Assay Method Development and Validation for Valsartan using high Performance liquid Chromatography

Dr. O. S. S. Chandana

Aditya College of Engineering, Surampalem, Andhra Pradesh, India-533437.

*Corresponding Author E-mail: osschandana@gmail.com

ABSTRACT:

Our main objective is to develop a simple, accurate, precise and stability indicating HPLC method for the determination of valsartan and its impurities. An inertsil ODS-3v; (150 × 4.6) mm; 5 µm column is used for the separation of drugs. The flow rate was maintained at 1.0 mL/min and the wave length used for detection was 230 nm. The linearity was observed in the range of 0.025-50µg/ml of spiked impurities in valsartan, impurity B and impurity C with a correlation coefficient of 0.990, 0.996 and 0.997 respectively. The drug undergoes degradation under acid, base, H₂O₂, thermal and humidity conditions. Linearity, accuracy, precision and robustness parameters for the suggested method were estimated for validation as per ICH guidelines. The developed method can be utilized in the analysis of valsartan tablets.

KEYWORDS: Valsartan, HPLC, stability indicating, Validation, force degradation studies.

1. INTRODUCTION:

Valsartan (trade name Diovan) is an angiotensin II receptor antagonist (commonly called an ARB, or angiotensin receptor blocker), that is selective for the type I (AT₁) angiotensin receptor. Valsartan is mainly used for treatment of high blood pressure, congestive heart failure, and to increase the chances of living longer after a heart attack. Valsartan is chemically N-(1-Oxopentyl)-N-[2'-(1H-tetrazol- 5-yl) [1, 1'-biphenyl]-4-yl] methyl]-L-valine, is a potent angiotensin receptor blocker belonging to BCS class II (Fig-1) [1]. Molecular formula and Molecular weight are C₂₄H₂₉N₅O₃and435.519g/mol.

Impurity-B is chemical known as(S-N-Butyryl-N-([2’-(1H-tetrazole-5-yl) biphen-4-yl] methyl)-valine. Impurity- C is chemically known as(S-N-Valeryl-N-([2’-(1H-tetrazole-5-yl) biphen-4-yl] methyl)-valine benzyl ester. The structure of valsartan and its impurities are shown in the figure1.

Valsartan Impurity-B

Impurity -C

Figure 1: Structure of Valsartan and its impurities

The development and validation of an analytical method is to ensure a specific, accurate and precise method for a particular analyte. The principal objective for that is to enhance the conditions and parameters, which should be observed in the evolution and establishment. Literature survey revealed that HPLC [2-4], LC-MS [5-8], protein precipitation [9], capillary electrophoresis [10] and simultaneous UV spectrophotometric methods [11-12] were reported for estimation of Valsartan alone or in combination with other drugs. Two stability indicating high performance liquid chromatography methods were reported for the determination of impurities and assay of Valsartan [13,14,15]. So far no method is available for the determination of Valsartan along with its process impurities. At present study attempts were made to develop simple, rapid, robust and economic method for the estimation of Valsartan along with its process impurities. The developed method was validated as per ICH guidelines. Due to short run time and simple mobile phase composition, the proposed method will be of immense help to the pharmaceutical industry for routine analysis of Valsartan along with process impurities in bulkdrug.

2. MATERIALS AND METHODS:

2.1 Reagents and Materials:

The reference sample of Valsartan and its impurities (b), (c) were supplied as a gift sample from Fortune laboratories, Kakinada, Andhra Pradesh. Milli-Q-water was used throughout this research. All other analytical reagents such as Ammonium formate, Acetonitrile, Hydrochloric acid, Sodium hydroxide and Hydrogen peroxide (30%) were obtained from Merck specialty chemicals, Mumbai, India.

2.2 INSTRUMENT:

This work has been performed on PEAK Chromatographic (HPLC) instrument. It has binary gradient pump (G1311A), G1314A variable wavelength detector (VWD), G1329A Auto Sampler and G1316A column compartment. Chromatogram was analysed using PEAK chromatographic chemistration version B.02.01.

