Novel RP-HPLC Method for Simultaneous Estimation of Doxofylline and
Sertraline in Bulk and Tablet Dosage Form
M.M. Eswarudu*, Y. Ravi Kumar and M. Chinna Eswaraiah
Department of Pharmaceutical Analysis, Anurag Pharmacy College,
Ananthagiri (V), Kodad (M),
Nalgonda (Dt), 508206, Telangana State, India.
*Corresponding Author E-mail:
eswarmunnangi@gmail.com
ABSTRACT:
The objective of this study was to
develop a simple, rapid, accurate and precise isocratic stability indicating
reversed-phase high-performance liquid chromatographic method for simultaneous
estimation of Doxofylline and Sertraline in tablet dosage form. The separation
method was carried out using Std XDB C18 column (150 x 4.6 mm; 5μ).The mobile phase used was a mixture of Acetonitrile and Potassium Di Hydrogen Phosphate buffer in the ratio of
45:55(v/v) delivered at an isocratic flow rate of 0.8mL/min. Column temperature
was 300c and eluents were monitored at 231nm using Waters 2695 HPLC instrument equipped with the waters
2998 PDA detector and Empower-2 software for data collecting and processing.
With the optimized method, the retention times of Doxofylline and Sertraline
were found to be 2.06 min and 4.14 min respectively, with theoretical plate
count and asymmetry as per the ICH limits. The method has shown
a good linearity in the concentration range of 100-600 μg/ml for
Doxofylline and 12.5-75μg/ml for Sertraline with regression coefficient (R2)
of 0.999 and 0.997.The percentage assays were
found to be 99.11 and 96.44 for Doxofylline and Sertraline. The
method was found to be accurate (with percentage mean recoveries 99.71 for
Doxofylline, and 99.10 for Sertraline), precise, robust, reliable, specific and
stable. The proposed method was validated in accordance with ICH guidelines and
hence this method can be successfully used for quantitative analysis of
Doxofylline and Sertraline in tablet formulations.
KEYWORDS: Doxofylline,
Sertraline, RP- HPLC, Validation, Tablet dosage
forms.
Doxofylline is a xanthine derivative drug used in the
treatment of asthma. It has antitussive and
bronchodilator effects, and acts as
a phosphodiesterase
inhibitor in animal and human studies, it has shown
similar efficacy to theophylline but with
significantly fewer side effects other
xanthines, doxofylline lacks any significant affinity for adenosine
receptors and does not produce stimulant effects.Chemically, Doxofylline is 7-(1, 3-dioxolan-2-methyl)-1,
3-dimethyl purine-2, 6-dione and the structure shown in figure-11.
Figure 1: Chemical structure of
Doxofylline
Sertraline is a selective
serotonin reuptake inhibitor (SSRI) used in the therapy of depression, anxiety
disorders and obsessive-compulsive disorder. Sertraline therapy can be
associated with transient asymptomatic elevations in serum amino transferase
levels and has been linked to rare instances of clinically apparent acute liver
injury. Chemically, Sertraline is (1S, 4S)-4-(3, 4- dichlorophenyl)-N-methyl-1,2,3,4tetra
hydronaphthalen-1-amine and the structure shown in figure-22.
Figure 2: Chemical structure of
Sertraline
The literature survey revealed that there is only one
RP-HPLC method was reported for the simultaneous determination of doxofylline
and sertraline till date3. But methods are available for the
quantification of doxofylline individually and with other combinations other
than sertraline4-17. Methods are available for the quantification of
sertraline individually and with other combinations other than doxofylline18-22.
Present study involves the development and validation of a RP-HPLC method for
the simultaneous determination of doxofylline and sertraline in bulk and its
pharmaceutical formulations. The method was validated as per the ICH guidelines
23-25.
