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www.wjpr.net Vol 8, Issue 9, 2019.
Kajal et al. World Journal of Pharmaceutical Research
1111
NEW LC–UV METHODS FOR PHARMACEUTICAL ANALYSIS OF
NOVEL BRONCHODILATOR DOXOFYLLINE AND
ANTIDEPRESSANT SERTRALINE COMBINATION METHOD IN
DIFFERENT DOSAGE FORM LIKE TABLET CAPSULE AND
SUSPENSION AND INJECTION
P. Kajal*, Dr. P. Arun, P. Shailendra, P. Bhavesh, D. Neelesh and V. Koushal
Department of Pharmacy, Shri Ram Group of Institution, ITI Madhotal Jabalpur-482002.
ABSTRACT
A sensitive & selective stability indicting RP-HPLC method has been
developed & validated for the analysis of Sertraline & Doxofylline in
table, sustained release (capsule) and syrup and injection and
suspension dosage form. Based on peak purity results, obtained from
the analysis of samples using described method, it can be concluded
that the absence of co-eluting peak along with the main peak of
Sertraline & Doxofylline indicated that the developed method is
specific for the estimation of Sertraline & Doxofylline. Further the
proposed RP-HPLC method has excellent sensitivity, precision and
reproducibility.
KEYWORDS: Qualitative analysis, sertraline, doxofylline, chromatography, validation,
tablet, capsule, injection suspension.
INTRODUCTION
Sertraline is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. It
was introduced to the market by Pfizer[1]
in 1991. Sertraline is primarily prescribed for major
depressive disorder in adult outpatients as well as obsessive–compulsive, panic, and social
anxiety disorders in both adults and children. In 2013, it was the most prescribed
antidepressant and second most prescribed psychiatric medication (after Alprazolam) on the
U.S. retail market, with over 41 million prescriptions.[2]
World Journal of Pharmaceutical Research SJIF Impact Factor 8.074
Volume 8, Issue 9, 1111-1123. Research Article ISSN 2277– 7105
Article Received on
07 June 2019,
Revised on 27 June 2019,
Accepted on 17 July 2019,
DOI: 10.20959/wjpr20199-15487
*Corresponding Author
P. Kajal
Department of Pharmacy,
Shri Ram Group of
Institution, ITI Madhotal
Jabalpur-482002.
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Chemically, Sertraline is (1S, 4S)-4-(3,4-dichlorophenyl)-N-methyl - 1, 2, 3, 4
tetrahydronaphthalen-1-amine and the structure as shown in shown in fig1.
Sertraline is an antidepressant of the selective serotonin reuptake inhibitor. It is primarily
prescribed for major depressive disorder in adult as well as obsessive-compulsive, panic and
social anxiety disorders in both adults and children.
Differences with other newer antidepressants are subtle and mostly confined to side effects. It
has a similar tolerability profile to other SSRIs, with the types of adverse events usually
including diarrhea, nausea, trembling, sexual dysfunction and weight gain. The incidence of
diarrhea was higher with sertraline in comparison to other SSRIs.[3]
Doxofylline (INN), (also known as doxophylline) is a xanthine derivative drug used in the
treatment of asthma.[4]
It has antitussive [citation needed] and bronchodilator effects, and acts as a phosphodiesterase
inhibitor.[5]
In animal and human studies, it has shown similar efficacy to theophylline but with
significantly fewer side effects.[6]
Unlike other xanthines, doxofylline lacks any significant affinity for adenosine receptors and
does not produce stimulant effects. This suggests that its antiasthmatic effects are mediated
by another mechanism, perhaps its actions on phosphodiesterase.[7]
Chemically, Doxofylline is 7-(1, 3-dioxolan-2-methyl)-1,3-dimethyl purine-2,6-dione and the
structure shown in figure-2.
