INTRODUCTION:

Nebivolol hydrochloride (NEB), α,α’-[Iminobis(methylene)]bis[6-fluoro-3,4-dihydro-2H-1-benzpyran-2-methanol], is a highly selective β1 adrenergic antagonist which produces nitric oxide mediated vasodilation¹. Spectrophotometric^2-6, HPLC^7-9and HPTLC¹⁰methods have been reported for analysis of NEB either alone or in combination with other drugs in pharmaceutical formulations. Hydrochlorothiazide (HCTZ), chemically 6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide with diuretic activity. HCTZ inhibits the absorption of sodium and chloride at the beginning of distal convoluted tubule¹. Spectrophotometric^11-12, HPLC^13-15, and LC–MS¹⁶ methods have been reported for analysis of HCTZ either alone or in combination with other drugs in pharmaceutical formulations and biological fluids. The structures of the drugs are shown in Fig. 1.

Fig 1. Chemical structure of nebivolol hydrochloride (a) and Hydrochlorothiazide (b)

As visible Spectrophotometric and HPTLC assays offer significant economic advantages over the techniques cited above, the aim of the present investigation was to develop simple and sensitive yet economical methods for the determination of NEB and HCTZ in dosage forms. The reported visible spectrophotometric method, although a couple of them sensitive, suffer from one or other disadvantage. Hence, there is a need to develop simple, selective and sensitive procedures for the determination of NEB in pharmaceuticals. In this paper, we describe the development and validation of spectrophotometric method for the determination of NEB in bulk drug and in tablet dosage form using ceric (IV) sulphate as a reagent and HPTLC method for simultaneous determination of NEB and HCTZ in bulk drug and in tablet dosage form. The methods developed offer the advantages of simplicity, speed, accuracy and precision.

EXPERIMENTAL:

Chemicals and reagent

Pure drugs of NEB and HCTZ were obtained from Emcure Pharmaceutical Limited, Pune and Torrent Pharmaceutical Ltd. Ahemdabad as a gift sample. The commercial formulation of NEB (Nebistar 5) and its combination with HCTZ are available in the ratio of 1:2.5 (Nebicard-H) (5/12.5mg) as tablets. All chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals, India, and distilled water was used to prepare all solutions.

INSTRUMENTATION:

Spectrophotometer:

A Jasco model V530 digital spectrophotometer with 1 cm matched quartz cells was used for absorbance measurements.

Preparation of reagents:

Cerium (IV) Sulphate (500μg/ml):

A 1000 μg/ml solution was first prepared by dissolving 100 mg of cerium (IV) sulphate (Loba-Chemie., Mumbai, India; content 99.9 %) in 1M sulphuric acid and diluting to the mark in a 100 ml calibrated flask with the same acid and filtered. This was appropriately diluted to get 500 μg/ml cerium (IV) sulphate solutions for use in spectrophotometric method .

Indigo carmine (200 μg/ml):

A 1000 μg/ml solution was first prepared by dissolving 111 mg of dye (Loba-Chemie., Mumbai, India; dye content 90 %) in water and diluting to the mark in a 100 ml calibrated flask and filtered. This was appropriately diluted to get 200 μg/ml solution with water.

Sulphuric acid (5 M):

A 272 ml of concentrated sulphuric acid (S. d. Fine Chem, Mumbai, India; Sp gr 1.84) was added to 728 ml water with cooling.

Preparation of standard solutions:

A stock standard solution containing 50 μg/ml NEB was prepared by dissolving 50mg of NEB in 10 ml of methanol and diluting to the mark in a 100 ml calibrated flask with water. This was diluted to get 50 μg/ml working concentration for the spectrophotometric study. For the chromatographic work, a stock standard solution of a concentration of 1 mg/ml of NEB and 1 mg/ml of HCTZ were freshly prepared separately using methanol.

