Rao J R et al.

/ Pharmacie Globale (IJCP) 2011, 7 (09)

Available online at www.pharmacie-globale.info

ISSN 0976-8157

Research Article

PHARMACIE GLOBALE INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR SIMULTANEOUS QUANTITATION OF PARACETAMOL AND DEXKETOPROFEN TROMETAMOL IN BULK DRUG AND FORMULATION
Toufik S Mulla, Janhavi R Rao*, Savita S Yadav, Vishal V Bharekar and Milindkumar P Rajput
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune, Maharashtra, India. Received: 12 April 2011; Revised: 23 June 2011; Accepted: 30 June 2011; Available online: 5 July 2011

ABSTRACT

An accurate, simple, sensitive and precise reverse phase high performance liquid chromatographic (RP-HPLC) method for the determination of paracetamol and dexketoprofen trometamol was developed and validated in the bulk drug and in tablet dosage form. Thermo Hypersil ODSC18 (250 mm 4.6 mm, 5.0 ) used as stationary phase and methanol: ammonium acetate buffer (65: 35 v/v) as mobile phase at a flow rate of 1.0 ml/min and the detection wavelength was 256 nm. The retention time for paracetamol and dexketoprofen trometamol was found to be 3.20 and 5.94 min, respectively. Proposed method was validated for precision, accuracy, linearity range and robustness. Keywords: Paracetamol, Dexketoprofen trometamol, HPLC, Validation.

INTRODUCTION

Paracetamol, N- (4-hydroxyphenyl) acetamide (Figure 1) has analgesic (pain reliever) and antipyretic (fever reducer). It is commonly used for the relief of headaches, other minor aches and pains, and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post surgical pain and providing palliative care in advanced cancer patients. The main mechanism of action of paracetamol is considered to be the inhibition of cyclooxygenase (COX), and recent findings suggest that it is highly selective for COX-2.1 Figure 1. Structure of Paracetamol

reported for paracetamol and dexketoprofen trometamol, stability LCDAD method for analysis of dexketoprofen trometamol, tramadol, and haloperidol3, RP-HPLC method for determination of paracetamol in combination with other drugs.4-8 As no method is reported for paracetamol and dexketoprofen trometamol in combination, the aim of the present study was to develop accurate, precise and selective reverse phase HPLC assay procedure for the analysis of paracetamol and dexketoprofen trometamol in bulk drug samples and in combined dosage formulation.

MATERIALS AND METHODS

Materials Emcure Pharmaceuticals Ltd. Pune, India, kindly supplied pure drug sample of paracetamol as a gift sample of Batch No: 01PAR9491 and dexketoprofen trometamol of Batch No: 509KETF005. It was used without further purification and certified to contain 99.92% (w/w) for paracetamol and 99.85% (w/w) for dexketoprofen trometamol on dried basis. All chemicals and reagents used were of HPLC grade and were purchased from Merck Chemicals, India. Instrumentation The HPLC system consisted of a Pump (model Jasco PU 2080), Intelligent LC pump with sampler programmed at 20 L capacity per injection was used. The detector consisted of UV/ VIS (Jasco UV 2075) model operated at a wavelength of 256 nm. Data was integrated using Jasco Borwin version 1.5, LC-Net II/ADC system. The column used was Thermo Hypersil ODSC18 (250 mm 4.6 mm, 5.0 ) from Germany. Preparation of standard stock solutions Standard stock solution of concentration 500 g/mL of paracetamol and 25 g/mL of for dexketoprofen trometamol was prepared using methanol. From the Pharmacie Globale (IJCP), Vol. 02, Issue 07

Dexketoprofen trometamol, (2S)-2-[3-(benzoyl) phenyl] propanoic acid (Figure 2). Dexketoprofen trometamol is a new, quick acting analgesic for the treatment of painful musculoskeletal conditions such as osteo-arthritis and low back pain. It is also used as a treatment for post-operative pain, toothache and dysmenorrhoea. It is the active optical isomer of ketoprofen.2 Figure 2. Structure of Dexketoprofen trometamol
O CH3 OH O

