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Year : 2016  |  Volume : 48  |  Issue : 5  |  Page : 550--554

A simple colorimetric method for estimation of tramadol hydrochloride in pure and tablet dosage forms

Scaria P Thomas1, Hari K. N. Sankar2,  
1 Department of Pharmacology, Government Medical College, Thiruvananthapuram, Kerala, India
2 Department of Pharmacology, Government Medical College, Kottayam, Kerala, India

Correspondence Address:
Dr. Scaria P Thomas
Department of Pharmacology, Government Medical College, Thiruvananthapuram, Kerala


Objective: The objective of this study was to develop and validate a simple method for estimation of tramadol hydrochloride (TH) in pure and pharmaceutical dosage forms using a colorimeter. Materials and Methods: TH on reaction with Eriochrome Black T in the presence of acetate buffer at pH 3.5 forms a colored complex. This complex was extracted with a fixed volume of chloroform. The optical density of this colored complex was measured against reagent blank using a colorimeter at 520 nm. Results: Beer«SQ»s law was obeyed with a good correlation coefficient (0.999) in the concentration range of 2.5 μg/ml to 10 μg/ml. Drug content estimation and recovery studies carried out on commercial tablet dosage forms demonstrated the accuracy of the method and that excipients do not cause interference. Precision and robustness were measured and found to be acceptable (% relative standard deviation <2%). Conclusion: The proposed method can be used for the rapid determination of TH content in tablets at a health-care provider level using already available staff and equipment.

How to cite this article:
Thomas SP, Sankar HK. A simple colorimetric method for estimation of tramadol hydrochloride in pure and tablet dosage forms.Indian J Pharmacol 2016;48:550-554

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Thomas SP, Sankar HK. A simple colorimetric method for estimation of tramadol hydrochloride in pure and tablet dosage forms. Indian J Pharmacol [serial online] 2016 [cited 2022 Jan 26 ];48:550-554
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Tramadol hydrochloride (TH) is the salt of tramadol which is a centrally acting analgesic that acts as a weak agonist at the μ-opioid receptor. A part of its analgesic effect is produced by inhibition of uptake of norepinephrine and serotonin. It is used in the management of moderate to moderately severe pain. Its respiratory depressant effect is less than that of morphine. The oral bioavailability of tramadol after a single oral dose is 68%. [1] Tramadol undergoes extensive hepatic metabolism by a number of pathways, including CYP2D6 and CYP3A4, as well as by conjugation with subsequent renal excretion. The primary O-demethylated metabolite is two to four times more potent than the parent drug and may account for part of the analgesic effect. The elimination half-life is 6 h for tramadol and 7.5 h [1] for its active metabolite. The important adverse effects include seizures and risk of serotonin syndrome. Tramadol-induced analgesia is not entirely reversible by naloxone, but tramadol-induced respiratory depression is reversed by naloxone. [1] The chemical name for TH is 2-(dimethylaminomethyl)-1-(3-methoxyphenyl) cyclohexanol hydrochloride. [2] Its molecular formula is C 16 H 25 NO 2 . The structural formula [2] is given in [Figure 1].{Figure 1}

TH is a white, crystalline, bitter, and odorless powder with a molecular weight of 299.8. It is readily soluble in water and ethanol and has a pKa of 9.41. [3] There are more than 35 commercial formulations of TH and are available as tablets containing 50 mg or 100 mg, capsules containing 50 mg or 100 mg, injections containing 25 mg or 50 mg per ml and 100 mg suppository.

Literature review reveals that several analytical methods have been reported for the estimation of TH alone or in combination with other drugs which include spectrophotometry, [4],[5],[6],[7],[8],[9],[10],[11],[12] spectrofluorimetry, [13],[14],[15] high-performance thin-layer chromatography, [16],[17],[18] high-performance liquid chromatography, [19],[20],[21] and gas chromatography-mass spectrometry. [22],[23] Even though these methods are accurate, they require costly equipments, costly reagents, and trained technicians for the operation of these equipments. The proposed study aims to develop and validate a method which overcomes these limitations and to utilize this method for estimating the TH content in tablet dosage forms.