MATERIALS AND METHODS:
Chromatographic conditions:
Separation
was performed with Waters HPLC equipped with pumps, auto sampler, PDA detector,
Empower-2 software was applied for data collecting and processing. The
separation was achieved on an STD XDB C18 column (150 x 4.6 mm, 5 μ). The
mobile phase consisted of Acetonitrile: Buffer (1ml of Triethylamine then pH adjusted to 3.5 with dilute ortho
phosphoric acid) in the ratio of 45:55 V/V. The flow rate was 0.8mL/min and UV
detection was performed at 231 nm. The mobile phase was shaken on an ultrasonic
bath for 20 min. The resulting transparent mobile phase was filtered through a
0.45 μ membrane filter. The injection volume was 10μL and all the experiments
were performed at temperature 300C.The run time was set at 8
minutes.
Chemicals and reagents used:
Pharmaceutical grade of Doxofylline
and Sertraline were kindly supplied as gift samples by Spectrum analytical
Labs, Hyderabad, India. Commercially available Doxoder tablets were purchased
from local market. Tablets claimed to contain 400 mg of Doxofylline and 50 mg of Sertraline have been utilized in the present work. Potassium
dihydrogen phosphate and ortho phosphoric acid of AR Grade were obtained from
S.D. Fine Chemicals Ltd., Mumbai, India. HPLC grade Acetonitrile and HPLC grade
water was purchased from Rankem, RFCL Gujarat, India.
Preparation of mobile phase solution:
Weighed
accurately 1.41grms of potassium dihyrogen phosphate and transferred into a
clean dry 1000ml volumetric flask, it dissolved in 900ml of HPLC graded water,
degas to sonicate and then added 1ml of triethyl amine. Finally make up to the
volume with HPLC water. PH adjusted to
3.5 with dilute orthophosphoric acid solution filtered this solutions through
0.45 μ filter paper and sonicated
for degas.
Preparation of standard stock
solution:
Accurately weighed and transferred
400 mg of Doxofylline and 50 mg of sertraline working standards into 10 ml
clean dry volumetric flask, add about 7 ml of methanol and sonicated for 5
minutes and dissolved it completely and make volume up to the mark with diluent
(mobile phase) and sonicated for 5minutes.
Preparation of diluted standard
solution:
From the above stock solution pipette
out 1ml into 50 ml clean dry volumetric flask and diluted up to the mark with
diluent.
Preparation of sample solution:
Twenty tablets were accurately
weighed, their mean weight was determined and they were mixed and finally
powdered in a clean dry motor and pestle. The tablet powder equivalent to 400
mg Doxofylline and 50mg of Sertraline transferred into a 100mL clean volumetric
flask. Add about 70 ml of diluent and sonicated to 20 minutes dissolve it
completely and make volume up to the mark with diluent. Mix well and filter
through 0.45 μ filter paper, from this solution 0.2mL pipette out and
transferred into a 10 ml volumetric flask and dilute up to the mark with
diluent and sonicated for 10 minutes.
Method validation:
The method was validated in
accordance with ICH guidelines. The parameters assessed were Linearity,
Accuracy, Precision, Limit of Detection (LOD), Limit of Quantification (LOQ),
Robustness, Ruggedness and Specificity etc.
Linearity:
Several aliquots of standard
solutions of Doxofylline and Sertraline
were taken into different 10 ml volumetric flasks and diluted up to the mark
with mobile phase such that the final concentrations of Doxofylline 100 to 600 μg/ml and Sertraline 12.5 to 75 μg/ml. Evaluation of the two drugs was performed with PDA detector at 231nm;
peak area was recorded for all the dilutions. The correlation coefficient (R2)
values 0.999 and 0.997 for Doxofylline and Sertraline respectively. The results
show that an excellent correlation exists between peak area and concentration
of drugs within the concentration range indicated.
