RP-HPLC Method for Simultaneous Estimation of Tramadol HCl and Paracetamol Bulk Drug and Its Combined Dosage Form

Rajesh Shukla*, R. Sivakumar, P.R. Vijayanand, P. Kumar Nallasivanand R. Venkatnarayanan

Dept. of Pharmaceutical Analysis, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, Tamilnadu.

*Corresponding Author E-mail: rajeshshukla2628@gmail.com

ABSTRACT:

This work is concerned with application of simple, accurate, precise and highly selective reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of Tramadol HCl and Paracetamol in combined dosage form. Chromatographic separation was achieved isocratically at room temperature on phenomenex C18 column (250 х 4.6 mm) with a mobile phase composed of 5 % Tri-fluoroaceticacid in water: Acetonitrile: Methanol in the ratio of 70:20:10 % v/v/v at flow rate of 1.0 ml/min. Detection is carried out using a UV detector at 254 nm. The retention time of Tramadol HCl and Paracetamol was found to be 3.890± 0.5 min and 1.990± 0.5 min. respectively. The method was found to be linear in the range of 0.1-10 μg/ml with mean recovery of 98.96 % for Tramadol HCl and 99.11 % for Paracetamol. The correlation coefficients for all components are close to 1. The developed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Thus the proposed method was successfully applied for simultaneous determination of Tramadol HCl and Paracetamol in routine analysis.

KEYWORDS: Tramadol HCl , Paracetamol, RP-HPLC

INTRODUCTION:

Tramadol HCl (TRD) (+)-cis-2-[(dimethylamino) methyl]-1-(m-methoxyphenyl) cyclohexanol hydrochloride is used to treat moderate and severe pain and most types of neuralgia, including trigeminal neuralgia.¹ TRD could be effective for alleviating symptoms of depression and anxiety because of its action on the noradrenergic and serotonergic systems, the involvement of which appear to play a part in its ability to alleviate the perception of pain. TRD is a synthetic, centrally acting opioid analgesic with a potent active opioid metabolite. In general, TRD has been found to be an opioid agonist with selectively for the μ receptor but with some weak affinity for the κ and δ receptors².

Paracetamol (PM) is a synthetic, nonopiate, centrally acting, antipyretic and analgesic³. The mechanism by which PM reduces fever and pain is still a source of debate. The reason for this confusion has largely been due to the fact that PM reduces the production of prostaglandins³.

Recently a combination of TRD and PM has been launched in market. In this combination, PM shows a synergistic effect with TRD. TRD was determined by several methods including gas chromatography (GC)⁴, liquid chromatography with UV detection (LC–UV)⁵, HPTLC⁶, derivative spectrophotometric⁷and GC-M⁸. PM was determined with or without combination of several drugs by HPLC, spectrophotometrically and HPTLC^9-13. Literature survey revealed that no HPLC method has been reported yet for the analysis of these two drugs in combination without preliminary separation that makes it worthwhile to pursue the present work.

MATERIALS AND METHODS:

TRM and PM were obtained as gift sample from Zydus cadila healthcare Ltd, Ahemedabad, India. All solvents were of HPLC grade obtained from Merck Research Laboratory, Mumbai, India. Fine chemicals were used for the study. A Shimadzu Binary Gradient system was used coupled with SPD 10A UV detector and Rheodyne injector with 20 μl fixed loop. Chromatographic analysis was performed using spinchrome software on a phenomenex C18 column (250x4.6mm, 5 μ particle size) at ambient temperature. The mobile phase consisting of 5% tri-fluoroaceticacid in water: acetonitrile: methanol in the ratio of 70:20:10% v/v/v, detection was carried out at 254 nm.

PREPARATION OF STOCK SOLUTION OF TRAMADOL HCl AND PARACETAMOL:

Standard stock solution of 1000 µg/ml of each TRM and PM were prepared by dissolving 100 mg of each drug in 70% methanol. Sub stock solution was prepared from stock solution by diluting each standard stock solution (10ml) up to 100 ml to get 100µg/ml of both drugs. The nominal concentration in range of 0.1-10 μg/ml and 0.5-20 μg/ml were prepared simultaneously for TRM and PM for calibration. Tablets of PM and TRM combination are available in 10:1.15 ratio. Working standards were prepared in the ratio of 10:1.15 from standard sub stock solution.

CHROMATOGRAPHIC CONDITIONS:

CALIBRATION CURVE:

An aliquot of 20 μl of the solution from each flask was injected two times. Calibration curves were constructed by plotting mean peak areas against the corresponding drug concentrations. TRM and PM were found to be linear in the range of 0.1- 10 μg/ml and 0.5-20 with coefficient of correlation (r²) 0.9995and 0.9990 for TRM and PM, respectively.

