1. INTRODUCTION:

simultaneous estimation of drug is very important as the new combined formulation approved in market. Simultaneous estimation of drug is more economical for estimation of combined drug formulation, and it is widely used for the quantification of drug combination^[1]. The principle of separation of normal phase and reverse phase mode is adsorption. When mixtures of components are introduced into a HPLC column, they travel according to their affinities towards the stationary phase. The components which has more affinity towards the stationary phase travels slower and vice versa^[2].

Valsartan is chemically (2S)-3-methyl-2-[N-({4-[2-(2H-1,2,3,4-tetrazol-5yl) phenyl] phenyl} methyl) pentanamido] butanoic acid (figure 1). Valsartan belongs to the angiotensin II receptor blocker (ARB) family of drugs. ARBs selectively bind to angiotensin receptor 1 (AT1) and prevent the protein angiotensin II from binding and exerting its hypertensive effects, which include vasoconstriction, stimulation and synthesis of aldosterone and ADH, cardiac stimulation, and renal reabsorption of sodium, among others. Overall, valsartan's physiologic effects lead to reduced blood pressure, lower aldosterone levels, reduced cardiac activity, and increased excretion of sodium ^{[3] [4]}.

Figure 1. Structure of valsartan (C₂₄H₂₉N₅O₃)

Amlodipine is chemically 3-ethyl 5-methyl 2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate (figure 2). Amlodipine, initially approved by the FDA in 1987, is a popular antihypertensive drug belonging to the group of drugs called dihydropyridine calcium channel blockers. Due to their selectivity for the peripheral blood vessels, dihydropyridine calcium channel blockers are associated with a lower incidence of myocardial depression and cardiac conduction abnormalities than other calcium channel blockers^[5]. Amlodipine is commonly used in the treatment of high blood pressure and angina. Amlodipine has antioxidant properties and an ability to enhance the production of nitric oxide (NO), an important vasodilator that decreases blood pressure. The option for single daily dosing of amlodipine is an attractive feature of this drug^{[6] [7]}.

Figure 2. Structure of amlodipine (C₂₀H₂₅ClN₂O₅)

The aim and objective of this study was to develop and validate a simple, precise, accurate and economical RP-HPLC method for the estimation of amlodipine and valsartan simultaneously as per the ICH guidelines^[8].

2. MATERIALS AND METHOD:

2.1. Chemical and apparatus:

HPLC grade methanol, HPLC grade water, orthophosphoric acid, water 1525 binary HPLC pump, water 2487 dual λ absorbance detector, digital ultrasonic cleaner (INDOSATI), volumetric flask, measuring cylinder, injector, Vacuum filter, syringe filter, micro pipette, Whatman filter paper.

2.2. HPLC instrumentation and condition:

The analysis was carried out on a HPLC system equipped with UV detector, pressure control by prominent pump and operated by empower3 software. C 18 column was used consisting diameter of 150 x 4.6mm, 5µm particle size was used for separation. Mobile phase used for separation was a mixture of two components i.e. methanol and water in the ration of 90;10 adjusting the pH to 3.2 with orthophosphoric acid. The flow rate was kept 1.0ml/min, system was carried out in a room temperature and eluents were detected by UV detector at the wavelength 254nm, the injection volume was 20µl.

2.3. Selection of solvents:

The solubility of both drugs is determined in verity of solvents as per pharmacopoeia standard. Solubility test was carried out in different solvents like acetonitrile HPLC grade, methanol HPLC grade, water HPLC grade, ethanol. From this, studies it was found that methanol and water which is a good solvent for both drugs.

2.4. Preparation of standard stock solution:

An accurately weighed quantity of 10mg was taken in a 10ml of volumetric flask. Small amount of mobile phase was added to it for dissolve and sonicated for 10 min and made the volume up to the mark (1000mcg/ml). From that solution 4ml was taken in a 100ml of volumetric flask and 80 ml of solvent was added to it. This was again sonicated for 10 mins and volume was diluted up-to the mark with solvent to get the concentration 40mcg/ml (working standard).

Figure 3. Diagram of dilution process

Note: Every step of mixing the solvent to inject sonication is most important.

2.5. Preparation of mobile phase:

mobile phase was prepared by using 90 volume of methanol with 10 volume of water HPLC grade was mixed and was adjusted the pH 3.2 with orthophosphoric acid. Sonicated the solvent for 10-15 mins. The mobile phase was then ultrasonicated, filtered through 0.45µm membrane filter paper with the help of vaccum filtration, degassed the content again.

