
than 2.0. All the system suitable parameters were passed and
were within the limits.
2.Specificity:

Chromatogram of blank.

Chromatogram of placebo
Standard Chromatogram

Cilnidipine
Discussion: Retention time of Sample ID
Cilnidipine was eluted at RT AREA

0.81min. We did not found BLANK No peak observed


and interfering peaks in blank 0.81

and placebo at retention times STANDARD


0.81
615.723

of these drugs in this method. PLACEBO No peak observed


0.81
So this method was said to be
specific.
3.Linearity:
LINEARITY SOLUTION DILUTIONS FINAL CONCENTRATION OBSERVED AREA
ml SSS diluted
2.50 50 ml 49.96 mcg/ml 307.267
to
ml SSS diluted
3.75 50 ml 74.94 mcg/ml 459.511
to
ml SSS diluted
5.00 50 ml 99.92 mcg/ml 606.063
to
ml SSS diluted
6.25 50 ml 124.90 mcg/ml 761.148
to
ml SSS diluted
7.50 50 ml 149.87 mcg/ml 916.826
to
   
X axis (Concentration) : 49.96 74.94 99.92 124.90 149.87  
Y axis (Observed area) : 307.267 459.511 606.063 761.148 916.826  

1000.000

900.000 916.826
f(x) = 6.09 x + 1.82
Discussion: Five linear 800.000 R² = 1
concentrations of Cilnidipine 700.000
761.148

was injected in a duplicate 600.000 606.063


manner. Average areas was 500.000
459.511
mentioned above and linearity 400.000
equations obtained for 300.000 307.267

Cilnidipine was y = 6.0884x + 200.000


1.8249. Correlation coefficient 100.000
obtained was 0.999 for the 0.000
Cilnidipine. 40.00 60.00 80.00 100.00 120.00 140.00 160.00
4.Precision:
4.1.System Precision:
Calculated
Assay
Conc. level Sample ID Sample wt. (mg) Sample Area
(in
percentage )
Sample -01 100.76 644.346 100.20
Sample -02 101.04 646.681 100.30
Sample -03 100.55 648.538 101.10
MIDDLE LEVEL (100%)
Sample -04 102.16 646.954 99.20
Sample -05 101.04 648.890 100.60
Sample -06 101.99 650.804 100.00
Average : 100.23

SD: 0.63
% RSD : 0.63

Discussion: From a single volumetric flask of working standard solution six injections
were given and the obtained areas were mentioned above. Average area, standard
deviation and % RSD was calculated for Cinidipine. % RSD obtained as 0.63% for
Cilnidipine.As the limit of Precision was less than “2” the system precision was
passed in this method
4.2.Intermediate precision :
Calculated Assay
Conc. level Sample ID Sample wt. (mg) Sample Area
(in percentage )

Sample -01 100.21 661.000 100.80


Sample -02 102.59 667.528 99.40
Sample -03 101.08 663.028 100.20
MIDDLE LEVEL (100%)
Sample -04 102.15 666.646 99.70
Sample -05 101.96 670.910 100.50
Sample -06 101.58 673.982 101.40
Average : 100.33

SD. : 0.73
% RSD : 0.73

Discussion: Multiple sampling from a sample stock solution was done and six
working sample solutions of same concentrations were prepared, each injection
from each working sample solution was given and obtained areas were mentioned
in the above table. Average area, standard deviation and % RSD was calculated for
Cilnidipineand obtained as 0.73% . As the limit of Precision was less than “2” the
system precision was passed in this method
5.Accuracy:
Sample
Conc. level Sample wt. (mg) Sample Area Calculated Assay (in percentage )
ID
Sample
51.18 319.786 101.10
-01
Sample
LOW LEVEL (50%) 52.34 321.460 99.40
-02
Sample
51.66 322.667 101.10
-03
Sample
101.79 627.188 99.70
-01
Sample
MIDDLE LEVEL (100%) 101.34 628.474 100.40
-02
Sample
100.69 630.618 101.40
-03
Sample
150.13 940.413 101.40
-01
Sample
HIGH LEVEL (150%) 152.66 941.413 99.80
-02
Sample
150.99 947.844 101.60
-03
Average : 100.66

SD. : 0.84
% RSD : 0.83

Discussion: Three levels of Accuracy samples were prepared by standard addition


method. Triplicate injections were given for each level of accuracy and mean
%Recovery was obtained as 99.40% and 101.60% for Cilnidipine.
6.Robustness:
Robustness data for Cilnidipine

%RSD of
S.No Condition
Cilnidipine

1 Flow rate (-) 0.60

2 Flow rate (+) 0.64

3 Wavelength (-) 0.75

4 Wavelength (+) 0.59


ROBUSTNESS - FLOW RATE DECREASE
Calculated
Conc. level Sample ID Sample wt. (mg) Sample Area Assay (in
percentage )

