INTRODUCTION:

Valsartan (VAL) is chemically N-[p-(o-1H-Tetrazol-5-ylphenyl)benzyl]-N-valeryl-L-valine. It is an Angiostensin II blocker and it is used as an antihypertensive agent. It inhibits the conversion of the inactive angiostensin I to the highly potent vasoconstrictor, angiostensin II ^[1]. Ramipril (RAMI) is chemically (1S, 5S, 7S)-8-[(2S)-2-[[(1S)-1ethoxycarbonyl- 3- phenyl- propyl] amino] propanoyl]-8 azabicyclo[3.3.0]octane-7-carboxylic acid. It is an angiostensin-converting enzyme inhibitor. Both the drugs, Valsartan and Ramipril are official in USP 2007^[2]. Ramipril is also official in BP 2007 ^[3] Detail survey of literature of Valsartan revealed several methods, viz., UV Spectrophotometry ^[4-8] and HPLC ^[9-12] methods for determination as single drug and in combined dosage forms. Similarly, literature survey of Ramipril revealed several methods, viz., HPLC ^[13-22], HPTLC ^[23-24] and UV Spectrophotometric ^[25-29] methods. A fixed dose combination containing Valsartan 80 mg and Ramipril 5 mg is available as a tablet in the market. Two HPLC methods were found to be reported for the determination of Valsartan and Ramipril in a combined dosage form ^[30-31]. The present work describes the development and validation of a simpler reverse phase high performance liquid chromatography method, which can be used to determine simultaneously these components in combined dosage form. The present RP-HPLC method was validated following the ICH guidelines.

MATERIAL AND METHODS:

Reagent and Chemicals:

Acetonitrile and Water of HPLC grade were procured from Ashnoj specialties Pvt. Ltd., Navi Mumbai. Working standard of Valsartan was provided by Lupin Research Park, Pune, India and Ramipril was provided by Mepro pharmaceuticals Pvt.Ltd., Wadhwan, India.

Equipment:

Chromatographic separation was performed on a Jasco chromatographic system equipped with a Jasco PU-2080 plus intelligent HPLC pump and Jasco MD-2010 plus multiwavelength detector and Rheodyne injector with 20 ml loop volume.

Chromatographic Conditions:

Hypersil GOLD CN column (250 mm x 4.6 mm) was used for the separation; mobile phase consisted of a mixture of acetonitrile and water [pH 3.0 adjusted with orthophosphoric acid (0.08%)] in the ratio (70:30v/v) was delivered at a flow rate of 1 ml/min with detection wavelength 217 nm. The mobile phase was filtered through a 0.45 m membrane filter and sonicated for 15 minutes. The injection volume was 20 ml. Analysis was performed at an ambient temperature.

Method Development:

Different columns (C₈ HiQ-sil 250 x 4.6mm and C₁₈HiQ-sil 250 x 4.6mm)were tried for the chromatographic run. The acceptable elution pattern or adequate resolution could not be obtained. Cyano column resulted in acceptable resolution of the drug peaks.

Different mobile phases containing water and acetonitrile in different proportions, and various pH were tried and finally acetonitrile: water [pH 3.0 adjusted with orthophosphoric acid (0.08%)] in the ratio (70:30 v/v) was selected as an appropriate mobile phase that resulted in good resolution and acceptable system suitability parameters for both Valsartan and Ramipril.

Fig 1: A Representative Chromatogram of Valsartan (4µg/ml) and Ramipril (20µg/ml)

Preparation of Working Standard Solutions:

Working Standard stock solutions of strength 0.4 mg/ml of Valsartan and Ramipril were prepared separately using acetonitrile. From standard stock solution of each drug, appropriate amount of respective solutions were mixed to get the required concentration of both the drugs and diluted with mobile phase, to obtain standard mixture solution.

System Suitability Studies:

The resolution, number of theoretical plates, retention time and peak asymmetry were calculated for the working standard solutions and is as shown in Table 1. The values obtained demonstrated the suitability of the system for the analysis of these drugs in combination. The typical chromatogram of standard solution is as shown in Fig. 1.

Calibration Curve:

Accurately measured volumes of working standard solution of Valsartan and Ramipril were transferred into a series of 10 ml volumetric flasks and diluted appropriately with mobile phase. 20 ml of each solution was injected under operating chromatographic conditions as described above. The calibration curves were plotted over a concentration range 2 mg/ml to 32 mg/ml for Valsartan and 10 mg/ml to 80 mg/ml for Ramipril. Calibration curves were obtained by plotting the area of drug peak versus concentration of drug. Regression equations were calculated.

Assay:

Preparation of sample solution:

Twenty tablets were weighed, each containing 80 mg Valsartan and 5 mg Ramipril were weighed and finely powdered. A quantity of powder equivalent to 80 mg Valsartan and 5 mg of Ramipril was weighed and transferred to 25 ml volumetric flask. Acetonitrile was added to the same flask and sonicated for 10 minutes. The volume was made up to 25 ml with acetonitrile. The solution was filtered using whatmann filter paper No.41. From the filtrate, appropriate dilutions were made using mobile phase to obtain the concentration in the Beer’s law range for both the drugs.

