Development and Validation of Simultaneous Equation Estimation Method For Hamycin And Ketoconazole In Cream Formulation By UV

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Int. J. Pharm. Res. Sci., 2014, 02(1), 60-70.

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Development and Validation of Simultaneous Equation Estimation Method
For Hamycin And Ketoconazole In Cream Formulation By U.V.
Spectrophotometric Method
Sunil Kumar1*, R. K. Nanda1, Dhananjay Babar2, Rajendra Patil2
1. Department of Quality Assurance Technique, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Science
and Research, Pimpri, Pune- 411018
2. Genpharma International Pvt. Ltd., Bhosari, Pune.
*
Corresponding author address:Mr. Sunil Kumar,Quality Assurance Department,Uttaranchal Biotech
Ltd.,Jainagar-3, DineshpurRoad,Rudrapur(U.S.Nagar),Uttarakhand.,India.,Email:pharmacistsk@rediffmail.com
--------------------------------------------------------------------------------------------------------------------------------------bases. It is an imidazole derivative with molecular
Abstract
A simple and accurate UV method has been
weight 531.44.[1,2] It inhibits cytochrome P450
developed for the simultaneous estimation of
dependent lanosterol C14 demethylase, which is
Hamycin and Ketoconazole cream formulation
responsible for production of ergosterol, a necessary
using
SHIMADZU
UV-Visible
1700
component in fungal cell wall synthesis.[3, 4]
spectrophotometer by simultaneous equation
Ketoconazole is a weak base with pKa values of
method, with Acetonitrile: 0.5% w/v Ammonium
2.94 and 6.51.[5] Ketoconazole is an antifungal
acetate (80:20v/v) as a solvent. The absorbance
drug approved by the US FDA in 1981. Only a few
maxima were found to be 381.5 nm for Hamycin
analytical methods for the determination of the drug
and 243.5 nm for Ketoconazole. The percentage
in biological samples and in the presence of other
purity of cream formulation was found to be
drugs have been reported.[6,7,8]
99.08% for Hamycin and 98.22% for Ketoconazole.
This method was also validated by checking the
accuracy, precision, LOQ, LOD and Ruggedness.
The %RSD shows within specification limits. The
linearity profile shows coefficient of variation 0.99
for both drugs.
KEYWORDS
Hamycin, Ketoconazole, Acetonitrile, Ammonium
acetate, UV, Simultaneous
INTRODUCTION
Ketoconazole is Chemically Cis-1-acetyl-4-[4-[2(2,4-dichlorophenyl)-2H-imidazolyl
methyl)-1,3dioxolan-4-yl] methoxy] phenyl]- piperazine is a
topical as well as systemic antifungal agent.
Ketoconazole is practically insoluble in water;
sparingly soluble in strong acid, soluble in strong

Fig No. 1: Structure of Ketoconazole
Hamycin is
a
polyene
antimycotic organic
compound. It is a heptaene antifungal compound
rather similar in chemical structure to amphotericin
B except
that
it
has
an
additional aromatic group bonded to the molecule.
Hamycin
is
obtained
from
a
strain
of Streptomyces bacteria
growing
in
soil
i.e., Streptomyces pimprina. This compound is
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being produced in India by Hindustan Antibiotics
Limited, located at Pimpri, Pune, Maharashtra,
India. It is useful as an antifungal antibiotic drug
for topical as well as systemic mycoses. It is Yellow
amorphous powder, no definite M.P., decompose
after 1600 C. UV max (80% methanol): 383 nm
(E1%1cm916). An amphoteric compd. Almost
insoluble in water, benzene, chloroform, dry lower
aliphatic alcohols, ether. Solution in basic solvents
such as pyridine, and in aqueous lower alcohols. In
conc. H2SO4 gives stable blue color, no coloration
with ferric chloride or with HCl. Hamycin is a rigid,
rod-shaped molecule that kills cells by disrupting
the cell membrane, causing leakage of electrolytes
and small molecules. Hamycin bind to ergosterol,
the major membrane lipid in fungal cells.[7,8]

