IAJPS 2016, 3 (5), 487-491
G.Soundarya et al
CODEN (USA): IAJPBB
ISSN 2349-7750
ISSN: 2349-7750
INDO AMERICAN JOURNAL OF
PHARMACEUTICAL SCIENCES
Research Article
METHOD DEVELOPMENT AND ANALYTICAL METHOD
VALIDATION OF ITRACONAZOLE BY USING UV- VISIBLE
SPECTROPHOTOMETRY
G. Soundarya*, P. Venkateswara Rao, CH. Chandrika, T. Praveena, S. Seetha Sravani,
U. Renuka Devi, S. Ravi, S. Santhi Kumari
*Department of Pharmaceutical Analysis, Sri Sivani College of Pharmacy, NH-5, Chilakapalem
Jn., Etcherla(M), Srikakulam (Dt), Andhra Pradesh 532402
Abstract:
A simple UV-spectrophotometric method was developed for the determination of Itraconazole in pure and its
pharmaceutical formulations. Itraconazole exhibited maximum absorption at 261nm in Methanol and obeyed
linearity in the concentration range of 2.5-25 g/ml. The proposed method was statistically validated.All the
proposed methods are simple, selective, reproducible, sensitive and accurate with good precision. Some of the
methods were proved to be superior to most of the reported methods. All these proposed methods for estimation of
selected drugs such as Itraconazole were successfully applied either in bulk or pharmaceutical formulations. The
proposed methods can be used as alternative methods to the reported ones for the routine determination of selected
drugs under the study in bulk and pharmaceutical dosage forms.
Keywords: Itraconazole, UV-Visible Method, Methanol.
Corresponding Author:
G. Soundarya,
QR code
Department of Pharmaceutical Analysis,
Sri Sivani College of Pharmacy,
NH-5, Chilakapalem Jn., Etcherla(M),
Srikakulam (Dt), Andhra Pradesh 532402
Cell.No:+918099186202
Email:[email protected]
Please cite this article in press as G. Soundarya et al, Method Development and Analytical Method Validation
of Itraconazole by Using UV- Visible Spectrophotometry, Indo Am. J. Pharm. Sci, 2016; 3(5).
www.iajps.com
Page 487
IAJPS 2016, 3 (5), 487-491
INTRODUCTION:
The UV-visible spectrophotometric methods which
fall in the wavelength region 200-800 nm and
fluorimetric methods (may fall in UV & Visible
regions) are very simple, cheap & easy to carry out
estimations of drugs in bulk form and their
formulations. The limitations of many colorimetric or
fluorimetric methods of analysis lie in the chemical
reaction upon which the procedures are based
rather than the instruments available. Many of the
reactions involve color or fluorescence of a particular
drug are quite selective or can be rendered selective
through the introduction of masking agents, control
of pH, use of solvent extraction technique,
adjustment of oxidation states or by prior removal of
interfering
ingredients
with
the
aid
of
chromatographic separate[1-3].
Itraconazole is a potent triazole antifungal agent that
is prescribed to patients with fungal infections used
for the treatment of mycoses. The drug may be given
orally or intravenously.1-3 The IUPAC nomenclature
of the drug is as follows: (2R,4S)-rel-1- (butan-2-yl)4-{4-[4-(4-{[(2R,4S)-2-(2,4-dichlorophenyl)-2-(1H1,2,4-triazol-1-ylmethyl)-1,3-ioxolan-4-yl]methoxy}-phenyl)-piperazinyl]phenyl}-4,5-dihydro1H-1,2,4-triazol-5-one.ITZ is used orally in the form
of capsules for treatment of dermatophyte infections,
occurrence of superficial fungal infections and
systemic fungal infections. For quality control and
stability testing of Itraconazole in pharmaceutical
formulations, limited methods have been published,
because the drug is not yet official in any
pharmacopoeia. Spectrofluorimetry method has been
used for assay of Itraconazole in raw material and in
dosage forms. RP-HPLC [4-6] method is used for
determination of Itraconazole in human plasma.4separation in this method was performed on
anoctadecylsilane column using fluorescence
detector. However, it has the disadvantage of being
time consuming. All these studies have furth
emphasized the need to perform rapid and sensitive
G.Soundarya et al
ISSN 2349-7750
quality-control
analysis
of
pharmaceutical
formulations containing Itraconazole. As these
methods are expensive, we have made an attempt to
develop a more precise, simple and economical
spectrophotometric method with greater precision,
accuracy and sensitivity for the analysis of
Itraconazole in bulk and dosage forms [7].
MATERIALS AND METHODS:
Itraconazole was obtained as gift sample from Elite
chemicals and all reagents were purchased from SD
Chemicals Chennai. All materials and reagents used
were in analytical grade.
Method Development
A simple UV-Visible Spectrophotometric method
was developed for the determination of Itraconazole
in pure and its pharmaceutical formulation.
