INTRODUCTION:

Tramadol hydrochloride is a narcotic analgesic drug. Tramadol is 4-phenyl-piperidine analogue (2-(dimethylaminomethyl)-1-(3-methoxyphenyl) cyclohexanol) of codeine. Chemical name of Tramadol hydrochloride is (1R, 2R)-2- [(Dimethyl amino) methyl]-1-(3-methoxy phenyl) cyclo hexan-1-ol. The molecular formula and molecular weight of Tramadol hydrochloride is C₁₆H₂₆ClN0₂ and 299.84 g respectively. Tramadol hydrochloride is white to off-white soluble in water and organic solvents, it possess pKa value of 9.41 and melting point of 120°C. Biological half life of Tramadol hydrochloride is 6-7 Hours with bioavailability of 70-80 %. Tramadol hydrochloride is actively soluble in water and ethanol. Tramadol hydrochloride is used to treat post operative, dental, cancer and acute musculo skeletal pain and adjuvant to NSAID therapy.^1-4

Tramadol, a centrally acting analgesic structurally related to codeine and morphine, consists of two enantiomers, both of which contribute to analgesic activity via different mechanisms. (+)-Tramadol and the metabolite (+)-O-desmethyl-tramadol (M1) are agonists of the mu opioid receptor. (+)-Tramadol inhibits serotonin reuptake and (-)-tramadol inhibits norepinephrine reuptake, enhancing inhibitory effects on pain transmission in the spinal cord. After oral administration, tramadol is rapidly and almost completely absorbed. Sustained-release tablets release the active ingredient over a period of 12 hours, reach peak concentrations after 4.9 hours and have a bioavailability of 87-95% compared with capsules. Tramadol is rapidly distributed in the body; plasma protein binding is about 20%.^1,2,5-10 Tramadol is mainly metabolised by O- and N-demethylation and by conjugation reactions forming glucuronides and sulfates. Tramadol and its metabolites are mainly excreted via the kidneys. The mean elimination half-life is about 6 hours.^11-14

The structure, chemical name, molecular formula and molecular weight of Tramadol hydrochloride and its impurities (A, B, C, D, E) are tabulated in Table 1.^6-13Joshi et al. formulated and evaluated matrix tablet of Tramadol Hydrochloride.¹⁵ Raval et al. formulated and evaluated mouth dissolving tablet of Tramadol Hydrochloride.¹⁶ Satinder et al. formulated and evaluated orodispersible tablet of Tramadol Hydrochloride.¹⁷ Kawai et al. determined the efficacy and safety of Tramadol Hydrochloride sustained-release bilayer tablet. ¹⁸ Vinay et al. determined Tramadol hydrochloride by acid-base titration.¹⁹ Nidhi et al. developed and validated ultraviolet spectroscopy for determination of Tramadol hydrochloride.²⁰ Ahir et al developed a RP-HPLC method for simultaneous estimation of Tramadol HCl, Paracetamol and Domperidone.²¹ Chandra et al. developed a method for simultaneous analysis of Aceclofenac, Paracetamol and Tramadol hydrochloride.²²Aminove et al. developed and validated stability indicating assay method for Lornoxicom and Tramadol in Tablet dosage form by RP-HPLC.²³Syamalambica developed and validated RP-HPLC Method for Estimation of Tramadol Hydrochloride and Paracetamol in Pharmaceutical Formulation.²⁴ Anil et al. developed UPLC Method for Tramadol HCl Impurities in the Tramadol Injection.²⁵Scaria at al. developed and validated RP-HPLC Method for simultaneous estimation of Paracetamol and Tramadol Hydrochloride.²⁶ Jigar et al. developed and validated RP-HPC method for the estimation of Diclofenac sodium, Tramadol hydrochloride and Chlorzoxazone from tablet.²⁷ Susmitha et al. developed and validated RP-HPLC method for the simultaneous estimation of Dexketoprofen Trometamol and Tramadol Hydrochloride.²⁸ Hemnat et al. developed RP-HPLC method for estimation of tramadol hydrochloride and paracetamol.²⁹ Shailesh et al. developed and validated RP- Hplc Method for the Simultaneous Estimation of Tramadol Hydrochloride and Dicyclomine.³⁰ Pereira et al. developed and validated RP-HPLC-PDA Method for determination of Paracetamol, Caffeine and Tramadol Hydrochloride.³¹ Augustin et al. optimized HPLC method for tramadol and O-desmethyl tramadol determination in human plasma.³² Hazem et al. determined tramadol hydrochloride in biological fluids and pharmaceutical preparations utilizing a modified carbon paste electrode.³³ A validated UV spectrophotomeric was developed for combination of drug of Tramadol and Ketorolac Tromethamine.³⁴ Beedha et al. formulated and characterized transdermal patch.³⁵ Rohan et al. formulated and evaluated fast dissolving tablet of Tramadol.³⁶ Research work is done on some more formulations and development of RP-HPLC methods.^37-40 Some more research is done related to Tramadol.^41-43

