Development and Validation of RP – HPLC Method for the estimation of Oxyclozanide in Pure and Pharmaceutical formulation

185
* Corresponding author: Anusha.G
E-mail address: anushagarnepudi1990@gmail.com
IJPAR |Volume 3 | Issue 2 | April –June -2014 ISSN: 2320-2831
Available Online at: www.ijpar.com
[Research article]
Development and Validation of RP – HPLC Method for the
estimation of Oxyclozanide in Pure and Pharmaceutical
formulation
Anusha.G*, T.Rattaiah Guptha, S.Manohar Babu.
Sims College of Pharmacy, Mangaldas Nagar, Mangalagiri Road, Guntur, A.P. India.
ABSTRACT
A simple, fast, precise, selective and accurate RP-HPLC method was developed and validated for the
simultaneous determination of oxyclozanide from pharmaceutical formulation. Chromatographic separation was
achieved gradient on a YMC c18 column (250 x 4.6 mm, 5 µ particle size) using a mobile phase acetonitrile and
water in the ratio of 80:20.the flow rate was 1.0ml / min and effluent was detected at 300nm.the retention time
of oxyclozanide was found to be 1.89min. Linearity was observed in the concentration range of 10 -100µg / ml
.The method was validated according to ICH guidelines with respect to specificity, linearity, accuracy, precision
and robustness. The method developed can be used for the routine analysis of oxyclozanide.
Keywords: Oxyclozanide, RP-HPLC, Development, Validation.
INTRODUCTION
Oxyclozanide is chemically 2, 3, 5-Trichloro-N-(3,
5-dichloro-2-hydroxyphenyl)-6-
Hydroxy benzamide used as anti helmenthic.
Figure 1: Chemical structure of oxyclozanide
Several HPLC, GC,
and LC/MS-MS methods have
been reported for the analysis of oxyclozanide in
plasma that suffer from either undesirably long
chromatographic run times and requirement for
gradient analysis or use of an internal standard.

Anusha.G, et al / Int. J. of Pharmacy and Analytical Research Vol-3(2) 2014 [185-191]
186
The objective of this study was to develop reverse
phase high performance liquid chromatography
method for the estimation of oxyclozanide in pure
and pharmaceutical dosage form without any
derivatization and having the short retention time.
This method was found to be linear, precise,
accurate, sensitive, specific, and robust, and
therefore suitable for routine analysis.
MATERIALS AND METHOD
HPLC Instrumentation and
Chromatographic conditions
The analytical separations were carried out on a
waters 2487 HPLC system equipped with Photo
Diode Array detector. The output of signal was
monitored and integrated using LC – solutions
2000 software. The analytical column was YMC
C18 (250 × 4.6mm, 5µ). Mobile phase consisted
Acetonitrile and water in the ratio of 80:20. Mobile
phase was mixed, filtered through 0.45µmembrane
filter and degassed under ultrasonication. The
mobile phase was used as diluent. The flow rate
was 1.0 ml/min and runtime was 5 minute. The
column was maintained at ambient temperature.
UV detection was measured at 300 nm and the
volume of sample injected was 10 μl.
Preparation of standard stock solution
25mg of oxyclozanide was weighed accurately and
dissolved in 25ml of mobile phase to get the
concentration of 1000 µg/ml. Resultant solution
was filtered through Whatman filter paper. The
standard chromatogram for oxyclozanide (100
μg/ml) was shown in figure 2.
Preparation of working standard solution
Working standard solutions of oxyclozanide were
prepared by accurately transferring the
(0.1,0.2,0.4,0.6,0.8 and 1.0 ml) aliquots of the
standard stock solution into a series of six 10 ml
volumetric flasks. The volume was made up to
mark with mobile phase to obtain concentration
range of 10 –100 µg/ml.
Preparation of sample solutions
20 tablets were weighed individually and their
average weight was calculated. Then the tablets
were grounded and weight equivalent to 141.25mg
of oxyclozanide was taken into 25mL volumetric
flask and then the sample was diluted to 25ml with
mobile phase to get concentration of 1000µg/ml
and used for analysis.
RESULTS AND DISCUSSION
HPLC method development and
optimization
To optimize the chromatographic conditions,
different columns, mobile phases, flow rates etc.,
were tested. Acetonitrile and water in the ratio of
80:20 was preferred as mobile phase because it
resulted in a greater response to oxyclozanide after
several preliminary investigatory runs compared
with the different mobile phase combinations. The
effect of the flow rate was studied in the range 0.9
to 1.5ml/min and 1.0ml/min was preferred to be
effective. Under these conditions, the analyte peak
obtained was well-defined and free from tailing.
The retention time (RT) was found to be 1.89 min.
The optimized chromatographic parameters were
listed in table 1
Fig 1. optimized chromatogram

