HPTLC Method Development and Validation for the Estimation of Nebivolol Hydrochloride

B.Dhandapani

A simple, fast, specific and precise High Performance Thin Layer Chromatographic Method (HPTLC) has been developed for estimation of Nebivolol hydrochloride in tablet dosage form.

The stationary phase silica gel G60F₂₅₄ was selected for separation and the sample was developed using a mixture of Ethyl acetate: Methanol: Ammonia in the ratio 8.5:1: 0.5 v/v as mobile phase. Quantification was carried out at 285 nm photometrically. The R_f value of Nebivolol hydrochloride was found to be 0.52 ± 0.02. Linearity was found to be in the concentration range of 250 to 1250 ng/spot of Nebivolol hydrochloride and the correlation coefficient value is 0.9994. The results of analysis were validated in terms of accuracy and precision. The LOD was found to be 20ng/spot, LOQ as 50 ng/spot and stability studies were made. The content uniformity test was carried out as per the USP specification. The proposed HPTLC method provides a faster and cost effective quantitative control for routine analysis of Nebivolol hydrochloride in its formulation.

Introduction:

Nebivololhydrochloride ¹ ( NEB-H ), is a benzopyran antihypertensive drug (β ₁ blocker)and chemically it is a α , α^’ – [ iminobis ( methylene )] bis [ 6-flouro-3,4,-dihydro-2H-1-benzopyran-2-methanol hydrochloride.

Literature survey showed no HPTLC method for the estimation of Nebivolol hydrochloride in tablet formulation. However, few analytical methods have so far been reported for its determination. Most of this methods report the estimation of nebivolol hydrochloride from tablet the biological samples particularly from plasma^2-7. Hence this paper reports a simple, precise, rapid and cost effective HPTLC method for the estimation of Nebivolol hydrochloride in its formulation.

Materials and Methods:

A CAMAG TLC system comprising of a Linomat-5 applicator and CAMAG TLC scanner and single pan balance of Shimadzu model was used, for the present study.

Stationary phase used was silica gel G60F₂₅₄, 20x10 cm TLC plate activated at 75 ^o C for 20 mins, the mobile phase used was Ethyl acetate: Methanol : Ammonia (8.5:1:0.5 v/v). The plates were developed by ascending method in a CAMAG twin trough glass chamber (20 x 10 cm) saturated with filter paper for 10 mins. Distance of solvent front 80 mm, band length 8mm, slit dimension 6.00 x 0.30 mm, detection wavelength 285 nm, temperature 26.4 ^oC and humidity 61% were used for the present study.

Procedure:

Accurately weighed 10 mg of Nebivolol hydrochloride, dissolved in methanol and the volume was made up to 10 ml with same (1 mg/ml). This was used as stock solution. Varying volumes from 2 to 10 ml of standard stock solution (250 – 1250 ng / spot) were spotted on precoated TLC plates using Linomat 5 applicator, plates were developed and scanned using CAMAG TLC scanner 3. The peak areas and R_f values were noted and a calibration graph was plotted [Peak area Vs Concentration (ng/spot)].

About twenty tablets of each formulation were weighed accurately, powdered and quantity equivalent to 10 mg of Nebivolol hydrochloride was dissolved in methanol and made up to the volume to 10 ml with methanol and filtered through whatmann filter paper. Aliquot (2, 4 and 6 ml) were spotted as sharp bands on the chromatographic plate using Linomat-5 applicator. The plate was developed and the spots were scanned, peak areas were noted. The amount of Nebivolol hydrochloride was calculated by using the recovery studies, performed by in which preanalysed samples were taken and standard drug was added at different levels carried out validation of proposed method. Results are shown in Table 1& 2.

The content uniformity test was carried out by taking 10 individual tablets of marketed formulation, which was then extracted with methanol and determined by applying content uniformity – single component mode option of WINCATS 4 software. The Nebivolol hydrochloride content of individual tablet unit (10 replicates) was calculated by comparing the peak area with that of standard with complies with USP specification of the content uniformity test of 85 – 115% and CV must be less than 6%.The recovery of the drugs from the sample matrix evaluated on the basis of standard addition method has been almost 100% indicating the accuracy of the method and non-interference of sample matrix Results are shown in Table 3.

Results and Discussion:

The developed method was validated as per ICH guidelines.  The observed percentage recoveries were 99.67, 101.65 which shows that the method is free from interference from excipients present in the formulation, as given in table I.

The analysis of Nebivolol hydrochloride in dosage forms by the developed HPTLC method resulted in good accuracy as in recovery studies. The estimated amount of Nebivolol hydrochloride present in tablet was in good agreement with the percentage label claim (4.98 and 5.099 mg / tab). The proposed method can be used for routine analysis of Nebivolol hydrochloride in pharmaceutical dosage forms.

Conclusion:

Considering the obtained data, it was possible to affirm that proposed method was precise, fast, simple and suitable for the accurate determination of Nebivolol hydrochloride in bulk drug and tablet dosage forms.

References:

1. Budavari, S., Eds., the Merck index, 12^th edition. Merck and Co., Inc., Whitehouse Station, NJ, 1994, 1103

2. R.woestenborghs,, methadol, sur. Biochem Analysis .18. 215 (1988).

3. RP-HPLC estimation of Nebivolol in dosage form Asian journal of chemistry 17, (2), 1259-1263- 2005.

4. HPLC Enantiomeric Resolution of Nebivolol on Normal and Reversed amylase based chiral phases, Pharmazie, 2001, 56, 214-216.

5. Aboul.Enein, H.X. Enantioseparation of some clinically used drugs by HPLC using chiral stationary phase, Biomed, Chromatogr. 2003, 17, 113-117.

6. Thevis, M:, Opfermann, G.; Schanzer, W.High speed determination of Beta blocker Blocking agents in Human Urine by Liquid chromatography / Tandem mass spectrometry, Biomed, Chromatogra., 2001, 15, (393-402).

7. Rabid Quantification of Nebivolol in Human plasma by Liquid Chromatography coupled with electro spray ionization Tandem mass spectroscopy. Journal of Pharmaceutical and Biomedical analysis Vol -39 Issue 5, 4, Oct-2005. 1006 -1013.

Table 1: Result of Analysis and % Recovery of Nebivolol Hydrochloride

· Average of 6 readings

Table II: Results of Method Validation Experiments of Nebivolol Hydrochloride

Table III: Content Uniformity Data for Nebivolol Hydrochlor

* Mean ± STD Deviation

UV Spectra of Nebivolol Hydrochloride:

Limit of Detection 20ng / spot

Chromatogram of Nebivololhydrochloride at 285nm

Mobile Phase: Ethyl Acetate: Methanol: Ammonia (8.5: 1; 0.5)

About Authors:

B.Dhandapani^*, K.Suresh Kumar, J.Dharuman, M.P.Kavitha and Abhaydharamsi.

Department of pharmaceutical analysis, K.M.C.H. College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore, Tamilnadu – 641035, India.

* Presenting author

B.Dhandapani.

^*For correspondence

Email: dhandapanirx@yahoo.co.in, dhandapanirx@gmail.com