Tramadol Hydrochloride is a costly drug; it has international market and had wider use as an analgesic. Aim is to develop and validate assay method which is more economical and should have its application to work place with high performance and relevant results. Development of assay method for Tramadol hydrochloride capsules for estimation of Tramadol hydrochloride related compounds (or impurities). Objectives are to develop simple, rapid, sensitive, precise and accurate RP-HPLC method for the assay of Tramadol hydrochloride capsules.^44-46

MATERIAL AND METHODS:

Chemicals and Reagents:

Tramadol Hydrochloride working standard; Tramadol Hydrochloride capsules 50mg; Tramadol Hydrochloride related substances or impurities (Impurity-A, Impurity-B, Impurity-C, Impurity-D and Impurity-E) of Synzeal grade. Acetonitrile (HPLC Grade), Trifluoroacetic acid (analytical reagent grade) and milli-Q water were used.

Preparation of buffer:

Transferred 2mL of Trifluoro acetic acid in 1000 mL volumetric flask. The volume was make up with Milli-Q water to obtain 0.2% of Trifluoro acetic acid buffer.

Preparation of mobile phase:

Acetonitrile and 0.2% of Trifluoro acetic acid were mixed in the ratio of 29.5:70.5% V/V respectively. Then the solution were filtered through 0.45µm Nylon membrane filter and degassed prior to use.

Preparation of diluent:

Mobile phase was employed as diluent for all solutions preparations.

Chromatographic conditions:

A high performance liquid chromatography equipped with a pump, an injection module, PDA detector and a suitable data capture device was used. Waters Empower software was used for HPLC data analysis. The optimized mobile phase is Acetonitrile and 0.2% of Triflouro acetic acid buffer in the ration of 29.5:70.5% V/V respectively, and pumped at a flow rate of 1.0 mL/ min. Separations done by isocratic mode of elution with a run time of 30min. The column employed is Thermo, Hypersil BDS C₈ (250mm × 4.6mm, 5µm) maintained at a temperature of 25°C. Injection volume and detector wavelength are 20µL and 270nm respectively.

Preparation of Solutions:

Preparation of Tramadol hydrochloride working standard solution:

Weighed accurately about 40mg of Tramadol hydrochloride working standard and was transferred into a 100 mL volumetric flask. About 50mL of diluent was added and sonicated. The volume was make up to obtain a concentration of about 400µg/mL (working standard stock solution). Solution was stirred by mechanical means for 15 minutes. Then the solution was filtered through Whatmann no 41 filter paper. 3mL of the filtrate was diluted to 20mL with diluent to obtain a concentration of about 60µg/mL of Tramadol hydrochloride.

Preparation of system suitability solution:

Weighed accurately about 5mg of Tramadol Hydrochloride, and Tramadol hydrochloride impurities (Impurity A, B, C, D and E) and transferred it into a 100 mL volumetric flask, stirred by mechanical means for 15 min. The volume was make up with diluent.

Preparation of Assay sample solution:

Weighed about 20 capsules. The contents of capsules were taken and the average fill weight of capsules content was determined. The capsules contents were crushed into a fine powder in a mortar with pestle. Accurately weighed a portion of the powdered capsules equivalent to about 37.5mg of Tramadol hydrochloride and was transferred into a 100mL volumetric flask. About 70mL of diluent was added and stirred by mechanical means for 30min to dissolve. The solution was allowed to equilibrate to room temperature, diluted to volume with diluent and mixed well. The sample solution was filtered through a Whatmann no 41 filter paper. Transferred 4mL of filtrate into a 25mL volumetric flask, diluted to volume with diluent and mixed well to obtain a concentration of about 60µg/mL of Tramadol hydrochloride. All the assay samples in replicate of six were prepared in the same manner as described above.