Anusha.G et al / Int. J. of Pharmacy and Analytical Research Vol-3(2) 2014 [185-191]
187
Table 1: Optimized chromatographic parameters
Optimized Chromatographic parameters
Elution gradient
Mobile phase acetonitrile water (80:20)
Column
YMCc18 column
Flow rate
1.0ml/min
Detection
300nm
Injection volume
10μl
Temperature
Ambient
Retention time
1.89min
Run time
5.0 min
Concentration 10 - 100μg/ml
Validation of the method
When method development and optimization are
complete, it is necessary to accomplish method
validation. The validation studies include linear
range (correlation coefficient), method precision
(RSD, %), method accuracy (% recovery and RSD,
%), sensitivity studies (LOD & LOQ), and
robustness.
System suitability studies
System-suitability tests are an integral part of
method development and are used to ensure
adequate performance of the chromatographic
system. Retention time (RT), tailing factor (T), and
peak asymmetry (AS), resolution (RS) were
evaluated. The system suitability test was
performed using five replicate injections of
standards before analysis of samples. The system
suitability method acceptance criteria set in each
validation run were: capacity factor > 2.0, tailing
factor ≤ 2.0 and theoretical plates > 2000. In all
cases, the relative standard deviation (R.S.D) for
the analytic peak area for two consecutive
injections was < 2.0%. System suitability
parameters were shown in table 2.
Table 2: System suitability parameters
Parameters Values
Retention time 1.89min
Linearity
The linearity of the method was evaluated by
preparing six series of standard solutions of
oxyclozanide in the range of 10 – 100 µg/ml in
mobile phase and injecting the solutions into the
HPLC system. Excellent correlation between
oxyclozanide peak area and concentration was
observed with R2
= 0.998 (Figure.3). The
regression equation was found to be Y =18861 x
+33523. Statistical data are presented in table 3 and
the calibration curve was shown in figure 3.
Table 3: Linearity results for oxyclozanide
s.no concentration Area
1 10 207406
2 20 437484
3 40 786045
4 60 1506426
5 80 1945004
6 100 2065987

188
Fig 2. Calibration curve for oxyclozanide
PRECISION
System precision: (Repeatability)
To study precision, five replicate standard solutions
of oxyclozanide (40 µg/ml) were prepared and
analyzed using the proposed method. The percent
relative standard deviation (% RSD) for peak
responses was calculated. Results of system
precision studies were shown in table 4.
Table 4: Results of system precision for oxyclozanide
s.no Retention time(min) Area(Mv. sec)
1 1.193 1023654
2 1.923 1.45897
3 1.857 1056488
4 1.857 1023654
5 1.913 1023564
6 1.848 1023654
Mean 1.8854 1032833.5
Standard deviation 0.047545968 14609.88216
% RSD 1.645189498 1.414543
Method precision: (Reproducibility)
The intraday and inter-day precision of the
proposed method was determined by analyzing the
corresponding responses 6 times on the same day
and on different days for concentration of sample
solutions of 100µg/ml. The result was reported in
terms of relative standard deviation (% RSD).
Results of method precision studies were shown in
table 5.
Table 5: Results of Method precision for oxyclozanide
s.no Peak area %labelled claim
1 803093 102
2 832580 99.3
3 830258 99.6
4 833425 98
5 803092 102
6 823695 99
y = 18861x + 33523
R² = 0.9987
0
500000
1000000
1500000
2000000
2500000
0 20 40 60 80 100 120
area
cocentration (µg/ml)