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Analytical methods are essential to characterize drug substances and drug products
composition during all stages of pharmaceutical development. For routine analytical purpose
it is always necessary to establish methods capable of analyzing huge number of samples in a
short time period with high accuracy and precision. The number of drugs, which may be
either new entities or partial structural modification of the existing ones, introduced into the
market is increasing every year. Very often there is a time lag from the date of introduction of
a drug into the market to the date of its inclusion in pharmacopoeias. Hence standards and
analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes
necessary, therefore to develop new analytical methods for such drugs. These products can
present challenges to the analytical chemist responsible for the development. Basic criteria
for new method development of drug analysis for sertraline and doxyfylling is:
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to
patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to
the interference caused by the formulation excipients for tabet, capsule suspension, syrup
and parental.
Analytical methods for a drug in combination with other drugs or individually may not be
available.
The existing analytical procedures may require expensive reagents and solvents.
It may also involve cumbersome extraction and separation procedures and these may not
be reliable.
The review of literature reveals that no method has been developed so far for simultaneous
estimation of Sertraline & Doxofylline by RP-HPLC method and in different dosage form
like tablet, capsule, syrup, suspension, injection is available with single method. Further, a
good HPLC method for the estimation of Sertraline & Doxofylline has not yet been cited in
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Kajal et al. World Journal of Pharmaceutical Research
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literature. Hence it is essential to develop a new HPLC method for this combination ,
individually which can be applied to different dosage forms at time in single run with same
method.
CHEMICALS / REAGENTS USED
S.N. Name Specifications
Manufacturer/Supplier Purity Grade
1. Doubled distilled water ---- ---- In house
2. Methanol 99.9% HPLC grade. Rankem Mumbai
3. Dipotassium hydrogen
phosphate 96% L.R. Sd fine-Chem ltd; Mumbai
4. Acetonitrile 99.9% HPLC Rankem Mumbai
5. Potassium dihydrogen
orthophosphate 99.9 L.R. Sd fine-Chem ltd; Mumbai
6. orthophosphoric acid 99.9 L.R. Sd fine-Chem ltd; Mumbai
7. Doxofylling Gift sample Wings biotech
8. Sertraline Hetero Drugs Limited,
Hyderabad,
INSTRUMENTS USED
Sr. no. Name of Instrument Instrument Model Name of manufacturer
1 UV-Visible double beam
spectrophotometer UV 1800 Elico India
2 HPLC 2996 Waters The system was controlled by Empower
2 software and it is used for the analysis.
3 Ultra sonicator -------- Entrech electronics limited
4 Melting point appraturs -------- Electronic india 934
5 Weighing balance
Contech
6 Vaccum pump and assembly Sigma
7 Millipore 0.2µm membrane
filter Millipore
8 Syringe filter 0.2µm
membrane filter Millipore
9. pH meter poloman
CHARACTERIZATION OF SERTRALINE & DOXOFYLLINE
Solubility of Sertraline: The solubility of drug sample was determined according to I.P.
199638
.
Two 10 ml and one 250 ml volumetric flasks were taken.
Solubility of Doxofylline: The solubility of drug sample was determined according to I.P.
199638
. Two 10 ml volumetric flasks were taken.
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Table Solubility of Sertraline and Doxophylline
Content Doxofylline Sertraline
Flask -1
10 mg doxofyline + 10 ml volumetric
flask +0.1 ml water add +mixed +drug
not dissolve + 0.4 ml of water add
+mixed + solubility state was noted
10 mg setraline+ 10 ml of volumetric flask +0.1 ml
water added + mixed one time +0.1 ml of water
added again volumetric flask + solubility state was
noted
Flask 2
10 mg doxofyline +10 ml volumetric
flask +0.1 ml mathenol +mixed drug
was slightly dissolved +0.1 ml of
methanol added + mixed solubility state
was noted.
10 mg setraline +10 ml volumetric + 0.1 ml
mathenol +mixed +0.1 ml of methanol again add +
solubility state was noted
Flask 3
10 mg setraline transfer to 250 ml volumetric flask
+10 ml acetonitrile + mixed +drug could not
dissolved +added 90 ml of acetonitrite +mixed the
solution for 2 min +drug not dissolve+ added 100 ml
water + the solubility state was noted
Fig. Characterization Of Sertraline & Doxofylline.
