ISSN-
2231–5640 (Print) www.asianpharmaonline.org
ISSN-
2231–5659 (Online)
RESEARCH ARTICLE
Development and
Validation of Spectrophotometric Method for Determination of Azelaic Acid
Kadam T.V. 1,
Darekar A.B.1, Gondkar S.B.1, Saudagar R.B.2
1Department of Pharmaceutics, R.G. Sapkal
College of Pharmacy, Anjaneri, Nashik-422213 Maharashtra India
2Department of Pharmaceutical Chemistry,
R.G. Sapkal College of Pharmacy, Anjaneri, Nashik-422213 Maharashtra India
*Corresponding Author E-mail:truptikadam91@gmail.com
ABSTRACT:
In these studies describes a simple, accurate, precise and cost
effective UV-visible spectrophotometric method for the estimation of Azelaic
acid in pure and pharmaceutical formulations. The method is based on the
measurement of absorbance of Azelaic acid solution in Phosphate buffer pH 6.8
at 204nm in the wavelength range of 200-400nm. The method obeys Beer’s
Lambert’s law in the selected concentration range10-50 μg/ml in selected
media. The slope, intercept and correlation coefficient were also calculated.
Results of percentage recovery study shows that the method was not affected by
the presence of common excipients in tablets. The parameters like linearity,
precision, accuracy, sensitivity study i.e. limit of detection and limit of
quantitation were studied according to International Conference on
Harmonization (ICH) guidelines. The developed method was validated in terms of
accuracy, precision, linearity, limit of detection and limit of quantitation
which proves suitability of proposed method for routine estimation of Azelaic
acid in pure and pharmaceutical formulations.
KEYWORDS: UV-visible spectrophotometer, method development, Azelaic acid.
1. INTRODUCTION:
Azelaic acid is
dermatologic agent. Chemically it is known as 1, 7- heptanedicarboxylic acid with molecular weight 188.22. Freely soluble
in phosphate buffer solution pH 6.8, 0.1N NaOH and elimination half-life 45 min
(oral) and 12 hrs (topical).It is thought that Azelaic acid
manifests its antibacterial effects by inhibiting the synthesis of cellular
protein in anaerobic and aerobic bacteria, especially Staphylococcus epidermidis and Propionibacterium acnes.
In aerobic bacteria, Azelaic acid
reversibly inhibits several oxidoreductive enzymes including tyrosinase,
mitochondrial enzymes of the respiratory chain, thioredoxin reductase,
5-alpha-reductase, and DNA polymerases.
In anaerobic bacteria, Azelaic acid impedes glycolysis.1,2 Literature survey revealed that most of the HPLC, GC
methods used hyphenated techniques with detectors such as mass spectrometry,
ultraviolet, Electrospray mass spectrometric techniques all these methods have
high sensitivity, but most of them highly expensive and are not easily
available in quality control laboratories The literature survey clearly
indicates there is no spectrophotometric method is available for the
quantification of Azelaic acid. The analytically useful functional groups in
Azelaic acid have not been fully exploited for designing suitable, visible
spectrophotometric methods and so still offer a scope to develop more visible
spectrophotometric methods with better sensitivity, selectivity, precision and
accuracy. The author has made an attempt in this direction and succeeds in
developing methods.3-5 so author’s objective is to develop accurate,
simple, UV spectrophotometric method which is free from extraction techniques,
and shorter time and highly sensitive technique.
EXPERIMENTAL MATERIALS:
Azelaic acid
was received as a gift sample from Cadila Pharmaceuticals Ltd., Thane,
India. All analytical grade chemicals
and solvents were supplied by S.D. Fine Chemicals, Mumbai, India. Distilled
water was used to prepare all solution. Freshly prepared solutions were always
employed.
Instruments:
Jasco V-630 UV-visible spectrophotometer with data processing
system were used. The sample solution were recorded in 1 cm matched quartz
cells was used for spectral an absorbance measurements. Shimadzu AUX-220
electronic balance was used for weighing the sample.
Standard Solution Selection of common solvent:
Phosphate buffer solution (PBS) pH 6.8 was selected as a common
solvent for developing spectral characteristics of drug. The selection was made
after using different solvents and their different normalities.
Preparation of standard drug solution:
Standard stock solution containing Azelaic acid was prepared by
dissolving 10 mg of Azelaic acid separately in 25 ml of PBS pH 6.8 shake for 5
min. and then final volume of the solutions was made up to 100 ml with same
solvents to get stock solution containing 100 µg/ml
Selection of λ max:
The standard stock solution was further
diluted with PBS pH 6.8 to get a 10 μg/ml of concentration (1 ml to 10
ml). The solution was scanned between 200 and 400 nm using PBS pH 6.8 as blank.