With the optimized chromatographic conditions, these mixed working standards were injected and the chromatogram was recorded. The retention times of Valsartan and Ramipril were found to be 3.68 ± 0.02 and 5.08 ± 0.02 respectively. The assay was calculated from the equation of regression line for each drug. The percentage assay of individual drug was calculated and presented in Table 2.

Method Validation

As per ICH guidelines, the method validation parameters checked were specificity, linearity, precision, accuracy, limit of detection, limit of Quantitation and Robustness.

Specificity

The specificity is the ability to access unequivocally the analyte in the presence of components which may be expected to be present. This has been confirmed by checking the peak purity profile using Jasco Borwin PDA software, version 1.5. The peak purity values for standard solution of Valsartan and Ramipril are 993.970 and 987.180 and for the sample solution are 999.657 and 904.965. A value more than 900 indicates no interference by any other component.

Linearity and Range:

The linearity of Valsartan and Ramipril was determined at five concentration levels ranging from 2 mg/ml to 32 mg/ml for Valsartan and 10 mg/ml to 80 mg/ml for Ramipril. The linear regression equations of the lines are:

For Valsartan y = 75217x + 23706, (r = 0.9999)

For ramipril y = 27588x + 27626, (r = 0.9993)

Precision:

The precision of the method was demonstrated by interday and intraday precision studies. For the intraday precision, injections of the three mixed standard solutions were repeated thrice in a day and % RSD was calculated. The intraday % RSD of Valsartan and Ramipril was found to be 0.218 % and 0.261% respectively. In the interday studies, injection for standard solutions was made on 3 consecutive days and % RSD was calculated. The interday % RSD for Valsartan and Ramipril were found to be 0.185 % and 0.317% respectively. From the data obtained, the developed RP-HPLC method was found to be precise.

Accuracy:

The accuracy was determined by recovery studies. The recovery studies were performed by standard addition method, at 80%, 100%, 120% level. Known concentration of working standard was added to the fixed concentration of the pre-analyzed tablet solution. Percent recovered was calculated using regression equation. For both the drugs, recovery was performed in same way and in triplicate. The percentage recovery were calculated and presented in Table 3.

Table 1: System Suitability Parameters

Sr. No.	Drug	RT	Plates	Resolution	Asymmetry
1.	Valsartan	3.68	10733	2.97	1.32
2.	Ramipril	5.08	5285	-	1.26

Table 2: Table for Assay

Drug	Amount present (mg/tab)	Amount found (mg/tab)	% Label Claim
Ramipril	5	4.989	99.78 ± 0.05
Valsartan	80	79.64	99.55 ± 0.96

Table 3: Recovery Studies

Drug	Level of Recovery	Amount present (mg)	Amount added (mg)	Amount Recovered	% Recovered + SD*
Ramipril	80	5	4	8.91	99.00 ± 0.18
	100	5	5	10.04	100.40 ± 0.08
	120	5	6	10.90	99.16 ± 0.02
Valsartan	80	80	64	144.7	100.48 ± 0.04
	100	80	80	158.8	99.25 ± 0.07
	120	80	96	96.54	100.56 ± 0.06

*Standard deviation of three determinations

Limit of Detection and Limit of Quantitation:

The Limit of detection (LOD) is the smallest concentration that can be detected but not necessarily quantified as an exact value. LOD was calculated using the following formula:

3.3 X Standard deviation of y intercept

LOD =

Slope of calibration curve

The LOQ is the lowest amount of analyte in the sample that can be quantitatively determined with suitable precision and accuracy. LOQ was calculated was calculated using the following formula:

10 X Standard deviation of y intercept

LOD =

Slope of calibration curve

Robustness:

Robustness of the method was determined by making slight deliberate changes in chromatographic conditions like 2% change in ratio of mobile phase constituents, ± 1⁰C change in room temperature and 0.05% change in flow rate. It was observed that there were no marked changes in the chromatogram. It suggests that the developed method is robust.

RESULTS AND DISCUSSION:

The proposed method was found to be simple and sensitive with linearity in the concentration range of 2 to 32 mg/ml for Valsartan and 10 to 80 mg/ml for Ramipril. The method was found to be accurate and precise as indicated by results of recovery studies and %RSD not more than 2%. LOD and LOQ for Valsartan were found to be 0.437 mg/ml and 1.430 mg/ml respectively and for Ramipril were 0.284 mg/ml and 0.861 mg/ml respectively. The proposed method was found to be specific as there is no interference from common tablet excipients like lactose, starch etc and Peak purity values for peaks of both Ramipril and Valsartan confirmed the specificity.

CONCLUSION:

The developed RP-HPLC method for the simultaneous determination of Valsartan and Ramipril can be used for routine analysis of both these components in combined dosage form.

ACKNOWLEDGEMENT:

Authors wish to thank Lupin Research Park, Pune, India and Mepro pharmaceuticals Pvt. Ltd, Wadhwan, India for providing the gift samples of Valsartan and Ramipril respectively for the work. We are thankful to Dr. K. G. Bothara, Principal, AISSMS College of pharmacy for providing the required facilities and instruments.

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