Preparation of sample solution for spectrophotometry:

Twenty tablets of the pharmaceutical formulations Nebistar 5 and Nebicard (containing 2.5 and 5 mg NEB) were weighed and ground into a fine powder. An amount of powder equivalent to 2.5 and 5 mg of NEB was accurately weighed into a 100 ml calibrated flasks, 10 ml methanol and 40 ml of water added, and shaken for about 20 min. The volume was diluted to the mark with water, mixed well and filtered using a Whatman No.41 filter paper. The filtrate contains (50 μg/mL) of NEB and a suitable aliquot was analyzed by spectrophotometry.

Preparation of sample solutions for chromatography:

Twenty tablets of the pharmaceutical formulation Nebicard-H and Nebilong-H (containing 5 mg NEB and 12.5 mg HCTZ) were assayed. They were crushed to a fine powder and an amount of the powder corresponding to approximately 5 mg Nebivolol hydrochloride and 12.5 mg hydrochlorothiazide was weighed in a 25mL volumetric flask. After addition of 10 mL methanol and sonication (30 min) the solution was diluted to volume with methanol and filtered through a Whatman no. 41 filter paper.

General procedures

Spectrophotometric method:

Varying aliquots (0.4- 2 ml) of a standard 50 μg/ml NEB solution were accurately measured into a series of 10 ml calibrated flasks and the volume was adjusted to 4.0 ml by adding requisite quantity of water. To each flask was added 1 ml of 5 M sulphuric acid followed by 1 ml of 500 μg/ml cerium (IV) sulphate solutions. The contents were mixed well and the flasks set-aside for 10 min. Then, 1 ml of indigo carmine solution was added; the volume was diluted to the mark with water, and mixed well. The absorbance of each solution was measured at 610 nm against a water blank after 5 min. The concentration of the unknown was read from the calibration graph or computed from the respective regression equation.

HPTLC method:

TLC was performed on aluminium foil plates coated with 0.2-mm layers of silica gel 60F254 (Merck). Before use plates were prewashed with methanol then dried and activated. Samples were applied to the plates, as 6-mm bands, by means of a Camag Linomat 5 sample applicator used at a constant application rate of 0.5 µL/s. Plates were developed with chloroform: toluene: methanol: ammonia (5:3:2:0.1, v/v/v/v) as mobile phase in a Camag twin-trough chamber previously saturated with mobile phase vapour for 20 min at room temperature (25 ± 2°C). The development distance was approximately 80 mm. After development the plates were scanned in absorbance mode at 278 nm by use of a Camag TLC Scanner 3 controlled by win CATS software. The slit dimensions were 5 mm × 0.45 mm and the source of radiation was a deuterium lamp emitting a continuous UV spectrum in the range 190–400 nm. From the standard stock solution, the mixed standard solution was prepared using the methanol to contain 90 μg/ml NEB and 30 μg/ml HCTZ, 2 to 7 μl was applied. Solution was spotted on the TLC plate to obtain the final concentration 180-630 ng/spot for NEB and 60-210 ng/spot HCTZ. Each concentration was applied six times to the TLC plate. The plate was then developed using the previously described mobile phase and the peak areas were plotted against the corresponding concentrations to obtain the calibration curves.

METHOD VALIDATION:

Precision

The intra-day precision (RSD, %) was assessed by analyzing standard drug solutions within the calibration range, three times on the same day. Inter- day precision (RSD %) was assessed by analyzing drug solutions within the calibration range on three different days over a period of a week.

Limits of Detection and Quantitation

To determine the limits of detection (LOD) and quantitation (LOQ), solutions of concentration in the lower part of the linear range of the calibration plot were used. LOD and LOQ were calculated using the equations LOD = 3.3 × N/B and LOQ = 10 × N/B, where N is the standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and B is the slope of the corresponding calibration plot.

Specificity

The specificity of the method was ascertained by analysis of drug standards and samples. The mobile phase resolved both the drugs very efficiently, as shown in Fig. 2. The identities of the bands for NEB and HCTZ were confirmed by comparing the R_F and spectra of the bands with those of standards.

