Development and Validation of RP-HPLC Method for Simultaneous Estimation of Nebivolol HCl and Indapamide in Tablet

Indapamide (INDA), chemically is a 4-chloro-N-(2-methyl-2, 3-dihydroindol-1-yl)-3-sulfamoyl-benzamide used as an antihypertensive agent and as a diuretic, which inhibits the reabsorption of sodium and calcium at the beginning of distal convoluted tubules. Nebivolol hydrochloride, chemically is a α, α’–(Imino bis (methylene)) bis (6- fluro-3, 4 dihydro-2-H-1-benzopyran-2-methanol) is an antihypertensive agent and α-adrenergic receptor blocker ^[1-3]. It produces sympatholytic effect on heart. Deep literature survey reveals that, HPLC ^[4-6], Spectrophotometric ^[7] method are reported for the estimation of INDA alone and combination with others. Spectrophotometric ^[8], HPLC ^[9-10], HPTLC ^[11] are reported for the estimation of NEBI. Simultaneous equation method and Q absorbance ratio method was found for Nebivolol HCl and Indapamide in combine dosage form ^[12]. RP-HPLC method was not found so in present study, simple, accurate, precise, rapid RP-HPLC method is developed for the simultaneous estimation of these two drugs in combined dosage form.

Objective:

• By literature survey it was found that there is no official method is available for Nebivolol HCl and Indapamide in combination. Reported methods (Simultaneous equation method and Q absorbance ratio method) are available for Nebivolol HCl and Indapamide in combination.
• By literature survey RPHPLC method was not found for combination of Nebivolol HCl and Indapamide so, there is a thought of interest to develop new RP-HPLC method for estimation of Nebivolol HCl and Indapamide.

MATERIALS AND METHODS

Instruments:

A Shimadzu RP-HPLC instrument (LC-20AT) equipped with an SPD-ZDA UV-Visible detector, manual injector with 20 μL loop, and C18 column (250 mm × 4.6 mm id, 5 μm particle size) and Sprinchrom software were used. Sartorius CP224S analytical balance, ultra sonic cleaner and pH meter were used during the study.

Materials: Nebivolol HCl and Indapamide were received as a gift sample from Vaibhav Laboratory, Ahmedabed. Acetonitrile of HPLC grade and orthophosphoric acid of HPLC grade were purchased from S.D fine chemicals Ltd. Mumbai. The water for RPHPLC was prepared by triple glass distillation and filtered through a nylon 0.45 μm –47 mm membrane filter (Gelman Laboratory, Mumbai,India). Pharmaceutical formulation of NEBI and INDA were purchased from local pharmacy.

Chromatographic conditions: The column used for chromatographic separations was C18 (250 mm x 4.6 mm i.d., 5 μm particle size). The analytical wavelength was set at 282 nm and samples 20 μl were injected. The chromatographic separations were accomplished using mobile phase comprised of 0.074 M potassium dihydrogen phosphate and acetonitrile in the proportion of 40:60 (v/v) with 2ml triethaylamine and an apparent pH adjusted to 3.0±0.1 using orthophosphoric acid, filtered through 0.45 μm filter (Millipore) and deaerated in ultrasonic bath. Mobile phase was pumped at a flow rate of 1.0 ml/min at ambient temperature.

Preparation of standard stalk solution:

For HPLC analysis 50 mg of Nebivolol HCl and 15 mg of Indapamide powder was weighed accurately using sartorius precision balance (readability 0.01 mg) and transferred in to 50 ml volumetric flask, dissolved and diluted to 50 ml with mobile phase.

Preparation of working standard solution:

From the standard stalk solution take 0.5, 0.75, 1, 1.25, 1.5 ml and transferred to separate 10 ml volumetric flask made up to mark with mobile phase for Nebivolol HCl and Indapamide.

Preparation of sample solution:

Twenty tablets were weighed accurately. Powder equivalent to 50 mg of Nebivolol HCl and 15 mg of Indapamide was weighed and transferred in a 50 ml volumetric flask and mobile phase was added. This solution was sonicated for 15 minutes and final volume was made to the mark with mobile phase. The solution was filtered through Whatman filter paper No. 41. The filtrate (5ml) was transferred in a 50 ml volumetric flask and diluted to the mark with mobile phase to obtain sample solution.

Method Validation:

The method was validated for various parameters as per ICH guidelines.

RESULTS AND DISCUSSION:

Table 1: Regression characteristics and validation parameters

Table 3: Parameters of chromatogram

Table 4: Estimation of NEBI and INDA tablets

CONCLUSION

Results are in good agreement with claim which indicates there is no interference of routinely used excipients. The proposed method was accurate, precise, simple, sensitive and rapid. The proposed method can be used for routine analysis of Nebivolol HCl and Indapamide in tablets.

Figure 1 HPLC Chromatogram of NEBI (100 µg/ml) and INDA (30 µg/ml)

Figure 2: Calibration curve of Nebivolol HCl

Figure 3: Calibration curve of Indapamide