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Simultaneous estimation of paracetamol and tramadol in tablet formulation by reverse phase HPLC method.

Introduction

Tramadol (TAM) is chemically [+ or -])cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanol hydrochloride used as CNS depressant and analgesic, for treating moderate to severe pain. Paracetamol (PAR), chemically 4-hydroxy acetanilide, is a centrally and peripherally acting non-opioid analgesic and antipyretic. A combination of these drugs, TAM (37.5 mg), and PAR (325 mg) is available as tablets for clinical practice. Paracetamol plus tramadol was an effective analgesic in dental and postsurgical pain, based on limited information. Many methods [1], [2], [3], [4], [5],[6],[7] have been described in the literature for the determination of TAM, PAR, individually. However, there is no HPLC method reported for the simultaneous determination of these drugs either as active pharmaceutical ingredient or from dosage forms. The present work describes a simple, precise and accurate reverse phase HPLC method for simultaneous estimation of PAR and TAM in combined dosage forms.

Materials and Methods

The drug samples, PAR, TAM and valdecoxib were obtained as gift samples from the Alpha Remedies Limited, Hingna, Nagpur, Technica Labs & Pharma Private Limited, Dehradun, Uttarakhand and Emcure Pharmaceuticals Pvt. Ltd., Pune. Pottasium dihydrogen phosphate AR, Methanol of HPLC grade, and methanol HPLC grade were supplied by S. D. Fine Chemicals, Mumbai. Water HPLC grade was obtained from a milli-Q RO water purification system.

A Waters model 515 HPLC double reciprocating pump, 2487 Dual UV detector, and Kromasil C18 column (5 [micro]m particle size) was used. The RP-HPLC system was equipped with Empower- 2 software for data processing. Separations were carried out on a Kromasil C18 column (250x4.6 mm) packed with 5 [mu] particle size as the stationary phase. The mobile phase consisting of methanol and 0.1M potassium dihydrogen phosphate buffer (60:40 %v/v), was pumped at a flow rate 1 ml per min, the detection was monitored at 273 nm and the run time was 10 min. The mobile phase was filtered through a 0.45 [mu] membrane filter and degassed before analysis. A rheodyne 7725 injector with a 20 [micro]l loop was used for the injection of samples.

Standard stock solution of PAR, TAM, and tramadol (100 [micro]g/ml) were prepared separately by using methanol as a solvent. From the standard stock solutions, mixed standard solution was prepared containing 32.5 [micro]g/ml of PAR, 3.75 [micro]g/ml of TAM and 2 [micro]g/ml of valdecoxib as internal standard.

Twenty tablets INSTREL (manufactured by Dr. Reddys Labs, Hyderabad) each containing 37.5 mg Tramadol and 325 mg Paracetamol were weighed and finely powdered. A quantity of powder equivalent to 7.5 mg Tramadol and 65 mg Paracetamol was accurately weighed and transferred to a 100 ml volumetric flask, dissolved in methanol of about 25 ml and the solution was filtered through Whatman filter paper no. 1(0.45 [mu]) and the volume was made up to the mark with the same solvent. To this 1 ml of Valdecoxib (2 [micro]g/ml) was added and then the mixture was sonicated for 20 min and then filtered. From the filtrate, 5.0 ml was further diluted to 50 ml mark with distilled water . Aliquots of this tablet solution were diluted to get the concentrations ~ 3.75 [micro]g/ml of Tramadol and ~ 32.5 [micro]g/ml of Paracetamol. A 20 [micro]l of sample solution was injected into sample injector for three times under chromatographic condition as described above. Area of each peak was measured at 273 nm. The ratio of peak area of drug to that of internal standard was calculated . Concentrations of Tramadol and Paracetamol in the formulation were calculated by comparing AUC of the sample with that of the standard. The mixed standard solution was subjected to proposed HPLC method of analysis for finding out intra and interday variations. The recovery studies were carried out by adding known amount of standard drug to the pre-analyzed samples and subjecting them to the proposed HPLC method of analysis.

