HPLC
HPLC
HPLC
1
Department of Pharmaceutical Analysis, A.M. Reddy Memorial College of Pharmacy, Petlurivaripalem,
Narasaraopet (Mdl), Guntur District – 522601. Andhrapradesh, India.
2
Patlolla Ramakrishna Reddy College of Pharmacy, Nandigama village, Patancheru, Medak District, Hyderabad -
502 319. Andhrapradesh, India.
3
Department of Pharmaceutical Analysis, Donbosco College of pharmacy, Pulladi gunta, Etukuru,Guntur, Andhra
Pradesh.
4
K.M.C.H. College of Pharmacy, Kovai Estate, Kalappatti Road, Coimbatore – 641035, Tamilnadu
Abstract
A simple reverse phase liquid chromatographic method has been developed and subsequently
validated for simultaneous determination of Ofloxacin and Ornidazole in combination. The separation was carried
out using a mobile phase consisting of 2mM phosphate buffer and Acetonitrile with pH 3.5 adjusted with ortho
phosphoric acid in the ratio of 70: 30%v/v. The column used was Phenomenex C 18, (250 mm x 4.6 mm i.d, 5m)
with flow rate of 1 ml / min using PDA detection at 293 nm. The described method was linear over a concentration
range of 5-50 g/ml and 12.5-125 g/ml for the assay of Ofloxacin and Ornidazole respectively. Gatifloxacin (50
g/ml) was used as internal standard. The retention times of Ofloxacin, Ornidazole and Gatifloxacin were found to
be 2.1, 2.5 and 5.5min respectively. Results of analysis were validated statistically and by recovery studies. The
limit of detection (LOD) and the limit of quantification (LOQ) for Ofloxacin and Ornidazole were found to be 5
and10 µg/ml 10 and 25 µg/ml respectively. The results of the study showed that the proposed RP-HPLC method
is simple, rapid, precise and accurate, which is useful for the routine determination of Ofloxacin and Ornidazole
bulk drug and in its pharmaceutical dosage form.
Keywords: Ofloxacin, Ornidazole and Gatifloxacin.
1. Introduction
Ofloxacin (OFL) is a synthetic broad spectrum antibacterial agent. Chemically ofloxacin 1 a fluorinated
carboxy quinolone, 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 non aqueous titration for estimation of ofloxacin. Literature surveys reveal
Spectrophotometric method atomic absorption spectrometry, spectroflurometry5-6, HPLC7 and microbiological
method8 for its determination. Ornidazole (ORN)1 is a 5-nitroimidazole derivative used as antiinfective agent. It is
not official in any Pharmacopoeia. Literature survey reveals that ornidazole is estimated by voltametry9 and HPLC10
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B.Dhandapani et. al. / International Journal of Pharma Sciences and Research (IJPSR)
Vol.1(1), 2010, 78-83
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 diarhoea, bacterial and protozoal
infections. This paper presents two simple, accurate and reproducible spectrophotometric methods for simultaneous
determination of ofloxacin and ornidazole in tablet dosage form. So far, no method has been reported for estimation
of OFL and ORN in combined dosage form by HPLC, hence we attempted to develop a simple, accurate, and
economical analytical method. This paper describes validated RP-HPLC for simultaneous estimation of OFL and
ORN in combination, using 2mM phosphate buffer and Acetonitrile with pH 3.5 adjusted with ortho phosphoric acid
in the ratio of 70: 30%v/v. The column used was Phenomenex C18, (250 mm x 4.6 mm i.d, 5m) with flow rate of 1
ml / min using PDA detection at 293 nm.
2. Experimental
2.1. Chemicals, reagents and Instrumental Conditions
Standard bulk drug sample Ofloxacin and Ornidazole and Gatifloxacin were provided by Micro
Laboratories Ltd., Bangalore. Tablets of combined dosage form were procured from the local market. All other
reagents used were of HPLC grade. Chromatographic separation was performed on a Shimadzu LC-20 AT HPLC
(Double pump) with Rheodyne 7725i type injector with 20µl loop capacity and SPD M20A, Prominence Diode
Array Detector. The wavelength of detection chosen was 293 nm. A reverse phase Phenomenex C18 column (250
mm × 4.6 mm, 5 μm) was used for the analysis. The mobile phase comprising of a mixture of 2mM phosphate
buffer and Acetonitrile with pH 3.5 adjusted with phosphoric acid in the ratio of 70: 30%v/v with a flow rate of
1ml/min. The injection volume was 20 μL.
