Chapter-5 Determination of nebivolol and its related...

Chapter-5

Determination of

nebivolol and its

related impurities by RP-HPLC method

5.1 Introduction

Nebivolol hydrochloride is α

benzopyran-2-methanol], a new antihypertensive drug, is a racemate of two enantiomers with

four chiral centers. Its molecular formula is C

The SRRR-enantiomer (d-nebivolol) is a potent and cardio selective fl 1

RSSS-enantiomer (l- nebivolol) has a favorable hemodynamic profile, in that normal energy

supply during exercise is not affected [1

Figure-5.1

Figure-5.1: Chemical

Nebivolol hydrochloride is a white to almost white powder that is soluble in methanol,

dimethylsulfoxide, and N, N-dimethylformamide, sparingly soluble in ethanol, propylene glycol,

and polyethylene glycol, and very

methylbenzene. BYSTOLIC tablets for oral administration contains nebivolol hydrochloride

equivalent to 2.5, 5, 10, and 20 mg of nebivolol base. In addition, BYSTOLIC contains the

following inactive ingredients: colloidal silicon dioxide, croscarmellose sodium, D&C Red #27

Lake, FD&C Blue #2 Lake, FD&C Yellow #6 Lake, hypromellose, lactose monohydrate,

Nebivolol hydrochloride is α, α-[Iminobis (methylene)] bis [6-fluoro-3, 4

a new antihypertensive drug, is a racemate of two enantiomers with

Its molecular formula is C22H25F2NO4 and molecular weight is 405

nebivolol) is a potent and cardio selective fl 1-adrenergic blocker. The

nebivolol) has a favorable hemodynamic profile, in that normal energy

supply during exercise is not affected [1- 3]. The chemical structure of nebivolol is shown in

5.1: Chemical structure of nebivolol


dimethylformamide, sparingly soluble in ethanol, propylene glycol,

and polyethylene glycol, and very slightly soluble in hexane, dichloromethane, and

BYSTOLIC tablets for oral administration contains nebivolol hydrochloride


ts: colloidal silicon dioxide, croscarmellose sodium, D&C Red #27


3, 4-dihydro-2H-1-

a new antihypertensive drug, is a racemate of two enantiomers with

and molecular weight is 405g/mole.

adrenergic blocker. The

nebivolol) has a favorable hemodynamic profile, in that normal energy

The chemical structure of nebivolol is shown in


dimethylformamide, sparingly soluble in ethanol, propylene glycol,

slightly soluble in hexane, dichloromethane, and

BYSTOLIC tablets for oral administration contains nebivolol hydrochloride


ts: colloidal silicon dioxide, croscarmellose sodium, D&C Red #27


magnesium stearate, microcrystalline cellulose, pregelatinized starch, polysorbate 80, and

sodium lauryl sulfate. The different brands of nebivolol tablets are shown in the Figur-5.2

Figure: 5.2 Different brands of nebivolol tablets

Nebivolol is used for treatment of hypertension through vascular endothelial nitric oxide

releasing capabilities and β1- antagonist action [4-6]. It is highly cardio selective under certain

circumstances. Nebivolol is unique as a beta-blocker. Unlike carvedilol, it has a nitric oxide

(NO) potentiating vasodilatory effect. The vasodilatation mechanism of nebivolol is shown in the

Figure-5.3. Along with labetalol, celiprolol and carvedilol, it is one of four beta blockers to cause

dilation of blood vessels in addition to effects on the heart. However, recent studies question the

clinical relevance of this property to nebivolol efficacy. 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.

In the available literature, many analytical procedures have been reported for the

quantitative determination of nebivolol in pure form as well as in pharmaceutical dosage

formulation by different analytical techniques like HPLC [7-31], spectrophotometry [32-43].

