INTRODUCTION:

Chemically Glibenclamide is 5-Chloro-N-[2-[4-[[[(cyclohexylamino)carbonyl]-amino]sulfonyl]phenyl]-ethyl]-2-methoxybenzamide is oral hypoglycemic drug sulfonyl urea-second generation)act by inhibiting ATP-sensitive potassium channels in pancreatic beta cells. This inhibition causes cell membrane depolarization, which causes voltage-dependent calcium channels to open, which causes an increase in intracellular calcium in the beta cell, which stimulates insulin release.

The literature survey reveals that Glibenclamide was analyzed by RPHPLC¹,spectrophotometer², LCMS³ was used for determination of glibenclamide.

Analysis is an important component in the formulation development of any drug molecule. It becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug sample. Our main concern is development and validation⁴ of UV spectrometric method.

MATERIALS AND METHOD:

Instruments and materials:

Instrument used were pharmaspec UV 1700 Schimadzu and AX200 analytical balance. Glibenclamide pure drug was obtained from MICRO LABS LTD as gift sample with 99.9%w/w assay value and was used without further purification. All chemical and reagents used were of analytical grade. Glyburide tablets were purchased from market.

Figure No.1- Chemical stracture of Glibenclamide

Molecular Formula: C₂₃H₂₈ClN₃O₅S

Molecular Weight: 494.00

Preparation of standard stock solution:

Standard drug solution of Glibenclamide was prepared by dissolving 10mg in 10ml chloroform (1mg/ml) and transfer 1ml of this solution to 10ml of volumetric flask, made upto mark with same solvent. For obtaining clear solution, solution was filtrated.

Preparation of calibration curve:

Aliquots of 0.5 to 3ml of portion of stock solution were transferred to series of 10ml volumetric flask and made up to mark with solvent (chloroform). Solutions were scanned in the range of 200 – 400nm against blank. The absorption maximum was found to be at 242nm against blank (Figure No: 2).The calibration curve was plotted. The optical characteristics are summarized in (Table No: 2)

Table No.1 - Calibration Curve

Sr. No:	Concentration	Absorbance
1.	0	0
2.	5	0.066
3.	10	0.2.7
4.	15	0.302
5.	20	0.432
6.	25	0.535
7.	30	0.598

Table.2 - Validation parameters

S. No.	Parameter	Result
1	Absorption maxima(nm)	242
2	Linearity Range (μg/ml)	5-30
3	Standard Regression Equation	y = 0.0211x - 0.0114
4	Correlation Coefficient (r2 )	0.9934
5	Molar absorptivity	17685.99
6	Accuracy (% recovery )	99.89
7	Precision	98.87% (Intra-day precision) and 99.60%(Inter-day precision)
8	Specificity	A 20μ g/ml solution of candidate drug in methanol at UV detection λ of 242 nm will show an absorbance value of 0.423
9	LOD (μg/ml)	0.578
10	LOQ (μg/ml)	1.926

Preparation of sample solution:

The proposed method was applied to analyze commercially available Glibenclamide tablet. Ten tablets were weighed and powdered. The amount of tablet powder equivalent to 25 mg of Glibenclamide was weighed accurately and transferred 100ml volumetric flask. Then 20ml chloroform was added and kept for 15 min frequent shaking and volume was made upto mark with chloroform. The solution was then filtered through whatmann filter paper #41. This filtrate was diluted suitably with solvent. To get the solution 20µg/ml concentration. The absorbance was measured against blank.

RESULT AND DISCUSSION:

Precision:

Assay of method precision (intra-day precision) was evaluated by carrying out three independent assays of test samples of Glibenclamide. The intermediate precision (inter-day precision) of the method was also evaluated using two different analysts, systems and different days in the same laboratory. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.37, 99.87 and 0.33, 99.60 respectively Table no.4

Accuracy (Recovery test):

Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts to tablet. The recovery was performed at three levels, 80,100and 120% of Glibenclamide standard concentration. The recovery samples were prepared in afore mentioned procedure. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The recovery values for Glibenclamide ranged from 99.97. Table no.3.

