Development of UV Spectrophotometric method of Idebenone in ...

Asian J. Research Chem. 2(2): April.-June, 2009
,
ISSN 0974-4169
www.ajrconline.org
RESEARCH ARTICLE
Development of UV Spectrophotometric method of Idebenone in Bulk and
Pharmaceutical Formulation.
Amit Rathi*, Dinesh Dhamecha, Saifee Maria and Mohd Hassan G Dehghan
Y.B.Chavan College of Pharmacy ,Dr. Rafiq Zakaria Campus, Maulana Azad Education Trust, Rauza Bagh ,
Aurangabad (MS) India. 431001
*Corresponding Author E-mail: amit.pharmaceutics@gmail.com
ABSTRACT
The present research work discusses the development of a UV estimation method for Idebenone. Simple, accurate,
cost efficient and reproducible spectrophotometric method has been developed for the estimation of Idebenone in
bulk and pharmaceutical dosage form. UV spectrophotometric method, which is based on measurement at
maximum wavelength ( max ) 282nm. The percentage recovery of Idebenone ranged from 99.68 to 99.82% in
pharmaceutical dosage form. Beers law was obeyed in the concentration range of 4-20µg/ml having line equation
y = 0.0485x - 0.0239 with correlation coefficient of 0.9978. Results of the analysis were validated statistically and
by recovery study.
KEY WORDS: UV spectrophotometry, Idebenone
INTRODUCTION:
Chemically Idebenone is 2,5-cyclohexadiene-1,4-
dione,5,6-dimethoxy -2-(-10-hydroxydecyl)-3-methyl 1
(figure no.1) , is a synthetic analogue of coenzyme Q 10
(CoQ 10 ), 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-MS 2 . HPLC was used for determination
of Idebenone 3 , its metabolites 4 and UV, colorimetric
method 5 .
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 validation 6 of UV
spectrophotometric method as per ICH guideline.
Received on 16.04.2009 Modified on 20.05.2009
Accepted on 09.06.2009 © AJRC All right reserved
Asian J. Research Chem. 2(2): April.-June, 2009 page 168-170
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)
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Asian J. Research Chem. 2(2): April.-June, 2009
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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).
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Asian J. Research Chem. 2(2): April.-June, 2009
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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.
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