development and validation of analytical method for

www.wjpps.com Vol 6, Issue 7, 2017.

924

Adnan et al. World Journal of Pharmacy and Pharmaceutical Sciences Adnan et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD

FOR SIMULTANEOUS ESTIMATION OF DIMETHYL FUMARATE

AND ONDANSETRON

Mohammed Asif Adnan* and Chandrashekhar Javali

Department of Pharmaceutical Chemistry, Government college of Pharmacy,

Bengaluru, Karnataka, India.

ABSTRACT

A simple, sensitive, precise rapid and accurate reverse phase high

performance liquid chromatography (RP- HPLC) method was

developed and validated for simultaneous estimation of Dimethyl

Fumarate and Ondansetron. The Chromatographic separation was

achieved by using Lichrospher® 100, C18(250 mm×4.6 mm, 5μ) as

stationary phase and mobile phase consists of Methanol: Acetonitrile:

phosphate buffer with pH 5.5 (50:20:30 v/v/v) with a flow rate of

1ml/min. The analysis was performed at ambient temperature and the

eluent was monitored at 239nm using UV detector. The retention time

of Dimethyl Fumarate & Ondansetron was found to be 3.1min and 6.9

min respectively and the calibration curves were linear (r2 = 0.9998

and 0.9997) over a concentration range of 100-1000μg/ml for

Dimethyl Fumarate & Ondansetron respectively. The Limit of detection (LOD) for Dimethyl

Fumarate & Ondansetron was observed to be 0.1μg/ml and 0.4μg/ml respectively, the limit of

quantitation (LOQ) was found to be 0.4μg/ml and 0.8μg/ml respectively. The developed

method was validated as per ICH guidelines using parameters like linearity, specificity,

system suitability, precision, ruggedness, robustness, accuracy. All the validation parameters

were found to be well within the acceptance criteria. Hence the proposed method can be used

for the routine analysis of Dimethyl Fumarate and Ondansetron in bulk and tablet dosage

forms.

KEYWORDS: Dimethyl Fumarate, Ondansetron RP-HPLC method, Validation.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.647

Volume 6, Issue 7, 924-936 Research Article ISSN 2278 – 4357

Article Received on

27 April 2017,

Revised on 17 May l 2017, Accepted on 07 June 2017,

DOI: 10.20959/wjpps20177-9470

*Corresponding Author

Mohammed Asif Adnan

Department of

Pharmaceutical

Chemistry, Government

college of Pharmacy,

Bengaluru, Karnataka,

India.


925


INTRODUCTION

Dimethyl Fumarate is a methyl ester of fumaric acid and chemically is (E)-2-Butenedoic acid

dimethyl ester. Dimethyl Fumarate was initially recognized as a very effective hypoxic cell

radiosensitizer. Later, Dimethyl Fumaratewas used in the treatment of psoriasis along with

three other fumaric acid esters and also used to treat, necrobiosis, lipoidica, granulose

annulare and sarcoidosis. Phase III clinical trails found that Dimethyl Fumarate successfully

reduced relapse rate and increased time to progression of disability in Multiple

Sclerosis(MS). The mechanism by which Dimethyl Fumarate is unknown. Dimethyl

Fumarate and its metabolite MonomethylFumarate activates the nuclear factor(erythroid-

derived 2)-like 2(Nrf2) pathway and acts as a nicotinic acid receptor agonist in vitro.[1,2]

Ondansetron is chemically 1,2,3,9-Tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-

yl)methyl]-4H-carbazol-4-one which a competitive serotonin 5-HT3 receptor antagonist used

mainly as an antiemetic (to treat nausea and vomiting) often following chemotherapy, which

affects both peripheral and central nerves and reduces the activity of the vagus nerve, thus

deactivating the vomiting centre in the medulla oblongata, and serotonin receptors in the

chemoreceptor trigger zone (CTZ). It is also used to treat nausea and vomiting occurring post

surgery.[3,4]

Dimethyl fumarate is used in effective treatment of Multiple Sclerosis but at the same time it

is responsible for the generation of nausea, vomiting, diarrhea, gastritis, gastroenteritis,

leucopenia, hypersensitivity, burning sensation and flushing.