and Ketoconazole pure form and its formulation can
be freely soluble in water and organic solvents.
Hence Acetonitrile: 0.5% w/v Ammonium acetate
(80:20v/v) was selected as solvent for UV
spectrometric determination.
Preparation of standard stock solutions
Standard stock solutions (100 µg mL-1) of Hamycin
and Ketoconazole were prepared by dissolving
separately 10 mg of drug each in 100 mL
Acetonitrile: 0.5% w/v Ammonium acetate
(80:20v/v). The working standard solutions of these
drugs were obtained by dilution of the respective
stock solution with Acetonitrile: 0.5% w/v
Ammonium acetate (80:20v/v).
Selection of analytical wavelengths
Appropriate dilutions were prepared for each drug
from the standard stock solution and scanned in the
spectrum mode from 400 nm to 200 nm. For
Hamycin and Ketoconazole, 381.5 nm and 243.5
nm were selected as λmax respectively. Fig. No.3
represents the spectra of Hamycin and Fig.No.4
represents the spectra of Ketoconazole.

Fig No. 2: Structure of Hamycin
MATERIALS AND METHODS
A SHIMADZU 1700 Double beam UV-VISIBLE
spectrophotometer with 1.0 cm matching quartz
cells, All the reagents used were of analytical grade
from Rankem, India. Reference standard of
Ketoconazole was supplied as gift sample from
Genpharma International Pvt. Ltd., Pune and
Hamycin was supplied as gift sample from
Hindustan Antibiotic Limited, Pune.
Analytical Method Development
Selection of Solvent
Selection of solvent was based on solubility and
stability of drug in solvent system as well as
extraction of drug from its formulation. Hamycin
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Fig. No. 3: Spectrum of Hamycin
for simultaneous equation method

Fig No.5: Plot of Time Vs.
Absorbance for Hamycin.
Fig. No. 4: Spectrum of Ketoconazole for
simultaneous equation method
Stability of the Drugs in the selected Solvent
The stability of the drugs in the selected solvent was
checked by measuring the absorbance of the drug
solutions at the selected wavelengths. Both the
drugs were found stable in Acetonitrile: 0.5% w/v
Ammonium acetate (80:20v/v). The analyte stability
was studied for 1 hour. Absorbance is constant
during this given time period as shown in Fig. No. 5
and 6 respectively.

Fig No. 6: Plot of Time Vs. Absorbance for
Ketoconazole.
Linearity profile
For each drug, appropriate aliquots of standard
stock solutions were transferred to a series of 10 ml
volumetric flasks. The volume was made up to the
mark with Acetonitrile: 0.5% w/v Ammonium
acetate (80:20v/v) to obtain working standard
solutions of appropriate concentrations. Triplicate
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dilutions of each concentration of the drugs were
prepared separately. The absorbances of the
working standard solutions of each concentration
were measured at the selected analytical
wavelengths in duplicate. The standard calibration
curves of Absorbance Vs Concentration were
plotted using the mean of these six independent

observations. The concentration range over which
the drugs obeyed Beer- Lambert’s law was found to
be between 5-30 g ml-1 for Hamycin and 10-50 g
ml-1 for Ketoconazole respectively. The calibration
curves for Hamycin and Ketoconazole are given in
Fig. No.7 and 8 respectively.

Absorbance

Hamycin at 381.5 nm
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

0.839
0.697
0.563
0.453

y = 0.027x + 0.012
R² = 0.997

0.308
0.141
0
0

5

10

15

20

25

30

35

Concentration (µg/ml)

Fig. No. 7: Standard calibration curve of Hamycin at 381.5 nm
Ketoconazole at 243.5 nm
1.4

Absorbance

1.2

1.201
1.019

1
0.8

y = 0.024x + 0.014
R² = 0.994

0.703

0.6

0.549

0.4
0.247

0.2
0

0
0

10

20

30

40

50

60

Concentration (µg/ml)

Fig. No. 8: Standard calibration curve of Ketoconazole at 243.5 nm.
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Table No.1: Linearity Profile
Parameters

