UV
Spectrophotometric Method Development and Validation of Fluticasone
Propionate
Rajashri R. Kulkarni1*, Dipti G. Phadtare2, Ravindra
B. Saudagar2
1Department of Quality Assurance Techniques, R. G. Sapkal College of Pharmacy, Anjaneri,
Nashik.
2Department of Pharmaceutical Chemistry, R. G. Sapkal College of Pharmacy, Anjaneri,
Nashik.
*Corresponding
Author E-mail: kulkarniraj1993@gmail.com
Analytical methods development and
validation play important roles in the discovery, development, and manufacture
of pharmaceuticals. Simple, precise and accurate UV spectroscopic method has
been developed and validated for estimation of Fluticasone
propionate. It is a selective agonist at the glucocorticoid
receptor. UV spectroscopic method which is based on measurement of absorption
of UV light, the spectra of Fluticasone propionate in
methanol showed maximum wavelength at 236nm and calibration curve were plotted
over the concentrations ranging from 2-22ug/ml of Fluticasone
propionate with correlation coefficient 0.9812 validation was performed as per
ICH Q2 (R1) guidelines for linearity, accuracy, precision and recovery. The
proposed method was validated.
KEYWORDS:
INTRODUCTION:
Chemically Fluticasone
propionate is S-(fluoromethyl) 6α, 9-difluoro-11 β-hydroxy-16α- methyl-3-oxo-17α-(1-oxopropoxy)
androsta-1,4-diene-17-carbothioate (Cutivate).. It is
a neutral, highly potent trifluorinated
corticosteroid based on the androstane nucleus. Fluticasone propionate is highly selective agonist at the glucocorticoid receptor with the negligible activity
androgen, estrogen or mineralocorticoid receptor,
thereby producing anti-inflammatory and vasoconstriction effect. It has been
shown to have a wide range of inhibitory effect on multiple cell types, e.g.
mast cell, eosinophills, neutrophills,
leukotrines and cytokinins
involves in inflammation.
Fluticasone mimics the naturally occurring hormone
produced by the adrenal glands, cortisol or
hydrocortisone. It is 3 to 5 fold more potent than dexamethasone
in receptor binding assays[1,2].
Fig.1 Chemical structure of Fluticasone propionate
Fluticasone propionate appears as a white or almost
white powder having 500.6gm. it is practically insoluble in water, sparingly
soluble in methylene chloride and slightly soluble in
alcohol and acetonitrile and soluble in dimethyl sulfoxide. A simple and
precise method was developed and validated by using solvents like methanol and acetonitrile respectively[3,4].
Instrumentation:
Double beam UV- visible
spectrophotometer, Make: JASCO spectrophotometer, model JASCO V630 connected to
a computer loaded with spectra manager software was used for all the
spectrophotometric measurements in all proposed spectrophotometric methods. It
consists of pair of 10 mm matched quartz cells was used for experiments. The
absorption spectra of reference and test solution were carried out in a 1 cm
quartz cell over the range 200-400 nm.
MATERIALS AND METHODS:
Preparation of stock solution of Fluticasone
propionate:
Standard Fluticasone
propionate of 10 mg was accurately weighed and transferred into a 100 ml volumetric
flask. Add small quantity of methanol in volumetric flask and dissolve the drug
into it and then adjust the volume upto 100 ml this
solution form 100 ppm of concentration and then
prepared further dilutions.
Selection of analytical concentration ranges:
From the standard stock
solutions of Fluticasone propionate (100 ug/ml), appropriate aliquots of 2 ppm,
4 ppm, 6 ppm, upto 24ppm.
Determination of λmax:
10ug/ml solution of Fluticasone propionate was prepared and scanned in UV range
of 200-400nm and spectrum was obtained. The λmax
was found to be at 249nm wavelength where absorbance was maximum at this
wavelength.
Validation parameters for Fluticasone
propionate:
Accuracy:
Accuracy is the closeness of
the results obtained by the method to the true value. This study can be done
with the help of preparing three concentrations of 14, 16 and 18 ppm each respectively.