Literature review reveals that methods have been

*Corresponding Author: Dr Janhavi R Rao, Professor, Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune-411038, Maharashtra, India. Contact no: +91-9822532662, Email: raojanhavi@rediffmail.com

Rao J R et al. / Pharmacie Globale (IJCP) 2011, 7 (09)

standard stock solution, the mixed standard solutions were prepared using methanol to contain 50 g/mL of paracetamol and 2.5 g/mL of dexketoprofen trometamol. Optimization of HPLC Method: The HPLC procedure was optimized with a view to develop a simultaneous assay method for paracetamol and dexketoprofen trometamol respectively. The mixed standard stock solution (50g/mL of paracetamol and 2.5 g/mL of dexketoprofen trometamol) was injected in HPLC system. For method optimization different ratios of methanol and ammonium acetate buffer were tried but it was found that methanol: ammonium acetate buffer in the ratio 65: 35 v/v, at flow rate 1 mL/min gives acceptable retention time (tR), plates and good resolution for paracetamol and dexketoprofen trometamol (Figure 3). Figure 3. HPLC chromatogram of standard paracetamol and dexketoprofen trometamol (50g/mL and 2.5 g/mL)

The parameters included variation of flow rate, percentage of methanol in the mobile phase and solvents from different lots were taken. Robustness of the method was done at three different concentration levels 3, 5, 7 g/mL and 5, 7, 9 g/mL for paracetamol and dexketoprofen trometamol respectively. Specificity: The specificity of the method towards the drug was established through study of resolution factor of the drug peak from the nearest resolving peak. The peak purity of paracetamol and dexketoprofen trometamol was determined by comparing the spectrum at three different regions of the spot i.e. peak start (S), peak apex (M) and peak end (E). Effect of excipients of formulation was studied for whether it interfered with the assay. Accuracy: Accuracy of the method was carried out by applying the method to drug sample (Paracetamol and Dexketoprofen trometamol combination tablet) to which know amount of paracetamol and dexketoprofen trometamol standard powder corresponding to 80, 100 and 120 % of label claim had been added (Standard addition method), mixed and the powder was extracted and analyzed by running chromatogram in optimized mobile phase. Analysis of a marketed formulation To determine the content of paracetamol and dexketoprofen trometamol in conventional tablet (Brand name: Infen-P, Label claim: 500 mg paracetamol and 25 mg dexketoprofen trometamol per tablet), twenty tablets were weighed, their mean weight determined and finely powdered. The weight of the tablet triturate equivalent to 500 mg of paracetamol and 25 mg of dexketoprofen trometamol was transferred into a 50 mL volumetric flask containing 30 mL methanol, sonicated for 30 min and diluted up to 50 mL with methanol. The resulting solution was centrifuged at 3000 rpm for 5 min and the drug content of the supernatant was determined (500 and 25 g/mL for paracetamol and dexketoprofen trometamol respectively). Supernatant was taken and after suitable dilution the sample solution was then filtered using 0.45micron filter (Millipore, Milford, MA). The above stock solution was further diluted to get sample solution of 100 and 5 g/mL for paracetamol and dexketoprofen trometamol respectively. A 20-L volume of sample solution was injected into HPLC system, under the conditions described above.

Validation of the method Validation of the optimized HPLC method was carried out according to the ICH guidelines with respect to the following parameters.9-11 Linearity: The mixed standard stock solution (3 g/mL of paracetamol and 5 g/mL of dexketoprofen trometamol) was further diluted to get paracetamol and dexketoprofen trometamol concentration in the range of 3-8 g/mL and 5-10 g/mL respectively. Linearity of the method was studied by injecting six concentrations of the drug prepared in the mobile phase in triplicate into the LC system keeping the injection volume constant. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs. Precision: The precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies were performed by analysis of three different concentrations 3, 5, 7 g/mL for paracetamol and 5, 7, 9 g/mL for dexketoprofen trometamol six times on the same day. Repeating studies on three different days checked the intermediate precision of the method. Limit of detection and limit of quantitation: Limits of detection (LOD) and quantification (LOQ) represent the concentration of the analyte that would yield signal to noise ratios of 3 for LOD and 10 for LOQ, respectively. To determine the LOD and LOQ, serial dilutions of mixed standard solution of paracetamol and dexketoprofen trometamol was made from the standard stock solution. The samples were injected in LC system and measured signal from the samples was compared with those of blank samples. Robustness of the method: To evaluate robustness of a HPLC method, few parameters were deliberately varied.

