INTRODUCTION:

Tramadol Hydrochloride (TRM) chemically known as (±)cis-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexanol hydrochloride, is centrally acting analgesic, stucturlly related to morphine. It is effectivley used to treat moderate to severe acute and chronic pain¹. It exerts its analgesic activity through a dual mechanism of action inhibiting transmission as well as perception of pain². A new fixed dose combination containing Tramadol hydrochloride (TRM) and Paracetamol (PCM) is available in the market in the tablet dosage form. European pharmacopoeia describes potentiometric³ method for assay of TRM hydrochloride. Literature survey reveals that reports are available for estimation of TRM hydrochloride by GC⁴ in plasma and brain tissue of mice and rats, using HPLC^5,6in plasma and urine, and spectrophotometry^7,8 in pharmaceutical formulations. A spectrophotometirc⁹method is reported for simultanious estimation of TRM hydrochloride and Chlorzoxazone in tablet dosage form. However, no method is yet reported for simultanious estimation of tramadol hydrochloride and paracetamol in combined dosage form. Hence, the authors have deveoped simple and accurate zero absorbance measurement spectroscopic method to estimate these two drugs from their combined dosage form.

MATERIALS AND METHOD:

All the chemicals used were of analytical grade. Commercial formulations, Domadol plus (Unichem) and Acuvin (Nicholas Piramal) containing TRM 37.5 mg and PCM 325 mg were procured from market. TRM hydrochloride and PCM, 100 mg each, were accurately weighed and dissolved separately in 100 ml of methanol. Ten ml of above solution of PCM was diluted separately to 100 ml with water to produce 100 µg/ml. Spectral and absorbance measurement were made on Shimadzu – 1601 UV/VIS Spectrophotometer by using 1 cm matched quartz cells.

Construction of Beer’s curve for TRM hydrochloride:

Aliquots (0.6 to 1.6 ml) of standard solution of TRM hydrochloride were transferred to a series of 10 ml volumetric flasks and diluted to mark with distilled water. The absorbance was measured at the λmax 272 nm, against a blank, and plotted versus the TRM hydrochloride concentration (60-160 μg/ml). The calibration curve was constructed, and the regression equation was calculated (Table 1).

Construction of Beer’s curve for PCM:

Aliquots (0.8 to 3.0 ml) of standard solution of PCM (100 μg/ml) were transferred to a series of 10 ml volumetric flasks and diluted to mark with distilled water. The absorbance was measured at the λmax 250 nm, against a blank, and plotted versus the TRM hydrochloride concentration (8-30 μg/ml). The calibration curve was constructed, and the regression equation was calculated (Table 1).

Fig. 1: UV spectra of TRM (272.0 nm) and PCM (250.0 nm) in combination.

Table 1: Method validation parameters

Parameters

Values

TRM

PCM

Linearity range (µg/ml)

Correlation coefficient (r)

Regression equation (y=mx+c)

Slope (m)

Intercept (c)

Limit of detection (LOD) (µg/ml)

Limit of quantification (LOQ) (µg/ml)

% relative standard deviation

60 – 160

0.9965

0.006

-18.26

0.930

8 - 30

0.9952

0.0498

-0.2655

1.26

Table 2: Intra-day and Inter-day Precision data for Paracetamol by proposed method (n = 5)

Sr. No.

Concentration

(µg/ml)

Intra-day precision %RSD

Inter-day precision %RSD

0.534

0.656

0.530

0.721

0.645

0.469

0.554

0.423

0.619

0.592

RSD – Relative standard deviation.

Analysis of commercial formulations:

Twenty tablets were weighed and finely powdered. Accurately weighed tablet powder equivalent to 100 mg was taken in 100 ml volumetric flask. To it 980.50 mg of pure TRM was added and sonicated for 5 min with 50 ml of methanol. The volume was made to mark. Aliquot portion of this solution was further diluted with water to achieve final concentration of 10 μg/ml for PCM and 100 μg/ml for TRM. The absorbances were noted at respective wavelengths. The concentration of each drug in tablet formulation was determined using proposed method.

