An Ion-Pair Chromatography Method for Simultaneous Estimation of Tramadol and Paracetamol in Combined Tablet Dosage Form

 

Jane Fatima Mary Titus*, A Thenmozhi, and D Sridharan

Department of Pharmaceutical Analysis, Arulmigu Kalasalingam College of Pharmacy, Anand Nagar, Krishankoil – 626 190. India.

*Corresponding Author E-mail: janepapers@rediffmail.com

 

ABSTRACT:

A simple, fast, and new ion pair chromatographic method was developed and validated, for the simultaneous estimation of Tramadol hydrochloride and paracetamol in pharmaceutical formulations. The developed method uses acetonitrile: 5% sodium lauryl sulphate: methanol in the ratio of 45:15:40 v/v with flow rate of 1mL /minute. The optimum separation was achieved in less than 5 minutes using WATERS Symmetry C18 column (250mm X 4.6mm, 5”m, i.d.) detection was carried out using UV detector, measuring the response at 217nm. Beer’s law was obeyed in the concentration range of 20-187.5”g/mL for tramadol and 20-1625 ”g/mL for paracetamol, with a detection limit of 4ng/mL for tramadol and 5ng/mL for paracetamol, and a quantitation limit of 15ng/mL for tramadol and 16ng/mL for paracetamol. Intra-day and Inter-day precision and accuracy of the methods have been established according to the current ICH guidelines. The regression co-efficient (r2) value for tramadol and paracetamol was found to be 0.9998 and 0.9996 respectively. The average recovery for tramadol was 100.03% and 100.28% for paracetamol.  No interferences were observed from the excipients.  The proposed method was found to be accurate, precise and rapid for the simultaneous estimation of tramadol and paracetamol.

 

KEYWORDS: Tramadol, Paracetamol, HPLC, simultaneous, ion pairing. 

 

 

INTRODUCTION:

Tramadol hydrochloride chemically known as (1RS, 2RS)- 2- [(dimethyl amino) methyl]-1-(3-methoxy phenyl) cyclohexanol hydrochloride, which produces relief of moderate to severe pain across the range of acute and chronic pain states by combining synergistically weak opioid and monoaminergically-mediated antinociceptive mechanisms1. Paracetamol chemically known as N-(4-hydroxy phenyl) acetamide reduces the production of prostaglandins2. Tramadol hydrochloride tablet is not official in any pharmacopoeia. According to literature survey, it was found that there are few analytical methods reported for tramadol hydrochloride and paracetamol either in individual or in combination with other drugs. Literature survey reveals that reports are available for the simultaneous estimation of tramadol and its active metabolite in human plasma3-4, simultaneous determination of tramadol and its impurities in oral drops5, estimation of tramadol hydrochloride by gas chromatography in plasma6 and brain tissue of mice and rats using HPLC7 and spectrophotometry, spectroflirimetric methods8 in pharmaceutical formulations.

 

A derivative spectrophotometric method9 was reported for simultaneous estimation of tramadol hydrochloride and chlorzoxane in tablet dosage form.

 

The aim of the present study was to develop a simple, sensitive, accurate, versatile, speedy and time–saving ion-pair HPLC method for the simultaneous estimation of tramadol and paracetamol in pharmaceutical formulations. The developed method has been validated by evaluation of the systems suitability, specificity, linearity, limits of detection and quantification, precision and accuracy. The validated method was applied to the commercially available pharmaceutical tablet dosage form containing tramadol and paracetamol.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

All chemicals used were of analytical reagent grade and HPLC grade acetonitrile and methanol (Merck, Ltd, Mumbai) were used. Double distilled water filtered through 0.45”m filter (Milli Pore) was used to prepare solutions; pharmaceutical grade tramadol and paracetamol were procured from Micro labs, Bangalore which was certified to be 99.7% and 99.6% pure respectively. Commercial formulations, Urgendol (WIN-Medicare private limited) and Acuvin (Nicholas Piramal) containing tramadol 37.5mg and paracetamol 325mg were procured from local market.

 

Table-1. Assay of tablets

Brand Name

TRAMADOL

PARACETAMOL

Label Claim mg/tab

Amount found* ± SD (mg/tab)

% assay ±SD

Label Claim mg/tab

Amount found* ± SD (mg/tab)

% assay ± SD

Urgendol

37.5

37.47± 0.02

99.92± 0.05

325

324.89± 0.05

99.96 ± 0.02

* Average of three readings.

