RP- HPLC Method for Simultaneous Estimation
of Ofloxacin and Ornidazole
from Bulk and Tablets
RS
Jadhav1, PN Kendre2, MH Kolhe1, S N Lateef2,
SM Shelke3, RK Godge1*.
1Dept. of
Pharmaceutics, Pravara Rural
2
Sanjivani
college of pharmaceutical Education and Research, Kopargaon.
3
Research Associate Orchid Pharmaceutical Ltd., Chennai.
ABSTRACT
A
simple, selective, rapid, precise and economical reverse phase high-pressure
liquid chromatographic method has been developed for the simultaneous
estimation of Ofloxacin and Ornidazole
from pharmaceutical formulation. The method was carried out on a Kromasil C18 (5 mm, 25 cm X 4.6 mm, i.d.)
column, with a mobile phase consisting acetonitrile:
phosphate buffer (pH 2.4) in the ratio 80: 20% V/V at a flow rate of 1.0
ml/min. Detection was carried out at 294 nm. The retention time of Ofloxacin and Ornidazole were
2.773 and 5.448min respectively. The developed method was validated in terms of
accuracy, precision, linearity, Limit of detection, Limit of quantitation. The proposed method can be used for
estimation of these drugs in combined dosage form for routine analysis.
Keywords: Ofloxacin, Ornidazole, RP-HPLC.
INTRODUCTION
Ofloxacin
is a synthetic broad spectrum antibacterial agent. Chemically ofloxacin1
a fluorinated carboxyquinolone, is a racemate, (±)- 9-fluro-2, 3-dihydro-3-methyl-10- (4-methyl-
1-piperazinyl)-7-oxo-7H-pyrido [1,2,3-de]-1,4-benzoxazine- 6-carboxylic acid.
It is official in BP2, USP3, and EP4. The
assay procedure mentioned in these pharmacopoeias uses nonaqueous
titration for estimation of ofloxacin. Literature
surveys reveals Spectrophotometric method5,6
atomic absorption spectrometry5, spectroflurometry5, HPLC7
and microbiological method8 for its determination.
Ornidazole1 is a 5-nitroimidazole
derivative used as anti-infective agent. It is not official in any Pharmacopoeia.
Literature survey reveals that ornidazole is
estimated by voltametry9 and HPLC10 methods for its
determination in dosage forms and biological fluids. Ofloxacin
and ornidazole in combined tablet dosage form is
available in the market, has gained increasing acceptance in diarrhooea, bacterial and protozoal
infections. Spectrophotometric11, 12 method have been established
for their simultaneous estimation in tablet dosage form. This paper presents a
simple, accurate and reproducible HPLC method for simultaneous determination of
ofloxacin and ornidazole in
tablet dosage form.
EXPERIMENTAL
Reagents and Chemicals
Acetonitrile (HPLC grade) and
was purchased from Merck specialties pvt. Ltd. (Worli,
Pharmaceutical
formulation:
Commercial
tablets, each containing Ofloxacin (200mg) and Ornidazole (250mg) (Oflab –
Oz); were procured from the local market.
Different mobile phases containing methanol,
water, Acetonitrile,
and different buffers in different proportion were tried and finally of acetonitrile :phosphate buffer (pH 2.4)in the ratio 80: 20%
V/V was selected as moile phase which gave good
resolution and acceptable peak parameters for both Ofloxacin
and Ornidazole.
Chromatographic
separation was performed on a Shimadzu (LC-2010 AHT) and 20ml sample injection loop and U.V. visible
absorbance detector. The output signal was monitored and integrated using the Shimadzel CLASS – VP software was used for the separation;
mobile phase of a mixture of Acetonitrile and
phosphate buffer was delivered at a flow rate of 1.0 ml/min with detection at
294nm. The mobile phase was filtered through a 0.2 m membrane filter
and degassed. The injection volume was 20 ml; analysis was performed at ambient
temperature.
|
Parameters |
Method |
|
Stationary phase
(column) |
C18
(5um, 25cm X 4.6mm,i.d.) |
|
Mobile phase |
Phosphate
buffer (pH= 2.4) : ACN (80:20) |
|
Flow rate (ml /
min ) |
1.0 |
|
column
temperature (0C) |
Ambient |
|
Volume of
injection (ml) |
20 |
|
Detection
wavelength (nm) |
294 |
|
Level
of %
Recovery |
%
Mean Recovery* |
%
R.S.D.† |
||
|
OFLOX |
ORNI |
OFLOX |
ORNI |
|
|
80 |
99.87 |
98.2 |
0.06993
|
0.3388 |
|
100 |
98.37 |
99.41 |
0.04553
|
0.4066 |
|
120 |
99.82 |
98.23 |
0.0766
|
0.28283 |
20 mg of ofloxacillin and 25 mg of ornidazole
working standard Weighed into 50 ml of volumetric flask and dilute with mobile
phase to 50 ml. Pipette out 5 ml of resulting solution in 50 ml volumetric
flask and dilute to 50 ml with mobile phase.
Calibration Curve
Linearity of the
system was investigated by serially diluting the stock solutions to give
concentrations in the range of 20 mg/ml to 100 mg/ml for Ofloxacin
and 25 mg/ml
to 125 mg/ml
for Ornidazole. An aliquot (20 ml) was injected
using mixture of Acetonitrile: phosphate buffer (80:20) v/v, as mobile
phase. Calibration curves were obtained by plotting the peak area vs.
concentration. The calibration curves are as shown in Fig.2 and Fig.3 for Ofloxacin and Ornidazole
respectively. The equations of the regression lines are
For Ofloxacin y =
81719x – 105961 (R2 = 0.999)
For Ornidazole y =105021x -99999 (R2 = 0.999)
Assay
Twenty Tablets,
each containing 200 mg of Ofloxacin and 250mg Ornidazole were weighed and finely powdered. A quantity of
powder equivalent to 50mg of Ofloxacin was weighed
and transferred to 50 ml volumetric flask. To this, acetonitrile
was added and sonicated for 10 min; the volume was
made up to 50 ml with acetonitrile to get solution of
1000 mg/ml.