Preparation of impurities stock solution:

Impurity A, B, C, D and E were prepared in the concentration of 100µg/mL individually, each by weighing about 20mg of Impurity into a 200mL volumetric flask. Add about 140mL of diluent was added and sonicated to dissolve. Volume was make up with diluent.

Preparation of Assay sample solution spiked with known impurities:

Accurately weighed a portion of the powdered capsules equivalent to 37.5mg of Tramadol Hydrochloride and was transferred into a 100mL volumetric flask. About 70 mL of diluent was added and stirred by mechanical means for 30 min to dissolve. The solution was allowed to equilibrate to room temperature. The sample solution was spiked with 1mL of each impurity A, B, C, D, and E solution (from 100µg/mL impurities stock solution). The volume was make up with diluent and mixed well. Filtered the sample solution through a Whatmann No 41 filter paper. Transferred 4mL of filtrate into a 25mL volumetric flask, diluted to volume with diluent and mixed well. The obtained concentration was about 60µg/mL of Tramadol hydrochloride and 0.16µg/mL of each impurity A, B, C, D and E.

Validation:

Method development and validation done as per ICH Q8, Q14 and Q2 (R2) guidelines.^34-36

System Suitability test:

The working standard solution of about 60µg/mL concentration was injected in five replicates for system suitability testing.

Specificity:

Specificity was determined by injecting a blank solution, placebo solution, Tramadol hydrochloride assay sample solution spiked with impurities.

Linearity and Range:

The working standard stock solution was diluted into six concentrations by diluent to obtain solutions in the range of about 6µg/mL to 121µg/mL of Tramadol hydrochloride. From the working standard stock solution (about 400µg/mL) serial dilutions were done by pipetting out of 3mL, 7.5mL, 7.5mL, 3.0mL, 4.5mL and 6.0mL and diluted to 200mL, 200mL, 100mL, 20mL, 20mL and 20mL with diluent to obtain concentrations of Tramadol hydrochloride of about 6, 15, 30, 60, 90 and 121µg/mL respectively. This concentration represents the range from 10% to 200% level of the target concentration. Impurity-A solutions were prepared in the range from LOQ level to 150% level of known impurity-A. Correlation coefficient was determined by regression analysis method.

Precision:

Precision was performed by injecting six assay sample solutions and %RSD was calculated. Repeatability was tested by injecting six test sample solutions prepared as per the above-mentioned procedure. Intermediate Precision (Ruggedness) was performed by two analysts within the same laboratory

Accuracy/ Recovery:

Recovery samples of Tramadol hydrochloride working standard were prepared at the levels of 50%, 100% and 120% of target concentration in triplicates i.e, to obtain concentrations of about 30µg/mL, 60µg/mL and 90 µg/mL of Tramadol hydrochloride. Impurity-A was subjected to recovery analysis at LOQ, 50, 100 and 200 % level in triplicates. %recovery was calculated and reported.

Robustness:

Robustness was performed for the factors of flow rate, temperature variation, and organic phase composition. Robustness for flow rate was done at 1.0mL/min ±0.1, i.e., at 0.9mL/min and 1.1mL/min. Robustness for temperature was done at 25°C±5°C, i.e., at 20°C and 30°C. Robustness for organic phase composition was done at 29.5:70.5% V/V (Acetonitrile: Buffer) ±2%, ie., 27.5:72.5% V/V (Acetonitrile: Buffer) and 31.5:68.5% V/V(Acetonitrile:Buffer). %RSD and system suitability parameters were evaluated for Tramadol hydrochloride and its capsule.

Filter Integrity Test:

Assay sample solutions were prepared in triplicates as per the above describe procedure and 400µg/mL sample solution was prepared. Solution was filtered through Whatmann filter paper. The filtrate was centrifuged. Supernatant liquid was filtered through 0.45µm Nylon (Millipore brand) filter, initially certain amount is discarded and remaining filtrate was diluted to obtain sample solution of about 60µg/mL concentration.

Stability Studies:

Tramadol hydrochloride working standard solution and assay sample solution was stored and analyzed at 4^th, 8^th, 12^th, 18^th and 24hour. These stored solutions were analyzed against freshly prepared working standard solution of Tramadol hydrochloride. % RSD, system suitability parameters and % assay were recorded and reported.