3. METHOD VALIDATION:

3.1. System suitability:

it was performed by injecting the blank solution once 100% test concentration standard solution for six times into HPLC system. The system suitability test was evaluated from the chromatograms obtained^[11].

3.2. Specificity:

it was determined by comparison between standard drug with sample. Fixed concentration of working standard of both standard drug and sample test solution were injected to HPLC system for six time were analysed. % of RSD was calculated from their peak area^[12].

3.3. Linearity:

linearity was demonstrated over the range of 8-28µg/ml of test concentration. The solution at six level of the concentration was prepared and 20µl of each solution were injected into the HPLC system to get the chromatograms. By plotting concentration against the peak area, the linearity curve was constructed and the regression equation was calculated by the method of least squares, the correlation coefficient, y-intercept and slope of the regression line were reported^[9].

3.4. Accuracy:

the closeness of agreement of between a test result and true value.

Accuracy (%) = 100 x test value /reference value

Method was established by performing recovery studies, recovery studies was performed by spiking sample solution with pure standard drug at three different concentration level. Mean recovery of the drug was calculated.

3.5. Precision:

Intraday and interday precision of the method were demonstrated by taking one of the test concentration. The concentration injected in triplicate in to HPLC system to obtained the chromatogram and peak areas were recorded from the obtained peaks. The average and the standard deviation of the peak areas at each concentration level were calculated^[10].

% of RSD can be calculated by 100 x SD/X

SD= standard deviation of “n” responses

X= mean of “n” responses

3.6. Limit of detection:

it can be defined as the lowest amount of the analyte in a sample which can be detected but not necessarily quantitated as an extract value, using a specific method under the required experimental conditions ^[13].

LOD = 3σ/S

where σ= standard deviation of the response

S = slope of the calibration curve

3.7. Limit of quantification:

it can be defined as the smallest concentration of analyte which gives a response that can be accurately quantified. It can be calculated by

LOD = 10σ/S

where σ = standard deviation of response

S = slope of the calibration curve

3.8. Robustness:

For the determination of method’s robustness, deliberate change in flow rate, mobile phase composition, pH, temperature was made to evaluate the impact of this variation on the method ^[14].

4. RESULT AND DISCUSSION:

4.1. optimization of chromatographic condition:

To develop a suitable RP-HPLC method for simultaneous estimation of amlodipine and valsartan, different chromatographic conditions were applied and optimized chromatographic conditions were developed (see figure 4).

Optimized chromatographic conditions are as follows:

Instrument: water 1525 binary HPLC pump, water 2487 dual λ absorbance detector and empower3 software.

Mobile phase: methanol and water along with orthophosphoric acid to adjust pH 3.2 (90:10 v/v)

Injection volume: 20 µl

Flow rate: 1ml/min

Detection wavelength: 254 nm

Runtime: 5 min

Temperature: room temperature

4.2. VALIDATION:

4.2.1 specificity:

Table 1: specificity data

	Avg standard area	Avg sample area	SD	RSD	interference
AMLO	164516.3	164225	1263.6	0.7	RSD found to be <2
VAL	212849.7	212512	1604.5	0.8	RSD found to be <2

Figure 4. Optimized chromatogram of amlodipine and valsartan

4.2.2. Linearity:

The calibration curve was constructed with concentration on X-axis and peak area on Y-axis to establish the linearity of drug. From the calibration curve, it was observed that the method was linear over the concentration range of 8-28 mcg/ml for both drugs. (see figure 5 and table 2)

Table 2: peak areas of linearity standard solution of amlodipine and valsartan

Sl. No	Concentration	Area of AMLO	Area of VAL
1	8	69910	114350
2	12	104130	152069
3	16	130424	178857
4	20	162764	209069
5	24	197749	244808
6	28	229592	279529

Figure 5: linearity plot of amlodipine(a) and valsartan(b) (8-28 mcg/ml) standard solution

4.2.3. Accuracy:

The recovery studies were performed to check the accuracy of the method at three levels 50%,100% and150%. The mean recovery of amlodipine was found to be 98.7% and valsartan was 99.5%. (see table 4 and 3)

Table3: recovery studies of valsartan

Level	Added amount (µg/ml)	Found amount (µg/ml)	Recovery	Average % recovery
50%	8	8.02	100.3	99.5
100%	16	15.85	99.1
150%	24	23.73	99.3

Table 4: recovery studies of amlodipine

Level	Added amount (µg/ml)	Found amount (µg/ml)	Recovery	Average % recovery
50%	8	7.93	99.2	98.7
100%	16	15.71	98.2
150%	24	23.7.	98.9

4.2.4. Precision:

precision of the method was studied by making repeated injections of the mixture of drugs. The coefficient variation CV was after six determination was 0.77% and 0.75% for amlodipine and valsartan at 20mcg/ml respectively. The % of RSD of peak area of chromatograms of AMLO and VAL is <2 for intraday as well as interday precision respectively (see table no 5 and 6).

