chapter-5 determination of nebivolol and its related...
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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
Nebivolol hydrochloride is a white to almost white powder that is soluble in methanol,
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
equivalent to 2.5, 5, 10, and 20 mg of nebivolol base. In addition, BYSTOLIC contains the
ts: colloidal silicon dioxide, croscarmellose sodium, D&C Red #27
Lake, FD&C Blue #2 Lake, FD&C Yellow #6 Lake, hypromellose, lactose monohydrate,
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
Nebivolol hydrochloride is a white to almost white powder that is soluble in methanol,
dimethylformamide, sparingly soluble in ethanol, propylene glycol,
slightly soluble in hexane, dichloromethane, and
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
ts: colloidal silicon dioxide, croscarmellose sodium, D&C Red #27
Lake, FD&C Blue #2 Lake, FD&C Yellow #6 Lake, hypromellose, lactose monohydrate,
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.
Column : Kinetex C18 column (75 x 4.6 mm; 2.6µ) particle size
Pump mode : Gradient
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-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.
Column : Kinetex C18 column (75 x 4.6 mm; 2.6µ) particle size
Pump mode : Gradient
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-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
Table-5.5: Precision study of nebivolol 10 mg tablets
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
Table-5.6: Precision study of nebivolol 25 mg tablets
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
Table-5.9: Results of intermediate precision of nebivolol 10 mg
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
Table-5.10: Results of intermediate precision of nebivolol 25 mg
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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