Godse A. M.*, Pishawikar S.A, Purake R. R, Killedar S.G, Puri U. A.
Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Dist- Kolhapur, Maharashtra, 416013, India
*Corresponding Author E-mail: godse43999@gmail.com
ABSTRACT:
A simple, accurate, sensitive and reliable method have been developed for the determination of Glibanclamide in bulk and Pharmaceutical formulation using extract of Beta vulgaris root. Phytochemical investigation shows presence of betanin group of compounds which have polyphenolic nature similar to synthetic dyes like methyl orange, bromocresol green etc. which are commonly used as colouring agent to form a colour complex with amide group containing compounds in developing visible spectroscopic method of analysis. In present work a successful attempt has been done to develop a colorimetric method for estimation of glibanclamide by forming a stable colour complex using extract of Beta vulgaris root on the basis of amide linkage present in glibanclamide. The absorbance of the pale yellow colored complex was measured spectrophotometrically at 533 nm against reagent blank. From Calibration graphs Beer’s law is followed in range of 2 to 20µg/ml with correlation coefficient 0.999587, while the LOD and LOQ was 0.27, 0.79 respectively.
KEYWORDS: Glibenclamide, spectrophotometry, Beta vulgaris root, Anthocyanine, validation
Glibenclamide is 1-[4-[2-(chloro-2-methoxy benzamido) ethyl]-benzenesulphonyl]-3cyclohexylurea,5-chloro-N- [2-[4[[[(cyclohexyl(amino)carbonyl]-amino]sulphonyl] phenyl] ethyl]-2-methoxy benzamide or 1-[[p-[-2-(5-chloro-oanisamido) ethyl]phenyl]-sulphonyl-3-cyclohexylurea,a sulphonyl urea derivative is a second generation oral hypoglycemic agent which is more potent than those of first group. The drug works by inhibiting ATP-sensitive potassium channels [4] in pancreatic beta cells. This inhibition causes cell membrane depolarization opening voltage-dependent calcium channel. This results in an increase in intracellular calcium in the beta cell and subsequent stimulation of insulin release. Also increased glucose uptake in the liver and utilization in the skeletal muscles. It is drug of choice for initiating treatment in noninsulin-dependent diabetes when diet and weight control fails.
Several assay techniques have been described for quantitative determination of glibenclamide in biological fluids; these include procedures based on high performance liquid chromatography (HPLC) fluorometry, radioimmunoassay and gas chromatography.
A few reports deal with the analysis of the drug in these dosage forms; such procedures include micellar electrokinetic capillary chromatography, RPHPLC, fluorometry, TLC-UV spectrophotometry, derivative spectrophotometry, UV spectrophotometry and colorimetry.
Betalains are natural water-soluble nitrogen-containing poly hydroxyl pigments, which comprise the red-violet beta cyanins and the yellow-orange betaxanthins. The major commercially exploited betalain crop is red beetroot (Beta vulgaris), whose main pigment is betanidin-5-O-β-glycosidase, or betanin, which is the most common betacyanin (Strack et al., 2003; Stintzing and Carle, 2004). In colorimetric method development use of synthetic poly hydroxy dyes is done as coloring agent hence it was decided to develop colorimetric method using extract.
The present work is aims to establish a simple, precise and reproducible colorimetric method for the quantitative estimation of Glibenclamide in bulk and tablet dosage forms by forming a colour complex with extract of Beta vulgaris roots. The method is validated as per ICH guidelines.
EXPERIMENTAL:
EXTRACTION OF BETANIN
The hydro alcoholic extracts was obtained by process of maceration for which cut pieces of beet root (0.5 kg) were soaked in alcohol-water at room temperature (25°C). Extract was subjected to drying process by evaporation on thermostatic water bath.
Instrument
A Jasco spectrophotometer (model: UV-630) having spectral bandwidth 2nm and wavelength accuracy ±1nm and Matched quartz cells having 10mm optical path length, was used to measure the λmax of plane extract and that of colour complex formed between glibanclamide and extracts. Same instrument was used in fixed wavelength mode for plotting calibration curve.
All chemicals of analytical grade and doubled distilled water was used for the preparation of solutions. The drug sample was procured from Franco-India pharmaceutical Ltd. Signal component tablet formulation of Glibenclamide (1mg) (formulation A Eurepa, manufactured by Torrent Pharmaceutical Ltd, Ahmadabad) were purchased from local market.
PREPARATION OF STANDARD STOCK SOLUTION:
The Standard stock solution of Glibenclamide was prepared by dissolving 20 mg of Glibanclamide in few ml of methanol by sonication and the final volume was made up to 100 ml with methanol to get stock solution having concentration 200 μg/ml.
REAGENT PREPARATION:
Solutions of extract were prepared in doubled distilled water.
PLOTTING CALIBRATION CURVE:
For plotting calibration curve first λmax of the colour-complex was estimated.. At this fixed wave length optimization of % and quantity of extract to be used was done. Optimized % and quantity was found to be 1% solution and 1 ml.
