INTRODUCTION:

By curing various fatal diseases, drugs play a vital role in the progress of the human civilization. Heartattack is one of the fatal and severe disease is threat for the entire world. In India, more than 62 million Indians are affected by Heart problems. Hence, it is necessary to control heart problem. Nebivolol and Valsartan combination the new drug combination used to cure heart attack¹.

Chemically, Nebivolol is known as ,1'-Bis(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)-2,2'-iminodiethanol It is an orally administered Nebivolol is unique as a beta-blocker. Nebivolol lowers blood pressure (BP) by reducing peripheral vascular resistance, and significantly increases stroke volume with preservation of cardiac output. The net hemodynamic effect of nebivolol is the result of a balance between the depressant effects of beta-blockade and an action that maintains cardiac output. Antihypertensive responses were significantly higher with nebivolol than with placebo in trials enrolling patient groups considered representative of the U.S. hypertensive population, in Black patients, and in those receiving concurrent treatment with other antihypertensive drugs.

The chemical name of valsartan is (S)-N-Valeryl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]-methyl}-valine Valsartan is an angiotensin-receptor blocker (ARB) that may be used to treat a variety of cardiac conditions including hypertension, diabetic nephropathy and heart failure. Valsartan lowers blood pressure by antagonizing the renin-angiotensin-aldosterone system (RAAS); it competes with angiotensin II for binding to the type-1 angiotensin II receptor (AT1) subtype and prevents the blood pressure increasing effects of angiotensin II. Unlike angiotensin-converting enzyme (ACE) inhibitors, ARBs do not have the adverse effect of dry cough. Valsartan may be used to treat hypertension, isolated systolic hypertension, left ventricular hypertrophy and diabetic nephropathy. It may also be used as an alternative agent for the treatment of heart failure, systolic dysfunction, myocardial infarction and coronary artery disease

Nebivolol and Valsartan is a new drug combination. Therefore, there are very few reports of quantitative estimatin methods for this new combination. Simultaneous Equation Method is typically applies for the estimation of drug combinations containing two or more than two drugs. This method is easy, simple and gives reproducible results as compared to other UV methods. Therefore, this is a attempt to develop simple, robust, reproducible method for the determination of efficacy and safety of Nebivolol and Valsartan combination. This method was fully validated according to International Conference on Harmonization (ICH) and ready for the application in routine analysis without interference of an excipients.

Published Papers on this drug combination and in combination with other drug by UV^9,10,11, HPLC.^12-18

Fig. No. 1. Structure of Nebivolol

Fig. No. 2. Structure of Valsartan

MATERIALS AND METHODS:

Instruments:

A Systronics HPLC-8600 chromatographic system is used for the quantitative analysis. It consists of a prominence solvent delivery module, a manual injector with a 20µL fixed loop, Pressure pump, UV-visible detector, operated by computer software Chemitochrom 2000. A separation was performed on Hibar^® (Merck Germany) RP-Purospher Star C18 column with dimensions 5µm, 4.6mm*, 250mm at an ambient temperature. A Fast Clean ultrasonicate sonicator was used for the degassing purpose. Weighing balance Sansui Vibra DJ-150S-S was used for the weighing of samples and reagents, pH meter (Equiptronics EQ 621) was used for the checking and maintaining pH of the mobile phase, and filter papers of Sartorius Stedim grade 292 was used for the filtration of mobile phase and other chemical reagents.

Chemicals and Reagents:

Nebivolol(5 mg) and Valsartan(80 mg) pure drugs were obtained as a gift sample from Lupin Pharma and Dolphin Pharma India. The combined formulation Torrent Pharma (5 mg/80 mg) of the two drugs purchased from Vitthalkrupa Medical, Bhusawal. Analytical grade methanol purchased from Merck Chemicals Pvt. Ltd. Mumbai.

Method Development:

Various pre-trials of the mobile phases has been carried out before selecting the proper mobile phase. Finally Acetonitrile,Water and Methanol with concentrations 25ml,35ml and 40 ml respectively, has been selected as a mobile phase and pH value for the same is 4.0. Flow rates between 0.5 ml/min and 1.5 ml/min has been studied. But, optimal signal can’t be obtained on those flow rates. So that, flow rate of 1.0 ml/min was tested, and it gave optimal signal with noise ratio and reasonable separation using C18 column. Total analysis time is less than 12 minutes. The maximum absorption of NEBI and VAL was detected at 270 nm. At this wavelength both Nebivolol and Valsartan were showed complete resolution.

Preparation of Buffer:

According to Indian Pharmacopoeia 2007, The assay of Phosphate buffer is as follows. Dissolve 5.04 gm of disodium hydrogen phosphate and 3.01 gm of potassium dihydrogen phosphate in sufficient water to produce 1000 ml. Adjust the pH with Orthophosphoric acid.

