Simultaneous Determination of Paracetamol and Diclofenac Potassium in Pharmaceutical Preparation by using High Performance Liquid Chromatography

 

Saad Antakli, Leon Nejem, Duha Shawa

Department of Chemistry, Faculty of Science, University of Aleppo, Syria

 

ABSTRACT:

An accurate, simple, reproducible and sensitive method for the determination of Paracetamol (Par) and Diclofenac potassium (Dic.p) in pharmaceutical formulations was developed and validated. (Par) and (Dic.p) were separated by using an isocratic mobile phase of acetonitrile:phosphoric acid (pH=2.43) in ratio of 60:40 (v/v) on a Shim-pack clc-C₈ column (25 cm x 4.6 mm i.d., 5 μm), with UV detection at 274 nm, pump flow rate was 1.0 mL/min and sample injection volume 20 μL. Under these conditions, separation of each two components was achieved in less than 8 min with a good resolution, whereas the retention times of (Par) and (Dic.p) were found to be 3.03 min and 6.68 min respectively. The proposed method was validated for linearity, accuracy, and precision, limit of detection and limit of quantification. The linear range of determination for (Par) and (Dic.p) in presence of Sodium propyl paraben (PP.Na) as internal standard were (2.5-500) µg/mL and (1-250) µg/mL respectively. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.48 𝜇g/mL and 1.47 𝜇g/mL for (Par), 0.26 𝜇g/mL and 0.80 𝜇g/mL for (Dic.p), respectively. The proposed method was successfully applied to analysis individual or mixture of (Par) and (Dic.p) in Syrian trademark drugs. All studied samples showed that the drug levels were conformed to united state pharmacopeia (USP) legislation. Finally, the developed method is simple, quick and suitable for routine quality control analysis of titled drugs in combination of tablet formulations.

 

 

 

INTRODUCTION:

Paracetamol:

N-(4-Hydroxyphenyl) acetamide or Paracetamol (Par), Fig. 1,a, is a white or almost white crystalline powder. Sparingly soluble in water, freely soluble in alcohol, very slightly soluble in methylene chloride¹.

 

(Par) is a p-aminophenol derivative that exhibits analgesic and antipyretic activity. It does not possess anti-inflammatory activity. (Par) is thought to produce analgesia through a central inhibition of prostaglandin synthesis.

 

Several methods have been applied in the literature for the determination of (Par) in dosage forms and in biological fluids. Techniques such as spectrophotometry^2,3,4,5,6,7, capillary electrophresis⁸, TLC⁹, high performance liquid chromatography (HPLC)^10,11,12,13.

 

Diclofenac potassium:

Potassium [2-[(2,6-dichlorophenyl) amino] phenyl] acetate or Diclofenac potassium (Dic.p), Fig. 1,b, is a white or slightly yellowish, slightly hygroscopic, crystalline powder¹. Sparingly soluble in water, freely soluble in methanol, soluble in ethanol, slightly soluble in acetone.

 

(Dic.p) is a non-steroidal anti-inflammatory drug (NSAID) that exhibits anti-inflammatory, analgesic, and antipyretic activities in animal models. The mechanism of action of (Dic.p) tablets, like that of other NSAIDs, is not completely understood but involves inhibition of cyclooxygenase (COX-1 and COX-2).

 

 

Fig.1: Structure of the analytes:

(a) Paracetamol (Par), (b) Diclofenac Potassium (Dic.p).

 

MATERIALS AND METHODS:

Apparatus:

HPLC analysis was performed on an YL 9100 HPLC system equipped with binary pump YL9111, vacuum degasser series YL9101, YL9130 column compartment and YL9120, UV/Vis. Detector (Korea). Chromatographic separations were obtained by using Shim-pack clc-C₈ column (25 cm×4.6 mm i.d., 5 µm) (Shimadzu, Japan). Ultrasonic bath (Daihan, USA), analytical balance TE64 Sartorius (Germany) sensitivity 0.01 mg. Germany digital pipettes (Isolab).