Itraconazole exhibiting maximum absorbance at
261nm in Methanol and obeyed linearity in the
concentration range of 2.5 to 25 g/ml. The proposed
method was statistically validated.
Instrumentation:
Analytical technologies ltd, T60 UV-Visible
Spectrophotometric method was performed with 1cm quartz cells
Selection of Solvent:
Methanol was selected an ideal solvent for
spectrophotometric analysis of Itraconazole.
Scanning and Determination of Maximum
Wavelength (lmax)
In order to ascertain the wavelengths of maximum
absorption (max) of the drug, different solutions of
the drug (10g/ml and 20g/ml) in Methanol were
scanned using UV-Visible spectrophotometer within
the wavelength region of 200380nm against
Methanol as blank. The resulting spectrum was
presented in Fig 1 and and the absorption curve
showed characteristic absorption maximum at 261
nm for Itraconazole.
Fig1: Absorption curve for Itraconazole ( max = 261nm)
www.iajps.com
Page 488
IAJPS 2016, 3 (5), 487-491
G.Soundarya et al
Preparation of Stock Solution
Standard stock solution of Itraconazole was prepared
by dissolving 10mg of Itraconazole drug in 10ml of
Methanol in 10ml of volumetric flask to get a
concentration of 1mg/ml (1000mg/ml) solutions.
Preparation of Working Standard Solutions and
construction of standard graph
The prepared stock solution was further diluted with
Methanol to get working standard solutions of
10mg/ml and 100mg/ml. To construct Beers law plot
for Itraconazole different aliquots of Itraconazole
were taken and diluted to 10 ml with Methanol to get
the working standard solutions as shown in the table
8.1. The absorbances of each solution were measured
at lmax 261 nm against Methanol as blank. The
results were shown in table. The standard graph for
Itraconazole was plotted by taking concentration of
ISSN 2349-7750
drug on x-axis and absorbance on y-axis and was
shown in Fig 8.1. The drug has obeyed Beers law in
the concentration range of 2.5-20mg/ml.
Estimation of Itraconazole in commercial
formulations
For analysis of commercial formulations, 10 capsules
containing Itraconazole were taken and powdered.
The powder equivalent to 0.047g of Itraconazole
was taken in a 10ml volumetric flask, containing 7ml
of Methanol and sonicated for 30 minutes. The
volume was made up to 10ml with Methanol and
filtered to get a solution of concentration 1000g/ml.
This was further diluted with Methanol to get a
concentration within the linearity range and the
absorbances were measured against the blank at
261nm.
Table 1 : Linearity table of Itraconazole (pure drug) in methanol at 261nm
S.No
Concentration
Absorbance
(g/ml)
1
2.5
0.114
2
5
0.224
3
7.5
0.345
4
10
0.442
5
12.5
0.569
6
15
0.672
7
17.5
0.78
8
20
0.856
Absorbance
Fig 2: Linearity graph of Itraconazole
1
0.8
0.6
0.4
0.2
0
y = 0.043x + 0.008
R = 0.999
10
20
Concentration
30
Table 2: Optical characteristics of proposed method.
Parameter
lmax (nm)
Beers Law limit (mg/ml)
Regression equation (Y)
Slope (a)
Intercept (b)
% Range of error
95% confidence limits
99% confidence limits
Correlation co-efficient
www.iajps.com
Itraconazole
261
2.5-25
0.050x-0.053
0.050
0.053
0.0021
0.0028
0.999
Page 489
IAJPS 2016, 3 (5), 487-491
G.Soundarya et al
ISSN 2349-7750
To determine the accuracy of the proposed method,
Validation
recovery studies were carried out by adding different
Precision
amounts (80%, 100% and 120%) of bulk samples of
The precision of the proposed method was
Itraconazole within the linearity range were taken and
ascertained by actual determination of six replicates
added to the pre-analyzed formulation of
of fixed concentration of the drug within the Beers
range and finding out the absorbances by the
concentration 10mg/ml. From that percentage
proposed method. From this absorbances Mean,
recovery values were calculated. The results were
Standard deviations, %R.S.D were calculated. The
shown in Table 5.
readings were shown in Table 4.
Accuracy
Table 3: Amount of Itraconazole in formulation by proposed method.
Formulation
Sl.No.
1.
Itromed-100
Drug
Labeled
Amount mg
Itraconazole
47
Table 4: Precision data
Concentration (g/ml)
S.no
Observed
Amount
mg MeanSD
44.9020.293
% Recovery
98.546
Absorbances (nm)
10
0.413
10
0.415
10
0.411
10
0.412
10
0.411
10
0.413
Mean
0.4125
S.D
% R.S.D
0.001751
0.070776
Table 5: Accuracy data
ACCURACY
Conc (bulk)
conc(formln)
Abs
%rec
10
0.726
5.872
10
0.729
5.895
10
0.728
5.887
10
10
0.836
6.710
10
10
0.839
6.737
10
10
0.838
6.729
12
10
0.912
7.296
12
10
0.919
7.349
12
10
0.918
7.345
www.iajps.com
Mean
Sdv
%rsd
5.88
0.011676
0.416
6.725
0.013868
0.377
7.329
0.028792
0.369
Page 490
IAJPS 2016, 3 (5), 487-491
SUMMARY:
Pharmaceutical analysis simply means analysis of
pharmaceuticals. Today pharmaceutical analysis
entails much more than the analysis of active
pharmaceutical ingredients or the formulated product.
