Validated RP- HPLC Method for the Quantitation of Nebivolol in Bulk and Pharmaceutical Dosage Forms
M.R. Santhosh Kumar*, K.P. Channa Basavaraj, C. Jose Gnana Babu and T. Tamizh Mani
Department of Pharmaceutical Analysis, Bharathi College of Pharmacy, Bharathi Nagara, Mandya District, Karnataka, India – 571422.
*Corresponding Author E-mail: Santhosh36524@gmail.com
ABSTRACT:
A simple, specific, accurate, precise and sensitive Reverse Phase High Performance Liquid Chromatographic method has been developed for the quantitation of Nebivolol in both pure and pharmaceutical dosage forms. A Phenomenex Gemini C-18, 5 µm column having 250 x 4.6 mm internal diameter in isocratic mode with mobile phase containing Acetonitrile : 50mM Ammonium acetate buffer (60 : 40 v/v) and adjust the pH to 3.5 by using glacial acetic acid. The flow rate was 1.0 ml / min and the effluents were monitored at 282 nm. The retention time was 3.783 min. The linearity was in the range of 20-100 µg / ml. This method was validated for linearity, precision, limit of detection, limit of quantitation, accuracy, ruggedness and robustness. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug.
KEYWORDS: RP-HPLC, Nebivolol, Validation.
INTRODUCTION:
Nebivolol is an anti-hypertensive drug used in the management of hypertension and it is not official in any Pharmacopoeia. The chemical name of Nebivolol is α, α1 - [Imino-bis-(methylene)]-bis-[6-fluoro-3, 4-dihydro-2H-1- benzopyran-2-methanol]. It has a molecular formula of C22H25F2NO4 and a molecular weight of 441.9 g / mol and its structure was given in Figure: 1. Nebivolol is a white or almost white powder. It is freely soluble in Methanol, Dimethyl sulfoxide and N, N-Dimethyl formamide and sparingly soluble in Water1-3. Several analytical methods that have been reported for the estimation of Nebivolol in biological fluids or pharmaceutical formulations include HPLC, HPTLC, LC, LC-TMS, UV-Visible Spectrophotometry and spectrofluorimetry4-20. The objective of the work was to develop simple, accurate, precise and economic RP-HPLC method with lesser run time to estimate the Nebivolol in bulk and pharmaceutical dosage forms.
MATERIALS AND METHODS:
The liquid chromatographic system consisted of following components: A Shimadzu HPLC model containing LC-20AT (VP series) pump, variable wavelength programmable UV / VIS detector SPD-20A (VP series) and Hamilton syringe (705 NR, 50 µL).
Chromatographic analysis was performed using Spinchrom software on a Phenomenex Gemini C-18 (250 x 4.6 mm, 5 µm) column. The mobile phase consisting of Acetonitrile : 50mM Ammonium acetate buffer (60 : 40 v/v) and adjust the pH to 3.5 by using glacial acetic acid was selected. The optimized chromatographic conditions are summarized in Table.1. The standard solution of Nebivolol was prepared by dissolving 10 mg in 100 ml of Mobile phase to give the concentration 100 µg / ml. The mobile phase and the solution were sonicated for 10 min and filtered using whatman filter paper No.1. The various dilutions of Nebivolol in the concentration of 20, 40, 60, 80 and 100 µg / ml were prepared. The solutions were injected using a 20 µl fixed loop in to the chromatographic system at the flow rate of 1.0 ml / min and the effluents were monitored at 282 nm, chromatograms were recorded. The Nebivolol was eluted at 3.783 min as shown in Figure: 2. The calibration curve was constructed by plotting average peak area versus concentration and was presented in Figure: 3 with its computed regression equation. The method was extended for determination of Nebivolol in pharmaceutical dosage form. The pharmaceutical dosage form containing 5 mg strength was taken.
30 tablets of each two different brands of Nebivolol (containing 5 mg) were weighed and powdered in glass mortar and the powder equivalent to 10 mg of Nebivolol was transferred into 100 ml volumetric flask containing 50 ml of mobile phase and flask was kept for Ultrasonication for 15 min, then it was diluted up to the mark with mobile phase and the solution was filtered through Whatman filter paper No. 41. From this solution various dilutions were made with the mobile phase, which were analysed. The concentration of the drug in tablet sample solution was calculated by comparing the peak area of standard. The proposed methods were validated as per the ICH guidelines21-23.
