INTRODUCTION:^1-3,7-30

A combined fixed dose formulation containing Moxifloxacin hydrochloride SR (MOX) and Cefixime trihydrate SR (CEF) (400mg+400mg) was approved in August-2011 by FDA for the treatment of lower respiratory tract infections. MOX is chemically known as 1-Cyclopropyl-6-fluoro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-1,4-dihydroquinoline- 3-carboxylic acid hydrochloride used as antibiotic or anti-infective agent which inhibits the enzymes topoisomerase II (DNA gyrase) and topoisomerase IV. Reversed Phase High Performance liquid chromatographic (RP HPLC) method for the determination of MOX has been reported in USP-35 NF-30 and BP-2009.

Cefixime trihydrate (CEF) chemically is a (6R,7R)-7-[[(Z)-2-(2-Aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl] amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene 2- carboxylic acid trihydrate used as an antibiotic or antibacterial agent which inhibits mucopeptide synthesis in the bacterial cell wall. Reversed Phase High Performance liquid chromatographic (RP HPLC) method for the determination of CEF has been reported in USP-35 NF-30, BP-2009,EP-2005and IP-2010. The thorough literature survey revealed that various analytical methods have been reported for estimation of Cefixime trihydrate and Moxifloxacin hydrochloride like spectrophotometric, HPTLC and HPLC individually or in combination with other drugs either as API or in plasma, serum, urine and pharmaceutical dosage forms.

Figure 1. Cefixime trihydrate

Figure 2. Moxifloxacin hydrochloride

EXPERIMENTAL:^1-30

Reagents and Chemicals

Acetonitrile HPLC grade, Moxifloxacin hydrochloride, Cefixime trihydrate, tetrabutyl ammonium hydrogen sulphate, tetrabutyl ammonium bromide, orthophosphoric acid, etc. were provided for the research work at and by Bharat Parenterals1 Ltd., Savli, Vadodara, Gujarat.

Apparatus and Chromatographic Conditions:

An electronic analytical balance (Shimadzu) was used for weighing the materials. Glassware used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven. A High Performance Liquid Chromatographic system with Rheodyne injection syringe with 20 μl loop volume and LC solutions data handling system (Shimadzu-LC 20) was used for the analysis equipped with Thermoscientific BDS HYPERSIL C18 (250mm X 4.6 mm i.d., 5 μm particle size) column which was used with a mobile phase of acetonitrile: tetrabutyl ammonium hydrogen sulphate (TBAHS) + tetrabutyl ammonium bromide (TBAB) buffer pH 3 (20:80 v/v) and adjusting pH 3 with orthophosphoric acid. Flow rate was kept at 1.5ml/min and elution was detected by UV detector at 280.8nm and the data were recorded using LC 20 solutions software and analysed by software LC solution (version 1.02).

Preparation of Standard Stock Solution:

Standard stock solution 1000 µg/ml of MOX and 1000 µg/ml of CEF were prepared by dissolving 100mg of MOX and 100mg of CEF in 100ml of mixture of diluent water: ACN (80:20v/v). The solutions were suitably diluted with diluent water: ACN (80:20v/v) to get mixed standard solution containing 10µg/ml of MOX and 10µg/ml of CEF.

Preparation of Sample Solution

Twenty tablets made up of synthetic mixture each containing 400mg of MOX and 400mg of CEF were weighed and powdered. Powder equivalent to 100mg of MOX and CEF was weighed accurately and taken into 100 ml volumetric flask. The drugs were extracted into diluent water: ACN (80:20v/v) and the volume was adjusted to 100ml, sonicated for 45min and then filtered through 0.45 µ membrane filter. From this solution, further dilutions were made using diluent water: ACN (80:20v/v) to get a final concentration of 100µg/ml of MOX and 100µg/ml of CEF. This solution was used for the estimation. The proposed method was validated as per ICH and FDA guidelines.

System Suitability Studies

The column efficiency, resolution and tailing factor were calculated for the standard solutions. The values (Table.1) obtained demonstrated the suitability of the system for the analysis of this drug combination.

Table 1. System suitability parameters:

Parameters MOX CEF

Theoretical plates 4775.133 5268.814

Resolution factor - 9.334

Tailing factor 1.21 0.918

Retention time (min) 3.078 5.263

MethodValidation⁶ Linearity

Several aliquots of standard solutions of MOX and CEF were taken in different 10ml volumetric flasks and diluted up to the mark with diluent water: ACN (80:20v/v) such that the final concentration of MOX and CEF was 10-60 µg/ml and 10-60 µg/ml respectively. Evaluation of two drugs was performed with UV detector at 280.8 nm and peak area recorded for all the peaks. This linearity was represented by a linear regression equation as follows.

