Indo American Journal of Pharmaceutical Research, 2013
Journal home page: http://www.iajpr.com/index.php/en/
ISSN NO: 2231-6876
INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH
Development And Validation Of UV-Spectrophotometric Method For The Estimation Of Terconazole In Bulk And Pharmaceutical Dosage Form Srilakshmi. M*, S. A. Rahaman, K. Shanthakumari. Department of pharmaceutical Analysis, Nirmala College of pharmaceutical sciences, Atmakuru (vill), Mangalgiri (Mdl) 522503, Andhra Pradesh,
ARTICLE INFO Article history Received 10/04/2013 Available online 28/04/213
Keywords Terconazole, UVspectrophotometry, Validation.
ABSTRACT A simple efficient, precise and accurate spectroscopic method has been developed and validated for quantitative estimation of Terconazole in bulk and pharmaceutical dosage form. Terconazole is slightly soluble in distilled water, so 0.1N HCl was used as solvent. Terconazole is dissolved in 0.1N HCl the resulting solution was then scanned in the UV range (200-400nm) in a 1cm quartz cell in a double beam UV spectrophotometer. The λmax of Terconazole was found to be 227nm.The method obeys Beers law in the concentration range from 2-10 μg/ml. The correlation coefficient was found to be 0.999 (r 2═ 0.999). The LOD and LOQ were found to be 0.265 and 0.804μg/ ml respectively. The result of estimation of marketed formulation (Terazol) was found to be 97.88%. The accuracy of the method was determined by recovery studies. The percentage recovery was found to be 99.76%. The method was validated statistically as per ICH guidelines. The method showed good reproducibility and recovery with % RSD less than 2. So, the proposed method was found to be simple, specific, precise, accuracy, linear, and rugged. Hence it can be applied for routine analysis of Terconazole in bulk drug and the Pharmaceutical formulations.
Corresponding author: Srilakshmi. M, Dept. of Pharmaceutical Analysis, Acharya Nagarjuna University, Guntur, 522007, Andhra Pradesh, India. E-mail:
[email protected]
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Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited .
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Please cite this article in press as Srilakshmi et al. Development And Validation Of UV-Spectrophotometric Method For The Estimation Of Terconazole In Bulk And Pharmaceutical Dosage Form. Indo American Journal of Pharm Research.2013:3(4).
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INTRODUCTION Terconazole is an anti-fungal medication, primarily used to treat vaginal fungal infections. It is commercially available in several dosage forms for topical administration. Topical terconazole formulations have been found to be effective in controlling vaginal infections without producing significant adverse reactions. Terconazole is an odorless, white powder having a molecular weight of 532.47g and melting point of 126.3°C. Its chemical formula is C26H31Cl2N5O3. The structural formula of terconazole is as follows:
The IUPAC name of Terconazole is 1-(4-{[(2R, 4S)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)1,3-dioxolan-4 yl] methoxy}phenyl)-4-(propan-2-yl) piperazine. It is sparingly soluble in ethanol, soluble in Butanol and insoluble in water. It is available as Terazol, Terazol 3, Terazol 7 and Tercospor.
Pharmacodynamics: Terconazole is a triazole antifungal agent available for intra-vaginal use. It is structurally related to Imidazole-derivative antifungal agents; although terconazole and other triazole have 3 nitrogen’s in the azoles’ ring. By inhibiting the 14-alpha-demethylase (lanosterol 14-alpha-demethylase), Terconazole inhibits ergosterol synthesis. Depletion of ergosterol in fungal membrane disrupts the structure and many functions of fungal membrane leading to inhibition of fungal growth.
Mechanism of action: It acts by disrupting normal fungal cell membrane permeability. It inhibits cytochrome P450 14-alphademethylase in susceptible fungi, which leads to a decrease in ergosterol concentration. Depletion of ergo sterol [1-4] will disrupts the structure and function of the fungal cell and cell growth. Literature survey reveals no validated method was found for its quantitative determination in bulk and pharmaceutical dosage forms, but it was used as an internal standard in estimation of some other drugs like [5-6] ketoconazole , Itraconazole [7], and in some enantiomeric separations [8], also in detection of some antifungal agents in illegal shampoo by HPLC
[9]
.
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The objective of this work was to develop sensitive and efficient analytical methods for quantitative determination of terconazole in bulk and pharmaceutical dosage forms. In this study, efforts were made to develop a simple, easy, and economic UV spectrophotometric method using 0.1N HCl as solvent for the determination of terconazole in the bulk drug and dosage form. The developed method was optimized validated as per the guidelines of International Conference on Harmonization and demonstrated excellent linearity, precision, accuracy for Terconazole.