Accuracy:
The accuracy of the method was
assessed by recovery studies of Doxofylline and Sertraline in the dosage form
at three concentration levels. A fixed amount of preanalyzed sample was taken
and standard drug was added at 50%, 100% and 150% levels. Each level was
repeated three times. The contents of Doxofylline and Sertraline per tablet
were calculated. The % mean recoveries of Doxofylline and Sertraline were
99.38% and 99.10% that shows there is no interference from excipients and the
lower values of % RSD of assay indicate the method is accurate.
Precision:
The precision was determined for both
the drugs Doxofylline and Sertraline in terms of intra-day and inter-day
precision. For intra-day precision evaluation, a standard solution of fixed
concentration was injected at various time intervals and % RSD for Doxofylline
and sertraline were 0.862% and 0.92% respectively (limit of % RSD < 2.0%).
In addition, the inter-day precision was studied by injecting the same
concentration of standard solution on consecutive days and the % RSD for
Doxofylline and Sertraline were 0.3% and 0.4% respectively (limit % RSD <
2.0%).
Limit of detection and limit of
quantification:
The limit of detection (LOD) and
limit of quantification (LOQ) of the developed method were determined by
injecting progressively low concentrations of the standard solutions using the
developed HPLC method. The LOD for Doxofylline and Sertraline were found to be
0.345μg/ml and 0.176μg/ml respectively. The LOQ for Doxofylline and
Sertraline were found to be 0.046μg/ml and 0.536μg/ml respectively.
Robustness:
Robustness of the method was
determined by making slight changes in the chromatographic conditions like
mobile phase composition and flow rate. It was observed that there were no
marked changes in the chromatograms, which demonstrated that the HPLC method so
developed is robust.
Ruggedness:
The ruggedness of the method was
determined by carrying out the experiment on different instruments by different
operators using different columns of similar types, which demonstrated that the
developed HPLC method is rugged.
Specificity:
Specificity is the ability of the
analytical method to measure the analyte response in the presence of
interferences including degradation products and related substances. No
interference from any of the excipients was found at retention times of the
examined drugs. These results demonstrate the absence of interference from
other materials in the pharmaceutical formulations and therefore confirm the
specificity of the proposed method.
Assay:
10 μl of each standard and
sample solution were injected and from the peak area of Doxofylline Sertraline
amount of each drug in samples were computed. The result of assay undertaken
yielded 99.11% and 96.44% of label claim of Doxofylline and Sertraline
respectively.
RESULTS
AND DISCUSSION:
The HPLC procedure was optimized with
a view to develop an accurate assay method for simultaneous estimation of
Doxofylline and Sertraline in tablet dosage form using STD XDB C18 column (150
x 4.6 mm; 5μ) in isocratic mode with mobile phase composition of Acetonitrile:
potassium dihydrogen phosphate buffer in the ratio of 45: 55 V/V. The use of 1%
Triethylamine and pH adjusted to 3.5 with dilute ortho phosphoric acid. The
flow rate was 0.8 ml/min and both the components were measured with PDA
detector at 231nm.The results of optimized HPLC conditions were shown in Table
1.
Table no. 1: Optimized chromatographic conditions of Doxofylline and
Sertraline
|
Parameter |
Condition |
|
Mobile
phase |
Acetonitrile
: Potassium Di Hydrogen phosphate buffer (45: 55V/V) |
|
Resolution |
6 |
|
Column |
Std
XDB, C18 column (150 x 4.6 mm; 5μ) |
|
Column
temperature |
300c |
|
Wave
length |
231nm |
|
Injection
volume |
10μl |
|
Flow
rate |
0.8
ml/min |
|
Run
time |
8 min |
The method was linear in the range of 100-600μg/ml
and 12.5-75μg/ml for Doxofylline and Sertraline with correlation coefficient of R2=1 and R2=0.999
for Doxofylline and Sertraline. Linear regression data for Doxofylline and
Sertraline were given in Table 2&3.The linearity curves for Doxofylline and
Sertraline were shown in Fig. 3&Fig. 4.