The solution was filtered using whatmann filter
paper. From the filtrate appropriate dilutions were made to obtain
concentration in the range of 20 to 100mg/ml for Ofloxacin
and 25 to 125 mg/ml
Ornidazole respectively.
As per the ICH guidelines, the method validation parameters
checked were linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.
The linearity of the method was determined for the formulation at five
concentration levels ranging from 20 to 100 mg/ml for Ofloxacin and 25
to 125 mg/m for Ornidazole. The equation for regression line for Ofloxacin was y =81719x – 105961 (R2 = 0.999)
and
For Ornidazole y =105021x -99999 (R2 =
0.999).
The results show that an excellent correlation exists between Peak area
and concentration of drugs within the concentration range indicated above.
Fig. 1:
Chromatogram of Ofloxacin (2.733 min), Ornidazole 5.403 min respectively.
|
Conc. (mg/ml) |
Ofloxacin |
Conc. (mg/ml) |
Ornidazole |
||
|
%
RSD |
%
RSD |
||||
|
Intra- day |
Inter- day |
Intra- day |
Inter- day |
||
|
20 |
0.16 |
0.22 |
25 |
0.53 |
0.62 |
|
40 |
0.29 |
0.30 |
50 |
0.69 |
0.76 |
|
60 |
0.2 |
0.27 |
75 |
0.31 |
0.36 |
|
80 |
0.31 |
0.25 |
100 |
0.92 |
1.5 |
|
100 |
0.32 |
0.37 |
125 |
0.13 |
0.2 |
RSD = Relative Standard
Deviation (n = 3).
|
Conc. (mg/ml) |
Ofloxacin |
Conc. (mg/ml) |
Ornidazole |
||
|
% RSD |
%
RSD |
||||
|
Intra- day |
Inter- day |
Intra- day |
Inter- day |
||
|
20 |
0.36 |
0.52 |
25 |
0.45 |
0.63 |
|
40 |
0.42 |
0.48 |
50 |
0.61 |
0.76 |
|
60 |
0.56 |
0.43 |
75 |
0.35 |
0.39 |
|
80 |
0.36 |
0.21 |
100 |
1.2 |
1.33 |
|
100 |
0.21 |
0.34 |
125 |
0.9 |
0.37 |
RSD = Relative Standard Deviation (n = 3).
Fig.2: Calibration curve
for Ofloxacin
The accuracy of the method was determined by
recovery experiments. The recovery studies were carried out at three levels of
80, 100 and 120% and the percentage recovery was calculated and presented in
Table 2. Recovery was within the range of 100 ± 2% which indicates accuracy of
the method.
The precision of the method was demonstrated by
inter day and intra day variation studies. In the intra day studies, 3 repeated
injections of standard and sample
solutions were made in a day and
percentage RSD were calculated. In the inter day variation studies, 3 repeated
injections of standard and sample solutions were made on 3 consecutive days and
percentage RSD were calculated and presented in Table 3 and 4. The data
obtained, %RSD not more than 1.5%, indicates that the developed RP-HPLC method
is precise.
The Limit of
Detection (LOD) is the smallest concentration of the analyte
that gives the measurable response. LOD was calculated using the following
formula
LOD = (3.3 x standard deviation)/ Slope of
calibration curve
The LOD for Ofloxacin and Ornidazole were
found to be 0.002 mg/ml
and 0.091 mg/ml,
respectively.
The Limit of
Quantification (LOQ) is the smallest concentration of the analyte,
which gives response that can be accurately quantified. LOQ was calculated
using the following formula
LOQ = (10 x
standard deviation) / Slope of calibration curve.
The LOQ was 0.0061 mg/ml and 0.2768 mg/ml for Ofloxacin and Ornidazole
respectively.
Robustness
Robustness
is checked by making slight deliberate change in the experimental procedures.
In the present method a deliberate change of Wavelength, pH, and flow rate was
made and the effects were noted. The method was found to be robust with respect
to change in wavelength, pH, and flow rate.
The proposed method
was found to be simple and linear in the concentration range of 20 to 100 mg/ml for Ofloxacin and 25 to 125mg/ml for Ornidazole
respectively. The method was found to be accurate and precise as indicated by
recovery studies and % RSD not more than 1.5. Moreover LOD and LOQ Ofloxacin were found to be 0.002 mg/ml and 0.0061 mg/ml, respectively
and for Ornidazole were 0.091 mg/ml and 0.2768 mg/ml, respectively.
Thus the method is specific and sensitive.
The proposed
RP-HPLC method for the simultaneous estimation of Ofloxacin
and Ornidazole in combined dosage forms was found to
be sensitive, accurate, precise, simple and rapid. Hence the present RP –HPLC
method may be used for routine analysis of the raw materials and formulations.
ACKNOWLEDGEMENT:
The author wish to
thanks to Concept Pharma Ltd. Aurangabad, India for
providing Working standard of Ofloxacin and Ornidazole and also thankful to Dr. S. R. Pattan for providing guidance for carrying research.
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Received on 04.06.2009
Accepted on 22.07.2009
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Research J. Science
and Tech. 1(1): July-Aug. 2009: 43-46