In order to minimize the adverse effect of DMF such as nausea, vomiting, Ondansetron can

be prescribed or given in combination with Dimethyl Fumarate. But no analytical method

was found for the simultaneous estimation of Dimethyl Fumarate and Ondansetron in

combination by HPLC method.

MATERIALS AND METHODS

Chemicals And Reagents

The reference samples of Dimethyl Fumarate was purchased from SIGMA ALDRICH &

Ondansetron was procured as a gift sample from DRUG TESTING LABORATORY,

Acetonitrile, Methanol, and Water were of HPLC grade. Potassium dihydrogen phosphate &

Disodium hydrogen phosphate used was of Analytical grade.


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STRUCTURE OF ONDANSETRON

STRUCTURE OF DIMETHYL FUMARATE

INSTRUMENT AND CHROMATOGRAPHY CONDITION

The High Performance Liquid Chromatography consisted of SHIMADZU-SPD-20A

prominence auto sampler fitted with UV Visible detector (SPD-20A) with SHIMADZU-LC-

20AT pump. The chromatogram was recorded using LC Solution software. The

Chromatographic separation was achieved by using Lichrospher® 100, C18(250 mm×4.6

mm, 5μ) as stationary phase and mobile phase consists of Methanol: Acetonitrile: phosphate

buffer with pH 5.5 (50:20:30 v/v/v) with a flow rate of 1ml/min. The analysis was performed

at ambient temperature and the eluent was monitored at 239nm using UV detector.

PREPARATION OF MOBILE PHASE

Volume of 500 mL HPLC grade Methanol, Volume of 200mL Acetonitrile was mixed with

300mL phosphate buffer, prepared by dissolving 13.61 gm of potassium dihydrogen

phosphate (Solution I) and 35.81gm of disodium hydrogen phosphate (Solution II) in 1000

mL of Millipore water and then mix 96.4mL of (Solution I) and 3.6mL of (Solution II),

filtered with 0.45μ filter paper and sonicated for 10 mins. Mobile phase was used as diluent.

Diluent Preparation: Mobile phase is used as diluent.


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PREPARATION OF STANDARD SOLUTION

Accurately 10 mg of DMF & OND were weighed into a clean and dry 10mL volumetric flask

separately dissolved with sufficient volume of diluent. The final volume was made up to

10mL with diluent to get the concentration of 1000μg/mL for DMF & OND.

PREPARATION OF WORKING STANDARD SOLUTION OF DMF & OND

1 mL of standard stock solution was pipetted out into 10mL volumetric flask and further

diluted with diluent to 10mL to get concentration of 100μg/mL.

DETERMINATION

Wavelength for detection was selected by examining the resulted solution that consists of

DMF & OND (10μg/mL) in SHIMADZU UV- Spectrometer (UV- 1800) instrument. The

maximum absorbance for DMF & OND was observed at 239nm and hence 239nm was

selected as wavelength of detection.

METHOD VALIDATION

The proposed method was validated in compliance with ICH guidelines for linearity,

accuracy, precision, specificity, robustness, and system suitability parameters by the

following procedures.

LINEARITY

Accurately 10 mg of DMF and OND was weighed into a clean and dry 10 mL volumetric

flask, dissolved with sufficient volume of diluent. The volume was made up to 10 mL with

diluent to get the concentration of 1000 μg/mL for DMF and OND.

Preparation of working standard solutions of DMF and OND

The various concentration of working standard solutions of DMF & OND was made by

pipetting 1.0mL, 2.0mL, 4.0mL 8.0mL and 10.0 mL from stock (I) separately into a series of

10mL volumetric flask and diluted to 10mL to get the final concentration of 100μg/mL,

200μg/mL, 400μg/mL, 800μg/mL and 1000μg/mL solutions respectively.

