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					Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010                          ISSN 0974 – 9446




  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
                                  Department of Pharmaceutical Analysis,
                                      J.S.S. College of Pharmacy,
                                    Ootacamund, Tamilnadu-643 001
Arunadevi Birajdar                               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 5µm). The method was linear over the
concentration range of 1.0-12.0 µg/mL for paracetmol and 0.1-1.2 µg/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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Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010                        ISSN 0974 – 9446


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
paracetamol’s rapid onset of efficacy with tramadol’s 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
liquid–liquid 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, HPLC–MS/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 µL. The detection carried by using UV
(Waters 2487) duel wavelength absorbance detector. The column used was C18 (25 cm Χ 4.6 mm i.d., 5
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Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010                  ISSN 0974 – 9446
µm 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 µg/mL
and paracetamol 1.0 - 12.0 µg/mL and each concentration solution contains 10 µg/mL of domperidone
as an internal standard.

  A volume of 20 µL 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 µg/mL of tramadol and paracetamol 6.5 µg/mL and 10 µg/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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Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010                         ISSN 0974 – 9446


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 µg/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 1.0–12.0 µg/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 author’s 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 author’s 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 paracetamol–tramadol, Clin Rheumatol , 2006, 25, 16.
 4.    Zeˇcevi´c M, Stankovi´c Z, Zivanovi´c LJ, Joci´c 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
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     Harmonization, Geneva, March 1996.


Table 1. Validation parameters of determination of Tramadol, Paracetamol by HPLC method

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



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Publication Ref No.: IJPRD/2009/PUB/ARTI/VOV-1/ISSUE-10/DEC/010                       ISSN 0974 – 9446

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     3.34 min,
      2) Peak of tramadol at 5.47 min, 3) Peak of domperidone (IS) at 7.54 min.

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