B.S.Sastry et.al [19] proposed reverse phase HPLC method for the determination of

nebivolol in pharmaceutical preparations. The drug was chromatographed on a C-18 column

using a mixture of water and methanol in the ratio (40:60) as mobile phase at a flow rate of 1.0

ml/min. Bavita Gaur et.al [21] proposed HPLC method for simultaneous estimation of nebivolol

and indapamide in their combined tablet dosage form. The chromatographic method was

standardized using a base deactivated silica (BDS) hypersil C18, 250 mm × 4.6 mm, 5µ column

with isocratic conditions using mobile phase containing potassium dihydrogen orthophosphate

buffer(pH 3.5), triethyl amine: acetonitrile in the ratio (40:0.5:60) at a flow rate of 1 ml/min

using UV detector at 286. A simple, sensitive, precise, accurate and specific high performance

thin layer chromatographic (HPTLC)method has been developed and validated for simultaneous

estimation of nebivolol and hydrochlorothiazide in tablets by Patel Satish Ambalal et.al[30]. The

stationary phase used was precoated silica gel 60F254 plate. The mobile phase was methanol:

chloroform: toluene: triethylamine (2.0:5.0:2.8:0.2, v/v/v/v). The detection of spots was carried

out densitometrically using a UV detector at 284 nm in absorbance mode. The method is based

upon determination of nebivolol at 281 nm and hydrochlorothiazide at 316.5 nm, in aqueous

methanol (20 % v/v). Absorbance Ratio spectrophotometric method was developed by RK.Patel

and JB. Patel [38] for simultaneous estimation of nebivolol HCl (NEB-H) and

hydrochlorothiazide (HCTZ) in bulk as well as in the pharmaceutical formulation. The method

uses the ratio of absorbance at two selected wavelengths, one which is an isoabsorptive point and

other being the λ-max of one of the two components. Nebivolol HCl and hydrochlorothiazide

have shown an isoabsorptive point at 287 nm in methanol. The second wavelength used is 271

nm, which is λ-max of hydrochlorothiazide. A new, simple, accurate and sensitive UV-

spectrophotometric absorption correction method has been developed for simultaneous

determination of nebivolol and hydrochlorothiazide in combined pharmaceutical dosage form by

PS Tarte et.al [41].

Nebivolol does not exist officially in any pharmacopoeia. A few reports are available in

literature on nebivolol drug, since this drug is being marketed in domestic and international

market. Though large number assay methods are available in literature for nebivolol, only very

few of them are standard, sensitive and selective. In view of the importance of nebivolol in drug

formulation in the treatment of various hypertension diseases, a more simple, sensitive, selective

and robust method is needed for its validation in bulk drug formulations. All the reported

methods used for the determination of nebivolol in bulk and formulations but there is no reported

method for the determination of nebivolol and its related impurities. We are now reporting a

simple sensitive and selective RP-HPLC method for the validation of nebivolol and its related

impurities which is a robust and rugged method.

5.2 Experimental:

5.2.1 Chemicals, Reagents and Samples:

The standard and samples of nebivolol and known related substances of nebivolol,

such as desfuoro impurity [1-(chroman-2-yl)-2-(2-(6fluorochroman-2-yl)-2-

hydroxyethylamino)ethanol hydrochloride], related compound-A [2S*{1R*,5R*(S*)}]-α, α`-

[Iminobis(methylene)] bis [6-fluoro-3,4-dihydro-2H-1-benzopyran-2-methanolhydrochloride]

and benzylated impurity [2,2`-(Benzylazanediyl)bis(1-(6-fluorochroman-2yl)ethanol)oxalate]

were received from Bio-Leo analytical labs, Hyderabad. HPLC grade methanol, acetonitrile,

potassium hydrogen phosphate, and potassium hydroxide were purchased from Merck, Mumbai,

India. High purity water was prepared by using Millipore Milli-Q plus water purification system.

The purity of all samples and impurities used in this study was greater than 99.0%.

5.2.2 Instrumentation:

For initial method development studies Waters prominence HPLC system was employed.

This was equipped with a quaternary UFLC LC-20AD pump, DGU-20A5 degasser, SPD-M20A

diode array detector, SIL-20AC auto sampler, CTO-20AC column oven and CBM-20A

communications bus module. Agilent 1200 series with high pressure liquid chromatographic

instrument provided with Auto sampler and VWD UV detector with thermostatted column

compartment connected with EZ Chrom software was employed for the validation of the drug

and its related impurities. The analysis was carried out on Kinetex C18, 75 cm x 4.6 mm column

with 2.6µm particle size.

