Validated RP-HPLC method for the estimation of nebivolol hydrochloride and valsartan in combined tablet dosage form

R. Anantha Kumar¹*, G. Raveendra Babu², M. Sowjanya³, M. Ramayyappa²

¹Assistant Professor, Nova College of Pharmacy, Jangareddy gudem, Andhra Pradesh, India.

²A.K.R.G. College of Pharmacy, Nallajerla, Andhra Pradesh, India.

³Vijaya Teja Degree College, Addanki, Andhra Pradesh, India.

*Corresponding Author E-mail: ananth2.pharma@gmail.com

ABSTRACT:

The intention of this work is to develop a rapid, precise, accurate and sensitive reverse phase liquid chromatographic technique for the simultaneous estimation of Nebivolol and Valsartan in tablet dosage form. The chromatographic method was standardized using Inertsil ODS column (250×4.6mm, 5μm particle size) with UV detection at 278nm and flow rate of 1ml/min. The mobile phase comprises of ACN: Buffer (pH adjusted to 3.5 with dilute Ortho Phosphoric acid) in the fraction of 60:40 v/v. The linearity of proposed method was investigated in the range of 12.5-62.5μg/ml (R²=0.999) for Nebivolol and 200-1000μg/ml(R²=0.999) for Valsartan duly. The limit of detection (LOD) was found to be 0.05μg/ml and 0.81μg/ml for Nebivolol and Valsartan duly. The limit of quantification (LOQ) was found to be 0.15μg/ml and 2.44μg/ml for Nebivolol and Valsartan respectively. The retention time of Nebivolol and Valsartan were ground in to be 4.400min and 2.568min respectively. The method was authentically suggested and % RSD was ground in to be less than 2 indicating high degree of accuracy and precision. Hence proposed method can be successfully graded for the simultaneous estimation of Nebivolol and Valsartan in marketed formulation.

KEYWORDS: Nebivolol, Valsartan, RP-HPLC, ICH guidelines, Validation.

INTRODUCTION:

Valsartan (fig. 2) is a new potent, mighty selective and orally active antihypertensive drug belonging to the family of angiotensin II type I receptor antagonists. Angiotensin II receptor type I antagonists have been commonly used in the therapy of hypertension, heart failure, myocardial infraction, and diabetic nephropathy.

It is a lipophilic medicine, and it was first introduced by Novartis and has a largemarket in developed and developing countries. It is available as single and in blend with other antihypertensive drugs.

Nebivolol (fig. 1) is a beta blocker with a distinctive function which differs it from other beta blockers. It increases the production of nitric oxide (NO) which produces vasodilatation and thereby enhance arterial compliance and decreases peripheral vascular resistance. It also reduces heart rate without improving maximal exercise tolerance. These effects are beneficial in hypertension and angina pectoris^1-8.

Valsartan is rapidly absorbed after oral administration. The volume of distribution at constant state has been estimated 17L and mean absolute bioavailability is 23%. Food decreases the exposure of Valsartan by about 40% and peak plasma concentration by around 50%. 94% - 97% of drug bound to serum protein mainly albumin. It is eliminated by non-renal route. Plasma clearance of Valsartan is 2L/h, and renal clearance is 0.62L/h^9,10.

Fig. 1: Chemical structure of Nebivolol HCl

Fig. 2: Chemical structure of Valsartan

The literature review tells that these different dosage forms of Nebivolol hydrochloride and Valsartan are examined by a different method including crystal structure elution, polarimetry, UV, IR, HPLC, LCMS, and further more techniques are useful in different types of analysis in the different dosage form. But now a day most crucial and better procedures in HPLC and GC by using HPLC simple Reverse Phase Chromatographic method expand for the determination of active content and relative impurities, while it is better to find stability indicating a method for analysis ^11-20. Hence, the developed method aims in separating the selected drugs simultaneously. The developed method was validated as per the ICH guideleines^21-24.

MATERIALS AND METHODS:

Chemicals and reagents:

API of Nebivolol and Valsartan were collected as gift sample from Analog Labs (Hyderabad, Andhra Pradesh). Acetonitrile and water of HPLC grade were obtained from Rankem lab ltd. Ortho Phosphoric acid AR grade was purchased from E.Merck chemicals Mumbai, India. All other reagents were of AR grade. The money oriented sample of Nebicard-V containing Nebivolol 5mg and Valsartan 80mg is bought from local market.