RESULTS AND DISCUSSION

The proposed method for simultaneous estimation of for paracetamol and dexketoprofen trometamol dosage forms was found to be simple, accurate, economical and rapid. The method was validated as per the ICH guidelines using methanol: ammonium acetate buffer (65: 35 v/v) as mobile phase. Linearity: Linearity was studied by preparing standard solutions at different concentration levels. The Linearity range for paracetamol and dexketoprofen trometamol were found to be 3-8 g/ml and 5-10 g/ml, respectively. The regression equation for paracetamol and dexketoprofen trometamol were found to bey = 16350x 29867 y = 20547x 84606 with coefficient of correlation (r) 0.999 and 0.9952, respectively. Precision: The results of the repeatability and intermediate precision experiments are shown in Table 1. Pharmacie Globale (IJCP), Vol. 02, Issue 07

Rao J R et al. / Pharmacie Globale (IJCP) 2011, 7 (09)

The developed method was found to be precise as the RSD values for repeatability and intermediate precision studies were < 2%, as recommended by ICH guidelines. Table 1. Precision studies
Concentration (g/mL) Measured concentration, RSD (%) Repeatability Intermediate precision (n= 6) (n= 6) 2.885, 1.528 5.189, 1.170 6.018, 1.321 4.244, 0.919 6.325, 0.763 8.404, 0.664

LOD and LOQ: Signal-to-noise ratios of 3:1 and 10:1 were obtained for the LOD and LOQ respectively. The LOD and LOQ were found to be 0.05 g/mL and 0.1 g/mL for paracetamol and 2 g/mL and 4 g/mL for dexketoprofen trometamol. Robustness of method: To evaluate the robustness of the developed RP-HPLC method, small deliberate variations in the optimized method parameters were done. The effect of change in flow rate, mobile phase ratio and solvents of different lots were studied. The method was found to be unaffected by small changes like 1 change in flow rate, 1 change in mobile phase and 1 change in solvent lot (Table 2).
Retention time Retention factor Asymmetry

Paracetamol 3 2.929, 1.926 5 5.586, 0.908 7 6.258, 0.951 Dexketoprofen trometamol 5 4.243, 0.419 7 6.325, 1.045 9 8.406, 0.899

Table 2. Robustness testinga (n = 3)

Factora Level Paracetamol A: Flow rate (mL/min) 0.9 -1 1.0 0 1.1 +1 Mean SD (n = 3) B: % of methanol in the mobile phase (v/v) 64 -1 65 0 66 +1 Mean SD (n = 3) C: Solvents of different lots First lot Second lot Mean SD (n = 3) Dexketoprofen trometamol A: Flow rate (mL/min) 0.9 -1 1.0 0 1.1 +1 Mean SD (n = 3) B: % of methanol in the mobile phase (v/v) 64 -1 65 0 66 +1 Mean SD (n = 3) C: Solvents of different lots First lot Second lot Mean SD (n = 3) a Three factors were slightly changed at three levels (-1, 0, 1)