5.2.3 Standard and Sample solutions

5.2.3.1. Preparation of standard solution

Accurately weighed and transferred 22.0 mg of nebivolol working standard into a 200 ml

clean and dry volumetric flask. To this, 120 ml of diluent were added, sonicated to dissolve and

finally made up the volume with diluent. 5 ml of this solution was further diluted to 100 ml with

diluent to get the working solutions.

5.2.3.2 Sample preparation

50 mg of nebivolol sample were weighed exactly and transferred into 50 mL clean and dry

volumetric flask and added 30 mL of diluent sonicated to dissolve for about 20 minutes and

made up the volume with diluent. The solution was filtered through 0.45 micron filter.

5.3 Evaluation of system suitability:

The system suitability was evaluated by injecting a known volume of sample containing a

known amount of nebivolol into chromatograph and calculated the number of theoretical plates

and asymmetry of the chromatogram. The number of theoretical plates was found to be greater

than 3000 and the tailing factor of nebivolol was calculated as not more than 2.0 which showed

that the selected column is suitable for the analysis.

5.4 Results and discussion:

5.4.1 Method development and optimization:

The method development was initiated with solubility study of nebivolol. Based on the

studies, acetonitrile: water (1:1v/v) was chosen as diluent for the preparation of sample solutions.

Nebivolol is polar in nature due to presence of –OH groups. Hence, a non polar C18 column

containing octadecyl chemically bonded to porous silica stationary phase was selected for

developing reverse phase high performance liquid chromatography. The nebivolol solution has a

pH 5.2.Therfore a buffer solution of pH about ±2.0 with respect the observed pH 5.2 is most

suitable for validation study. Hence buffer solution of pH 3.4 was chosen for chromatographic

studies. From the molecular structure, it was concluded that, there are chromophore groups

(double bonds) present in nebivolol, hence there is possibility for its UV–Visible detection. The

UV experiment performed nebivolol showed that nebivolol shows maximum absorbance at

280nm.

To arrive at the optimal chromatographic conditions suitable for the validation of nebivolol

and its related impurities, various trail chromatograms were recorded with different conditions.

Trail-1

The trail-1 was performed by using isocratic mode using buffer and acetonitrile in the ratio

(80:20v/v) as mobile phase. The other conditions are as follows

Column : Kinetex C18 column (75 x 4.6 mm; 2.6µ) particle size

Pump mode : Isocratic

Flow rate : 1.0 ml/min

Detection wavelength : UV , 280 nm

Injection Volume : 10µl

Run time : 23 min

Buffer : 3.4g of tetra butyl ammonium hydrogen sulphate were dissolved in

1000ml of HPLC grade water.(pH-3.4)

Mobile phase : Buffer : Acetonitrile (80:20V/V)

In this trail there was no elution of nebivolol and its related impurities peaks up to a run time of

50 minutes.

Trail-2

To over the limitations of the above trail method, the experiment was repeated by using a

second mobile phase (mobile phase-B) in the form of acetonitrile and water in the ratio 80:20 v/v

and remaining conditions same as above.


Pump mode : Gradient




Run time : 23 min



Mobile phase-A : Buffer : Acetonitrile (80:20V/V)

Mobile phase-B : Acetonitrile : Water (80:20V/V)

In this trail nebivolol peak was eluted at 10.85 and well separated from its related impurities but

impurities could not separate.

Trail-3

To achieve separation of impurities of nebivolol the compositions of both mobile phase-A

and mobile phase-B were changed. The buffer solution (pH 3.4) and acetonitrile in the ratio

95:5(v/v) was used as mobile phase-A and a 95:5 (v/v) mixture of acetonitrile and water was

taken as mobile phase-B.