Instrumentation

The LC system comprises of Waters model 2695, UV-Visible detector. The output signals were monitored and integrated using Empower2 software. Melter Electronic Balance together with Solomon pH meter were used.

Chromatographic conditions:

The chromatographic separation was attained on Inertsil ODS (250×4.6mm, 5μm) column using mobile phase consisting of ACN: Buffer in ratio of 60:40v/v. The buffer (0.01N disodium hydrogen phosphate) pH is adjusted to 3.5 with dilute ortho phosphoric acid and filtered through 0.45μm membrane filter. The column wasmaintained at room temperature (25ºC) and the flow rate is 1ml/min. Prior to inject of the solutions the column is stabilised for atleast 30 minutes with the mobile phase flowing through the system. The volume of sample injected was 20μl.The UV-Visible detector was set at wavelength 278nm.Under described experimental conditions, all the peaks were well defined and free from tailing. A typical chromatogram of Nebivolol and Valsartan sample is shown in figure 3. The parameters are tabulated in table 1.

Table 1: Chromatographic Conditions

Mobile phase	ACN: Buffer (60:40%, v/v)
Diluent	Mobile phase
Column	Inertsil C18 (250 mm x 4.6 mm, 5 µm)
Column temp.	Ambient
Wave length	278 nm
Injection vol.	20 µl
Flow rate	1 ml/min.
Run time	10 min.
Retention times	2.568 min for VAL; 4.400 min for NEB

Fig. 3: Chromatogram of Nebivolol and Valsartan sample

Preparation of Standard solution:

5mg of Nebivolol working standard and 80mg of Valsartan working standard was weighed and transferred into 100ml volumetric flask, to it 50 ml of diluent (mobile phase) was added and sonicated to dissolve. Then the solution was made upto mark with diluent. Further the solution was diluted to get a concentration of 50μg/ml of Nebivolol and 800μg/ml of Valsartan. The solutions of concentration range 12.5-62.5μg/ml of Nebivolol and 200-1000μg/ml of Valsartan were prepared and linearity was determined.

Preparation of Sample solution

About 20 tablets were taken and their average weight is determined. These pre-weighed 20 tablets are finely grinded. Of the grinded powder, sample quantitatively equivalent to 5mg Nebivolol and 80mg Valsartan is transferred into 100ml volumetric flask and 50ml of diluent is added, sonicated to dissolve for 10 minutes. Then the solution is made upto mark with diluent. Further the solution is filtered through 0.45μm membrane filter.10ml of above filtrate to is diluted to 100ml with diluent to get a concentration of 50μg/ml of Nebivolol and 800 μg/ml of Valsartan⁶.

Validation of the method:

The analytical method was validated as per ICH guidelines^7-9 with respect to parameters such as linearity, accuracy, precision, assay, ruggedness, robustness, limit of detection and limit of quantification as follows.

Linearity:

Linearity of this procedure was evaluated by linear regression analysis, calculated by least square method and considered by preparing standard solutions of Nebivolol and Valsartan at different concentrations. The peak areas of Nebivolol or Valsartan were plotted versus their respective concentrations. The response was found to be linear over the concentration range of 12.5-62.5μg/ml for Nebivolol and 200-1000μg/ml for Valsartan. The correlation coefficient (R²) for both Nebivolol and Valsartan were estimated to be 0.999. The data is given in table2 and depicted in fig. 4 and 5.

Fig. 4: Linearity of Nebivolol

Fig. 5: Linearity of Valsartan

Accuracy:

Accuracy was done in triplicate for various concentrations of Nebivolol and Valsartan equal to 50%, 100% and 150% of standard amount, was injected into the HPLC system as per test procedure. The average % recovery of Nebivolol and Valsartan was calculated. The data was given in the table 3.

Table 3: Accuracy and % recovery of each analyte

Accuracy Level %	Mean recovery of Nebivolol (%)	Mean recovery of Valsartan (%)
50	101.60	99.61
100	100.11	100.86
150	101.25	100.61

Precision:

Method repeatability:

Six sample solutions of the same concentration were prepared and injected into the HPLC system as per test procedure.

Intermediate Precision:

Two analysts as per test procedure conducted the study. For Analyst-1 Method Repeatability and for Analyst-2 six sample solutions of same concentration were prepared and injected into the HPLC system as per test procedure. The results were given in table 4.