Fig. 1: Chemical Structure of Nebivolol
Fig. 2: Typical RP-HPLC Chromatogram of Nebivolol by the proposed method.
Table 1: Optimized Chromatographic conditions for the proposed method
Optimized condition |
|
Column Mobile phase
Flow rate Injection volume Detection Temperature Retention time Run time |
Phenomenex Gemini C-18 (250 X 4.6 mm,5µ) Acetonitrile:50mM Ammonium acetate (60:40 v/v, pH 3.5) 1.0 ml / min 10 µl 282 nm in UV detector Ambient 3.783 min 10 min |
Fig. 3: Calibration curve of Nebivolol by the proposed method.
RESULTS AND DISCUSSION
A suitability test was applied to representative chromatograms for various parameters. The results obtained were within acceptable limits (Table 2). Thus, the system meets suitable criteria. The calibration curve was obtained for a series of concentration in the range of 20-100 µg / ml and it was found to be linear. The data of regression analysis of the calibration curves are shown in Table 3. The precision was measured in terms of repeatability, which was determined by sufficient number of aliquots of a homogenous sample. The % RSD was found and lying with in 2. This showed that the precision of the method was satisfactory. The accuracy of the method was inferred by establishing the precision and linearity studies of the standard. The % RSD was less than 2.0. This showed that the recoveries of Nebivolol by the proposed methods are satisfactory. Ruggedness and Robustness were determined and the % RSD values were calculated from precision study was less than 2.0. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by the proposed methods. The results of validation parameters are summarized in Table 4. The results of capsule analysis and recovery studies obtained by the proposed method were validated by statistical evaluation and are given in Table: 5.
Table 2: System Suitability Test Parameters for the proposed method
Parameters |
Values |
Required limits |
Retention time Theoretical plates Asymmetric factor Tailing factor |
3.783 ± 0.004 5033.166 ± 321.92 1.227 ± 0.048 1.1472 ± 0.2150 |
RSD ≤ 1% N > 2000 As ≤ 1.5 T ≤ 2 |
Mean ± S.D. from five determinations
Table 3: Regression analysis of the Calibration curve for the proposed method
Values |
|
Linearity range (µg / ml) Correlation coefficient (r2) Regression equation Slope Intercept |
20-100 0.9996 Y= 3243.4 X + 5051.4 3243.4 5051.4 |
Table 4: Summary of Validation Parameters for the proposed method
Values |
|
Limit of detection (µg / ml) |
0.0064 |
Limit of quantitation (µg / ml) |
0.0193 |
aPrecision ( % RSD) Intra-day precision |
0.385 |
Inter-day precision |
0.416 |
aRuggedness ( % RSD) Analyst I |
0.358 |
Analyst II |
0.296 |
aRobustness ( % RSD) Changed condition I ( ratio of mobile phase) 65 : 35 (Acetonitrile : Buffer) |
1.129 |
60 : 40 (Acetonitrile : Buffer) |
0.615 |
70 : 30 (Acetonitrile : Buffer) |
0.862 |
Changed condition II (pH of mobile phase) 3.3 |
0.966 |
3.5 |
0.630 |
3.7 |
0.825 |
aMean of six determinations, RSD indicates relative Standard deviation
Table 5: Assay Results of Nebivolol tablets using proposed method
Brand used |
Labelled amount (mg) |
Amount found (mg) |
% Recovery ± SD** |
Tab-A |
5 |
4.962 |
99.24 ± 0.101 |
Tab-B |
5 |
4.981 |
99.62 ± 0.156 |
** Standard deviation of six determinations
CONCLUSION:
Thus it can be concluded that the method developed in the present investigation was simple, sensitive, accurate, rugged, robust, rapid and precise. Hence, the above said method can be successfully applied for the estimation of Nebivolol in pharmaceutical dosage forms.
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Received on 23.04.2010 Modified on 13.05.2010
Accepted on 29.05.2010 © RJPT All right reserved
Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1167-1169