INTRODUCTION:

Ramipril¹ is a 2-aza-bicyclo [3.3.0]-octane-3-carboxylic acid derivative. It is a white, crystalline substance soluble in polar organic solvents like methanol and buffered aqueous solutions.

It is practically insoluble in water, very slightly soluble in water. Ramipril melts between 105°C and 112°C. Ramipril chemical name is (2S, 3aS, 6aS) 1[(S) N(S) 1-carboxy-3-phenyl propyl] alanyl) octa hydro cyclo penta [b] pyrrole-2-carboxylic acid, 1ethyl ester. It has the chemical formula as C₂₃H₃₂N₂O₅ chemical formula and molecular weight about 416.51 g/mol. Its structural formula is shown in fig 1.

Figure 1: Chemical Structure of Ramipril

Ramipril and ramiprilat inhibit angiotensin-converting enzyme (ACE) in human subjects and animals. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin I to the vasoconstrictor substance, angiotensin II. Angiotensin II also stimulates aldosterone secretion by the adrenal cortex. Inhibition of ACE results in decreased plasma angiotensin II, which leads to decreased vasopressor activity and to decreased aldosterone secretion.

Amlodipine besylate² is white crystalline powder, freely soluble in polar organic solvents, slightly soluble in water. Chemically it is 3-Ethyl-5-methyl (±)-2-[(2-aminoethoxy)methyl]4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate, monobenzenesulphonate. It has chemical formula as C₂₀H₂₅CIN₂O₅•C₆H₆O₃S and has molecular weight about 567.10 g/mol. Its structural formula is shown in fig 2.

Figure 2: Chemical Structure of Amlodipine besylate

Amlodipine is a dihydropyridine calcium antagonist (calcium ion antagonist or slow-channel blocker) that inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle. Experimental data suggest that amlodipine binds to both dihydropyridine and nondihydropyridine binding sites. The contractile processes of cardiac muscle and vascular smooth muscle are dependent upon the movement of extracellular calcium ions into these cells through specific ion channels. Amlodipine inhibits calcium ion influx across cell membranes selectively, with a greater effect smooth muscle cells than on cardiac muscle cells effect.

Impurity profiling studies of drug substances and drug products in pharmaceutical industries play a vital role during drug/process development. Gathering of analytical data on impurities is important for early stage of pharmaceutical development. During the chemical process development of bulk drug, several impurities aroused from precursors, side products formed in the reaction and impurities related to the reagents used may be present at low levels^3,4. Separation of all these impurities including degradation impurities in a single analytical HPLC method is a challenging job. Moreover, stringent ICH guidelines are forcing to monitor and control the level of impurities to the specified limit in drug substances and drug products and hence there is a practical and scientific need to develop a suitable and efficient analytical method for analysis.

The literature survey reveals that only few methods were available for estimation of selected drugs simultaneously and individually by different analytical methods like RP-HPLC^5-11. Based on literature we observed that there is no stability indicating RP-HPLC method available for simultaneous estimation of selected drugs. So there is a need to develop a new accurate stability indicating RP-HPLC simultaneous estimation of selected drugs. The present work explains the development and validation of stability indicating RP-HPLC method for simultaneous estimation of Ramipril and amlodipine besylate.

EXPRERMENTAL METHODOLOGY:

Materials:

Pharmaceutical grade Ramipril and amlodipine besylate were obtaiend form Dr. Reddy’s Institute of life sciences, Hyderabad. The Ammonium nitrate (Merck), Docusate Sodium (Merck), Methanol (Merck/HPLC grade), Acetonirile (Merck/HPLC grade), Glacial Acetic Acid (Merck/HPLC grade) were purchased from market and double distilled water was collected from in-house production. The commercially available Asomex-R (Emcure) each tablets (contain 2.5mg of Ramipril and 2.5mg of amlodipine besylate) were purchased from market for analysis.

Instruments

Instruments:

The instruments that are used for method development and validation are Agilent technologies 1200 series system connected to UV detector, operated by Chemstation software. The supporting instruments like Ultrasonicator (Bandelin), pH meter (Polomon-1365), Analytical balance (Sarotorious AE-160) were used.

