INTRODUCTION:

Tramadol is a antibiotic belonging to the chemical class of pencillins derivatives Chemically it is (2S,5R,6R)-6-[3-(2,6-dichlorophenyl)-5-methyl-1,2-oxazole-4-amido]-3,3- dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.^1-3 Tramadol exerts a bactericidal action against penicillin-susceptible microorganisms during the state of active multiplication. All penicillins inhibit the biosynthesis of the bacterial cell wall. By binding to specific penicillin-binding proteins (PBPs) located inside the bacterial cell wall, Tramadol inhibits the third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins; it is possible that Tramadol interferes with an autolysin inhibitor. Tramadol and Aceclofenac are formulated together in the form of a tablet.

Literature survey revealed no method reported for simultaneous determination of the two drugs. The present RP-HPLC method uses simple mobile phase ratio, higher sensitivity and analysis will complete before 7 min. Therefore the present study was to determine both drugs concurrently by sensitive, accurate, rapid and precise RP-HPLC^4-9 method for routine analysis.

Fig.No.1.chemical structure of Tramadol.

Aceclofenac is a anti- bacterial agent. : (6R,7R)-7-[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-(methoxyimino) acetamido]-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid^1-3. The molecular formula is C₁₅H₁₇N₅O₆S_2;the molecular weight is 427.455; the structural formula is.

Fig.No.2.Chemical structure of Aceclofenac

MATERIALS AND METHODS:

Reagents and chemicals

Methanol HPLC grade was procured from E. Merck Ltd, Mumbai. Methanol, Acetonitrile. Fine chemicals, Hyderabad. Water HPLC grade was prepared using Millipore purification system. Tramadol and Aceclofenac reference standards procured from Active Pharma labs, Hyderabad.

Instrumentation

The HPLC system consists of water Empower -2690/5 having photodiode array detector system, which was connected with the help of Empower 3000 software for data integration and processing. Inertsil ODS‐3V (250 X 4.6 mm) 5μ column was used for the analysis.

HPLC conditions

The contents of the mobile phase were Methanol and Water in the ratio of 50:50 v/v. These were filtered through 0.45μ membrane filter and degassed by sonication before use. The flow rate of mobile phase was optimized to 1.0 ml / min. The run time was set at 7 min and column temperature was maintained at ambient. The volume of injection was 20 μl, and the eluent was detected at 290 nm. Each of standard and test preparations was injected into the column and the responses recorded (Fig.05 and Fig.06.).

METHOD

The RP-HPLC Method of Aceclofenac and Tramadol were achieved by isocratic elution technique with PDA Detector. Aceclofenac and Tramadol were determined at 290nm respectively with the concentration range of 0.20-0.8g/ml for both Aceclofenac and Tramadol respectively.fig.03 and04. For analysis of tablet formulation the tablet powder equivalent to 25 mg was taken, dissolved in 25 ml volumetric flask and made up to 25ml with Methanol. The solution was sonicated for 15min, centrifuged at 100 rpm for 15 min and filtered through Whatman filter paper No.41. From clear solution, further dilutions were made to get 10 µg/ml of Aceclofenac and Tramadol theoretically.

Figure 3: Calibration Graph of Aceclofenac

Figure 4: Calibration Graph of Tramadol

Figure 5: Typical chromatograms for recovery studies

For recovery studies, to the reanalyzed formulation, solutions of raw material containing different concentrations were added and the amount of drug recovered was calculated. The procedure was repeated as per the analysis of formulation. The amount of drug recovered was calculated by using slope and intercept values from the calibration graph. Finally the method was validated as per ICH guide linesfor precision, accuracy, specificity, linearity, reproducibility, limit of detection and limit of quantification.

Figure 6: Typical chromatogram of Tramadol and Aceclofenac

RESULTS AND DISCUSSION:

A simple, selective, rapid and precise validated RP-HPLC Method for Simultaneous Estimation of Aceclofenac and Tramadol in bulk material and in pharmaceutical formulation has been developed and validated. The correlation coefficient was found to be 0.9997and0.9998 Aceclofenac for and Tramadol respectively.fig.03 and fig.04. In this method the % purity were found to be 98.0 – 101.50 respectively. The recovery studies range is.99.98-100.03% and 99.94 – 100.01 % for Tramadol and Aceclofenac, respectively. Detection limit was found to be 0.17for Tramadol and 0.36 for Aceclofenac. The Intraday and Inter day analysis carried out for precision. The ruggedness study was performed.

In First order Derivative method the % purity were found to be 98.0 – 101.50 respectively, respectively. The recovery studies range is 99.98-100.03% and 99.94 – 100.01 %. Table 1.The Intraday and Inter day analysis carried out for precision. The ruggedness study was performed. The method was validated for statistical analysis

Table 1: Recovery Studies

(a)Data of Accuracy (Recovery) for Tramadol

Concentration % of spiked level	Amount added (ppm)	Amount found (ppm)	% Recovery	Statistical Analysis of % Recovery
50% Injection 1	20	19.98	99.93	MEAN	100.01
50% Injection 2	20	20.02	100.10
50% Injection 3	20	20.0	100.00	%RSD	0.09
100 % Injection 1	40	39.99	99.98	MEAN	100.10
100 % Injection 2	40	40.10	100.26
100% Injection 3	40	40.03	100.08	%RSD	0.14
150% Injection 1	60	60.02	100.03	MEAN	100.00
150% Injection 2	60	60.01	100.01
150% Injection 3	60	59.99	99.98	%RSD	0.03

(b)Data of Accuracy (Recovery) for Aceclofenac

Concentration % of spiked level	Amount added (ppm)	Amount found (ppm)	% Recovery	Statistical Analysis of % Recovery
50 % Injection 1	20	19.99	99.94	MEAN	99.94
50 % Injection 2	20	19.98	99.95
50 % Injection 3	20	19.98	99.95	%RSD	0.01
100 % Injection 1	40	39.99	99.98	MEAN	100.04
100 % Injection 2	40	40.00	100.01
100 % Injection 3	40	40.05	100.13	%RSD	0.08
150 % Injection 1	60	59.99	99.98	MEAN	99.97
150% Injection 2	60	59.98	99.97
150% Injection 3	60	59.99	99.98	%RSD	0.01

CONCLUSION

The developed RP-HPLC method was validated and the statistical validation was performed with the simplicity and ease of operation ensures that the validated method can successfully used for routine Analysis of Aceclofenac and Tramadol in bulk and tablet dosage formulation.

ACKNOWLEDGEMENTS:

The authors are thankful to principal and Chairman of Teegala Ram Reddy College of Pharmacy Meerpet-Hyderabad and Also thank full to Chandra Labs for their kind help and providing all necessary facilities.

REFERENCE:

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Received on 25.06.2015 Accepted on 30.07.2015

Asian J. Res. Pharm. Sci. 5(3): July-Sept.; Page 135-138

DOI: 10.5958/2231-5659.2015.00021.1