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Accurately weighed 100 mg of Sertraline and 100 mg of Doxofylline were transferred to two
different 100 ml volumetric flask. About 40 ml of mobile phase was added and sonicated to
dissolve. The volume was made up to mark with same solvent.
Method development
Standard stock solution preparation
Weigh and transfer 40mg of Doxofylline working standard and 5mg of Sertraline working
standard into a 10 ml clean dry volumetric flask, add 7 ml of diluent, sonicated for 5 minutes
and make up to the final volume with diluents.[8]
Standard preparation Transfer 1 ml from the above stock solution was taken into a 10 ml
volumetric flask and dilute to volume with diluent. The standard solution consists of
400μg/ml of Doxofylline and 50μg/ml of Sertraline, respectively.
Sample preparation for combined Tablet Finely grind pre-weighed twenty tablets.
Transfer grinded sample quantitatively equivalent to 40mg Doxofylline and 5mg of Sertraline
into a 100 ml volumetric flask, add 70 ml of diluent, sonicate to dissolve for 25 min, and the
dilute to volume with diluent. Further filter the solution through filter paper. From the filtered
solution 1 ml was pipetted out into a 10 ml volumetric flask and the volume made up to 10 ml
with diluent then the solution consist of 400μg/ml of Doxofylline and 50μg/ml of Sertraline.
Individual Tablet containing Doxophylline 20 tablets of DOXOCARE of care
pharmaceutical Pvt ltd (400 mg) was weighed, powdered and transfer it into 100 ml
volumetric flask which contains 60 ml of methanol. The resulting solution was centrifuged at
3000 rpm for 5 min. The supernatant liquid contains 4000 µg/ml of Doxophylline is filtered
through a 0.45µ membrane filter. Then, the sample solutions are diluted to 40µg/ml by using
a methanol solvent.
Individual Tablet containing Sertraline Finely grind pre-weighed twenty tablets of
sertraline 50mg tablet of Aurobindo Pharma - Milpharm Ltd. Transfer grinded sample
quantitatively equivalent to 5mg of Sertraline into a 100 ml volumetric flask, add 70 ml of
diluent, sonicate to dissolve for 25 min, and the dilute to volume with diluent. Further filter
the solution through filter paper. From the filtered solution 1 ml was pipetted out into a 10 ml
volumetric flask and the volume made up to 10 ml with diluent then the solution consist of
50μg/ml of Sertraline.
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Sample preparation for Doxophylline syrup 1.0g of Doxfree syrup was weighed in 10 mL
volumetric flask, filled with 80% of the volume with methanol, sonicated for 15 min to
homogenize the solution in ultrasonic bath, and after cooling to room temperature the flask
was filled up to the mark. According to this procedure, the final solution is supposed to have
a concentration of 100 mg/mL doxophylline.
Sample Preparation Sustained release The drug content in each formulation was
determined by triturating 20 tablets and powder equivalent to 800 mg was dissolved in 100ml
of phosphate buffer pH 3.0, followed by sonication for 10 minutes. The solution was filtered
through a 0.45µ membrane.
Sample Preparation Suspension 5 ml sample (Coxylate oral suspension equivalent to 100
mg of Doxophylline) into a volumetric flask & dissolved in 15 ml mobile phase with the aid
of ultrasonic bath and made volume up to 50 ml with mobile phase(10mg/ml).
Sample preparation of injection Vials of doxophylline injection (100 mg each) were
reconstituted with the entire contents of their accompanying solvent vials and carefully
homogenized to avoid the formation of foam. This process yielded a nominal drug
concentration of 10 mg/mL. Doxophylline 10 mg/mL solution either to german remedies
(Doxolin injection) vial of polyolefin containers or to glass bottlesc to which had been added
the appropriate volume of 0.9% sodium chloride injection to result in a final volume of 50 ml.