Preparation of Mobile Phase:

Acetonitrile, Water and Methanol were taken in the ratio of 25:35:40

Preparation of Standard Solution and Stock Solution:

A) Valsartan Standard Stock Solution [V]:

An accurately weighed quantity of VAL (80 mg) was taken in 100 mL volumetric flask and was dissolved in methanol (80 mL). Then the volume was made up to the mark using methanol to get VAL standard stock solution (80µg/mL). Stock solution was filtered through a 0.45 µm membrane filter paper. The working standard solution of VAL was prepared from suitable aliquots of stock solution.

B) Nebivolol Standard Stock Solution [N]:

An accurately weighed quantity of NEBI (5 mg) was taken in 10 mL volumetric flask and was dissolved in methanol (8 mL) with ultrasonication. Then the volume made up to the mark using methanol to get NEBI standard stock solution [N] (5 µg/mL). Stock solution was filtered through a 0.45 µm membrane filter paper. The working standard solution of NEBI was prepared from suitable aliquots of stock solution.

Maintained Chromatographic conditions:

Mobile Phase: Acetonitrile,Water and Methanol

HPLC Column: Octadecylsilyl C18 Column (5µm, 4.6mm* 250mm)

Detection wavelength: 270 nm

Flow rate: 1ml/min

Column Temperature: Ambient

Run Time: 12 minutes

Injection Volume: 20 µL

Diluent: Methanol

Method Validation:

System Suitability: The standard solution was injected into the Chromatographic system. Percentage relative standard deviations have been found satisfactory. System suitability results are tabulated in table.

Calibration curve (Linearity):

Linearity of an analytical method is its ability to elicit test results that are directly proportional to concentration of analyte in sample within given range, this was studied by analyzing five different concentrations of drug ranging from 5µgm/ml to 25µgm/ml (5µgm/ml, 10µgm/ml, 15µgm/ml, 20µgm/ml, 25µgm/ml) for Nebivolol and 20µgm/ml to 120µgm/ml (20µgm/ml, 40µgm/ml, 80µgm/ml, 100µgm/ml, 120µgm/ml) Valsartan were transferred to series of 10mL volumetric flasks and the contents of the flasks were diluted upto the mark with diluent. A 20µL aliquot of each solution was injected into the chromatographic system. The conditions including the flow rate 1ml/min and detection wavelength was set to the 270 nm and the run time program was set to the 12 minutes. A calibration curve for each drug was obtained by plotting area under the peak versus concentration. Linearity results tabulated in Table 2. Linearity graphs for Nebivolol and Valsartan are presented in Figure 3 and 4 respectively.

Table No. 1 Regression Analysis of the calibration curves for Nebivolol and Valsartan in the proposed HPLC method.

Parameters	Nebivolol	Valsartan
Linearity Range (µgm/ml)	5 – 25µgm/ml	20 – 120 µgm/ml
Detection Wavelength	270 nm
Slope ± S.D.	50.882	32.023
Intercept ± S.D.	4.51	43.89
Correlation Coefficient (R²)	0.9999	0.9966

Table No. 2 Summary of the validation parameters for the proposed HPLC Method:

Parameters	Nebivolol	Valsartan
LOD	0.0287	0.0295
LOQ	0.0089	0.08703
Accuracy %	99.6	99.2
Repeatability
Precision (RSD %)	0.1680	2511
Interday, n=3	0.1290	0.8525
Intraday, n=3	0.3371	1.5665

LOD = Limit of Detection

LOQ = Limit of Quantification

RSD = Relative Standard Deviation

Figure No. 3 Typical liquid chromatogram obtained for a 20 µL injection of a binary mixture of NEBI and VAL

Figure No. 4 Calibration curve of Nebivolol

Figure No. 5 Calibration curve of Valsartan

Accuracy (% Recovery):

Accuracy refers to the closeness of a measured value to a standard value. Accuracy studies were carried out by adding a known amount of pure drugs of NEBI and VAL to the pre-analyzed sample solution. The percentage recovery studies was carried out by spiking 80%, 100% and 120% of respective drug, each level was injected 3 times, shown in Table 4. According to the results the method is capable to estimate both drug components accurately in the tablet dosage form at a time and the results were within acceptable limits, i.e, above 99 % and below 101 %.

Table No. 3 Assay results for the combination dosage form using the proposed HPLC method.

Formulation	Nebivolol	Valsartan
Nebicart-V	99.59 ± 0.3158	97.098 ±0.4372

Table No. 4 System suitability test parameters for NEBI and VAL for the proposed HPLC method.

System suitability parameters	Proposed method
System suitability parameters	VAL	NEBI
Retention Time (t_R)	10.2	4.59
Number of Theoretical plates	3369	2488
Assymetry factor	4.01	1.29
Resolution factor (R)	10.717	0

Precision (Repeatability):

The precision of the method has been evaluated by injecting the six replicate sample preparations. The percentage assay for both Nebivolol and Lingaliptin were calculated and tabulated in Table 3. The % R.S.D. values of the results corresponding to the peak area and retention time were expressed for intra-day precision and on 3 days for inter-day precision.

