VALIDATION AND APPLICATION OF RP-HPLC
METHOD FOR QUANTIFICATION OF ENROFLOXACIN IN PURE AND VETERINARY DOSAGE FORMS
INTRODUCTION
Enrofloxacin is a synthetic chemotherapeutic agent from the class
of the fluoroquinolone carboxylic acid derivative which was developed
exclusively for use in animals [1]. Chemically it is a
1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1, 4-dihydro-4-oxo-3-quinoline
carboxylic acid. It inhibits the bacterial DNA gyrase (a type-II
topoisomerase), thereby preventing DNA supercoiling and DNA synthesis [2, 3].
It exhibits a wide spectrum of antimicrobial activity against gram-negative
bacteria and intracellular organisms such as Rickettsia, Chlamydia, and
Mycoplasma, and a tighter spectrum of activity against gram-positive organism’s
drug with a wide spectrum of activity against bacteria [4].
Fig. 1: Chemical structure of enrofloxacin
International trade in aquaculture has been growing continuously
in recent years, and is expected to persist in the future. The variety of
farmed aquatic species is now diversifying and fish farming is intensifying. At
the same time, increased incidence of disease, also due to intensive
aquaculture, requires more intense use of veterinary drugs and chemicals.
During the last few years enrofloxacin has received growing attention because
of its potential efficiency for the treatment of diseases in fish [5, 6].
The methods available for Enrofloxacin estimation are thin-layer
chromatography (TLC)-fluorescence, capillary electrophoresis–fluorescence,
Spectrophotometry [7, 8], high-performance liquid chromatography (HPLC)-UV or
fluorescence [9-17], HPLC-Mass spectrometry (MS) or gas chromatography (GC)-MS
[18]. The majority of these methods have been developed to analyze biological
samples such as serum or urine. The extensive literature surveys revealed that
few methods have been reported for the estimation of Enrofloxacin in pure and
dosage forms.
There is a wide scope for the development of new analytical
methods for the estimation of the Enrofloxacin. This paper presents simple,
sensitive, accurate and reproducible RP-HPLC methods that were developed for
the estimation of Enrofloxacin in bulk and veterinary dosage forms.
MATERIALS
AND METHODS
Chemicals and reagents
The standard drug of Enrofloxacin was collected as gift samples
from Sura labs, Hyderabad. The commercial tablet dosage form was procured from
a local pharmacy. Acetonitrile (HPLC grade), Methanol (HPLC grade), water (HPLC
grade), and orthophosphoric acid used for the preparation of the mobile phase
were a product of Merck.
Instruments used
The chromatographic system used to perform development and
validation of this assay method was comprised of a LC-10ATvp binary pump, an
SPD-20Avp UV-Visible detector and a Hamilton manual injector with 20μl loop
(Shimadzu, Kyoto, Japan) connected to a multi-instrument data acquisition and
data processing system (LC Solution, Shimadzu).
Experimental
Chromatographic conditions
Column Phenomenex C18 column (250 mm x 4.6 mm i.
d,5 µm) was used for separation and the mobile phase, acetonitrile and water
(80:20), was pumped at a flow rate of 1 ml/min. It was filtered through
0.20/0.45μm filter and degassed before use. The elution was monitored at 270 nm
and the injection volume was 20μL. The oven temperature was 40 °C.
Preparation of mobile phase
A mixture of acetonitrile and water at a ratio of 80:20 was
prepared with vigorous shaking. The solution was sonicated and degas before
use.
Preparation of standard solutions
10 mg of Enrofloxacin was weighed accurately and transferred to 10
ml standard flask, add 1 ml methanol for dissolving, then add mobile phase
(acetonitrile and water-80:20) and the volume made up to 10 ml to get a
concentration of 1000μg/ml (Stock solution A).
From Stock solution–A, different volume of standard solutions was
taken and prepare 0.1, 0.2, 0.3, 0.4, 0.5, 0.6μg/ml concentration of
enrofloxacin solutions.
Preparation of formulation solutions
Twenty tablets were weighed and finely powdered. From the powdered
tablet, a quantity of powder equivalent to 10 mg of enrofloxacin was weighed
and extracted with mobile phase having acetonitrile and water (80:20) and
finally made up with mobile phase to get a concentration of 1000μg/ml. From this,
prepare 10μg/ml of working solution. 20μl of each concentration of the drug
were injected into the HPLC system and their chromatograms were recorded. Peak
areas were recorded for all the peaks and a standard calibration curve of area
under curve against concentration was plotted.
Method validation
The optimized chromatographic method was completely validated
according to the procedures described in ICH guidelines Q2 (R1) for the
validation of analytical methods [19-21].