Accuracy

Analysed samples were overapplied with an extra 80, 100, and 120% of the drugs from standard solutions of NEB and also in combination with HCTZ and the mixtures were reanalyzed by use of the method. The experiment was conducted in triplicate. This was done to check for the recovery of the drug at different levels in the formulation.

Robustness

Robustness was assessed by deliberately changing the chromatographic conditions and studying the effects on the results obtained.

RESULTS AND DISCUSSION:

Method Development

Spectrophotometric method: The ability of cerium (IV) sulphate to cause oxidation of NEB and bleach the colour of indigo carmine dye and it has been used for the indirect spectrophotometric assay of NEB. In this method, the drug was reacted with a measured excess of cerium (IV) sulphate in acid medium and the unreacted oxidant was determined by reacting with indigo carmine followed by absorbance measurement at 610 nm. In this method, the absorbance increased linearly with increasing concentration.

NEB, when added in increasing amounts to a fixed amount of cerium (IV) sulphate, consumed the latter and there occurred a concomitant fall in its concentration. When fixed amount of dye was added to decreasing amounts of oxidant, a concomitant increase in the concentration of dye resulted. This was observed as a proportional increase in absorbance at λmax with increasing concentration of NEB (Fig. 3).

Fig 3. Beer’s law curve for Spectrophotometric method

Preliminary experiment was performed to fix the maximum concentration of the dye that could be determined spectrophotometrically, and it was found to be 200 μg/ml for indigo carmine. For 200μg/ml indigo carmine, 500 μg/ml cerium (IV) sulphate was sufficient to bleach the blue colour in acid conditions. Hence, different amounts of NEB were reacted with 1.0 ml of 500 μg/ml oxidant before determining the residual cerium (IV) sulphate as described under the procedure. The reaction was carried out in sulphuric acid medium. One ml of 5 M acid was used in the assay procedure. For quantitative reaction between NEB and cerium (IV) sulphate a contact time of 10 min was found sufficient in this method and constant absorbance readings were obtained when contact times were extended upto 30 min. The standing time of 5 min was necessary for the bleaching of dye colour by the residual oxidant. The measured colour was stable for hours in the presence of reaction product.

Chromatographic method: The TLC procedure was optimized for simultaneous determination of NEB and HCTZ.

The mobile phase chloroform: toluene: methanol: ammonia (5:3:2:0.1, v/v/v/v) resulted in good resolution, and sharp and symmetrical peaks of R_F 0.30 for NEB and 0.42 for HCTZ. It was observed that prewashing of TLC plates with methanol (followed by drying and activation) and pre-saturation of TLC chamber with mobile phase for 20min (optimum chamber saturation time) ensured good reproducibility and peak shape of both drugs.

VALIDATION

Linearity, LOD and LOQ:

In the spectrophotometric method, Beer’s law was obeyed over the concentration range 2-10 μg/ml. The linear plot gave the regression equation: Y = 0.009X + 0.545 (r² = 0.999, n=6). The limits of detection and quantification were established according ICH guidelines and were calculated to be 0.5 μg/ml and 2 μg/ml respectively. For TLC method linear regression data for the calibration plots revealed good linear relationships between response and concentration over the ranges 180– 630 ng/spot for NEB and 60–210 ng/spot for HCTZ. The linear regression equations were Y = 3.011X + 120.8 (r2 = 0.9990) for NEB and Y = 28.60X - 618.2 (r2 = 0.998) for HCTZ. The limits of detection and quantification were 140 and 160 ng/spot, respectively, for NEB and 30 and 50 ng/spot for HCTZ. This indicates the method is sufficiently sensitive.

