INTRODUCTION:

Nebivolol chemically, 1-(6-fluorochroman-2-yl)-{[2-(6-fluorochroman-2-yl)-2-hydroxy-ethyl] amino} ethanol, is a third-generation vasodilating cardio selective β-blocking agent. Its molecular formula is C₂₂H₂₅F₂NO₄•HCl and it has a molecular weight of 405.435 g/mol.^{1, 2} Indapamide chemically, 4-chloro-N-(2-methyl-2,3-dihydroindol-1-yl)- 3-sulfamoyl-benzamide, is a non-thiazide sulphonamide diuretic. Its molecular formula is C₁₆H₁₆CIN₃O₃S and it has a molecular weight of 365.84 g/mol.³ The combination is used for the treatment of Hypertension.

Figure1: Indapamide

Figure 2: Nebivolol

A recent literature survey revealed that few methods are available for the determination of Nebivolol hydrochloride in pure, pharmaceutical dosage forms and/or biological fluids, it includes high-performance liquid chromatography (HPLC)^5-8, high-performance thin-layer chromatographic (HPTLC)^9-11, UV spectrophotometric^12,13, Spectro- fluorometric¹⁴ method. Numerous different analytical methods have been developed for quantitative determination of Indapamide in pure, pharmaceutical dosage forms and/or biological fluids. These methods include high performance liquid chromatography^15-18, high-performance thin layer chromatography^19,20 and Spectrophotometry^21,24 method. However, there is no method for the simultaneous determination of these two drugs by high performance liquid chromatography (HPLC). The intended purpose of this investigation was to develop and validate a sensitive, precise, accurate and specific HPLC method for the simultaneous estimation of Nebivolol and Indapamide in pharmaceutical dosage form.

MATERIAL AND METHODS:

Materials and Reagent:

All the materials and reagents used were of Analytical grade supplied from Rankem. Indapamide was gifted by Dishman pharmaceuticals and chemicals Ltd, Ahmedabad. Formulation, NEBULA-D tablets, was procured from local market.

Dosage form:

Procured from local market containing combination of Indapamide (1.5 mg) as sustained release and Nebivolol (5 mg) as immediate release.

Chromatographic conditions:

Isocratic HPLC system Shimadzu LC20AT VP series HPLC pumps, and SPD 20A VP UV-Visible detector.

· Column : Inertsil ODS C-18 (150 x 4.6 mm), 5 μ

· Detection : 282 nm

· Injection Volume : 20 μl

· Flow Rate : 1 ml/min

· Temperature: ambient room temperature

· Run Time : 8 minute

· Mobile Phase : Buffer: Acetonitrile (60:40)

· Diluent: Mobile phase

Preparation of solutions:

Buffer Solution:

Dissolve 150 mg of potassium dihydrogen phosphate in 100 ml of water (HPLC grade). Then add 0.15 ml of triethylamine and adjust pH of this buffer solution to 3 with O-phosphoric acid.

Mobile Phase:

Buffer:Acetonitrile (60:40 v/v):.

Preparation of Standard Solution:

The standard stock solutions were prepared by transferring 25 mg of Nebivolol in a 50 ml volumetric flask and 15 mg of Indapamide in 50 ml volumetric flask. Then volume was made upto 50 ml with mobile phase to get concentration of 500 μg/ml of Nebivolol and 300 μg/ml of Indapamide.

Sample Preparation:

Accurately 20 intact tablets were weighed to determine average weight of tablets. Then tablets were finely crushed and tablet powder equivalent to 5 mg Nebivolol and 1.5 mg Indapamide was transferred into 50 ml volumetric flask. Then 30 ml diluent was added to flask and sonicated for 15 minute with intermittent shaking. Volume was made upto 50 ml to obtain solution of Nebivolol 100 μg/ml and Indapamide 30 μg/ml.