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Objective:
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MATERIALS AND METHODS Instruments: UV-Visible Double Beam Spectrophotometer (Thermo Scientific, Evolution 201) with 1cm matched Quartz cells, micro pipette of variable volume and Digital Balance.
Chemicals: Standard Solution of Terconazole: Standard Terconazole of 10mg was accurately weighed and transferred to 10ml volumetric flask. It was dissolved properly and diluted to mark with 0.1N HCl to obtain concentration of 1mg/ml. This solution was used as stock solution. From this, working standard solution and suitable dilutions were prepared.
Preparation of 0.1N HCl: Accurately measured 8.5 ml of concentrated HCl and transferred to 1000ml volumetric flask. Then added required amount of water and made up to mark with distilled water.
Determination of Absorption Maxima: By the appropriate dilution of standard drug solution with 0.1N HCl, solution contain 10µg/ml of terconazole was scanned in the range of 200-400nm to determine the wavelength of maximum Absorption. Drug showed Absorption maxima at 227nm. (Fig-1)
Fig. 1: Absorption maxima of terconazole
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Aliquots of standard solution of terconazole ranging from 0.2-1ml (1ml=100µg) were transferred in to a series of 10ml volumetric flasks. To each flask required amount of 0.1N HCl was added and finally the volume in each flask was brought up to the 10ml with the 0.1N HCl. Then Absorbance’s were measured at 227nm against the reagent blank. Then Calibration data was indicated in (Table-1) and calibration curve was plotted by taking concentration on X-axis and Absorbance on Y-axis which is shown in (Fig-2).
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Procedure:
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0.8
y = 0.043x + 0.295 R² = 0.999
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0
2
4
6
8
10
12
Fig. 2: Calibration Curve of Standard Terconazole
Analysis of pharmaceutical dosage form: An accurately weighed amount of cream equivalent to 10 mg Terconazole (2.5gm of cream, since each gm contains 4mg of drug) was placed in a 100 ml volumetric flask, taking care to avoid sticking cream to the walls of the volumetric flask, and 40 ml methanol was added. After the flask was shaken for 30 min until the cream is dissolved completely, then the volume was made up to 100 ml with 0.1N HCl. This solution was filtered through filter paper to remove some un-dissolved excipients. The first 10 ml of the filtrate was discarded. After filtration, from this 0.4ml was taken and diluted to 10 ml with 0.1N HCl which gives 4µg/ml solution and the absorbance of the solution was measured at 227 nm. Table-2: Results obtained in the determination of terconazole in creams: Sample
Amount declared (mg/g)
Amount found (mg/g)
Amount found (10 mg/g)
Confidence limit
Terconazole
4 mg
3.915 mg
9.788 mg
97.88 %
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An aliquot of concentration of 2-14µg/ml were prepared in duplicate, but linearity was found to be between 2-10µg/ml concentrations. The linearity was calculated by the Least Square Regression Method.
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Validation Parameters of Method: Linearity:
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Precision: The precision of the assay was determined by repeatability and reported as RSD%. For this 4µg/ml concentration was taken and measured 6 times in day and same was measured in the next day. The RSD% was calculated. Table-3 Precision: 4µg/ml concentration was taken Sr. No.
Intra-day Precision
% RSD
1
0.448
0.418
2
0.444
0.412
3
0.447
4
0.442
0.414
5
0.445
0.415
6
0.443
0.409
0.560 %
Inter-day Precision
0.410
% RSD
Mean % RSD
0.8103 %
0.6851 %
Accuracy: The accuracy of the method was evaluated through Standard Addition Method. In this, known amount of standard Terconazole was added in pre-analyzed sample. This was done for 6µg/ml concentration taking it as 100% for 3 times. It was done for 80%, 100%, 120% and the Recovery studies were performed and finally the RSD% was calculated. Table-4 Accuracy: 6µg/ml concentration was taken as 100 % Spike Level
Mean Absorbance
Amount Found
% Recovery
Mean % RSD
6
80 %
0.759
10.62
96.99 %
0.2976 %
6
100 %
0.819
11.98
99.76 %
0.5208 %
6
120 %
0.870
13.15
99.27 %
0.4827 %
Toatal Mean % RSD
0.4337 %
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Conc. (µg/ml)
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Robustness: It is a study of small but deliberate variations in method parameters such as Absorption Maxima, P H and ratio of mobile phase solvents. In this present work the Absorption maxima was decreased and increased 2nm and carried the process for 8µg/ml solution for 6 times. The RSD% was calculated. Table-5 Robustness: 8µg/ml concentration was taken Sr. No
At 225nm (-2nm)
% RSD
1
0.666
0.542
2
0.667
0.542
3
0.661
4
0.666
0.546
5
0.661
0.544
6
0.662
0.546
0.441 %
At 229nm (+2nm)
0.541
% RSD
Mean % RSD
0.411 %
0.426 %
Ruggedness: It is the degree of reproducibility of test results obtained by the variety of conditions like different analysts, reagents, laboratories, days, equipment etc. The present work was performed by the change of analyst. Then nearly same results were obtained which are similar to that of first analyst.