Method Development:

Preparation of standard stock solution:

Weigh accurately 5mg of Ramipril and 5mg of amlodipine besylate pure drugs individually and transferred to clean and dry100 ml volumetric flask, add 3/4th volume of diluents, then sonicated for 5 minutes and make up to the final volume up to the mark with diluents. The resulting solution contains each 50 µg/ml solution of Ramipril and amlodipine besylate.

Preparation of Mobile Phase:

Preparation 5mM Ammonium Acetate Buffer: Weigh accurately 6.75gm of Ammonium acetate and transferred to1000ml volumetric flask of water and volume was made up to 1000ml with double distilled water. Adjust the pH to 3.2 using ortho phosphoric acid. The buffer was filtered through 0.45μ filters to remove all fine particles and ultrasonicate for about 10 to 15 min to remove the dissolved gases.

The mobile phase was prepared by mixing 5mM Ammonium acetate buffer, Acetonitril and 0.1% formic acid in the ratio of 45: 55: 0.5. The prepared solution was filtered through 0.45μ filters to remove all fine particles and ultrasonicate for about 10 to 15 min to remove the dissolved gases.

Preparation of sample solutions:

Weigh accurately 20 tablets individually, transfer powder equivalent to 5mg of Ramipril and 5mg of amlodipine besylate clean and dry100 ml volumetric flask, add 3/4th volume of diluents, then sonicated for 10-15 minutes and make up to the final volume up to the mark with diluents. Filter the resulting solution, collect the filtrate and make the necessary dilution with diluents.

Optimized Chromatographic Conditions:

Chromatographic separation was achieved by using X-Bridge C₁₈ (150x4.6 mm, 5µ) column as stationary phase and composition of 5mM Ammonium acetate: 0.1%formic acid (45): ACN (55) as mobile phase. Flow rate was maintained at 1 ml/min at 40⁰C temperature and the injection volume used was 20μl. The detection was carried out at 240 nm. Dilute the standard and sample solutions with diluent filtered through Whattman filter paper (0.45μm) and degassed before use. Typical chromatogram of standard drug and Sample were shown in Fig. 3.

METHOD VALIDATION

The method was validated for its linearity range, accuracy, precision, sensitivity and specificity. Method validation is carried out as per ICH guidelines.^12-13

System suitability

A standard solution was prepared by using ramipril and amlodipine besylate working standards as per test method and was injected five times into the HPLC system.

The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from five replicate injections for ramipril and amlodipine besylate retention times and peak areas

Acceptance criteria

· The % RSD for the retention times of principal peak from 5 replicate injections of each Standard solution should be not more than 2.0 %

· The % RSD for the peak area responses of principal peak from 5 replicate injections of each standard Solution should be not more than 2.0%.

· The number of theoretical plates (N) for ramipril and amlodipine besylate peaks is NLT 2000.

· The Tailing factor (T) for ramipril and amlodipine besylate peaks is NMT 2.0

Observation

The %RSD for retention times and peak areas were found to be within the limit.

Specificity

Blank and sample were prepared and are injected into chromatographic system.

Acceptance criteria

The peaks of blank should not interfere with peaks of ramipril and amlodipine besylate

Observation

No peaks were interfered with ramipril and amlodipine besylate

Linearity

Five linear concentrations of ramipril (4-12μl) and amlodipine besylate(4-12μl) were prepared and injected. Regression equation of the ramipril and amlodipine besylate were found to be y = 689.9x + 8.947 and y = 1327.x + 99.17 and regression co-efficient was 0.999

Acceptance criteria

Correlation Coefficient should be not less than 0.9990.

Observation

The linear fit of the system was illustrated graphically

Precision

Repeatability

a. System precision: Standard solution prepared as per test method and injected six times.

b. Method precision: Prepared six sample preparations individually using single as per test method and injected each solution.

Acceptance criteria

· The % relative standard deviation of individual samples from the six units should be not more than 2.0%.

· The assay of ramipril and amlodipine besylate should be not less than 98% and not more than 102.0%.

Observation

Test results are showing that the test method is precise.

Intermediate precision (analyst to analyst variability)

A study was conducted by two analysts as per test method.

