INTRODUCTION:

Nebivolol is a β₁ receptor blocker with nitric oxide potentiating vasodilatory effect used in treatment of hypertension. Chemically it is 1-(6-fluorochroman-2-yl)-{[2-(6-fluorochroman-2-yl)-2-hydroxy-ethyl] amino} ethanol.^[1] Indapamide is a non-thiazide sulphonamide diuretic drug. Chemically it is 4-chloro-N-(2-methyl-2, 3-dihydroindol-1-yl) - 3-sulfamoyl-benzamide.^[2,3,4] It is used in the treatment of hypertension as well as decompensate cardiac failure.^[5,6] Structures of both of the drugs (NEBI and INDA) are shown in the figure 1. Indapamide is official in IP^[1],USP^[2], EP^[3] and BP^[4] whereas Nebivolol is official in only IP. Both the drugs are marketed as combined tablet dosage form in the ratio (1.5:5 INDA: NEBI) has gained increase in treatment of hypertension.

Nebivolol

Indapamide

Figure 1: Structure of Nebivolol and Indapamide

Literature survey revealed that a number of analytical methods have been reported for estimation of INDA in combination with other drugs are spectrophotometry, HPLC, RP-HPLC, HPTLC^{[7,
8, 9]} .The analytical methods reported for estimation of NEBI in combination with other drugs are spectrophotometry, HPLC, RP-HPLC, HPTLC. ^[10-
13]

The analytical methods reported for estimation of NEBI and INDA simultaneously are simultaneous equation method, absorbance ratio^[14]. Derivative spectrophotometric method has been not reported for simultaneous estimation of NEBI and INDA. Present work describes simple, accurate, reproducible, and rapid method for simultaneous estimation of NEBI and INDA in tablet formulation by derivative spectrophotometry method. The proposed method is optimized and validated as per the ICH guidelines ^[15].

MATERIALS AND METHODS:

Apparatus:

Instrument used was an UV-Visible double beam spectrophotometer, SHIMADZU (model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on Shimadzu analytical balance (Model AU-220).

Reagents and chemicals:

Pure drug samples of NEBI and INDA were obtained from reputed pharmaceutical company. Methanol LR was used as solvent. Calibrated glass wares were used throughout the work.

Marketed formulation:

Combined tablet formulation (NEBULA-D TAB) was procured from local market. Each tablet contains 5 mg nebivolol and 1.5 mg indapamide.

Preparation of standard stock solution:

Accurately weighed quantity of NEBI (50 mg) and INDA (50 mg) was transferred to two separate 50 ml volumetric flasks, dissolved in methanol and diluted to the mark with same solvent. (Stock solutions: 1000μg/ml of NEBI and 1000μg/ml of INDA).

Preparation of working standard solution:

100μg/ml of NEBI solution was prepared by diluting 5.0 ml of stock solution with methanol in 50 ml volumetric flask up to the mark. 100μg/ml of INDA solution was prepared by diluting 5.0 ml of stock solution with methanol in 50 ml volumetric flask up to the mark.

Procedure for determination of wavelength for measurement:

1.8 ml of working standard solution of NEBI (100μg/ml) and 0.6 ml of working standard solution of INDA (100μg/ml) were pipette out into two separate 10 ml volumetric flask and volume was adjusted to the mark with methanol to get 18μg/ml of NEBI and 6μg/ml of INDA. Each solution was scanned between 200-400 nm against methanol as a reagent blank for zero order spectra (figure 2). The first order derivative spectra of each solution were obtained using smoothing (∆ λ = 4 nm). The zero crossing points were found to be 244.90 nm and 271.40 nm for NEBI and INDA respectively (figure 3 and 4). Wavelengths selected for quantitation were 244.90 nm for Indapamide (zero crossing point for nebivolol) and 271.40 nm for Nebivolol (zero crossing point for indapamide).

Figure 2: Overlain zero order spectra:

(1) INDA (6μg/ml) and

(2) NEBI (18μg/ml) in methanol.

Calibration curves for NEBI and INDA:

Standard NEBI solution from 6-30μg/ml were prepared by pipetting out 0.6, 1.2, 1.8, 2.4, and 3.0 ml of the working standard solution of NEBI (100μg/ml) into series of10 ml volumetric flasks and the volume was adjusted to mark with methanol. Absorbance of each solution was measured at 271.40 nm using first derivative spectrophotometry. A calibration curve was prepared by plotting absorbance against respective concentration (figure 5). Standard INDA

solution from 2-10μg/ml were prepared by pipetting out 0.2,0.4, 0.6, 0.8 and 1.0ml of the working standard stock solution of INDA (100μg/ml) into series of 10ml volumetric flasks and the volume was adjusted to mark with methanol. Absorbance of each solution was measured at 244.90 nm using first order derivative spectrophotometry. A calibration curve was obtained by plotting absorbance against respective concentration (figure 6).