DETERMINATION

The working standard solutions of DMF and OND ranging from 100 μg/mL to 1000 μg/mL

were injected into a chromatograph at flow rate of 1 mL/min. Retention time and peak area

obtained were recorded and standard calibration curve was plotted for DMF and OND and


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linearity equation was derived. The Correlation coefficient, % curve fitting were also

calculated. The results obtained were shown in Table 1.

TABLE 1: LINEARITY DATA FOR DMF & OND

Sl.

No

Volume of DMF &

OND working standard

solution (mL)

Final Volume

(mL)

Concentration

(µg/mL) DMF Peak area* OND Peak area*

1 1 10 100 225455 529180

2 2 10 200 475891 1028645

3 4 10 400 960885 2004596

4 8 10 800 1843299 3896470

5 10 10 1000 2311949 4900010

*Average of three determination

TABLE 2: LINEARITY REPORT OF DMF & OND

Parameters DMF OND Acceptancecriteria

Linearity range 100-1000 µg/mL 100-1000 µg/mL -

Regression equation y=2306.9x + 8385.9 y = 4859.5x + 35008 -

Correlationcoefficient 0.9997 0.9998 NLT 0.997

Percentage curve Fitting 99.97 99.98 NLT 99%

Slope 2318 4906 -

CALIBRATION CURVE OF DIMETHYL FUMARATE AND ONDANSETRON.

ACCURACY

Preparation of sample stock solution

Twenty tablets each containing 100 mg of DMF and 100mg of OND was weighed and finely

powered. Powder equivalent to 10 mg of DMF and 10mg of OND was taken and transferred

into a clean, dry 10 mL volumetric flask. The powder was first dissolved in diluent and


929


sonicated for 10 mins. The resulting mixture was then filtered through whatmann filter no

0.45 μ. The final volume of filtrate was made up to 10 mL with diluent.

Preparation of standard stock solution

Accurately weighed 10 mg of standard drug DMF and OND was transferred into a clean, dry

10 mL volumetric flask and the volume was made up to 10 mL with diluent to get the

concentration of 1000 μg/mL of DMF and OND.

Preparation of standard and sample mixture

Level I (80%): volume of 0.5 mL sample stock solution, 0.3 mL of standard solution was

transferred to 10 mL volumetric flask and volume was made up to mark with diluent (three

replicates).

Level II (100%)

Volume of 0.5 mL sample stock solution, 0.5 mL working standard stock solution was


replicates).

Level III (120%)

Volume of 0.5 mL sample stock solution, 0.7 mL of working standard stock solution was


replicates).

Determination

The resulting mixture was injected repeatedly into the chromatograph, the peak area and

chromatogram obtained were recorded and the % recovery of standard DMF and OND was

calculated. The results obtained are presented in Table 3 & 4.