RESULTS AND DISCUSSION:

Determination of wavelength:

A microgram concentration solution of Tramadol hydrochloride was prepared in diluent and its (λ_max) was determined by UV-Visible spectrophotometer in spectrum mode.

System Suitability test and Specificity:

No peaks were detected in the retention time window of Tramadol Hydrochloride from the injection of placebo solution. No peaks were detected at Rt of Tramadol hydrochloride and its impurities in blank and placebo. All impurities, blank and placebo peaks are adequately resolved from Tramadol Hydrochloride peak. The Rt (Retention time), RRT (Relative retention time) and peak purity of Tramadol hydrochloride and its impurities are given in Table 2. The system suitability results of Tramadol hydrochloride are given in Table 3. The chromatogram of blank, placebo, Tramadol hydrochloride working standard and assay sample spiked with impurities are represented in figure 2.

Linearity and Range:

The method was found to be linear for Tramadol hydrochloride (correlation coefficient of 0.999) over a range of 10% to 200% of the target concentration which corresponds to about 6µg/mL to 12µg/mL. The linearity results of Tramadol hydrochloride are given in Table 3. The method was found to be linear for Impurity-A (correlation coefficient of 0.996) over a range from LOQ level to 150% level of the target concentration which corresponds to about 0.07µg/mL to 6.4µg/mL. The linearity results of Impurity-A are given in Table 3. The LOD and LOQ values of Tramadol hydrochloride were found to be 0.80 and 2.42µg/mL respectively, as given in table 3. The LOD and LOQ values of Impurity-A were found to be 0.51 and 1.54µg/mL respectively, as given in table 3. Linearity plot of Tramadol hydrochloride and Impurity-A are represented in Figure 1.

Table 3. Validation Results

S. No.

Parameter

Obtained results

System suitability

Blank Interference

Placebo Interference

% RSD of standard solution, at 100% level i.e., 61µg/mL (Five replicates)

USP Plate count

Tailing factor

No interference was observed at Rt of Tramadol standard and its impurities

0.3 (Acceptance criteria-Not more than 2%)

9309

1.3

Linearity Range

For Tramadol standard

For Impurity-A

10 % to 200 % level of target concentration i.e., 6 to 121 µg/mL

LOQ to 150 % level of target concentration i.e., 0.07 to 6.4 µg/mL

Regression equation

For Tramadol standard

For Impurity-A

Y = 6853 x – 858.5

Y = 6905 x – 754.4

Correlation coefficient

For Tramadol standard

For Impurity-A

0.999

0.996

Y intercept at 100 % level

For Tramadol standard

For Impurity-A

0.0

-1.05

LOD and signal to noise ratio (S/N)

For Tramadol standard

For Impurity-A

LOD: 0.80, S/N-4.5

LOD: 0.51, S/N-2.6

LOQ and signal to noise ratio (S/N)

For Tramadol standard

For Impurity-A

LOQ: 2.42, S/N-11.3

LOQ: 1.54, S/N-10.5

Precision- Repeatability

Assay samples-Mean % ± RSD

99.7 ± 0.6

Recovery % ± SD

For Tramadol

50 % Level, Mean Recovery % ± RSD

100% Level, Mean Recovery % ± RSD

150% Level, Mean Recovery % ± RSD

For Impurity-A

LOQ Level, Mean Recovery % ± RSD

50 % Level, Mean Recovery % ± RSD

100% Level, Mean Recovery % ± RSD

150% Level, Mean Recovery % ± RSD

101.8 ± 0.4

99.0 ± 0.3

99.3 ± 0.6

86 ± 0.0

91 ± 0.7

94 ± 1.4

93 ± 0.6

10.

Precision- Intermediate Precision/ Ruggedness

Assay samples-Mean % ± RSD

100.3 ± 0.8

Table-5. Solution stability – Standard and sample solution

Station	Standard	Sample
	% Found	% Found	% difference
Initial	99.7	100.3	NA
4^th Hour	99.8	100.4	0.1
8^th Hour	99.9	100.5	0.2
12^th Hour	100.1	100.6	0.3
18^th Hour	100.3	100.7	0.4
24^th hour	100.3	100.9	0.6

Filter Integrity Test:

Filter integrity test results for Tramadol hydrochloride standard solution are reported in Table 6 and all the values were found to be within limits.