HAMYCIN

KETOCONAZOLE

Beers-Lambert’s range

5-30 g ml-1

10-50 g ml-1

Regression equation

Y=0.027x + 0.012

Y=0.024x + 0.014

Slope*

0.027

0.024

Intercept*

0.012

0.014

Correlation coefficient*

0.997

0.994

*Average of six determinations.
Simultaneous Estimation of Hamycin and
Ketoconazole
Determination of absorbtivity values of drugs at
selected wavelengths
Suitable aliquots of standard stock solutions of
Hamycin and Ketoconazole each were diluted with
Acetonitrile: 0.5% w/v Ammonium acetate
(80:20v/v) to obtain working standard solutions of
concentrations within the Beer-Lambert’s range.
The absorbance of each resulting solution was
measured at 381.5 nm and 243.5 nm.
The Absorbtivity values for Hamycin and
Ketoconazole were calculated from the following
formula:
Absorbtivity 

The standard absorbtivity values of drugs at
the selected wavelengths are shown in Table No.2.
Table No.2: Standard Absorptivity values of
Hamycin and Ketoconazole for simultaneous
equation method
Drug
Absorptivity* (g/lit) at
381.5 nm
243.5 nm
24.7
7.6
Hamycin
30
Ketoconazole 0
*Average of six determinations
Setting up of Simultaneous Equations
Using the mean of standard absorbtivity values,
estimation of both drugs were done by following
simultaneous equation

Absorbance
Conc.(g/lit)

A2 aY1 – A1 aY2
cx

……………. (1)

=

aX2 aY1 – aX1 aY2
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A1 aX2 – A2 aX1
cY

…………....... (2)

=

aX2 aY1 – aX1 aY2
concentration of 10 µg ml-1 of Hamycin and 40 µg
Analysis of the formulations
Cream equivalent to 10 mg of Hamycin and 40 mg
ml-1 of Ketoconazole respectively. The sample
was weighed and dissolved in 100 mL Acetonitrile:
solutions were analyzed as per the procedure for
0.5% w/v Ammonium acetate (80:20v/v) with the
mixed standards. Solutions were scanned in the
aid of ultrasonication for 30 min. The solution was
range of 400 – 200 nm and their absorbances were
then filtered through Whatmann filter paper No.42.
recorded at the selected wavelengths. The
From the above solution 1 ml was taken and diluted
concentrations of each drug in sample solutions
further with Acetonitrile: 0.5% w/v Ammonium
were calculated using equations (1) and (2).
acetate (80:20v/v) up to 10ml to obtain final
Table No.3: Analysis of formulations by Simultaneous Equation method.
Sr. Amount Present
Amount Found
% Amount Found
-1
-1
No. (µg ml )
(µg ml )
Hamycin Ketoconazole Hamycin Ketoconazole Hamycin Ketoconazole
10
40
9.76
39.16
97.6
97.9
1.
10
40
9.83
39.27
98.3
98.18
2.
10
40
9.91
39.19
99.1
97.98
3.
10
40
10.08
39.25
100.8
98.13
4.
10
40
9.83
39.47
98.3
98.67
5.
10
40
10.04
39.39
100.4
98.47
6.

RESULTS AND DISCUSSIONS
The percentage content of both drugs in formulation
Hamycin and Ketoconazole were found to be
99.08% and 98.22% respectively.
Method Validation
Purpose: Validation is a process of establishing
documented evidence, which provides a high degree
of assurance that a specific activity will consistently
produce a desired result or product
meeting its predetermined specifications and quality
characteristics.

Accuracy
Solutions were prepared in triplicate at levels 80%,
100% and 120% of test concentration using
Hamycin and Ketoconazole working Standard as
per the test method and taken absorbance of each
solution in triplicate. The recovery results showed
that the proposed method has an acceptable level of
accuracy for Hamycin and Ketoconazole which is
from 80% - 120% of test concentration is form 98
% - 102 %. Results were shown in table 4 and 5.

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Level of % Mean*
%
HAM
KETO
Recovery

S.D.*
HAM

KETO

HAM

KETO

99.0
98.65
99.04

0.0665
0.0556
0.1670

0.0850
0.1059
0.18

0.3731
0.2818
0.7664

0.1192
0.134
0.2075

80%
100%
120%

98.98
98.40
98.53

% R.S.D.*

Table No. 4: Recovery Studies for formulation

Level of Amount
%
present
Recovery (mg/2gm)
HAM KETO
80%
10
40
100%
10
40
120%
10
40

Amount
of
standard added
(mg)
HAM
KETO
8
32
10
40
12
48

Total
amount % Recovery*
recovered (mg)
HAM
17.82
19.73
21.79

KETO
71.27
78.72
86.71

HAM
99.0
98.65
99.04

KETO
98.98
98.40
98.53

Table No. 5: Statistical Validation for Recovery Studies
*Average of three determinations
The % R.S.D. was found to be less than 2%.
Precision
Repeatability
To check the degree of repeatability of the method,
six samples of the formulations were analyzed as
per the procedure given under formulation. For each
of the developed UV-spectrophotometric methods
the standard deviation and % Relative Standard
Deviation (% R.S.D.) were calculated. The results
of the same are given in Table No.6.