930

Adnan et al. World Journal of Pharmacy and Pharmaceutical Sciences

Adnan et al. World Journal of Pharmacy and Pharmaceutical Sciences

TABLE 3: RECOVERY STUDY DATA FOR DMF & OND

Level Rep

licate

Standard

Conc µg/m L

Sample

Conc µg/m L

Peak

Area of

DMF

Peak

Area of

OND

Conc found

µg/m L of

DMF

Conc found

µg/m L of

OND

Amt of

standard

recovered of

DMF

Amt of

standard

recovered of

OND

%

Recovery of

DMF

%

Recovery of

OND

80%

I 3 5 180364 423344 7.92 7.85 2.91 2.95 97.00 98.33

II 3 5 180585 415269 7.89 7.82 2.88 2.91 96.00 97.00

III 3 5 181266 428875 7.95 7.89 2.95 2.93 98.33 97.66

100%

I 5 5 225576 529476 9.85 9.91 4.81 4.86 96.22 97.20

II 5 5 225755 539687 9.92 9.89 4.87 4.88 97.40 97.60

III 5 5 225989 513648 9.95 9.93 4.93 4.91 98.60 98.20

120%

I 7 5 270658 635425 11.79 11.85 6.79 6.88 97.00 98.28

II 7 5 265598 648579 11.85 11.91 6.81 6.83 97.28 97.57

III 7 5 296851 639980 11.78 11.88 6.89 6.93 98.42 99.00

TABLE 4: REPORT OF RECOVERY STUDIES FOR DMF & OND

Level Mean %

Recovery of DMF

Mean %

Recovery of OND

Acceptance

Criteria

80% 97.11 97.66 90-110%

100% 97.40 97.66 90-110%

120% 97.56 98.28 90-110%


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PRECISION

1 mL of standard solution of DMF and OND was transferred into a 10 mL volumetric flask

and final volume was then made up to 10 mL with diluent to get a concentration of 100μg/mL

of DMF and OND.

DETERMINATION

Successive six injections of working standard solution (six replicates) were injected into a

HPLC chromatograph, the peak area and chromatograms obtained were recorded. The %

relative standard deviation was calculated for peak areas and retention time of replicates. The

results and chromatogram obtained were shown in Table 5.

TABLE 5: REPORT OF PRECISION FOR DMF & OND.

Precision Parameters % RSDOF

DMF

% RSDOF

OND

Acceptance

Criteria

System Precision 0.14 0.78 < 2.0%

Method Precision 0.08 0.04 < 2.0%

Intraday Precision 0.03 0.01 < 2.0%

Inter day Precision 0.03 0.01 < 2.0%

SPECIFICITY

The diluent, working standard of DMF and OND were injected separately into the

chromatograph to examine that the DMF and OND peak is not affected by the mobile phase

and diluent and the chromatogram was recorded and is presented in Fig 1-2.

Chromatogram of only diluent was taken to check the interference of diluent with the peaks

of DMF and OND at the retention time of respective drugs. There was no peak detected at

retention time of DMF 3.1 min and OND 6.9 min. so, proposed method is specific in nature.


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LOD and LOQ

LOD and LOQ for DMF, OND by this method were evaluated on the basis of signal-to-noise

ratio method described in ICH guidelines. A signal-to noise ratio between 3 or 2:1 is

generally considered acceptable for estimating the detection limit. A typical signal-to-noise

ratio required for LOQ is 10:1. Using the proposed HPLC method, the LOD and LOQ values

were calculated and are given in Table 6.

TABLE 6: DATA FOR LOD AND LOQ OF DMF & OND

Parameter

DMF OND

Peak

Area

Concentration

in µg/mL

Peak

Area

Concentration

in µg/mL

LOD 2257 0.1 5296 0.4

LOQ 8825 0.4 20356 0.8

ROBUSTNESS

To evaluate the robustness of the developed RP-HPLC method, small deliberate variations in

the optimized parameters were made in chromatographic conditions like of flow rate, mobile

phase ratio and wavelength. The effect of change in flow rate, mobile phase ratio and

wavelength of detection on retention time and tailing factor were examined. The values

obtained are mentioned in Table 7, 8, 9. The method was found to be unaffected by the small

changes like ± 0.1 mL/min in flow-rate of mobile phase and change in mobile phase ratio

from 50:20:30 to 60:20:20 & 50:30:20 and ± 2 nm in detection wavelength.