Filter integrity test results for assay sample solution are reported in Table 7 and all the values were found to be within limits.

Table-6. Filter study results for Standard solution

Parameter	Whatmann No 41	0.45µ Nylon membrane	Centrifuge
Resolution	3.0	3.0	2.9
Relative Retention time	0.88	0.87	0.88
Theoretical plates	8272	8090	7879
Tailing Factor	1.6	1.6	1.6
%RSD	0.1	0.2	0.0
System performance	0.1	0.1	0.3
Similarity Factor	0.98	0.98	1.01

Table 7. Filter study results for Sample solution

Replicate	%Assay
Replicate	Whatmann No 41	Centrifuge	0.45µ Nylon
1	100.4	99.6	100.0
2	100.6	101.5	101.8
3	101.1	103.9	100.1
Avg	100.7	101.7	100.6
Difference	NA	1.7	0.1

CONCLUSION:

The method is developed for the qualitative and quantitative analysis of Tramadol Hydrochloride and its related compounds in capsule dosage form. The developed method is found to be rapid, accurate, precise, selective and reproducible. The developed RP-HPLC method is specific and no interference was observed at the Tramadol hydrochloride or impurities peak, and the peak purity index was found to be 1.000. The obtained validation results for Tramadol hydrochloride and Impurity-A reveals that the developed method is found to be specific, linear, precise, accurate, robust and rugged. The developed method is analyzed for stability and filter integrity testing and all the values were within acceptable range. The method can be employed in quality control laboratory for regular analysis of Tramadol hydrochloride in capsules.

DECLARATION OF INTEREST:

We declare no conflicts of interest.

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Received on 30.07.2025 Revised on 29.08.2025

Accepted on 15.09.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):279-287.

DOI: 10.52711/2231-5675.2025.00044

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Name of compound	Structure	Chemical Name	Other details
Tramadol hydrochloride		(1R,2R)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl) cyclohexan-1-ol; hydrochloride	Molecular Formula: C₁₆H₂₅NO_2.HCl Molecular weight: 299.836
Impurity A or trans-Tramadol		(1R,2S)-2-((dimethylamino)methyl)-1-(3-methoxyphenyl) cyclohexanol hydrochloride	Molecular Formula: C₁₆H₂₅NO₂ : HCl Molecular weight: 263.4: 36.5
Impurity B		[2-(3-methoxyphenyl) cyclohex-1-en-1-yl]-N, N-dimethylmethanamine hydrochloride	Molecular Formula: C₁₆H₂₃NO: HCl Molecular weight: 245.4: 36.5
Impurity C or 1,6-dehydro Tramado		(1RS)-[2-(3-methoxyphenyl) cyclohex-2-enyl]-N, N-dimethylmethanamine hydrochloride	Molecular Formula: C₁₆H₂₃NO: HCl Molecular weight: 245.4: 36.5
Impurity D or O-Desmethyl Tramadol		2-[(Dimethylamino)methyl]-1-(3-hydroxyphenyl) cyclohexanol hydrochloride	Molecular Formula: C₁₅H₂₃NO₂ : HCl Molecular weight: 249.4: 36.5
Impurity D		(2RS)-2-[(dimethylamino) methyl] cyclohexanone hydrochloride	Molecular Formula: C₉H₁₈ClNO Molecular weight: 191.7

Name	RT (min)	RRT	Peak purity
Impurity-E	3.49	0.55	1.0000
Impurity-D	4.23	0.67	1.0000
Impurity-A	5.69	0.89	1.0000
Tramadol hydrochloride	6.36	1.00	1.0000
Impurity-C	14.88	2.34	1.0000
Impurity-B	17.35	2.73	1.0000

Robustness	Parameter	Theoretical plates	Tailing Factor	%RSD	% Assay
Developed Method		9309	1.3	0.3	99.7
Flow rate	0.9 ml/min	10966	1.3	0.1	99.4
Flow rate	1.1 ml/min	9988	1.3	0.0	99.5
Column temperature	Low temp(20°c)	10322	1.3	0.1	100.7
Column temperature	High temp(30°c)	10638	1.3	0.2	101.0
Mobile Phase composition	72.5% Buffer: 27.5% ACN	10971	1.3	0.3	99.8
Mobile Phase composition	68.5% Buffer: 31.5% ACN	9771	1.3	0.1	99.9