Development
and Validation of UV Spectroscopic Method for Simultaneous Estimation of Doxofylline and Terbutaline Sulphate in Combined Dosage Form
Patel Juhee J.*, Mrs. Henal
Chorawala, Dr. Zarna R. Dedania, Dr. S.M. Vijendraswamy
Bhagwan Mahavir College of Pharmacy, (215) Sr. No. 149, Near Ashirwad Villa, New City Light Road,
B/H Heena Bunglow’s, Vesu, Bharthana, Surat-395017,
Gujarat.
*Corresponding Author E-mail: dollypatel26791@gmail.com
ABSTRACT:
Two simple spectrophotometric methods have been developed for
simultaneous estimation of Doxofylline and Terbutaline sulphate in tablet
dosage form. The method is developed using 0.1M NaOH
as a solvent. Method (I) is the Q- absorbance ratio method, two wavelengths are
selected, one being the isoabsorptive point 294.1 nm
and other being the absorption maxima of Doxofylline
at 273 nm. Method (II) is the Absorbance correction method, two wavelengths are
selected, one being absorption maxima of Doxofylline
at 273 nm and other being the corrected absorbance at 302.96 nm. The Linearity
was observed in the concentration range of 10-50 µg/ml for Doxofylline
and 2-8 µg/ml for Terbutaline sulphate
respectively, correlation coefficient (r 2 < 1). The accuracy and precision
were determined and found to comply with ICH guidelines. Both the methods
showed good reproducibility and recovery with % RSD in the desired range. The
proposed methods can be successfully applied for the routine analysis of both
the drugs from tablet dosage form.
KEYWORDS: Doxofylline, Terbutaline sulphate, Q- absorbance ratio method, Absorption
correction, Beer–Lambert’s law, UV visible spectrophotometric.
INTRODUCTION:
Spectrophotometric methods
are a large group of analytical methods that are based on atomic and molecular
spectroscopy. Spectroscopy is a branch of science dealing with the study of
interaction of electromagnetic radiation with matter. The kind and amount of
radiation absorbed by the molecule depends on the number of molecule
interacting with the radiation. [1, 2]
A fixed dose combination of Doxofylline and Terbutaline sulphate is available for the treatment of asthma.
Chemically DOXO known as 7-(1,3-dioxolan-2-ylmethyl)-1,3-dimethyl- 3,7-dihydro-1H-purine-2,6-dione
and TBS known as 5-[2-(tert-butylamino)-1-hydroxyethyl]- benzene-1,3-diol sulfate
(2:1) (salt). Doxofylline is a new methyl xanthine derivative used in obstructive airway diseases.
Doxofylline has significantly fewer side effects,
making the drug immensely beneficial to the patients. Terbutaline
sulphate is widely used as a bronchodilator for the
treatment of bronchial asthma, chronic bronchitis, and emphysema. Terbutaline sulphate stimulates
the α-adrenergic
receptors of the sympathetic nervous system and has little or no effect on the
adrenergic receptors. [3]
Figure 1: Structure of Doxofylline
Figure 2: Structure of Terbutaline sulphate
In
February 2009, a fixed-dose combination of Doxofylline
and Terbutaline sulphate
was approved by DCG (I) in India. Co-administration of Doxofylline
with Terbutaline sulphate
gives better bronchodilation with a lower degree of
skeletal muscle tremor than a higher dose of terbutaline
sulphate by mouth alone. Therefore, a fixed-dose
combination of Doxofylline and Terbutaline
sulphate is a better
alternative for the treatment of acute and chronic asthma.
As per literature review RP-HPLC method, HPLC method and
simultaneous equation method using UV spectroscopy have been reported for the
estimation of Doxofylline and Terbutaline
sulphate in combination of other drugs. No any other spectroscopic method
published and reported for simultaneous estimation of Doxofylline
and Terbutaline sulphate
combined dosage form as per the literature survey. [4-7]
So, best attempt will be to achieve a simple, sensitive, accurate and
cost-effective UV spectrophotometric methods (Q- absorbance ratio method and
absorbance correction method) of Doxofylline and Terbutaline sulphate in their
combined dosage form and to validate the as per ICH guidelines.[8]
MATERIALS AND
METHODS:
Materials:
Pure Doxofylline
was obtained from Ami Pharmaceutical, Vadodara and
pure Terbutaline sulphate
was obtained from Brundavan Laboratories, Hyderabad
as a gift sample. Methanol, sodium hydroxide were
purchased from Merck Chemicals, India.