Pump mode : Gradient




Run time : 23 min



Mobile phase-A : Buffer : Acetonitrile (95:5V/V)

Mobile phase-B : Acetonitrile : Water (95:5 V/V)

In this trail all related impurities and nebivolol were well separated with excellent peak shapes.

Finally, satisfactory separation with better peak shape was achieved within a reasonable

retention time in a gradient mode with flow rate of 1.0mL/min at ambient temperature.

5.5 Method validation:

In order to determine nebivolol and its related substances, the method was validated as per

the ICH guidelines individually in terms of system suitability, specificity, precision, accuracy,

linearity, robustness, limit of detection and limit of quantification (LOD and LOQ) and solution

stability.

5.5.1 System suitability test:

System suitability was studied by injecting diluent as blank, placebo, standard solution

and nebivolol solution into the HPLC system. The system suitability results are given in the

Table-5.1.

Tabel-5.1 System suitability results of nebivolol

System suitability parameters Result Acceptance criteria as per USP

Theoretical plates 54397 >3000

Tailing factor 0.98 <2.0

.

5.5.2 Specificity of the method with related substances:

Specificity is the ability of the method to accurately measure the analyte response in the

presence of all potential sample components. The response of the analyte in test mixtures

containing the analyte and all the potential components is compared with the response of a

solution containing only the analyte. For specificity determination diluent and all related

substances of nebivolol such as desfuoro impurity, related compound-A and benzylated impurity

were injected along with nebivolol and the chromatogram was recorded. The peak homogeneity

was verified for nebivolol and its related substances using EZ Chrom software. The summary of

specificity experiment results is shown in the Table-5.2.The specific chromatogram is given in

Figure-5.4.

Table-5.2: Results of specificity experiment

S.No. Name of the analyte/ impurity Peak purity

1 Nebivolol 1.00000

2 Desfluoro impurity 1.00000

3 Related compound-A 1.00000

4 Benzylated impurity 1.00000

5.5.3 Precision:

5.5.3.1 System precision:

Six replicate aliquots of sample solution of nebivolol spiked with impurities were

injected into RP-HPLC system and the chromatograms were recorded for checking the

performance of the system under the chromatographic conditions on the day tested and

calculated the relative standard deviation for the percentage of impurity. The RSD value of

impurities was obtained as ≤3.15%. System precision results of different strengths of tablets

were summarized in Tables 5.3-5.6

Table-5.3: Precision study of nebivolol 2.5 mg tablets

S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.48 0.49 0.51

2 0.50 0.50 0.51

3 0.48 0.48 0.50

4 0.49 0.51 0.53

5 0.49 0.50 0.52

6 0.52 0.51 0.50

Average 0.49 0.50 0.51

SD 0.02 0.01 0.01

%RSD 3.15 2.32 1.94

Table-5.4: Precision study of nebivolol 5 mg tablets

S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.49 0.49 0.51

2 0.52 0.51 0.50

3 0.52 0.52 0.52

4 0.50 0.51 0.51

5 0.51 0.50 0.52

6 0.49 0.50 0.52

Average 0.51 0.51 0.51

SD 0.02 0.01 0.01

%RSD 3.01 2.29 1.55


S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.51 0.52 0.49

2 0.50 0.52 0.52

3 0.50 0.51 0.52

4 0.50 0.51 0.53

5 0.51 0.51 0.51

6 0.50 0.51 0.52

Average 0.50 0.51 0.52

SD 0.02 0.01 0.01

%RSD 0.92 1.17 2.52


S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.52 0.51 0.51

2 0.52 0.52 0.51

3 0.49 0.48 0.50

4 0.52 0.51 0.53

5 0.52 0.52 0.52

6 0.51 0.52 0.50

Average 0.52 0.51 0.51

SD 0.01 0.01 0.01

%RSD 2.92 2.80 1.94

5.5.3.2 Intermediate Precision:

Intermediate precision also called as ruggedness .The intermediate precision is the inter-day

variation. It is defined as the degree of reproducibility obtained by following the same procedure

as mentioned for method precision experiment. The ruggedness of test method was demonstrated

by carrying out precision study in six preparations of sample, a single batch sample by different

analysts, different columns, on different days and using different instruments and calculating the

percentage of impurities. The RSD % of impurities from six spiked sample preparations was ≤

3.49%. The validated intermediate precision results of different strengths of tablets are given in

the Tables-5.7-5.10

Table-5.7: Results of intermediate precision of nebivolol 2.5 mg

S.No.