Table
no.2: Linearity results of Doxofylline
|
S.no |
% of
test |
Concentration (µg/ml) |
Area |
|
1 |
25 |
100 |
1990564 |
|
2 |
50 |
200 |
4273464 |
|
3 |
75 |
300 |
6403423 |
|
4 |
100 |
400 |
8657588 |
|
5 |
125 |
500 |
10440148 |
|
6 |
150 |
600 |
12629785 |
Table no.3: Linearity results of Sertraline
|
S.no |
% of test |
Concentration (µg/ml) |
Area |
|
1 |
25 |
12.5 |
393653 |
|
2 |
50 |
25 |
951622 |
|
3 |
75 |
37.5 |
1456381 |
|
4 |
100 |
50 |
1984703 |
|
5 |
125 |
62.5 |
2398170 |
|
6 |
150 |
75 |
2868752 |
Figure 3: Linearity curve of
Doxofylline
Figure 4: Linearity curve of
Sertraline
The mean % recoveries were found to
be 99.38% for Doxofylline and 99.10% for Sertraline which indicate the method
is accurate. The accuracy results were shown in Table 4&5.
Table no.4: Accuracy data of
Doxofylline
|
S. no |
Spike Level |
µg/ml added |
µg/ml found |
%Recovery |
Mean % recovery |
|
1 |
50% |
100 |
98.84 |
98.84 |
98.68% |
|
2 |
50% |
100 |
98.50 |
98.50 |
|
|
3 |
50% |
100 |
98.70 |
98.70 |
|
|
1 |
100% |
400 |
398.60 |
99.65 |
99.59% |
|
2 |
100% |
400 |
398.19 |
9954 |
|
|
3 |
100% |
400 |
398.37 |
99.59 |
|
|
1 |
150% |
600 |
599.22 |
99.87 |
99.87% |
|
2 |
150% |
600 |
599.99 |
99.83 |
|
|
3 |
150% |
600 |
599.59 |
98.93 |
Table no.5: Accuracy data of
Sertraline
|
S.no |
Spike
Level |
µg/ml
added |
µg/ml
found |
%
Recovery |
Mean%
recovery |
|
1 |
50% |
25 |
24.85 |
99.41 |
99.42% |
|
2 |
50% |
25 |
24.75 |
97.00 |
|
|
3 |
50% |
25 |
24.96 |
99.86 |
|
|
1 |
100% |
50 |
49.06 |
98.12 |
98.95% |
|
2 |
100% |
50 |
49.71 |
99.43 |
|
|
3 |
100% |
50 |
49.65 |
99.31 |
|
|
1 |
150% |
75 |
74.36 |
98.14 |
98.94% |
|
2 |
150% |
75 |
74.06 |
98.75 |
|
|
3 |
150% |
75 |
74.21 |
98.95 |
The %RSD for intra-day precision and
inter-day precision for were found to be Doxofylline 0.862 and 0.92% for
Sertraline were found to be 0.3% and 0.4% respectively, which indicate the
method is precise. The precision results were shown in Table 6 &7.