2.3 Chromatographic Conditions:

The liquid chromatography is equipped with a 230 nm UV detector

Column : Inertsil ODS-3V, 4.6 ｘ150 mm, 5μm

Flow Rate : 1.0 ml/min

Injection Volume : 20 μL

Run Time : 35 min

Column oven temperature : 30°C

Gradient Program :

Table-1: Gradient programming of binary mobile phase

Time (in min)	% MP-A	% MP-B
0	40	60
26	20	80
27	40	60
35	40	60

2.4 Preparation of mobile phase-A:

About 1.36g of monobasic potassium phosphate and 0.87g of dipotassium hydrogen

Phosphatewere dissolved in 1000mL of Milli-Q water, mixed well and adjusted its pH to 3.0

With dilute orthophosphoric acid and then solution filtered through 0.45µm membrane filter followed by degassing by sonicator for 10 minutes.

2.5 Preparation of Mobile phase-B:

Milli-Q water and acetonitrile were mixed in the ratio of 200:800 v/v respectively and then degassed in a sonicator for about 10 minutes.

2.6 Preparation of diluent:

Water and acetonitrile are mixed in the ratio of 500: 500 v/v respectively.

2.7 System suitability solution preparation:

About 2mg of USP Valsartan related compound-B and 20mg ofValsartan working or reference standard was weighed accurately and transferred into a 100ml volumetric flask, then diluted to volume with diluent.5.0 ml of the above solution was transferredinto a 50ml volumetric flask and diluted to volume with diluent.

2.8 Standard Solution Preparation:

About 50mg of Valsartan working or reference standard was weighed accurately into a 250ml volumetric flask, dissolved and diluted to volume with diluent. Then 5.0 ml of above standard stock solution was transferred into a 100ml volumetric flask, diluted with diluent to volume and mixed well. Now 4.0ml of above solution was transferred into a 100ml volumetric flask, diluted with diluent to the volume.

2.9 Preparation of Sensitivity solution:

5.0ml of standard solution was transferred into a 20ml volumetric flask and diluted with diluent to the volume and mixed well.

3.0 Test solution preparation:

Not less than 20 tablets were weighed and crushed to a fine powder in mortar with pestle. An accurately weighed portion of tablet powder equivalent to 200mg of Valsartan was transferred into a 200mL volumetric flask. To this 120ml of diluent was added and sonicated for 20 minutes with intermediate shaking in a sonicator. Now the flask was cooled to room temperature and diluted to volume with diluent and mix well. Some portion of the sample solution was centrifuged at 3500rpm for 15 minutes. From this 5.0ml of the solution was pipetted into a 25ml volumetric flask and diluted to volume with diluent and mixed well.

The chromatograms for optimized method is shown in Fig2

Figure 2: Chromatograms for optimized method

3.0 RESULTS AND DISCUSSION:

METHOD VALIDATION:

A Gradient reverse – phase HPLC procedure was suggested as a suitable method for the analysis of Valsartan and its impurities. From the results of optimized method, 7.16, 5.41 and 21.26 minutes were the retention times for Valsartan, impurity B and impurity C respectively. System suitability parameters like theoretical plate, % relative standard deviation and tailing factor for the Valsartan, Impurity B and Impurity C were reported. Resolution values of between Valsartan and Impurity C and between Valsartan and impurity B were found to be 36.49 and 7.07 respectively. Relative retention time values of Valsartan, impurity B and impurity C were found to be 0.76, 1.00 and 2.97 respectively.

3.1 SYSTEM SUITABILITY PARAMETERS:

Standard solutions were prepared as per optimised procedure and injected into the HPLC system as per optimized method. Evaluated system suitability parameters and Summarized in the table -2

Table 2: System suitability results

System suitability parameters		Observed value	Acceptance criteria
Theoretical Plates	Valsartan	11108	Should be NLT 2000
	Impurity-B	6004
	Impurity-C	4534
%RSD	Valsartan	0.44	Should be NMT 5.0
	Impurity-B	1.3
	Impurity-C	1.5
Tailing factor	Valsartan	1.0	Should be NMT 2.0
	Impurity-B	1.2
	Impurity-C	0.9

3.2 Method Precision:

The method precision of test method was evaluated by analyzing five standard preparation and test preparations. Test preparations was prepared by spiking test preparation with Valsartan impurities blend solution to get 0.2% of each impurity with respect to test concentration and analyzed as per test method. Method precision results were given in percentage content. The individual results of Valsartan and its impurities were reported in the table 3.0

Table 3.0: Method precision data for Valsartan and its impurities:

