Development and Validation of a Forced Degradation UPLC Method for the Simultaneous Determination of Nebivolol HCl and Valsartan in Bulk and Pharmaceutical Dosage Form

 

S. Sangeetha*, S. Alexandar, M. V. Kumudhavalli, M. Kumar

Department of Pharmaceutical Chemistry, Vinayaka Mission’s College of Pharmacy, Salem - 636008,

Vinayaka Mission’s Research Foundation (Deemed to be University) Tamilnadu, India.

 

ABSTRACT:

An excellent method with simple, precise was developed for Nebivolol HCl and Valsartan by using Forced degradation UPLC method. The column used was C-₁₈ BEH _ 1.7µm x 2.1 x 50mm in ambient temperature. Flow rate was 0.8ml/min, wavelength of 278nm, mobile phase used was acetonitrile : Buffer (50:50). Buffer used 0.01 N Disodium hydrogen phosphate with pH 3.5 adjusted by OPA. Run time 4 min. The percentage purity and RT of Nebivolol HCl and Valsartan was found to be  99.72 and 99.30 and 1.193 and 1.827  respectively. The validation parameters was carried out, linearity of Nebivolol HCl was found to be (1μgm/ml to 8μgm/ml) R²= 0.998 and Valsartan was found to be (16μgm/ml to 128.8μgm/ml) R²= 0.999. Intermediate precision, Robustness, LOD LOQ was within the limit as per ICH guidelines. Recovery studies taken place in 80%, 100% and 120. Forced Degradation was carried out in three conditions acidic, basic and peroxide condition, degradation takes at basic and peroxide. As per literature review there is no method developed for Nebivolol HCl and Valsartan in Forced degradation UPLC method. So we made attempt to develop the Nebivolol HCl and Valsartan in UPLC method.

 

 

 

INTRODUCTION: 

Nebivolol hydrochloride (NEB)¹ is (±) [2R* R* R* (S *)] œ, œ [iminobis (methylene)] bis- [6- fluoro- 3, 4 - dihydro2H- 1- benzopyran- 2-methanol] β1-adrenergicblocker 1,3,4 . It is a beta blocker used to treat high blood pressure and heart failure. It is generally less preferred than a number of other blood pressure medication. Valsartan² Chemically, is (2S)-3-methyl-2-[N- ({4- [2- (2H- 1, 2, 3, 4-tetrazol-5-yl)phenyl] phenyl} methyl) pentanamido] butanoic acid. By blocking the action of angiotensin, valsartan dilates blood vessels and reduces blood pressure. Valsartan is  a‎ Angiotensin II receptor antagonist medication used to treat high blood pressure, heart failure, and diabetic kidney disease. Valsartan¹³ is an ARB that selectively inhibits the binding of angiotensin II to AT1, which is found in many tissues such as vascular smooth muscle and the adrenal glands.

 

 

Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the safety and efficacy of the drug product. The FDA and ICH guidances state the requirement of stability testing data to understand how the quality of a drug substance and drug product changes with time under the influence of various environmental factor^3,4  . The drug was subjected to acid, base, oxidation,  which enabled separation and detection of degradation products from basic and oxidation stress¹² .Ultra Performance Liquid Chromatography (UPLC) system is an innovative product that brought revolution in high performance liquid chromatography by outperforming conventional high performance liquid chromatography (HPLC). UPLC decreases sample run times up to a factor of 10, uses up to 95 percent less solvent and significantly improves productivity in the laboratory^5,6.

 

HPLC and UV methods are reported for it^16-21. There is, however, no work reported on combination of these two drugs. In the present communication, we propose fast, simple and accurate spectrophotometric method for simultaneous estimation of both the drugs in tablet dosage form¹⁵.

 

Structure of Nebivolol HCl

 

Structure of Valsartan

 

MATERIALS AND METHOD:

HPLC Water- Milli-Q grade, Acetonitrile- Fisher scientific, Methanol- Fisher scientific, Potassium di hydrogen phosphate- Merck, Ortho phosphoric acid- Sigma, Hydrochloric acid- Merck. These solvents were throughout method development and validation. Nebivolol HCl and Valsartan  are gift sample from Aurobindo Pharmaceuticals Andhra Pradesh..