From the spectrum obtained, 204 nm was selected as λmax for the analysis
of Azelaic acid. Standard stock solution was further diluted to obtain 10-50
µg/ml with PBS pH 6.8. Calibration curve was plotted in the concentration range
of 10-50 µg/ml of Azelaic acid using PBS pH 6.8 as blank.
Validation of the proposed
method 6, 7
The proposed method was validated according to the International
Conference on Harmonization (ICH) guidelines.
Linearity (calibration
curve)
The developed method validated as per ICH guidelines. The plot of
absorbance verses concentration is shown in figure no.1 for PBS pH 6.8. It can
be seen that plots are linear in the concentration range 10-50 µg/ml with
correlation coefficients (r2) is 0.994.
Precision (repeatability)
Intraday and interday precision was determined by measurement of
the absorbance for three times on same day and on three different days. The
relative standard deviation for replicates of sample solution was less than 2%
which meet the acceptance criteria for established method.
Accuracy (recovery study):
Recovery study was carried out by adding a known quantity of pure
drug to the preanalysed formulations and the proposed method was followed. From
the amount of drug found, percentage recovery was calculated as per ICH
guidelines.
Sensitivity:
Sensitivity study was carried out where limit of detection (LOD)
and limit of quantification (LOQ) were determined using following equation.
LOD = 3.3* σ
/ S
LOQ = 10* σ
/ S
Where, σ = standard deviation of the response.
S = slope of calibration curve
RESULT
AND DISCUSSION:
The maximum absorption for Azelaic acid in PBS pH 6.8 was observed
at 204 nm. The high values of correlation coefficient in PBS pH 6.8 indicate
linearity for Azelaic acid in PBS pH 6.8. Beer’s law was obeyed for PBS pH 6.8
in the range of10-50 µg/ml the accuracy of method was determined by calculating
mean percentage recovery and % relative error. The percentage recovery ranges
from 98 µg/ml and % relative errors was within 2% in PBS pH 6.8 and are
presented in table no. 4. Precision was calculated as repeatability, inter and
intraday variation for Azelaic acid, percentage RSD was found to be less than
1. The repeatability data are presented in table no. 2 and table no. 3. LOD and
LOQ were found to be 0.099 µg/ml and 0.30 µg/ml respectively for detection of
Azelaic acid in PBS pH 6.8. The proposed method was found to be simple,
accurate, precise and rapid for the routine determination of Azelaic acid in
pure and pharmaceutical formulation
Table no.1: Optical
Parameters for Determination of azelaic acid
Precision:
|
Sr.no.
|
Data
|
Results
|
|
1
|
Wavelength (nm)
|
204
|
|
2
|
Slope
|
0.0022
|
|
3
|
Correlation
coefficient
|
0.994
|
|
4
|
Intercept
|
0.067
|
|
5
|
LOD (µg/ml)
|
0.099
|
|
6
|
LOQ(µg/ml)
|
0.30
|
CONCLUSION:
Simple spectrophotometric method for determination of Azelaic acid
have been developed and validated as per ICH guidelines. The proposed method is
found to be simple, rapid, sensitive, accurate and reproducible also can be
used for the routine quality control analysis of Azelaic acid in bulk and
pharmaceutical formulations.
REFERENCES:
1.
Indian Pharmacopoeia – 2014, Volume II, Government of India
Ministry of Health And Family Welfare, Published by the Indian Pharmacopoeia
Commission, Ghaziabad, pn.1113-14.
2.
http://www.drugbank.ca/drugs/DB00548.Accessed On 13 Aug.
2014 At 12.25pm.
3.
Medikondu Kishore, M.
Jayaprakash, T. Vijayabhaskara Reddy. Spectrophotometric Determination of
Azelaic Acid in Pharmaceutical Formulations.Journal of Pharmacy Research 2010; 3(12):3090-3092.
4.
Muhammed
Alzweiri, Yusuf M. Al-Hiari, Talal Aburjai, Osama Abdel-Aldaem. On-Column Approach in the HPLC-UV Analysis of Non-chromophoric Compounds
Using Azelaic Acid as a Model. Jordan
Journal of Pharmaceutical Sciences 2012; 5(3): 243-50.
5.
Mahdi Garelnabi, Dmitry Litvinov, Sampath Parthasarathy. Evaluation
of a gas chromatography method for azelaic acid determination in selected
biological samples. North American Journal of Medical Sciences 2010;
2(9): 397-402.
6. Q2A: Text On; Validation of Analytical
Procedures. In: International Conference on Harmonization. Federal Register,
60(40):11260-11262 (1995)
7. Q2B: Text On; Validation of Analytical
Procedures. In: International Conference on Harmonization. Federal Register,
62(96):27463-27467 (1997)
