INTRODUCTION:

Granisetron hydrochloride is a selective 5-HT₃ receptor antagonists. Chemically it is 1-methyl-n-(9-methyl-9-azabicyclo(3.3.1)non-3-yl)-1h-indazole-3-carboxamidendo-monohydrochloride. Serotonin receptors of the 5-HT₃ type are located peripherally on vagal nerve terminals, enteric neurons in the GI tract, and centrally in the chemoreceptor trigger zone, which may have beneficial therapeutic effects in the treatment of vomiting and nausea resulting from cancer therapy^1-3.

It has an improved side effect and tolerability profile, a lower risk of drug interactions and a longer duration of action than other 5-HT₃ receptor antagonists. It is also an effective and well tolerated agent in the management of chemotherapy- induced, radiotherapy- induced and post–operative nausea and vomiting in adults and children^4,5.

A survey of literature revealed that the following analytical methods were reported for determination of Granisetron hydrochloride in biological samples including liquid chromatography with fluorescence^6-12 and tandem mass spectrometric detector methods^13-14. Only a single spectrophotometric method has been reported for Granisetron hydrochloride³. In the present investigation an attempt has been made to develop a more simple and precise methods for estimation of Granisetron hydrochloride as compared to existing one in pure and tablet formulations with good accuracy, precision, simplicity and economy.

MATERIALS AND METHODS:

Instruments:

An UV – Visible double beam spectrophotometer of make Jasco, model V- 530 with a pair of 1 cm matched quartz cell, spectral band width of 2 nm and Shimazdu balance, AUX- 220 were used for experimental purpose.

Reagents:

Phosphate buffer (pH 7.0) and distilled water were used.

Pure Drugs: (Working Standards )

Pure Drug sample of Granisetron hydrochloride was procured from Sun Pharma Ltd, Silvassa as gift sample.

Marketed Formulation:

Tablet formulation – Graniset (Sun pharma Ltd) and Graniforce (Mankind)

Stock solution:

An accurately weighed 10mg of Granisetron hydrochloride was transferred to 100ml volumetric flask, dissolved and made upto the volume with phosphate buffer (pH 7.0) to obtain a concentration of 100µg/ml.

PROCEDURE:

Method A: Absorption Maxima Method:

Working standard solutions of Granisetron hydrochloride were prepared by diluting different volumes (2, 4, 6, 8, And 10 µg/ml) of stock solution in a 10ml volumetric flask to give solutions in the concentration range of 2-10 µg/ml and made up the volume with distilled water. Solutions were scanned in the UV range. The λmax by UV spectrum of Granisetron hydrochloride in phosphate buffer, in the range 200-400 nm was found to be 302 nm (Figure 1). Spectral characteristics of Granisetron hydrochloride are given in Table I. The calibration curve was found to be linear in the concentration range of 2-10 µg/ml.

Fig. 1: UV spectrum of Granisetron hydrochloride

Method B– First order derivative spectroscopic method:

The first order derivative spectra at n=1 (method B), showed a sharp peak at 241.5 nm (Figure 2), which was selected for its quantitation. The absorbance difference at n=1 (dA/dλ) is calculated by the inbuilt software of the instrument which was directly proportional to the concentration of the standard solution. The standard drug solution was diluted so as to get the final concentration in the range of 2-10 µg/ml and scanned in the first order derivative spectra. The calibration curve of dA/dλ against concentration of the drug showed linearity.

Fig.2: First order derivative spectrum of Granisetron hydrochloride

Method C– Area under curve (AUC) method:

The AUC method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelength 299 and 305 nm (Figure 3). Area calculation processing item calculates the area bound by the curve and the horizontal axis. The horizontal axis is selected by entering the wavelength range over which the area has to be calculated. The wavelength range is selected on the basis of repeated observations so as to get the linearity between area under curve and concentration. Suitable dilutions of standard stock solution (100 µg/ml) of the drug were prepared and scanned in the spectrum mode from the wavelength range 400-200 nm and the calibration curve was plotted. By using the calibration curve, the concentration of the sample solution can be determined.

Fig. 3: Wavelength range selected for AUC method of Granisetron hydrochloride

Table I: Optical Characteristics and other parameters of Granisetron hydrochloride

Parameters	Method A	Method B	Method C
λmax (nm) / wavelength range	302	241.5	299-305
Beer’s law limits (µg/ml)	2-10	2-10	2-10
Molar absorptivity (L/mol/cm)	1.1038 x 10⁴	1.0051 x 10⁴	5.4951 x 10⁴
Coefficient of Correlation (r²)	0.9994	0.9989	0.9994
Regression Equation, Y=mx + c
a. Slope (m) b. Intercept (c)	0.030 0.0127	0.0253 0.0161	0.1495 0.0639
LOD (µg/ml)	0.187	1.460	0.0507
LOQ (µg/ml)	0.5666	4.426	0.1538

*Average of six determinations, In Y=mx + c, Y is absorbance and x is concentration

Table II : Analysis of Tablet Formulation

Tablet Formulation	Label Claim (mg)	Amount Found (% label claim ± S.D.)	% CV
Brand A	1.0	100.5+ 0.52	0.178
Brand B	1.0	100.3+ 0.49	0.329

*Here Average + standard deviation of six determinations and % CV is coefficient of variance

Table III: Recovery Studies of Granisetron hydrochloride

Method

Tablet

Formulation

Label Claim

(mg)

Amount

Added (%)

Total amount

added(mg)

Amount

recovered(mg)