189
Intermediate precision
The intermediate precision of the proposed method
was determined by performing the method by two
analysts (Analyst 1 and Analyst 2) for
concentration of sample solutions 40 µg/ml. The
percent relative standard deviation (% RSD) for
peak responses was calculated. The results for
intermediate precision were shown in table 6.
Table 6: Results of Intermediate precision for oxyclozanide
OXYCLOZANIDE
S.NO
ANALYST – 1 ANALYST – 2
AREA (Mv.sec) Retention time AREA (Mv.sec) Retention time
1 1088512 1.857 786045 1.857
2 1088836 1.857 774744 1.865
3 1085985 1.857 785396 1.857
4 1045311 1.848 774744 1.865
5 1055614 1.873 803093 1.865
MEAN 1072852 1.8584 785011 1.601
S.D 20789.75 0.009044 10393 0.00438178
% RSD 1.937803 0.486673 1.323 0.235453007
ACURACY
Accuracy of the method was confirmed by the
standard addition method, which was carried out by
performing recovery studies at 2 different
concentrations 40 and 60 µg/ml of these expected,
in accordance with ICH guidelines, by
replicate analysis (n=3). Known amount of
standard drug solution (40µg/ml) was added to a
pre analyzed sample solution (40 and 60 µg/ml)
and percentage drug content was measured. The
closeness of obtained value to the true value
indicates that the proposed method is accurate.
Recovery studies were shown in table 7.
% Recovery = [(Ct –Cpa)/ Cs] × 100.
Where,
Ct = Total concentration of analyte
Cpa = Concentration of pre-analysed sample
Cs = Concentration of standard added to pre-analysed sample.
Table 7: Results of recovery studies for oxyclozanide by using RP –HPLC method
s.no std level Amount added Total recovery recovered %recovery
1 40 40 40 81.3 41.3 103.25
2 40 40 40 83.93 43.93 109.825
3 40 40 40 81.39 41.39 103.475
4 40 60 60 98.3 58.3 97.1
5 40 60 60 102.46 62.46 104.1
6 40 60 60 100.8 60.8 101.3
ROBUSTNESS
The robustness study was performed to evaluate the
influence of small but deliberate variation in the
chromatographic condition. The robustness was
checked by changing parameters like flow rate of
mobile phase and detection wavelength
 Change in the detection wavelength by ± 2nm
(298 nm and 300 nm)

190
 Change in flow rate by ± 0.1 ml/minute (1.2
ml/min and 0.9 ml/minute)
After each change, sample solution was injected
and % assay with system suitability parameters
were checked.
Robustness values were given in table 8
Table 8: Results of Robustness for oxyclozanide
parameter Rt(min) Area(mvsec)
Flow rate(ml/min)1.2 1.90 1485965
0.9 1.892 1300145
Wavelength(nm)302 2.08 1328815
298 1.986 1215141
Limit of Detection and Quantitation
Detection and Quantitation limit were calculated by
the method based on the standard deviation () and
slope of the calibration plot, using the formula
Limit of Detection   × 3.3/S
Limit of Quantitation   × 10/S
Where  = the standard deviation of the response.
S = the slope of the calibration curve (of the analyte).
Table 9: Results of LOD, LOQ for oxyclozanide
S.No LOD LOQ
1 0.081 0.075702
Specificity
Specificity of an analytical method is its ability to
measure the analyte accurately and specifically in
the presence of component that may be expected to
be present in the sample matrix. Chromatograms of
standard and sample solutions were compared in
order to provide an indication of specificity of the
method.
Assay of pharmaceutical formulation
The proposed validated method was successfully
applied to determine oxyclozanide in their
pharmaceutical dosage form and the % Assay
results were shown in table 10.
Table 10: Results of % assay by using RP – HPLC method
Drug Sl.No Amount found(mg) Test area Standard area % Assay
(AT /AS*100)
Oxyclozanide 1 999.99 774744 785396.563 98.64%
2 999.99 803093 102.25%
3 999.99 786045 100.08%
CONCLUSION
A simple, rapid, accurate, and precise RP-HPLC
method for the analysis of oxyclozanide in pure
and in pharmaceutical dosage forms had been
developed and validated in accordance with ICH
guidelines. The RP-HPLC method developed is
cost-effective due to short retention time which
enabled analysis of oxyclozanide samples with a
small amount of mobile phase. From the % RSD
values of precision and recovery studies the method
was found to be precise and accurate. The low
detection and quantification limits achieved

191
indicate the method is very sensitive. The
robustness data gathered during method validation
showed that the method is not susceptible to small
changes in chromatographic conditions. The
proposed RP-HPLC method developed by the
author is suitable for routine analysis and quality
assessment of oxyclozanide in pharmaceutical
products.
Table 12: Summary of validated parameters for proposed method
Parameter Result
Linearity range 10 – 100 µg/ml
Regression equation Y = 18861 x +33523
Slope 18861
Intercept 33523
Correlation coefficient 0.998
System precision (% RSD,n=5)1.4145
Intermediate precision (% RSD, n=5) 0.23
LOD (µg/ml) 0.081
LOQ (µg/ml) 0.075
% Recovery (Accuracy, n =3) 100%
% Assay (% Assay, n=3) 100%
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Development and Validation of RP – HPLC Method for the estimation of Oxyclozanide in Pure and Pharmaceutical formulation

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Development and Validation of RP – HPLC Method for the estimation of Oxyclozanide in Pure and Pharmaceutical formulation