RP-HPLC Method for Simultaneous Determination of Ofloxacin and Satranidazole in Tablet Dosage Form

 

T Venkatachalam*1, S Narendiran1, P Kalai Selvi1 and  P Dheen Kumar2

1Dept. of Pharmaceutical Chemistry, JKKMMRF College of Pharmacy, Komara Palayam -638183.Tamil Nadu, India.

2 Dept. of Pharmaceutical Chemistry,   Maharaji College of Pharmacy, Chennai 600090.Tamil Nadu, India.

*Corresponding Author E-mail: venkatachalammpharm@yahoo.co.in

 

ABSTRACT

A simple, precise, accurate and rapid HPLC method has been developed, and validated for the determination of ofloxacin and satranidazole simultaneously ,in combined  dosage form. Acetonitrile and ammonium di-hydrogen orthophosphate buffer with ph7.0 (50%:50%)is used as the mobile phase ,300 is the detection wavelength for this study. The applicability of the method for simultaneous determinations of oflaxacin and satranidazole  was verified by the determination of these compounds in marketed tablets .Results of the analysis were validated statistically, and by recovery studies (99.43-101.28 %). The recovery and RSD values with in limits given in ICH guidelines method developments indicates that the suitability  of proposed for the routine  determination of the these compounds in tablets .the validation parameters; linearity (r=9999) sensitivity     (LOD (1.0 -1.5x10-4), LOQ(3.0-4.5×10-4) accuracy(99.43 -101.28  %) and reproducibility were found to satisfactory. The proposed method can be successfully used to determine the drug contents of marketed formulation.

 

KEYWORDS:  RP-HPLC, Oflaxacin, Satranidazole, Validation

 

 

INTRODUCTION:

Ofloxacin, chemically (+/-) -9- Fluoro-2,3 Dihydro-3-Methyl -10- (4-Methyl-1-Piperazinyl) -7- Oxo-7H-Pyrrido[1,2,3-de]-1,4-Benzoxazine-6-Carboxylic acid, it is used as an Antibiotic1,3,4,5. Satranidazole chemically, 1-Methyl Sulfonyl -3-(1-methyl -5- Nitroimidazole -2-yl) -2-Imidazolidinone. It is used as an anti Protozoa2.

 

Literature survey6,7,8 revealed that there is no analytical method reported for estimation of oflaxacin and satranidazole in combination reported methods were discussing  the analysis  either  individually or in combination  with  other  by spectrophotometrically and HPLC in biological fluids and pharmaceutical formulations. The combination of oflaxacin and satranidazole as recently has been introduced in to the market. Our study made an attempt to develop, simple, accurate and reproducible RP-HPLC method for the routine analysis of these drug simultaneously in tablet dosage form.

 

EXPERIMENTAL:

Chemicals:

All the chemicals and solvents were of analytical reagent grade. Acetonitrile and H3PO3 were obtained from MERK (Germany), HPLC water (Quligens–India) were used. Standards for oflaxacin and satranidazole were obtained from Moral labs, Hosur, India. Sample Capsules were obtained from Moral Labs, Hosur, India. Ammonium dihydrogen orthophosphate, Triethylamine, ortho-phosphoric acid were got from Sd fine chemicals from Mumbai.

 

Chromatographic equipments and conditions:

RP-HPLC analysis was performed on a waters alliance 2695 separations module and PDA detection, equipped with Empower chromatography software. Detection was carried out at 300nm using C18 column, 4.6 X 250mm, 5µl.  was used for the separation of these compounds.

 

Mobile Phase:

Buffer: 11.5gm of Ammonium di-hydrogen orthophosphate and 1ml of triethylamine in 1000ml water and pH adjusted to 7.0 with ortho-phosphoric acid Mobile phase - Buffer: Acetonitrile (50:50%v/v).

 

Standard Preparations:

Accurately, about 100mg of Ofloxacin and 150mg of Satranidazole was accurately weighed and transferred separately volumetrically into 100ml volumetric flask, dissolved in mobile phase and diluted to volume with same solvent to get the stock solution. From this suitable dilutions are made to obtain a final concentration of 100μg/ml of Ofloxacin and 150μg/ml of Satranidazole. The stock solution was filtered through a 0.2µ membrane filter paper.