Desfluoro

impurity

Related

compound-A Benzylated

impurity

1 0.48 0.49 0.51

2 0.50 0.50 0.51

3 0.48 0.48 0.50

4 0.49 0.51 0.53

5 0.49 0.50 0.52

6 0.52 0.51 0.50

Average 0.49 0.50 0.51

SD 0.02 0.01 0.01

%RSD 3.15 2.32 1.94

Table-5.8: Results of intermediate precision of nebivolol 5 mg

S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.49 0.49 0.51

2 0.52 0.52 0.50

3 0.52 0.51 0.52

4 0.50 0.52 0.51

5 0.51 0.50 0.52

6 0.49 0.50 0.52

Average 0.51 0.51 0.51

SD 0.02 0.01 0.01

%RSD 3.01 2.29 1.55


S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.51 0.52 0.49

2 0.50 0.52 0.52

3 0.50 0.51 0.52

4 0.50 0.51 0.51

5 0.51 0.51 0.52

6 0.50 0.51 0.52

Average 0.50 0.52 0.51

SD 0.00 0.01 0.01

%RSD 0.92 1.17 2.52


S.No.

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

1 0.52 0.51 0.48

2 0.52 0.52 0.52

3 0.49 0.48 0.50

4 0.52 0.51 0.51

5 0.52 0.52 0.51

6 0.51 0.52 0.54

Average 0.51 0.51 0.51

SD 0.01 0.01 0.02

%RSD 2.92 2.80 3.49

5.5.4 Accuracy:

The accuracy of the proposed method was tested by preparing sample solutions with known

quantities of impurities of nebivolol at the level of LOQ, 50%, 100%, 150% and 200% of target

concentration (i.e. 0.5% of test concentration).The chromatograms were recorded and the

recovery percentages were evaluated from the peak areas. The results are shown in Table-

5.11.The mean recovery values of all known impurities ranged between 98.24% and 105.40%

Table-5.11: Accuracy results of nebivolol and its impurity

% Mean recovery

Spike level

Desfluoro

impurity

Related

compound-A

Benzylated

impurity

LOQ level

101.01

99.32

99.85

50%

99.34

99.11

99.82

100%

101.89

99.92

99.97

150%

103.63

98.24

102.14

200%

105.40

104.14

102.88

5.5.5 Linearity:

Linearity between the concentration of analyte and peak area was evaluated based on the

residual standard deviation of a regression line and slope. Six different aliquots of standard

solutions (LOQ, 25, 50,100,150 and 200% of test concentration) were injected into the RP-

HPLC chromatograph and the chromatograms were recorded. A plot of peak area of the resultant

chromatogram versus concentration was drawn and the data was subjected to statistical analysis

using a linear-regression model. The statistical parameters of linear curve, slope, intercept,

residual standard deviation and correlation coeffient values were calculated and shown in Tables-

5.12-5.15. The resultant linear plots obtained for these data are given in Figure-5.5. The data was

subjected to statistatical analysis and the results of these analyses are presented in Table-5.16.