Acceptance criteria

The individual assays of ramipril and amlodipine besylate should be not less than 98% and not more than 102% and %RSD of assay should be NMT 2.0% by both analysts.

Observation

Individual %assays and % RSD of Assay are within limit and passes the intermediate precision.

Accuracy (Recovery)

A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of ramipril and amlodipine besylate into each volumetric flask for each spike level to get the concentration of ramipril and amlodipine besylate equivalent to 80% to 120% of the labeled amount as per the test method. The average % recovery of ramipril and amlodipine was calculated.

Acceptance criteria

The mean % recovery of ramipril and amlodipine besylate at each spike level should be not less than 98.0% and not more than 102.0%.

Observation

(Amount Found)

% Recovery =---------------------------- X 100

(Amount Added)

The recovery results indicating that the test method has an acceptable level of accuracy.

Robustness

Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there were no recognized change in the result and are within range as per ICH Guide lines

Acceptance criteria

The Tailing Factor of ramipril and amlodipine besylate standards should be NMT 2.0 for Variation in Flow.

Limit of Detection and Quantitation (LOD and LOQ):

From the linearity data calculate the limit of detection and quantitation, using the following formula.

LOD= 3.3σ/S

σ = Standard deviation of the response

S = Slope of the calibration curve of the analyte

LOQ= 10σ/S

σ = Standard deviation of the response.

S = slope of the calibration curve of the analyte.

Forced Degradation

A study was performed to determine the major degradation products formed under stressed conditions like Acid degradation, base degradation, oxidative degradation, thermal degradation and photolytic degradation according to ICH^14-15 guidelines using above developed method conditions. The results of forced degradation studies shown in Fig. 4, 5 and Table 2

Oxidation

To 1 ml of stock solution of ramipril andamlodipine besylate , 5 ml of 6% hydrogen peroxide was added separately. The solutions were kept for 1hr at 800C. For HPLC study ,the resultant solution was diluted to obtain 5μg/ml and10μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies

To 1 ml of stock solution of ramipril and amlodipine besylate 5ml of 0.5N Hydrochloric acid was added and refluxed for 1hr at 800c.the solution were neutralized with 0.5 N NaOH dilute up to the mark with mobile phase The resultant solution was diluted to obtain 5μg/ml and 10μg/ml solution and10μl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkali Degradation Studies

To 1 ml of stock of ramipril and amlodipine besylate 5ml of 0.5N NaOH was added and refluxed for 1hr at 800c.the solution were neutralized with 0.5 N HCL dilute up to the mark with mobile phase The resultant solution was diluted to obtain 25μg/ml and 10μg/ml solution and 10μl were injected into the system and the chromatograms were recorded to assess the stability of sample

Thermal Degradation Studies

The standard drug solution was placed were exposed in oven at 80°C for1hr.the solid were allowed to cool and 5mg each of RAM and AML were weighed transferred to separate volumetric flask in 50ml.dissolved in few ml of methanol .the solutions were further diluted by mobile phase. To study dry heat degradation .For HPLC study ,the resultant solution was diluted to 5μg/ml and 10μg/ml solution and10μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability studies

The photochemical stability of the drug was also studied by exposing the 5μg/ml and 10μg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 5μg/ml and 10μg/ml solutions and 10μl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acceptance criteria

The peak purity of ramipril and amlodipine besylate peak in all stressed samples should be within the limits.

Observation

The peak purity of ramipril and amlodipine besylate peak in all stressed samples is found to be within the acceptance criteria.

RESULTS AND DISCUSSION

Method Validation

System suitability

Table 2: System suitability data

Parameters	Ramipril	Amlodipine besylate
% RSD for five replicate injections of standard	0.50	0.64
Tailing factor	1.12	1.03
Theoretical plates	3733	3852
% Check standard recovery	99.66	101.73

Conclusion: % RSD peak area was found to be within limit. So the method is system suitable

Specificity

Table 3: Blank and Placebo interference

Sample No.	Peak found at RT of analyte peak		Acceptance criteria
Sample No.	Blank	Sample	There is no interference of Blank and placebo peak
1	Nil	Nil
2	Nil	Nil