 

Chemical regents:

The solvents and materials were used as analytical grade: water, ethanol (Merck, Germany), acetonitrile (Isolab, Germany), all were HPLC grade. Phosphoric acid (Isolab, Germany), and sodium propyl paraben (PP.Na), purity 99.22 %, (Clariant, UK).

 

Stock standard preparation:

25 mg/L of (Par), (Dic.p) and (PP.Na) were prepared as standard solutions by dissolving appropriate weights of these materials in ethanol, by taking the purity of the material on consideration.

 

Calibration Curve:

To construct the calibration curve, five standard solutions for each concentration were prepared and the area of peaks was measured of each solution five times.

 

Samples preparation:

Four Syrian products were studied:

·      Ten "Agilomox" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 500 mg (Par) and 50 mg (Dic.p) was accurately weighed, then dissolved in 100 mL volumetric flask with ethanol. The sample solution was filtered through a filter (Whatman 3, England). Then 0.5 mL was taken to 25 mL volumetric flask and adjusted to volume with mobile phase.

·      Ten "Supergesic" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 500 mg (Par) and 50 mg (Dic.p) was accurately weighed, then dissolved in 100 mL volumetric flask with ethanol. The sample solution was filtered through a filter (Whatman 3, England). Then 0.5 mL was taken to 25 mL volumetric flask and adjusted to volume with mobile phase.

·      Ten "Paracetamol Ultra Medica" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 500 mg (Par) was accurately weighed, then dissolved in 100 mL volumetric flask with ethanol. The sample solution was filtered through a filter (Whatman 3, England). Then 0.5 mL was taken to 25 mL volumetric flask and adjusted to volume with mobile phase.

·      Ten "Voltarile" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 50 mg (Dic.p) was accurately weighed, then dissolved in 100 mLvolumetric flask with ethanol. The sample solution was filtered through a filter (Whatman 3, England). Then 0.5 mL was taken to 25 mL volumetric flask and adjusted to volume with mobile phase.

 

RESULTS AND DISCUSSION:

Chromatograms of the standard solutions (as internal standard) of mixture (Par), (Dic.p) and (PP.Na) at five different standard concentrations, which every concentration was injected five times under optimized method conditions showed that (Par) was well separated from (Dic.p) with good resolution, Fig. 2, and the time of analysis was achieved in less than 8 min.

 

 

Fig.2: Chromatogram of standard solutions showing separated peaks of:

1-(Par) 25 mg/L, 2-(PP.Na) 25 mg/L, 3-(Dic.p) 25 mg/L.

 

Optimization of the HPLC conditions:

Mobile phase effect:

The aim of this study was to develop HPLC assay for the analysis of (Par) and (Dic.p) drugs in pharmaceutical dosage form.

 

Initial studies to develop HPLC assay involved the use of C₁₈ and C₈ column with various mobile phases containing polar solvent like methanol and acetonitrile with aqueous solutions for some salts (sodium dihydrogen ortho-phosphate dehydrate, di-sodium hydrogen ortho-phosphate anhydrous and sodium acetate) and different acids (formic acid, acetic acid and phosphoric acid).

 

The C₈ column was chosen for further studies since it produced fine and symmetrical peaks.

 

The final selective HPLC mobile phase consisting of acetonitrile/phosphoric acid (pH=2.43).

 

The effect of composition of the mobile phase on the retention time of (Par), (Dic.p) and the internal standard (PP.Na) was investigated Fig. 3.

 

An increase in the percentage of acetonitrile decreases the retention of compounds (Par) and (Dic.p). Increasing acetonitrile percentage to more than 85% (Par) peak is eluted with the solvent front, while at acetonitrile percentage lower than 55 % the elution of (Dic.p) peak is delayed.

 

The optimum acetonitrile percentage was found to be 60 %.