RESULTS AND DISCUSSION:

The main objective of this work was to develop and validate RP- HPLC method for simultaneous estimation of Nebivolol and Indapamide in solid dosage form. The method has provided adequate separation for Nebivolol and Indapamide from their dosage form. Separation was obtained by using Inertsil ODS C-18 (150 x 4.6 mm), 5μ column at room temperature and using a mobile phase Buffer: acetonitrile (100 ml buffer + 0.15ml Triethylamine, pH adjusted to 3 with ortho-phosphoric acid) in a ratio of 60:40 v/v at a flow rate 1.0 ml/min and wavelength for detection was 282 nm (Figure 3).

Figure 3: Overlay spectra of Nebivolol and Indapamide

Figure 4: Chromatographic separation of standard Solution of Nebivolol and Indapamide

Figure 5:Chromatographic separation of test Solution of Nebivolol and Indapamide

METHOD VALIDATION:

The method was validated in accordance with ICH guideline Q2 (R1).²⁵

System Suitability:

System suitability tests were carried out on freshly prepared working standard solution of Nebivolol and Indapamide. 10 μl of working standard solution was injected in to the chromatograph under the proposed chromatographic conditions and parameters like Resolution (R), Number of Theoretical Plates, Capacity factor and Tailing factor (T) were studied to evaluate the suitability of system.

Table 1: System suitability parameter

Parameter	Nebivolol	Indapamide
Retention Time (min)	3.42±0.002	5.71±0.002
Theoretical Plates	4781	6546
Asymmetry	1.4	1.4
Capacity Factor	1.21	2.47
Resolution	7.39

Linearity and Range:

Linearity was studied by preparing standard solutions at 9 different concentrations. Each concentration was repeated 6 times. From the stock solution of Nebivolol 0.5,1,1.5-4.5 ml and from Indapamide 0.25,0.5,0.75-2.25 ml solution were taken, mixed and diluted upto 10 ml to get the concentration range of 25-225 µg/ml of Nebivolol and 7.5-67.5 µg/ml of Indapamide.

Precision:

Intra-day precision was determined by analyzing the three different concentrations 25, 100, 225 μg/ml for Nebivolol and 7.5, 30, 67.5 μg/ml for Indapamide, for six times in the same day. Day to day variability was assessed using above mentioned three concentrations analyzed on six different days.

Accuracy:

Recovery study was performed by addition of known amounts of standard drugs to a known concentration of commercial pharmaceutical product (standard addition method). Standard drug was added at three different concentrations 80, 100, 120% to pre-analyzed sample and mixture were analyzed by proposed method.

Figure 6: Calibration curve

Sensitivity:

The sensitivity of proposed method was estimated in terms of the Limit of Quantitation (LOQ) and Limit of Detection (LOD). The LOQ and LOD were calculated using following equations,

LOD = 3.3σ/S

LOQ = 10σ/S

Where σ = Standard deviation of response

S = Slope of calibration curve.

Table 2: Calibration Data for Nebivolol and Indapamide

Sr. No.	NEBIVOLOL			INDAPAMIDE
Sr. No.	Concentration (µg/ml)	Mean (Area) ± S.D. (n=6)	% R.S.D	Concentration (µg/ml)	Mean (Area) ± S.D. (n=6)	% R.S.D
1	25	448.44 ± 4.83	1.08	7.5	119.44 ± 1.65	1.38
2	50	824.41 ± 9.35	1.13	15	221.64 ± 3.90	1.76
3	75	1353.82 ± 9.73	0.72	22.5	349.41 ± 4.94	1.41
4	100	1679.49 ± 5.38	0.32	30	452.88 ± 6.63	1.46
5	125	2094.23 ± 9.22	0.44	37.5	562.22 ± 6.31	1.12
6	150	2543.14 ± 18.91	0.74	45	685.94 ± 9.35	1.36
7	175	2986.41 ± 24.40	0.82	52.5	781.79 ± 14.95	1.91
8	200	3380.23 ± 22.24	0.66	60	899.38 ± 11.39	1.27
9	225	3796.00 ± 50.34	1.33	67.5	1040.65 ± 14.42	1.39

Robustness:

By introducing small deliberate changes in the mobile phase composition and flow rate, the effects on the results were examined. Study was carried out at 30 µg/ml and 100 µg/ml concentration of Indapamide and Nebivolol respectively.