Linearity and range
The linearity of a method is its ability to obtain test results
that are directly proportional to the sample concentration over a given range
of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6μg/ml should be investigated. The relationship
between sample concentration and detector response (peak area or height) was
used to make this determination. Calibration curves for each method was plotted
and the obtained data were subjected to regression analysis.
Precision
The reproducibility of the proposed method was determined by
performing the assay for the same day (intra-day assay precision) and on three
different days (inter-day assay precision). Precision studies was performed by
preparing six determinations in the concentration of 0.5μg/ml. The results of
repeatability, intra-day and inter-day precision were expressed in % RSD.
Accuracy
In order to ensure the suitability and reliability of the proposed
method, recovery studies were carried out. The accuracy study was carried out
by the analysis of standard additions at three levels that is, multi-level
recovery studies. To a fixed equivalent quantity of formulation powder as well
as a synthetic mixture, a known quantity of standard enrofloxacin added at 50%,
100% and 150% level and the contents was re-analyzed. The % recovery and %RSD
were calculated.
Limit of detection and limit of quantification (LOD and LOQ)
The LOD and LOQ were separately determined based on the standard
calibration curve. The residual standard deviation of the y-intercept of
regression lines may be used to calculate LOD and LOQ. LOD=3.3*D/S and
LOQ=10*D/S, where D is the standard deviation of the intercept of the
regression line and S is the slope of the calibration curve.
Ruggedness
The ruggedness of an analytical method is the degree of
reproducibility of test results obtained by the analysis of the same samples
under a variety of conditions, such as different laboratories, analysts,
instruments. Ruggedness of the proposed method was determined by analysis of
aliquots from homogenous slot by two analyst using the same operational and
environmental conditions.
Robustness
The robustness of an analytical procedure is the measure of its
capacity to remain unaffected by small, but deliberate, variations in method
parameters and provides an indication of its reliability during normal usage.
The proposed method parameters were the change of volumetric flasks (10 ml, 50
ml and 100 ml) were performed. The robustness of was determined by different
wavelengths and mobile phase flow rate. The result is expressed in Percentage
RSD.
System suitability
A system suitability test of the chromatographic system was
performed to ensure the validity of the analytical procedure. Five replicate
injections of standard preparation were injected and tailing factor,
theoretical plate and % RSD of peak area were determined
RESULTS
Method development
In this proposed method, RP-HPLC method based on UV detection was
developed and validated for the estimation of Enrofloxacin in pure and dosage
forms. Phenomenex make Shimadzu C18 column (250 mm × 4.6 mm
i.d., 5μm particle size) column and mobile phase composition consisting of a
mixture of Acetonitrile: Water (80:20v/v) found to be giving satisfactory
results. For the selection of organic constituent of mobile phase, acetonitrile
was chosen to reduce the longer retention time and to attain good peak shape.
[fig. 2] represent the wavelength selection [fig. 3] represent the
chromatograms of standard.
Determination of wavelength of maximum absorption
Fig. 2:
HPLC scan spectrum for enrofloxacin chromatogram of standard
Fig. 3: Chromatogram for enrofloxacin [Std]
Method validation parameters
System suitability
All the efficiency parameters like theoretical plates were
observed to be more than 7000 for Enrofloxacin. The peak tailing was not more
than 2.0. The %RSD for the five replicate injections was not more than 2.0 and
ensured that the entire testing system and chemicals used could generate an
accurate and precise result by showing all the efficiency parameters within the
specified limits. Reported the results in tables 1.
Linearity
For linearity, seven points calibration curve [table 2] was
obtained in a concentration range from 0.1-0.6 µg/ml for Enrofloxacin. The
response of the drug was found to be linear in the investigation concentration
range and the linear regression equation for Enrofloxacin was y =
866919x+6888.7 with a correlation coefficient 0.9991 [fig. 4]. Where x is the
concentration in µg/ml and y is the peak area in the absorbance unit.
Table 1: System suitability parameters of enrofloxacin
Parameter |
Enrofloxacin |
Theoretical
plate |
7854 |
Tailing factor |
0.9 |
Retention time |
3.405 |
Peak symmetry |
1.26 |
Area of peak |
434592 |
Fig. 4: Calibration curve of enrofloxacin
Table 2: Linearity data for enrofloxacin
Parameters |
Data’s |
λmax |
270 nm |
Linearity(µg/ml) |
0.1-0.6 |
Correlation
coefficient(r²) |
0.9991 |
Slope(m) |
866919 |
Intercept(c) |
6888.7 |
λmax=Maximum absorbance, µg/ml =microgram per millilitre
LOD and LOQ
The limit of detection and limit of quantification were evaluated
by serial dilutions of Enrofloxacin stock solution. The LOD value for
enrofloxacin was 0.001µg/ml and LOQ was found to be 0.03µg/ml. LOD and LOQ
study values were shown in table [table 3]
Accuracy
The HPLC area responses for accuracy determination are depicted in
[table 4]. The result shown that the best recoveries (98-102 %) of the spiked
drug were obtained at each added concentration, indicating that the method was
accurate.