Injection	Valsartan	Impurity-B	Impurity-C	Amount of valsartan in mg	Impurity-B (mg) present in Valsartan	Impurity-C(mg.) present in Valsartan	Percentage of impurity B present in spiked sample	Percentage of impurity C present in spiked sample
1	98.84	98.22	98.38	49.418	0.098	0.098	0.196	0.197
2	101.40	99.18	100.62	50.698	0.099	0.101	0.198	0.201
3	99.94	102.04	100.84	49.970	0.102	0.101	0.204	0.202
4	103.78	100.13	103.31	51.892	0.100	0.103	0.200	0.207
5	98.45	99.70	99.50	49.225	0.100	0.099	0.199	0.199
6	101.03	101.08	98.15	50.514	0.101	0.098	0.202	0.196
Mean	100.57	100.06	100.13	50.286	0.100	0.100	0.200	0.200
Standard deviation	1.96	1.36	1.91	0.978	0.001	0.002	0.003	0.004
% Relative standard deviation	1.94	1.36	1.91	1.944	1.361	1.906	1.361	1.906

3.3 Force degradation studies:

Blank and placebo solutions were prepared and injected into HPLC system. Blank and Placebo solutions were analyzed as per test method. Forced degradation study was conducted to demonstrate the effective separation of degradants from valsartan. Valsartan was exposed to the following stress conditions such as refluxed with 1M HCl solution for about 24 hours at 70ºC (Acid). Refluxed with 1M NaOH solution for about 24 hours at 70ºC (Base). Treated with 3% Hydrogen peroxide (H₂O₂) for 24 hours at 70ºC (Peroxide).Dry heat at 85°C for about 24 hrs.

Force degradation studies are resulted in the table-4

Table 4. Forced degradation studies

S. No	Valsartan	% of Degradation	% of Assay	Mass balance
1	Unstressed API	0.141	98.06	98.20
2	Unstressed tablet valsartan	0.129	99.58	99.71
3	Acid stressed API	0.116	98.89	99.01
4	Acid stressed capsule valsartan	0.114	96.98	97.10
5	Base stressed API	0.136	98.06	98.19
6	Base stressed capsule valsartan	0.309	98.79	99.10
7	Thermal stressed API	0.145	98.96	99.10
8	Thermal stressed capsule valsartan	0.134	97.29	97.43
9	H₂O₂ stressed API	0.278	98.37	98.65
10	H₂O₂ stressed capsule valsartan	0.784	98.08	98.87
11	Humidity stressed API	0.147	99.38	99.46
12	Humidity stressed capsule valsartan	0.201	99.98	100.02

3.4: Limit of detection and limit of quantitation:

Limit of Detection and Limit of Quantitation were established based on signal to noise ratio. A series of injections of blank solution were injected and average noise was calculated. Limit of Detection for each impurity was established by identifying the concentration which gives signal to noise ratio about 3. Limit of Quantitation was established by identifying the concentration which gives signal to noise ratio about 10.

Table 5: LOD and LOQ Values

Name	Concentration		Signal to Noise Ratio
Name	LOD	LOQ	LOD	LOQ
Valsartan	0.0004	0.0012	3.1	9.3
Impurity-B	0.0002	0.0006	3.2	9.6
Impurity-C	0.0001	0.0003	3.3	9.9

3.5 Accuracy (%Recovery):

A study of accuracy of Valsartan impurities from spiked samples of test preparation was conducted. Samples were prepared in triplicate at each level by spiking test preparation with 50%, 80%, 100%, 150% and 200% of target concentration (i.e., 0.2% of each impurity) of Valsartan impurities. The closeness of obtained value to true value indicates that the proposed method was accurate. The recovery data for accuracy studies was shown in table-6.

3.6 Linearity:

Linearity was established by plotting a graph between concentration versus peak area and the correlation coefficient was determined. A series of solutions of Valsartan and its impurities with concentrations ranging from 50%, 75%, 100%, 125% and 150% were prepared and injected into the HPLC system. The concentrations of above percentage solutions were mentioned. Excellent correlation was obtained between peak area and concentration withr² =0.99, 0.996 and 0.997 for valsartan, impurity B and impurity C respectively. The calibration curves for linearity for valsartan and its impurities are shown in figures 2,3and 4.