Table-5.12: Results of linearity experiment nebivolol

Table-5.13: Results of linearity experiment desfluoro impurity

S.No. Linearity level Amount of nebivolol (ppm) Average peak area

1 LOQ 0.08 114989

2 25% 1.41 227449

3 50% 2.282 371894

4 100% 5.63 750794

5 150% 8.46 1128334

6 200% 11.26 1561769

S.No. Linearity level Amount of desfluoro impurity (ppm) Average peak area

1 LOQ 0.10 8434

2 25% 1.25 114989

3 50% 2.50 230904

4 100% 5.00 467109

5 150% 7.50 694620

6 200% 10.00 954198

S.No. Linearity level Amount of related compound-A (ppm) Average peak area

Table-5.14: Results of linearity experiment related compound-A

Table-5.15: Results of linearity experiment benzylated impurity

Table-5.16: Summarized statistical results of nebivolol and its impurities

1 LOQ 0.11 9827

2 25% 1.25 119340

3 50% 2.50 243108

4 100% 5.00 489656

5 150% 7.50 725734

6 200% 10.00 987816

S.No. Linearity level Amount of benzylated impurity (ppm) Average peak area

1 LOQ 0.06 4942

2 25% 1.25 107285

3 50% 2.50 207780

4 100% 5.00 415464

5 150% 7.50 610422

6 200% 10.00 827097

Nebivolol

Desfluoro

impurity Related

compound-A

Benzylated

impurity

Correlation coefficient 0.9992 0.9998 0.9999 0.9999

Slope 136058 98870 98443 84649

% of Y-Intercept 2895.02 5190.9 3244.09 1985.59

Residual sum square 2.9295 x 109 2.7300 x 10

8 1.3705 x 10

8 9.8463 x 10

7

Residual standard

deviation 27063 8261 5854 4961

The values of different statistatical analysis like correlation coeffient, Y- intercept, residual sum

square and relative standard deviation conforms high precision and accuracy of the proposed

method.

5.5.6 Robustness:

The robustness of an analytical method is a measure of its capacity to remain unaffected

by small changes but deliberate variations in method parameters and provides an indication of its

reliability during normal usage. The study was carried out with respect to change in flow rate,

column temperature. The chromatographic conditions were maintained same as per test method

in each case. From the obtained results, it was observed that there was no much variation in

retention time, theoretical plates and asymmetry for nebivolol peak, obtained at different

deliberately varied conditions from the test method. Hence it can be concluded that the method is

robust for all the varied conditions. The complete robustness results are shown in Table-5.17.

Table-5.17: Robustness results of nebivolol

Robust Condition

Theoretical plates Asymmetry

As per test method 54397 0.98

Flow changed to 0.9ml/min 45407 0.96

Flow changed to 1.1ml/min 53205 0.97

Column Temperature changed to 20°C 53664 1.03

Column Temperature changed to 30°C 54747 1.02

5.5.7 Solution Stability:

The stability of sample solution was tested by recording the chromatograms of freshly

prepared sample solutions of nebivolol at different time intervals i.e. after 24 hours and 48 hours

by keeping the sample temperature at 25°C. It was noticed that no much variation in the % of

impurities and related compound-A. From this observation, it was concluded that sample

solution was stable for 48 hours at ambient temperature (25°C). The solution stability results are

shown in Table-5.18

Table-5.18: Solution stability results

Impurity name Initial After 24 hours After 48 hours

% of desfluoro Impurity 0.00 0.00 0.00

% of difference - NIL NIL

% of related compound-A 0.13 0.12 0.14

% of difference - 0.01 0.01

% benzylated impurity 0.00 0.00 0.00

% of difference - NIL NIL

% of total impurities 0.13 0.12 0.14

% of difference - 0.01 0.01

5.5.8 Limit of Detection and Limit of Quantification:

The detection limit and limit of quantification of nebivolol and related impurities were

determined by diluting known concentrations. The simplest method to calculate limit of

detection and limit of quantification is signal to noise ratio method. The quantification limits and

detection limits of nebivolol and its impurities are given in Tables 5.19 and 5.20.

Table-5.19: LOQ values of nebivolol and its impurities

S.No. Name of the component S/N ratio Amount of analyte

1 Nebivolol 10.09 0.0079

2 Desfluoro impurity 10.14 0.0096

3 Related compound-A 9.70 0.0108

4 Benzylated impurity 10.08 0.0058

Table-5.20: LOD values of nebivolol and its impurities

S.No. Name of the component S/N ratio Amount of analyte

1 Nebivolol 2.56 0.0024

2 Desfluoro impurity 3.01 0.0029

3 Related compound-A 2.98 0.0032

4 Benzylated impurity 2.73 0.0017

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Chapter-5 Determination of nebivolol and its related...

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