INTRODUCTION:

Valsartan (VAL), a competitive and selective antagonist, at the angiotensin II receptor subtype derivative. Chemically, it is N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl) [1,1'-biphenyl]-4-yl] methyl]-L-valine. Nebivolol (NEB) is a, third-generation vasodilating cardioselective [beta]-blocking agent. Chemically, it is 1-(6-fluorochroman-2-yl) -2-[[2-(6-fluorochroman-2-yl)-2-hydroxy-ethyl] amino] ethanol. The advantages expected from this combination are synergistic effects such as inhibition of different physiologic pathways, blockage of the Renin-Angiotensin Aldosterone system at different points, cardio protection, and trans-inhibition of the reciprocal receptor. This drug combination is used as an antihypertensive agent.

A literature survey reveals that several HPLC methods are reported for the determination of valsartan in biological fluids^[1-6] or in tablet dosage form.^[7] Some spectrophotometric, HPTLC and HPLC methods also reported for the estimation of valsartan in combination with mainly hydrochlorothiazide^[8-11] or amlodipine^[12]. Several analytical procedures have been described for the individual determination of nebivolol or in combined with hydrochlorothiazide, most frequently by using HPLC, HPTLC, or spectrophotometer.^[13-18] Senthamil et. al., reported a LC/MS/MS method for simultaneous determination of nebivolol and valsartan in human plasma.^[19]

Doshi et. al.,^[20] only reported liquid chromatographic method for the determination of nebivolol and valsartan in fixed dose combination. However the method employed tedious mobile phase preparation and used of column oven temperature, which is not necessary for the separation of analytes in this combination.

In this section we report a simple, rapid and precise liquid chromatographic method for the separation of nebivolol and valsartan and to apply the method for simultaneous quantitative determination of these drugs in combined dosage form.

EXPERIMENTAL:

Working Standard and Chemicals:

The formulations, Nebicard-V tablet (containing 80 mg of valsartan and 5 mg nebivolol; manufactured by Torrent Research Centre), were procured from pharmacies. Valsartan working standard was obtained from Lupin Ltd, nebivolol from Torrent research centre.

Acetonitrile and Methanol were of HPLC grade purchased from E-Merck (India) Ltd. Glacial acetic acid was of spectrochem Pvt. Ltd. Potassium Dihydrogen ortho phosphate was of AR grade purchased from Qualigens Ltd.

Instrument:

The analysis was carried on the integrated HPLC system (Agilent 1100 series, Germany) consisted of G1311A quaternary pump with G1379A degasser, G1329A autosampler (1 – 100 µL) with G1330B autosampler thermostat, G1316A column compartment with temperature controller and G1314A VWD UV detector. Chromatograms were processed and the results were analyzed by using Chemstation software (Rev.10.01).

Table 1A: Tablet assay of Valsartan: 80 mg

Obs. No.	Area of STD	Area of Test sample	Content In mg	% Assay
1	211.781	209.835	79.225	99.032
2	213.300	211.835	79.411	99.264
3	212.780	210.835	79.229	99.036
4	213.619	213.835	80.041	100.051
5	214.119	211.835	79.107	98.884
6	214.518	213.835	79.705	99.632
Mean	213.353	212.002	79.453	99.316
SD	0.982	1.602	0.356	0.445
%RSD	0.460	0.756	0.448	0.448

Table 1B: Tablet Assay of Nebivolol: 5 mg

Obs No.	Area of STD	Area of Test sample	Content In mg	% Assay
1	23.001	22.823	4.951	99.028
2	21.913	21.652	4.931	98.611
3	22.994	22.831	4.955	99.093
4	24.075	23.965	4.967	99.344
5	22.156	22.453	5.057	101.138
6	23.237	23.061	4.952	99.044
Mean	22.896	22.798	4.969	99.376
SD	0.780	0.757	0.045	0.895
%RSD	3.406	3.320	0.900	0.900

Optimized Chromatographic Conditions:

Analytical Column: Kromasil 100-5C₁₈, (250 X 4.6 mm, 5 µm).

Mobile Phase : 20 mM potassium dihydrogen orthophosphate: acetonitrile (43:57) containing 0.1% glacial acetic acid.

UV Detection : 282 nm

Flow Rate : 1 ml/min

Injection Volume : 5 µl

Temperature : Ambient

Run Time : 8.0 min.

Retention Time : Nebivolol ~ 3.11 Valsartan ~ 5.46

Working Standard Solution Preparation:

Valsartan and Nebivolol standard stock solution-I and II (1mg/mL) were prepared by dissolving 10 mg of the drug in 10 mL methanol and further dilutions were prepared in mobile phase to obtain calibration standards in the concentration range of 40-96 µg/mL and 2.5-6.0 µg/mL respectively.

Linearity:

Eight different concentrations (40-96) µg/mL for valsartan and (2.5-6.0) µg/mL for nebivolol were prepared for linearity studies. The responses were measured as peak areas and plotted against concentrations. Linear regression least square fit data obtained from the above calibration curve. The respective slopes (m), intercept (b) and correlation co-efficient (r) are also obtained. A typical HPLC chromatogram is shown in Figure 1.