Sample -01 100.76 786.140 99.80

Sample -02 100.67 790.388 100.40

Sample -03 102.09 797.648 99.90


MIDDLE LEVEL (100%)
Sample -04 100.46 792.679 100.90

Sample -05 102.04 801.276 100.40

Sample -06 101.47 801.827 101.10

Average : 100.42

SD. : 0.52
% RSD : 0.52
ROBUSTNESS - FLOW RATE INCREASE
Calcula
ted
Sample wt. Assay
Conc. level Sample ID Sample Area
(mg) (in
percent
age )
Sample -01 100.76 534.910 100.00
Sample -02 100.67 534.196 99.90
Sample -03 102.09 534.891 98.70
MIDDLE LEVEL (100%)
Sample -04 100.46 538.136 100.90
Sample -05 102.04 537.966 99.30
Sample -06 101.47 540.887 100.40
Average : 99.87

SD. : 0.78
% RSD : 0.78
ROBUSTNESS - WAVE LENGTH DECREASE
Sample Sample wt. Sample Calculated Assay (in
Conc. level
ID (mg) Area percentage )
Sample
100.76 625.782 101.00
-01
Sample
100.67 628.438 101.50
-02
Sample
102.09 627.665 100.00
MIDDLE LEVEL -03
(100%) Sample
100.46 623.890 101.00
-04
Sample
102.04 633.627 101.00
-05
Sample
101.47 629.161 100.90
-06
Average : 100.90

SD. : 0.49
% RSD : 0.49
ROBUSTNESS WAVE LENGTH INCREASE
Sample wt. Sample Calculated Assay
Conc. level Sample ID
(mg) Area (in percentage )

Sample -01 100.76 827.333 99.90


Sample -02 100.67 826.775 99.90
Sample -03 102.09 835.849 99.60
MIDDLE LEVEL (100%)
Sample -04 100.46 832.609 100.80
Sample -05 102.04 828.921 98.80
Sample -06 101.47 842.390 101.00
Average : 100.00

SD. : 0.81
% RSD : 0.81

Discussion: Robustness conditions like Flow minus (0.4ml/min), Flow plus


(0.8ml/min), Wavelength minus and Wavelength plus was maintained and
samples were injected in duplicate manner. System suitability parameters were
not much affected and all the parameters were passed. %RSD was within the
limit.
7.Assay:
LABEL CLAIM:
   
CILNIDIPINE : 10 mg  
   
Average wt.of tablet : 100.0 mg  
   

CALCULATION :

   

Content in mg  

   
602.076 50.28 5 100 99.36
X X X X X 100.00  
597.604 50 50 101.06 100

   

.= 9.96 mg  

 
 

Content in % Limit : 98 % to 102 %  

.= 99..96 %  

 
 
Assay Chromatogram

Assay: Assay was performed with the above formulation.


Average

% Assay for Cilnidipine obtained was 99.96 % .


SUMMARY AND CONCLUSION
SUMMARY AND CONCLUSION:
Parameters Cilnidipine LIMIT
Linearity:
y = 6.0884x +
Regression equation R<1
1.8249(r2=0.99)
(Y=mx+c)
Assay (% mean assay) 99.96% 90-110%
No interference of any
Specificity Specific
peak
System precision %RSD 0.63 NMT 2.0%
Method precision
0.73 NMT 2.0%
%RSD

Accuracy %recovery 99.40% --101.60%


98-102%
FM 0.52
FP 0.78
Robustness WM 0.81 %RSD NMT 2.0
WP 0.49
Conclusion:
• A simple, Accurate, precise method was developed for the
estimation of the Cilnidipine in solid dosage form.
Retention time of Cilnidipine was found to be 0.81min .
%RSD of the Cilnidipine was found to be 0.61. %Recovery
was obtained as 99.40%--101.60% for Cilnidipine.
Regression equation of Cilnidipine is y = 6.0884x + 1.8249.
Retention times were decreased and that run time was
decreased, so the method developed was simple and
economical that can be adopted in regular Quality control
test in Industries.
BIBLIOGRAPHY:
• https://www.drugbank.ca/drugs/DB09232
• https://pubchem.ncbi.nlm.nih.gov/compound/5282138
• http://www.drugsupdate.com/generic/view/1129/Cilnidipine
• https://www.drugs.com/international/cilnidipine.html
• Simultaneous UV Spectrophotometric methods for estimation of Cilnidipine and
Telmisartan in Tablet Dosage Form. Inventi Rapid: Pharm Analysis & Quality Assurance,
2013.
• https://www.researchgate.net/journal1491_International_Journal_of_Pharmacy_and_Pharm
aceutical_Sciences
• O’Neil MJ, The Merk Index, An Encyclopedia of Chemicals, Drugs and Biologicals,
14th ed. Merck & Co. Inc.;2006:2275,6839
• P. Chaudhari, A. Bhalerao. Method validation for spectrophotometric estimation of
cilnidipine. International Journal of Pharmacy and Pharmaceutical Sciences 2012;4(5):96-
98.
• Mohammed Safhi, Spectrophotometric method for the estimation of cilnidipine in bulk and
pharmaceutical dosage forms. Oriental Journal of Chemistry 2013;29 (1):131-34.
• M.Haripriya, Neethu Antony, P. Jayasekhar. Development and validation of uv
spectrophotometric method for the simultaneous estimation of cilnidipine and telmisartan
in tablet dosage form utilising simultaneous equation and absorbance ratio method.
International Journal of Pharmacy and Biological Sciences 2013;3(1):343-48.
THANK YOU

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