INTRODUCTION:

Validation is defined as establishing document evidences which provides a high degree assurance that a specific process will consistently produce a product meeting it’s pre-determined specification and quality characteristics^1-5.

This medication is used to treat Amlodipine is a medicine used to treat high blood pressure (hypertension). If you have high blood pressure, taking amlodipine helps prevent future heart disease, heart attacks and strokes. Amlodipine is also used to prevent chest pain caused by heart disease (angina). Perindopril is used to treat high blood pressure (hypertension)^6-8. Lowering high blood pressure helps prevent strokes, heart attacks, and kidney problems. This medication is also used to prevent heart attacks in patients with a certain type of heart disease (stable coronary artery disease).^9-10

MATERIAL AND METHODS:

HPLC, WATERS, software: Empower 2, 2695 separation module. 996 PDA detector. pH meter-Lab India, Weighing machine-Sartorius, Pipettes and Burettes Borosil, Beakers Borosil, Digital ultra sonicator Enertech, Amlodipine, Perindopril, Water and Methanol for HPLC-LICHROSOLV (MERCK) Anhydrous di hydrogen phosphate- Finar chemicals, Phosphate Buffer -Finar chemicals, Citric Acid- Finarchemicals

Experimental Work:

The experimental work of stability indicating HPLC for the estimation of amlodipine, perindopril

HPLC Method Development:

Mobile Phase Optimization:

Initially the mobile phase tried was Water: Methanol and ACN: Phosphate Buffer ACN: Methanol with varying proportions. Finally, the mobile phase was optimized to phosphate buffer (pH 3), Methanol in proportion 30:70 v/v respectively.

Optimization of Column:

The method was performed with various columns like C18 column ODS column, Zodiac column, and Xterra C18 column. Phenomenex Luna C18 (4.6 x 150mm, 5mm) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

Optimized Chromatographic Conditions:

Instrument used: Waters HPLC with auto sampler and PDA detector 996 model.

Temperature: Ambient

Column: Phenomenex Luna C18 (4.6mm×250mm) 5µm

Buffer: Phosphate buffer (pH-3)-Dissolve 0.9g of anhydrous di hydrogen phosphate and 1.298g of Citric acid mono hydrate in sufficient water to produce 1000ml. Adjust the pH-3 by using ortho phosphoric acid.

pH: 3.0

Mobile phase: Methanol: Phosphate Buffer pH-3 (70:30v/v)

Flow rate: 1ml per min

Wavelength: 230nm

Injection volume: 10ml

Run time: 6min.

Preparation of Phosphate buffer (pH-3.0):

Dissolve 0.9g of anhydrous dihydrogen phosphate and 1.298g of Citric acid mono hydrate in sufficient water to produce 1000mL. Adjust the pH-3 by using ortho phosphoric acid.

Preparation of mobile phase:

Accurately measured 700ml (70%) of Methanol and 300 ml of Phosphate buffer pH-3 (30%) were mixed and degassed in digital ultrasonicater for 15 minutes and then filtered through 0.45µ filter under vacuum filtration.

Diluent Preparation:

The Mobile phase was used as the diluent.

Preparation of Standard Solution:

Accurately weigh and transfer 10mg of Amlodipine and 10mg of Perindopril working standard into a 10ml and 10ml of clean dry volumetric flasks add about 10ml and 10ml of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further pipette 0.48ml of Amlodipine from stock solution and 0.3ml of Perindopril into a 10ml volumetric flask and dilute up to the mark with diluents.

Preparation of Sample Solution:

Accurately weigh 10 combination tablets crush in mortar and pestle and transfer equivalent to 10mg of Amlodipine, Perindopril (marketed formulation-dose of Perindopril is 300mg, Dose of Amlodipine is 150mg in combination tablet) sample into a 10ml clean dry volumetric flask 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 0.48ml of above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. The standard and sample solutions of 30µg/ml of Perindopril, 48µg/ml of Amlodipine are injected for five times and the peak areas were recorded.

Chromatographic peaks:

Fig:1 chromatographic peaks of amlodipine and perindopril

Table1: Peak Results for Trail-1:

S. No	Peak Name	Rt	Area	Height	USP Resolution	USP Tailing	USP Plate count
1	Amlodipine	3.525	242286	20725		0.85	1644

Table: 2 Peak Results for Trail-2

S. No	Peak Name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Amlodipine	3.914	16388	16388		1.3	1152
2	Perindopril	5.218	627642	48723	2.1	2.3	746

Table:3 Peak Results for Trail-3

S. No	Peak name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Amlodipine	1.676	331978	70137		0.69	278
2	Perindopril	2.012	522972	83622	9.63	1.00	9218

Table:4 Peak Results for Trail-4

S. No	Peak Name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Amlodipine	1.966	175068	15332		1.13	936
2	perindopril	2.264	792682	36779	8.06	0.97	863

Table: 5 Peak Results for Trail-5

S. No.	Peak name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Amlodipine	1.870	5664027	299752			2314
2	Perindopril	2.499	5033532	210321	4.6	1.3	2921

Observation:

This trial shows improper separation of sample peaks in the chromatogram, so more trials were required for obtaining good peaks.

Observation:

This trial show improper separation of sample peaks and show very less plate count, improper baseline in the chromatogram, so more trials were required for obtaining good peaks.

Observation:

This trial shows improper baseline and show less plate count in the chromatogram, so more trials were required for obtaining peaks.

Observation:

This trial shows improper baseline and peak in the chromatogram, so more trials were required for obtaining good peaks.

Observation:

From the above chromatogram it was observed that the Amlodipine and Perindopril peaks are well separated and they shows proper retention time, resolution, peak tail and plate count. So it’s optimized trial.