Tablet formulations, namely OXOBIT-TR and PHYLEX-TR (Lexus) were purchased from
a local market. The marketed formulations have a composition of 400 mg of Doxofylline, 5 mg of Terbutaline sulphate and excipients (q.s).
Instrument:
UV-visible
double beam spectrophotometer (Shimadzu, 1800, Japan) with matched quartz cells
corresponding to 1 cm path length.
Preparation of 0.1N NaOH [9]:
0.1N NaOH
was prepared by dissolving 4 gm Sodium Hydroxide pellets in 1000 ml volumetric
flask using distilled water and then volume was make up by distilled water.
Preparation of standard stock solution:
Standard stock solutions
(1000 µg/ml) of DOXO and TBS were prepared separately by dissolving 100 mg
of DOXO and TBS, respectively in 100 ml
0.1N NaOH.
Preparation of working standard solution
of Doxofylline and Terbutaline sulphate:
100
µg/ml of DOXO
and TBS solution was prepared by diluting 10 ml stock solution up to 100 ml with
0.1 N NaOH The solutions were prepared by pipetting out 1, 2, 3, 4, 5 ml of the working standard
solution of DOXO (100 µg/ml) and 0.2,
0.4, 0.6, 0.8, 1 ml of the working standard solution of TBS (100 µg/ml) into a
series of 10.0 ml volumetric flasks. This series consisted of five
concentrations of standard DOXO solution ranging from 10 – 50 μg/ml and TBS solution ranging from 2- 10 μg/ml.
Preparation of sample solution:
Twenty
tablets were weighed and powdered. Powder equivalent to 80 mg of DOXO was weighed
and transferred into a 100 ml of volumetric flask, volume adjusted up to mark
with 0.1 N NaOH. The mixture sonicated
for 10 minutes and filtered through Whatman filter paper no.42, discarding
first few ml of filtrate. 1ml of this filtrate diluted to 10 ml with 0.1 N NaOH Further 1 ml diluted to 10 ml of 0.1 N NaOH. The solution was scanned against 0.1 N NaOH as blank in a range of 200-400 nm with medium scan
speed. The spectrum was obtained. The concentration of DOXO and TBS can be
obtained by using equation of straight line.
Calibration Curve Procedure:
Aliquots of standard stock
solutions of DOXO and TBS were taken in volumetric flasks and diluted with 0.1N
NaOH to get final concentrations in range of 10-50
µg/ml for DOXO and 2-8 µg/ml of TBS. The solution was scanned in the range of
200 to 400 nm against 0.1N NaOH as blank, the
wavelengths were found to be 273 nm λmax of
DOXO and iso-absorptive point of DOXO and TBS at
294.1nm for Method I and 273 nm λmax of
DOXO and corrected absorbance of TBS at 302.96 nm for method Method II
Method I:
Q- absorbance ratio method:
Absorbance ratio method uses
the ratio of absorbances at two selected wavelengths
one at iso-absorptive point and other being the λmax of one of the two components. In Absorbance ratio
method overlain spectra showed that, DOXO and TBS shown isobestic
absorbance point at 294.1 nm wavelength. Weres TBS
and DOXO has same absorbance at 273 nm which is λmax of
DOXO, at this wavelength, both drugs shows considerable absorbance. So selected wavelength were 273 nm and 294.1 nm.
Relative concentration of two
drugs in the sample was calculated using following equations.
Cx = [(QM-Qy)/
(Qx-Qy)] × A1/ax1
Cy = [(QM-Qx)/ (Qy-Qx)] × A1/ay1
Figure 3: Overlain UV
spectra of Doxofylline (50 μg/ml)
and Terbutaline sulphate (10 μg/ml)
Figure 4: Overlain uv spectra of Doxofylline
(50 μg/ml) and Terbutaline
sulphate (10 μg/ml)
Method II:
Absorbance correction method:
In absorbance correction
method overlain spectra showed that, DOXO has zero absorbances
at 302.96 nm Weres TBS has substantial absorbance.Thus TBS was estimated directly at 302.96 nm
without interference of DOXO At 273 nm which is λmax of DOXO, at this
wavelength, both drugs shows considerable absorbance. So selected wavelength
were 273 nm and 302.96 nm
Method validation:
Linearity and range:
The
linearity of response was determined in concentration range of 10-50 μg/ml DOXO and 2-10 μg/ml
TBS. The calibration curve of absorbance vs
concentration plotted, correlation coefficient and regression line equations
for DOXO and TBS were obtained using Microsoft excel. Linearity is expressed in
terms of correlation co-efficient of linear regression line.