TABLE 7: ROBUSTNESS DATA OF DMF & OND WITH CHANGE IN FLOW RATE

Change in Flowrate

ml/min

Peak area* of

DMF

%

Assay

Peak area* of

OND % Assay

0.9 224785 99.76 519136 96.19

1.0 225563 100.04 529597 99.77

1.1 223565 103.11 536379 101.56

TABLE 8: ROBUSTNESS DATA OF DMF & OND WITH CHANGE IN RATIO OF

MOBILE PHASE

Change in Mobile

phase ratio v/v

Peak area*

of DMF % Assay

Peak area* of

OND % Assay

60:20:20 225169 99.87 519569 98.18

50:20:30 225509 100.02 529290 100.02

50:30:20 225963 100.22 533815 100.87


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TABLE 9: ROBUSTNESS DATA OF DMF & OND WITH CHANGE IN

WAVELENGTH

Change in

wavelength in nm

Peak area*

of DMF % Assay

Peak area*

of OND % Assay

237 215519 95.59 508985 96.18

239 225496 100.01 519325 98.13

241 235691 104.54 548836 103.71

SYSTEM SUITABILITY

Six replicate of sample containing DMF & OND were given to evaluate equipment,

electronics, analytical operations and samples suitability. Parameters calculated for system

suitability were %RSD of retention time and area, number of theoretical plates and

Resolution. The results are given in Table 10.

TABLE 10: DATA FOR SYSTEM SUITABILITY PARAMETER FOR DMF & OND.

System Suitability

Factor DMF OND

Acceptance

Criteria

Theoretical plates* 4547.999 5232.982 > 2000

HETP (mm)* 32.982 28.664 -

Tailing factor 1.124 1.143 < 2

Resolution 0.000 13.549 > 2

RUGGEDNESS

Intermediate precision expresses the variations within laboratories variations: (different days,

different analysts, different equipment etc.). The Intermediate precision was performed for

DMF and OND by different analyst on different instrument using different lot of column on

different day. The % RSD for the same was calculated for Intermediate precision. The results

are given in Table 11 & 12.

TABLE 11: INTERMEDIATE PRECISION DATA OF ANALYST 1

REPLICATES DMF OND

AREA % ASSAY AREA % ASSAY

1 225739 100.12 529213 100.00

2 225631 100.07 529410 100.04

3 225519 100.02 529150 99.99

4 225499 100.01 529211 100.00

5 225691 100.10 529199 100.00

6 225418 99.98 529256 100.00

MEAN 225583 100.05 529240 100.00

Standard Deviation 0.0546 0.0549 0.0169 0.0175

% RSD 0.05 0.05 0.02 0.02

*Average of six determinations.


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TABLE 12: INTERMEDIATE PRECISION DATA ANALYST 2

REPLICATES DMF OND

AREA % ASSAY AREA % ASSAY

1 225643 100.08 529279 100.01

2 225750 100.13 529329 100.02

3 225529 100.03 529131 99.99

4 225510 100.02 529309 100.02

5 225681 100.12 529756 100.10

6 225519 100.02 528165 99.80

MEAN 225605 100.05 529165 99.99

Standard Deviation 0.0443 0.0454 0.0999 0.0996

% RSD 0.04 0.05 0.10 0.10

*Average of Six determinations.

RESULT AND DISCUSSION

Optimized chromatography condition: Chromatographic conditions were screened for mobile

phase composition, wavelength proportion and flow rate Finally, mobile phase of Methanol:

Acetonitrile: phosphate buffer with pH 5.5 (50:20:30 v/v/v) was optimized to give symmetric

peak with short runtime at UV detection wavelength of 239 nm and flow rate at 1mL/min was

found to be appropriate with adequate separation between the two drugs. Chromatogram of

DMF & OND at optimized chromatographic condition was recorded, the runtime was 12.5

min and the retention times of DMF & OND were found to be 3.1 and 6.9 min respectively.

CONCLUSION

The proposed HPLC method was found to be economical, simple, sensitive, accurate, precise,

specific and robust and can be used for the routine quality control analysis of DMF & OND

in bulk as well as in tablet formulation.

ACKNOWLEDGEMENT

The author is highly thankful to the Government College of Pharmacy, Bengaluru, Karnataka

for providing all the laboratory facilities. I would also express our gratitude to Drug Testing

Laboratory, Bengaluru, for providing me the gift sample of Ondansetron.

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development and validation of analytical method for

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