Precision:
The precision of an
analytical method is the degree of agreement among individual test results,
when the method is applied repeatedly to multiple sampling of homogeneous
samples. It provides an indication of random error results and is expressed as
relative standard deviations [% RSD].
Robustness:
The solution were prepared
and analysed with change in the analytical conditions
like change in wavelength, change in solvent etc. This can be studied with the
help of using minimum six concentrations. Absorbance can be calculated using UV
spectrophotometer.
Limit of detection [LOD] and limit of quantitation [LOQ]:
The sensitivity of the
proposed method for the measurement of Fluticasone
propionate was estimated in terms of limit of detection [LOD] and limit of quantitation [LOQ]. The LOD and LOQ were calculated by
using the slope and SD of response [intercept]. The mean slope value and the SD
of response were obtained from the calibration curve. The LOD and LOQ
calculations were done and reported.
Linearity, Range and Calibration:
The method can be validated
by using ICH guidelines. The linearity of analytical method is its ability to
elicit test results that are directly proportional to the concentration of analyte in the sample within the range. The range of the
analytical method is the interval between the upper and the lower levels that
have been demonstrated to be determined within a suitable level of precision,
accuracy and linearity.
EXPERIMENTATION:
Table 1. Calibration of Fluticasone propionate-
|
Sr.
no. |
Concentration
(ppm) |
Absorbance |
|
1 |
2 |
0.2918 |
|
2 |
4 |
0.3288 |
|
3 |
6 |
0.4796 |
|
4 |
8 |
0.5800 |
|
5 |
10 |
0.6450 |
|
6 |
12 |
0.6779 |
|
7 |
14 |
0.7844 |
|
8 |
16 |
0.8354 |
|
9 |
18 |
0.9374 |
|
10 |
20 |
0.9618 |
|
11 |
22 |
1.0090 |
Fig.2 Calibration curve of Fluticasone propionate in methanol
Table 2. Accuracy-
|
Sample % |
Amount of Standard Drug
Added ( μg/ml) |
Absorbance |
Absorptivity |
[x-x*]2 |
Standard Deviation |
% Relative Standard
Deviation |
|
80 |
14 14 14 |
0.7834 0.7969 0.7844 |
5.5957 5.6921 5.6028 |
0.001190 0.003831 0.000750 |
0.05371 |
0.0095 |
|
100 |
16 16 16 |
0.8520 0.8253 0.8354 |
5.3250 5.1581 5.2212 |
0.00815 0.00586 0.000182 |
0.08423 |
0.0160 |
|
120 |
18 18 18 |
0.9374 0.9210 0.9366 |
5.2077 5.1166 5.2033 |
0.001017 0.003504 0.000756 |
0.05136 |
0.00992 |
Table 3. Precision-
|
Concentration % |
Absorbance
Mean |
Standard
Deviation |
%
Relative Standard Deviation |
|
Intraday Precision(n=6) |
|||
|
14 |
0.7312 |
0.3824 |
0.07335 |
|
14 |
0.7204 |
0.3795 |
0.07281 |
|
14 |
0.7001 |
0.3741 |
0.07178 |
|
14 |
0.7322 |
0.3826 |
0.07340 |
|
14 |
0.7681 |
0.3919 |
0.07518 |
|
14 |
0.7270 |
0.3813 |
0.07314 |
|
Interday Precision(n=6) |
|||
|
14 |
0.7834 |
0.3958 |
0.07077 |
|
14 |
0.7969 |
0.3992 |
0.07137 |
|
14 |
0.7844 |
0.3960 |
0.07081 |
|
14 |
0.7813 |
0.3952 |
0.07067 |
|
14 |
0.7842 |
0.3960 |
0.07080 |
|
14 |
0.7681 |
0.3919 |
0.07007 |
Table 4. Change in
wavelength:
|
Sr.