 

 

Fig.3: Mobile phase effect on the retention time of: (Par) 25 mg/L, (Dic.p) 25 mg/L and (PP.Na) 25 mg/L.

 

pH effect of mobile phase:

The effect of pH in the chromatographic elution of both compounds (Par) and (Dic.p) was investigated by changes in the pH values of the aqueous component of the mobile phase from (2.2 to 6), Fig. 4.

 

For experimental pH values from (2.2 to 4.3) the drugs are eluted in order of (Par), (PP.Na) and (Dic.p), but for pH values from (4.3 to 6) the order was (Dic.p), (Par) and (PP.Na). pH=2.43 was chosen for the optimum separation of these compounds.

 

Fig.4: pH effect of mobile phase acetonitrile:phosphoric acid in ratio of 60:40 (v/v) on the retention time of (Par) 25 mg/L, (Dic.p) 25 mg/L and (PP.Na) 25 mg/L.

 

Flow rate effect:

The optimum flow rate was identified by changing flow rate from (0.8 to 1.3) mL/min. It was concluded that each two peaks were completely separated, fine and symmetrical at the flow rate (1 mL/min) which also corresponds to the deviation point as seen in Fig. 5.

 

Fig.5: Flow rate effect of mobile phase acetonitrile:phosphoric acid (pH=2.43) in ratio of 60:40 (v/v) on the retention time of (Par) 25 mg/L, (Dic.p) 25 mg/L and (PP.Na) 25 mg/L.

 

Chromatographic conditions:

Table 1 presents the chromatographic method conditions, which were applied for simultaneous determination of (Par) and (Dic.p) in presence an internal standard (PP.Na).

 

Table 1: (Par) and (Dic.p) separation chromatographic conditions.

 

METHODS VALIDATION:

Linearity:

System linearity was determined by analysis of five replicates of five standards concentrations of (Par) and (Dic.p) as it is seen in Fig. 6, 7.

 

Linear relationship was obtained between the peak area ratio of (Par) or (Dic.p) to the internal standard (PP.Na) in function to (Par) or (Dic.p) concentrations.

 

 

Fig. 6: Linear relationship for (Par):

C₁: 2.5 𝜇g/mL, C₂: 25 𝜇g/mL, C₃: 50 𝜇g/mL,

C₄: 150 𝜇g/mL, C₅: 250 𝜇g/mL, C₆: 350 𝜇g/mL,

C₇: 500 𝜇g/mL.

n=5 for each concentration.

 

 

Fig. 7: Linear relationship for (Dic.p):

C₁: 1 𝜇g/mL, C₂: 10 𝜇g/mL, C₃: 25 𝜇g/mL,

C₄: 100 𝜇g/mL, C₅: 150 𝜇g/mL, C₆: 200 𝜇g/mL,

C₇: 250 𝜇g/mL.

n=5 for each concentration.

Accuracy/recover:

The accuracy of the method was checked by evaluating the experimental concentration of the solutions prepared for the linearity test versus the nominal concentration. Good recovery of )Par( and )Dic.p( was observed as shown in Table 2.

 

Precision:

System precision:

Five replicates (n=5) of a standard mixture solution 0.1 mg/mL )Par( and 0.05 mg/mL )Dic.p) were analyzed to assess system precision. The RSD % of peak area response in Table 2 showed the satisfactory repeatability of the system (<2 %).

 

Method precision:

Five replicates (n=5) of sample solutions were analyzed in the same day to determine method precision. The low RSD % (<2 %) showed the suitability of the method for the determination of (Par) and (Dic.p) in pharmaceutical formulation, the method precision was summarized in Table 3.

 

Limit of detection (LOD) and Limit of quantification (LOQ):

The (LOD) and (LOQ) were obtained from the calibration curves. The (LOD) and (LOQ) were calculated based on the standard deviation of the intercept (SD=δ) and the slope (S) of the calibration curves using the formulae 3.3 δ/S and 10 δ/S respectively. The (LOD) and (LOQ) concentrations were reported in Table 2.

