development and validation of analytical method for
TRANSCRIPT
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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.
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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
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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
transferred to 10 mL volumetric flask and volume was made up to mark with diluent (three
replicates).
Level III (120%)
Volume of 0.5 mL sample stock solution, 0.7 mL of working standard stock solution was
transferred to 10 mL volumetric flask and volume was made up to mark with diluent (three
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.
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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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