Figure 6: chromatogram of specificity

LINEARITY:

Table 4: LINEARITY DATA

S. No	RAMIPRIL		AMLODIPINE
S. No	Concentration(μg/ml)	Peak Area*	Concentration (μg/ml)	Peak Area*
1	4	696.06	4	1397.25
2	6	1383.41	6	2764.80
3	8	2094.30	8	4122.73
4	10	2765.41	10	5412.50
5	12	3454.80	12	6710.80

* Mean of Three detrminations

Figure 7: Calibration Curve of Ramipril

Figure 8: Calibration Curve of Amlodipine Besylate

PRECISION:

Table 5: Intra Day Precision

Conc.

(µg/ml)

Ramipril

Amlodipine Besylate

Mean*

% RSD

Mean*

% RSD

1329.57

0.11

0.12

2758.38

0.19

0.2

* Mean of Six determinations

Table 6: Inter Day Precision

Conc.

(µg/ml)

Ramipril

Amlodipine Besylate

Mean*

% RSD

Mean*

% RSD

1362.13

0.58

0.61

2789.39

0.34

0.35

* Mean of Six determinations

Accuracy

Table 7: Results of Accuracy

Conc. (%)	Ramipril			Amlodipine Besylate
Conc. (%)	% Recovery*	% RSD	% Recovery*		% RSD
80	98.68	0.46	99.40		0.87
100	101.09	1.03	100.13		0.44
120	99.79	0.86	99.46		0.37

* Mean of Three determinations

Robustness:

Table 8: Robustness data of Ramipril and Amlodipine besylate

S. NO	Robustness condition	Ramipril %RSD	Amlodipine besylate %RSD
1	Flow rate (0.9ml/min)	0.43	0.54
2	Flow rate(1.1ml/min)	0.26	0.27
3	Mobile phase ratio (45:24:31)	0.69	0.43
4	Mobile phase ratio(52:26:22)	1.4	1.2
5	Temperature (28ºc)	0.69	0.43
6	Temperature (32ºc)	0.26	0.27

LOD and LOQ

Table 9: LOD and LOQ results for Ramipril and Amlodipine Besylate

S.NO	Drug Name	LOD (µg/ml)	LOQ (µg/ml)
01	Ramipril	0.5	0.5
02	Amlodipine Besylate	1.9	2.0

FORCED DEGRADATION STUDIES:

Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated.

Table 10: Forced Degradation Data

S. NO	Degradation Condition	Area of ramipril	Area of amlodipine	Tailing factor
1	Acid	8926	67957	1.24
2	Alkali	3608	6122	1.01
3	Oxidation	9107	7086	1.46
4	Thermal	9233	7137	0.99
5	photo	9932	7646	1.31

SUMMARY AND CONCLUSION:

Table 11: Summary

Parameters	RAMIPRIL	AMLODIPINE
Calibration range (μg/ml)	4-12μg/ml	4-12μg/ml
Optimized wavelength	240nm	240nm
Retention time	5.7	7.061
Regression equation (Y*)	y = 344.9x - 681	y = 663.7x - 1228
Correlation coefficient(r2)	0.999	0.999
Precision (% RSD*)	0.434	0.549
%Recovery	96.66	97.73
Limit of Detection (mcg / ml)	0.5μg/ml	0.5μg/ml
Limit of Quantification (mcg / ml)	2μg/ml	2μg/ml

CONCLUSION:

A simple and reproducible RP-HPLC method was developed and validated as per ICH guidelines for estimation of ramipril and amlodipine besylate in tablet dosage form. Quantitative estimation of ramipril and amlodipine besylate was done by RP-HPLC using a mobile phase consisting of 5mm ammonium acetate : 0.1%formic acid : acetonitrile 50:25:25 v/v, an X-bridge C 18 150* 4.6mm, 5μm column as a stationary phase with UV detection at 240 nm. After development of the method, it was validated for various analytical parameters such as specificity, system suitability, accuracy, linearity, precision, LOQ, LOD and robustness, as per ICH guidelines. The values of theoretical plates, tailing factor, retention time and peak area were found to be within limits, hence it is concluded that the system is suitable for the assay of Ramipril and amlodipine besylate in pharmaceutical formulation. The method was found to be highly specific and precise because it did not show any interference from sample and blank. From the linearity studies, it was observed that the method shows linearity in a range from 4μg/ml to 12μg/ml. The precision values were within limits, hence the method is precise one. In accuracy studies, % recovery was calculated and found to be within limits (98%-102%). The robustness of the method was checked in terms of varying flow rate, column temperature, column flow rate column temperature, mobile phase. The standard was able to give system suitability parameters within limit, which indicates that the method is robust. Therefore, it could be concluded that the proposed method can be used for routine analysis of ramipril and amlodipine besylate tablet dosage form.