Table 2 present

 

Specificity:

It was determined (Par) and (Dic.p) in all samples without any interference, peaks were completely separated with good resolution and specificity, Fig. 8, 9. Confirming the specificity of the method, the peaks of (Par) and (Dic.p) in the samples were identified by comparing the retention time with that of the standards.

 

 

 

Table 2: Some of the validation parameters for (Par) and (Dic.p).

Component	Linearity (µg/mL)	Regression equation	Correlation coefficient	Percentage (%)	*Precision (RSD%)	LOQ (µg/mL)	LOD (µg/mL)
(Par)	2.5 - 500	y=0.0075x+0.0811	0.9996	99.37	0.87	1.47	0.48
(Dic.p)	1 - 250	y=0.0200x+0.0584	0.9998	100.10	1.12	0.80	0.26

*n=5

 

 

Fig.8: Chromatogram of “Agilomox” tablets showing separated peaks of: 1-(Par), 2-(PP.Na) as internal standard 25 mg/L, 3-(Dic.p).

 

 

Fig.9: Chromatogram of “Supergesic” tablets showing separated peaks of: 1-(Par), 2-(PP.Na) as internal standard 25 mg/L, 3-(Dic.p).

 

 

Fig.10: Chromatogram of “Paracetamol Ultra Medica” tablets showing separated peaks of: 1-(Par), 2-(PP.Na) as internal standard 25 mg/L.

 

 

Fig.11: Chromatogram of “Voltarile-K” tablets showing separated peaks of: 1-(PP.Na) as internal standard 25 mg/L, 2-(Dic.p).

 

Application of the proposed method for estimation in Syrian tablets formulation drugs:

The applicability of HPLC-UV method was investigated for pharmaceutical preparations Fig (8-11). In all the preparations, the amount of (Par) and (Dic.p) was obtained by direct measurement using the linear relationship curve. The total analysis time was less than 8 min with good resolution, good peak shapes and minimal tailing. Four pharmaceutical formulations containing (Par), (Dic.p) or (Par) and (Dic.p) were analyzed. Fig. 8, 9 illustrates typical chromatograms of (Par) and (Dic.p) in ''Agilomox'' and ''Supergesic'' tablets product respectively. Fig. 10 illustrates a typical chromatogram of (Par) in ''Paracetamol Ultra Medica'' tablets product. Fig. 11 illustrates a typical chromatogram of (Dic.p) in ''voltarile-K'' tablets product. The dosages of (Par) and (Dic.p) were conformed to USP legislation¹.

 

In general, the dosage of (Par) is higher than those of (Dic.p) in all analyzed products. the concentrations of the detected pharmaceutical compounds in analysed products were within the allowed limits according to USP legislation.

 

 

 

 

 

Table 3: Recoveries and amount of (Par) 500 mg and (Dic.p) 50 mg in Syrian pharmaceutical products on C₈ column.

Product	Company	Pharmaceutical compounds	(mg/dose)	Per.%	RSD%	Rec.(%)
Supergesic (tablets)	Kanawati	(Par)	499.23	99.85	0.89	100.71
		(Dic.p)	49.82	99.64	1.02	99.18
Agilomox (tablets)	Hama	(Par)	499.3	99.86	1.24	100.73
		(Dic.p)	50.02	100.04	0.97	99.97
paracetamol Ultra Medica (tablets)	Ultra Medica	(Par)	499.39	99.88	0.91	100.39
		(Dic.p)	-	-	-	-
Voltarile-K (tablets)	Alrazi	(Par)	-	-	-	-
		(Dic.p)	50.42	100.84	1.21	100.86

 

CONCLUSION:

Proposed HPLC methods are rapid, direct, specific, and precise for simultaneous determination of paracetamol and diclofenac potassium in pharmaceutical formulation.

The levels of pharmaceutical compounds were within the permissible limits set by the USB legislation¹.