Y_MOX= 35847x – 17001 (R² = 0.999) and

Y_CEF= 35834x - 56310 (R² = 0.999) (Table 2).

Recovery Studies

To study the accuracy and reproducibility of the proposed method recovery experiments were carried out. A fixed amount of pre-analyzed sample was taken and standard drug was added at 80% and 100% and 120% levels. Each level was repeated three times. The mean recoveries of MOX and CEF were in range of 98% and 102% that shows there is no interference from excipients (Table 2).

Precision Studies

The Intraday and Interday precision were determined by analyzing a set of laboratory sample (n=3) with each of the three concentration. Results of Precision Studies were shown in Table 2.

Limit of Detection and Limit of Quantification

The limit of Detection (LOD) and limit of Quantification (LOQ) of the developed method were determined by standard deviation of response and slope of calibration curve (Table 2).

Robustness

The robustness of the method was determined by making slight changes in the chromatographic conditions. It was observed that there were no marked changes in the chromatograms, which demonstrated that the RP-HPLC method developed was robust (Table 2).

Solution Stability

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 24 hrs at room temperature. The result showed that for both solutions the retention time and peak area of MOX and CEF remained almost unchanged (% RSD less than 2.0) and no significant degradation within the indicated period was found, thus it was concluded that both the solutions were stable for at least 24 hrs which was sufficient to complete the whole analytical process (Table 2).

Table 2. Summary of Validation Parameters

Validation parameters	Moxifloxacin HCl	Cefixime trihydrate	Specifications
Linearity and range	10-60μg/ml	10-60μg/ml	-
Slope	35847	35834	-
Intercept	17001	56310	-
Regression coefficient(R²)	0.999	0.999	≥0.999
Recovery study(n=3)	98.88±0.6040	100.70±0.5760	98-102%
Intraday precision (n=3)	1.5479%	1.1459%	%RSD NMT 2
Inter day precision (n=3)	1.4878%	1.7298%	%RSD NMT 2
LOD*	0.9294575	0.873272	-
LOQ*	2.8165379	2.646279	-
Stability of analytical solution	24 hrs	24 hrs	-
Specificity	Specific	Specific	-

*Average of six determinations

RESULTS AND DISCUSSION:

Estimation of MOX and CEF in Tablet Dosage Form:

The HPLC procedure was optimized with a view to develop accurate and stable assay method. Both the pure drugs MOX and CEF were run in different mobile phase compositions. The flow rate was also varied from 1.3 mL to 1.7 mL/ min. Thermo Scientific BDS HYPERSIL C18 (250mm X 4.6 mm i.d., 5 μm particle size) column was used with a mobile phase of acetonitrile: tetrabutyl ammonium hydrogen sulphate (TBAHS) + tetrabutyl ammonium bromide(TBAB) buffer pH 3 (20:80 v/v) and adjusting pH 3 with orthophosphoric acid. Flow rate was kept at 1.5ml/min and elution was detected by UV detector at 280.8nm and analysed by software LC solution (version 1.02) gave sharp and symmetrical peak with retention time 3.078 and 5.263 min for MOX and CEF respectively. The typical chromatogram of the sample is shown in Fig. 1.The assay procedures were repeated for six times and the percentage of individual drugs found in formulations, standard deviation, and % R.S.D were calculated and presented in Table 3.

Table 3. Analysis of Tablet Dosage Form*

Drug

Labeled claim

(mg/

tablet)

Amount found

(mg/

tablet)

% Label Claim*

Standard deviation

%RSD

MOX

400

425.6

106.4

0.0354

0.7068

CEF

400

399.6

99.9

0.0078

0.5196

*Average of six determinations

Figure 3. Chromatogram of MOX and CEF.

CONCLUSION:

The proposed method gives good resolution between MOX and CEF within short analysis time (<10min). The method is very simple, rapid and no complicated sample preparation is needed. High percent of recovery shows the method is free from interference of excipients present in the formulations.

ACKNOWLEDGEMENT:

The authors are grateful to Bharat Parenterals Ltd., Savli, Vadodara for giving an opportunity to execute a research work at their premises and for providing all the necessities for the same and special thanks to my mentor Dr. Virendra C. Bhatt.

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Received on 13.04.2013 Modified on 22.04.2013

Asian J. Research Chem. 6(5): May 2013; Page 498-501