Three identical polyolefin containers and one control glass bottle for each concentration were
prepared. All test solutions were stored at 19–21°C and protected from light.
RESULTS AND DISCUSSION
System Suitability The system suitability tests were conducted before performing the
validation and the parameters were within the acceptance criteria like retention times of
Doxofylline and Sertraline were 2.82 minutes and 3.93 minutes, respectively. The plate count
was >2000, peak tailing was <2 and the %RSD of peak areas of standard were ≤ 2.(Table 1),
(fig. 3). Hence the proposed method was successfully applied to routine analysis without any
problems.[9]
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Table 1: System suitability of Doxofylline and Sertraline.
S NO Doxofylline setraline
1 RT(min) AREA USP plate
count
usp
tailling
RT
min AREA
USP
PLATE
USP
TALING
2 2.803 3670119 8104 1.17 3.924 362952 7009 1.16
3 2.807 3624766 8093 1.17 3.932 363964 7387 1.21
4 2.808 3639058 8158 1.17 3.932 363266 7404 1.19
5 2.818 3645506 8217 1.16 3.934 363258 7235 1.15
Mean 2.823 3599764 8210 1.16 3.949 366194 7413 1.14
SD
3635842
363927
% RSD 25994.94 1320.58
0.71 0.36
Linearity The linearity of Doxofylline and Sertraline were prepared in the range of 100-
600μg/ml and 12.5-75μg/ml. These were represented by linear regression equation
(Doxofylline) y=9205x+3043 (r2=0.998), (Sertraline) y=7412x+1130 (r
2=0.99). From the
calibration curve the regression line for both drugs was linear.
Doxofylline
Concentration (μg/ml) Mean Area
(n=3)
100 959750
200 1805512
300 2765110
400 3624173
500 4700041
600 5494993
Sertraline
Concentration
(μg/ml)
Mean
Area (n=3)
12.5 92562
25 188687
37.5 278100
50 364097
62.5 475030
75 551182
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Precision
Injected standard preparation six times in same concentration in to the system. The precision
of analytical method expresses closeness of agreement between a series of measurements
obtained from multiple sampling of the homogenous under the prescribed conditions.
Reproducibility and Repeatability for Doxofylline and Sertraline was shown in (Table 3).
This indicated the method was highly precise.
Table 3: Interday and Intraday precision study for Doxophylline and Sertraline.
DAY DOXOFYL
LINE
SERTRAL
LINE
SAME DAY
08/05/19
DOXOFYL
LINF
SERTRAL
LINE
(100mg) (25mg) (100mg) (25mg)
8/5/2019 959840 188678 1 956750 188786
9/5/2019 959750 188256 2 957650 187695
10/5/2019 957509 182865 3 955560 188578
11/5/2019 950597 183754 4 956543 185887
12/5/2019 954386 184673 5 955436 184226
13/05/2019 953475 188365 6 957568 183145
mean 955926.16 186098.5 mean 956584.5 186386.16
%RSD 0.38 1.41 %RSD 0.09 1.26
SD 3720.90 2624.11 SD 948.71 2353.59
Accuracy
The percentage recoveries for Doxofylline and Sertraline was found to be 98-120% and the
%RSD for Doxoylline and Sertraline was found to be 0.34 and 0.63. The results of recovery
studies was shown in (Table 4).
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Table 4: Accuracy for Doxophylline and Sertraline.
ACCURANCY
LEVEL (%) DOXOFYLLINE SETRALLINE
SAMPLE
CONC
ADDED
CONC
FOUND
CONC
%
RECOVERY
SAMPLE
CONC
ADDED
CONC
FOUND
CONC
%
RECOVERY
(ug/ml) (ug/ml) (ug/ml)
(ug/ml) (ug/ml) (ug/ml)
50% 400 200 202.22 101.11 50 25 24.81 99.27
400 200 202.08 101.04 50 25 25.13 100.54
400 200 201.46 100.73 50 25 25.26 101.07
400 400 411.84 101.47 50 50 50.68 101.37
100% 400 400 401.36 100.34 50 50 50.6 101.2
400 400 401.12 100.28 50 50 50.04 100.09
400 600 600.72 100.12 50 75 74.7 99.61
150% 400 600 602.76 100.46 50 75 75.41 100.55
400 600 599.82 99.97 50 75 75.6 100.81
MEAN
100.61
100.5
SD
0.5053
0.7198
%RSD
0.5
0.71
Robustness
Robustness data for Doxofylline and Sertraline by changing the parameters like flow rate,
temperature and mobile phase ratio. It was shown in (Table 5).
