NEW LC UV METHODS FOR PHARMACEUTICAL ANALYSIS OF …

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.



1112

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.



1113

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



1114

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.



1115

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.



1116

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.

https://www.medicines.org.uk/emc/product/5760/smpc#companyDetails



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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]



1118

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).



1120

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).



1121

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).



1122

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



1123

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