Recovery

RSD

Brand A

80%

100%

120%

0.8

1.0

1.2

0.817 + 0.32

1.006 + 0.42

1.21 + 0.32

101%

101.1%

0.51

0.34

0.23

Brand B

1.0

80%

100%

120%

0.8

1.0

1.2

0.805 + 0.42

1.206 + 0.32

1.006 + 0.42

100.9%

100.03%

100.04%

0.42

0.31

0.22

Brand A

1.0

80%

100%

120%

0.8

1.0

1.2

0.799 + 0.52

1.004 + 0.31

1.008 + 0.59

99.98 %

100.04%

98.99%

0.32

0.51

0.43

Brand B

1.0

80%

100%

120%

0.8

1.0

1.2

0.779 + 0.52

1.004 + 0.31

1.21 + 0.32

98.98%

100.04%

100.9%

0.34

0.50

0.41

Brand A

1.0

80%

100%

120%

0.8

1.0

1.2

0.811 + 0.32

1.006 + 0.42

1.202 + 0.32

101%

101.1%

0.56

0.61

0.42

Brand B

1.0

80%

100%

120%

0.8

1.0

1.2

0.803 + 0.42

1.006 + 0.42

1.19 + 0.52

100.6%

101.1%

99.99%

0.52

0.49

0.37

*Average value + standard deviation of three determinations

All these three methods were checked by analysing the samples with known concentration. As the result obtained were satisfactory, the method was applied for the pharmaceutical formulations.

Analysis of Tablet Formulation:

Graniset and Graniforce these two commercial brands of Granisetron hydrochloride (1 mg strength) were procured. Each brand contained a label claim of 1 mg of Granisetron hydrochloride per tablet. Twenty such tablets of each brand were weighed and powdered for analysis. The tablet powder equivalent to 10 mg of Granisetron hydrochloride was weighed accurately, dissolved in phosphate buffer and the volume of solution was made upto 100 ml with buffer. Ultra sonicator was used for 40 min for complete dissolution. The solution was diluted upto 100 ml and filtered through Whatman filter paper no. 40 to get 100 µg/ml. Further dilutions were made to obtain six replicates of 10 µg/ml solution and the concentration was calculated by using the calibration curve for these three methods. The results of the analysis of tablet formulations is summarized in Table II.

VALIDATION OF METHODS:

The proposed method is validated in terms of accuracy, linearity, precision (Inter day, Intraday) and reproducibility. The results are presented in Table III.

Recovery studies

Accuracy: The accuracy of the method was proved by carrying out recovery study. For that, known concentration of standard drug was added to a preanalysed tablet sample at three different levels namely 80%, 100% and 120% then average recovery was calculated. As evident from the results recorded in Table III, the accuracy of the methods was found in the range of 98 - 102 % for the drug, which indicates that the proposed methods is accurate for the analysis of drug. The effect of common excipients was tested for possible interference by analysing synthetic mixture of the drug. The percent recovery clearly indicates that the method does not suffer from the interference of excipients.

Linearity: Granisetron hydrochloride exhibited maximum absorbtion at 302 nm and obeyed Beer’s law in the concentration range 2-10 µg/ml. A calibration curve was taking absorbance against concentration in the range of 2-10 µg/ml. It has shown linear relationship for all three methods.

Precision: Repeatability is performed by intra and inter day precision. Intraday precision was determined by analyzing the three different concentrations of drug for three times in the same day. Inter day precision was determined by analyzing the three different concentrations of the drug for three days in a week. Result of intra-day and inter-day precision is expressed in % RSD. Percent RSD for Intraday assay precision was found to be 0.0905, 0.0153 and 0.0436 for Method A, B and C respectively. Interday assay precision was found to be 0.0504, 0.0552 and 0.0460 for Method A, B and C respectively. The precision was determined and found to be lower than 1.0.

RESULTS AND DISCUSSION:

The proposed methods for determination of Granisetron hydrochloride in tablet formulation were found to be simple, accurate, economical and rapid. Granisetron hydrochloride exhibited maximum absorption at 302 nm (Method A); in the first order derivative spectra, sharp peak at 241.5 nm (Method B) and area under curve in range of 299-305 nm (Method C) and obeyed Beer’s Law in the concentration range of 2-10 µg/ml. The proposed method showed linear regression, Y= 0.03x + 0.0127; Y= 0.0253x + 0.0161 and Y=0.1495x + 0.0639 with a correlation coefficient (r²) of 0.9994, 0.9989 and 0.9994 for method A, B and C respectively. The optical characteristics such as molar absorptivity, LOD, LOQ are given in Table I. Relative standard deviation of 0.178 and 0.329 was observed on analysis of six replicate samples of the two brands A and B respectively (Table II). The proposed methods were applied to two marketed formulations. The amount of drug found in formulations is well agreed with label claim. The results obtained are more accurate, precise and validated as compared to existing ones.

CONCLUSION:

The present study comprises an Absorbance maxima method, First order derivative method and Area under curve method to determine Granisetron hydrochloride from pure and it’s tablet formulations. From the study of validation parameters namely accuracy, precision (Intra day and Inter day precision), reproducibility, linearity and range, it was observed that the methods are specific, accurate, precise and reproducible. The proposed method can be applied to routine analysis in quality control laboratories.

ACKNOWLEDGEMENTS:

The authors are very thankful to Sun Pharma Ltd, Silvassa for providing gift sample of Granisetron hydrochloride to carry out this work.

REFERENCES:

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Received on 30.04.2010 Modified on 10.05.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 928-931