2.1. Productos químicos, reactivos y condiciones instrumentales
Micro Laboratories Ltd., Bangalore proporcionó muestras de fármaco a granel estándar de ofloxacina y ornidazol y
gatifloxacina. Las tabletas de forma de dosificación combinada se adquirieron en el mercado local. Todos los demás
reactivos utilizados fueron de grado HPLC. La separación cromatográfica se realizó en un Shimadzu LC-20 AT
HPLC (doble bomba) con inyector tipo Rheodyne 7725i con capacidad de bucle de 20 µl y detector de matriz de
diodos de prominencia SPD M20A. La longitud de onda de detección elegida fue de 293 nm. Para el análisis se utilizó
una columna Phenomenex C18 de fase inversa (250 mm × 4,6 mm, 5 μm). La fase móvil compuesta por una mezcla
de tampón fosfato 2mM y Acetonitrilo con pH 3,5 ajustado con ácido fosfórico en la relación 70:30%v/v con un
caudal de 1ml/min. El volumen de inyección fue de 20 μL.
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Vol.1(1), 2010, 78-83
3.2.4 Specificity
The specificity of the method was checked for the interference of impurities in the analysis of a blank
solution (without any sample) and then a drug solution of 20 μg/mL was injected into the column, under optimized
chromatographic conditions, to demonstrate the separation of both OFL and ORN from any of the impurities, if
present. As there was no interference of impurities and also no change in the retention time, the method was found
to be specific and also confirmed with the results of analysis of formulation.
3.2.5 Robustness
To determine the robustness of the method, experimental conditions such as the composition of the mobile
phase, pH of the mobile phase, and flow rate of the mobile phase were altered and the chromatographic
characteristics were evaluated. No significant change was observed.
3.2.6 LOD and LOQ
Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 ∂ /S and 10 ∂/S,
respectively as per ICH guidelines, where ∂ is the standard deviation of the response ( y-intercept) and S is the slope
of the calibration plot.
The results of validation parameters and System suitability parameters were shown in Table 2.
Table 2:
Validation Parameters OFL ORN
Linearity range ( µg / ml) 5-50 12.5-125
r 0.9998 0.9991
LOD (ng /ml) 5 10
LOQ (ng /ml) 10 25
Intra day (% RSD)* 0.879 0.945
Inter day (% RSD)* 0.254 0.364
Repeatability (% RSD)* 0.3251 0.4250
Accuracy 99.50 – 102.17 99.38-100.53
Peak purity index 1.0000 1.0000
Resolution factor ( Rs) - 19.618
Asymmetry factor (As) 0.95
No. of theoretical plates (N) 3059 12500
Capacity factor (K’) - 0.330
High equivalent to theoretical 49.029 11.999
plates (HETP)
Tailing factor 1.149 1.140
Separation factor 7.902
Each value is a mean of six observations.
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Vol.1(1), 2010, 78-83
ornidzol/5.532
300
250
ofloxcin/2.161
200
150
gatifloxin/2.588
100
50
As there was no interference of impurities and also no change in the retention time, the method was found to be
specific and the respective peak purity curve and overlay UV Spectrum were shown in Fig 2, 3, 4 & 5.
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Vol.1(1), 2010, 78-83
mAU
0.8 Purity Curv e Zero Line Peak
350
0.7
300
0.6
0.5 250
0.4 200
0.3 150
0.2
100
0.1
50
0.0
0
2.1 2.2 2.3 2.4 min
Fig-2 Peak Purity Curve of Ofloxacin
175.0 175.0
0.4 0.4
150.0 150.0
100.0 100.0
0.2 0.2
75.0 75.0
0.1 50.0 0.1 50.0
25.0 25.0
0.0 0.0
0.0 0.0
2.55 2.60 2.65 2.70 min 2.55 2.60 2.65 2.70 min
Fig-3 Peak Purity Curve of Ornidazole Fig-4 Peak Purity Curve of Gatifloxacin
mAU
800
295
700
600
500
227
400
327
300
319
293
257
200
232
217
326
100
1
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Vol.1(1), 2010, 78-83
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