J. Chil. Chem. Soc, 55, N° 2 (2010), págs.: 223-226

 

A STABILITY INDICATING HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF VALSARTAN AND RAMIPRIL IN BINARY COMBINATION

 

K. S. LAKSHMI AND LAKSHMI SIVASUBRAMANIAN*

Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Kattankulathur — 603 203, Tamilnadu, South India

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ABSTRACT

A simple, sensitive and validated HPLC method has been developed to determine valsartan and ramipril simultaneously in synthetic mixture. Chromatographic separation was achieved on a C-18 column using a mixture of acetonitrile and water in the ratio 55:45 (v/v), pH adjusted to 3.6 with 88% orthophosphoric acid at a wavelength of 215 nm. Linearity of the method was found to be in the concentration range of 50-250 µg/ml for valsartan andl00-500 µg/ml for ramipril with correlation coefficient greater than 0.999. The total eluting time for the two components is less than five minutes. The method can be used for simultaneous determination of valsartan and ramipril.

Keywords: HPLC, Valsartan, Ramipril

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INTRODUCTION

Valsartan [fig 1] chemically designated as (S)-3-methyl-2-[N({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)pentanamido]butanoic acid belong to angiotensin II receptor antagonist used for the treatment of hypertension¹. A number of analytical methods have been developed for its determination in pharmaceutical formulations or in biofluids either alone or in combination with other drugs, which includesliquid chromatography-tandem mass spectrometry², HPLC³ and a spectrophotometric analysis⁴.

Ramipril [fig 2], (2S,3aS,6aS)-1-[(2S)-2{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-y1]amino}propanoyl]-octahydrocyclopenta[b]pyrrole-2-carboxylic acid is a prodrug which is rapidly hydrolyzed with the cleavage of an ester group through hepatic metabolism forming an active metabolite ie, ramiprilat. This prodrug itself is a poor inhibitor of angiotensin converting enzyme (ACE) but its active metabolite has a higher affinity for ACE, thus blocking the conversion of the angiotensin I to the angiotensin II, a highly potent vasopressor activity^5,6. The drug is officially listed in British Pharmacopoeia⁷, which describes a potentiometric titration procedure for its assay in bulk and dosage forms. The determination of ramipril along with hydrochlorothiazide in binary mixture was performed by derivative compensation technique⁸ as well as zero crossing derivative technique^9,10 . Few visible spectrophotometric^11-13 and HPLC methods^14,15 have also been reported for the assay of this drug in commercial dosage forms.

According to the information collected from literature there is no method reported for the simultaneous determination of valsartan and ramipril. In the present work we are therefore focused to achieve the optimum chromatographic conditions for the simultaneous determination of valsartan and ramipril in synthetic mixture.

We describe a simple, sensitive and validated HPLC method with total run time less than ten minutes for the simultaneous determination of valsartan and ramipril. The developed method can be applied successfully to quality control and for other analytical purposes.

1.     Chemicals and Reagents

Valsartan and ramipril reference substance with claimed purity of 99.6% and 99.72% respectively were obtained from Madras Pharmaceuticals, Chennai. Acetonitrile (HPLC grade) and orthophosphoric acid (Analytical reagent grade) were purchased from Merck (Mumbai, India). All excipients used were of pharmaceutical grade. Water used was prepared in the laboratory using Milli-Q system (Millipore, USA).

2.     Apparatus and Chromatographic Conditions

HPLC apparatus consisting of Shimadzu LC-10A system equipped with LC-10 AT dual pump, an SPD 10A variable wavelength detector (set at 215 nm), a spincobiotech software and rheodyne injection valve with a 20 µl loop was used for development and evaluation of this method. A Hypersil C18 column (250 x 4.6 mm id, 5 µm particle size) was selected. The mobile phase was composedof a mixture of acetonitrile and water in the ratio 55:45 (v/v) and the pH adjusted to 3.6 with 88% ortho phosphoric acid. The flow rate was lml/ min and the system was operated at room temperature 25+/-2°C

3.     Preparation of standard solution

A stock solution of valsartan and ramipril was prepared at about 0.5 mg/ml and 1 mg/ml respectively in mobile phase.

4.     Linearity

Linearity of the proposed method was checked by analyzing five solutions in the range of 50-250 µg/ml for valsartan and 100-500 µg/ml for ramipril. Each level was prepared in triplicate.

5.     Accuracy

Method accuracy was performed by adding known amounts of valsartan and ramipril to the pre-analysed synthetic mixture solution and then comparing the added concentration with the found concentration. Three levels of solutions were made which correspond to 50, 100 and 150% of the nominal analytical concentration (100 µg/ml for Valsartan and 200 µg/ml for Ramipril). Each level was prepared in triplicate.

6.     Selectivity

The selectivity of the proposed method was checked by making a synthetic mixture of both the analytes with commonly occurring excipients that are found in most tablet formulations and then comparing the chromatogram with the chromatogram of the reference standard. Synthetic mixture containing 100 mg of ramipril, 50 mg of valsartan and 20 mg each of starch, lactose, magnesium stearate and tale which are present as excipients in the pharmaceutical formulation were accurately weighed and transferred in to a 100 mi volumetric flask. The mixture was shaken well with 70ml mobile phase and then the volume was made with mobile phase and filtered.About 1 ml of the filtrate was transferred in to 10 ml volumetric flask and mobile phase was then added to volume to obtain a final concentration containing 100 µg/ml ramipril and 50 µg/ml valsartan.

