INTRODUCTION:

Valsartan, chemically designated as (S)-3-methyl-2-[N({4-[2-(2H-1,2,3,4-tetrazol-5-yl) phenyl]phenyl}methyl) pentanamido] butanoic acid (Figure - 1a), belong to the category of angiotensin II receptor antagonist used for the treatment of hypertension⁽¹⁾. A number of analytical methods have been developed for its determination in pharmaceutical formulations and in bio-fluids either alone or in combination with other drugs, which includes liquid chromatography-tandem mass spectrometry⁽²⁾, HPLC⁽³⁾ and a Spectrophotometric analysis⁽⁴⁾. Aliskiren, chemically designed as (2(S), 4(S), 5(S), 7(S)-N- (2-carbamoyl- 2- methylpropyl) -5-amino-4-hydroxy2,7 diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy) phenyl] octanamide hemifumarate) ⁽⁵⁾(Figure - 1b).The first oral direct rennin inhibitor approved for clinical use, exhibits a novel and advantageous pharmacokinetic and pharmacodynamic profile for the long-term treatment of hypertension. Aliskiren blocks the renin system at its rate-limiting step by directly inhibiting the catalytic activity of renin, thereby reducing generation of angiotensin I and angiotens in II. There are so many analytical techniques are available for the determination of Aliskiren and Valsartan by using HPLC-UV detection individually or in combination with other hypertensive drug ^(6-15).

According to the information collected from literature there is no method reported for the simultaneous determination of Valsartan and Aliskiren. In the present work, we have therefore focused to achieve the optimum chromatographic conditions for the simultaneous determination of Valsartan and Aliskiren in synthetic mixture. We describe a simple, sensitive and validated HPLC method with total run time less than 5 minutes for the simultaneous determination of Valsartan and Aliskiren. The developed method can be applied successfully to quality control and for other analytical purposes.

MATERIALS AND METHODS:

Standard drugs

Aliskiren Hemifumarate of purity 99% w/w procured from Hetero Drugs Ltd. Valsartan of purity 99%w/w procured from Hetero Drugs Ltd as a gift sample.

Chemicals and reagents

Methanol-HPLC grade (Merck), Acetonitrile -HPLC grade (Merck) Water for HPLC-milli-Q grade (Merck), Potassium dihydrogen phosphate (Merck) Orthophosphoric acid.

Apparatus

Single pan balance (Mettler), _PH meter (Thermo), Sonicator (Shimadzu),

HPLC – (Waters, UV – 2489).Waters – C8 (250 x 4.6mm), 5μmm, column,

Membrane filter (0.45μ) Centrifugation Apparatus, Volumetric flask borosilicate, Pipettes (borosilicate)

Preparation of standard solutions:

Accurately weighed amount of 150mg Aliskiren Hemifumarate and 160mg Valsartan were taken to a 100 ml cleaned and dried volumetric flask. This was then diluted with 70ml of diluents and sonicates to dissolve it completely and make volume up to the mark with the same solvent. Further, an amount of 0.3 ml Aliskirene Hemifumarate and Valsartan of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluents.

Sample Solution Preparation:

Weigh and accurately equivalent weight 150mg Aliskiren Hemifumarate and 160mg Valsartan were weighed and transferred to 100ml volumetric flask, it was dissolved with diluent and the volume was made up to the mark with same solvent. Further 0.3 ml of above solution was diluted to 10ml with the diluent to get 45 µg/ml Aliskirene and 48 µg/ml Valsartan.

Optimized chromatographic conditions

Equipment: High performance liquid chromatography equipped with Auto Sampler Waters Empower- 2, UV detector 2489

Column: Waters C8 (4.6 x 150mm, 5mm, Make: Waters) or equivalent

Mobile phase: Buffer (pH=4): Methanol: Acetonitrile in the ratio of 40:15:45v/v

Flow rate: 1 ml per min

Wavelength: 220 nm

Injection volume: 10 ml

Column oven: Ambient

Run time: 18min

Technique: Isocratic.

Preparation of 20mM Potassium dihydrogen Ortho phosphate (pH= 4)

Accurately weighed 2.72 grams of KH₂PO₄ was taken in a 1000ml volumetric flask, dissolved and diluted to 1000ml with HPLC water and the volume was adjusted to pH 4 with Orthophosphoric acid.

Method Validation:

The suggested analytical method was validated according to international guidelines with respect to certain parameters such as, precision, accuracy, linearity, robustness, ruggedness, LOD and LOQ.

Precision: Method precision was determined both in terms of repeatability (injection and analysis) and intermediate precision. In order to determine injection repeatability, samples spiked with 150mg of Aliskiren Hemifumarate, 160mg of Valsartan were injected 5 times into HPLC system and repeatability of the retention time and peak area was determined and expressed as mean and %RSD calculated from the data obtained.