Precision
The result of repeatability of std and inter-day and intra-day
precision study are shown in [table 5]. The developed method was found to be
precise as the % RSD values for the repeatability and inter and intra-day
precision studies were<0.096 % and<0.268 %, respectively, which confirms
that method was precise.
Table 3: LOD and LOQ for enrofloxacin
Method |
Detection
wavelength (nm) |
LOD
(μg/ml) |
LOQ
(μg/ml) |
Proposed
Method |
270 nm |
0.001 |
0.03 |
LOD=Limit of Detection, LOQ=Limit of Quantification, µg/ml
=microgram per millilitre
Table 4: Accuracy data for enrofloxacin
Level
of addition (%pure drug) n=3 |
Con.
of drug in formulations (µg/ml) |
Conc.
drug (µg/ml) of pure |
Total
conc. of the drug found (µg/ml) |
%
Analytical recovery |
Data |
50% 50% 50% |
0.5 0.5 0.5 |
0.25 0.25 0.25 |
0.749 0.749 0.748 |
99.86 99.86 99.73 |
Mean=99.816 SD=0.075 %RSD=0.075 |
100% 100% 100% |
0.5 0.5 0.5 |
0.5 0.5 0.5 |
0.998 1.006 1.003 |
99.8 100.6 100.3 |
Mean=100.233 SD=0.4041 %RSD=0.403 |
150% 150% 150% |
0.5 0.5 0.5 |
0.75 0.75 0.75 |
1.248 1.246 1.243 |
99.84 99.68 99.44 |
Mean=99.653 SD=0.2013 %RSD=0.202 |
SD =Standard deviation, RSD= Relative standard deviation,
n=number, µg/ml =microgram per millilitre
Table 5: Precision for enrofloxacin
Intraday
[n=6] |
Interday
[n=6] |
||||||
Conc. (µg/ml) |
Peak area (1) |
Peak area (2) |
Peak area (3) |
Peak area (D-1) |
Peak area (D-2) |
Peak area (D-3) |
|
0.5 |
435524 |
437134 |
438548 |
436159 |
438546 |
441562 |
|
0.5 |
434258 |
437232 |
437562 |
436298 |
439258 |
439265 |
|
0.5 |
434582 |
437689 |
438556 |
436623 |
439236 |
439874 |
|
0.5 |
435692 |
436313 |
437584 |
436325 |
439658 |
440321 |
|
0.5 |
435268 |
437819 |
437685 |
437819 |
439684 |
440687 |
|
0.5 |
435879 |
437132 |
439665 |
437132 |
438987 |
441698 |
|
Mean |
435200.5 |
437068.3 |
438266.7 |
436726 |
439228.2 |
440567.8 |
|
SD |
642.289 |
702.160 |
826.936 |
638.054 |
428.253 |
950.741 |
|
%RSD |
0.147 |
0.160 |
0.188 |
0.146 |
0.097 |
0.215 |
SD =Standard deviation, RSD= Relative standard deviation,
n=number, µg/ml =microgram per millilitre
Ruggedness
The robustness method was studied and the results are given in
[table 6]. The ruggedness of the methods was conducted by two different analyst
and %RSD was calculated. Variation in percentage content was found to be within
the limit so the method is rugged.
Robustness
The result of the robustness study of the developed assay method
was established in [table 7 and 8]. The result shown that during all variance
conditions, assay value of the test preparation solution was not affected and
it was in accordance with that of actual. Hence the analytical method would be
concluded as robust.
Table 6: Ruggedness of enrofloxacin
S. No. |
Conc.
(µg/ml) (n=6) |
Analyst-1 |
Analyst-2 |
1 |
0.5 |
434524 |
434351 |
2 |
434258 |
433547 |
|
3 |
433582 |
433554 |
|
4 |
434692 |
434968 |
|
5 |
434268 |
434321 |
|
6 |
434879 |
434877 |
|
Mean |
434367.2 |
434269.7 |
|
S. D |
453.943 |
466.473 |
|
%RSD |
0.104 |
0.114 |
SD =Standard deviation, RSD= Relative standard deviation,
n=number, µg/ml =microgram per millilitre
Table 7: Robustness studies of enrofloxacin (Change flow rate)
S. No. |
Flow
rate ml/min (n=3) |
Peak
area |
Statistical
data |
Retention
time |
1 |
1.0 ml/min. |
434524 435598 434587 |
Mean=434903 SD=602.711 %RSD=0.138 |
3.401 3.402 3.402 |
2 |
0.5 ml/min. |
432587 432987 431214 |
Mean=432262.67 SD=929.933 %RSD=0.215 |
3.408 3.408 3.411 |
SD =Standard deviation, RSD= Relative standard deviation,
n=number, µg/ml =microgram per millilitre
Table 8: Robustness of enrofloxacin at change in wavelength
S. No. |
Wavelength
[n=3] |
Peak
area |
Statistical
data |
Retention
time |
1 |
270 nm |
434692 434268 434879 |
Mean=100.266 SD=0.2730 %RSD=0.272 |
3.402 3.402 3.401 |
2 |
269 nm |
528471 528478 527458 |
Mean=100.266 SD=0.2730 %RSD=0.272 |
5.405 5.405 5.408 |
SD =Standard deviation, RSD= Relative standard deviation,
n=number, µg/ml =microgram per millilitre
Estimation
The content of enrofloxacin in the marketed brands was determined.