Precision:
Variations
of results within the same day (intraday), variation of the results between
days (inter day) were analyzed and its %RSD for each observation was
calculated. For Intraday and Interday precision, Synthetic mixture of drugs
containing DOXO (μg/ml) and TBS (μg/ml)
equivalent to 20 DOXO:4 TBS, 30 DOXO:6 TBS, 40 DOXO:8 TBS were determined 3 times a day interval of 1 hour, and different day
simultaneously and %RSD was calculated.
For
Intraday and Interday precision, three levels of assay were selected. At each
level three times assay was carried out and result was expressed as %RSD.
Accuracy:
Accuracy
of the method was confirmed by recovery study from marketed formulation at
three level of standard addition. Percentage recovery for DOXO and TBS were
found out. Recovery between 98%-102% justifies the accuracy of the method. Accurately
weighed quantity of pre analysed tablet 80 mg of DOXO
and 1 mg of TBS was taken in 100 volumetric flask. To
above flask API of both drug in 80%, 100%, and 120%
were added and continued assay procedure.
LOD and LOQ:
The sensitivity of the method
was determined with respect to limit of detection (LOD) and limit of quantitation (LOQ). The LOD was calculated by below eq.
LOD:
3.3
LOQ:
10 
RESULTS AND DISCUSSION:
A simple, economic, precise,
accurate method for simultaneous estimation of Doxofylline
and Terbutaline sulphate
was developed. This developed method was validated according to ICH guidelines.
Linearity and range:
For Method I:
|
Figure 5: Calibration curve for Doxofylline
(10-50μg/ml) at 273 nm and
294.1 nm
|
Figure 6: Calibration curve for Terbutaline
sulphate (2-10 μg/ml) at 273 nm and 294.1 nm
|
For Method II:
|
Figure 7: Calibration curve for Doxofylline
(10-50 μg/ml) at 273nm
|
Figure 8: Calibration curve for Terbutaline sulphate (2-10 μg/ml) at 273 nm and
302.93 nm
|
Table 1: Optical parameters and regression characteristic for Doxofylline and Terbutaline sulphate
|
|
Method I
|
Method II
|
|
parameters
|
DOXO
|
TBS
|
DOXO
|
TBS
|
|
273 nm
|
294.1 nm
|
273 nm
|
294.1 nm
|
273 nm
|
273 nm
|
302.93nm
|
|
r2
|
0.999
|
0.999
|
0.998
|
0.998
|
0.999
|
0.998
|
0.998
|
|
equation
|
Y=0.034X-
0.033
|
Y=0.005X+
0.024
|
Y=0.003X+
0.059
|
Y=0.027X+
0.022
|
Y=0.034X-
0.033
|
Y=0.008X+
0.032
|
Y=0.010X+
0.05
|
|
absorptivity
|
321.4
|
45.37
|
171
|
229.01
|
321.43
|
162.43
|
218.84
|
Table 2: Results of Accuracy
|
|
Method I
|
Method II
|
|
DOXO
|
TBS
|
DOXO
|
TBS
|
|
Amount Taken (µg/ml)
|
80
|
1
|
80
|
1
|
|
Amount Added
|
%
|
80
|
100
|
120
|
80
|
100
|
120
|
80
|
100
|
120
|
80
|
100
|
120
|
|
µg/ml
|
64
|
80
|
96
|
0.8
|
1
|
1.2
|
64
|
80
|
96
|
0.8
|
1
|
1.2
|
|
% Recovery*
|
100.