 

Table No 1: Analytical Parameters

Parameters

Ofloxacin

Satranidazole

Linear dynamic range

80-120(µg/ml)

120-180(µg/ml)

Retention time

3.1

4.3

Resolution

6.404

Theoretical plates

4280

8287

Tailing Factor

1.783

1.414

R value

0.9999

0.9999

LOD (µg/mL)

1

1.5

LOQ (µg/mL)

3

4.5

 

Sample Solution Preparations:

The content of twenty tablets were accurately weighed and powdered. The powder equivalent to 100mg of Ofloxacin was accurately weighed and transferred to a 100ml volumetric flask.  Dissolved with mobile phase and made the volume with same solvent. The resulting solution was filtered through a nylon membrane filter (0.45μ) and first few ml of the filtrate was rejected. It is then suitably diluted to yield 100μg/ml of Ofloxacin and 150μg/ml of Satranidazole which are used for the estimation.

 

Optimization of Chromatographic conditions:

The optimum composition of the mobile phase containing Acetonitrile : Buffer (50:50) was selected because it was found to give a peak for both Ofloxacin and Satranidazole with minimal tailing.  The flow rate was set to 2.0 ml/min and UV detection was carried out at 300nm.  All determinations were performed at ambient column temperature.  The separation was carried out on a C18 column (4.6 x 250 mm, 5 µm).

 

Linearity:

From stock solution diluted to obtain the concentrations ranging from 80-120μg/ml for Ofloxacin and 120-180μg/ml for Satranidazole (80 to 120% of target level). 20µl of each of standard solutions were injected and peak areas were measured. The result of the linearity studies are given in Table1.

 

RESULTS AND DISCUSSION:

The procedure for the simultaneous analysis of oflaxacin and satranidazole using isocratic RP-HPLC has been reported. This work was focused on  optimization of the conditions for simple and rapid as well as low cost effective analysis including a selection of the proper column or mobile phase to obtain satisfactory results. To obtain satisfactory resolution and to avoid peak tailing of compounds an optimization of the proposed method was carried using the different mobile phases. Mobile phases of various compositions of Acetonitrile - water were also tested. The best results were obtained using the mobile phase Acetonitrile – ammonium di-hydrogen orthophosphate buffer (50:50) adjusted to pH 7.0 with ortho-phosphoric acid.

 

Spectral Data:

 

Fig - 1: Figure 1.Chromatogram of combined standard - Ofloxacin and Satranidazol (100 mcg/ml and 150 mcg/ml).

 

Fig - 2: Calibration curve for Ofloxacin.

 

The most reproductive results were obtained with 300nm using C18 column, 4.6 x 250mm, 5µl.  column was used for the separation of these compounds. Detection was performed at 300nm in the sensitivity range of 0.01 AUFS using acetonitrile –ammonium di hydrogen ortho phosphate buffer (50:50 v/v) as a mobile phase at  a flow rate of 2.0ml min-1. System suitability tests were performed and chromatographic parameters such as asymmetry factor, tailing factor, resolution, no. of theoretical plates,   were calculated from experimental data were given in   table-1.

 

The validity of the liquid chromatographic assay was established through a study of linearity, sensitivity, system precision, accuracy, robustness and ruggedness. Fig1-2 and 3.

 

The linearity was established with a series of working solution prepared by diluting the stock solution with dilution to the final concentration. Each concentration was injected in triplicate and the mean value of peak area was taken for the calibration curve.