Fig. No.2 - Determination of λ max of Glibenclamide by UV scanning.

Linearity:

The linearity of the response of the drug was verified at 5 to 40μg/ml concentrations, but linearity was found to be between 5-30μg/ml concentrations. The calibration graphs were obtained by plotting the absorbance versus the concentration data and were treated by linear regression analysis Table no.2. The equation of the calibration curve for Glibenclamide obtained y = 0.0211x - 0.0114, the calibration curve was found to be linear in the aforementioned concentrations. The correlation coefficient (r2) of determination was 0.9934.

Limit of detection (LOD) and limit of quantification (LOQ):

The LOD and LOQ of Glibenclamide were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines .The LOD and LOQ was found to be as in Table no.2.

Determination of Active Ingredients in Tablets:

The validated method was applied to the determination of Glibenclamide in Tablets. Ten tablets were assayed and the results are shown in Table no. 3 indicating that the amount of drug in tablet samples met with requirements (99–101% of the label claim).

CONCLUSION:

The developed method was found to be simple, sensitive, accurate, precise, reproducible, and can be used for routine quality control analysis of glibenclamide in bulk and pharmaceutical formulation.

Figure No.3 - Determination of linearity of Glibenclamide

ACKNOWLEDGEMENT:

We are thankful to MICRO LAB Ltd. for providing the gift sample of Glibenclamide. We would also like to thank Mr. Ishari Ganesh, Chancellor, VEL’S University, Chennai 117 for providing all the facilities to complete our work successfully. I thank to VEL’S University authorities for providing excellent laboratory and library facilities.

Table No. 4 - Determination of Precision

Sample number	Assay of Glibenclamide as % of labeled amount
	Analyst-I (Intra-day precision)	Analyst-II (Inter-day precision)
1	99.42	99.70
2	99.63	99.23
3	99.58	99.57
4	99.10	99.88
5	100.12	99.98
6	99.20	99.25
Mean	99.87	99.60
RSD	0.37	0.33

REFERENCES:

1. SD Rajendran, BK Philip, R Gopinath, B Suresh J. S. S. College of Pharmacy, Ootacamund - 643 001, India RPHPLC method for the estimation of glibenclamide in human serum Year : 2007 | Volume : 69 | Issue : 6 | Page : 796-799.

2. Sankar, D. G. Kumar, J. M. R. Latha, P. V. M. Spectrophotometric Estimation of Glibenclamide, ASIAN JOURNAL OF CHEMISTRY, 2005, VOL 17; NUMB 2, pages 1334-1336.

3. Hans H. Maurer, Carsten Kratzsch, Thomas Kraemer, Frank T. Peters and Armin A. Weber validated quantification of oral ant diabetics of the sulfonylurea-type in plasma by atmospheric pressure chemical ionization liquid chromatography–mass spectrometry (LCMS).

4. Validation of Analytical Procedure: Methodology (Q2B) Harmonized tripartite Guidelines.

5. Mee-Kyung Kima., Su-Eon Jina., Jeong Sook Parkb

6. J.Pharmaceut.Biomed.Anal. 2005, 37,603–609.

7. Jabera A.M.Y., Sherifeb H.A. Al., Omarib, M.M., J.Pharmaceut.Biomed.Anal.. 2004, 36,341–350.

Received on 23.09.2009 Modified on 09.11.2009

Asian J. Research Chem. 3(2): April- June 2010; Page 316-318

Ingredient	Tablet amount(µg/ml)	Level of Addition (%)	Amount added (mg)	Drug found (mg/ml)	% Recovery	Average % recovery
Glibenclamiode*	20	80	17.4	4.97	99.4	99.49
	20	100	20	4.98	99.6
	20	120	22.6	4.96	99.2