Table 6: Accuracy for Valsartan

	Valsartan	Impurity B	Impurity C	Valsartan		Impurity B		Impurity C
Samples	Amount in mg			Mean % recovery	%RSD	Mean % recovery	%RSD	Mean % recovery	%RSD
50%	52.22± 0.425	0.10± 0.001	0.10± 0.001	104.43± 0.850	0.81	96.83± 0.764	0.79	96.77± 1.365	1.41
80%	48.10± 0.826	0.15± 0.004	0.15± 0.004	96.20± 1.652	1.72	95.07± 2.572	2.71	95.33± 2.309	2.42
100%	48.67± 0.764	0.20± 0.002	0.21± 0.002	97.33± 1.528	1.57	97.67± 1.155	1.18	102.67± 1.155	1.12
150%	49.03± 0.785	0.30± 0.002	0.29± 0.007	98.07± 1.570	1.60	98.83± 0.569	0.58	97.43± 2.205	2.26
200%	48.17± 0.764	0.39± 0.007	0.41± 0.005	96.33± 1.528	1.59	97.00± 1.732	1.79	101.77± 1.365	1.34

Table 7: Linearity data

Valsartan		Impurity A		Impurity B
Concentration of Valsartan	Mean± Standard deviation	Concentration of Impurity A	Mean± Standard deviation	Concentration of Impurity B	Mean± Standard deviation
5	9652.00±78.94	0.005	952.33 ±9.50	0.005	749.33±4.04
7.5	10388.00±43.51	0.0075	1048.00±5.00	0.0075	813.33±2.52
10	11863.00±157.88	0.01	1159.00±6.00	0.01	890.33±2.52
12.5	12640.00±95.54	0.0125	1227.00±15.10	0.0125	940.67±2.08
15	13681.00±41.33	0.015	1348.00±6.00	0.015	994.33±5.03
Slope	412.40±1.53	Slope	38813.33±2040.52	Slope	24693.33±454.90
Intercept	7520.80±89.95	Intercept	758.73±17.90	Intercept	630.67±4.39
Correlation Coefficient	0.99±0.00	Correlation Coefficient	0.996±0.00	Correlation Coefficient	0.997±0.00

3.7 Robustness:

Robustness was carried with different mobile phases and column oven temperatures. Column oven temperature can be used from 25°C to 35°C. % RSD varies from 0.66%-0.23%.

Solution Stability:

Bench top Stability of diluted standard solution and Test preparation:

Similarity factor at 24 hrs for Stability of Diluted standard preparation at ambient temperature about (25ºC) were found to be 0.99, 1.00 and 1.01 for Valsartan, impurity B and impurity C respectively. Results were reported in the table 8.

Refrigerator Stability of diluted standard solution and Test preparation:

Similarity factor at 24 hrs were found to be 1.01, 1.00 and 1.00 for Valsartan, impurity A and impurity B under refrigerator temperature respectively. Results were reported in the table 9.

Figure 3: Linearity curve for Valsartan and its impurities

Table 8: Stability of test preparation at ambient temperature about (25ºC)

Time in hours	Valsartan				Percentage of impurity B present in Valsartan				Percentage of impurity C present in Valsartan
	% content		Difference from Initial		% Impurity		Difference from Initial		% Impurity		Difference from Initial
	Sol-1	Sol-2	Difference from Initial		Sol-1	Sol-2	Difference from Initial		Sol-1	Sol-2	Difference from Initial
Initial	98.01	101.09	NA		0.196	0.202	NA		0.198	0.198	NA
24	99.05	102.29	1.04	0.198	0.205	0.201	0.199	0.195	0.18	0.172	0.018	0.026

Sol = solution; NA = not applicable

Table 9: Stability of test preparation at refrigerator temperature about (8ºC)

Time in hours	Valsartan				Percentage of impurity B present in Valsartan				Percentage of impurity C present in Valsartan
	% content		Difference from Initial		% Impurity		Difference from Initial		% Impurity		Difference from Initial
	Sol-1	Sol-2	Difference from Initial		Sol-1	Sol-2	Difference from Initial		Sol-1	Sol-2	Difference from Initial
Initial	99.15	99.16	NA		0.203	0.199	NA		0.205	0.201	NA
24	99.62	97.47	0.47	0.200	0.200	0.02	0.003	0.001	0.203	0.2	0.002	0.001

Sol = solution; NA = not applicable

4.0 CONCLUSION:

A simple HPLC method was developed for the determination and method validation of Valsartan along with impurities. The accuracy, precision, specificity, robustness, and linearity are within the acceptance limits. Hence this HPLC method can be used for routine pharmaceutical analysis is the most simple and cost effective method.

