Simultaneous Quantitation of Nebivolol Hydrochloride and Benidipine Hydrochloride from Tablet Formulation using Validated Stability Indicating HPLC Method

 

Vrushali Tambe1, Shreya Patil1, Archana Karnik2, Rekha Bhalerao1,

Supriya Savale1, Aarti Patil1

1Progressive Educations Society’s, Modern College of Pharmacy, Moshi, Pune - 412105, Maharashtra, India.

2SCES’s Indira College of Pharmacy (now Indira University, School of Pharmacy),

Tathawade, Pune - 411033, Maharashtra, India.

*Corresponding Author E-mail: rekhabhalerao2016@gmail.com

 

ABSTRACT:

A robust validated stability-demonstrating high-performance liquid chromatography (HPLC) method is proposed to simultaneously quantify Nebivolol HCl and Benidipine HCl in a tablet formulation. The method was designed to accurately quantify the active pharmaceutical ingredients (APIs) in the presence of their degradation products, ensuring its application for stability studies. The separation was successfully done on C18 column using Methanol, H2O and Acetonitrile in a 70:15:15 (v/v/v) mobile phase ratio with flow rate of 1.0mL/min at 220nm wavelength. The method was validated according to the guidelines of International Conference on Harmonisation (ICH) for accuracy, precision, linearity, specificity, robustness, and limits of detection (LOD) and quantitation (LOQ). Both APIs showed good linearity within the concentration range of 250-750 mcg/mL for Nebivolol hydrochloride and 200-600mcg/mL for Benidipine hydrochloride in the mixture, with correlation coefficients of 0.9994 and 0.9985 respectively. The degradation studies were performed under various stress conditions including acidic, basic, oxidative and photolytic environments. The degradation products were separated from the APIs, confirming the method's stability-indicating capability. Nebivolol HCl was susceptible to oxidative stress while Benidipine HCl was found to underwent degradation under acidic and oxidative conditions. This methods effectiveness was demonstrated by its application to the assay of commercial tablet formulation, demonstrating its reliability for routine quality control and stability testing of combined Nebivolol HCl and Benidipine HCl formulations.

 

KEYWORDS: Stability-indicating, High-Performance Liquid Chromatography, Nebivolol Hydrochloride, Benidipine Hydrochloride.

 

 

INTRODUCTION: 

Hypertension, a major cardiovascular risk factor, often necessitates combination drug therapies for effective management, particularly in patients with comorbidities. Fixed-dose combinations like (NBH) and Benidipine hydrochloride (BNH) are increasingly utilized due to their synergistic effects and improved patient adherence.

 

NBH, a third-generation beta-blocker, offers cardioselectivity and nitric oxide-mediated vasodilation, thus enhancing antihypertensive efficacy and vascular health.1 NBH is also available in transdermal patches with 92% penetrating efficiency.2 It is selective antagonist of β1 adrenoreceptor with vasodilating properties.3 BNH, a dihydropyridine calcium channel blocker, provides prolonged antihypertensive action with protective effects on renal and cardiovascular systems, making it a preferred choice in FDCs for hypertensive therapy.4 Practitioners mostly prescribed Calcium channel blocker drugs followed by Angiotensin receptor blocker.5 For such FDCs, reliable analytical methods are critical for quality control and regulatory compliance. High-Performance Liquid Chromatography (HPLC) has proven to be a robust technique for the analysis of pharmaceutical formulations. Existing methods have demonstrated its efficacy in the simultaneous quantification of NBH with other antihypertensive agents such as Hydrochlorothiazide and Valsartan.6,7 Similarly, HPLC methods for BNH in combination with drugs like Telmisartan and Chlorthalidone highlights the versatility of the technique in analysing multi-component formulations.8,9 The stability of pharmaceutical products is a critical aspect of their development, requiring methods that not only quantify the APIs but also identify and quantify degradation products under stress conditions. Stability-indicating methods are essential in ensuring drug safety and efficacy throughout the shelf life. Prior studies have focused on the stability of NBH and BNH individually and in combination.10,11

 

Estimation of NBH in biological fluids or formulations is addressed by several analytical methods like HPLC, HPTLC, LC, LC-TMS.12 Simultaneous quantification of Amlodipine besylate and NBH in pharmaceutical dosage is done by  RP-HPLC method using UV detection.13 However, there is a notable gap in the development of a comprehensive, validated stability-indicating method for the simultaneous estimation of NBH and BNH in tablet dosage forms. This study aims to address this gap.