DETERMINATION OF TRAMADOL HCL AND PARACETAMOL IN THEIR COMBINED DOSAGE FORMS:

Twenty tablets (brand name-Ultracet, manufactured by Johnson and Johnson Ltd.) were taken and their average weight was determined. They were crushed to fine powder containing the equivalent to 1.154 mg of TRM and 10 mg of PM were transferred to 100 ml volumetric flask. It was then dissolved in 50 ml of mobile phase by intermittent shaking for 4-5 min. The volume was made up to 100 ml by same mobile phase, and the solution was filtered through Whatman filter paper No.41. Appropriate dilutions were prepared from the above stock solution to get the final concentration of PM 10µg/ml and TM 1.154 µg/ml and the amount of drug was determined. Results of the triplicate analysis are given in Table 1.

TABLE 1: ASSAY RESULTS OF COMBINED DOSAGE FORM

Formulation

Label Claim gm/tab

Amount found gm/tab ± RSD*

%Assay*

±RSD*

Paracetamol

0.325

0.321 ± 0.47

98.87 ± 0.47

Tramadol HCl

0.0375

0.0371 ± 0.41

99.01 ± 0.41

* denotes average of five determinations. TRM and PM denote, Tramadol HCl and Paracetamol respectively

The method was validated for statistical parameters i.e. precision, accuracy, specificity, linearity, stability, of analytical solutions and ruggedness criteria. Results of the method validation experiments are given in Table 2. The precision of the method was determined by knowing percentage RSD of means of three replicate solutions of all the three independent samples.

PRECISION:

The intra day precision study of TRM and PM was carried out by estimating the corresponding responses six times on the same day and the results are reported in terms of relative standard deviation (RSD, Table 2).

ACCURACY:

The accuracy of method is determined by adding known amount of standard to that of sample (above and below the normal level) at 3 different levels to cover both above and below (80% to 120%) the normal levels expected in the sample.

SPECIFICITY:

The specificity of the RP-HPLC method was determined by complete separation of TRM and PM as shown in Fig No.1 with parameters like retention time (t_R), resolution (R_s) and tailing factor (T). Here tailing factor for peaks of TRM and PM was less than 2% and resolution was satisfactory. The peaks obtained for TRM and PM were sharp and have clear base line separation.

DETECTION LIMIT AND QUANTIFICATION LIMIT:

The LOD and LOQ were determined by injecting minimum concentration of the drugs. The LOQ were found to be 100ng and 500ng for TRD and PM respectively. The standard deviation of y-intercepts of regression lines were determined and kept in the following equation for the determination of detection limit and quantification limit. Detection limit = 3.3 σ/s; quantification limit = 10 σ/s; Where σ is the standard deviation of y intercepts of regression lines and s is the slope of the calibration curve.

RESULT AND DISCUSSION:

Optimization of the mobile phase was performed based on resolution, asymmetric factor and peak area obtained for both TRM and PM. The mobile phase 5% tri-fluoroacetic acid in water: acetonitrile: methanol in the ratio of 70:20:10% v/v/v was found to be satisfactory and gave two symmetric and well resolved peaks for TRM and PM. The resolution between TRM and PM was found to be 1.9 which indicates good separation of both the compounds. The retention time for TRM and PM were 3.89 min and 1.990 min, respectively (Fig. no 1). The asymmetric factors for TRM and PM were 0.98 and 0.97, respectively.

Fig. 1 A TYPICAL CHROMATOGRAM OF TRAMADOL HCl (TRM) AND PARACETAMOL (PM)

The calibration curve for TRM was obtained by plotting the peak area of TRM versus the concentrations of TRM over the range of 0.1- 10 μg/ml, and it was found to be linear with r² = 0.9995. Similarly, the calibration curve for PM was obtained over the range of 0.5-20 μg/ml and was found to be linear with r² = 0.9990.

TABLE 2: VALIDATION AND SYSTEM SUITABILITY PARAMETERS

Parameter	Tramadol HCl	Paracetamol
Calibration range (μg/ml)	0.1-10	0.5-20
Correlation Coefficient (r²) ± S.D*	0.9995	0.9990
Retention time (min.) ± S.D*	3.89 ± 0.5	1.990 ± 0.5
Resolution factor	1.9
Tailing factor*	0.96	0.95
Asymmetry	0.98	0.97
No. of theoretical plate*	19226	18768
Limit of quantification (ng/ml)	100	500
Precision (RSD*, %) Intraday (n=5)	0.57	0.49
Repeatability (RSD*, %) n=5	0.79	0.61

* Mean of five determinations (n=5).