5.0 CONFLICTS OF INTEREST:

“This manuscript has not been previously published and is not under consideration in the same or substantially similar form in any other peer-reviewed media.” The authors have no conflict of interest to declare with respect to financial, personal or other relationships with other people or organizations.

6.0 ACKNOWLEDGEMENT:

The author would like to thank Fortune laboratories, Kakinada for providing instrument facility and technical guidance.

7.0 REFERENCES:

1. Budavari S, the Merck index, Merck and Co. Press. Edn 12, Whitehouse Station, New Jersey, 1997.

2. Kocyiğit KB, Unsalan S, Rollas S. Determination and validation of Ketoprofen, Pantoprazole and Valsartan together in human plasma by high performance liquid chromatography. Pharmazie 2006; 61: 586-589.

3. Daneshtalab N, Lewanczuk RZ, Jamali F. High performance liquid chromatographic analysis of angiotensin II receptor antagonist Valsartan using a liquid extraction method. Journal of Chromatography B, Analytical Technology and Biomedical Life Science 2002; 766: 345-349.

4. Gonzalez L, Lopez JA, Alonso RM, Jimenez RM. Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high- performance liquid chromatography with fluorimetric detection. Journal of Chromatography A2002; 949: 49-60.

5. Koseki N, Kawashita H, Haraa H, Niina M, Tanaka M, Kawai R, Nagae Y, Masuda N. Development and validation of a method for quantitative determination of Valsartan in human plasma by liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2007; 43: 1769-1774.

6. Hao, Yingwu W, Yao J, Yunbiao T, Jiang W, Limei Z, Jingkai G A liquid Chromatography/tandem mass spectrometry method for the simultaneous quantification of Valsartan and Hydrochlorothiazide in human plasma. Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences 2007; 852: 436-442.

7. Selvan PS, Gowda KV, Mandal U, Solomon WDS, Pal TK. Simultaneous determination of fixed dose combination of Nebivolol and Valsartan in human plasma by liquid chromatographic-tandem mass spectrometry and its application to pharmacokinetic study. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences 2007; 858: 143-150.

8. Jing N, Bingren X, Yuqi F, Danhua.. Isolation and Identification of Process Impurities in Crude Valsartan by HPLC, Mass Spectrometry, and Nuclear Magnetic ResonanceSpectroscopy. Journal of Liquid Chromatography & Related Technologies 2006; 29(4): 553 -568.

9. Macek J, Klima J, Ptacek P Rapid determination of Valsartan inhuman plasma by protein precipitation and high performance liquid chromatography. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences 2006; 832: 169-172.

10. Hillaerty S, Bossche VW. Simultaneous determination of Hydrochlorothiazide and several Angiotensin-II-receptor antagonists by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis 2003; 31: 329-339.

11. Satana E, Altmay S, Goger NG, Ozkan SA, Senturk Z. Simultaneous determination of Valsartan and Hydrochlorothiazide in tablets by first-derivative ultraviolet Spectrophotometry and LC. Journal of Pharmaceutical and Biomedical Analysis 2001; 25: 1009-1013.

12. Tatar S, SaglikS.Comparision of UV and second derivative spectrophotometric and LC methods for the determination of Valsartan in pharmaceutical formulation. Journal of Pharmaceutical and Biomedical Analysis 2002;30: 371-375.

13. Agrahari V, Kabra V, Gupta S, Nema RK, Nagar M, Karthikeya C, Trivedi P. Determination of inherent stability of Valsartan by stress degradation and its validation by HPLC. International Journal of Pharmaceutical and Clinical Research 2009; 1:77-81.

14. Bhatia M. Sudesh and Kokil S. Uttamrao Determination and validation of valsartan and its degradation products by isocratic HPLC. J. Chem. Metrl. 2009; 3: 1-12.

15. ICH. Stability Testing of New Drug Substances and Products. International Conference on Harmonization, IFPMA, Geneva, 2003.

Received on 27.02.2020 Modified on 18.03.2020

Asian J. Pharm. Ana. 2020; 10(2): 103-108.

DOI: 10.5958/2231-5675.2020.00018.6