Sample Preparation:

Twenty tablets were weighed and finely powdered. Tablet powder equivalent to 80 mg of valsartan and 5 mg of nebivolol was accurately weighed and transferred to a 100 ml volumetric flask. To this solution was added about 50 ml of methanol and flask was sonicated for 15 min. The flask was shaken and volume was made up to the mark with methanol. The above solution was then filtered through 0.45µ whatman filter paper. 1 mL of the filtrate was diluted to 100 mL with mobile phase to obtain 100% level of pharmaceutical preparation i.e. 80 µg/mL of valsartan and 5 µg/mL of nebivolol.

Assay:

The 5 µl sample solution was injected under the optimized chromatographic conditions and areas were measured. The quantitation was carried out by keeping these values to the straight line equation of calibration curve. Results of the assay are tabulated in Table 1A and 1B.

Recovery:

Recovery experiments were carried out to check for the presence of positive or negative interferences from excipients present in the formulation and to study the accuracy and precision of the method. Recovery experiment was performed by the standard addition method.^[21] The recovery of the standard was studied at the three different levels viz. 80%, 100% and 120% of the estimated amount of drug. Each set of recovery of added standard was calculated. The results of recovery experiment are tabulated in Table 2.

The limit of quantitation (LOQ) and limit of detection (LOD):

The limit of detection (LOD) and quantification (LOQ) were evaluated from calibration curves plotted in concentration ranges of 0.40-0.96 µg/ml for valsartan and 0.25-0.60 µg/ml for nebivolol, with formula LOD = 3.3 syx/S and LOQ = 10 syx/S (where syx = residual error and S= slope of the calibration curve). The LOD and LOQ for each drug were thus obtained. Thereafter, the standard drug solutions at each value of LOD and LOQ concentration were injected six times and % RSD of area of the replicate injections were calculated.

Robustness:

Robustness of the method was ascertained by evaluating the effect of deliberate change pH of the mobile phase by adding different volumes of glacial acetic acid, proportion of organic solvent in mobile phase and flow rate. The pH of mobile phase was varied within a range of ± 0.2 unit of the optimize pH (4.0) by adding different volumes of glacial acetic acid. While proportion of organic solvent (acetonitrile) was varied in the range of ± 2%. The mobile phase were employed, keeping the other chromatographic conditions constant, to evaluate the influence of pH and organic solvent on resolution between two drugs. Keeping other chromatographic conditions optimized different flow rates ± 0.1 ml/min were employed to check influence of flow rate on the proposed method.

RESULTS AND DISCUSSION:

The Reverse Phase High Performance Liquid Chromatography method was optimized with a view to developed a simple assay method. Pure drugs chromatogram was run in different mobile phase containing methanol, acetonitrile, water and different buffers in different ratios. Buffer concentrations ranging from 10 to 50 mM phosphate buffer were tried, and the best results were obtained with a 20 mM concentration. Glacial acetic acid was added to the buffer to decrease peak asymmetry. For the organic constituent of the mobile phase, acetonitrile was chosen to reduce the longer retention time and provide the best peak shape. Different columns such as C₈, C₁₈ with different dimension were used. Finally C₁₈ column and mobile phase containing 43:57 buffer and acetonitrile with 0.1% acetic acid were selected. System suitability parameter was tested which indicate that the proposed LC method permitted adequate resolution of the mixture components within the reasonable run-time. In addition high column efficiency was indicated from the large number of theoretical plates (>3000). The degree of asymmetry was also evaluated using the tailing factor, which did not exceed the critical value (1.5) indicating acceptable degree of peak asymmetry. The optimum wavelength for detection was found to be 282 nm, wherein both the analytes were detected. The linearity of the calibration curves indicates the suitability of the method over a wide range of concentration. Regression analysis of the calibration data for valsartan and nebivolol showed that the dependant variable (peak area) and the independent variable (concentration) were represented by the equations: y = m x + b was found to y = 2.6696 x + (-6.5834) and y = 4.0645 x +(-0.5758) for valsartan and nebivolol respectively. The correlation of coefficient (r²) obtained was found to be 0.99972 and 0.99971 for valsartan and nebivolol respectively. The sensitivity of the method was found to be good from LOD and LOQ of the analytes. The average % recovery for valsartan and nebivolol shows that method is free from interference like excipients present in the formulation. The low value of the relative standard deviation for valsartan and nebivolol indicate high precision of the method.

CONCLUSION:

In the present study the attempt has been undertaken to development simple, economical, sensitive and accurate analytical HPLC method for the simultaneous estimation of these drugs without their prior separation. The method gives good resolution between both the compounds with a short analysis time (8 min.). The method was validated and found to be simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from the interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of valsartan and nebivolol in their combined dosage form.

ACKNOWLEDGEMENT:

Authors are thankful to Mr. Sudhir S. Jadhav and the staff of Shri C. B. Patel research centre for their valuable contribution throughout the research.

