INTRODUCTION:

Tioconazole is an imidazole antifungal used to treat fungal and yeast infections. Topical formulations are used for ringworm, jock itch, athlete's foot, and tinea versicolor or "sun fungus". Tioconazole interacts with 14-alpha demethylase, a cytochrome P-450 enzyme that converts lanosterol to ergosterol, an essential component of the yeast membrane. In this way, tioconazole inhibits ergosterol synthesis, resulting in increased cellular permeability. (1,2)

Tioconazole is a antifungal medicated of the imidazole class used to treat infections caused by a fungus or yeast. Tioconazole ointments serve to treat women's vaginal infections. They are available in one day doses, as opposed to the 7-day treatments more common in use in the past.

Tioconazole topical (skin) preparations are also available for ringworm, jock itch, athlete's foot, and tinea versicolor or "sun fungus".

It was patented in 1975 and approved for medical use in 1982. The literature reveal that various analytical technique viz, High performance liquid chromatography (HPLC), Ultra-visible spectroscopy (UV), Ultra performance liquid chromatography (UPLC).Our aim was to develop proper method which estimates both the analytes in shorter time and to develop low cost method.

Gel is a semi-solid soft material that can have properties ranging from soft and weak to hard and tough. Gel it is a thick clear and liquid substance especially one used on the hair or body.(7,9)

Fig No 1: Tioconazole

MATERIALS AND METHODS:

Apparatus and Equipment:

A Shimadzu UV-visible spectrophotometer UV-1800 was used for all absorbance measurement with 1 CM paired quartz cell. High performance liquid chromatography (HPLC) HPLC-3000 Series.

Reagents and chemical:

Pharmaceutical grade tioconazole was kindly supplied as a gift sample by USV pharmaceutical Pvt. Ltd Mumbai. The procured drug standards were standardized by measurement of physical properties like melting point, infrared spectrum UV absorption spectrum and comparing with the reported literature.

Preparation of standard stock solution:

An accurately weighed quantity 10 mg of the Tioconazole were transferred in three different 10ml volumetric flasks dissolved with sufficient quantity of diluent (mobile phase) and volume was made up to the mark with diluent. This gave 1000μg/ml standard stock solution for Tioconazole, respectively and scanned in range of 200-400nm. (6)

Preparation of sample solution and formulation analysis:

10mg tioconazole drug weighed and dissolved in 10 ml methanol and phosphate buffer (95:05) and sonicated for 15 min in 100 ml volumetric flask; volume was make up to the mark.

Fig. No. 2 Linearity curve of tioconazole

Selection of wavelength:

By appropriate dilution of standard stock solution. Solution containing 10µg/ml, these solution were scanned in range 200-400nm separately. Tioconazole showed lambda max at 220nm in methanol and phosphate buffer.

Theoretical aspect:

Method validation:

The method was validated with respect to linearity, precision, accuracy, specificity, and robustness and ruggedness, limit of detection, and limit of quantification accordingly to ICH guidelines.

Linearity:

The linearity was determined by analyzing in depended level of calibration range 10-50µg/ml for tioconazole. Absorbance of each solution was recovered against methanol at 220nm. The calibration curve absorbance vs. concentrations was plotted and correlation coefficient regression line equation was obtained for determination of tioconazole.

Precision:

Method repeatability was determined by three times repetition of assay procedure. For intra-day precision method was repeated 3 times in a day and the average % RSD was determined. Similarly the method was repeated on 3 different day’s for inter-day precision and average % RSD was determined. Precision of analyst was determined by repeating study by another analyst working in the laboratory.

Accuracy:

The accuracy studies were carried out at different concentration by spiking a known concentration of standard drug to the placebo at three levels (80%, 100%, 10%) on three different preparation and analyzed by the developed methods.

Specificity:

Specificity is a procedure to detect quantitatively the analyst in the presence of component that may be expected to be present in the sample matrix commonly used excipient in gel preparation were spiked in a pre-working quantity of drug and then absorbance was measured and calculation done to determined the quantity of the drugs.

Robustness:

The robustness of proposed method was tested by changing parameters such as wavelength range, slit width, temperature and shaking time. None of the variables significantly affected the absorbance of the drugs indicating that are method could be considered as robust.