Retention time of Amlodipine–1.870min

Retention time of Perindopril – 2.499min

System Suitability:

Acceptance criteria

It was found from above data that all the system suitability parameters for developed method were within the limit.

Table:7Assay results

S. No	Name of compound	Label claim	Amount taken (from combination tablet)	% purity
1	Amlodipine	5mg	4.92	99.852%
2	Perindopril	4mg	3.86	99.764%

The % purity of Amlodipine and Perindopril in pharmaceutical dosage form was found to be 99.9% and 99.9% respectively

ACCURACY:

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the % recovery was calculated.

Table:8 Accuracy (recovery) data for Amlodipine

% Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	2771991	15	14.9	98%	99.1%
100%	5664027	30	29.99	99.9%
150%	8337191	45	44.95	99.6%

Acceptance Criteria:

The % Recovery for each level should be between 98.0 to 102.0%.

Table:9 Accuracy (recovery) data for Perindopril

% Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	2426681	24	23.9	98%	98.8%
100%	5033532	48	47.92	99.2%
150%	7419721	72	71.9	99.3%

Acceptance Criteria:

The percentage recovery was found to be within the limit (98-102%).

The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence method is accurate.

Linearity:

The linearity range was found to lie from 10µg/ml to 50µg/ml of Amlodipine, 16µg/ml to 80µg/ml of Perindopril

Linearity Results: (forAmlodipine)

Fig:2 Calibration Graph of Amlodipine

Table:10 Linearity Results for Amlodipine

S. No	Linearity Level	Concentration (ppm)	Area
1	I	10	892464
2	II	20	1866364
3	III	30	2777423
4	IV	40	3709213
5	V	50	4601317
Correlation Coefficient			0.999

Acceptance Criteria:

Correlation coefficient should be not less than 0.999.

Linearity Results: (for Perindopril)

Fig:3 Calibration Graph of perindopril

Table: 11 Linearity Results for Perindopril

S. No.	Linearity Level	Concentration (ppm)	Area
1	I	10	920032
2	II	20	1752782
3	III	30	2521426
4	IV	40	3326009
5	V	50	4217393
Correlation Coefficient			0.999

Acceptance Criteria:

Correlation coefficient should be not less than 0.99.

CONCLUSION:

High performance liquid chromatography is at present one of the most sophisticated tool of the analysis. The estimation of Amlodipine and Perindopril was done by RP-HPLC. The Phosphate buffer was pH-3 and the mobile phase was optimized with consists of Methanol: Phosphate buffer (pH-3) mixed in the ratio of 70:30% v/v. A Phenomenex Luna column C18 (4.6 x 150mm, 5mm) or equivalent chemically bonded to porous silica particles was used as stationary phase. The solutions were chromatographed at a constant flow rate of 1 ml/min. The linearity range of Amlodipine and Perindopril were found to be from 10-50mg/ml, 16-80mg/ml respectively. Linear regression coefficient was not more than 0.999, 0.999.

The values of %RSD are less than 2% indicating accuracy and precision of the method. The percentage recovery varies from 99.9-99.9% of Amlodipine and Perindopril. LOD and LOQ were found to be within limits.

REFERENCES:

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2. Md. Ahsanul Haque, Mohammad Shahriar, Most. Nazma Parvin, S. M. Ashraful Islam. Validated RP-HPLC Method for Estimation of Ranitidine Hydrochloride, Domperidone and Naproxen in Solid Dosage Form. Asian J. Pharm. Ana. 1(3): July-Sept. 2011; Page 59-63.

3. Sharmin Reza Chowdhury, MahfuzaMaleque, Mahbubul Hoque Shihan. Development and Validation of a Simple RP-HPLC Method for Determination of Caffeine in Pharmaceutical Dosage Forms. Asian J. Pharm. Ana. 2(1): Jan.-Mar. 2012; Page 01-04

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5. D. Sridharan, Umarani A. Thenmozhi, L. Pavan Kumar, AswaniDuttChintalapati, M. Venkata Ramanaiah, YelikaPhanikishore. Development and Validation of UV Spectrophotometric Method of Darifenacin Hydrobromide in Bulk and Tablet Dosage Form. Asian J. Pharm. Ana. 1(3): July-Sept. 2011; Page 43-45.

6. L. Satyanarayana, S.V. Naidu, M. Narasimha Rao, Reddy Suma Latha. The Estimation of Nilotinib in Capsule dosage form by RP-HPLC. Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 100-102.

7. A. Thenmozhi, D. Sridharan, S. Veeramani, M. Palanivelu. An RP-HPLC Method for the estimation of Dexibuprofen in Pharmaceutical Tablet Dosage Form. Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 98-99.

8. Mahmoud M. Sebaiy, Abdullah A. El-Shanawany, Sobhy M. El-Adl, Lobna M. Abdel-Aziz, Hisham A. Hashem. Rapid RP-HPLC Method for Simultaneous Estimation of Norfloxacin and Tinidazole in Tablet Dosage Form. Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 79-84.

9. Md. Ahsanul Haque, Mohammad Shahriar, Most. Nazma Parvin, S. M. Ashraful Islam. Validated RP-HPLC Method for Estimation of Ranitidine Hydrochloride, Domperidone and Naproxen in Solid Dosage Form. Asian J. Pharm. Ana. 1(3): July-Sept. 2011; Page 59-63.

10. Akhilesh Gupta, Jaydeep Singh Yadav, Swati Rawat, Mayuri Gandhi. Method Development and Hydrolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J. Pharm. Ana. 1(1): Jan.-Mar. 2011; Page 14-18.

Received on 26.05.2022 Modified on 29.07.2022

Res. J. Pharma. Dosage Forms and Tech.2022; 14(4):289-292.

DOI: 10.52711/0975-4377.2022.00047