Simultaneous Determination of Ofloxacin and Ornidazole in Solid Dosage Form by RP-HPLC and HPTLC Techniques

1. Jain SK, De Fillips RA. Medicinal Plants of India. Michigan, USA: Reference Publication Inc; 1991. p. 227-30. 2. Bhakuni DS, Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN. Screening of Indian plants for biological activity: Part II. Indian J Exp Biol 1969;7:250-62. 3. Prajapati ND, Kumar U. Agro’s Dictionary of Medicinal Plants. Jodhpur, India: Agrobios; 2003. p. 264. 4. Sandhya B, Thomas S, Isabel W, Shenbarathai R. Ethnomedicinal plants used by the Valaiyan community of Piranmalai hills (reserved forest) Tamilnadu, India: A pilot study. Afr J Trad CAM 2006;3:101-14. 5. Ghazanfar SA. Handbook of Arabian Medicinal Plants. Boca Raton FL: CRC Press; 1994. p. 342. 6. Ayatollahi SA, Ahamad Z, Malik A. Fernane-type triterpene from Sericostoma paucifl orum. J Nat Prod 1991;54:570-2. 7. Ayatollahi SA, Ahamad Z, Afza N, Malik A. A triterpene from Sericostoma paucifl orum. Phytochem 1992;31:2899-901. 8. Ayatollahi SA, Ahamad Z, Malik A, Afza N, Bader A. Hopane type triterpenoid from Sericostoma paucifl orum. Fitoterapia 1992;63:304-7. 9. Boyanava L, Gergova G, Nikolov R, Derejian S, Lazarova E, Katsarov N, et al. Activity of Bulgarian propalis against 94 Helicobacter pylori strains in vitro by agar well diffusion, agar dilution and disc diffusion methods. J Med Microbiol 2005;54:481-3.