Intermediate precision (Intra-day and Inter-day
precision)
The Intra and Inter-day precision was determined
by assay of the sample solutions on the same day at
different time intervals and on different days
respectively. The S.D. and % R.S.D. were
calculated and are presented in Table No.7.

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Table No. 6: Statistical Evaluation for Repeatability
Drugs
% Mean*

S.D.*

% R.S.D.*

Hamycin

98.90

0.0456

0.46

Ketoconazole

98.40

0.1111

0.28

*Average of six determinations
Table No. 7: Statistical validation for Intermediate Precision
Drug

Intra-day Precision*

%
Mean
98.8
Hamycin
Ketoconazole 98.4

S.D.
0.0753
0.1237

%
R.S.D.
0.76
0.31

Inter-day Precision*
%
Mean
98.6
98.25

S.D.
0.0694
0.1267

%
R.S.D.
0.70
0.32

Ruggedness study
Ruggedness study was performed by changing the analysts and instrument used. The S.D. and % R.S.D. were
calculated and are presented in Table No. 8.

Table No. 8: Ruggedness Study

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Parameter

% Mean*

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S.D.*

HAM

KETO

HAM

Instrument

98.8

98.42

0.0683

Analyst

99.2

98.87

0.0420

Limit of detection (LOD) and Limit of
quantitation (LOQ)
The LOD and LOQ were separately determined
based on the standard deviation of response of the

% R.S.D.*
KETO

HAM

KETO

0.1360

0.69

0.35

0.3365

0.42

0.85

calibration curve. The standard deviation of yintercept and slope of the calibration curves were
used to calculate the LOD and LOQ. The results of
the same are shown in Table No. 9.

Table No. 9: LOD and LOQ
Limit of Detection (LOD)

Limit of Quantitation (LOQ)

Hamycin

Ketoconazole

Hamycin

Ketoconazole

0.2296

0.2712

0.7407

0.875

Conclusion
A simple, accurate, specific, precise, rugged and
economical
UV-spectroscopic
method
(Simultaneous equation method) was developed and
validated for formulation. Linear relationships were
obtained between response and amount of drug
with high correlation coefficients (r2) in the
range 5-30µg mL-1 for Hamycin (r2 =0.997) and
10-50 µg mL-1 for Ketoconazole (r2 =0.994) . The
results of validation studies of the developed
methods suggest that the developed UV
spectrophotometric methods can be employed
successfully for the simultaneous estimation
Hamycin and Ketoconazole in formulation.

Acknowledgements
I express my special thanks to Dr. P. D.
Patil, Vice-Chancellor, Dr. D. Y. Patil University,
Pimpri, Pune and Chairman Dr. D. Y. Patil Vidya
Pratishthan Society, Pimpri, Pune for providing
excellent infrastructural facility for undertaking this
research work.
My sincere thanks to Dr. Sohan S.
Chitlange, Principal, Padm. Dr. D. Y. Patil Institute
of Pharmaceutical Sciences and Research, Pimpri,
Pune for his constant support and guidance.
I owe a deep sense of gratitude and
indebtedness to Mr. Sambhaji Patil, Plant Head,
Genpharma International Pvt. Ltd, Pune
for
providing me with all the excellent facilities for
completion of my research work.
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Words are less to express my deep heartfelt
gratitude to my guide Dr. R. K. Nanda, for his
constant guidance, encouragement with which he
has equipped me to complete this project. I extend
my deepest sense of gratitude for his inspiration and
time he has spared despite his very busy schedule,
will always be a part of immortal reminiscences and
remain idol throughout my life.
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Development and Validation of Simultaneous Equation Estimation Method For Hamycin And Ketoconazole In Cream Formulation By UV