INTRODUCTION:

Chemically Idebenone is 2,5-cyclohexadiene-1,4-dione,5,6-dimethoxy -2-(-10-hydroxydecyl)-3-methyl¹ (figure no.1) , is a synthetic analogue of coenzyme Q₁₀ (CoQ₁₀), a vital cell membrane antioxidant and essential constituent of the adenosine-triphosphate (ATP) producing mitochondrial Electron Transport Chain (ETC) , it is also used as antioxidant by protecting against multiple free radical pathway modulation , regulation of inflammatory markers, and in treatment of photodamage skin.

The literature survey reveals that Idebenone was analyzed by GC-MS or LC-MS². HPLC was used for determination of Idebenone³, its metabolites⁴ and UV, colorimetric method⁵.

Analysis is an important component in the formulation development of any drug molecule. It becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug samples. Our main concern is development and validation⁶ of UV spectrophotometric method as per ICH guideline.

MATERIALS AND METHOD:

Instrument and materials:

Instrument used were JASCO V-630 double beam UV/Visible Spectrophotometer and schimadzu AX200 analytical balance. Idebenone pure drug was obtain from International Specialty Products (India) Pvt. Ltd. as gift sample with 99.9% w/w assay value and was used without further purification. All chemicals and reagents used were of analytical grade. Idebenone tablets were purchased from market.

Preparation of standard stock solution:

Standard drug solution of Idebenone was prepared by dissolving 10mg Idebenone in 20ml absolute ethanol and transfers it to 100ml volumetric flask and volume was made upto mark with phosphate buffer pH 6.8 to obtain stock solution of 100µg/ml concentration. For obtaining clear solution, solution was ultrasonicated.

Preparation of calibration curve:

Aliquots of 0.2 to 2 ml portion of stock solutions were transferred to series of 10 ml volumetric flasks, and volume made up to mark with solvent (20% v/v of absolute ethanol in phosphate buffer pH 6.8). Solutions were scanned in the range of 200-400 nm against blank .The absorption maxima were found to be at 282 nm against blank (Figure No.2). The calibration curve was plotted. The optical characteristics are summarized in (Table No.2)

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 (mg/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 (mg/ml)	4-20
3	Standard Regression Equation	y = 0.0485x - 0.0239
4	Correlation Coefficient (r²)	r²= 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 mg/ml solution of candidate drug in solvent (20%v/v ethanol in 6.8-pH phosphate buffer) at UV detection l of 282 nm will show an absorbance value of 0.5777
10	Sandell’s Sensitivity ⁸ (mg/cm²/0.001 absorbance unit)	0.022735
11	LOD (mg/ml)	1.6261
12	LOQ (mg/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 (inter-day 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

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 λ_max of Idebenone by UV scanning.

Linearity:

The linearity of the response of the drug was veriﬁed at 2 to 40 μg/ml concentrations, but linearity was found to be between 4-20 μg/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 (r²) of determination was 0.9978).

Table No.4 - determination of Precision

Sample number	Assay of Idebenone as % of labeled amount
	Analyst-I (Intra-day precision)	Analyst-II (Inter-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

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).

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.

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. Uv-spectrophotometric 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.

Received on 16.04.2009 Modified on 20.05.2009

Asian J. Research Chem. 2(2): April.-June, 2009 page 168-170