Table 4: Precision data for NEBandVAL

	NEB			VAL
	Mean	Std. Dev.	%RSD	Mean	Std. Dev.	%RSD
Method precision	236102	1837.3	0.78	1689472	9071.0	0.54
Intermediate precision	237124	1952.2	0.98	1725143	8996.5	0.98

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ were performed on samples containing concentration of analytes, based on calibration curve method. Standard solution of Nebivolol and Valsartan were injected in six replicates. Average peak area of six analytes was plotted against concentration.LOD and LOQ were calculated using the formula LOD=3.3 σ/S and LOQ=10 σ/S. The LOD and LOQ were ground in to be 0.05μg/ml, 0.81μg/ml and 0.15μg/ml, 2.44μg/ml for Nebivolol and Valsartan respectively.

Table 5. LOD and LOQ of NEB and VAL

Drug	LOD (µg/mL)	LOQ (µg/mL)
Nebivolol	0.05	0.15
Valsartan	0.81	2.44

Assay:

The validated method was put in for the determination of Nebivolol and Valsartan in commercially obtainable Nebicard-V tablets. The results of assay (n=3) undertaken yielded 99.99% (%RSD=0.07) of Nebivolol and 99.79 % (%RSD= 0.18%) of Valsartan.

Robustness:

Robustness was done by small deliberate changes in chromatographic conditions and retention time of Nebivolol and Valsartan was marked. The factors selected were flow rate and mobile phase. The results remained unaffected by small variation in these parameters as shown in table 6.

Table 6. Robustness Parameters of NEB and VAL

Parameter	Nebivolol			Valsartan
Parameter	Mean area	STDV	%RSD	Mean area	STDV	% RSD
Initial sample	236102	1837.3	0.778	1689472	9071.0	0.54
Flow (+0.1mL/min)	282188	1291.2	0.52	2015472	6644.1	0.32
Flow (-0.1mL/min)	234001	4347.9	0.91	1634642	1193.3	0.12
Org. Phase 10 % more	1208735	6397.19	0.53	647092	6356.2	0.98
Org. Phase 10 % less	1321776	4357.19	0.33	604022	2356.8	0.79

Table 7: System suitability parameters of NEBandVAL

Drug

Theoretical plates

Tailing factor

Retention time

Resolution

Nebivolol

7866

0.88

4.642

---

9.15

Valsartan

9174

0.76

2.412

System Suitability:

System suitability and chromatographic measures were validated such as number of theoretical plates, asymmetry factor and tailing factor. The results are given in the table 7.

CONCLUSION:

The proposed method was ground in to be linear over concentration range of 12.5-62.5µg/ml and 200-1000µg/ml for Nebivolol and Valsartan respectively. System suitability parameters indicate good resolution for both the peaks >2. The method was ground in to be accurate and precise as indicated by the results of recovery studies and precision studies whose % RSD is not more than 2%. There were no marked changes in the chromatograms which committed the ruggedness of the method. The standard deviation of %assay for sample was calculated, for each parameter in robustness studies the relative standard deviation was ground in less than 2%. The low %RSD value confirms the robustness of method. The suggested method was ground in to be rapid, precise, accurate and sensitive. It makes use of lesser amounts of solvents and has shorter retention times than surviving methods. Many samples can be suitably scrutinized by this method. Hence developed method can be used for normal analysis of Nebivolol and Valsartan in tablet dosage form.

Table 8. Summary of system suitability and validation parameters of LAM and RAL

Parameter	Results
Parameter	NEB	VAL and PIP
Linearity range (μg/mL)	62.5-125	200-1000
Correlation coefficient	0.999	0.999
Theoretical plates (N)	7868	9174
Tailing factor	0.88	0.76
LOD (μg/mL)	0.05	0.81
LOQ (μg/mL)	0.15	2.44

ACKNOWLEGEMENT:

The authors are thankful to Analog Labs (Hyderabad, Andhra Pradesh) for providing API of Nebivolol and Valsartan.

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Received on 14.10.2020 Modified on 08.01.2021

Asian Journal of Research in Chemistry. 2021; 14(3):168-172.

DOI: 10.52711/0974-4150.2021.00031

S. No	Conc. Taken in mg/ml (NEB)	Conc. Taken in mg/ml (VAL)	Peak area of NEB	Peak area of VAL
1	12.5	200	59389	437281
2	25.0	400	115200	845700
3	37.5	600	168090	1198897
4	50.0	800	225013	1605023
5	62.5	1000	282758	2023178