Table No: 1. Precision results for Aliskiren and Valsartan

Injection	Area of Aliskiren	Area of Valsartan
Injection-1	1006046	5294078
Injection-2	1003598	5292758
Injection-3	1006183	5310288
Injection-4	1009363	5330655
Injection-5	1002628	5296927
Average	1005564	5304941
Standard Deviation	2623.6	15972.5
%RSD	0.26	0.30

Table No: 2. ID-Precision results for ALSK and Valsartan

Injection	Area of Aliskiren	Area of Valsartan
Injection-1	1026256	5437315
Injection-2	1028738	5447109
Injection-3	1029051	5455962
Injection-4	1032135	5471662
Injection-5	1035894	5492850
Average	1030415	5460979
Standard Deviation	3706.4	21836.9
%RSD	0.36	0.40

Robustness: The robustness of the developed method was investigated by evaluating the influence of small deliberate variations in procedure variables like flow rate (±5%) and change in organic composition.

Ruggedness: The ruggedness of the method was investigated by evaluating the influence of different analyst, different time intervals.

Stability: The stability studies of Aliskiren Hemifumarate, valsartan samples were carried out over a period of 48 h at 25 0C (room temperature under laboratory light), 2–80C (refrigerator) and standard solutions for one month at 2–80C.

Figure – 2: Chromatogram for Aliskiren and Valsartan

Table No: 3. Accuracy of Aliskiren and Valsartan

Drug	% Level	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
Aliskiren	50%	4999737	7.45	7.30	98.1%	99.0%
	100%	1017257	15.0	14.8	99.1%
	150%	1537895	22.5	22.4	99.9%
	50%	2647781	7.68	7.79	101.5%	101.6%
Valasrtan	100%	5377240	15.6	15.8	101.4%
Valasrtan	150%	8094886	23.4	23.8	101.8%

RESULTS AND DISCUSSION:

Sample preparation: Several organic solvents were tried for the preparation of stock solutions of all analytes. Aliskire highly soluble in water, freely soluble in methanol and Soluble in phosphate buffer, n-Octanol, acetonitrile. Valsartan was soluble in ethanol and methanol, acetonitrile and slightly soluble in water Finally, methanol, phosphate buffer and acetonitrile mixture was chosen as solvent for the present work.

Accuracy: Accuracy was determined in terms of percent recovery. Sample solution spiked with the analytes at three different concentration levels 22.5, 45, 67.5 mg/ml of Aliskiren Hemifumarate, 24, 48, 72mg/ml of Valsartan. Another set of standard mixtures at the same concentration levels was also prepared with the diluents. Sample and standard solutions are injected onto the HPLC system in triplicate. Percentage recoveries of Aliskiren Hemifumarate, Valsartan were calculated.

Linearity: The linearity of the method was established by spiking a series of standard mixtures of Aliskiren Hemifumarate (15-75μg/ml), Valsartan (16-80μg/ml). Above solutions are injected onto the HPLC system. Construct the Calibration curves for standard solutions by plotting their response (peak area of the analytes) against their respective concentrations. Linear regression was applied and slope (a), intercept (b), correlation coefficient (r) and standard error (Es) were determined.

Limit of detection and Limit of quantification: Detection and quantification limits were determined through dilution method using S/N approach by injecting a 10μl sample. LOD was considered as the minimum concentration with a signal to noise ratio of at least three (S/N˜3), while LOQ was taken as a minimum concentration with a signal to noise ratio of at least ten (S/N˜10).

Method validation:

Precision: Precision data representing both repeatability (injection and analysis) and intermediate precision (intra-day and inter-days reproducibility) are summarized in Table No: 1 and Table No: 2, respectively. The %RSD values for both intra-day and inter-days were less than 2.0%, which indicates that the proposed method is precise.

Accuracy: Average recoveries of both the drugs are 99.6%, 100%, 99.7%, at 50%, 100% and 150% concentrations level respectively. The percentage recoveries of all the drugs are within the limits 99-101%. So the method is accurate, accuracy data for Aliskiren and Valsartan are presented in Table: 3.

Linearity:

The response was found linear over a concentration range of 15-45 ppm of Aliskiren and 16-48 ppm of Valsartan. The correlation co-efficient were found to be 0.998 and 0.999 for Aliskiren andValsartan respectively. So the method is linear, data is presented in Table - 4 and 5. Linearity curve of Aliskire and valsarten is given in Figure: 3 and 4.