Literature survey revealed that OFL is official in USP [3] .Several methods are reported for the determination of OFL by spectrofluorimetry [4] and HPLC by UV detection [5] and by fluorimetric detection [6] .ORN is not an official drug.Several methods are reported for determination of ORN by spectrophotometry [7] , calorimetry [8] , GC [9] in solid dosage form and in biological fluids.Few methods are also reported for simultaneous estimation of OFL and ORN by HPLC [10] and HPTLC [11] .
In present paper, we report the HPLC and HPTLC methods for simultaneous determination of OFL and ORN in pure laboratory mixture and in tablet dosage form and their comparison.Combinations of ORN and OFL in solid dosage form are marketed in India as antiprotozoal and antibacterial.The marketed formulation selected for present study was Ofl ox OZ of Cipla Ltd., Jaipur, India.
ORN and OFL standards were supplied by Jenburkt Pharmaceutical Ltd., Sihor, Gujarat, India.HPLC grade water, methanol and acetonitrile were purchased from Loba Chemicals, Mumbai, India.Dichloromethane, ammonia and potassium dihydrogen phosphate used were of analytical grade.
The mobile phase was filtered through Whatman fi lter paper No. 41 and degassed prior to use.The elution was monitored at 318 nm.The injection volume was 20 μl.
Solutions of the test substance were applied to silica gel 60 F 254 TLC plates.The plate was placed in a chromatographic tank previously saturated for 10 min with developing mobile phase; dichloromethane:methanol:25% ammonia solution (9.5:1:3 drops v/v).The plate was developed by normal vertical developing tank at ambient temperature for a distance of 90 mm. the spots were detected under a UV lamp and scanned densitometrically at 318 nm.
Accurately weighed amount of standards of OFL (100 mg) and ORN (250 mg) were transferred to 100 ml volumetric flask, separately and dissolved into and diluted up to the mark with methanol.The resulting solutions were sonicated for 15 min and filtered through Whatman fi lter paper No. 41.All solutions were prepared freshly.
An aliquot portion of the standard stock solution of OFL and ORN were further diluted with mobile phase to get the series of concentration of 2-40 μg/ml for OFL and 5-100 μg/ml for ORN.20 μl of each solution was injected under operating chromatographic conditions described above.Calibration curve was constructed by plotting peak areas versus concentration and the regression equation was calculated.
The limit of detection was calculated and it was found to be 20 μg/ml and 50 μg/ml for OFL and ORN, respectively.Limit of quantifi cation for OFL and ORN was found to be 60 μg/ml and 150 μg/ml, respectively.
An aliquot portion of standard stock solution of OFL and ORN were further diluted to get 0.01 μg/ml for OFL and 0.025 μg/ml for ORN.Each solution was applied as band ranging from 2-20 μl on TLC plate with Linomat V.The plate was developed in twin trough glass chamber under operating conditions described above.After development the plate was air dried and evaluated densitometrically at wavelength of 318 nm.Calibration curve was constructed by plotting peak areas versus concentration and the regression equation was calculated.
The laboratory mixture was prepared in the ratio of 1:2.5 w/w for OFL and ORN, respectively.From the stock solution aliquot portion was further diluted with mobile phase to get concentration 10 μg/ml for OFL and 25 μg/ml for ORN, for RP-HPLC method and 0.050 μg/ml for OFL and 0.125 μg/ml for ORN, for HPTLC method.
Twenty tablets were accurately weighed and finely powdered.An accurately weighed amount equivalent to 100 mg of OFL (equivalent to 250 mg of ORN) was transferred into 100 ml volumetric fl ask, along with 50 ml of methanol.The fl ask was mechanically shaken for 10 min; fi nally volume was made to mark with methanol, and fi ltered.
The stock solution was diluted with mobile phase to get concentration of 10 μg/ml for OFL (25 μg/ ml for ORN) for RP-HPLC method.Further, the stock solution was diluted with methanol to get concentration of 50 μg/ml for OFL (125 μg/ml for ORN).Two microlitres of this solution was applied on TLC plate with Linomat V.The plate was developed in twin trough glass chamber under operating conditions described above and calculations were performed using peak area.
A solvent system that would give dense and compact spots with appropriate and significantly different Rf values was desired for quantifi cation of combination by HPTLC.The mobile phase consisting of dichloromethane:methanol:25% ammonia solution (9.5:1:3drops v/v) gave the mean Rf value of 0.16 and 0.56, 0.78 (isomers), respectively for OFL and ORN.In HPLC conditions were optimized to obtain an adequate separation of eluted compounds.Initially, various mobile phase compositions were tried, but only acetonitrile:methanol:0.025 M phosphate buffer, pH 3.0 (30:10:60 % v/v/v) as the mobile phase at a flow rate of 1 ml/min at ambient temperature was found to give desired separation between OFL and ORN.The average retention times for OFL and ORN was found to be 4.04 min and 5.83 min, 6.77 min (isomers), respectively.Quantifi cation was achieved in HPTLC as well as in HPLC with UV detection at 318 nm.Typical chromatograms for OFL and ORN for marketed formulations are shown in Figs. 1 and 2.
