Determination of tramadol in capsules by high performance thin layer chromatography - densitometry Venkateshwarlu K 1,3, Reddy YN 3 *, Srisailam K 2, Rajkumar V 2 and Pai MG 1 1 Department of Pharmaceutical Analysis, Goa College of Pharmacy, Panaji, Goa, India 2 Vaagdevi College of Pharmacy, Ramnagar, Hanamkonda, Warangal, AP, India 3 University College of Pharmaceutical Sciences, Kakatiya University, Warangal, AP, India *For correspondence - yellu_nr@yahoo.com Abstract A simple, sensitive and reproducible high performance thin layer chromatographic method for the determination of tramadol in capsules is developed using silica gel plates with fluorescent indicators. The system is equipped with an automated sample applicator and the detection was performed at 254 nm by using HPTLC. The mobile phase consists of methanol and ammonia in the ratio of 19:1. The retention factor for tramadol is 0.67. The limit of detection of tramadol was found to be 13 µg/ml and the limit of quantitation is 15 µg/ml. The method shows good linearity in the range of 18 to 100 µg/ml (R 2 = 0.993). The intra and inter day assay (n=5)variations in the linear range are less than 2%. A pharmaceutical capsule product containing tramadol was analyzed to test the applicability of new method. The percentage of tramadol in the capsules was found to be from 93.5 to105 Key words Tramadol, HPTLC of Tramadol, Tramadol capsules, Densitometry Introduction High performance thin layer chromatography (HPTLC) is a versatile technique for the analysis of a large number of chemical substances including drugs. The technique is easily and conveniently used for routine quality control analysis (1). Analysis of drug samples by HPTLC saves time and reduces cost, when compared to HPLC analysis, particularly during handling of a large number of samples. Multiple number of samples can be analysed in a single run allows one to save time and thus cost of analysis. The sample preparation will be easier with respect to the tedious process involved in HPLC analysis. Tramadol, [(1S,2R)-2-[(dimethylamino) methyl]-1-(3-methoxyphenyl)cyclohexanol] (Fig. 1), is an analgesic that acts as a weak agonist at all types of opioid receptors with some selectivity for the mu-receptors. It has excellent analgesic activity with little respiratory depression. The drug is indicated in the treatment of moderate to severe pain. Tramadol is available in the form of oral drops, tablets, capsules and injections (2). Previous works indicate that tramadol content in these formulations can be determined by spectrophotometry (3), spectrofluorometry (4), HPLC (5), GC (6), GC- MS & LC-MS (7), capillary electrophoresis (8) and HPTLC (9). Few researchers have reported the determination of tramadol in various matrices by Krzek and Starek (9) performed identification and HPTLC determination of tramadol and its impurities in pharmaceutical preparations. Krzek and Starek (9) performed the separation on silica gel-coated chromatographic plates (HPTLC)
422 Venkateshwarlu et al using two mobile phases: (I) chloroformmethanol-glacial acetic acid (9:2:0.1, v/v/v); (II) chloroform-toluene-ethanol (9:8:1, v/v/v). The UV densitometry was carried out at ëmax of 270 nm. The developed method was of high sensitivity and low detection and determination limits ranging from 0.044 to 0.35 ìg. For individual constituents the recovery ranged from 93.23 to 99.66%. Ahrens et al (10) reported an advanced fibre optical scanning densitometry in HPTLC determination of tramadol in biological samples. The sensitivity of the assay is found to be in the range of 250-1000 ng/spot. Fig. 1: Structure of Tramadol The present investigation was undertaken for determination of tramadol in capsules by HPTLC densitometry. This paper reports a simple, sensitive, and reproducible HPTLC method for determination of tramadol and its applicability in the estimation in marketed capsules. The developed method can be used to estimate tramadol in quality control during and after manufacturing of the dosage forms where large number of samples can be handled simultaneously. The present method was found to be more sensitive than the reported HPTLC methods. Materials and Methods Tramadol was obtained from Unichem Laboratories Ltd, Goa, India. Precoated HPTLC plates were made of silica gel 60 F254 of size 10 cm 20 cm purchased from Merck (Mumbai, India). All the solvents were of analytical grade purchased from Merck (Mumbai, India). Capsules of Tramazac (Cadila Healthcare Pvt Ltd, Ahmedabad, India) were purchased from a local pharmacy. Capsules were selected for the study since the active constituent can be extracted with much ease. Preparation of standard solutions Tramadol (100 mg) was accurately weighed into a 100 ml volumetric flask, dissolved in methanol (~20 ml), and the solution was diluted to volume with the same solvent to get a standard solution of 1 mg/ml. One milliliter of this solution was diluted 10 times with methanol in volumetric flask to prepare the working standard solution (0.1 mg/ml). The working standard solutions containing 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 150, 200, 250 and 300 µg/ml of tramadol were prepared for calibration curve. HPTLC conditions Analysis was performed on silica gel 60F254 plates of 50 x 50 mm size. The standard solutions were applied by using a Desaga automated sample applicator (Desaga, Heidelberg, Germany) equipped with 100 ìl syringe. The settings were: band length, 6 mm; application rate, 4 ìl/s; table speed (scan speed), 10 mm/s; distance between bands, 2 mm; distance from the plate edge, 10 mm; and distance from the bottom of the plate, 20 mm. The volumes applied for each analysis were 30 ìl which gave 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600, 900, 1200, 1500, 3000, 4500, 6000, 7500 and 9000 ng/spot of tramadol. The plates were developed to a distance of 4.5 cm beyond the
