Comparative study of analytical method development of fluconazole in tablets and capsule by ultraviolet spectrophotometric method
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1 Research Article Comparative study of analytical method development of fluconazole in tablets and capsule by ultraviolet spectrophotometric method Shital S. Patil*, Ubhale Ravindra, Yeole Tejal, Tayade Laxmi, P. A. Salunke, R. S. Wagh, S. D. Barhate ABSTRACT Objective: This comparative study is aimed to develop and validate a simple, accurate, economic, and precise ultraviolet (UV) spectroscopic method for tablets and capsules of fluconazole. Percentage purity of fluconazole in tablets and capsule was determined using bracketing method. Method: The method involves the use of easily available inexpensive solvents that is distilled water. UV-visible spectrophotometer (Shimadzu, model 1800) which has two matched quartz cells with 1 cm light path and a detection wavelength of 261 nm using a UV detector. Results: A linear response was observed over the concentration range 5-25 μg/ml of fluconazole. Detection limit for fluconazole tablet and capsule was found to be and 1.35, respectively. Quantitation limit for fluconazole tablet and capsule was found 2.71 and 4.1, respectively. The percentage purity was found % and 97.29% for tablet and capsule, respectively. Conclusion: The comparative study of proposed method gives analytical results which satisfy all validation parameters for both tablet and capsules. The method was successfully validated in accordance to ICH guidelines acceptance criteria for linearity, accuracy, precision, robustness, and ruggedness. Good precision and accuracy which prove the reliability of proposed method in different dosage form. Hence, this method can be used routinely for quantitative estimation of fluconazole in solid dosage form (tablet and capsule) by UV spectrophotometer. The literature survey revealed that various research works are done with fluconazole in bulk, tablet, capsule, and any other formulations. The present work gives detail account on comparative study of tablet and capsule for analytical method development and validation. KEY WORDS: Analytical method validation; Bracketing method; Fluconazole; Ultraviolet spectrophotometric method INTRODUCTION The present comparative study aimed to develop and validate a simple, accurate, economic, and precise analytical method for the marketed formulations of fluconazole in tablet and capsule by ultraviolet (UV) spectrophotometer. Percentage purity of fluconazole in tablets and capsule was determined using bracketing method. The assay bracketing method is applied occasionally a linear, but nonproportional relationship between concentration and absorbance occurs which is indicated by significant positive or negative intercept in a Beer s law plot. A two-point bracketing standardization is therefore required to determine the concentration of the sample solution. The concentration of one of the standard solutions is greater than that of the sample while the other Access this article online Website: ISSN: standard solution has a lower concentration than the sample. The concentration of a substance in the sample solution is given by the following equation. [1] C = (A A 1)(C 1 C 2) + test test std std std Cstd1(Astd1 Astd 2) A A std1 std2 Fluconazole is a triazole antifungal drug which in semi-sensitive fungi inhibits cytochrome P450- dependent enzymes, resulting in ergosterol synthesis in fungal cell members. Fluconazole is used to prevent and treat a variety of fungal and yeast infections. It shows activity against species of Candida sp. and it is indicated in cases of oropharyngeal candidiasis, esophageal, vaginal, and deep infection. [2] Fluconazole is highly absorbed by the gastrointestinal tract and spreads easily by body fluids. The main side effects related to the use of fluconazole are nausea, vomiting, headache, rash, abdominal pain, diarrhea, and alopecia in patients undergoing prolonged treatment. [3] The Department of Quality Assurance, Shree Sureshdada Jain Institute of Pharmaceutical Education and Research, Jamner, Maharashtra, India *Corresponding author: Shital S. Patil, Shree Sureshdada Jain Institute of Pharmaceutical Education and Research, Jamner, Maharashtra, India. Phone: sspatil26888@gmail.com Received on: ; Revised on: ; Accepted on:
2 detailed literature revealed that in the form of raw material, pharmaceutical formulations, or biological material, fluconazole can be determined by methods such as titration, spectrophotometry, and thin-layer, gas, and liquid chromatography. In this article, the analytical method is developed and validated for both tablet and capsule in comparative manner (Figure 1). [4] The apparent volume of distribution of fluconazole approximates that of total body water. Plasma protein binding is low (11-12%) and is constant over the concentration range tested ( mg/l). The pharmacokinetic properties of fluconazole are similar following administration by the intravenous or oral routes and do not appear to be affected by gastric ph. The bioavailability of orally administered fluconazole is over 90% compared with intravenous administration. Essentially, the entire administered drug reaches systemic circulation; thus, there is no evidence of the first-pass metabolism of the drug. [3] MATERIALS AND METHODS Chemicals and Reagents Analytically, pure sample of fluconazole with purities greater than 95% was procured form market. Tablet formulations (Fluka 150) and capsule