INTRODUCTION:

Paracetamol chemically 4-hydroxy acetanilide is a centrally and peripherally acting analgesic and antipyretic agent. Paracetamol is classified as a mild analgesic. Aceclofenac, {[2-(2', 6'-dichlorophenyl) amino] phenyl acetoxyacetic acid} is a new phenyl acetic acid derivative with potent analgesic and anti-inflammatory properties and improved gastric tolerance. Aceclofenac is the glycolic acid ester of Diclofenac. Paracetamol is official in Indian¹, United States² and European Pharmacopoeia³. Aceclofenac is official in Indian¹and European Pharmacopoeia². The tablet combination dosage form in the ratio of 325:100mg Paracetamol and Aceclofenac respectively.

Many formulations are available with many trade names. numorous methods have been reported simultaneously or in combination with other drugs including simultaneous estimation of Paracetamol and aceclofenac by spectrophotometric⁴, simultaneous estimation by HPLC^5-9, Simultaneous estimation of rabeprazole and aceclofenac by HPLC^10-12, simultaneous estimation of thiocolchicoside and aceclofenac RP-UPLC¹³, simultaneous estimation of tizanidine and aceclofenac by HPLC¹⁴, simultaneous estimation of aceclofenac, paracetamol and chlorzozazone by HPLC, HPTLC and UHPLC^15-23, simultaneous estimation of paracetamol, aceclofenac and rabeprazole sodium by HPLC and UV^24-26, Simultaneous Estimation of Paracetamol, Aceclofenac and Serratiopeptidase by HPLC^27-29, Paracetamol, Aceclofenac and Thiocolchicoside by HPLC and UV^30,33, Simultaneous Determination of Aceclofenac, Paracetamol and Tramadol Hydrochloride by HPLC^34-36, Simultaneous Determination of Some Anti-Inflammatory and Analgesic Drugs UHPLC³⁷, from literature^38-42 reveals Diclofenac is the degradation of Aceclofenac. So this present proposed method validated as per ICH guidelines⁴³. This proposed method simultaneously estimates the Paracetamol and Aceclofenac in presence of Diclofenac in tablet formulation.

MATERIAL AND METHODS:

Instrumentation:

The separation was carried out on Shimadzu HPLC system with quaternary gradient and UV and 20MP PDA detectors, LC solutions software and Kromasil C18 (150mm x 4.6mm, 5µm) column.

Chemicals and reagents:

The working standards of Paracetamol, Aceclofenac and Diclofenac were provided as gift samples from Bio-Leo Analytical Labs., Hyderabad. Marketed formulation was purchased from local market. Potassium dihydrogen orthophosphate, Water and Acetonitrile of HPLC grade from Rankem.

HPLC Conditions:

The mobile phase consisting of 0.05M potassium dihydrogen phosphate and acetonitrile in the ratio 40:60 was filtered through 0.45µm PVDF membrane filter before use, degassed pumped into the column at a flow rate of 1.0mL/min. The column temperature was 40°C. The detection was monitored at 275nm and the run time was 10 min. The injection volume was 10µL. the column was equilibrated for 30 min prior to injection of the drug solutions with the mobile phase.

Preparation of system suitability Solution:

Accurately transferred 10mg each of Paracetamol, Aceclofenac and Diclofenac into 100ml volumetric flask dissolve and diluted to volume with mobile phase.

Preparation of Standard stock Solutions:

Paracetamol: Weighed Accurately 32.5mg transferred into 100mL of volumetric flask dissolve and diluted to volume with diluent and sonicate for 10 min.

Aceclofeanc:

Weighed Accurately 10.0mg transferred into 100mL of volumetric flask dissolve and diluted to volume with diluent and sonicate for 10 min.

Preparation of Standard Solution:

Accurately transferred 10ml each of paracetamol and aceclofenac stock solutions into 100ml volumetric flask dissolve and diluted to volume with mobile phase.

Preparation of Sample Solution:

Take accurately sample powder equivalent to 32.5mg of Paracetamol and 10mg of Aceclofenac and transfer it into 100ml of volumetric flask dissolve and diluted to volume with mobile phase sonicate for 10 min. From the above solution take 10ml into 100ml volumetric flask make up the volume with mobile phase.

System Suitability Studies:

System suitability testing is an integral part of analytical procedure, the parameters like resolution, tailing factor, theoretical plates and area %RSD determined for system suitability solution and five replicate injections for standard solution. The results shown in Table-1.

Table 1: System suitability results

Parameters	Paracetamol	Aceclofenac	Diclofenac	Limit
For SST solution
Retention time	2.362	3.235	6.388	--
Resolution (R)	-	5.247	14.595	R > 2
Tailing factor (T)	1.37	1.26	1.10	T < 2
Theoretical plates (N)	4075	4888	10708	N > 2000
For standard solution
Tailing factor (T)	1.37	1.26	--	T < 2
Retention time	2.360	3.236	--	--
Resolution ( R )	-	5.273	--	R > 2
Theoretical plates (N)	4091	4915	--	N > 2000
Area %RSD	0.05	0.20	--	<1.0 for n≥5

Figure 1: System suitability chromatogram

Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present and to provide an exact content or potency of the analyte in the sample. The test solution monitored on PDA (Figure.2)

Figure 2: test chromatogram

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three concentration levels with triplicate preparations. The percentage recovery, mean recovery and standard deviation of the percentage recovery were calculated. (Table-2&3).

