Development of UV Spectrophotometric method of Idebenone in ...

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Asian J. Research Chem. 2(2): April.-June, 2009 , Preparation of sample solution: The proposed method was applied to analyse commercially available Idebenone tablet. Ten tablets were weighed and powdered. The amount of tablet powder equivalent to 10 mg of Idebenone was weighed accurately and transfer to 100ml volumetric flask then 20 ml absolute ethanol was added and kept for 15 min with frequent shaking and volume was made up to mark with phosphate buffer pH 6.8.The solution was then filtered through Whattman filter paper #41.This filtrate was diluted suitably with solvent (20% v/v absolute ethanol in phosphate buffer pH 6.8) to get the solution of 12µg/ml concentration .The absorbance was measured against solution blank. Table No.1 - Calibration Curve parameter Sr. No. Concentration (µg/ml) Absorbance (average) Standard deviation 1 0 0 0 2 2 0.0713 ±0.005859 3 4 0.1552 ±0.001724 4 6 0.2552 ±0.0009 5 8 0.3629 ±0.001206 6 10 0.4766 ±0.014856 7 12 0.5577 0 8 14 0.6332 ±0.010201 9 16 0.7384 ±0.006341 10 18 0.8584 ±0.009174 11 20 0.9651 ±0.007279 Table.2 - Validation parameters Sr. No. Parameter Result 1. Absorption maxima(nm) 282 2. Linearity Range (µg/ml) 4-20 3 Standard Regression y = 0.0485x - 0.0239 Equation 4 Correlation Coefficient (r2 ) r 2 = 0. 9978 5 Molar absorptivity 14818.71 6 A( 1% , 1cm ) 443.088 7 Accuracy (% recovery ±SD) 99.74 % ± 0.07211 8 Precision (% CV) 99.67 %, 99.68 % 9 Specificity A 12 µg/ml solution of candidate drug in solvent (20%v/v ethanol in 6.8-pH phosphate buffer) at UV detection λ of 282 nm will show an absorbance value of 0.5777 10 Sandell’s Sensitivity 8 0.022735 (µg/cm 2 /0.001 absorbance unit) 11 LOD (µg/ml) 1.6261 12 LOQ (µg/ml) 4.9278 The drug content of the preparation was calculated using standard calibration curve. Amount of drug estimated by this method is given in (Table No.3). RESULT AND DISCUSSION: Precision: Assay of method precision (intra-day precision) was evaluated by carrying out six independent assays of test samples of Idebenone. The intermediate precision (interday precision) of the method was also evaluated using two different analysts, systems and different days in the same laboratory. The relative standard deviation (RSD) and assay values obtained by two analysts were 0.28, 99.67 and 0.26, 99.68 respectively (Table no.4). Fig. No.1 - Chemical structure of Idebenone O MeO MeO O CH 3 Accuracy (Recovery Test) 7 : Accuracy of the method was studied by recovery experiments. The recovery experiments were performed by adding known amounts of the drugs in the placebo. The recovery was performed at three levels, 80,100and 120% of Idebenone standard concentration. The recovery samples were prepared in afore mentioned procedure. Three samples were prepared for each recovery level. The solutions were then analyzed, and the percentage recoveries were calculated from the calibration curve. The recovery values for Idebenone ranged from 99.68 to 99.82 % (Table no.3). Fig. No.2 - Determination of max of Idebenone by UV scanning. 282 nm 1.4 1 Abs 0.5 OH -0.1 200 250 300 350 400 Wavelength [nm] Linearity: The linearity of the response of the drug was verified at 2 to 40 g/ml concentrations, but linearity was found to be between 4-20 g/ml concentrations. The calibration curve was obtained by plotting the absorbance versus the concentration data and was treated by linear regression analysis (Table no.2). The equation of the calibration curve for Idebenone obtained was y = 0.0485x - 0.0239, the calibration curve was found to be linear in the aforementioned concentrations (The correlation coefficient (r 2 ) of determination was 0.9978). 169
Asian J. Research Chem. 2(2): April.-June, 2009 , Table No.3 - Determination of Accuracy by percentage recovery method Ingredient Tablet amount Level of Amount added Amount recovered (µg/ml) addition (%) (µg/ml) (µg/ml) %Recovery 12 80 9.6 21.53 99.68 Idebenone 12 100 12 23.93 99.72 12 120 14.4 26.35 99.82 Average % Recovery 99.74 ± 0.07211 Table No.4 - Determination of Precision Sample Assay of Idebenone as % of labeled amount number Analyst-I (Intra-day precision) 1 99.72 99.77 2 99.93 99.97 3 99.78 99.71 4 99.90 99.88 5 99.48 99.51 6 99.20 99.25 Mean 99.67 99.68 SD 0.28 0.26 Analyst-II (Inter-day precision) Limit of Detection (LOD) and Limit of Quantification (LOQ): The LOD and LOQ of Idebenone were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines. The LOD and LOQ for Idebenone are described in (Table no. 2). Determination of Active Ingredients in Tablets: The validated method was applied to the determination of Idebenone in Tablets. Six tablets were assayed and the results are shown in (Table no. 3) indicating that the amount of drug in tablet samples met with requirements (98–102% of the label claim). REFERENCES: 1. Nagaoka A.Idebenone. New Cardiovascular Drugs. 1987; 217-235. 2. Wempe MF, Lightner JW, Zoeller EL, Rice PJ. Investigating idebenone and idebenone linoleate metabolism: in vitro pig ear and mouse melanocyte studies .J Cos Dermat. 2009; 8(1): 63-73. 3. Kim HJ, Yoon KA, Hahn M, Park ES, Chi SC. Preparation and In Vitro Evaluation of Self- Microemulsifying Drug Delivery Systems Containing Idebenone .Drug Devlop Ind Pharm. 2000; 26 : 523-529. 4. Wakabayashi H, Nakajima M, Yamato S, Shimada K . Determination of idebenone in rat serum and brain by high-performance liquid chromatography using platinum catalyst reduction and electrochemical detection. J Chromatogr. 1992; 573:154-157. 5. Francis M, Sane RT, Khatri A, Pathak AR. Uvspectrophotometric and colorimetric methods for determination of idebenone from bulk drug and its pharmaceutical formulation.Ind Drugs. 2001; 38: 642- 645 . 6. Validation of Analytical Procedures: Methodology (Q2B) Harmonized Tripartite Guidelines. 7. ICH (2005) Text on validation of analytical procedures. Q2B. 8. Khopkar SM . Basic Concepts of Analytical Chemistry .3rd ed., New Age International Pblisher. 2008; 277-278. CONCLUSIONS: The developed method was found to be simple, sensitive, accurate, precise, reproducible, and can be used for routine quality control analysis of Idebenone in bulk and pharmaceutical formulation ACKNOWLEDGEMENT: We would like to thank Mrs Fatma Rafiq Zakaria, Hon’ble Chairman of Maulana Azad Educational Trust, Dr Rafiq Zakaria Campus for providing all the facilities. We are also thankful to International Specialty Products PVT.LTD.,Hyderabad for giving us gift sample of Idebenone drug. 170
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