formulations (Fumycin) were procured from the medical store, Maharashtra, India, with labeled amount of 150 mg of fluconazole. Distilled water was obtained from the laboratory (Figure 2). Methods Selection of solvent Distilled water was selected as an ideal solvent for spectrophotometric analysis of fluconazole. [6,7] absorbance of the resulting solution was scanned in UV spectrometer under the range of nm the suitable wavelength was found at 261 nm for fluconazole. The UV spectrum for fluconazole is shown in Figure 3. Preparation of standard stock solution Accurately weighed 10 mg fluconazole in a volumetric flask and dissolved and diluted up to the mark with water to give a stock solution having strength 1000 μg/ml. 100 μg/ml working standard solution was prepared by diluting 1 ml of stock solution to 10 ml with distilled water. Preparation of sample solution for tablet To measure the fluconazole content of tablet (label claim 150 mg fluconazole per Tablet, Fluka 150), 20 tablets were weighed, the mean weight was determined. A weight of the powder equivalent to 100 mg fluconazole was transferred to a 100 ml volumetric flask containing 50 ml distilled water and then diluted to 100 ml with distilled water (1000 µg/ml). The solution was filtered and 1 ml of filtered solution was diluted tenfold to furnish a concentration of 100 µg/ml. Preparation of sample solution for capsule To measure the fluconazole content of capsule (label claim 150 mg fluconazole per capsule, Fumycin capsules), 20 capsules were weighed, the mean weight was determined. A weight of the powder equivalent to 100 mg fluconazole was transferred to a 100 ml volumetric flask containing 50 ml distilled water and then diluted to 100 ml with distilled water Determination of wavelength The stock solution was diluted with distilled water to obtain a solution of concentration 10 μg/ml. The Figure 2: Ultraviolet spectrophotometer Figure 1: Chemical Structure of fluconazole [5] Figure 3: Ultraviolet spectrums for fluconazole 2
3 (1000 µg/ml). The solution was filtered and 1 ml of filtered solution was diluted tenfold to furnish a concentration of 100 µg/ml. Validation of UV spectrophotometric method The validation study was consisted of selectivity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantitation (LOQ), as well as linearity. Analytical method development and validation were carried out for two different marketed formulations such as tablet and capsule for fluconazole in the following manner. Assay (Bracketing Method) For standard solution The range was selected 5 µg/ml (STD 2), 10 µg/ml (test), and 15 µg/ml (STD 1). The standard solution of 5 µg/ ml and 15 µg/ ml and test solution of 10 µg/ml was prepared. Accurately 10 mg of pure fluconazole powder was weighed and dissolved in 10 ml of distilled water. Pipette out 1 ml and transfer in 10 ml volumetric flask dilute to 10 ml, with distilled water for 100 µg/ml. Dilutions of 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, and 25 µg/ml were prepared. For tablet test solution Twenty tablets of fluconazole were weighed accurately. Equivalent weight to g of powder fluconazole tablet was transferred in 10 ml in a volumetric flask and dissolved it in distilled water and made the volume with distilled water. Dilutions of 5 µg/ml, 10 µg/ml, and 15 µg/ml were prepared with distilled water for resulting STD 1 and STD 2 solutions, respectively. 1 ml into 10 ml distilled water for test solution. Take the absorbance of test and standard solution. For capsule solution Twenty capsules of fluconazole were weighed accurately. Equivalent weight to g of powder fluconazole capsule transfers it 10 ml in a volumetric flask. Dissolve it in distilled water and make up the volume with distilled water. Dilutions of 5 µg/ml, 10 µg/ml, and 15 µg/ml were prepared with distilled water for resulting STD 1 and STD 2 solutions, respectively. 1 ml into 10 ml distilled water for test solution. Take the absorbance of test and standard solution. The method validated for linearity, assay and accuracy, precision, repeatability, LOD, and LOQ as discussed and resulted in the following section. [8] RESULT AND DISCUSSION Selection of Wavelength and UV Spectrum This method was established to achieve quantity estimation accurately. To have a high sensitivity for each target analyte, the wavelength of 261 nm was used for bulk drug fluconazole and UV spectrum for the same as shown in Figure 4. Linearity The linearity was determined by analyzing six independent levels of calibration curve in the range of 5-25 μg/ml. Absorbance of each solution against distilled water was recorded at curve of absorbance versus concentration was plotted, and correlation coefficient and regression line equation for fluconazole were determined. Calibration curve was plotted in the range of 5-25 µg/ml. Linearity was found R 2 = for Bulk drug and observations are shown in Table 1 with calibration curve plotted in Figure 5. Linearity of fluconazole in tablet and capsule Calibration curve was plotted in the range of 5-25 µg/ml for tablet and capsule. Linearity was found R 2 = for tablet and capsule, respectively, and observations are shown in Table 2 with calibration curve plotted in Figure 4. Validation Assay Assay was carried out with bracketing method and % drug recovered was found to be and in percent, absorbance is shown in Table 3. Accuracy Accuracy was determined by performing recovery studies by spiking different concentrations of pure drug in the preanalyzed powder for infusion samples Figure 4: Linearity curve for tablet and capsule