REFERENCES:

1. Danestalab N, Lewanczuk RZ, High Performance liquid chromatographic analysis of angiotensin II receptor antagonist Valsartan using Liquid extraction method, J. of Chromatogr B Analyt Technol Biomed Life Sci. 2002, Jan 25, 766(2), 345-9.

2. Gonzalez L, Lopez J A, Alonso R M, Jimenez R M, Fast screening method for the determination of angiotensin II receptor antagonist in human plasma by high-performance liquid chromatography with fluorimetric detection, J. Chromatogr. A, 2002, 949, 49-60.

3. Sechaud R, Graf P, Bigler H, Gruendi E, Letzkus M, Merz M. Bioequivalence study of a valsartan tablet and a capsule formulation after single dosing in healthy volunteers using a replicated crossover design. Int J Clin Pharmacol Ther. 2002; 40: 35–40.

4. Macek J, Klima J, Ptacek P. Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography.J chromatogr B. 2006; 832: 169–172.

5. Nozomu koseki, Hiroto Kawashita, Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography-tandem mass spectrometry. J. Pharmaceutical and Biomedical Analysis, 2007, 43, 1769–1774.

6. Zarghi A, Shafaati A, Foroutan S M, Movahed H, Rapid Quantification of Valsartan in Human Plasma by Liquid Chromatography using Monolithic Column and a Fluorescence Detection : Application for Pharmacokinetic Studies, Sci Pham., 2008, 76, 439-450.

7. Sevgi Tatar, Serap Saglik. Comparison of UV- and second derivative- spectrophotometric and LC methods for the determination of valsartan in pharmaceutical formulation. J. Pharmaceutical and Biomedical Analysis, 2002, 30, 371-375.

8. Kadam B., Bari S. Quantitative analysis of Valsartan and Hydrochlorothiazide in tablets by High Performance Thin-Layer Chromatography with Ultraviolet absorption densitometry. Acta Chromatographia, 2007, 18.

9. Eda Atana, Adi Altnay, Simultaneous determination of Valsartan and Hydrochlorothiazide in tablets by first order-derivative spectrophotometry and LC. J. Pharmaceutical and Biomedical Analysis, 2001, 25, 5-6, 1009-1013.

10. Tian DF, Tian XL, Simultaneous determination of Valsartan and Hydrochlorothiazide in tablets by RP-HPLC. Indian J. Pharma. Sci. 2008, 70, 372-374.

11. Nevin Erk. Spectrophotometric analysis of Valsartan and Hydrochlorothiazide. Analytical letters. 2002, 35, 283-302.

12. Chitlange SS, Bagri Kiran, Stability indicating RP-HPLC methods for simultaneous estimation of Valsartan and Amlodipine in capsule formulation. Asian J. Research Chem. 2008, 1(1), 15-18.

13. Patel LJ, Suhagia BN, RP-HPLC and HPTLC methods for the estimation of nebivolol hydrochloride in tablet dosage form. Indian J. Pharma. Sci. 2007, 69, 594-596.

14. Shah DA, Bhatt KC, Stability indicating RP-HPLC methods estimation of Nebivolol HCL in pharmaceutical formulations. Indian J. Pharma. Sci. 2008, 70, 591-595.

15. Kachhadia P., Doshi A., Development and validation of a stability-indicating column high-performance liquid chromatographic assay method for determination of nebivolol in tablet formulation. J. AOAC Internat. 2008, 91, 557-561.

16. Raddy T., Sita Devi P., Validation of a high-performance thin layer chromatographic method with denstiometric detection, for quantitative analysis of Nebivolol HCL in tablet formulation. J. Planner Chromatogr. 2007, 20,149-152.

17. Shah, Dimal A., Determination of Nebivolol hydrochloride and Hydrochlorothiazide in tablets by first order spectrophotometry and liquid chromatography. J. AOAC Internat. 2008, 91, 1075-1082.

18. Tarte PS, Wate SP, Simultaneous Estimation of Nebivolol and Hydrochlorothiazide in Combined Tablet Dosage Form by Multicomponent Mode of Analysis. Asian J. Research Chem. 2008, 1(2), 77-79.

19. Senthamil P., Gowda K., Simultaneous determination of fixed dose combination of nebivolol and valsartan in human plasma by liquid chromatographic-tandem mass spectrometry and its application to pharmacokinetic study. , J. Chromatogr. B. 2007, 858, (1-2), 143-150.

20. Doshi A., Bhagwan S., Determination of Nebivolol and Valsartan in a fixed-dose combination by liquid chromatography. J. AOAC Internat. 2008, 91,(2), 292-298.

21. ICH Q2A, Text on validation of analytical procedures, International Conference on Harmonization tripartite guidelines, adapted 27 Oct 1994B.

Drug	Original amount (mg)	Added Amount (mg)	Total amount (mg)	Recovery found	%RSD (n=6)
VAL	80	64	142.85	0.338	99.20
	80	80	161.02	0.446	100.64
	80	96	176.58	0.649	100.33
NEB	5	4	9.106	0.644	101.17
	5	5	10.065	0.479	100.65
	5	6	11.191	1.206	101.73