 

Instrumentations:

The UPLC method development and validation was done using Waters Acquity UPLC BEH Column. The dissolution apparatus Distek, UV-Visible spectrophotometer -Perkin elimer, Analytical balance-Sartorius.

 

ASSAY:

Test Solution Preparation:

Preparation of stock:

10 tablets were weighed and powered.Powered tablets transferred equivalent to about 5mg of Nebivolol HCl and 80mg of Valsartan into 50mL volumetric flask. Add about 10mL of mobile phase and sonicate for15min. Dilute to volume with diluent and mix well.

 

Preparation of standard:

Pipette out 10 mL of theabove stock solution into a 100 mL volumetric flask. Dilute to volume with diluent and mix well and to get final Concentration of about 100µg/mL Filter the above solution through 0.45µm PVDF membrane filter.

 

Chromatographic condition:

In the present study column used C-₁₈ BEH - 1.7µm x 2.1 x 50mm in ambient temperature. Flow rate was 0.8 ml/min, wavelength of 278nm, mobile phase used was Acetonitrile Buffer (50:50). Buffer used 0.01N Disodium hydrogen phosphate with  pH 3.5 adjusted by Ortho phosphoric acid. Run time 4 min. The retention time was found to be 1.193 and 1.827 for Nebivolol  HCl and Valsartan.

 

Validation Parameter:

·       System Suitability

·       Specificity

·       Linearity and Range

·       Precision

·       Ruggedness

·       Accuracy

·       Robustness

·       Limit of Detection and Limit of Quantitation

·       Solution stability

 

System Suitability:

System suitability was performed by injecting 5 replicate of injections Nevibolol and valsartan. Nevibolol HCl and Valsartan was diluted by mobile phase to the final concentration of 50μgm/ml and 80μgm/ml respectively. It was performed to determine the resolution, theoretical plates, tailing factor, repeatability of retention time etc. All parameters are within the range as per the ICH guidelines.

 

Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. It was performed to identify the any impurity may present, it was done by using standard, sample, placebo dilutions of both Nevibolol HCl and valsartan.

 

Calibration Curve ( Linearity):

Linearity of Nevibolol HCl was found to be (1μgm/ml to 8μgm/ml), 25.1mg of Nevibolol HCl was taken which is diluted to 50ml from that 0.2ml to 1.6ml was taken and make upto 100ml. Valsartan was found to be (16μgm/ml to 128.8μgm/ml), 40.1mg of valsartan was taken and diluted with mobile phase to 50ml from that 2ml to 16 ml was taken which is diluted to 100ml. %RSD of both Nevibolol HCl and valsartan was found to be 0.998 and 0.999 respectively. Tab 02, Fig 02,03.

 

Precision:

Intermediate precision was carried in both Nebivolol HCl and Valsartan,Precision of Nebivolol HCl standard dilution was (50μgm/ml) and sample dilution was (20 μgm/ml) ,Valsartan standard dilution (10μgm/ml) and sample dilution was (20μgm/ml) to determine repeatability of method development. With the same solution precision was carried out next day also. %RSD was with in the limit as per ICH guidelines. Tab 03

 

Accuracy:

Nebivolol HCl and Valsartan accuracy was study done in 80%, 100% and 120%. Recovery and percentage purity of Nebivolol HCl and valsartan was found in the range of 4.98 to 5.0 mg, 99.72% to 100.9% and 79.17 to 79.21 mg, 98.97 % to 99.02 % respectively. Tab 04

 

Robustness:

Robustness study was done by changing the pH, wavelength, flow rate in both the drug. % RSD was with in the limit as per ICH guidelines. Tab

 

LOD and LOQ:

Limit of detection was carried out in Nebivolol HCl and Valsartan the value are found to be 2.96 and 4.32 respectively.Limit of quantificationwas carried out in Nebivolol HCl and Valsartan the value are found to be 10.37 and 10.69 respectively.

 

Forced Degradation Study:

Acid Degradation Study (2N HCl)

The sample solution of Nebivolol HCl and Valsartan 50μgm/ml and 80μgm/ml respectivelyare expose to acidic condition for 30 minutes but the there is no degradation takes place.