 

Table-2.  System suitability parameters

S. No.

PARAMETERS

OBTAINED VALUES

TRAMADOL

PARACETAMOL

1.

Theoretical plates (N)

5121

6965

2.

Tailing factor (T)

1.06

1.01

3.

Asymmetry

1.16

1.18

4.

% RSD of Peak Retention Time

0.156%

0.104%

 

Instrumentation:

The chromatographic system consisted of a WATERS separation module equipped with a WATERS 2487 photodiode array detector; the volume of injection was 20”L. For weighing the substance, Shimadzu AX200 balance was used. Chromatogram and data were recorded by means of WATERS Empower-2 chromatographic software.

 

Optimized chromatographic conditions:

The mobile phase used was acetonitrile: 5% sodium lauryl sulphate : methanol in the volume of ratio 45:15:40 v/v.

 

Analytical column:               WATERS symmetry C18                                                                                 column (250 x 4.6mm) 5 ”

Detection wavelength:        217nm

Flow rate:                               1mL/min

Injection volume:                 20”L

Temperature:                         25șC ± 3 șC

Run time:                               5 minutes

Retention time:                     (A) Paracetamol - 1.1± 0.1min

                                                (B) Tramadol - 2.4± 0.1min

 

Fig.1.  A Typical HPTLC chromatogram of f Reference standard containing Tramadol (37.5 mcg/ml) and Paracetamol (325 mcg/ml)

 

 

Table.3. Summary of validation parameters

Parameters

Data

Tramadol

Paracetamol

Linearity range

20 – 187.5”g/mL

20 - 1625”g/mL

Correlation coefficient

0.9998

0.9996

Limit of detection

4 ng/mL

5 ng/mL

Limit of quantitation

15ng/mL

16ng/mL

%Recovery (n=6)

50%± SD

100%± SD

150%± SD

.

97.64±1.28

99.41±0.40

99.11±0.32

 

98.94±1.64

99.62±0.08

99.67±0.04

Precision (%RSD)

Intraday

Interday

Ist day

2nd day

 

0.7010

 

0.8348

1.2813

 

0.3373

 

0.8432

1.3827

Repeatability of application (n=6)

0.2944

0. 2713

 

Preparation of standard solutions:

37.5mg of tramadol and 325mg of paracetamol were weighed accurately and dissolved separately in mobile phase; the volume was made up to 100mL with mobile phase. From thus 10mL of each solution is transferred to a 100mL volumetric phase to get 37.5”g/mL concentration of tramadol and 325”g/mL concentration of paracetamol.

 

Sample preparation:

Twenty tablets were weighed and average weight was calculated. A quantity of powder equivalent to 37.5mg of tramadol and 325mg paracetamol were accurately weighed into a 100mL calibrated flask; the volume was made up to 100mL with mobile phase. The solution was sonicated for 10 minutes and filtered through Whatman filter paper No.41. Aliquot portion of this solution was diluted with mobile phase to achieve final concentration of 37.5 ”g/mL for tramadol and 325”g/mL for paracetamol.

 

Assay:

From the above sample solution 20 ”L was injected triplicate into the chromatographic system along with same concentration of standard solution and chromatogram was recorded. The peak area values of tramadol and paracetamol were calculated. The amount of tramadol and paracetamol in that solution were then estimated using calibration curve method. Results of assay are tabulated in Table 1.

 

RESULTS AND DISCUSSION:

The present study was undertaken to develop a sensitive, precise and accurate ion pair chromatographic   method for the simultaneous estimation of tramadol and paracetamol in tablet dosage form. The peak shape and resolution were found to be good when the mobile phase comprising of the acetonitrile, 5% sodium lauryl sulphate and methanol in the ratio 45:15:40 % v/v at flow rate of 1mL/minute. In this 15mL of 5% sodium lauryl sulphate (25mM) acting as ion paring reagent. The mechanism involved is interactions of ions. The ionic analyte is electrostatically attracted to the charged end of the ion-pair reagent (SLS) and an ion pair is formed. The charge neutralization coupled with the non-polar tail of the ion-pair reagent causes the charged analyte (Tramadol) to be retained by the non-polar stationary phase and hence tramadol was eluted later.

