ESTIMATION OF NEBIVOLOL HYDROCHLORIDE BY USING RP-HPLC

Department of pharmaceutical analysis ,M.A.M college of pharmacy ,kesanupalli. PRESENTED BY: G.Vamsi Krishna Murthy, B.Phani Saila Raja.

INTRODUCTION:Nebivolol Hydrochloride is a antihypertensive agent and it acts by blocking the -receptors . The dosage form select for the study contains Nebivolol Hydrochloride available as tablets. In the view of the literature cited, RAPID QUANTIFICATION OF nebivolol in human plasma by liquid chromatography coupled with electrospray ionization tendency mass spectrometry and HPLC enantiomeric resolution of nebivolol on normal and reversed amylase based chiral phases have been reported so far. How ever no analytical method has been reported for its determination in dosage form. Hence the study is aim to develop Reverse phase high performance liquid chromatographic method for the analysis of drug in the dosage from. objective of the work:1. Development of Rapid, sensitive and accurate reverse phase HPLC method. 2. Recovery study for reverse phase HPLC method. 3. To analyse the developed method for their accuracy, precision, reproducibility and to validate the results using statistical parameters. INSTRUMENT:-

INSTRUMENTS:

High performance liquid chromatography, shimadzu pumps LC-10 ATVP equipped with Hamilton 100 naduz Schweiz, Rheodyne valve injector with 20 micro litre fixed loop, UV V is detector SPD 10 ATVP (Shimadzu)

REAGENTS AND CHEMICALS

1. 2. 3. 4. 5. Potassium Dihydrogen Orthophsophate : AR Grade Acetonitrite Water 0.45 membrane filter Potassium Hydroxide : HPLC Grade : Milli Q grade : Millipore PVDF grade : AR Grade

PREPARATION OF PHOSPHATE BUFFER PH 7.0

Dissolve 1.38 grams of potassium dihydrogen orthophosphate in 1 litre of water. Adjust the PH to 7.0 0.05 with 1 M potassium hydroxide. Filter through 0.05 membrane filter.

PREPARATION OF MOBILE PHASE:

Prepared and filter and degassed mixture of phosphate buffer PH 7.0 and acetonitrile in the ration of 60 :40 v/v.

CHROMATOGRAPHIC CONDITIONS:
Column Pump mode Temperature : Nitrile Column : : Isocratic Ambient

Flow rate Detection Injection volume

: : :

1.6 ml / minute 230 nm 20 micro litres

LINERARITY AND CALIBRATION

Linearity was assessed by performing single measurement at several analyte concentrations. A minimum of five concentrations have recommended for linearity studies. Varying quantities of standard stock solution was diluted with the mobile phase to give a concentration of 160 240 g / ml. The injections were made at an interval of 10 minutes and the peak areas were determined. A calibration curve was constructed by plotting the peak area obtained against concentrations. These exists a linear relationship in the graphs showing ranging 160 240 g / ml. From the data obtained, correlation coefficient, intercept and slope was calculated to provide mathematical estimates of degree of linearity.

STANDARD SOLUTION
Weight and transfer accurately 51.5 mg of Nebivolol HCl working standard into a 100 ml clean dry volumetric flask, add about 500 ml of diluent sonicate to dissolve, make up to the volume with diluent. Fitter through 0.45 membrane filter.

SAMPLE SOLUTION
The content of twenty tablets was accurately weighed. The powder equivalent to 50 mg was accurately weighed and transferred to 100 ml volumetric flask. About 20 ml of acetonitile was added and kept in sonicate for 15 minutes, made upto 50 ml with mobile phase. This solution was filtered through a membrane filter and the volume was made upto the mark to get the stock solution. From this suitable dilutions are made to obtain the concentration of 200 g / ml.

PROCEDURE

Twenty micro litres of the sample solution was injected under the chromatographic conditions described above and the chromatogram was shown in figure. Each solution was run six times at an interval of 10 minutes to ensure the complete elution of the previous one. The amount of drug present in tablet formulation was calculated by comparing the peak area ratio from the standard.
Amount of drug content present in the sample sample peak area std dilution Potency Avg .wt s tan dard peak area sam. dilution 100

% Recovery

Concentrat ion found practicall y potency 100 Concentrat ion found theoretic ally 100

System stability parameters: They are used to verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. Resolution: It is a measure of relative separation of two peaks. Number of theoretical plates (n): It is a measure of column efficiency. If the number of the theoretical plates is high, the column is said to be highly efficient vice versa important for the detection of the trace components. Tailing factor (t): As peak asymmetry increases integration and hence precision becomes less reliable. These system stability tests were performed by collecting data from replicate injections of standard solution having concentration at 200 g / ml of Nebivolol HCl. Results and discussion: 1)Linearity data of nebivolol hydrochloride 2)system suitability data of nebivolol Hcl
Concentration S.No. 1. 2. 3. 4. 5. (g / ml) 160 180 200 220 240 35776211 39800287 44801623 48210947 52890421 1. 2. 3. Peak area S.No. Parameters Retention (minutes Tailing factor Number theoretical plates of time Nebivolol HCl 7.25 1.36 13,528

Conclusion: The proposed RP HPLC method was simple, precise, rapid, accurate. The linearity, reproducibility and recovery data confirms no major interference due to excipients in the tablet in the assay determination so this method can be used for routine quality control analysis of this drug in dosage formulations.