 

Base Degradation Study (2N NaOH)

The sample solution of Nebivolol HCl and Valsartan 50μgm/ml and 80μgm/ml respectivelyare expose to basic condition for 30 minutes there was degradation takes place, Extra peak were obtained and changes in RT.

 

Peroxide Degradation Study (2N H₂O₂)

The sample solution of Nebivolol HCl and Valsartan 50μgm/ml and 80μgm/ml respectively are expose to a peroxide condition for 30 minutes there was degradation takes place, extra peak were obtained.

 

Table:1 Assay of Nevibolol HCl and Valsartan

Commercial Formulation	Drug	Standard area	Sample Area	Label Claim (mg)	Amount Present (mg)	% Purity
Byvalson	NevibololHcl	90204	88841	5mg	4.986 mg	99.72
	Valsartan	1361535	1364168	80 mg	79.174 mg	99.30

 

Fig: 01 Chromtagram of Nebivolol HCl and Valsartan

 

 

Table 2: Linearity and Range

S. No	Nevibolol HCl		Valsartan
	Conc (mcg/mL)	Mean area	Conc (mcg/mL)	Mean area
1	1	16120	16.1	271190
2	2	31891	32.2	533214
3	3	50250	48.3	827780
4	4	65767	64.4	1081726
5	5	86857	80.5	1361544
6	6	102373	96.6	1639361
7	7	121200	112.7	1912479
8	8	138120	128.8	2183451

Fig 02 Linearity of Nebivolol HCl

 

Fig 03 Linearity of Valsartan

 

Table 3: IntermediatePrecision Results for 5/80 mg Tablets

S. No	Nebivolol HCl			Valsartan
	mg/ tab	AUC	% Assay	Mg/ tab	AUC	% Assay
1	4.98	88841	99.72	79.17	1364168	98.97
2	4.94	87461	99.00	79.70	1361928	99.63
3	5.00	89176	100.09	79.21	1364915	99.02
4	4.89	86504	98.00	80.14	1368279	100.18
5	4.90	86410	98.02	80.32	1369571	100.41
6	4.95	87388	99.06	80.26	1369456	100.33
Mean	4.94	87630	99.76	79.80	1366386	99.76
%RSD		0.87			0.65

 

Table 4: Accuracy of Nebivolol HCl and Valsartan

% Conc	Nebivolol HCl				Valsartan
	mg/tab	AUC		% Assay	Mg/ tab	AUC		% Assay
		STD	Sample			STD	Sample
80%	4.98	88841	66753	99.72	79.17	1364168	1064794	98.97
100%	4.94	87461	86255	99.00	79.70	1361928	1390645	99.63
120%	5.00	89176	103942	100.09	79.21	1364915	166855	99.02

 

Table 5: Robustness of Nebivolol HCl and Valsartan

S. No	Nebivolol HCl						Valsartan
	Std area	Sample area		content	assay	%RSD	Std area	Sample area	content	assay	% RSD
Flow rate (0.7 ml)	91622		91284	4.96	99.21	0.51	1473254	1475231	79.8	99.80	0.25
Flow rate (0.9 ml)	80974		80854	4.96	99.029	0.73	1473254	1470750	79.5	99.50	0.26
Wavelength276nm	79406		79650	5.003	100.06	0.61	1537073	1543867	80.1	100.22	0.16
Wavelength 280nm	90910		91177	5.0024	100.05	0.98	1229902	123492	79.9	99.99	0.88
pH(3.45)	85675		85777	4.99	99.99	0.73	1365133	1370167	80.03	100.5	0.24
pH (3.55)	86427		86379	4.98	99.81	0.45	137839	1378405	79.74	99.87	0.18

 

 

Fig 04: Base Degradation chromtogram

 

 

Fig 05: Peroxide Degradation chromtogram

 

CONCLUSION:

The present study was method development and validation of Nebivolol HCl and Valsartan by uplc method with forced degradation study. Since no method was developed in UPLC, RT of Nebivolol HCl and Valsartan was found to be 1.193 and 1.827 respectively. In forced degradation study both Nebivolol HCl and Valsartan degradated by basic and peroxide condition. Analytical method validation plays a fundamental role in pharmaceutical industry for releasing the commercial batch and long term stability data¹⁴. So this study is useful for routine pharmaceutical analysis.

 

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Accepted on 16.10.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1002-1006.