423 origin with methanol ammonia in the ratio of 19:1 in a vapor-equilibrated Desaga chamber. The vapor equilibration time was 25 min. After development, the plates were air-dried for 5 min. The sample and standard zones were quantitated by scanning at 254 nm with Densitometer CD60 (Desaga). The linear regression calibration curve of zone weights and scan areas were plotted by using Desaga ProQuant version 1.03.200. Method validation The standard solutions were chromatographed for inter and intraday assay variation (n = 5). The calibration curves were obtained by plotting the peak area against concentration for linearity of tramadol in the above concentration range. The accuracy of the method was determined using external standard addition. Known amounts of standard drugs were added at four different levels, and each determination was carried out in triplicate. The limit of detection (LOD) and quantitation (LOQ) were obtained. Application to the analysis of marketed Capsules A marketed product of tramadol capsules (each containing 50 mg of tramadol hydrochloride) was selected for the analysis using the procedure described above. Ten capsules were taken and the contents were weighed. Accurately weighed powder equivalent to 5 mg of tramadol was transferred to a 25 ml volumetric flask, dissolved in methanol (~10 ml), and shaken for 15 min. The solution was then diluted to volume with the same solvent, mixed, and finally filtered through Whatmann No. 42 filter paper (Whatmann, Middlesex, UK). A sample (1 ml) of the filtrate was serially diluted to get a concentration of 20 ìg/ml of tramadol, and this solution was used for analysis. The analysis was done in triplicate, and the amount of tramadol was calculated from the calibration curve. Results and Discussion After the development, the HPTLC plates containing fluorescent indicator are shown as compact bands of tramadol on green background when viewed under 254 nm UV light (Fig. 2). The retention factor value for tramadol was found to be 0.67. The densitogram of tramadol obtained using Densitometer CD60 is shown in Figure 3. The LOD and LOQ of tramadol in the series of solutions studied were found to be 13 and 15 ìg/ ml respectively. The method has shown good linearity in the range of 18 ìg/ml to 100 ìg/ml (R 2 = 0.993). The intra and inter day assay (n = 5) variations in the linear range were found to be less than 2% for tramadol. The developed method was found to be accurate which is evident from the statistical data. The RSD was found to be less than 2% which discloses the fact that the developed method is precise. Fig. 2: Bands showing Tramadol after development
424 Tramadol Determination by HPLC absence of the excipients peaks in control densitogram) in the results when the capsules were analysed. Fig. 3: Densitogram of Tramadol A pharmaceutical capsule product containing tramadol was analyzed to test the applicability of the new method. The percentage of tramadol in the five determinations from capsules powder studied is shown in Table 1 which ranged from 93.5 to 105%. Table 1: Percentage of tramadol in capsules from five determinations S. No. Tramadol Theoretical amount present Percentage obtained 1. 20 µg 98.0 2. 20 µg 105.0 3. 20 µg 95.5 4. 20 µg 102.0 5. 20 µg 93.5 Conclusion The HPTLC method developed for the determination of tramadol in capsules is accurate, precise, rapid, and selective. It can, therefore, be easily and conveniently used for routine quality control analysis, particularly when large numbers of samples are encountered. The developed method was found to be specific as there was no interference of the excipients (based on the References 1. Venkateshwarlu, K., Venishetty, R.K.,Yellu, N. R., Keshetty, S and Pai, M. G. (2007). Development of HPTLC-UV absorption densitometry method for the analysis of Alprazolam and sertraline in combination and its application in the evaluation of marketed preparations. Journal of Chromatographic Science, 45: 537-539. 2. Anonymous. (1985). The Martindale Extra Pharmacopoeia, EP 31: 1742-1744. 3. Abdellatef, H.E. (2002). Kinetic spectrophotometric determination of tramadol hydrochloride in pharmaceutical formulation. Journal of Pharmaceutical and Biomedical Analysis, 29: 835-842. 4. Abdellatef, H.E., El-Henawee, M.M., El- Sayed H.M. and Ayad, M.M. (2006). Spectrophotometric and spectrofluorimetric methods for analysis of tramadol,acebutolol and dothiepin in pharmaceutical preparations. Spectrochimica Acta A Molecular and Biomolecular Spectroscopy. 65(5):1087-1092 5. Negro, S., Salama, A., Sánchez, Y., Azuara, M.L. and Barcia, E. (2007). Compatibility and stability of tramadol and dexamethasone in solution and its use in terminally ill patients. Journal of Clinical Pharmacology and Therapeutics 32(5): 441-444. 6. Tao, Q., Stone, D.J.Jr., Borenstein, M.R., Jean-Bart, V., Codd, E.E., Coogan, T.P., Desai-Krieger, D., Liao, S. and Raffa, R.B. (2001). Gas chromatographic method using
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