7.     Robustness

Robustness¹⁶ of the method was performed by intentionally modifying the chromatographic conditions such as composition and flow rate of the mobile phase and the detection wavelength. The chromatographic parameters of each analyte such as retention time, tailing factor, and number of theoretical plates were measured at each changed conditions.

8.     Precision

For evaluating the within-day precision, results of five replícate analyses of three different concentrations of samples were calculated on a single day. The between-day precision was calculated from the same samples analyzed on six different days.

9.     LOD and LOQ

For calculating the LOD and LOQ values, solutions with known concentrations of analytes were injected into the HPLC system. The limit of detection (LOD) and limit of quantification (LOQ) were then measured by calculating the mínimum level at which the analytes can be readily detected (signal to noise ratio of 3:1) and quantified (signal to noise ratio of 10:1) with aecuracy respectively.

10.     Forced Degradation studies¹⁷:

Drugs were allowed to hydrolyze in base (0.1M NaOH), acid (0.1M HC1) and different strengths of hydrogen peroxide (30%, 3%, 1%). Drugs were also studied for its thermal degradation at 80°C and 105°C. Photo degradation was also analyzed in two forms, dry powder and in solution form. An accurately weighed 50 mg of valsartan and 100 mg of ramipril powder was dissolved in methanol. To each flask 5ml of respective base and acid were added and refluxed for 12hrs. Accurately weighed quantity of sample was also kept in different strengths of hydrogen peroxide solution for specified period of time. The samples were withdrawn at different time interval, allowed to cool to room temperature and treated as follows.

The acid and base samples were neutralized to pH 7. The thermal and photo degradation sample and hydrogen peroxide samples were used as such. All the samples were further diluted with mobile phase to get a concentration of 100 µg/ml of ramipril and 50 µg/ml. Blankwas also treated in the same way. The results of stability studies are presented in Table 4 and Fig 4.

RESULTS AND DISCUSSION

In the present work, conditions were optimized for the development and validation of a simple and aecurate HPLC method for the simultaneous determination of valsartan and ramipril in synthetic mixture form. Method development was started with acetonitrile and water in the ratio 50:50 (v/v). At this composition although both components were eluted, resolution was not good. The acetonitrile content of the mobile phase was then increased and the pH was adjusted with 88% of orthophosphoric acid. At the composition of 55:45 and pH adjusted to 3.6, both components were eluted with good resolution.

The most appropriate mobile phase composition was thus found to be acetonitrile and water in the ratio of 55:45 (v/v) and pH adjusted to 3.6 with 88% of orthophosphoric acid. Under the described experimental conditions, peaks that belong to ramipril and valsartan were obtained at retention times of 1.91 and 4.82 minutes respectively as shown in Fig 3.

The developed chromatographic method was validated using ICH guidelines [16]. Validation parameters performed include linearity, limit of detection and quantitation, selectivity, robustness, aecuracy and repeatability. The calibration curve was linear overthe concentration range of 100-500 µg/ml for ramipril and 50-250 µg/ml for valsartan. The correlation coefficient in both cases was found to be greater than 0.996, which manifests a linear relationship between concentration and peak area. The linear regression equation for ramipril was found to be y = 77901x + 470357 with correlation coefficient equal to 0.9995. The linear regression equation for valsartan was found to be y = 224846x +708111, with value of correlation coefficient equal to 0.9994.

In this study, the LOD was found to be 0.0126 µg/ml and 0.0280 µg/ml for ramipril and valsartan respectively. The LOQ was found to be 0.0383 ug/ mi and 0.0849 µg/ml for ramipril and valsartan respectively. The recovery and relative standard deviation for each of the analytes are given in Table 1.

The results of within day and between day precision are presented in Table 2. The chromatogram of valsatan and ramipril in synthetic mixtures is given in Fig 3 showing the selectivity of the proposed method. Robustness of the method was performed by intentionally modifying the chromatographic conditions. The results showed that the variance of the conditions had no appreciable effeets to that of actual. The results of the robustness study and system suitability parameters are given in Table 3a & 3b.

During the study it was observed that upon treatment of ramipril and valsartan with base (0.1M NaOH), acid (0.1M HC1) and different strengths of hydrogen peroxide (30%, 3%, 1%) the degradation was observed in acid, whereas no degradation was observed with different strengths of hydrogen peroxide. Table 4 indicates the extent of degradation of both the drugs under various stress conditions. Figure 4a to f and Figure 5a to e shows the chromatograms of forced degraded samples. Further it is important to note that from the chromatograms (Figs. 4a to f and Figs. 5a to e), it is evident that although the degrade peaks are observed, under the applied stress conditions like acid and oxidative degradation states. The drug is stable under alkali and other stress conditions.

CONCLUSION

A simple and accurate reverse phase HPLC method has been developed for the simultaneous determination of ramipril and valsartan. The method was validated by testing its linearity, accuracy, precision, limits of detection and quantitation, selectivity and robustness. The run time of less than ten minutes allows its application for the routine determination of ramipril and valsartan. Further, the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility. Even though no attempt was made to identify the degraded products, proposed method can be used as a stability indicating method for assay of ramipril and valsartan in combined dosage forms.

 

ACKNOWLEDGEMENT

The authors are grateful to M/S Madras Pharmaceuticals Ltd., Chennai for providing the gift samples of ramipril and valsartan.

 

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(Received: August 17, 2009 - Accepted: March 25, 2010)

* e-mail: lakshmiss@hotmail.com