 

HPLC is an efficient column chromatographic technique in which the pressurized mobile phase is passed through the column at high speed.14 The complexities available with the chemical structures of various drug substances under analysis necessitate a meticulous approach to method development in HPLC.15 The method is designed in compliance with ICH guidelines, ensuring its reliability and reproducibility across stress conditions such as oxidative, photolytic, and hydrolytic degradation.16,17 The analytical methods are validated according to ICH Q2B guidelines where the linearity, precision and accuracy of the analyte is determined.18,19,20

 

MATERIALS AND METHODS:

Materials and Instruments:

NBH and BNH were procured from Chromein Laboratories, Pune. All HPLC grade solvents like Methanol, H2O, Acetonitrile as well as AR grade chemicals like Formic Acid, Ammonium Acetate, Hydrogen Peroxide, Hydrochloric acid and Sodium Hydroxide were all sourced from Advent Chembio. Waters Alliance 2695 HPLC system with a PDA Detector 2969 and Empower 2 software was used for method development. Tablet formulation containing 4mg Benidipine HCL and 5mg Nebivolol HCL was purchased from local market.

 

 

 

 

Method Development:

Various combinations of methanol, water and acetonitrile were carried out to get optimum resolution between BNH, NBH and their degradation products. The separation was performed on Zorbax XDB C18 (5micron, 150*4.6mm) column using Methanol, H2O and Acetonitrile as a mobile phase in a 70:15:15 (v/v/v) ratio with flow rate of 1.0mL/min at 220nm wavelength.

 

Method Validation:

Specificity: Forced Degradation Studies:

NBH and BNH standard stock solution were prepared separately by dissolving 50mg of each drug in 25mL of a diluent yielding 2000µg/ml solution. In all cases, 3ml of standard stock of NBH and 2.4ml of standard stock of BNH was separately taken. About 3ml of specified stressor was added. Forced Degradation Study of NBH and BNH was performed individually under following conditions.

 

Acidic Degradation:

i.    By using 0.1N HCL: Solution was refluxed at 80 °C. At the intervals of 1hr, 8hrs, 16hrs, 0.5ml of the stress sample was withdrawn and subsequently diluted to 1ml with diluent.

ii.   By using 0.5N HCL: Solution was refluxed at 80 °C. At intervals 4hr, 8hrs, and 12hrs, the 0.5ml of the stress sample was withdrawn and subsequently diluted to 1 ml with diluent.

 

Alkali Degradation

i. By using 0.1N NaOH: Solution was refluxed at           80 °C. At intervals 8hrs, and 12hrs, 0.5ml of the stress sample was withdrawn and subsequently diluted to 1 ml with diluent.

ii. By using 0.5N NaOH: Solution was refluxed at             80 °C. At intervals 8 hrs, and 12hrs, 0.5ml of the stress sample was withdrawn and subsequently diluted to 1 ml with diluent.

 

Oxidative Degradation:

i.    By using 3 % H2O2: Solution was refluxed at 80°C. At intervals 8hrs, and 16hrs, 0.5ml of the stress sample was withdrawn and subsequently diluted to 1ml with diluent.

ii.   By using 5% H2O2: Solution was refluxed at 80°C. At intervals 2hrs, and 4hrs, 0.5ml of the stress sample was withdrawn and subsequently diluted to 1ml with diluent.

iii. By using 10% H2O2: Solution was refluxed at 80 °C. At intervals 2hrs, and 4 hrs, 0.5ml of the stress sample was withdrawn and subsequently diluted to 1 ml with diluent.

 

 

 

Photolytic Degradation:

NBH and BNH was kept in a white glass airtight container for 8 days in sunlight, and later on, 5mg of the drug was taken in a 10ml volumetric flask, and it was prepared by using the diluent. Later on, the 500 and 400 µg/ml concentration sample solution was prepared for NBH and BNH respectively.

 

For stress testing of mixture, 3ml of mixed standard (1000mcg/ml of NBH, and 800mcg/ml of BNH) solution was used.  

 

Linearity, range, LOD, LOQ:

About 50mg of NBH and 40mg of BNH was diluted to 50ml of Diluent (Methanol: Water: Acetonitrile 35:7.5:7.5 v/v/v) resulting into 1000µg/ml of NBH and 800µg/ml of BNH. The solution was further diluted to 25-150% of the targeted strength corresponding to 125-750µg/ml of NBH and 100-600µg/ml of BNH. The response of the solutions was further statistically treated.

 

Accuracy:

Accuracy was performed for various concentrations of mixture equivalent to 50, 100, and 150% of the test concentration.

 

For testing the accuracy at 50%, weighed powder equivalent to 2.5mg of NBH and 2mg of BNH. Transferred it to a 10mL volumetric flask containing 7 mL of methanol. Sonicated it for 15minutes, then made up the volume to 10mL using H2O and acetonitrile to achieve a concentration of 250µg/mL of NBH and 200 µg/mL of BNH. Then filtered this solution through an HPLC filter and analysed. At 100%, weighed powder equivalent to 5mg of NBH and 4mg of BNH. Similarly at 150%, weighed powder equivalent to 7.5mg of NBH and 6mg of BNH and above-mentioned process was executed.

