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: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010

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METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF PARACETAMOL AND TRAMADOL IN SOLD DOSAGE FORM BY RP-HPLC Arunadevi S. Birajdar* 1,S. N. Meyyanathan1, B. Suresh1.
1

Arunadevi Birajdar

Department of Pharmaceutical Analysis, J.S.S. College of Pharmacy, Ootacamund, Tamilnadu-643 001 INDIA E-mail: aruna_birajdar@rediffmail.com

Abstract A high-performance liquid chromatographic method has been developed for the simultaneous analysis of paracetamol and tramadol in combined solid dosage form. The mobile phase consisting of acetonitrile- 0.26 % triethylamine buffer (pH 7.3) in ratio of (45:55 % v/v) was delivered at the flow rate of 1.0 mL/min and UV detection was carried out at 264 nm. The separation was achieved using C18 reverse-phase column (250 X 4.6 mm I.D., particle size 5m). The method was linear over the concentration range of 1.0-12.0 g/mL for paracetmol and 0.1-1.2 g/mL for tramadol. Domperidone was used as an internal standard (IS). The analytical recovery obtained was 99.88%. The validation of method carried out as per ICH guidelines. The described HPLC method was successfully employed for the analysis of pharmaceutical formulations containing combined dosage form and can be employed for bioequivalence study in future for the same formulations. Keywords:- RP-HPLC, Paracetamol, Tramadol, Validation.

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INTRODUCTION AND MATERIAL AND METHODS Introduction Paracetamol, N-acetyl-p-aminophenol is a commonly used analgesic and antipyretic drug, present in different pharmaceutical formulations. Tramadol hydrochloride, (() trans-2-[(dimethylamino)methyl]-1(3-methoxyphenyl)-cyclohexanol) is a synthetic, centrally acting, analgesic agent, used for the relief of moderate to chronic pain and has no clinically relevant cardiovascular or respiratory depressant activity1. This combination has demonstrated genuine synergy in animal studies and also combines with paracetamols rapid onset of efficacy with tramadols prolonged analgesic effect. Numerous studies have confirmed the efficacy and tolerability of paracetamol plus tramadol in both acute and chronic pain. As a single-dose treatment for acute post-operative pain, this combination delivers rapid and sustained pain relief that is greater than either agent alone 2. A literature review reveals that only a few methods have been developed for the quantification of individual drug tramadol as by HPLC3, determination of tramadol in human plasma and urine by HPLC4, HPLC method for tramadol in human plasma using liquidliquid extraction5, HPLC and Enantioselective HPLC method for tramadol and o-desmethyl tramadol determination in human plasma and urine6-7. For paracetamol several number of methods are reported individual and in combination with other drugs as spectrophotometric determination of paracetamol in tablets and oral solutions8, fluorimetric assay for 4-aminophenol in paracetamol formulations9, HPLCMS/MS method for the selective determination of paracetamol metabolites in mouse urine10, HPLC assay for the determination of paracetamol, pseudoephedrine hydrochloride and triprolidine hydrochloride11, HPLC assay for phenylpropanolamine hydrochloride, caffeine, paracetamol, glycerylguaiacolate and chlorpheniramine maleate tablet 12. There were no simple and reproducible methods so far reported for simultaneous determination of tramadol and paracetamol by RP- HPLC in solid dosage form. It is essential to develop simple, precise, accurate HPLC method for simultaneous determination of both drugs in solid dosage form. Therefore, in this study we developed reproducible method which can be used in laboratory. HPLC method can be applied in future for estimation same drugs from biological samples. The validation of this method carried out as per ICH guidelines 13-14. The chemical structures of tramadol, paracetamol and domperidone are as given in (Fig. 1). MATERIAL AND METHODS Experimental Reagents and Materials Pharmaceutical grade Tramadol, Paracetamol were kindly supplied as a gift sample by Shreechem Pharmaceuticals Pvt Ltd. New Mumbai, India. Domperidone (IS) procured from Apex drugs and intermediates Medka (Andrapradesh, India) All chemicals and solvents of HPLC grade and were purchased from Qualigens fine Chemicals, Mumbai, India. Water HPLC grade was obtained from a Milli-QRO water purification system. Instrumentation of HPLC LC system used consisted of pump model (Waters 1515 isocratic solvent delivery system) with universal loop injector (Rheodyne 7725 i) of injection capacity 20 L. The detection carried by using UV (Waters 2487) duel wavelength absorbance detector. The column used was C18 (25 cm 4.6 mm i.d., 5 International Journal of Pharma Research and Development Online 2 2

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Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010 ISSN 0974 9446 m particle size) Phenomenex, USA, at ambient temperature. Different mobile phases were tested in order to find the best conditions, for separating both the drugs simultaneously.