Figure 3: Overlain first order derivative spectra:

(1) INDA (6μg/ml) and

(2) NEBI (18μg/ml) in methanol.

Figure 4: Overlain first order derivative spectra of NEBI (6-30μg/ml) and INDA (2-10μg/ml) in methanol.

Assay of tablet formulation:

Twenty tablets were weighed and crushed to obtain a fine powder. An accurately weighed tablet powder equivalent to about 5mg of NEBI and 1.5mg of INDA was transferred to 100 ml volumetric flask and the volume was made up to the mark using methanol as solvent. The solution was sonicated for 20minutes and filtered through whatman Filter Paper No. 42. First few ml of filtrate were discarded. Ten ml of the solution from above filtrate was diluted to 100 ml with methanol. One ml of above solution was further diluted to 10 ml with methanol. The absorbance of the resulting solution was measured using first order derivative spectrophotometry at 271.40nm for NEBI and 244.90nm for INDA. The concentration of each drug was calculated using equation of straight line.

Figure 5: Calibration curve of standard NEBI at 271.40 nm by first order derivative spectrophotometry.

Figure 6: Calibration curve of standard INDA at 244.90 nm by first order derivative spectrophotometry.

Method validation:

Linearity and range:

Aliquots of standard stock solutions of NEBI and INDA were taken in volumetric flasks and diluted with methanol to get final concentrations in range of 6-30μg/ml for NEBI and 2-10 μg/ml for INDA. This calibration range was prepared five times and absorbances were measured at respective wavelengths for each drug separately.

Precision:

Precision of the method was determined by performing interday variation, intraday variation and method repeatability studies. . In interday precision, the absorbance of standard solutions of NEBI (6-30μg/ml) and INDA (2-10μg/ml) were measured on three consecutive days. In intraday variation the absorbances were measured three times in a day. Repeatability study, one concentration of both the drugs was measured five times.

Recovery studies:

To study the accuracy of the proposed method, recovery studies were carried out by standard addition method at five different levels. A known amount of drug was added to preanalyzed tablet powder and percentage recoveries were calculated.

Ruggedness:

Analysis by proposed method was done by two different analysts and ruggedness was determined.

RESULTS AND DISCUSSION:

The proposed method was validated as per ICH guideline. The plot of absorbances versus respective concentrations of NEBI and INDA were found to be linear in the concentration range of 6-30 μg/ml and 2-10 μg/ml respectively with correlation coefficient 0.9998 at 271.40 nm and 0.9986 at 244.90nm as shown in Table 1 and Figure 5, 6. Precision was calculated as repeatability, intraday and interday variations and %CV (Coefficient of variance) was found to be in the range (Table 1).

Table 1: Validation Parameters

PARAMETERS	NEBI	INDA
Linearity range	6-30μg/ml	2-10μg/ml
Correlation Coefficient	0.9998	0.9986
Precision	% CV
Repeatability (n=5) Intraday (n=3) Interday (n=3)	0.83 0.33 – 1.69 0.31 – 1.88	1.48 0.39 – 2.32 0.35 – 2.06
% Recovery	100.62 – 102.09 %	98.92 – 101.21 %
Ruggedness % CV (n=3)	1.24	1.55

NEBI-nebivolol; INDA-indapamide; CV- Coefficient of variance

Table 2: Recovery studies

Drug

Amount of drug in sample

(μg/ml)

Amount of Std drug added

(μg/ml)

Total amount of

Drug

(μg/ml)

Spiked amount of drug found (μg/ml)

Mean ± S.D (n=3)

%Recovery*

Mean

%Recovery

NEBI

12.19 ± 0.14

18.15 ± 0.08

24.14 ± 0.16

30.62 ± 0.24

36.57 ± 0.16

101.62

100.84

100.62

102.09

101.60

101.35

INDA

3.6

6.6

9.6

12.6

15.6

3.55± 0.09

6.59± 0.09

9.51 ± 0.03

12.75 ± 0.07

15.61 ± 0.03

98.92

99.88

99.12

101.21

100.10

99.84

*Mean of Three estimations; NEBI-nebivolol; INDA- indapamide; SD-Standard Deviation.