Appropriate aliquots of standard drug solution were taken into a series of 10 ml volumetric flasks. To each volumetric flask freshly prepared solution of 1% extract of Beta vulgaris roots was added in 1 ml quantity, for stability purpose 0.2ml phosphate buffer of pH 7.6 was added and kept aside for 10 minutes for completion of formation of color complex. The final volume was made up to 10 ml with water to get final concentrations of 2 to 20 μg /ml. (fig no.1) The absorbance of the pale yellow coloured complex was measured at 605 nm against reagent blank. Absorbances are given in (Table no 1). Optical characteristics are shown in (Table no2).
ANALYSIS OF FORMULATION:
Twenty tablets were accurately weighed and average weight was calculated. The tablets were ground to a fine powder. An accurately weighed tablet powder equivalent to 5 mg of Glibenclamide was dissolved in 5 ml of methanol by sonication. The solution was then filtered through whatman filter paper No.41. Appropriate aliquots within the Beer's law limit were analyzed by the proposed method using the procedure described earlier. The concentration of Glibanclamide present in the sample solution was calculated by using the formula:
Abs = A + B* C
Where, A = 0.000432, B=0.002046 and C = concentration of Glibenclamide Result is summarised in table no.2
Table No. 1: Absorbance values for calibration curve of Glibenclamide at 605 nm
|
Sr. No. |
Concentration (µg/ml) |
Absorbance |
|
1 |
2 |
0.1486 |
|
2 |
4 |
0.2022 |
|
3 |
6 |
0.2537 |
|
4 |
8 |
0.3111 |
|
5 |
10 |
0.3712 |
|
6 |
12 |
0.4237 |
|
7 |
14 |
0.4780 |
|
8 |
16 |
0.5345 |
|
9 |
18 |
0.5963 |
|
10 |
20 |
0.6635 |
Table No. 2: Optical characteristics
|
Parameters |
Values for glibenclamide reagent complex |
|
Beer’s law limit (μg/ml) |
2- 20 |
|
Correlation coefficient |
0.999587 |
|
Regression equation (Y*) |
|
|
Slope (B) |
0.002046 |
|
Intercept (A) |
0.086593 |
|
LOD(µg/ml) |
0.2681 |
|
LOQ(µg/ml) |
0.7962 |
Y= A + B*C, where C is the concentration in µg/ml and Y is absorbance unit.
VALIDATION OF PROPOSED METHOD:
LINEARITY STUDY
A calibration curve was constructed at optimum experimental conditions using absorbance values at 605 nm in concentration range of 2 to 20 µg/ml. High value of the coefficient of correlation (r=0.999587) indicates a good linearity and adherence of the method to Beer’s law.
RECOVERY STUDIES
To study validity and reproducibility of the proposed method, recovery studies was carried out by adding known amount of drug to pre-analysed sample at four different levels and the percentage recoveries were calculated ( Table 3).
REPETABILITY
Repeatability is performed by intra-day and inter-day precision. Intraday precision was determined by analyzing absorbance of three different concentration of drug at three times in same day. Inter day precision was determined by analyzing absorbance of same solutions on day two. (Table no 3)
ROBUSTNESS
Robustness is performed by Using MeOH: Water (90:10). Average of nine determinations used for robustness. (Table no 4)
Fig no 1 Possible mechanism action of colorimetric reaction
Fig No. 2 Overlain spectra of glibenclamide – reagent complex
FTIR of Extracted Pigment :
FTIR for Complex formed Between extract and glibenclamide
Table no 3: Result of recovery study and repeatability
|
Analyte |
Label claim (mg/tab) |
% Recovery estimated* (Mean ± S. D.) |
R.S.D. |
Repeatability % Label claim estimated*(Mean ± R.S.D.) |
|
Glibanclamide |
5 |
99.54 ± 0.89 |
0.88 |
99.65 ± 1.06 |
* Average of nine determinations; R.S.D., relative standard deviation
Table No. 4: Results of robustness (Analysis Using MeOH: Water (90:10)
|
Label claim (mg/tab) |
% Label claim estimated * (Mean ± %SD ) |
R.S.D. |
|
5 |
99.25±0.65 |
0.66 |
*Average of nine determinations; R.S.D., Relative Standard Deviation
DETERMINATION OF LOQ AND LOD
Limit of detection and limit of quantification for pure drug of six determinations was determined by analyzing the three different concentration of drug for two times. (Table no 2)
RESULT AND DISCUSSION:
A colorimetric method for estimation of Glibenclamide by forming a stable complex with extract of Beta vulgaris root on the basis of amide linkage present in Glibenclamide was successfully done. The absorbance of the pale yellow colored complex was measured spectrophotometrically at 605 nm against reagent blank. From Calibration graphs Beer’s law is followed in range of 2to 20µg/ml with correlation coefficient 0. 0.999587 with robustness.
CONCLUSION:
Simple, specific and rapid spectrophotometric procedure described in this paper can be an alternative to the complex and time consuming method for assay of Glibanclamide in commercial samples. The method doesn’t require heating for development of colour and give instant coloration, thus giving an additional advantage compared to other methods.
ACKNOWLEDGEMENT:
Authors are thankful to Torrent pharmaceutical Ltd. India, for providing the free gift samples of Glibenclamide authors are also thankful to Principal, Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing necessary facilities for this work.
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Received on 16.03.2012 Modified on 02.04.2012
Accepted on 08.04.2012 © AJRC All right reserved
Asian J. Research Chem. 5(5): May 2012; Page 591-594