The optimal composition of the mobile phase was consisted of acetonitrile and 0.26 % v/v triethyleamine buffer (pH adjusted to 7.3 with 1% v/v ortho-phosphoric acid) in the ratio of 45:55 % v/v. The flow rate was 1 mL/min and UV detection was carried out at 264 nm due to better absorbance of each component spectrum observed. Domperidone was used as an internal standard. Preparation of working solutions Stock solution was prepared by dissolving 10 mg of tramadol and paracetamol in 100 mL volumetric flask separately with mixture of methanol and water (1:1).

Stock solution of domperidone (internal standard) was prepared by dissolving 10 mg of standard in separate 100 ml volumetric flask with same mixture of methanol: water (1:1). All solutions were stored at + 200C, these solutions were shown to be stable during the period of study. From the above stock solutions, dilutions were made to working standard the concentration range of tramadol 0.1 - 1.2 g/mL and paracetamol 1.0 - 12.0 g/mL and each concentration solution contains 10 g/mL of domperidone as an internal standard. A volume of 20 L of each working standard was injected into column. All measurements were repeated three times for each concentration and calibration curve was constructed by plotting the peak area ratios of analyte to internal standard versus the corresponding drug concentration.

Analysis of tablets To determine the content of tramadol and paracetamol simultaneously in tablets (label claim: 37.5 mg tramadol and paracetamol 325 mg); twenty tablets were weighed; their average weight determined and were finely powdered. The correct amount of powder equivalent to one tablet was dissolved in mixture of water and methanol (1:1) by stirring and sonicated for 30 min. The excipients were separated by filtration. After filtration, an appropriate amount of internal standard was added and diluted up to mark with methanol. Further dilutions are made with mobile phase to get working standard solution containing 0.75 g/mL of tramadol and paracetamol 6.5 g/mL and 10 g/mL of domperidone (internal standard). This sample solution injected thrice and recorded chromatogram. The amount of tramadol and paracetamol were determined. The results are reported in Table 1. To check the accuracy of the developed methods and to study the interference of formulation additives, analytical recovery experiments were carried out by standard addition method at 80, 100 and 120 % level. From the total amount of drug found, the percentage recovery was calculated. The results are reported in Table 2.

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Results and Discussion For HPLC method, chromatographic conditions were optimized to obtain, an adequate separation of eluted compounds. Initially, various mobile phase compositions were tried for better separation of drugs with internal standard. Mobile phase and flow rate selection was based on peak parameters (height, tailing, theoretical plates, capacity factor, run time etc). The mobile phase was consisted of acetonitrile and 0.26 % v/v triethyleamine buffer pH 7.3, with ratio of (45:55 % v/v) at 1 mL/min flow rate was quite satisfactory. Domperidone was used as an internal standard, neutralizing the error inherent in sample injection, eliminating random errors. The optimum wavelength fixed for detection was 264 nm at which better detector response for drugs were obtained.

System suitability tests are an integral part of chromatographic method. They are used to verify the reproducibility of the chromatographic system. The calibration was linear for tramadol at concentration range of 0.1 1.20 g/mL, with regression 0.998, intercept + 0.0087 and slope 0.0358 shown in (Fig. 1). The calibration was linear for paracetamol at concentration range of 1.012.0 g/mL, with regression 0.998, intercept +0.102 and slope 0.0340 respectively shown in (Fig.1). A typical chromatogram for tramadol, paracetamol and domperidone (internal standard) for standard solution was shown in (Fig.1). A typical chromatogram for tramadol, paracetamol and domperidone (internal standard) for sample solution was shown as in (Fig.5). The average retention time for tramadol, paracetamol and domperidone (IS) was found to be 3.204 0.03, 5.270 0.05 and 7.526 0.02 min, respectively. Sample to sample precision and accuracy were evaluated using, three samples of three different concentrations, which were prepared and analyzed on same day. Day to day variability was assessed using three concentrations analyzed on three different days, over a period of one week. These results show the accuracy and reproducibility of the assay. Thus, it was concluded that there was no significant difference on the assay, which was tested on an intra day and inter day basis. The % R.S.D. values was found to be less than 3% shows that proposed method provides acceptable intra day and inter day variation of tramadol and paracetamol with precision and accuracy reported in Table 1. The mean recoveries were found in the range of 98.20 99.08 %.