3.30 3.22 3.11 3.21 0.07 3.28 3.20 3.12 3.20 0.05 3.20 3.11 3.15 0.01

1.40 1.36 1.28 1.34 0.03 0.32 0.28 0.24 0.28 0.02 0.28 0.24 0.26 0.01

1.12 1.11 1.13 1.12 0.04 1.10 1.12 1.13 1.11 0.03 1.15 1.17 1.16 0.01

6.00 5.90 5.71 5.87 0.05 6.00 5.90 5.82 5.90 0.06 5.90 5.82 5.86 0.01

1.40 1.36 1.28 1.34 0.01 1.42 1.36 1.32 1.36 0.02 1.36 1.32 1.34 0.01

1.15 1.11 1.07 1.11 0.04 1.16 1.11 1.06 1.11 0.05 1.11 1.10 1.10 0.01

Specificity: The specificity of the method was evaluated to ensure separation of paracetamol and dexketoprofen trometamol and was demonstrated by assaying samples of paracetamol and dexketoprofen trometamol. The method demonstrated good resolution between the peaks and was found to be free of interference. For demonstrating the specificity of the method for drug formulation, the drug was spiked, wherein the excipients used in different Table 3. Recovery studies (n = 6)
Label claim (mg/tablet) Paracetamol 500 500 500 Dexketoprofen trometamol 25 25 25 Amount added (mg) 400 (80%) 500 (100%) 600 (120%) 20 (80%) 25 (100%) 30 (120%)

formulation products did not interfere with the drug peak and thus the method was specific for paracetamol and dexketoprofen trometamol. RecoveryStudies: As shown from the data in Table 3, good recoveries of the paracetamol and dexketoprofen trometamol in at various added the range from 98.82100.35 % were obtained concentrations.
Total amount (mg) 900 1000 1100 45 50 55 Amount Recovered (mg) 899.54 1000.25 1100.62 45.16 49.91 55.12 % Recovery 99.94 100.02 100.05 100.35 99.82 100.21

Analysis of a formulation Experimental results of the amount of paracetamol and dexketoprofen trometamol in tablets, expressed as a percentage of label claims were in good agreement with

the label claims thereby suggesting that there is no interference from any of the excipients, which are normally present. The drug content was found to be 100.02 % for paracetamol and 100.00 % for Pharmacie Globale (IJCP), Vol. 02, Issue 07

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dexketoprofen trometamol. Two different lots of paracetamol and dexketoprofen trometamol combination Table 4. Analysis of commercial formulation
Paracetamol (500 mg) 1st Lot 2nd Lot Dexketoprofen trometamol (25 mg) 1st Lot 2nd Lot

tablets were analyzed using the proposed procedures as shown in Table 4.

Paracetamol found (mg per tablet) Recovery (%) 100.02 100.03 Recovery (%) 99.80 100.20

Mean SD (n= 6) 500.12 1.16 500.15 1.04 Mean SD (n= 6) 24.95 1.06 25.08 0.94

Dexketoprofen trometamol found (mg per tablet)

CONCLUSION

The proposed method is rapid, simple and sensitive and hence can be used in routine for simultaneous determination of paracetamol and dexketoprofen trometamol in bulk as well as in pharmaceutical preparations. Statistical analysis of the results has been carried out revealing high accuracy and good precision.

ACKNOWLEDGMENT

The authors would like to thank, Emcure Pharmaceuticals Ltd. Pune, India for providing a gift sample of standard paracetamol and dexketoprofen trometamol. The authors also would like to thank, Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Pune, India for providing necessary facilities to carry out the work. RP-HPLC Method. International Journal of PharmTech Research. 2010; 2(2):1330-1333. 7. Battu P R, Reddy M S; RP-HPLC Method for Simultaneous Estimation of Paracetamol and Ibuprofen in Tablets. Asian Journal of Research Chem. 2009; 2(1):70-72. 8. Borkar D D, Godse V P, Bafana Y S et al. Simultaneous Estimation of Paracetamol and Promethazine Hydrochloride in Pharmaceutical Formulations by a RP-HPLC Method. International Journal of ChemTech Research. 2009; 1(3):667-670. 9. Q2 (R1) Validation of Analytical Procedure, Test and Methodology. International Conference on Harmonization ICH. Geneva, 2005. 10. Q2A Validation of Analytical Procedures: Consensus Guidelines. ICH Harmonized Tripartite Guidelines ICH. 1994. 11. Q2B Validation of Analytical Procedures: Methodology, Consensus Guidelines. ICH Harmonized Tripartite Guidelines. 1996.

REFERENCES

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