The concept of colored complex formation with Eriochrome Black T (EBT) and its extraction into chloroform layer employed in this study is based on an extractive spectrophotometric method for TH estimation. [4] Significant differences from the spectrophotometric method [4] include the use of a colorimeter and glass cuvettes, different wavelength, elimination of time and resource-consuming drying process, using magnetic stirrer for better mixing, and numerous modifications to ensure accurate and precise results even in the hands of a first-time user. These modifications also ensure that this method is economical and provide results in a shorter span of time. Additional validation parameters were also evaluated.

 Materials and Methods

Instruments and Reagents

The measurements were made on a Photochem-Micro Digital 8 Filter Colorimeter (Photo Electric Instruments, Jodhpur, India) using glass cuvettes of 1 cm path length. The pure standard of TH was obtained from Sigma-Aldrich, India. EBT was purchased from Spectrum Reagents and Chemicals Pvt, Ltd, Cochin, India. Commercially available tablet dosage forms of TH were used in the study. All glassware used for measuring volumes were Class A. Electronic balance with 0.1 mg sensitivity (Shimadzu, Japan) was used. Eutech CyberScan pH meter was used to measure the pH of buffer solution. Analytical grade chemicals and distilled water (DW) were used. Personal protection equipment used include super nitrile gloves, apron, goggles (3M India), and half face respirator (3M India).

Preparation of Standard Solution

One hundred milligrams of TH was dissolved in 100 ml of DW in a volumetric flask and sonicated for 10 min. From this, 10 ml was taken and made up to 100 ml to obtain a standard solution containing 100 μg/ml.

Preparation of Sample Solution

Twenty tablets of commercially available TH 100 mg tablets were weighed and powdered. Tablet powder equivalent to one tablet was weighed and dissolved in 100 ml DW. This solution was sonicated and filtered through 0.4 μ nylon filter using a vacuum pump. Tramadol, being readily soluble in water, [3] gets completely filtered while the particulate matter gets trapped by the nylon filter. A volume of 10 ml filtered drug solution was made up to 100 ml and analyzed.

Preparation of Eriochrome Black T Solution and Acetate Buffer

One hundred milligrams of EBT was dissolved in 100 ml DW. To this solution, 25 ml of chloroform was added and stirred well to remove any impurity soluble in chloroform. This was allowed to stand for 10 min to allow the layers to separate. The upper layer of EBT solution was used in the analysis. One gram of anhydrous sodium acetate was dissolved in 230 ml of DW; pH adjusted to 3.5 using diluted glacial acetic acid and made up to 250 ml in a measuring cylinder to obtain the buffer solution.

Assay Procedure

To a series of labeled 10 ml volumetric flasks, 250 μl, 500 μl, 750 μl, and 1000 μl of standard solution were added with a micropipette. To each of these, 0.5 ml of acetate buffer and 1.5 ml of EBT solution were added. The drug-buffer-EBT sequence was followed always. The volume was made up to 10 ml with DW. The contents of volumetric flasks were transferred to a series of labeled 100 ml beakers. To each of these beakers, 10 ml of chloroform was also added. The colored aqueous and colorless chloroform layers thus formed were mixed well for 2 min using a magnetic stirrer to let the colored complex move from the aqueous to chloroform layer. After mixing, the contents of the beakers were transferred into a series of labeled separating funnels with glass stopcocks and mounted on stands. The mixed solutions were allowed to stand for 10 min for separation of the aqueous and colored chloroform layers. The latter could be seen in the bottom layer and was drained into labeled 50 ml beakers. The optical density (OD) of these colored chloroform layers was read on a colorimeter with the filter set to 520 nm and using glass cuvettes against a reagent blank treated similarly. Calibration curve was plotted with OD values (Y-axis) against the concentration of the drug (X-axis). OD of the sample solution was also measured in a similar manner and the drug content in the tablet was calculated.