Validation of the method¹⁰:

The developed method was validated in terms of linearity, accuracy, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement.

RESULT AND DISCUSSION:

The standard solution was scanned from 200 to 400 nm to select the wavelength for estimation. As shown in fig. 1, 272 nm was then selected for determination of TRM hydrochloride (where PCM shows zero absorbance) and 250 nm was selected for determination of PCM (were TRM shows zero absorbance). Different solutions of TRM and PCM were run in entire range from 200 to 400 nm and absorbance at 272 nm were plotted against the concentrations of TRM and the absorbance at 250 nm were plotted against the concentrations of PCM. The method obeys Beer’s law in the concentration range of 8 – 30 μg/ml for PCM and 60 – 160 μg/ml for TRM with correlation coefficients, 0.9952 and 0.9965, respectively.

Table 3: Intra-day and Inter-day Precision data for Tramadol HCl by proposed method (n = 5)

Sr. No.

Concentration

(µg/ml)

Intra-day precision %RSD

Inter-day precision %RSD

100

120

130

140

150

0.365

0.543

0.565

0.628

0.641

0.407

0.525

0.443

0.612

0.530

RSD – Relative standard deviation.

The intra-day precision was determined by analyzing standard solutions in the concentration range of 10 – 18 μg/ml for PCM and 100 – 150 μg/ml for TRM, five times on the same day while inter-day precision was determined by analyzing corresponding standards daily for 3 day over a period of one week. The intra-day and inter-day coefficients of variation are given in Table 2 and 3, respectively.

Recovery studies of the drugs were carried out for the accuracy parameter. These studies were carried out at three levels i.e. multiple level recovery studies. % Recovery was found to be within the limits as listed in Table 4. The assay values for the marketed formulation were found to be within the limits and are well agreed with label claim (Table 4). The low RSD value indicated the suitability of the method for routine analysis of drugs in pharmaceutical dosage forms.

CONCLUSION:

The method described in this paper for the determination of tramadol Hydrochloride and paracetamol is simple, accurate, sensitive and reproducible. The amounts found in formulations are well agreed with label claim. The proposed method can be applied to routine analysis in quality-control laboratories.

REFERENCES:

1. Gustein HG. and Akil H, Opioid analgesics. In: Hardman JG, Limbird LE and Gilman AG, editors. Goodman and Gillman’s The Pharmacological basis of Therapeutics. 10^th ed. McGraw-Hill: London; 2001. p. 569 - 620

2. Rang HP, Dale NM, Ritter JM and Moore RK, editors. Antiinflamatory Agents: Rang and Dale’s Pharmacology. 5^th ed. Church Livingstone: London; 2003. p. 580 -5

3. European Pharmacopoeia, 5th Edi., European Department for the Quality of Medicines within the Council of Europe, Strasbourg, 2005, 2607

4. Tao,Q, Stone DJ, Borenstein MR, Jean B, Valerie C, Ellen E, Timothy P, Desao K D, Liao S and Raffa RB: 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, 2001; 763: 165

5. Paderson RS, Brosen K and Nielsen F: Enantioselective HPLC Method for Quantitative Determination of Tramadol and O-Desmethyltramadol in Plasma and Urine, Chromatographia, 2003; 57: 279

6. Meyyanathan SN, Pradeep K and Suresh B: Analysis of Tramodol in Pharmaceutical Preparations by High Performance Thin Layer Chromatography, J Separation Sci. 2003; 26: 1359 - 62

7. Gan S H. and Ismil R: Validation of High Performance Liquid Chromatographic method for Tramadol and O-desmethyltramadol in Human Plasma using Solid Phase Extraction, J Chromatogr B, 2001; 759: 325

8. Mashru RC and Banerjee SK: Development and Validation of spectrophotometric methods for estimation of Tramadol hydrochloride. Eastern Pharmacist, 1998;41: 141

9. Puranik M, Hirudkar A, Wadher SJ And 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 (6): 737 – 739

10. Validation of Analytical Procedures, Methodology, ICH harmonized tripartite guidelines, 1996, 1.

Received on 20.03.2009 Modified on 18.05.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 17-19