 

 

 

Table No 2: Accuracy (Recovery Studies)

% Target

Ofloxacin (% Recovery)

Mean

% RSD

Satranidazole  (% Recovery)

Mean

% RSD

80%

101.29

101.28

0.128

99.74

99.63

0.638

101.41

100.65

101.15

99.42

100%

99.94

99.93

0.13

99.63

99.8

0.503

99.8

100.37

100.06

99.41

120%

99.57

99.48

0.334

100.57

100.5

0.431

99.77

100.04

99.12

100.9

 

 

 

 

 

 

 

 

 

 

 

Table No 3: The Determination of Ofloxacin and Satranidazole in tablet dosage form

Components

Label Claim (mg/tablet)

N

Amount Present

Percentage Label Claim (%w/v)

Ofloxacin

100

6

99.64

100.23

Satranidazole

150

6

148.91

99.97

 

 

 

 

 

A linear response in peak area ratios was observed over the concentration range 80-120µg/ml for oflaxacin and 120 –180 µg/ml for satranidazole. The linear regression equations are Y= 45.59284x + 1.0105 (r = 0.9999) for oflaxacin, Y= 15.57286x – 4.42909 (r = 0.9999) for Satranidazole. The limits of detection were found to be 1µg/ml, 1.5µg/ml for oflaxacin and satranidazole respectively.

 

The limits of quantification were found to be 3µg/ml for Ofloxacin, 4.5 µg/ml for satranidazole. The result system precision and method precision were determined by 6 replicate injection of mixed standard solution of various concentrations was studied and the results were given in  table -3.

 

The accuracy of HPLC methods was confirmed by recovery by spiking 80%, 100% and 120% of pure drugs to the pre analyzed samples the recovery values for oflaxacin 99.48%w/v -  101.28w/v, satranidazole 99.63%w/v - 100.50%w/v respectively as given in table -2.

 

The robustness was performed by changing the detection wavelength ±5nm, flow rate ±10% and the results were interpreted by statistical analysis by calculating RSD values. All the results were with in the acceptance criteria of not more than 2%.

 

The ruggedness of the method was determined by the same assay by different instrument, different analyst and on different days and were in the acceptance criteria of not more than 2%.

 

The result is shown that the proposed RP-HPLC method provides better sensitivity in the assay, as well as significantly shorter analysis time.

 

CONCLUSION:

RP-HPLC method id simple, rapid and sensitive, therefore suitable for the routine analysis of Oflaxacin and Satranidazole in its oral dosage form.

 

Fig - 3:  Calibration curve for Satranidazole

 

REFERENCES:

1.        Indian pharmacopoeia, 4th ed., VOL. 1, GVOT of India, the controller of  publications, New Delhi,1996.

2.        Gowrishankar.R Phodke RP, Oza SD Talwalker S, Satranidazole: experimental evaluation of activity against anaerobic bacteria in vitro and in animal models of anaerobic infection J. Antimicrob Chemother.1985 Vol.15(4). pp 463-70.

3.        Gandhimathi M Ravi TK, Shukla Nilima, Validated HPTLC method for simultaneous estimation of ofloxacin and ornidazole in tablet dosage form. Indian Journal of Pharmaceutical Sciences 2006 Vol.68,pp838-840.

4.        Breg Garcia M.A.,Solans  C, Calvo  a, Royo M, Hernandez E,Rey  R,and Bregante M.A.,the simultaneous and determination of five quinolone antibiotics using reversed-phase HPLC application to stability studies on ofloxacin tablet formulations, J.Pharm. Biomedical. Anal., 2005,39,769-775.

5.        Jing Yu Shen, Mi Ra kim, Chang Joo Lee,In Seon Kim, Kang Bong Lee and Jae Han Shim, Determination of ofloxacin in biological fluids using HPLC with fluorimetric detection .Fresenius J.Anal.Chem.,1986,324,354.

6.        ICH.Q2A Text on validation of Analytical procedures, International Conference of Harmonization, Oct 1994.

7.        ICH, Q3B, Validation of Analytical procedures Methodology, International Conference on Harmonization. Nov 1996.

8.        Mruthyunayaswamy B.H..,Potil S M.M.,Raju S.A,Spectrophotometric methods for the estimation of satranidazole in pharmaceuticals formulations,Indian.J .pharmaceutical science ,2001,63(s),433-436.            

 

 

 

 

Received on  20.07.2009        Modified on 11.09.2009

Accepted on 08.10.2009        © AJRC All right reserved

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 469-471