Conclusion The new HPLC method developed and validated for simultaneous determination of tramadol and paracetamol in combined pharmaceutical dosage form and assured the satisfactory precision and accuracy and also determining lower concentration of each drug in its solid dosage form. The method was found to be simple, accurate, economical, rapid and they can be applied for routine analysis in laboratories and is suitable for the quality control of the raw materials, formulations, dissolution studies and can be employed for bioequivalence studies for the same formulation.

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Acknowledgement The authors thank to Shreechem Pharmaceuticals Pvt ltd. New Mumbai, India. for providing gift samples of Tramdol and paracetamol also to Apex drugs and intermediates Medka ( A.P.) for providing a gift sample of Domperidone. The authors are thankful to Mr. Supe (Drug Inspector). The authors are grateful to His Holiness Jagadguru Sri Sri Shivarathree Deshikendra Mahaswamigalavaru of Sri Suttur Mutt, Mysore and AICTE (QIP) cell for providing facilities to carry out this work.

References 1. The Martindale 35th ed. The complete drug reference, 2006, published pharmaceutical press, lambeta high street, londan SEI 75M, UK. 2. Perrot S, Krause D, Crozes P, Nai'm C, Treatment Study Clinical Theraputics, 2006, 28, 10. 3. A. Stephan and Schug, Combination analgesia in 2005 a rational approach: focus on paracetamoltramadol, Clin Rheumatol , 2006, 25, 16. 4. Zecevic M, Stankovic Z, Zivanovic LJ, Jocic B, J. Chromatogr. A, 2006, 1119, 251. 5. Ebrahimzadeh H, Yamini Y, Sedighi A, Rouini M R, J. Chromatogr. B., 2008, 863, 229. 6. Gana S H, Ismaila R, Wan Adnanb WA, Wanc Z, J. Chromatogr. B.,2002, 772,123. 7. Pedersen R S, Brosen K, Nielsen E, Application to Clinical Studies, 2003, 5, 279. 8. M. Knochen, J. Giglio and F. Boaventura, J. Pharma. Biomed. Anal., 2003, 33, 191. 9. Dejaegher B, Bloomfield M S, Smeyers-Verbeke J Y, Heyden V Talanta, 2008, 75 , 258. 10. Hewavitharana A K, Lee S, Dawson PA, Markovich D, Shaw P N, Anal. Biochem., 2008, 374,106. 11. Jamil akhtar M, khan S, hafiz M, J. Pham. Biomed. Anal., 1994, 12, 379. 12. Indrayanto G, Sunarto A, Adriani Y, J. Pharm. Biomed. Anal., 1995, 13, 1555. 13. ICH, Q2A validation of analytical procedure, Methodology International Conference on Harmonization, Geneva, October 1994 14. ICH, Q2B Validation of analytical procedure, Methodology International Conference on Harmonization, Geneva, March 1996.

Table 1. Validation parameters of determination of Tramadol, Paracetamol by HPLC method Validation parameters Tramadol Linearity and range (g/mL) Correlation coefficient Standard deviation LOD (g/mL) LOQ (g/mL) Accuracy (%) Precision RSD (%) Inter-day Intra-day 0.1 -1.2 0.998 0.0124 0.015 0.05 99.98 1.40 1.56 RP-HPLC Paracetamol 1-12 0.998 0.118 0.12 0.35 100.28 1.65 2.54

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Table 2. Results of analysis of formulation and recovery studies ______________________________________________________________________ Drugs Amount mg/ tablet %Recovery a RSDb

Labeled Found _______________________________________________________________________ Tramadol 37.50 37.44 99.84 0.164 Paracetamol 325 326 100.20 0.090 ________________________________________________________________________

a. Average of 6 determination. b. Relative standard deviation.

Figure captions Fig. 1. Typical chromatogram of sample solution with IS: 1) Peak of paracetamol at 3.34 min 2) Peak of tramadol at 5.47 min 3) Peak of domperidone (IS) at 7.54 min.

Fig. 1 Typical chromatogram of sample solution with IS: 1) Peak of paracetamol at 2) Peak of tramadol at 5.47 min, 3) Peak of domperidone (IS) at 7.54 min.
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3.34 min,

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