REFERENCES:

1. http://www.wikipeedia.org/wiki/ramipril

2. http://www.wikipedia.org/wiki/amlodipinebesylate

3. SB Bari, BR Kadam, YS Jaiswal and AA Shirkhedkar. Impurity Profile: Significance in Active Pharmaceutical Ingredient. Eurasian Journal of Analytical Chemistry. 2 (1); 2007: 177-189.

4. S Gorog. The Importance and the Challenges of Impurity Profiling in Modern Pharmaceutical Analysis. Trends in Analytical Chemistry. 25 (8); 2006: 156-164.

5. S Arunkumar, D Sridharan, A Thenmozhi, M Mahes. Simultaneous estimation of ramipril and amlodipine in bulk and tablet dosage form by RP-HPLC method, international journal of institutional pharmacy and life sciences. 1 (2); 2011: 28-32.

6. Jignesh Patel and Mandev Patel. RP-HPLC method development and validation for the simultaneous estimation of ramipril and amlodipine besylate in capsule dosage form, Journal of Chemical and Pharmaceutical Research. 6 (6); 2014: 725-733.

7. B Naveen, T Ganesh, B Kavitha and G Nagarjuna Reddy. Development and Validation of Ramipril and Amlodipine by RP-HPLC method in bulk and pharmaceutical dosage form, International Journal of Chemical and Natural Science Vol. 1, No. 1 (2013): 17-20.

8. rajesh sharma, sunil khanna, and ganesh p. Mishra development and validation of RP-HPLC method for simultaneous estimation of ramipril, aspirin and atorvastatin in pharmaceutical preparations, E-Journal of Chemistry http://www.ejchem.net. 9 (4); 2012: 2177-2184.

9. Rajitha V, Bishnupada Biswal, D Nagarjuna Reddy and B Ramesh. Method Development and Validation of Telmisartan and Amlodipine Besylate by RP-HPLC in Tablet Dosage Form, International Journal of Pharma Sciences. 3 (5); 2013: 365-369.

10. Sayyed Hussain, Rashid R Munjewar, Mazahar Farooqui. Development and Validation of a Simultaneous HPLC Method for Quantification of Amlodipine Besylate and Metoprolol Tartrate in Tablets, Journal of PharmaSciTech. 1 (2); 2012:1-5.

11. Kanakapura Basavaiah et al, Spectrophotometric and High Performance Liquid Chromatographic determination of Amlodipine Besylate in pharmaceuticals, Science Asia. 31; 2005: 13-21.

12. International Conference on Harmonisation, 1996. ICH, Validation of Analytical Procedures Methodology, ICH – Q2B, p1-3

13. ICH Guidelines, Q2 (R1) Validation of Analytical Procedures Text and Methodology, 2005; p1-6.

14. ICH guidelines Q1A (R2). Stability Testing of New Drug Substances and Products (revision 2), November 2003.

15. Reynolds DW, Facchine KL, Mullaney JF, Alsante KM, Hatajik TD, Motto MG. Available guidance and best practices for conducting forced degradation studies. Pharm Tech. 2002:48-56.

Received on 16.09.2016 Accepted on 27.10.2016

Asian J. Pharm. Res. 2016; 6(4): 242-249.

DOI: 10.5958/2231-5691.2016.00034.4

Drug	Peak area of standard*	Peak area of sample*	%RSD	% Recovery	Tailing factor*
RAMIPRIL	2076.23	2068.96	0.586	99.65	0.89
AMLODIPINE BESYLATE	1419.72	1430.08	0.364	100.73	0.97