No. |
Concentration [ppm] |
Absorbance
|
Difference
|
[d-d*]2 |
Standard
deviation |
|
|
|
|
236nm |
240nm |
|
|
|
|
1. |
10 |
0.6450 |
0.6069 |
0.0381 |
0.00000132 |
|
|
2. |
12 |
0.6779 |
0.6373 |
0.0406 |
0.0000136 |
|
|
3. |
14 |
0.7844 |
0.7681 |
0.0163 |
0.000424 |
0.01334 |
|
4. |
16 |
0.8354 |
0.8620 |
0.0266 |
0.000106 |
|
|
5. |
18 |
0.9374 |
0.8884 |
0.0490 |
0.000146 |
|
|
6. |
20 |
0.9618 |
0.9107 |
0.0511 |
0.000201 |
|
RESULT AND DISCUSSION:
The method can be validated
using ICH guidelines in order to determine linearity, range, accuracy,
precision and robustness of the method.
The standard stock solution
of Fluticasone propionate was prepared and this can
be scanned from 200-400 nm and absorption spectra were recorded at 236nm
wavelength in UV spectrophotometer using methanol as a solvent.
Linearity:
The absorbance of the
solutions of Fluticasone propionate was determined at
a wavelength 236nm. The correlation coefficient was found to be 0.9812 and the
regression equation was found to be Y= 0.037x +0.2406 and this can be shown in
table no. 1.
Precision:
Precision can be done for all
type of drugs and the limit of RSD value was found to be not more than 2. Here
RSD was found to be 0.070 to 0.071 and 0.073 to 0.075 and this can be shown in
table no. 3.
Accuracy:
Accuracy can be determined by
performing recovery studies at 3 levels that is taking 3 different
concentrations which has replicates and this can be studies using absorbance.
Limit of detection and limit of quantitation:
The parameters LOD and LOQ
were determined on the basis of response and slope the regression equation LOD
and LOQ values are 0.4595 and 1.3925.
Limit of detection:
LOD = 3.3 * Standard
deviation
Slope
= 0.4595
Limit of quantitation:
LOQ = 10 * Standard
deviation
Slope
= 1.3925
CONCLUSION:
According to ICH guidelines
slope, intercept and correlation coefficient can be calculated using
calibration curve were shown in fig.1.the results show that the methods are
reasonable precise. The developed UV spectrophotometric method was found to be
simple, economic, easy, accurate, precise, reproducible and highly sensitive
and can be used for routine estimation of Fluticasone
propionate in bulk.
Table 5. Summary of UV
spectrophotometric method validation:
|
PARAMETERS |
RESULTS |
|
Λmax |
236nm |
|
Range |
2-22ug/ml |
|
Correlation coefficient |
0.9812 |
|
Regression equation |
Y= 0.037x +0.2406 |
|
Slope [m] |
0.037 |
|
Intercept [c] |
0.2406 |
|
Precision [%RSD] |
0.070 – 0.075 |
|
LOD |
0.4595 |
|
LOQ |
1.3925 |
|
Accuracy [%RSD] |
0.0160 for 100% |
ACKNOWLEDGEMENTS:
The authors are grateful to
R. G. Sapkal College of Pharmacy for providing
necessary research facilities to carry out the research work and to Glenmark Pharmaceuticals, India for providing the gift
sample of the drug.
REFERENCES:
1. John H. Block,
John M. Beale, Wilson and Gisvolds textbook of
organic medicinal and pharmaceutical chemistry, 11th edition,
Lippincott Williams and Wilkins, page no. 812 -813.
2. Clarks analysis of
drugs and poisons, 3rd edited by Anthony Moffat, volume 2. Page no.
1057.
3. Remington the
science and practice of pharmacy, 21st edition volume 1. Lippincott
Williams and Wilkins. Page no. 643.
4. US 797262626, July
5, 2011. Inventor- Imtiaz Chaudry,
having patent on Fluticasone propionate
pharmaceutical nasal formulation.
Received on 17.03.2016 Accepted on 25.04.2016
© Asian Pharma
Press All Right Reserved
Asian J. Res.
Pharm. Sci. 2016; 6(2): 135-138
DOI: 10.5958/2231-5659.2016.00019.9