Ruggedness:

The ruggedness of proposed method was determined by analyzing aliquots from homogeneous lot in different under graduate laboratories using similar operation and environmental condition, data is presented in table no.1 (5,9)

Table No 1: Optical characteristics, precision study and result of formulation analysis

Parameter		Tioconazole
Wavelength(nm)		220nm
Beer’s law limit (µg/ml)		10-50µg/ml
Regression equation	Slope (m)	76684
	Intercept(c)	482808
Correlation coefficient (r)		0.9992
Precision (%RSD)	Intra-day	0.28%
		0.46%
	Inter-day
Formulation analysis (% Assay)		99.46%
LOD (µg/ml)		0.00187028
LOQ(µg/ml)		0.0056675447
Robustness (%RSD)	Change in wavelength	0.36399313
	Change in pH	0.28996909

Fig No.3 Representative chromatogram of Tioconazole in Methanol: phosphate buffer (pH3 95:05 %v/v)

RESULT:

For the method linearity was observed in the concentration range 10-50µg/ml for tioconazole. Precision was calculated as inter and intraday variation (%RSD is less than 1) for the drug. The precision, reproducibility data, robustness data are also presented in table 1.

Table No 2: Accuracy Studies

Sr. No	Conc.	Area	Mean	SD	%SD	% RSD
1	10	251247	251950	1091	0.433	0.43
	10	253208
	10	251397
2	30	1803747	1803908	4400	0.243	0.24
	30	1799591
	30	1808388
3	50	3330456	3325173	5860	0.176	0.17
	50	3326194
	50	3318869

DISCUSSION:

The proposed method was found to be simple, accurate and rapid for the routine determination of tioconazole. To study conducted as per the ICH guideline.

ACKNOWLEDGMENT:

The authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to USV Pharmacutical Pvt. Ltd. Mumbai, India, for providing pharmaceutical grade tioconazole API as a gift sample.

REFERENCES:

1. ICH Harmonized Triplicate Guidelines, “Validation of analytical procedures: text and methodology, Q2 (R1),” in International conference on Harmoniazation of Technical Requirements for Registration of pharmaceuticals for Human use,2005

2. International Conference of Harmonization (ICH) of Technical Requirements for the Registration of pharmaceuticals for Human use, validation of analytical procedures: Methodology, Adopted in Geneva (1996)

3. N. Padmaja and G. Veerabhadram 2017 Method development and validation of RP-HPLC method for the estimation of empagliflozin in APL, International Journal of pharmaceutical sciences and research, 724-727

4. Anjan Paudel, Ameeduzzafar, Farhan Jalees, Ahmad, etal. “Stress degradation studies on candesartan cilexetil bulk drug and development of validated method by UV spectrophotometry in marketed tablet.” World journal of pharmaceutical research, volume 3, 2014, 3975-3986.

5. Indian Pharmacopoeia 2014, volume 2, published by the Indian Pharmacopoeia commission Ghaziabad 1^st January 2014.

6. Katiyar manoj kumar, Kushwaha Dharmendra, Shukla R.N., “Specific and stability indicating assay method of candesartan cilexetil in presence of process and degradation impurities”, International journal of pharmaceutical innovations, vol.2, Sept.-Oct.2012, 1031- 2249.

7. Ayaman Abo EI-Maaty Mohamed Ahmed, Dr. Ehab Farouk Elkady., “Development and validation of advanced analytical technique for the determination of some drugs in pharmaceutical mixture or in the presence of their degradation products., Faculty of pharmacy cairo university, 2018.

8. Beckett A.H. and Stenlake J.B. (eds.), “Practical Pharmaceutical Chemistry”, 4th ed. 2-parts, New Delhi: CBS Publishers and Distributors.

9. Snyder L. R., Kirkland J. and Glajch L. J., (1997), Practical HPLC Method Development, 2nd ed. New York: John Wiley & Sons. pp. 1-19, 292

Received on 18.06.2019 Accepted on 11.07.2019

Asian J. Pharm. Ana. 2019; 9(4):229-231.

DOI: 10.5958/2231-5675.2019.00039.5