 

Robustness:

In a Robustness study, test solution containing 500 µg/mL of NBH and 400µg/mL of BNH was assayed with a change in flow rate by ±0.1ml/min i.e., 0.9 ml/min, 1.0ml/min and 1.1ml/min and change in column temp. by ±2ºC i.e., 28ºC, 30ºC, 32ºC.

 

Assay:

Weighed powder equivalent to 5mg of NBH and 4mg of BNH. Transferred it to a 10mL volumetric flask containing 7mL of methanol. Sonicated it for 15 minutes, then made up the volume to 10mL using H2O and acetonitrile to achieve a concentration of 500µg/mL of NBH and 400µg/mL of BNH. Then filtered this solution through an HPLC filter and assayed.

 

 

RESULTS AND DISCUSSION:

Method Development:

A representative chromatogram is shown in Fig. No. 1. The UV spectrum of drugs is shown in Fig 2. Key peaks at 281nm and 236.5nm indicate the presence of NBH and BNH, respectively. By selecting 220nm, it ensures that the PDA detector can effectively monitor both drugs simultaneously, leading to accurate and reliable analytical results. The retention time (plate count; Tailing factor) of NBH and BNH was found to be 12.326mins (17563.35; 1.66) and 16.090mins (164172.31; 1.32) respectively.

 

 

Fig. No. 1: Chromatogram of optimized method with Rt of NBH and BNH 12.326 and 16.090 mins respectively.

 

    

Fig No. 2: Overlay Spectra of NBH and BNH

 

Method validation:

The method specificity was demonstrated in terms of forced degradation studies. The chromatogram in Figure No 3b shows significant degradation of BNH under acidic conditions, with the formation of three degradation products while NBH was found to be stable. The degradation of BNH was found to be 5.79%. Peaks 1, 2, and 3 correspond to degradation products with retention times of 7.405, 7.801, and 9.090 minutes, respectively. The chromatogram 3f shows chromatogram under oxidative conditions (5% H2O2,          80 °C for 8hours). Peaks 1 correspond to the degradation product with a retention time of 10.542 minutes. Significant degradation was observed under oxidative conditions. The degradation of NBH and BNH was found to be 10.12 and 11.28% respectively. Both drugs were stable under alkaline and photolytic conditions. The origin of degradation products was confirmed on the basis of degradation carried out for individual drug.

 

 

 

 

a: Chromatogram of Stress Sample of a combination of NBH and BNH in 0.5 HCl (At 4 Hrs)

b: Chromatogram of Stress Sample of a Combination of NBH and BNH 0.5N HCl (At 8 Hrs)

 

 

c: Chromatogram of Stress Sample of a Combination of NBH and BNH in 0.5N NaOH (At 4 Hrs)

d: Chromatogram of Stress Sample of a combination of NBH and BNH in 0.5N NaOH (At 12 Hrs)

 

 

e: Chromatogram of Stress Sample of a combination of NBH and BNH in 5% H2O2 (At 4 Hrs)

f: Chromatogram of Stress Sample of a combination of NBH and BNH in 5% H2O2 (At 8 Hrs)

Fig No. 3: Chromatogram of Stress Sample of a combination of NBH and BNH

 

Different concentration solutions of the NBH and BNH were prepared and injected to study linearity. The area versus concentration calibration plot has been generated. The calibration plot of NBH and BNH is shown in Fig. No. 4 and 5 respectively.  Linear regression data for calibration curves showed a good relationship over the concentration range of 250 to 750 mcg/mL for NBH and 200 to 600 mcg/mL for BNH in the mixture. Summary of Linearity parameters are given in Table No.1.

 

 

Fig. No. 4: Calibration Curve of NBH

 

Fig No. 5: Calibration Curve of BNH                           

 

Table No. 1: Validation parameters of developed HPLC method

Parameters

NBH

BNH

Detection λ max

220ml

220nm

Linearity Range

750-250mcg/mL

600-200mcg/mL

Slope

9664.2

20177

Intercept

38966

368308

Correlation coefficient

0.9994

0.9985

Regression equation

y = 9664.2x + 38966

y = 20177x + 368308

Limit of Detection

18.31 mcg/mL

23.67mcg/mL

Limit of Quantitation

55.50  mcg/mL

71.75mcg/mL

 

The method was found to be precise as %RSD of peak area was found to be less than 2% in intermediate precision study (Table No.2).

 

Table No. 2: Results of Precision

Sr. No.

Peak area of NBH

Peak area of BNH

1.

4821248

8438209

2.

4943149

8319286

3.

4950175

8382810

4.

4857134

8396115

5.

4969128

8437132

6.