The developed method was validated in terms of linearity and range, limit of detection, limit of quantifi cation, recovery study, and inter day study, intra day study and study by different analysts.The linear regression data in HPTLC showed a good linear relationship over a concentration range 50-250 ng/spot for OFL and 20-100 ng/spot for ORN.The correlation coefficients obtained were 0.9994 and 0.9989 for OFL and ORN, respectively.Repeatability of method was determined by six times spotting 10 μl of standard drug solution on TLC plate, after development the separated spots were scanned six times without changing position, measurement of peak areas was performed and from the peak areas the % RSD was determined.For OFL and ORN % RSD was found to be 0.0086 and 0.0063, respectively System suitability tests are used to verify the reproducibility of the chromatographic system.To ascertain effectiveness of HPLC method, system suitability tests were carried out on freshly prepared standard stock solutions.The parameters obtained are shown in Table 1.The calibration curve was linear in  concentration range of 2-40 μg/ml and 5-100 μg/ml, with regression 0.9994 and 0.9913, slope 0.6165 and 0.4314 for OFL and ORN, respectively.
Recovery studies were carried out to study accuracy and precision of the method.In HPTLC, these studies were carried out on plate at three levels i.e. multiple level recovery studies.Two microlitres of pre-analyzed sample preparation having concentration of 0.050 μg/μl for OFL and 0.125 μg/μl of ORN was applied three times on TLC plate as a band of 6 mm.These bands were spiked on plate with OFL and ORN standard stock solution and analyzed by the method.In HPLC, recovery study was carried out at 80%, 100% and 120% level.To the powder formulations the pure standard drugs were added and dilutions were made and analyzed by the method.The % recovery was calculated by using formula, % recovery = (T-A)/S×100 where, T is total amount of the drug estimated, A is the amount of drug contributed by tablet powder and S is the amount of pure drug added.The result of recovery studies for both OFL and ORN by both HPLC and HPTLC was found to be around 99-100%, indicating that the methods are free from interference from excipients (Table 2).
Sample to sample precision and accuracy by both the techniques were evaluated using, three samples of three different concentrations, which were prepared and analyzed on same day.Day to day variability was assessed using three samples of three different concentrations analyzed on three different days, over a period of one week.The results show the accuracy and reproducibility of the assay.Thus, it was concluded that there was no significant difference on the assay, which was tested on an intra-day and inter-day basis.The % RSD values shows that proposed method provides acceptable intra-day and inter-day variation for OFL and ORN (Table 3).
From the above results it can be concluded that the HPTLC method is simple, accurate, most economic and less time consuming technique while RP-HPLC method is most accurate, precise, specifi c and very sensitive and can be used for routine analysis of OFL and ORN in their combined dosage form.Pregabalin, an antiepileptic drug similar to gabapentin produces its actions by binding to the alpha2-delta (α2δ) subunit of the voltage-gated calcium channels [1] .The infl uence of fl uctuating temperature and humidity conditions that might occur during transportation of drug products can be estimated using stability analysis of a drug [2] .Few reports on determining pregabalin content in pharmaceuticals have been published, involving spectrophotometric, spectrofluorimetric methods [3] and precolumn derivatization method using internal standard [4,5] .Synthesis and characterization of pregabalin lactose degradation product was reported [6] .Determination of pregabalin without pre-column derivatisation using RP-HPLC has not been reported thus far.Therefore, in the present investigation an attempt has been made to determine pregabalin in solid dosages form using RP-HPLC without precolumn derivatization of analyte and without internal standard.The assay is calibrated over the range of 500 μg/ml to 1500 μg/ml and without derivatization of analyte also the proposed method can quantify (LOQ) at least 0.61 μg/ml and can detect (LOD) at least 0.23 μg/ml.The developed method has been validated showing the method accuracy, linearity and reproducibility.Validation procedure was mainly based on the ICH guideline [7] .

Fig. 1 :
Fig. 1: HPLC pattern of OFL and ORN Typical chromatogram of sample obtained from tablet containing OFL and ORN.HPLC retention times of OFL was found to be 4.04 min (1), for ORN I 5.82 min (2) and for ORN II 6.77 min (3).

Fig. 2 :
Fig. 2: HPTLC pattern of OFL and ORN Typical chromatogram of sample obtained from tablet containing OFL and ORN.HPTLC R f of OFL was found to be 0.16 (a), for ORN I 0.56 (b) and for ORN II 0.78 (c).

TABLE 2 : ANALYSIS DATA AND RECOVERY STUDIES
*Mean of six determinations; SD is standard deviation and RSD is relative standard deviation.OFL, ofl oxacin; ORN, isomer of ornidazole.

TABLE 1 : SYSTEM SUITABILITY PARAMETERS FOR RP- HPLC
Mean of fi ve determinations.OFL for ofl oxacin; ORN I and II for isomer of ornidazole *