±0.9
|
100.90
±1
|
100.37
±2
|
100.08
±1
|
101.93
±1
|
100.66
±1
|
101.41±1
|
99.9
±2
|
100.8
±2
|
101
±1.75
|
100.5
±1
|
100.1
±2
|
|
Mean Recovery ±S.D.
|
100.42.±0.2
|
100.89.±1.2
|
100.56.±1.6
|
100.53.±1.1
|
*Average of three determinations
Table 3: Intraday Precision and Interday Precision
|
Method
|
Drug
|
Precision
%R.S.D.
|
|
Intraday n=3
|
Interday*
|
|
I
|
DOXO
|
1.28
|
1.36
|
|
TBS
|
1.43
|
1.35
|
|
II
|
DOXO
|
1.12
|
1.52
|
|
TBS
|
1.13
|
1.49
|
*Average of three
determinations
Table 4: LOD and LOQ
|
|
Method I
|
Method II
|
|
DOXO
|
TBS
|
DOXO
|
TBS
|
|
LOD
|
0.16 μg/ml
|
0.41μg/ml
|
0.16μg/ml
|
0.19 μg/ml
|
|
LOQ
|
0.48 μg/ml
|
1.27 μg/ml
|
0.48 μg/ml
|
0.58 μg/ml
|
Table 5: recovery data of marketed formulation
|
Formulation
|
Tablet content
taken(mg)
|
Amount
found(mg)
|
Assay
%estimated (n=3 MEAN±SD)
|
|
Method I
|
|
Oxobittr
|
DOXO
|
TBS
|
DOXO
|
TBS
|
DOXO
|
TBS
|
|
80
|
1
|
81.32
|
1.02
|
101.65 ±0.95%
|
102.3 ±0.74%
|
|
Method II
|
|
Oxobittr
|
80
|
1
|
79.81
|
1.02
|
99.76 ±0.94%
|
102 ±0.84%
|
CONCLUSION:
The proposed
spectrophotometric methods are simple, rapid, accurate, precise, and economic
and validated in terms of linearity, accuracy, precision, specificity and
reproducibility. These two methods can be successfully used for simultaneous
estimation of Doxofylline and Terbutaline
sulphate in pure and tablet dosage form.
ACKNOWLEDGEMENTS
:
The authors are heartily
thankful to Ami Pharma. Ltd. Vadodara and Brundavan
Laboratories, Hyderabad for providing gift standard sample of pure Doxofylline and Terbutaline sulphate.
REFERENCES:
1.
Chatwal GR, Anand S. Ultraviolet-spectroscopy-Instrumental Methods of
Chemical Analysis. 2nd ed., Himalaya
Publishing House, Mumbai: 2000, p.
180-98.
2.
Davidson AG, Beckett AH, Stenlake
JB. Ultraviolet-Visible absorption spectrophotometry,
Practical Pharmaceutical Chemistry. 4th ed., CBS Publishers, New Delhi: 2002, p. 264-271, 275-300.
3.
Tripathi KD. Essential of
Medical Pharmacology. 6th ed., Jaypee Brothers
Medical Publishers, New Delhi: 2009,
p. 151-161.
4.
Nanaware D, Bhusari V and Dhaneshwar
S, “validated hplc method for simultaneous quantitation of Doxofylline and Terbutaline sulphate in bulk drug
and formulation.” Asian J Pharm
Clin Res. 2013: 6(2); pp 237-241.
5.
Solanki V S, Muralikrishna K S and Shelar V,
“development and validation of uv spectroscopic
method for simultaneous estimation of Doxofylline and
Terbutaline sulphate in
combined dosage form.” Inventi J.
Pvt.Ltd. 2011: pp 440-402.
6.
Oza M, Kakadiya J and Oza
C, “Development and Validation of Solvent Extraction Spectrophotometric Method
for Simultaneous Estimation of Doxofylline and Terbutaline sulphate In their Combined Dosage Form.” Amer. J. Pharm.
Tech. 2014.
7.
Samanthula G, yadiki K, saladi
S, gutala S and Surendranath
KV. “Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Doxofylline and Terbutalinesulphate
in Pharmaceutical Formulations.” Sci
Pharm. 2013: 81; pp 969–982.
8.
ICH Harmonized Tripartite Guidelines, Validation of
Analytical Procedures: Text and Methodology, Q2 (R1), Geneva, Nov 2005
9.
Indian Pharmacopoeia, Government of India Ministry of
Health and Family Welfare, Ghaziabad, India: The Indian Pharmacopoeia
Commission. 2010; vol. II, 2010, p.
611