Limit of detection (LOD): It is the smallest quantity of an analyte that can be detected, and not necessarily determined, in a quantitative fashion. It was calculated by the following formula; LOD =
Where; S.D=Standard Deviation
Limit of Quantization (LOQ): It is the lowest concentration of an analyte in a sample that may be determined with acceptable accuracy and precision. It was calculated by the following formula;
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Where; S.D=Standard Deviation
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LOQ =
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Table-6 Validation Parameters Sr. No. Parameter
Result
1
Absorption (nm)
227nm
2
Linearity Range (µg/ml)
2-10(µg/ml)
3
Standard Regression Equation
Y=0.043x+0.295
4
Correlation Coefficient (r2)
0.999
5
Accuracy (%RSD)
0.4337%
6
Precision (%RSD)
0.6851%
7
Robustness (%RSD)
0.4261%
8
Detection Limit
0.265 (µg/ml)
9
Quantization Limit
0.804(µg/ml)
RESULTS AND DISCUSSION A simple, selective, accurate, precise spectroscopic method for the estimation of Terconazole in bulk and pharmaceutical dosage form has been developed and validated. The linearity range in the concentration range of 2-10μg/ ml (r2═ 0.999).It indicated that the concentrations of Terconazole had good linearity. The LOD and LOQ were found to be 0.265 and 0.804μg/ ml respectively. The amount of Terconazole was calculated as 97.88%. Further the precision of the method was confirmed by the repeatable analysis of solution. The % RSD was found to be 0.6851%. It indicated that the method has good precision. The percentage recovery was found to be in the range of 96.99-99.76%.The procedure was repeated for 3 times by taking 6µg/ml as 100%.The recovery was calculated for 80%, 100% and 120%. The % RSD was found to be 0.4337%.The low % RSD value indicated that there is no interference due to excipients used in formulation. Hence, the accuracy of the method was confirmed.
CONCLUSION
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ACKNOWLEDGEMENTS The authors would like to acknowledge the staff and management of the Nirmala College of Pharmacy, Atmakuru (GNT Dist) for providing all the facilities to carry out the present research work.
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The proposed method is simple, accurate, precise and selective for the estimation of Terconazole in bulk and pharmaceutical dosage forms. The method is economical, rapid and do not require any sophisticated instruments contrast to chromatographic method. Hence it can be effectively applied for the routine analysis of Terconazole in bulk and marketed formulation.
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REFERENCES: 1) 2) 3) 4)
Available from: http//www.drug bank.com Available from: http//www.en.wikipedia.org/ Terconazole Available from: http//www.Rx list .com/ Terconazole Terconazole cream:http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=426c71b4-39cf-4d12-ad6a8f64a7c15490 5) Erika Rosa Maria Kedor-Hackmann*, Maria Ines Rocha Miritello Santoro: First-derivative ultraviolet spectrophotometric and high performance Liquid chromatographic determination of ketoconazole in pharmaceutical emulsions: Brazilian Journal of Pharmaceutical Sciences vol. 42, n. 1, Jan. /mar., 2006 6) Turner CA, Turner A, and Warnock DW: High performance liquid chromatographic determination of ketoconazole in human serum: J Antimicrob Chemother. 1986 Dec; 18 (6):757-63. 7) Grabowski T, Swierczewska A, Borucka B, Sawicka R, Sasinowska-Motyl M, Gumułka SW: Chromatographic/mass spectrometric method for the estimation of Itraconazole and its metabolite in human plasma: Pub med: Arzneimittelforschung. 2009; 59(8):422-8. Doi: 10.1055/s-0031-1296418. 8) Maria Castro-Puyana, Antonio L Crego, and M Luisa Marina: Enantiomeric separation of ketoconazole and terconazole antifungal by electro kinetic chromatography: Electrophoresis . 10/2005; 26(20):3960-8. DOI:10.1002/elps.200500100 9) LU Dan, SHEN Lin-Mei: Detection of Antifungal Agents in Illegal Shampoo by HPLC: Chinese Journal of Pharmaceutical 2006-12. 10) ICH, Q2A Validation of analytical procedure, Methodology International conference on Harmonization, Geneva, October 1994.
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