The percentages of individual drugs found in formulations, amount and relative
standard deviation in formulations were calculated. The result of
analysis [table 9] shows that the amount of drugs present in the formulation
has a very good correlation with the label claim of the formulation. The
chromatogram of enrofloxacin shown in fig. 5.
Table 9: Estimation of enrofloxacin
Tablet
brand name |
Labelled
amount (g) |
Estimated
amount (mean±SD) |
%
Label claim |
%RSD |
Brand-A |
0.05 |
0.0503±0.00011 |
100.6% |
0.198 |
Brand-B |
0.05 |
0.0498±0.00048 |
99.6% |
0.240 |
Amount of Enrofloxacin is expressed as mean±SD (n = 3)
Fig. 5:
Chromatogram for enrofloxacin [Sample]
DISCUSSION
The methods available for Enrofloxacin estimation are thin-layer
chromatography (TLC)-fluorescence, capillary electrophoresis–fluorescence,
Spectrophotometry [7, 8], high-performance liquid chromatography (HPLC)-UV or
fluorescence [9-17], HPLC-Mass spectrometry (MS) or gas chromatography (GC)-MS
[18]. The majority of these methods have been developed to analyze biological
samples such as serum or urine.
We present a simple, new, fast, sensitive, precise and accurate
RP-HPLC analytical method for quantification of Enrofloxacin present in the
marketed formulation using reverse-phase Phenomenex C18 column
(250 mm x 4.6 mm i. d,5 µm) with a mobile phase composed of acetonitrile: water
in the ratio of 80:20 v/v. The method is highly specific as there was no interference
observed between chromatograms of blank, standard and sample. The analysis was
carried out in an isocratic mode at the flow rate of 1.0 ml/min, at 25 °C and
detection was carried out at 270 nm. Good linearity with the coefficient of
correlation 0.9991 indicated that the proposed method was linear within the
range of 0.1-0.6µg/ml compared to prevaling method having linearity with a
coefficient of correlation 0.9961 and flow rate of 3.0 ml/min [12]. Accuracy
results displayed good reproducibility with % RSD below 2%. The recovery of
analyte in the prevailing methods was only 93% when compared with our present
work [8, 9]. The method was found to be accurate as the values observed for
Brand A (100.6%) and Brand B (99.23%), respectively which was well within the
range of 98-102% as shown in table 9. The results for precision were expressed
in % RSD as tabulated in table 5. Whereas, The %RSD for intra and inter-day
precision of enrofloxacin was observed below 2%. The low values of % RSD
indicates that the method is precise and also the LOD and LOQ values showed the
method was sensitive for the quantification of enrofloxacin in pure and
veterinary formulation.
CONCLUSION
The established present method revealed that it is simple,
selective, specific, and can generate accurate and precise results. Moreover,
the shorter duration of analysis time and lesser mobile phase consumption
confirmed that the method is rapid and economical. The result of analysis shows
that the amount of drugs present in the formulation has a very good correlation
with the label claim of the formulation and %RSD will be less than 2 for all
the validation parameters. Recoveries studies revealed that results within the
specified limits. Hence the proposed method was found to be satisfactory and
could be used for the routine analysis of enrofloxacin in their marketed
formulation.
ACKNOWLEDGEMENT
The authors are thankful to Mr. P. P. Prem Krishnan, Managing
Trustee, KTN College of Pharmacy, Chalavara, Ottappalam, Palakkad, Kerala for
providing the necessary infrastructure and facility to carry out the research
work.
FUNDING
Nil
AUTHORS
CONTRIBUTIONS
We declare that it’s an original research work which was carried
out by Mrs. Aswathy SR and Devi Swapna PV, Mrs Anjali Sruthy S and Mr. Gopinath
U were carried out the validation of proposed method. Dr. Muhas C proofread the
manuscript, suggested the necessary corrections, and helped in writing the
manuscript.
CONFLICT
OF INTERESTS
Declared none
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