% RSD results show that the method is precise and can be used to estimate the drug components in the tablet dosage form.

Intermediate Precision (Reproducibility):

The intraday and interday precisions of the proposed method were determined by estimating the corresponding responses 5 times on the same day and on 5 different days for present method. The results are reported in terms of relative standard deviation (RSD).

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

LOD and LOQ of the drug were calculated using the equations according to International Conference on Harmonization (ICH) guidelines.

Robustness:

Robustness of the method was determined by making slight changes in chromatographic conditions. Effect of percentage of methanol in mobile phase on the retention time and slight changes in flow rate were applied as variable parameters. Flow rate varied at three levels (-1, 0, 1). One factor at the time was changed to estimate the effect. Thus standard solution at varied pH (pH 2.9, 3.0 and 4.0) three pH levels was performed.

Specificity:

Specificity of an analytical method is its ability to measure accurately, and specifically, the concentration of analyte without interference from other API, diluents, mobile phase, Specificity was checked by determining NEBI, VAL in laboratory prepared binary mixture and in binary mixture containing different degradation products.

System Suitability Test:

In the system suitability test, the binary solution of 5 µg/ml of NEBI, 80 µg/ml of VAL (n=6) was prepared and injected. Then the system suitability parameters like retention time, theoretical plates, tailing factor and resolution were calculated from the chromatogram.

Results and Discussion:

The absorption spectra of NEBI and VAL greatly overlap; so conventional determination of these compounds in mixture is not possible. To optimize the LC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for NEBI, and VAL were obtained with a mobile phase consisting of Acetonitrile:Methanol:Water (25:35:40 v/v), pH 4.0 adjusted using 10% o-phosphoric acid. Quantification of the drugs was performed at 270 nm. Resolution of the components with clear baseline separation was obtained.

Validation of the proposed method:

Linearity:

Linear correlation was obtained between peak areas and concentrations of NEBI, and VAL in range of 5µgm/ml to 80µgm/ml, for both drug compounds. The linearity of calibration curves was found to be acceptable over the concentration ranges of 10 µgm/ml-50 µgm/ml for Nebivolol and Valsartan, with a R²values 0.9999 and 0.9996. The results show that good correlation existed between the peak area and concentration of the analysts.

Accuracy:

The recovery experiments were performed by the standard addition method. The recoveries obtained were 99.62 and 99.78% for NEBI and VAL, respectively. The high values indicate that the method was accurate.

Precision:

Precision study was carried out using parameter like method repeatability study which showed that results were within acceptable limit 0.1680 and 0.2511i.e. % RSD below 2.0 indicating that the method is reproducible. The results are shown in (Table No.2)

Intermediate Precision:

The intraday RSD values for NEBI and VAL were 0.3371, 1.5665 and respectively. The interday RSD values for NEBI, and VAL were 0.1290, 0.8525 respectively. The % RSD (< 2%) values indicate that the method was sufficiently precise (Table 2).

LOD and LOQ

LOD values for NEBI and VAL were found to be 0.0287 µg/mL, 0.0295 µg /mL, respectively. LOQ values for NEBI and VAL were found to be 0.08703 µg /mL, 0.0089 µg /mL, respectively (Table 2). These data showed that the method was sensitive enough for the determination of NEBI and VAL.

Robustness:

The method was found to be robust with no significant changes on test result upon change of analytical conditions like different flow rate, % methanol in mobile phase and pH of mobile phase with the standard deviation was found to bellow 1 and % RSD is less than 2 for all results. It was found that under small deliberate changes of chromatographic factors, there was no considerable change in under study parameters.

System Suitability Test:

A binary solution of 5µg/mL of NEBI and 80µg/mL VAL (n=5) was prepared and same was injected, then the system suitability parameters were calculated from the chromatogram. The parameters, retention times, resolution factor, tailing factor and theoretical plates were evaluated. The results (Table 4) obtained from system suitability tests are in agreement with the official requirements.

CONCLUSION:

The proposed LC method presented in this paper has advantages of simplicity, accuracy, precision and convenience for separation and quantitation of NEBI, and VAL in combination and can be used for the assay of their respective dosage form. Moreover, the proposed HPLC method is a stability indicating assay method that can determine NEBI and VAL in presence of their degradation products. Thus, the proposed HPLC method can be used for the quality control of NEBI and VAL in typical laboratories.

ACKNOWLEDGEMENTS:

The authors wish to thank to Boehringer Ingelheim Pharmaceuticals Pvt. Ltd., and Vikram Pharmacy Jalgaon for supplying generous gift samples of pure drugs. Hereby, the authors declare that there is no conflict of interests regarding the publication of this paper.

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Received on 21.09.2022 Accepted on 15.10.2022

Int. J. Tech. 2022; 12(2):25-31.

DOI: 10.52711/2231-3915.2022.00005