4 within the analytical concentration range of the proposed method at three different set at level of 80%, 100%, and 120%. The amount of fluconazole was calculated at each level and % recoveries were computed. The percentage recovery study was carried out for 80%, 100%, and 120%. All observations are shown in Table 4. The accepted limits of mean recovery are 100 ± 2 and all observed data were within the required range, which indicates good Table 1: Linearity of Flz in bulk S. No. Concentration Absorbance recovery values, and hence, the accuracy of the method developed. The % RSD for tablet and capsule was found to be 0.48 and 0.52, respectively, which is <2%. The % RSD for tablet and capsule was found to be and 1.58, respectively, which is <2% The % RSD for tablet and capsule was found to be 1.29 and 1.29, respectively, which is <2% Precision The precision of the method was obtained by taking absorbance of same preparations as intraday and interday for tablet and capsule. The precision of this method reflected by relative standard deviation of replicates is not more than 2%. The absorbance and Table 2: Linearity of Flz in tablet and capsule S. No. Tablet Capsule Concentration Absorbance Concentration Absorbance Table 3: Assay Dosage form Concentration (µg/ml) Absorbance % Assay Tablet STD STD Test Capsule STD STD Test Table 4: Accuracy Conc. added Abs found Tablet received % received Abs found Capsule received % received 80% Mean Mean SD 0.47 SD 0.47 %RSD 0.48 %RSD Mean Mean SD 1.53 SD 1.53 %RSD 1.58 %RSD Mean Mean SD 1.27 SD 1.27 %RSD 1.29 %RSD 1.29 SD: Standard deviation, RSD: Relative standard deviation 4
5 Table 5: Intraday precision Conc Tablet Capsule Absorbance Mean %RSD Absorbance Mean %RSD I II III I II III RSD: Relative standard deviation Table 6: Interday precision Conc Tablet Capsule Absorbance Mean %RSD Absorbance Mean %RSD I II III I II III RSD: Relative standard deviation Table 7: Repeatability Tablet their relative standard deviation are shown in Tables 5 and 6 for tablet and capsule. The relative standard deviation for interday and intraday was found <2% for tablet and capsule. Repeatability Repeatability was carried out using a minimum of six determinations at one of the test concentrations. Repeatability was conducted on same concentration of tablet and capsule formulations; the percent relative standard deviations were found not more than 2% for both formulations and results are shown in Table 7. The repeatability study in terms of relative standard deviation was found not more than 2% for tablet and capsule. LOD AND LOQ The LOD and LOQ were estimated from the set of five calibration curves used to determine method linearity. LOD = 3.3*σ/S and LOQ = 10*σ/S Capsule Conc. Abs Conc. Abs Mean 0.04 Mean 0.04 %RSD 1.97 %RSD 1.99 RSD: Relative standard deviation Table 8: LOD and LOQ (tablet) Conc. Abs I II III Mean±SD ± ± ± ± ±0.001 Average SD SD: Standard deviation, LOD: Limit of detection, LOQ: Limit of quantitation Table 9: LOD and LOQ (capsule) Conc. Abs I II III Mean±SD ± ± ± ± ± Average SD SD: Standard deviation, LOD: Limit of detection, LOQ: Limit of quantitation Where, σ = the standard deviation of y-intercepts of regression lines S = the slope of the calibration curve LOD and LOQ for tablet and capsule, observations and results are given in Tables 8 and 9, respectively. The average SD was found to be and , respectively, for tablet and capsule. The LOD and LOQ for tablet formulations were found to be and 2.71, respectively. The LOD and LOQ for capsule formulations were found to be 1.35 and 4.10, respectively.
6 REFERENCES Figure 5: Linearity of fluconazole in bulk CONCLUSION The developed analytical method is simple, accurate, and precise with eco-friendly solvent (distilled water) and validated for all parameters in accordance with ICH guidelines. The different dosage forms of fluconazole give acceptable results with minimum linearity range. The percentage purity was determined using simple bracketing method which is simple and accurate for validation assay. The validation data demonstrate good precision and accuracy which prove the reliability of proposed method in different dosage form (tablet and capsule). Hence, this method can be used routinely for quantitative estimation of fluconazole in solid dosage form (tablet and capsule). 1. Bari SB, Sonawane LV, Bharud DN, Poul BN. Practical Handbook of Pharmaceutical Analysis. 3 rd ed. Pune: Nirali Prakashan; p Sweetman SC. Martindale: The Complete Drug Reference. 33 rd ed. London: Pharmaceutical Press; p Barar FS. Chemotherapy of viral diseases (Antifungal drugs). In: Essentials of Pharmacotherapeutics. New Delhi: Chand and Company Ltd.; p Corrêa JC, Salgado HR. Review of fluconazole properties and analytical methods for its determination. J Crit Rev Anal Chem. 2011;41(3): Available from: [Last accessed on 2017 Mar 03]. 6. Singh A, Sharma PK, Majumdar DK. Development and validation of different UV-spectrophotometric methods for the estimation of fluconazole in bulk and in solid dosage form. Indian J Chem Technol 2011;18: Jebaliya H, Patel M, Jadeja Y, Dabhi B, Shah A. A comparative validation study of fluconazole by HPLC and UPLC with forced degradation study. Chromatogr Res Int 2013;2013: Pharmaceuticals for Human Use, ICH Harmonisation Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2 (R1), Complementary Guideline on Methodology Dated 06 November 1996, Incorporated in November 2005, London. 6
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