Aceclofenac is chemically designated as 2-(2, 6-dichlorophenyl) amino phenyl acetyl oxyacetic acid. It is used as anti-inflammatory and analgesic agent. Paracetamol is chemically N - (4-hydroxyphenyl) acetamide and is used as analgesic and anti-pyretic agent. 

Reagents and Chemicals:

Methanol HPLC grade,Water HPLC grade, Reference standards of aceclofenac and paracetamol 

Apparatus and Chromatographic Conditions:

Chromatographic separation was performed on a Jasco HPLC system consisting of Jasco PU-2080 pump, Jasco UV 2010 photo diode array detector, Rheodyne injection

syringe with 20 μl loop volume and windows based chrompass software. 

COLUMN : ODS C18 RP-Column (Intersile 4.6 mm x 25 cm, 10 μm) 

Mode: Isocratic

Flow rate of 1 mL/min 

Mobile Phase: Methanol: water (70:30 v/v) 

Preparation of Standard Stock Solution:

The standard stock solution 1mg/ml of aceclofenac and paracetamol were prepared separately by dissolving 50 mg of each drug in 50 ml mixture of methanol and water(70:30 v/v). From the standard stock solution, mixed standard solution was prepared to contain 100 μg/mL of aceclofenac and 100 μg/mL of paracetamol.

Preparation of Sample Solution

Twenty tablets each containing 100 mg of aceclofenac and 500 mg of paracetamol were weighed and powder equivalent to 100mg of paracetamol and 20 mg of aceclofenac was weighed accurately (0.1002 gm ) and transferred to a conical flask and extracted thrice with 20 ml mixture of methanol and water (70:30). The combined extracts were transferred to a volumetric flask and the volume adjusted to 100 ml with mobile phase. From this solution, 10 ml was pipetted and transferred to 100 ml volumetric flask and made volume up to the mark with mobile phase to get the concentration 100 μg/ml of paracetamol and 20 μg/ml of aceclofenac. Further dilutions were made using mobile phase to get the final concentration of 10 μg/ml of paracetamol and 2 μg/ml aceclofenac

Linearity

Several aliquots of standard solutions of aceclofenac and paracetamol were taken in different 10ml volumetric flasks and diluted up to the mark with mobile phase such that the final concentration of aceclofenac and paracetamol is 2-50 μg/mL and 5-50 μg/mL respectively. Evaluation of two drugs were performed with PDA detector at 275 nm, peak area recorded for all the peaks and are given in the Table I. The slope and intercept value for calibration curve was y = 947.82x + 1.1033 (R2 = 0.9989) for aceclofenac and y = 502.37x + 84.858 (R2 = 0.09987) for paracetamol. The results show that an excellent correlation exists between peak area and concentration of drugs within the concentration range indicated above. 

Assay

20 μl of standard and sample solutions were injected into an injector of liquid chromatograph, from the peak area of aceclofenac and paracetamol amount of drug in samples were computed. The values are given in Table II. 

Estimation of Aceclofenac and Paracetamol in Dosage Forms:

The HPLC procedure was optimized with a view to develop accurate and stable assay method. Both the pure drugs aceclofenac and paracetamol were run in different

mobile phase compositions with different C18 columns [Kromacil 25cm x 4.6mm i.d. 5 micron), ODS C18(Intersile 25cm x4.6mm i.d.,10 micron)]. The flow rate

was also varied from 0.5 mL to 1.3 mL/min. ODS C18 column with a mobile phase of mixture of methanol and water (70:30 v/v), delivered at a flow rate of 1.0 mL/min

with a detection at 275 nm gave sharp and symmetrical peak with retention time 1.8 and 2.7 min for aceclofenac and paracetamol respectively. The typical chromatogram of the sample is shown in Fig. 1.The overlaid UV spectrum of aceclofenac and paracetamol is shown in Fig. 2. The assay procedures were repeated for six times and the percentage of individual drugs found in formulations, standard deviation, % R.S.D. and standard error were calculated and presented in Table II.

Limit of Detection and Limit of Quantification

The limit of Detection (LOD) and limit of Quantification (LOQ) of the developed method were determined by injecting progressively low concentrations of the standard solutions using the developed RP-HPLC method. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD for aceclofenac and paracetamol was found to be 10 ng/ml and 5 ng/ml, respectively. The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ was 30 ng/ml and 15 ng/ml for aceclofenac and paracetamol respectively Table III.

The ruggedness of the method was determined by carrying out the experiment on different instruments like Jasco HPLC and Agilent HPLC by different operators using different columns of similar type like Hypersil C18, Intersile C18. 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, are rugged and robust

Solution Stability

In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 5 h at room temperature. The

result show that for both solutions, the retention time and peak area of aceclofenac and paracetamol remained almost unchanged (% R.S.D. less than 2.0) and no significant degradation within the indicated period, thus indicated that both solutions were stable for at least 5 h, which was sufficient to complete the whole analytical process.

System Suitability Studies

The column efficiency, resolution and peak asymmetry were calculated for the standard solutions Table III. The values obtained demonstrated the suitability of the system for the analysis of this drug combination, system suitability parameters may fall within ± 3% standard deviation range during routine performance of the method.

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 50% and 100% levels. Each level was repeated three times. The contents of aceclofenac and paracetamol per tablet found by proposed method is shown in Table IV. The lower values of RSD of assay indicate the method is accurate. The mean recoveries of aceclofenac and paracetamol were in range of 100.77% and 100.66% that shows there is no interference from excipients.

CONCLUSION:

The proposed method gives good resolution between aceclofenac and paracetamol within short analysis time (

complicated sample preparation is needed. High percent of recovery shows the method is free from interference of excipients present in the formulations.