TABLE 3: RECOVERY STUDIES OF TRM AND PM IN COMBINED DOSAGE FORM

S. No.	% Drug Added	PM		TRM
S. No.	% Drug Added	% Drug Recovered ± % RSD*	% Recovery ± % RSD*	% Drug Recovered ± % RSD*	% Recovery ± % RSD*
1	80	79.20 ± 0.83	98.99± 0.57	79.06 ± 0.34	98.83 ± 0.34
2	100	99.20 ± 0.36	99.20± 0.36	99.10 ± 0.83	99.10 ± 0.83
3	120	118.97 ± 0.42	99.14 ± 0.42	118.67 ± 0.51	98.97 ±0 51

The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 2. The quantification limit for TRD and PM were 100 and 500 ng/ml respectively.

The recoveries of TRM and PM were found to be in the range of 98.83 -99.10 % and 98.99-99.20%, respectively. The system suitability test parameters are shown in Table 2.The chromatographic method was applied to the determination of TRM and PM in their combined dosage form (Tablet formulation). The result for TRM and PM were comparable with the corresponding labeled amounts (Table 1).

Proposed study describes a new RP-HPLC method for estimation of TRM and PM combination in mixture using simple mobile phase. The method gives good resolution between both the compounds with a short analysis time. The method was validated and found to be simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of TRM and PM in their combined dosage form.

ACKNOWLEDGEMENTS:

The authors are grateful to the Management, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, for providing the required facilities and also to ZYDUS CADILA PVT. LTD. AHMEDABAD, as the gift sample of drug the gift sample of Tramadol HCl and Paracetamol.

REFERENCES:

1. Puranik M, Hirudkar A, Wadher SJ, Yeole PG. Development and validation of spectrophotometric methods for simultaneous estimation of tramadol hydrochloride and chlorzoxazone in tablet dosage form, Indian J. Pharm. Sci., 2006; 68: 737-739.

2. The Merck Index, Merk Research Lab, Division Merck and Inc. U.S.A., 2002; 11: 7002.

3. Wadher SJ, Pathankar PR, Puranik M, Ganjiwale RO and Yeole PG. Simultaneous spectrophotometric estimation of paracetamol and metoclopramide hydrochloride in solid dosage form. Indian J. Pharm. Sci., 2008; 70: 393-395.

4. Tao Q, Stone DJ, Borenstein M.R, Jean-Bart V, Codd EE, Coogan TP, Desai-Krieger D, Lia S and Raffa R. B. Gas chromatographic method using nitrogen–phosphorus detection for the measurement of tramadol and its O-Desmethyl metabolite in plasma and brain tissue of mice and rats. J. Chromatogr., B: Biomed. Sci. Appli., 2001; 763: 165-171.

5. Kartinasari WF, Palupi T and G. Indrayanto., HPLC determination and validation of tramadol hydrochloride in capsules. J. Liq. Chromatogr. Relat. Technol, 2005; 27: 737–744.

6. Venkateshwarlu K, Reddy Y N, Srisailam K, Rajkumar V and Pai MG. Determination of tramadol in capsules by high performance thin layer chromatography –densitometry. Curr. Trends Biotechnol. Pharm., 2008; 3: 421 -425.

7. Srinivasan KK, Alex J, Shirwaikar AA, Jacob S, Sunil Kumar MR, and Prabu SL. Simultaneous derivative spectrophotometric estimation of aceclofenac and tramadol with paracetamol in combination solid dosage forms. Indian J. Pharm. Sci. 2007; 69: 540-545.

8. Staerk U and Kulpmann WR. High-temperature solid-phase micro extraction procedure for the detection of drugs by gas chromatography–mass spectrometry. J. Chromatogr. B. 2000; 745: 399–411.

9. Chan K and Mc Cann JF. Improved gas-liquid chromatography-electron-capture detection technique for the determination of paracetamol in human plasma and urine Journal of of sample preparation for the determination of paracetamol in plasma by HPLC. Chromatogr. Biomed. Appli. 1979; 164: 394-398.

10. Gandhi SV, Barhate NS, Patel BR, Panchal DD and Bothara KG. A validated densitometric method for analysis of aceclofenac and paracetamol as the bulk drugs and in combined tablet dosage forms. Acta Chromatogr. 2008; 20: 175–182.

11. Sullivan C and Sherma J. Development and validation of an HPTLC-densitometry method for assay of caffeine and acetaminophen in multicomponent extra strength analgesic tablets. J. Liq. Chromatogr. Relat. Technol. 2003; 26: 3453 – 3462.

12. Afkhami A, Sarlak N and Zarei AR. Spectrophotometric determination of salicylamide and paracetamol in biological samples and pharmaceutical formulations by a differential kinetic method. Acta Chim. Slov. , 2006; 53: 357–362.

13. Kucuk A and Kadıoglu Y.Determination of Tramadol hydrochloride in ampoule dosage forms by using UV Spectrophotometric and HPLC-DAD methods in methanol and water media. Il Farmaco. 2005; 60: 163-169.

Received on 28.12.2009 Modified on 30.01.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 552-554