Precision and Accuracy:

The precision and accuracy of the methods were expressed as relative standard deviation (RSD, %). The results listed in Table 1 and 2 revealing good precision and accuracies and common additives and excipients did not interfere in the determination. This was further confirmed by the fact that no more than the stoichiometric amount of cerium (IV) was consumed when the tablet extract was treated with cerium (IV) under the described experimental conditions

Table 1: Intra-day and inter-day precision of the method (n=3)

Spectrophotometric method

HPTLC method

NEB

μg/ml

Amount found %

% RSD

NEB

ng/spot

Amount found %

% RSD

HCTZ

ng/spot

Amount found %

% RSD

Intra-day precision

100.5

99.98

100.33

1.41

1.36

1.24

270

450

630

99.82

99.05

100.44

1.11

1.51

1.31

150

210

100.17

99.17

101.5

1.24

1.02

0.98

Inter-day precision

99.45

100.2

99.83

1.40

0.43

1.46

270

450

630

100.8

99.97

101.1

1.41

1.54

1.21

150

210

100.3

99.39

101.2

1.44

1.30

1.21

Table 2: Results from recovery studies

Drugs

Label claim(mg per tablet)

Amount of drug

Standard added (%)

Amount of drug

standard recovered (%)^a

RSD (%)

Spectrophoto

metric method NEB

100

120

99.21

99.55

99.36

0.47

1.02

0.63

HPTLC method

NEB

100

120

99.72

100.3

99.76

0.20

0.29

0.23

HPTLC method

HCTZ

12.5

100

120

99.56

99.85

100.53

0.16

0.19

0.18

a Mean from three estimates at each level

Table 3a: Robustness of the spectrophotometric method^a

Condition

NEB

% Recovery

RSD (%)

Ceric sulphate Concentration

450 µg/mL

500 µg/mL

550 µg/mL

99.69

100.2

100.38

1.38

1.09

1.69

Sulphuric acid Concentration

4 M

5 M

6 M

99.25

100.2

101.2

1.41

1.09

1.40

Reaction Time

08 min

10 min

12 min

102.0

100.2

98.5

1.60

1.09

0.90

^aMean from three estimates

Table 3b: Robustness of the HPTLC method^a

Condition	NEB		HCTZ
Condition	% Recovery	RSD(%)	% Recovery	RSD(%)
Mobile phase composition
Chloroform: toluene: methanol: ammonia 6: 2: 2: 0.1 (v/v)	99.74	0.17	99.63	1.10
Chloroform: toluene: methanol: ammonia 4: 3: 3: 0.1 (v/v)	99.86	0.12	99.92	1.05
Mobile phase volume
10. 1 mL	100.3	0.29	99.85	0.19
20. 2 mL	99.78	1.20	99.45	0.50
Development distance
7 cm	99.89	0.95	99.38	0.61
7.5 cm	99.36	0.73	99.25	0.79
8 cm	100.3	0.29	99.85	0.19
Duration of saturation
20 min	100.3	0.29	99.85	0.19
25 min	99.43	0.56	99.68	0.76
30 min	99.26	0.77	98.76	0.32
Time from application to chromatography +20	99.34	0.66	99.16	0.94
Time from chromatography to scanning +20	99.89	0.96	99.83	1.05

^aMean from three estimates

Table 4: Results of analysis of dosage form

Drugs

Brand Name

Label claim(mg per tablet)

% Found*

RSD (%)

Spectrophotometric method NEB

Nebicard

Nebistar

2.5

99.83

99.57

1.43

0.90

HPTLC method

NEB

Nebicard-H

Nebilong-H

100.1

99.89

1.47

0.94

HPTLC method

HCTZ

Nebicard-H

Nebilong-H

12.5

99.45

99.34

0.68

0.59

* Mean value of five determinations

ACKNOWLEDGMENT:

The authors are grateful to Emcure Pharmaceutical Limited, Pune and Torrent Pharmaceutical Ltd. Ahemdabad, providing samples of Hydrochlorothiazide and Nebivolol HCL as gift sample. The authors are also grateful to Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune, India, for providing excellent facilities for carrying out this research work.

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Received on 03.09.2011 Modified on 22.09.2011

Asian J. Research Chem. 5(5): May 2012; Page 576-581