 

The presence of active substances in actual quantities in pharmaceutical products was conformed to the paracetamol and diclofenac potassium formulations.

 

The described method is suitable for routine analysis and quality control of pharmaceutical preparation containing these two components, either alone or in combination.

 

ACKNOWLEDGEMENT:

The Ministry of High Education in Syria financially and technically supported this work through department of Chemistry, Faculty of Science, University of Aleppo, Syria.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1      United State Pharmacopeia 34. 2011.

2      Naveed S, Rehman H, Qamar F, Zainab S. Development of a spectrophotometric method for the assay of diclofenac potassium. Journal of Innovations in Pharmaceuticals and Biological Sciences. 2015; 2 (1): 7-11.

3      Derkar GK, Chimkode RM, Gaurav K, Yogesh K, Rohini J, Anita G, Ashwini K. Development and validation of UV-visible spectrophotometric methods for the estimation of paracetamol and diclofenac sodium in bulk and tablet dosage form. International Journal of Pharmaceutical Research & Analysis. 2015; 5 (1): 52-57.

4      Pandya EJ, Kapupara P, Shah KV. Development and validation of simultaneous estimation of diclofenac potassium, paracetamol and serratiopeptidase by first order derivative UV spectroscopy method in pharmaceutical formulation. Journal of Chemical and Pharmaceutical Research. 2014; 6 (5): 912-924.

5      Lakshmi S, Lakshmi KS, Tintu T. Simultaneous spectrophotometric estimation of paracetamol and lornoxicam in tablet dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 2010; 2 (4): 166-168.

6      Chintan RP, Ritu VK, Prachi VK, Harish AR, Nargund LVG. Spectrophotometric estimation of metaxalone and diclofenac potassium by multicomponent analytical method from tablet dosage form. Journal of Analytical & Bioanalytical Techniques. 2012; 3 (3).

7      Kalyankar T, Wadher SJ, Gholve R, Anitha K. UV Spectrophotometric estimation of diclofenac potassium and omeprazole magnesium in bulk and combined tablet dosage form. International Journal of MediPharm Research. 2016; 2 (1): 42-53.

8      Solangi A, Memon S, Mallah A, Memon N, Khuhawar MY, Bhanger MI. Determination of ceftriaxone, ceftizoxime, paracetamol, and diclofenac sodium by capillary zone electrophoresis in pharmaceutical formulations and in human blood serum. Turk. J. Chem. 2010; 34: 921-933.

9      Chhalotiya UK, Patel DB, Shah DA, Mehta FA, Bhatt KK. TLC Method for simultaneous quantification of chlorzoxazone, paracetamol, famotidine and diclofenac potassium in their combined dosage form. Austin Chromatogr. 2017; 4 (1).

10    Badgujar MA, Pingale SG, Mangaonkar KV. Simultaneous determination of paracetamol, chlorzoxazone and diclofenac sodium in tablet dosage form by high performance liquid chromatography. Journal of Chemistry. 2011; 8(3): 1206-1211.

11    Samlayawala ZA, Koradia SK. Development and validation of RP-HPLC method for simultaneous estimation of paracetamol, diclofenac potassium and famotidine in its combined pharmaceutical dosage form. International Journal of Pharmaceutical Research. 2014; 6 (4): 95-99.

12    Khatal LD, Rajage AD, More HN, Dhaneshwar SR, Polawar AR. Validated HPLC method for simultaneous quantitation of paracetamol, diclofenac potassium and famotidine in bulk drug and formulation. Int. J. Pharm Drug Anal. 2014; 2 (8): 633-640.

13    Gowramma B, Rajan S, Muralidharan S, Meyyanathan SN, Suresh B. A validated RP-HPLC method for simultaneous estimation of paracetamol and diclofenac potassium in pharmaceutical formulation. International Journal of Chem. Tech. Research. 2010; 2 (1): 676-680.

 

 

Accepted on 24.07.2018         © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(8): 3523-3528.