The present study was carried out to develop a simple and rapid HPLC method for the simultaneous estimation of PAR and TAM, using most widely used Kromasil [C.sub.18] column. The retention time of TAM, PAR and valdecoxib was found to be, 5.65, 3.15 and 8.22 min, respectively. The assay concentration of 32.5 [micro]g/ml of PAR and 3.75 [micro]g/ml TAM was selected according to the labelled claim. The peaks were well resolved and the resolution between PAR and valdecoxib was found to be 9.21 whereas between valdecoxib and TAM was 4.75. The peak of internal standard was 8.22 min and it was well resolved from the other analytes. The asymmetry factors of all the peaks were lesser than 2.0 and it showed that all peaks were symmetrical in shape. The precision of the proposed method was lesser than 2% for all the three drugs including internal standard when it was injected 6 times and there was good repeatability of the proposed method. The % CV of PAR, TAM was found to be 0.49%, 0.89%, respectively and it showed that the method was highly precise. The regression equation was found to be linear in the 80 to 120% of assay concentration. Accuracy of method was calculated by % mean recovery studies (n=3). The recovery studies were carried out by the addition of standard analyte to the preanalysed sample.

Results and Discussion

The concentrations of standard spiked to the sample were 26-39 [micro]g/ml of PAR and 34.5 [micro]g/ml of DMP. The recovery studies are showed in the [Table - 1]. The mean % recovery was found to be 99.81% for PAR and 98.98% for TAM. Assay of the combination in tablet dosage form was found to be TAM 99.73% and 99.14% of PAR. The estimated amount was within the acceptable limits of the labelled claim of the formulation. The total run time of the proposed method was 20 min and no peaks were found after 10 min. The finally estimated drug amount of TAM and PAR in tablet formulation were showed in the [Table - 2]. The proposed HPLC method was simple and precise because of the commonly used buffer, easier extraction procedures and shorter runtime. The proposed method is highly accurate which showed good recovery of the drug samples and there was no interference from the excipients at the retention time of all three drugs which showed that it was specific and the analysis was less time consuming. The proposed method can be used in routine quality control of combined dosage form containing PAR and TAM in tablets.

References

[1] Srinivasa KK et al, Simultaneous derivative spectrophotometric estimation of aceclofenac and tramadol in combination solid dosage forms, Indian J Pharm Sci, 2007;69(4) :540-545

[2] Garg G. et al, Simultaneous spectrophotometric estimation of Tramadol and paracetamol in tablets, Indian J Pharm Sci, 2007; 69(5):692-694

[3] RP-HPLC method for Simultaneous estimation of aceclofenac and paracetamol in tablets, Indian J Pharm Sci, 2007; 69(1):137-140

[4] Dayer P, Desmeules J, Collart L (1997). "[Pharmacology of tramadol]". Drugs 53 Suppl 2: 18-24.

[5] Zarapkar, SS. and Dhanvate AA., Indian Drugs, 32 (Aug) : p 405-408. 1995.

[6] Wiliam O,Foye, edt. Principles of medicinal chemistry, 3rd Edn., Varghese Bombay, 1989.

[7] Kartik A et al, RP-HPLC Simultenous estimation of pracetamol and domperidone in tablet formulation, Indian J Pharm Sci,2007;69(1):142-144.

Kapil Kalra *, Sharmistha Naik, Garima Jarmal and Neeti Mishra

Kanak Manjari Institute of Pharmaceutical Sciences, Rourkela, Orissa

* Correspondence Author E-mail: kapil752003@yahoo.com
Table 1: Recovery Studies.

                      Concentration
                        of added
      Concentration      drug in
       present in      preanalysed
         tablet         solution                       Statistical
      ([micro]g/ml)   ([micro]g/ml)  % Recovery        Analysis
S.
No   TAM      PAR    TAM      PAR    TAM      PAR      PAR

1    3.75     32.5   3        26     99.96    100.33   Mean = 99.81%
2    3.75     32.5   3        26     100.11   99.33    S.D = 0.44
3    3.75     32.5   3        26     100.04   100.33   % R.S.D = 0.441

4    3.75     32.5   3.75     32.5   99.97    100.03   TAM
5    3.75     32.5   3.75     32.5   99.94    100
6    3.75     32.5   3.75     32.5   99.94    100.03   Mean = 99.98%
7    3.75     32.5   4.5      39     99.97    99.56    S.D = 0.0561
8    3.75     32.5   4.5      39     99.95    99.11    % R.S.D = 0.057
9    3.75     32.5   4.5      39     99.97    99.56

Table 2: Estimation of TAM and PAR in Pharmaceutical Combination
formulation.

                Labelled amount       Observed        % Amount
Formulation          (mg)           amount * (mg)      found

Paracetamol           325              324.80          99.02
Tramadol             37.5               37.4           99.73
(INSTREL)
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Article Details
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Title Annotation:high performance liquid chromatography
Author:Kalra, Kapil; Naik, Sharmistha; Jarmal, Garima; Mishra, Neeti
Publication:International Journal of Applied Chemistry
Article Type:Report
Geographic Code:1USA
Date:May 1, 2009
Words:1464
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