INTRODUCTION:

Griseofulvin, chemically is (2S,6'R)- 7-chloro- 2',4,6-trimethoxy- 6'-methyl- 3H,4'H-spiro [1-benzofuran-2,1'-cyclohex[2]ene]- 3,4'-dione^1,2,3,4. This is a less toxic systemic antifungal antibiotic^{12; 13}.The drug binds to tubulin, interfering with microtubule function, thus inhibiting mitosis. Literature survey reveals that there are some analytical method for estimation of griseofulvin such as liquid chromatography–UV–mass spectrometry⁵, PMR⁶, LC–MS/MS⁷, HPLC⁸, Mass spectrometry ESI-IT/MS⁹ spectrophotometric¹⁰, Polarimetric¹⁰ gas-liquid chromatography¹¹. But there is no prompt analytical method for the determination of griseofulvin in formulation. This paper describes simple, rapid, accurate, reproducible and economical method for the estimation of griseofulvin.

MATERIALS AND METHODS:

Instrumentation:

A double-beam Shimadzu UV–Visible spectrophotometer, model UV-1700 with 1-cm quartz cells attached with printer of ESPON LQ 1150 II.

Materials and reagents:

Griseofulvin reference standard was kindly provided by Glaxo SmithKline Pharmaceuticals Ltd. Mumbai as a gift sample.

And solvents acetone, ethanol, HCl was used of analytical grade from loba.chem. The pharmaceutical preparations of griseofulvin is Dermonorm tab® from Glaxo SmithKline Pharmaceuticals Ltd.

Standard solutions and calibration:

Stock standard solutions of Griseofulvin were prepared by accurately weigh and dissolving 10 mg in 100 ml solvent to achieve concentration of 100µg/ml.

The standard solutions were prepared by dilution of the stock standard solutions with solvent to obtain the concentration range of 0.5µg/ml to 3.5µg/ml respectively and spectra curve was taken at 263.5nm (fig-1)

Analysis of the tablet formulation:¹⁴

Twenty tablets were weighed and ﬁnely powdered. A portion of the powder equivalent to about 10 mg of griseofulvin was weighed accurately, dissolved and diluted to 100 ml with solvent. The sample solution was ﬁltered. Further dilution was carried out with solvent. The general procedures for described under calibration were followed and the concentrations of griseofulvin were calculated at 263.5nm. (Fig-2, 3)

VALIDATION:^{15, 16}

The methods were validated with respect to accuracy linearity, limit of detection (LOD) and limit of quantitation (LOQ).

Figure 1: Spectra of Griseofulvin at 263.5nm

Figure 2: Calibration curve of Griseofulvin at 263.5nm

Figure 3: Calibration curve of Griseofulvin for AUC.

Accuracy (recovery test):

To ascertain the accuracy of proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percent recovery for griseofulvin, by all the methods, was found in the range of 100.45%-102.11% and for AUC method 99.67%-99.86 %( Table no.III)

Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of griseofulvin. Beer-Lambert’s concentration range was found to be 0.5-3.5 µg/ml.

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

The LOD and LOQ of griseofulvin were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines¹⁵.The LOD and LOQ was found to be as in table no. I

Table No.I: Optical characteristics and Other Parameters

Sr. No.	Parameters	Method
Sr. No.	Parameters	Zero order	AUC
1	λmax (nm) / wavelength range (nm)	263.5	258.5-268.5
2	Beer’s-Lambert’s range (µg/ml)	0.5-3.5	0.5-3.5
3	Slope	0.285	2.705
4	Coefficient of Correlation	0.999798	0.999833
5	Y - Intercept	0.004	0.048
6	Sandell’s Sensitivity (µg/cm2/0.001)	0.003508	0.000369
7	Molar absorptivity (lit/mol/cm)	100564. 677683	954900. 593585
8	LOD (µg/ml)	0.0463	0.05853
9	LOQ (µg/ml)	0.1403	0.17738

RESULT:

The methods discussed in the present work provide a convenient and accurate way for analysis of griseofulvin in its pharmaceutical dosage form. Absorbance maxima of griseofulvin at 263.5 nm (zero order) and in the AUC method 285.5-268.5 was selected for the analysis. Linearity for detector response was observed in the concentration range of 0.5-3.5 μg/ml for all under study method. Standard deviation for six determinations of tablet sample, by all study method, was found to be less than ± 2.0 indicating the precision of the methods (Table no.II)

Table No.II: Result of Analysis of griseofulvin in marketed tablet formulation

Method

Lable Claim

(mg)

Amount Found*

(mg)

% Estimated*

S.D.*

(±)

R.S.D.*

Zero order

250

253.5

101.4

0.0055785

0.0055015

AUC

250

245.45

98.18

0.08827

0.032918

Where, * indicates mean of six determinations.

