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Sejal K. Patel, Sumeet I. Chhabra*
Department of Pharmaceutical Analysis,
Centre For Health Science Studies, Ganpat University,
Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India

A simple, sensitive, accurate, precise and rapid reverse phase high performance liquid chromatographic method has been developed and validated for the simultaneous determination of cefixime trihydrate and levofloxacin hemihydrate in tablet formulation. The chromatographic separation was performed on ACE 5 C18 column (150 mm × 4.6 mm i.d, 5 μm particle size). Mobile phase consisted of acetonitrile, water and methanol in the ratio of 65: 15: 25, v/v/v at a flow rate of 1.0 ml/min. The detection wavelength was set at 289 nm. The proposed method was validated for linearity, accuracy, precision, LOD and LOQ. The calibration was linear over the concentration range of 2-24 μg/ml for cefixime trihydrate and 2-30 μg/ml for levofloxacin hemihydrate. The retention times were found to be 1.9 ± 0.26 min for cefixime trihydrate and 3.6 ± 0.32 min for levofloxacin hemihydrate. The mean recoveries were 100.8 ± 0.54 and 100.1 ± 0.76 for cefixime trihydrate and levofloxacin hemihydrate, respectively. The method can be easily adopted for quality control analysis.


Cefixime trihydrate (CEFI)is a well known cephalosporin antibiotic drug1. Chemicallyit is (6R,7R)-7-[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-[(carboxymethoxy)imino]acetamido]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid(Figure 1). It is official in Indian Pharmacopoeia (IP), United States Pharmacopoeia (USP), British Pharmacopoeia (BP), and Japanese Pharmacopoeia (JP). IP describes Liquid Chromatographic method for its estimation.   Literature survey reveals liquid chromatography-voltametric determination for determination of cefixime in human plasma3. Literature survey also reveals High Performance Liquid Chromatography and High Performance Capillary Electrophoresis method for the determination of Cefixime in Human Plasma4.

Levofloxacin hemihydrate(LEVO)1 is chemically (2S)-7-fluoro-2-methyl-6-(4-methylpiperazin-1-yl)-10-oxo-4-oxa-1-azatricyclo[^{5,13}]trideca-5(13),6,8,11-tetraene-11-carboxylic acid(Figure 2). Levofloxacin (LEVO) is official in Indian Pharmacopoeia (IP)5. IP describes Potentiometry detection method for determination of LEVO alone. Literature survey also reveals HPTLC13-19, RP-HPLC20 and HPLC21-26 method for the determination of LEVO with other drugs combination. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of CEFI and LEVO in their tablet formulation.. Literature survey does not reveal any simple chromatographic method for simultaneous estimation of EPE and PCM in synthetic mixture or dosage forms. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective RP-HPLC method for simultaneous estimation of both drugs in tablet formulation.


RP-HPLC instrument (Shimadzu, LC-2010CHT, Japan) equipped with a UV-Visible detector and a photodiode array detector, auto sampler, ACE 5 C18 column (150 x 4.6 mm, 5 µ particle size) was used. Chromatograms were automatically obtained by LC-Solution system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India), Nylon 0.45 µm – 0.47 mm membrane filter. Whatman filter paper no. 41 (Millipore, USA), Digital pH meter (LI 712 pH analyzer, Elico Ltd., Ahemedabad) were used in the study.

Reagent and materials
PCM bulk powder was kindly gifted by Acme Pharmaceuticals Ltd. Mehsana, Gujarat, India. EPE bulk powder was kindly gifted by Sun Pharmaceuticals Ltd. Vadodara, Gujarat, India..HPLC grade methanol (Merck Ltd., Mumbai, India), HPLC grade acetonitrile (Finar Chemicals Ltd.,Mumbai, India), NaH2PO4 and Na2HPO4 (S. D. Fine Chemicals Ltd., Mumbai, India)were used in the study. Water for RP-HPLC was prepared by triple glass distillation and filtered through a nylon 0.45 µm – 47 mm membrane filter.

Preparation of phosphate buffer solution (pH 6.0)
Phosphate buffer (pH 6) was prepared by dissolving 1.56 g sodium dihydrogen ortho phosphate and 0.35 g disodium hydrogen phosphate in 1000 ml HPLC-grade water.

Preparation of standard stock solutions
An accurately weighed standard EPE and PCM powder (10 mg) were weighed and transferred to 100 ml separate volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a solution containing 100 μg/ml of each EPE and PCM.

Preparation of synthetic mixture
Eperisone (20 mg) and Paracetamol (200 mg) were taken and then both the drug were mixed with routinely used excipients like starch, lactose, magnesium stearate, and Talc in suitable proportion. Total 500 mg of mixture was prepared and used as synthetic mixture.