Table 3: Results of simultaneous estimation of NEBI and INDA in marketed Formulation.

Brand

Labelled

mg/tablet

Obtained % of

Label claim*

NEBI

INDA

NEBI

INDA

NEBULA- D

1.5

101.58

99.79

*Average of five determinations

The %CV of ruggedness for NEBI was 1.24 %, while for INDA it was found to be 1.55 %. The accuracy of method was determined at 50, 75, 100,125 and 150 % level. The %recovery ranges from 100.62 – 102.09 % for NEBI and 98.92 – 101.21 % for INDA (Table 2).

The derivative spectrophotometric method can be successfully used for simultaneous estimation of NEBI and INDA in their combined tablet dosage form. Marketed tablets were analyzed and results obtained were within the range of 98 – 102% (Table 3).

CONCLUSION:

The proposed method gives accurate and precise results for determination of NEBI and INDA in marketed tablet formulation and is easily applied for routine analysis. The method is simple, accurate, precise and rapid. The proposed method was successfully applied for the estimation of these drugs in commercial tablets.

ACKNOWLEDGEMENT:

The authors are thankful to the Principal of Maliba Pharmacy College for providing the necessary facilities.

REFERENCES:

1) Indian Pharmacopoeia 2010, 6^th edition; Vol-II, III. Government of India, Ministry of Health and Family Welfare. Published by Indian Pharmacopoeia Commission. p.1489,1785

2) United state pharmacopoeia 2009, 32^nd edition; National formulary 27, Vol-II. USP Convention Rockville; p.2623

3) European Pharmacopoeia 2011, 7^th edition. Developed by the European Directorate for the Quality of Medicines and health (EDQM); p.2232

4) British Pharmacopoeia 2010. Department of Health. London: HMSO Publication; p.1127

5) Parfit K. Martindale, The complete drug reference, 35th edition, vol. 1. London Pharmaceutical press 2007; p.1314,1347

6) Richard D.H., Mary J.M., “Lippincott’s illustrated review: Pharmacology”, 3^rd edn, Lippincott williams and wilkins, 2006, 230-235

7) Jyoti P, Chandrakul K. Development of new spectroscopic methods for determination of indapamide in bulk and pharmaceutical formulation. International Journal of Chem-Tech Research 2011; vol. 3, Issue 2, p.755-760

8) Amit P, Kishor C. Method development and validation of simultaneous estimation of telmisartan and indapamide by RP-HPLC in pharmaceutical dosage form. Asian Journal of Biochemical and Pharmaceutical Research 2011;vol.1, p. 39-49

9) Mrinalini D, Mohit D. Simultaneous estimation of perindopril and indapamide in bulk drug and tablet dosage form by HPTLC. International Journal of Comprehensive Pharmacy 2011; vol.1, Issue 1

10) Kumbhar S, Shivsharan U. A validated HPTLC method for simultaneous quantification of nebivolol and hydrochlorthiazide in bulk and tablet formulation. International Journal of Pharmaceutical Science and Drug Research 2011; vol. 3, Issue 1, p. 62-66

11) Mrinalini D, Darshna C. Stability indicating HPTLC method for estimation of nebivolol hcl and amlodipine besylate in combine dosage form. Eurasian Journal of Analytical Chemistry 2011; vol.5, Issue 2, p.161-169

12) Srinivasulu D, Ramana H. RP-HPLC method for analysis of nebivolol in pharmaceutical dosage form. Journal of Pharmaceutical Research and Healthcare 2009; vol. 1, Issue 1, p.25-33

13) Kunal B, Darshan S. Stability indicating RP-HPLC estimation of nebivolol HCl in pharmaceutical formulation. Indian Journal of Pharmaceutical Sciences 2008; vol. 70, Issue 5, p. 591-595

14) Paresh P, Hetal P. Two spectroscopic methods for simultaneous estimation of indapamide and nebivolol hydrochloride in combined dosage form. International Journal of Pharmaceutical Research 2011; vol. 3,Issue 2, p. 55-57

15) Validation of Analytical Procedures: Text and Methodology, Proceeding of the International Conference on Harmonisation (ICH), Geneva, 2005.

Received on 29.11.2011 Modified on 10.12.2011

Asian J. Research Chem. 5(2): February 2012; Page 282-285