Table III:- Recovery study data.

Method	Level of % Recovery	% Recovery	(±) S.D.*	RSD*
Zero order	80	101.11	0.967	0.964
	100	102.5	0.80	0.796
	120	100.45	0.865	0.860
AUC	80	99.86	0..0100	0.00205
	100	99.67	0.0318	0.0058
	120	99.71	0.07295	0.0122

Where, * indicates mean of six determinations.

CONCLUSIONS:

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

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal Dr. S. B. Bhise, Government College of Pharmacy, Karad, Dist. Satara, Maharashtra for providing necessary facilities and Glaxo SmithKline Pharmaceuticals Ltd. MUMBAI (M.S.) India for providing gift samples of griseofulvin.

REFERENCE:

1. Indian Pharmacopoeia 2007, edition 5^th, published by The Controller of Publications, New Delhi, Page no. 1174-1175.

2. The united states pharmacopoeia 24, The national formulary 19, Asian edition 2007, published by united state pharmacopeial convention Inc. Page no. 788

3. http://en.wikipedia.org/wiki/Griseofulvin.

4. Eve Finkelstein, Boaz Amichai*, Marcel0 H. Grunwald, Griseofulvin and its uses, International Journal of Antimicrobial Agents 6 (1996) 189-194.

5. Kristian Fog Nielsen, Jørn Smedsgaard., Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography–UV–mass spectrometry methodology., Journal of Chromatography A, Volume 1002, Issues 1-2, 20 June 2003, Pages 111-136.

6. Elsayed A. Aboutabl, M. M. A. Hassan., PMR assay of natural products in pharmaceuticals-III Assay of Griseofulvin., Talanta, Volume 27, Issue 8, August 1980, Pages 679-681.

7. Hiren N. Mistri , Arvind G. Jangid , Mallika Sanyal , Pranav Shrivastav., Electrospray ionization LC–MS/MS validated method to quantify griseofulvin in human plasma and its application to bioequivalence study, Journal of Chromatography B, 850 (2007) 318–326.

8. Yoshitsugu Fujioka, Yukiko Metsugi, Ken-ichi Ogawara, Kazutaka Higaki, Toshikiro Kimura., Evaluation of in vivo dissolution behavior and GI transit of griseofulvin, a BCS class II drug, International Journal of Pharmaceutics Volume 352, Issues 1-2, 20 March 2008, Pages 36-43

9. K.E. Petita, F. Mondeguerb, M.F. Roquebertc, J.F. Biarda, Y.F. Pouchusa., Detection of griseofulvin in a marine strain of Penicillium waksmanii by ion trap mass spectrometry, Journal of Microbiological Methods 58 (2004) 59– 65.

10. Bershtein Ya., I., Bob, T.G., Raigorodskaya Ya., V., ,”Metrological estimation of spectrophotometric and polarimetric methods for determination of griseofulvin levels in finished products”, Antibiotiki 26 (3), pp. 93-96.

11. Iguchi, S., Yamamoto, M., Goromaru, T., Quantitative determination of griseofulvin by gas-liquid chromatography Journal of Chromatography A (1966), 24 (C), pp. 182-185.

12. V. Venkata Dasu, R. V. Muralidhar, T. Panda. Analytical techniques for Griseofulvin, Bioprocess and Biosystems Engineering, Volume 22, Number 3 / March, 2000, 201-204.

13. Shishu , N. Aggarwal., Preparation of hydrogels of griseofulvin for dermal application, International Journal of Pharmaceutics 326 (2006) 20–24.

14. Beckett A.H., Stenlake J.B., Practical pharmaceutical chemistry, 4^th edition ,part two, Page No.276-292.

15. Validation of Analytical Procedures, Methodology, ICH Harmonised tripartite guidelines; 1996:1.

16. Khopkar SM. Basic concepts of analytical chemistry. New age international publisher. 2008; 3rd edition: 277-278.

Received on 17.10.2009 Modified on 09.12.2009

Asian J. Research Chem. 3(2): April- June 2010; Page 404-406