Table no. 6: Inter-day precision data
of proposed method
|
S. no |
Doxofylline |
Sertraline |
||||
|
|
RT |
Area |
RT |
Area |
||
|
1 |
2.06 |
17003541 |
4.151 |
1901289 |
||
|
2 |
2.069 |
17062261 |
4.156 |
1935395 |
||
|
3 |
2.070 |
17084990 |
4.156 |
1913443 |
||
|
4 |
2.070 |
17097936 |
4.159 |
1886970 |
||
|
5 |
2.070 |
17401513 |
4.159 |
1891048 |
||
|
6 |
2.071 |
17115424 |
4.160 |
1879289 |
||
|
Average |
|
17114862 |
|
1904572 |
||
|
S.D |
|
147599 |
|
17664 |
||
|
%R.S.D |
|
0.862 |
|
0.92 |
||
Table no. 7: Intra- day precision
data of proposed method
|
S.no |
Doxofylline |
Sertraline |
||
|
RT |
Area |
RT |
Area |
|
|
1 |
2.068 |
17160404 |
4.151 |
1909991 |
|
2 |
2.069 |
17157293 |
4.156 |
1917052 |
|
3 |
2.069 |
17052753 |
4.156 |
1918659 |
|
4 |
2.069 |
17096680 |
4.159 |
1916970 |
|
5 |
2.070 |
17096712 |
4.159 |
1912088 |
|
6 |
2.072 |
17115101 |
4.160 |
1900943 |
|
Average |
|
17116446 |
|
1911747 |
|
S.D |
|
44861.1 |
|
6997.1 |
|
%R.S.D |
|
0.3 |
|
0.4 |
The retention times of Doxofylline
and Sertraline was 2.06 min and 4.14 minutes respectively. The number of
theoretical plates calculated was 3153 for Doxofylline and 2566 for Sertraline
and symmetry factor was 1.20 for Doxofylline and 1.59 for Sertraline, which
indicates efficient performance of the column. The LOD for Doxofylline and
Sertraline were found to be 0.345μg/ml and 0.176μg/ml respectively. The
LOQ for Doxofylline and Sertraline were found to be 0.046μg/ml and
0.536μg/ml respectively, which indicate the sensitivity of the method. The
summary of system suitability parameters and validation parameters were shown
in Table 8.
Table
no.8: System suitability parameters of the method
|
Parameter |
Observed
values |
|
|
Doxofylline |
Sertraline |
|
|
Retention time (min) |
2.068 |
4.140 |
|
Linearity (μg/ml) |
100-600 |
12.5-75 |
|
Theoretical plates |
3153 |
2566 |
|
Symmetry factor |
1.20 |
1.59 |
|
LOD (μg/ml) |
0.0345 |
0.176 |
|
LOQ (μg/ml) |
0.046 |
0.536 |
|
Resolution(Rs) |
6 |
|
Validated method was applied for the
determination of Doxofylline and Sertraline in commercial formulations. The %
assay was found to be 99.11% and 99.44% for Doxofylline and Sertraline
respectively and the assay results were shown in Table 9.
Typical chromatogram and UV spectra
of standards showing the separation of the drugs Doxofylline and Sertraline was
shown in Fig. 5& 6 No interfering peaks were found in the chromatogram of
the formulation within the run time indicating that excipients used in tablet
formulations did not interfere with the estimation of the drug by the proposed
HPLC method.
CONCLUSION:
The developed HPLC method is simple,
specific, accurate and precise for the simultaneous estimation of Doxofylline
and Sertraline tablet dosage form. The developed method provides good
resolution between Doxofylline and Setraline. It was successfully validated in
terms of linearity, accuracy, precision, specificity, robustness, LOD, LOQ and
system suitability in accordance with ICH guidelines. Thus the described method
is suitable for routine analysis and quality control of pharmaceutical
preparations containing these drugs in combinations.
Figure 5: Typical
chromatogram of Doxofylline and Sertraline
Figure 6: UV
Spectra of Doxofylline and Sertraline (λmax 231)
Table no.9: Assay results of proposed method
|
Formulation |
Label Claim |
Amount Found |
% Assay |
|||
|
DOXOER |
Doxofylline |
Sertraline |
Doxofylline |
Sertraline |
Doxofylline |
Sertraline |
|
400mg |
50mg |
396.78mg/tab |
47.48mg/tab |
99.11 |
96.44 |
|
ACKNOWLEDGEMENTS:
The authors are thankful to Management of Anurag Pharmacy College, for providing all types of
facilities for the research, and also thankful to Spectrum Analytical Lab pvt.
Limited, Hyderabad, India for providing the gift samples of the drugs and
research facilities for this work.
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Received on 22.07.2015 Modified on 16.08.2015
Accepted on 22.08.2015 © AJRC All right reserved
Asian J. Research
Chem. 8(8): August 2015; Page 539-544
DOI: 10.5958/0974-4150.2015.00086.3