Table 5: shows Robustness for Doxophylline and sertraline.
PARAMETER
DOXOFYLLINE 400 μg/ml SETRALINE 50 μg/ml
MEAN
AREA SD %RSD
MEAN
AREA SD %RSD
FLOW RATE 0.9ml/min 3634346 183091.1 0.5 354211 4409.3 1.2
1.0ml/min 3635842 25994.9 0.7 363927 1320.5 0.3
1.1ml/min 3598463 14749 0.4 351526 4316.3 1.2
TEMPERATURE 25 C 3524817 8670.3 0.2 346874 1168.8 0.3
30 C 3635842 25994.9 0.7 363927 1320.5 0.3
35 C 3559288 4145 0.1 348047 3457.3 1
MOBILE PHASE 29.71 3526011 10358.5 0.3 347571 182.4 0.1
30.7 3635842 25994.9 0.7 363927 1320.5 0.3
31.69 3555832 25306.5 0.7 348190 3463.4 1
Limit of detection and limit of quantification
The values of LOD and LOQ were calculated by using slope and Y-intercept. The LOD and
LOQ values for Doxofylline was found to be 1.28 and 3.38μg/ml and Sertraline was found to
be 0.09 and 0.2μg/ml, respectively (Table 6).
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Table 6: Shows the: LOD and LOQ for Doxophylline and Sertraline.
S.NO DOXOFYLLINE SERTRALLINE
100(ug/ml) 12.5(ug/ml)
1 959750 92562
2 958640 92131
3 957520 92121
4 952110 92013
5 954010 92146
6 951220 92415
mean 523886.5 92231.33333
SD 3578.342726 209.8882242
LOD 1.28 0.093
LOQ 3.3887 0.283
Assay
The content of Doxofylline and Sertraline in the pharmaceutical dosage forms by using the
developed method. The percentage purity of Doxofylline and Sertraline was found to be
99.85% and 99.96% and %RSD values for both Doxofylline and Sertraline was within limit
of ≤2 (Table 7).[10]
Fig. 3: HPLC chromatograph of standard solution (Doxofylline and Sertraline).
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ASSAY OF SERTRALINE & DOXOFYLLINE IN DOSAGE FORM
Assay was performed and Results obtained are tabulated below:
Table 12: Assay of SERTRALINE & DOXOFYLLINE in different dosage form.
SNo Brand
Name
Company
name
Label Drug
content
Doxophylline
Label
Drug
content
Sertraline
Found Drug
content
Doxophylline
Found Drug
content
Sertraline
Label area
Doxophylline
Label
area
Found area
Doxophylline
Found
area
1 Doxoder (Wonder
healthcare) 400mg 50mg 399.42 ± 0.28 49.98 ± 0.44 3624173 364097 3660414 361439
2 doxfree
Syrup
unichem
Pvt ltd,
Ranbaxy
100mg/5ml, NA 100 mg NA 181,208 NA 183020 NA
3
Doxovent
Sustained
release
Glenmark
pharmaceuti
cal Ltd
majesta
800mG NA 800mg NA 7248346 NA 7320829 NA
4 Coxylate
suspension
symbiosis
pharmaceuti
cal pvt ltd
100mg/5ml Na 100mg NA 181,210 NA 183022 NA
5 Doxolin
injection
German
remedies 10mg/ml NA 10 mg NA 90605 NA 91511 NA
6 Zoloft
sertraline Na 25,50mg NA NA NA 182,048
180719
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