Table No: 4. Linearity results for Aliskiren

S.No	Linearity Level	Concentration	Area
1	I	15ppm	583820
2	II	30ppm	800947
3	III	45ppm	1012427
4	IV	60ppm	1186638
5	V	75ppm	1415611
Correlation Coefficient			0.998

Figure – 3: Calibration graph of Aliskiren Hemifumarate

Table No: 5. Linearity results for Valsartan

S.No	Linearity Level	Concentration	Area
1	I	16ppm	3090740
2	II	32ppm	4237420
3	III	48ppm	5352248
4	IV	64ppm	6259440
5	V	80ppm	7451001
Correlation Coefficient			0.999

Figure – 4: Calibration graph of Valsartan

Table No: 6. Study of Robustness results for Aliskiren (Flow Rate)

S.No	Flow Rate (ml/min)	System Suitability Results
S.No	Flow Rate (ml/min)	USP Plate Count	USP Tailing
1	0.8	5970.2	1.1
2	1.0	6418.2	1.0
3	1.2	4855.5	1.2

Table – 7: Study of Robustness results for Valsartan (Flow Rate)

S.No	Flow Rate (ml/min)	System Suitability Results
S.No	Flow Rate (ml/min)	USP Plate Count	USP Tailing
1	0.8	7251.5	1.0
2	1.0	7640.2	1.1
3	1.2	6369.3	0.9

Table - 8: Study of Robustness (Effect of Mobile Phase)

Aliskiren	Change in Organic Composition in the Mobile Phase	System Suitability Results
Aliskiren	Change in Organic Composition in the Mobile Phase	USP Plate Count	UUSP Tailing
1	10% less	6844.0	0.9
2	Actual	6418.2	1.2
3	10% more	6451.9	1.1
Valsartan	Change in Organic Composition in the Mobile P	System Suitability Results
Valsartan	Change in Organic Composition in the Mobile P	USP Plate Count	USP Tailing
1	10% less	7643.0	0.8
2	Actual	7640.2	1.1
3	10% more	7266.3	1.1

Limit of detection:

The LOD for Aliskiren and Valsartan standard solutions were found to be 0.02.µg/ml , 0.005µg/ml respectively given in Figure No: 5.

Robustness:

Minor deliberate changes in different experimental parameters such as flow rate (±5%) and organic composition (±5% units) did not significantly affect the recoveries, peak area and retention time of all the above drugs indicating that the proposed method is robust which is mentioned in Table – 6, 7 and 8.

Ruggedness:

The method is rugged by different analyst, different time intervals and the method did not significantly affect the recoveries, peak area and retention time of all the above drugs indicating that the proposed method is rugged.

Limit of quantification:

The LOQ Ramipril and Valsartan standard solutions were found to be 0.07µg/ml, 0.017µg/ml respectively given in Figure - 6.

Figure – 5: Chromatogram of Limit of Detection

Figure – 6: Chromatogram for Limit of Quantification

Table No: 9. Validation summary of Aliskiren and Valsartan

SL. No	Parameter	Aliskiren	Valsartan	ICH acceptance limit
1	Accuracy	99.0%	101.6%	98-102%
2	Precision	0.26	0.30	%RSD < 2
3	Correlation coefficient	0.998	0.998	Not less than 0.999
4	LOD	S/N =2.93	S/N =2.97	S/N =3
5	LOQ	S/N =9.90	S/N =9.90	S/N =10
6	USP Resolution	....	4.9	Not less than 2
7	USP Tailing	1.0	1.2	Less than 2
8	USP Plate count	6418.0	7460.1	Not less than 2000

Stability:

Results from the stability studies of samples and standard solutions indicated that samples were stable for 48 h when stored at room temperature (25⁰C), refrigerator (2–8⁰C) and while the standard solutions demonstrated stability for one month at 2–8 ⁰C. A validation summary is given in Table - 9 which includes all parameters at a glens.

CONCLUSION:

A novel, simple, rapid and cost effective RP-HPLC/UV method was successfully developed for simultaneous determination of Aliskiren, valsartan. The proposed method was optimized and validated for the various experimental parameters. Influence of pH of the mobile phase, column oven temperature and various particulate columns on the analysis of Aliskiren was evaluated. All the analytes were well resolved and separated in less than 5 min. Our developed method will be applied for assessing the pharmacokinetics and drug–drug interaction studies of these anti-hypertension drugs with other commonly prescribed drugs. This method could also be used for the analysis of these drugs in pharmaceutical preparations and routine laboratory analysis with slight modification in the extraction procedure. Overall, the proposed method provides high throughput for simultaneous determination of Aliskiren and Valsartan with excellent accuracy, precision, selectivity and reproducibility.

AKNOWLEDGEMENT:

We are very thankful to authorities of Nalanda College of Pharmacy for providing the facilities to complete this research work.

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Received on 30.11.2012 Modified on 14.12.2012

Asian J. Research Chem. 6(1): January 2013; Page 19-23