INTRODUCTION:

Gliclazide (1-(3-azabicyclo [3.3.0] oct- 3- yl) - 3- p- tolylsulfonylurea (Figure 1) urea is an oral hypoglycemic agent used in the treatment of type-II diabetes mellitus. It belongs to the sulfonylurea class which act by stimulating β cells of the pancreas to release insulin. It reduces blood glucose levels by correcting both defective insulin secretion and peripheral insulin resistance, increasing the sensitivity of ß- cells to glucose, decreasing hepatic glucose production, and increasing glucose clearance. It also has anti-platelet adhesive activity and reduces levels of free radicals, thereby preventing vascular complications. It also has been reported to reduce plasma cholesterol and triglyceride levels after repeated administration^1-2.

Figure 1: Chemical structure of gliclazide.

From the literature survey, it was found that gliclazide was estimated by analytical methods such as Spectrophotometry^3-7, HPLC^8-12and HPTLC¹³ in single form or combination with other drugs. The aim of the present study is to develop a simple, precise and accurate spectrophotometric method for the estimation of gliclazide in pharmaceutical dosage form as per ICH guidelines.

MATERIALS AND METHODS:

Instruments and Reagents:

Agilant Cary UV – 60 spectrophotometer with 1cm matched quartz cells were used for the estimation. Ultra sonicator (ANM-USC 100) and electronic balance (Infra 201 LEC) were used for the experiment. Gliclazide was obtained as a gift sample from Dr.Reddy’s Laboratory, Hyderabad. Two brands of gliclazide tablets were procured from local manufactured by Aristo Pharmaceutical (Glycigon) and Cadila Pharmaceutical (Glyloc). All the reagents were of analytical grade. Glass double distilled water was used throughout the experiment.

Selection of solvent:

The solubility of Gliclazide is determined in variety of solvents as per pharmacopeia standard. Solubility test was carried out in different solvents like distilled water, methanol, ethanol, 0.1N sodium hydroxide. From the solubility studies it was found that gliclazide is soluble in distilled water, methanol and 0.1N sodium hydroxide. In this study methanol and distilled water (50:50) were selected as solvent.

Preparation of standard stock solution:

An accurately weighed quantity of 50mg was taken in a 50ml volumetric flask. 10ml of methanol was added to it. Then 20ml of solvent was added and sonicated for 15min. Then the volume was adjusted with solvent to get the concentration of 1000µg/ml. From this 10ml was taken into a 100ml volumetric flask and 80ml of solvent was added to it. This was sonicated for 10min and volume was diluted upto the mark with solvent to get a concentration of 100µg/ml.

Determination of λ_max:

The standard solution of gliclazide (10µg/ml) was scanned in UV region (200-400nm) and the spectrum was recorded. Solvent was used as blank. It was seen that at 224.05nm maximum absorbance was found, shown in figure 2. Therefore that 224.05nm was selected for this study.

Figure 2: Absorbance spectra of gliclazide

Method Validation:¹⁴

The objective of method validation is to demonstrate that the method is suitable for its intended purpose. The method was validated for linearity, precision, accuracy, robustness, ruggedness, LOD, LOQ, and specificity as per ICH guidelines.

Linearity:

From the standard stock solution, the various dilutions in the concentration of 2, 4, 6, 8, 10, 12, 14. 16,18, 20 were prepared. The solutions were scanned at 224.05 nm and the absorbance was recorded, given in table 1. From this calibration curve was obtained by plotting absorbance versus concentration of gliclazide and the linearity graph was represented in figure 3. The correlation coefficient was found to be 0.997.

Figure 3: Linearitygraph of gliclazide

Table 1: Linearity parameter for gliclazide

Concentration	Absorbance
2	0.0572
4	0.1534
6	0.2513
8	0.3169
10	0.4356
12	0.5011
14	0.6162
16	0.6912
18	0.8253
20	0.9126

Precision:

Repeatability of method was checked by scanning 10µg/ml of solution for 6 times. Intra-day precision was determined by checking the absorbance of (10µg/ml) on the same day. Inter-day precision was determined by checking the absorbance of (10µg/ml) on three different days. The %RSD was found to be 0.74% for intra-day and 0.95% inter-day as shown in table 2.

Table 2: Precision parameter for gliclazide

Scans	Intra-day	Inter-day
1	0.4352	0.4322
2	0.4390	0.4393
3	0.4362	0.4358
4	0.4298	0.4286
5	0.4349	0.4295
6	0.4321	0.4305
Mean	0.4345	0.4327
SD	0.003214	0.00413
% RSD	0.74	0.95

Accuracy:

Accuracy study was conducted by spiking at three different concentration levels (80%, 100%, 120%). At each level samples were scanned and from the absorbance recovery percentage was determined and presented in table 3.