Table 3: Linear Regression Data for calibration curves

Parameter	Nebivolol	Indapamide
Linearity range	25-225 µg/ml	7.5-67.5 µg/ml
r²	0.9992	0.9990
Slope	16.79 ± 0.18	15.15 ± 0.16
Intercept	24.03 ± 17.32	4.47 ± 2.99

Table 4: Intraday Precision for Nebivolol and Indapamide

Concentration (µg/ml)		Area ± S.D.(n=6)		% RSD
Nebivolol	Indapamide	Nebivolol	Indapamide	Nebivolol	Indapamide
25	7.5	471.61±8.47	128.25 ±2.34	1.80	1.83
100	30	1846.71 ±16.72	502.19 ±3.58	0.91	0.71
225	67.5	4202.79 ±41.56	1146.74 ± 16.31	0.99	1.42

Table 5: Interday Precision for Nebivolol and Indapamide

Concentration (µg/ml)		Area ± S.D.(n=6)		% RSD
Nebivolol	Indapamide	Nebivolol	Indapamide	Nebivolol	Indapamide
25	7.5	450.36 ± 8.17	117.62 ± 2.16	1.82	1.84
100	30	1654.94 ± 17.81	444.47 ± 6.84	1.08	1.54
225	67.5	3790.92 ± 37.65	1035.50 ± 15.18	0.99	1.47

Table 6: LOD and LOQ for Nebivolol and Indapamide

Parameter	Nebivolol	Indapamide
LOD (µg/ml)	3.40	0.65
LOQ (µg/ml)	10.32	1.97

Table 7: Accuracy study of Nebivolol

Set	Sample Concentration (µg/ml)	Standard Added (µg/ml)	Mean % Recovery	% RSD
80	100	80	99.10 ± 0.68	0.68
100	100	100	100.06 ± 0.94	0.94
120	100	120	100.37 ± 0.69	0.68

Table 8: Accuracy study of Indapamide

Set	Sample Concentration (µg/ml)	Standard Added (µg/ml)	Mean % Recovery	% RSD
80	30	24	101.73 ± 0.39	0.38
100	30	30	99.38 ± 1.41	1.41
120	30	36	99.76 ± 0.59	0.59

Table 9: Robustness study

Robust condition	Area ± SD		% RSD
Robust condition	NEB	IND	NEB	IND
Flow rate: 0.8 ml/min	1721.89 ± 6.83	447.20 ± 2.42	0.40	0.54
Flow rate: 1.2 ml/min	1678.01 ± 7.25	454.08 ± 5.60	0.43	1.23
Buffer-ACN (58:42 v/v)	1687.45 ± 9.94	451.31 ± 6.18	0.59	1.37
Buffer-ACN (62:38 v/v)	1713.99 ± 11.05	460.45 ± 2.99	0.64	0.65

Application of Proposed Method to Tablet Formulation

The prepared test sample solutions were chromatographed for 10 min using mobile phase at a flow rate 1 ml/min. From the peak area obtained in the chromatogram, the amounts of both drugs were calculated.

Table 10: Results of assay

Component	Amount found (n=3)	%RSD
Nebivolol	100.79 ± 0.75	0.75
Indapamide	100.09 ± 1.11	1.11

CONCLUSION:

This HPLC method for simultaneous analysis of Nebivolol and Indapamide in tablet dosage forms is simple, economic, accurate, precise, specific, robust, and rapid. It does not suffer from interference from common excipients present in the pharmaceutical preparation and can be conveniently adopted for quality-control analysis.

ACKNOWLEDGMENT:

The authors are thankful to Molecule Laboratory, Ahmadabad for providing necessary facilities. The authors are also thankful to Dishman Pharmaceuticals and Chemicals Ltd, Ahmadabad for providing gift sample of Indapamide.

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Received on 29.05.2012 Accepted on 03.04.2014

Asian J. Pharm. Ana. 4(3): July-Sept. 2014; Page 98-102