The pharmaceutical industry is under increased
scrutiny from the government and the public
interested groups to contain costs and at consistently
deliver to market safe, efficacious product that fulfill
unmet medical needs. The pharmaceutical analyst
plays a major rule in assuring identity, safety,
efficacy, purity, and quality of a drug product. The
need for pharmaceutical analysis is driven largely by
regulatory requirements. The commonly used tests of
pharmaceutical analysis generally entail compendia
testing method development, setting specifications
and method validation. Analytical testing is one of
the more interesting ways for scientists to take part in
quality process by providing actual data on the
identity, content and purity of the drug products. New
methods are now being development with a great
deal of consideration to worldwide harmonization. As
a result, new products can be assured to have
comparable quality and can be brought to
international markets faster.
Pharmaceutical analysis occupies a pivotal role in
statuary certification of drugs and their formulations
either by the industry or by the regulatory authorities.
In industry, the quality assurance and quality control
departments play major role in bringing out a safe
and effective drug or dosage form. The current good
manufacturing practices (CGMP) and the Food Drug
Administration (FDA) guidelines insist for adoption
of sound methods of analysis with greater sensitivity
and reproducibility. Therefore, the complexity of
problems encountered in pharmaceutical analysis
with the importance of achieving the selectivity,
speed, low cost, simplicity, sensitivity, specificity,
precision and accuracy in estimation of drugs.
UV-Spectrophotometric method development:
A simple UV-spectrophotometric method was
developed for the determination of Itraconazole in
pure and its
pharmaceutical
formulations.
Itraconazole exhibited maximum absorption at
261nm in Methanol and obeyed linearity in the
concentration range of 2.5-20 g/ml. The proposed
method was statistically validated.
All the proposed methods are simple, selective,
reproducible, sensitive and accurate with good
precision. Some of the methods were proved to be
superior to most of the reported methods. All these
proposed methods for estimation of selected drugs
such as Itraconazole were successfully applied either
in bulk or pharmaceutical formulations.
The proposed methods can be used as alternative
methods to the reported ones for the routine
www.iajps.com
G.Soundarya et al
ISSN 2349-7750
determination of selected drugs under the study in
bulk and pharmaceutical dosage forms.
CONCLUSION:
The proposed method was simple, sensitive and
reliable with good precision and accuracy. The
proposed method is specific while estimating the
commercial formulations without interference of
excipients and other additives. Hence, this method
can be used for the routine determination of
Itraconazole in bulk samples and Pharmaceutical
formulations.
ACKNOWLEDGEMENT:
The author was very thankful to Management and
Principal of Sri Sivani College of Pharmacy for
providing all necessary material and equipments for
carryingout of the research work.
REFERENCES:
1.Rang HP, Dale MM, Ritter JM and Flower RJ.
Pharmacology. 6th Edn. Elsevier publication house,New
Delhi; 2003, 666-671.
2.Joel GH, Goodman and Gilmans the Pharmacological
basis of therapeutics. 10th Edn. McGraw hill publishers,
New York; 2001, 356-362.
3.Tripathi KD, Essential of Medical Pharmacology. 5th
Edn. Jaypee brothers medical publishers. New Delhi;
2003, 245-249.
4.Al-Rawithi H, Sameer M, Hussein A, Rajaa Al-Moshen,
Ibrahim, Raines, Dale, et al. Determination of Itraconazole
and Hydroxyitraconazole in Plasma by High- Performance
Liquid Chromatography with Fluorescence Detection,
Therapeutic Drug Monitoring, 2001; 23, 445-448.
5.Khoschsorur G, Frueehwirth F, Zelzer S. Isocratic High
Performance Liquid Chromatographic Method with UV
detection for simultaneous determination of voriconazole
and itraconazole and its hydroxyl metabolite in human
serum. Antimicrobial agents and Chemotherapy, 2005; 49,
3569-3571.
6.Srivatsan V, Dasgupta A, Kale P, Datla R, Soni D, Patel
M, Patel R, and Mavadhiya, C. Simultaneous determination
of itraconazole and hydroxyitraconazole in human plasma
by highperformance liquid chromatography, Journal of
Chromatography, 2004; 1031, 307 313.
7.El-Enany N, El-Sherbilny D, Belal F. Spectrofluorimetric
Determination of Itraconazole in Dosage Forms and Spiked
Human Plasma. Journal of the Chinese Chemical Society,
2007; 54, 375-382.
Page 491