Table 5: intraday precision

injection (20mcg/ml)	Area amlodipine	Area valsartan	Interference
1	164511	214511	% RSD was found to be <2
2	164321	212321
3	164521	211521
4	164542	211542
5	164985	211985
6	164218	215218
Mean	164516.3	212849.7
Standard deviation	1263.6	1604.5
CV	0.77%	0.75%
RSD	0.7	0.8

Table 6: Interday precision

AMLO
	8 mcg/ml	12 mcg/ml	16 mcg/ml	20 mcg/ml	24 mcg/ml	28 mcg/ml
Day 1	69554	104130	179845	164985	242985	228592
Day 2	69889	102121	179750	165858	243584	228452
Day 3	69425	104215	178050	164594	245421	229712
Mean	69622.667	103488.7	179215	165145.7	243996.7	228918.7
STD DEV	239.5002	1185.196	1010.037	647.1355	1269.348	690.6036
%RSD	0.3439	1.145243	0.56359	0.391857	0.520232	0.301681
VAL
Day 1	114955	152070	178895	206045	244755	279535
Day 2	115542	153054	175845	203175	242298	275145
Day 3	114384	152019	175548	209451	244345	279751
Mean	114960.3	152381	176762.7	206223.7	243799.3	278143.7
SD	579.0184	583.3927	1852.616	3141.812	1316.255	2599.166
%RSD	0.503668	0.382851	1.048081	1.523498	0.539893	0.934469

Interference: % RSD was found to be <2

4.2.5. Limit of detection and limit of quantification:

(LOD and LOQ) of amlodipine and valsartan were determined by calibration curve method. Solution of both were prepared in the range of 8-28 mcg/ml and injected triplicate (see table no 7)

Formulas: LOD = 3.3 x SD/SLOPE

LOQ = 10 X SD/SLOPE

Table 7: Result of LOD AND LOQ of amlodipine and valsartan

	AMLO	VAL
SD	1263.6	1604.5
SLOPE	7940.1	8102.3
LOD	0.525167	0.6535
LOQ	1.591416	1.980302

4.2.6 robustness:

The standard chromatogram of the drugs was within limit for variation in flow rate (0.2 ml) and flow rate within the range of 0.8-1.0ml was allowed and variation in wavelength (2nm). The % RSD values is <2.0%, hence the method is proved to be robust.

Table 8: Result of robustness

Parameters	Retention time	AMLO %RSD	Retention time	VAL %RSD	Interference
Flow rate
1.0 ml/min	1.17 min	0.7	2.51	0.8	%RSD was found to be <2
0.8 ml/min	1.7 min	0.4	2.69	0.5	%RSD was found to be <2
Wavelength
245 nm	1.16 min	0.6	2.53	0.78	%RSD was found to be <2
249 nm	1.13 min	0.45	2.5	0.52	%RSD was found to be <2

4.2.7 system suitability:

SD, %RSD were calculated by performing the system suitability test the retention time of 1.5 min and 2.5 min were exhibited by the chromatograms. From the system suitability studies, it was observed that %RSD of peak area was to be 0.10 for amlodipine and 0.08 for valsartan standard drug. See table no 9.

Table 9: data of system suitability

Injection	Retention time (AMLO)	Peak area	Retention time (VAL)	Peak area
1	1.13	164348	2.11	211121
2	1.11	164215	2.19	211543
3	1.18	164454	2.09	211254
4	1.09	164154	2.19	211010
5	1.17	164521	2.12	211130
6	1.19	164121	2.13	211150
SD		164.569		184.51
%RSD		0.10		0.08

5. CONCLUSION:

The validation procedure of a newly developed RP-HPLC method for simultaneous estimation of amlodipine and valsartan was validated as per ICH guidelines. The method was validated in the purpose for routine analysis of the raw materials as well as the use of the pharmaceutical practices. The method was found to be rapid, precise, sensitive, accurate, reproducible, robust and specific of the bulk and formulation of amlodipine and valsartan. Since the method is validated successfully it can be assigned as a good economical method, which shows the accomplished sustained release of the drug.