 

Fig.2   A Typical HPTLC chromatogram of  Tablet containing Tramadol (37.5 mcg/ml) and Paracetamol (325 mcg/ml)

 

Fig-3. Calibration curve of Tramadol.

 

This developed method was validated as per ICH guidelines. Linearity was studied in the concentration range from 20 to 187. 5”g/mL for tramadol and 20 to 1625”g/mL for paracetamol. Both the drugs showed good linearity in the tested range (fig. 3 and 4). The area response obeyed the equation y=mx+c.  The regression co-efficient (R2) value for tramadol and paracetamol was found to be 0.9998 and 0.9996 respectively.   The mean recovery for tramadol was 100.03% and 100.28% for paracetamol, which is largely within the 90-110% range that is considered acceptable and it revels that the method is accurate. The sample recoveries in formulation were in good agreement with their respective label claims, indicating non-interference of excipients in the estimation.  Precision was measured as repeatability and intermediate precision. The percentage co-efficient of variation (%CV or %RSD) for tramadol and paracetamol was less than 2%, compared with an acceptance range of 5% which indicated the method has good reproducibility. Intermediate precision was studied by running the whole method on the same day as well as on different days. The % CV for tramadol and paracetamol was not greater than 2.5%.  Specificity was checked by comparing retention times obtained in the standard mixture with those from the samples. The minimal difference between retention times (<0.1%) allows confident, highly specific peak identification.  In order to demonstrate the stability of both standard and sample solutions during analysis, both solutions were analyzed over a period of 5 hour at room temperature. The results showed that for both solutions, the retention time and peak area of tramadol and paracetamol remained almost unchanged and no significant degradation with in the indicated period, thus indicated that both solutions were stable for at least 5 hour, which was sufficient to complete the whole analytical process. The system suitability parameters like column efficiency, resolution, capacity factor, selectivity factor and peak asymmetry factor were calculated as per ICH guidelines and are present in table-2

 

Fig-4. Calibration curve of Acetaminophen

 

Different validation parameters for the proposed HPTLC method for determining Tramadol and Paracetamol content were summarized in Table - 3 and chromatogram of Tramadol and Paracetamol after separation is shown in Figure - 1 and 2.


The result obtained was in agreement with the labeled value of Tramadol and Paracetamol in dosage form. The determined validation parameters are in the acceptable ranges.

 

CONCLUSION:

The proposed ion-pair chromatographic method is rapid, accurate and precise for simultaneous determination of tramadol and paracetamol in tablet dosage forms and can be used for routine quality control of these drugs in formulation.

ACKNOWLEDGMENT:

The authors are thankful to Micro labs, Bangalore, for providing reference standards and Pharma Fabrikon Pvt Ltd for facility to carry out the research work.

 

REFERENCE:

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2.        Goodman and Gillman's (2001), The Pharmacological basis of Therapeutics 10 th   edition. McGraw-Hill: London; p.569-620.

3.        Campanero M.A., Calahorra B., Garcia-quetglas E., Escolar M and Honorato J.Chromatographia. 1998,vol.48, page 354-358.

4.        Mohammead-Reza Rouini; Yalda Hosseinzadeh Ardakani; Faezeh soltani; J Chromatography B: Analyt Technol Biomed Life Sci. 2006, Jan 18;830(2):207-211.

5.        Zecevic M. Kadioglu Y, Celebi F., J.Chromatogr B, Analyt Technol Biomed life sci. 2005 Feb 25’ 816(1-2), page 203-208.

6.        Sha YF, Shen S, and Duan GL, Talenta, 2000, 32, 84-88.

7.        Shaikh K.A, and Devkhile, A.B Journal of chromatographic Science, August 2008, volume 46, number 7, page 649-652.

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9.        Manisha Puranik, Hirudkar A, Wadher SJ, and Yeole PG, Indian journal of pharmaceutical sciences, 2006, vol 119,page 251-256.

 

 

 

 

 

Received on 24.08.2009          Modified on 23.10.2009

Accepted on 21.11.2009         © RJPT All right reserved

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 179-182