Table 3: Accuracy parameter for gliclazide

Level of recovery	Amount added (mg)	Amount found (mg)	% Recovery	Mean recovery
80%	8	7.94	99.25	99.29%
100%	10	9.98	99.8
120%	12	11.86	98.83

Robustness:

To determine robustness of the method two parameters (wavelength and diluent composition) were made slightly different from the selected wavelength and diluent composition. No significant difference was found in the absorbance and hence the proposed method was considered as robust which is shown in table 4.

Table 4: Robustness parameter for gliclazide

Method parameters

Adjusted to

Average absorbance

S.D

%RSD

Wave length (224.05nm)

225

0.4312

0.00721

1.67

Diluent composition

(50:50)

45:55

0.4378

0.00654

1.49

Ruggedness

The ruggedness of the developed method was checked by analysing the samples by different analysts at different days at similar operational condition. The statistical analysis showed no significant differences were observed between results obtained employing different analysts, given in table 5.

Table 5: Ruggedness parameter for gliclazide

Analyst	Days	Average absorbance	S.D	% RSD
1	1	0.4286	0.00782	1.82
2	2	0.4299	0.00764	1.77
3	3	0.4310	0.00824	1.91

Limit of detection and Limit of quantification:

Limit of detection is the lowest amount of an analyte in a sample that can be detected, but not necessarily quantified, under the stated experimental conditions. Limit of quantification is the lowest amount of analyte in a sample that can be quantified under stated experimental conditions. The LOD and LOQ for gliclazide were found to be 0.15µg/ml and 0.50µg/ml.

Specificity:

A solution containing mixture of tablet excipients were prepared using the sample preparation procedure to evaluate the possible interference of the excipients. From the absorbance result no interference was observed from the excipients present in the formulation, indicated that the method is specific.

Assay of gliclazide tablets:

Two different brands of gliclazide were analysed using the validated method. For the analysis six replicates of each brand were assayed. 20 tablets of gliclazide were weighed and finely powdered. An accurately weighed quantity of powder equivalent to 50mg of gliclazide was taken in a 50ml volumetric flask. 10ml of methanol was added to it followed by 20ml of solvent. The solution was sonicated for 15 min and then filtered through Whatmann filter paper (No.41) and volume adjusted with the solvent. From this further dilution was made to get final concentration of 10µg/ml. The results were presented in table 6.

Table 6: Assay for gliclazide tablets

Sample

Label claim(mg)

Amount found(mg)

(± S.D)

% Amount found

Glycigon (Aristo)

9.83±0.35

98.3

Glyloc(Cadila)

9.94±0.25

99.4

RESULTS AND DISCUSSION:

The method was validated and developed as per ICH guidelines. The method was validated in terms of linearity, precision, accuracy, robustness, ruggedness, LOD, LOQ and specificity. Beer’s law is obeyed over the concentration range of 2-20 µg/ml, using regression analysis the linear equation Y= 0.047x-0.042 with correlation coefficient of r²=0.0997. The precision results shows % RSD of less than 2 at each level, which indicate clearly that the method is precise enough for the analysis of gliclazide. The accuracy of the method was checked by recovery studies. The high recovery values indicated the accuracy of the developed method. The robustness and ruggedness studies reveals that the method is enough robust and rugged. The LOD and LOQ values indicate sensitivity of the method. There was no interference observed from the excipients present in the formulation, indicated that the method is specific. Determination of gliclazide in tablet formulation of two brands showed content of gliclazide was very close to the labelled amount. The %RSD values in all the parameters were within the acceptable limit. The optical characteristics of the method are represented in table 7.

Table 7: Optical characteristics of the proposed developed method.

Parameter	Value
λ_max(nm)	224.05
Beer’s range(µg/ml)	2-20
Correlation coefficient(r²)	0.0997
Regression equation (Y= mX+C)	Y= 0.047x-0.042
Intercept (C)	-0.042
Slope (m)	0.047
LOD (µg/ml) &LOQ (µg/ml)	0.15 &0.50
Precision (%RSD)	0.95

CONCLUSION:

A validated UV Spectrophotometric method has been developed for the estimation of gliclazide in bulk as well as pharmaceutical dosage form. The developed method was found to be simple, accurate, precised, specific, reproducible and linear over the concentration range studied. The proposed method can be used for routine analysis of gliclazide in bulk as well as pharmaceutical formulations.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Reddy’s Laboratory Hyderabad, India for providing the gift sample of gliclazide for research work. We are thankful to the principal and management, Maharajah’s College of Pharmacy, Vizianagaram for providing necessary facilities to carry out this research work.

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Received on 15.05.2016 Accepted on 30.06.2016

Asian J. Pharm. Ana. 2016; 6(3): 143-146.

DOI: 10.5958/2231-5675.2016.00022.3