6. REFERENCE:

1. Ashish Chauhan, Bharti Mittu and Priyanka Chauhan. Analytical method development and validation: a concise review. Journal of Analytical and Bioanalytical Techniques. 2015; 6(1): 1-5

2. Azim Md. Sabir, Mitra Moloy, Bhasin Parmindar S. HPLC method development and validation: a review. international research journal of pharmacy. 2013; 4(4): 39-46

3. S. B. Wankhede, S. B. Wadkar, K. C. Raka and S. S. Chitlange. Simultaneous estimation of amlodipine besilate and Olmesartan medoxomil in pharmaceutical dosage form. International journal of pharmaceutical sciences. 2009; 71(5): 563-567

4. P.S. Jain, M.K. Patel, A P Gorle, A J Chaudhari and S J Surana. Stability indicating method for the simultaneous estimation of Olmesartan medoxomil, amlodipine besylate and hydrochlorothiazide by RP-HPLC method in tablet dosage form. Journal of chromatographic science. 2012; 50: 680-687

5. shah SK, Asnani AJ, Kawade DP, Dangre SC, Arora SK, Yende SR. simultaneous quantitative analysis of Olmesartan medoxomil and amlodipine besylate in plasma by high performance liquid chromatography technique. Journal of young pharmacists. 2012; 4(2): 88-94

6. Silvana E. Vignaduzzo, Patricia M. Castellano a and Teodoro S. Kaufman. Development and validation of an HPLC method for the simultaneous determination of amlodipine, hydrochlorothiazide, and valsartan in tablets of their novel triple combination and binary pharmaceutical associations. Journal of Liquid Chromatography and Related Technologies. 2011; 34(19): 34-19

7. Rasha A. Shaalan a and Tarek S. Belal. gradient HPLC-DAD determination of the antihypertensive mixture of amlodipine besylate, valsartan, and hydrochlorothiazide in combined pharmaceutical tablets. Journal of Liquid Chromatography and Related Technologies. 2012; 35(2): 215-230

8. Mustafa Celebier, Mustafa Sinan Kaynak, Sacide Altınoz1, Selma Sahin. HPLC method development for the simultaneous analysis of amlodipine and valsartan in combined dosage forms and in vitro dissolution studies. Brazilian Journal of Pharmaceutical Sciences. 2010; 46(4): 761-768

9. Nesrin K. Ramadan a, Heba M. Mohamed a and Azza A. Moustafa. Rapid and Highly Sensitive HPLC and TLC Methods for Quantitation of Amlodipine Besilate and Valsartan in Bulk Powder and in Pharmaceutical Dosage Forms and in Human Plasma. Analytical Letters. 2010; 43(4): 570-581

10. Nitesh Maheshwari. Validated reverse phase high performance liquid chromatography method for simultaneous estimation of valsartan potassium and amlodipine besylate in tablet dosage form. International journal of drug development and research. 2013; 5(3): 237-244

11. Asmita Y. Kamble, Mahadeo V. Mahadik, Laxman D. Khatal and Sunil R. Dhaneshwar. Validated HPLC and HPTLC method for simultaneous quantitation of amlodipine besylate and Olmesartan medoxomil in bulk drug and formulation. Analytical Letters. 2010; 43(2): 251-258

12. Susheel John Varghese a and Thengungal Kochupappy Ravi. Quantitative simultaneous determination of amlodipine, valsartan, and hydrochlorothiazide in "Exforge HCT" tablets using high-performance liquid chromatography and high-performance thin-layer chromatography. Journal of Liquid Chromatography and Related Technologies. 2011; 34(12): 981-994

13. Swamy, Ravindra, Sowmya. A new RP-HPLC method development and validation for the simultaneous estimation of amlodipine and valsartan in tablet dosage forms. Asian journal of pharmaceutical analysis. AJP Ana. 2014; 4(3): 103-07

14. Koralla, Konidala, Rao, Begum. Stability indicating RP-HPLC method for simultaneous estimation of ramipril and amlodipine besylate in pharmaceutical dosage form. Asian journal of pharmaceutical research. AJP Ana. 2016; 6(4): 142-49

Received on 30.06.2020 Modified on 14.07.2020

Research J. Science and Tech. 2020; 12(3):183-189.

DOI: 10.5958/2349-2988.2020.00025.X