4823122

8318197

Average

4893992.667

8381958.167

± SD

67662.5

53670.7

%RSD

1.382

0.640

 

Recovery studies at three different levels i.e. 50 %, 100 %, and 150 % has established the accuracy of the proposed method. The value of drug content for NBH was found to be within the range of 98% to 101% w/w and for BNH, it is 99% to 100% w/w. %RSD value less than 2 indicates that the proposed method is an accurate method for determination of both drugs. The results are given in Table No. 3.

 

Table No. 3: Accuracy Results of NBH and BNH

Level

NBH

BNH

Spiked Amt

Amt Recovered

% Recovery

Mean Amount Recovered ± SD (% RSD)

Spiked Amt

Amt Recovered

% Recovery

Mean Amount Recovered ± SD (% RSD)

 

50%

250

244.1

 

97.3

244.9 +1.35 (0.551)

200

195.5

 

99.7

197.1 +1.45 (0.73)

250

244.2

200

198.3

250

246.5

200

197.6

 

100%

500

497.8

 

98.6

496.4 +1.73 (0.348)

400

399.9

 

99.9

400.1 +1.32 (0.329)

500

494.5

400

399.0

500

497.1

400

401.6

 

150%

750

750.3

 

99.76

748.5 +1.66 (0.22)

600

606.5

 

100.5

603.7 + 2.40 (0.397)

750

748.3

600

602.1

750

747.0

600

602.6

 

Table No. 4: Result of Robustness by change in flow rate by ±0.1ml/min

Sr. No

Peak Area of NBH

Peak Area of BNH

Rate of Flow

(0.9ml/min)

Rate of Flow

(1.0ml/min)

Rate of Flow

(1.1ml/min)

Rate of Flow

(0.9ml/min)

Rate of Flow

(1.0ml/min)

Rate of Flow

(1.1ml/min)

1

4850000

4850175

4995085

8098385

8438029

9085646

2

4855000

4843147

4981241

8071625

8419217

9063218

3

4855734

4818297

4954128

8111448

8472189

9012451

Mean

4853578

4837206

4976818

8093819

8443145

9053771

± SD

31202.9

16748.74

20833.6

20300.29

26854.02

37500.69

% RSD

0.642

0.346

0.418

0.250

0.318

0.414

Drug Content

498.1

496.4

510.9

382.8

400.2

430.5

% Drug Content

99.62

99.28

102.1

95.7

100

107.6

 

Table No. 5: Result of Robustness by change in temp. by ±2°C

Sr. No

Peak Area of NBH

Peak Area of BNH

Temp (28ºC)

Temp (30ºC)

Temp (32ºC)

Temp (28ºC)

Temp (30ºC)

Temp (32ºC)

1

5062786

4850175

5158949

8689812

8489812

8737982

2

5121412

4843147

5114312

8523146

8423146

8631891

3

5078159

4818297

5177136

8671131

8471131

8712853

Mean

5087452

4837206

5150132

8628029

8461363

8694242

± SD

30397.8

16748.74

32326.6

91310.9

34389.67

55440.0

% RSD

0.597

0.346

0.627

1.05

0.406

0.637

Drug Content

522

496.4

528

409.3

401.1

412

% Drug Content

104.4

99.28

105.6

102.3

100.2

103

Robustness was established with addressing the impact of change in column temperature and flow rate on signal. % RSD of response of NBH and BNH was found to be less than 2% which is within the limit. The results are addressed in Table No. 4 and 5.

 

The marketed formulation was successfully estimated by this validated method. The % assay value for NBH and BNH was 100.1% and 99.4% w/w respectively.

 

CONCLUSION:

The present study successfully developed and validated a stability-indicating HPLC method for the simultaneous analysis of NBH and BNH in tablet formulation. The method was rigorously tested and met all the parameters of validation in tune with ICH guidelines. The developed HPLC method demonstrated excellent resolution between NBH, BNH and their degradation products, confirming its stability-demonstrating capability. The Rt’s were efficient, allowing for rapid analysis, which is crucial for high-throughput pharmaceutical quality control settings. The method was linear over the concentration ranges tested for both NBH and BNH with correlation coefficients exceeding 0.99.

 

The precision, expressed as % RSD, was within acceptable limits. Recovery studies (close to 100%) has demonstrated the accuracy of the method. The method was robust, with small deliberate variations in flow rate and column temperature showing no significant impact on the results. BNH was found to underwent degradation under acidic and oxidative conditions while NBH was susceptible to oxidative stress. Its stability-indicating nature ensures that it can effectively separate the API’s from their potential degradation products, making it invaluable for stability testing and routine quality control.

 

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Received on 09.12.2024      Revised on 24.06.2025

Accepted on 15.11.2025      Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):257-262.

DOI: 10.52711/0974-360X.2026.00036

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