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for EPE and PCM was obtained with a mobile phase consisting of phosphate buffer (pH 6): acetonitrile: methanol (10: 30: 60, v/v/v)at a flow rate 0.7 ml/min to get better reproducibility and repeatability. Quantification was carried out at 255 nm based on peak area. Complete resolution of the peaks with clear baseline was obtained (Figure 3). System suitability test parameters for EPE and PCM for the proposed method are reported in Table 1. Overlain UV spectrum showed that both drugs showed good absorbance at 255 nm, hence the wavelength of 255 nm was selected for quantification of EPE and PCM (Figure 4). 

Method Validation

Calibration Curve (Linearity)
Calibration curves were constructed by plotting peak areas Vs concentrations of EPE and PCM and the regression equations were calculated. The calibration curves were plotted over the concentration range 2-50 µg/ml for EPE and 2- 50µg/ml for PCM. Accurately measured standard working solutions of EPE (0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 ml) and DIC (0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 ml) from100 µg/ml of stock solution were transferred to a series of 10 ml of volumetric flasks and diluted to the mark with methanol.  20 µl of each solution were injected under the operating chromatographic conditions described above.

Accuracy (% Recovery)
The accuracy of the method was determined by calculating recovery of EPE and PCM by the standard addition method. Known amounts of standard solutions of EPE and PCM (50, 100, and 150 %) were added to pre quantified sample solutions of EPE and PCM. The amounts of EPE and PCM were estimated by applying obtained values to the regression equation of the calibration curve.

Method Precision (% Repeatability)
The precision of the Method was checked by repeatedly injecting six sample solutions of EPE (10 μg/ml) and PCM (10 μg/ml) under the same chromatographic condition and measurements of peak area, retention time and tailing factor. Percentage relative standard deviation (RSD) or % coefficient of variation (CV) should not be more than 2.

Intermediate Precision (Reproducibility)
The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of sample solutions of EPE (10, 20, 30 μg /ml) and PCM (10, 20, 30 μg /ml). The results were reported in terms of relative standard deviation (RSD).

Limit of Detection and Limit of Quantification
LOD and the LOQ of the drug were calculated using the following equations as per International Conference on Harmonization (ICH) guidelines27.

LOD = 3.3 × σ/S
LOQ = 10 × σ/s

Where   σ?= the standard deviation of the response

S = Slope of calibration curve.

Analysis of TOL and DIC in synthetic mixture
Eperisone hydrochloride (20 mg) and Paracetamol (200 mg) standard drug powder were accurately weighed and then mixed with commonly used formulation excipients like starch, lactose, magnesium stearate and talc. The mixture was then transferred to 100 ml volumetric flask containing 50 ml methanol and sonicated for 20 min.The solution was filtered through Whatman filter paper No. 41 and the volume was adjusted up to the mark with methanol.This solution (0.1 ml) was taken in to a 10 ml volumetric flask and the volume was adjusted up to mark with methanol to get a final concentration of EPE(2 µg/ml) and PCM (20 µg/ml) . Aliquot (20 µl) of sample solution was injected under the operating chromatographic condition as described above and peak area was determined for both drugs. From these area values, the concentrations of EPE and PCM were determined using respective calibration graph. The analysis procedure was repeated six times with synthetic mixture.

A RP-HPLC method was developed and validated for the determination of EPE and PCM in synthetic mixture on ACE 5 C18 column (150 mm x 4.6 mm i.d., 5 µm particle size) with variable wavelength detection at 255 nm. The retention time of PCM and EPE was 2.2 ± 0.14 min and 3.9 ± 0.13 min,respectively. System suitability test parameters for method are summarized in Table 1. Linear correlation was obtained between area and concentration of EPE and PCM in the concentration range of 2–50 µg/ml for both drugs (Figure 5 & 6). The low RSD value of interday (0.40-1.25 % for EPE and 0.32-0.93 % for PCM) and intraday (0.41-0.80 % for EPE and 0.35-0.64 % for PCM) at 255 nm, reveal that proposed method is precise. The limit of detection (LOD) and limit of quantification (LOQ) for EPE and PCM were found to be 0.4962 and 1.5036 µg/ml and 0.2239 and 0.6784µg/ml, respectively. These data show that method is sensitive for the determination of EPE and PCM. The recovery experiment was performed by the standard addition method. The mean recoveries were 99.2 ± 0.59 and 98.5 ± 0.43 for EPE and PCM, respectively (Table 2). The results of recovery studies indicate that the proposed method is highly accurate. The proposed validated method was successfully applied to determine EPE and PCM in synthetic mixture. No interference of the excipients with the retention time of drugs appeared (Figure 7); hence the proposed method is applicable for the routine simultaneous estimation of EPE and PCM (Table 3). The regression analysis data and summary of validation parameters is summarized in Table 4.

In this proposed RP-HPLC method, the linearity is observed in the concentration range of 2-50 µg/ml for both drugs with co-efficient of correlation, (r2) = 0.999 and (r2) = 0.999 for EPE and PCM, respectively at 255 nm. The results of the analysis of synthetic mixture by the proposed method are highly reproducible and reliable. The method can be used for the routine analysis of the EPE and PCM in mixture without any interference of excipients.



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