Method Validation

The developed method was validated according to the ICH guidelines, [24] in terms of linearity, accuracy, recovery, precision, and robustness. The accuracy of the method was assessed by spiking a sample solution, the mean concentration of which was estimated in the previous step to be 100.8 μg/10 ml, with a known quantity of standard (25 μg added to 10 ml of sample) and the analysis was performed six times. The percentage recovery was then calculated. Precision was determined by intraday variability (repeatability) and interday variability (intermediate-precision) studies. For intraday variability testing, a fixed concentration (100 μg/10 ml) of standard solution was prepared and analyzed six times a day. For interday variability testing, a fixed concentration (100 μg/10 ml) of the standard solution was prepared and analyzed two times a day on 3 consecutive days by different analysts. Robustness, which provides an indication to the reliability of the method, was assessed by making small changes in the method,i.e., changing pH of the buffer by ±0.1 and measuring the effect on OD of a fixed concentration of standard (100 μg/10 ml). Limit of detection (LOD) and limit of quantitation (LOQ) were determined mathematically based on standard error and slope of calibration curve values. LOD is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.



The chloroform layer can be seen to take up the colored complex from the aqueous layer after stirring and the intensity of color increased as the concentration of drug increased. This trend was confirmed on measuring the OD [Table 1]. The linearity of the data can be inferred from the calibration curve plotted with the concentration of drug solution on X-axis and OD at 520 nm on Y-axis [Figure 2].{Figure 2}{Table 1}

The Beer's law limits, regression equation, correlation coefficient, LOD, and LOQ were calculated [Table 2]. The linearity of calibration curve can also be appreciated by the high value of correlation coefficient. The mathematically calculated LOD and LOQ values showed that the method is sensitive and that even lower concentrations could be estimated using this method.{Table 2}

Accuracy and Recovery

The amount of drug found in the commercial pharmaceutical tablet formulation was found to be in good agreement with the labeled amount [Table 3]. Spiking with 25 μg pure drug yielded a recovery of 99.5% [Table 4]. This indicates that the method is accurate and unaffected by excipients in the tablet.{Table 3}{Table 4}


Intraday precision (repeatability) estimated after six measurements of a fixed concentration of the standard solution on a single day was within 2% relative standard deviation (RSD) [Table 5]. Intermediate precision calculated after estimating six times the drug content in a fixed concentration of the standard solution on different days by different analysts was also within 2% RSD. These results demonstrate the precision of the method and its reproducibility in varying settings.{Table 5}


Small changes in pH of acetate buffer (±0.1) did not significantly affect the OD readings. This indicates the reliability of the method.


The principle of this method is that a solution of TH on reaction with EBT in the presence of acetate buffer at pH 3.5 forms an ion-pair complex (colored) which was extracted using chloroform. The OD of this chloroform layer was proportionate to the quantity of TH in the solution. The method used in this study can complete the estimation of TH content in tablet dosage forms in <3 h. All other methods described in the references mentioned above except spectrophotometry and spectrofluorimetry require more than 6 h for completing the estimation (including initialization of equipment and shutting down procedures) and need costly reagents. Since the method involves the use of chloroform, it is essential that proper personal protection equipment (mentioned in materials and methods) are used. Glassware containing chloroform should always be kept closed. The experiment should be done in a spacious and well-ventilated room. The glassware used, especially which come into contact with the colored complex, should be properly washed immediately after the experiment to prevent staining of the glassware. All the steps involved have been well optimized and details provided include the use of glassware and equipment for easy implementation in a health-care provider set-up with minimal investment.


The validation parameters discussed above show that the proposed method is reliable, repeatable, reproducible, accurate, and is not affected by excipients in tablet formulation. The results also demonstrate that the proposed method is ideal for the estimation of TH in pure drug form and tablet dosage form utilizing minimum resources and time. The added advantage is that it can be performed using a colorimeter which is available in all laboratories and hospitals, thereby facilitating the quality assessment of TH tablets dispensed.


We express our heartfelt gratitude to the faculty, nonteaching staff, and junior residents in the Department of Pharmacology, Government Medical College, Kottayam, Kerala, for their whole-hearted cooperation. The authors are thankful to the State Board of Medical Research - Institutional Review Committee, Kottayam, Kerala, for funding this study.

Financial Support and Sponsorship

The study was funded by the State Board of Medical Research, Kerala, India.

Conflicts of Interest

There are no conflicts of interest.


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