Table 2: Paracetamol accuracy results:

Spiked Level	Sample amount added (mcg/ml)	Sample amount recovered (mcg/ml)	% recovery	mean % recovery	% RSD
50%	16.255	16.260	100.03	100.09	0.19
50%	16.234	16.283	100.30
50%	16.271	16.260	99.93
100%	32.532	32.548	100.05	99.97	0.11
100%	32.563	32.570	100.02
100%	32.606	32.554	99.84
150%	48.734	48.510	99.54	99.26	0.25
150%	48.718	48.255	99.05
150%	48.681	48.287	99.19

Table 3: Aceclofenac accuracy results:

Spiked Level	Sample amount added (mcg/ml)	Sample amount recovered (mcg/ml)	% recovery	mean % recovery	% RSD
50%	5.002	4.984	99.64	99.73	0.11
50%	4.995	4.988	99.86
50%	5.007	4.991	99.70
100%	10.010	9.989	99.79	99.71	0.09
100%	10.020	9.993	99.74
100%	10.033	9.993	99.61
150%	14.995	14.950	99.70	99.35	0.30
150%	14.990	14.869	99.19
150%	14.979	14.855	99.17

Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The % assay calculated for six different preparations of the sample (Table 4)

Table 4: precision results:

S.No	% Assay (Paracetamol)	% Assay (Aceclofenac)
Preparation-1	99.97	99.67
Preparation-2	100.02	99.72
Preparation-3	100.14	99.91
Preparation-4	100.04	99.68
Preparation-5	100.23	99.96
Preparation-6	99.93	99.67
Average Assay:	100.06	99.97
Std.dev	0.11	0.13
%RSD	0.11	0.13

Linearity Range:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

linearity was determined by plotting peak area against concentration of solutions. The linearity of Paracetamol and Aceclofenac determined in the concentration ranges of 16.25-48.75µg/mL and 5-15µg/mL respectively. The Paracetamol regression equation is y = 10245x with a coefficient of correlation (R²)of 0.999. The Aceclofenac regression equation is y = 17903x-46.49 with a coefficient of correlation (R²)of 1.0 and shown in Fig 3&4 and the overlay linearity chromatograms shown in Fig.5. The range of the analytical method determined for Paracetamol and Aceclofenac (Table 5)

Figure 3: Paracetamol calibration curve

Figure 4: Aceclofenac calibration curve

Figure 5: Overlay linearity chromatograms

Table 5: range results

Parameter	Unit	Paracetamol	Aceclofenac
Range	µg/mL	16.25-48.75	5.0-15.0
Linarity	Correlation coefficient (r²)	0.999	1.0
Accuracy at 50%	% recovery	100.09	99.73
Accuracy at 150%	% recovery	99.26	99.35
Precision at 50%	% RSD	0.19	0.11
Precision at 150%	% RSD	0.25	0.30

Robustness and ruggedness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

Robustness was determined a change the flow rate ±0.1ml/minute, column oven ±5°C, organic phase in mobile phase ±5% and different batch of the column with different analyst.

RESULTS AND DISCUSSIONS:

System suitability results were given by Table.1 and system suitability parameters are retention time, resolution, tailing, resolution and plate count were within the acceptance limits. so the system is suitable for the analysis. The test chromatogram extracted on PDA detector the peak paracetamol and aceclofenac purities found 1.0 respectively indicates no interference, so the method is specific. Paracetamol recovery was found 99.26 to 100.09% and %RSD was 0.11 to 0.19, Aceclofenac recovery was found 99.35 to 99.73% and %RSD was 0.09 to 0.30 indicates the method is accurate 50 to 150% of the target concentration. Six different %assay preparation values of the same homogeneous samples found 99.93 to 100.14% for paracetamol and 99.67 to 99.97% for aceclofenac indicates the method is precise. Paracetamol Linear correlation was found to be y=10245 x, correlation coefficient was 0.999 and aceclofenac was y=17903x-46.49 and correlation coefficient was 1.0 indicates the method is linear across the target concentration. The method unaffected due to deliberate changes in flow rate, column oven temperature and different batch column with diferent analyst proves the method is robust and rugged. The LOD and LOQ values were found to be 0.03 and 0.11µg/mL for Paracetamol and 0.02 and 0.06 µg/mL for Aceclofenac respectively. The proposed range 50 to 150% of the target concentration i.e, 16.25 to 48.75 µg/mL for Paracetamol and 5.0 to 15.0 µg/mL for Aceclofenac found linear, accurage and precise (Table 5).

CONCLUSION:

A new, specific, selective, linear, accurate and precise isocratic RP-HPLC method was developed for the estimation of Paracetamol and Aceclofenac in their pharmaceutical tablet formulation in presence of Diclofenac. The proposed method was successfully separated all the three compounds. The Proposed method is specific, selective and stability-indicating power. Hence the developed method could be adapted to regular quality control analysis and stability analysis.

ACKNOWLEDGEMENT:

The authors are thankful to Department of science & humanities, M/s Bharat Institute of Higher education and research, Chennai India for encouragement.

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Received on 21.11.2019 Modified on 24.01.2020

Research J. Pharm. and Tech 2020; 13(9):4334-4338.

DOI: 10.5958/0974-360X.2020.00766.0