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 Table of Contents  
Year : 2014  |  Volume : 6  |  Issue : 4  |  Page : 246-252  

RP-LC gradient elution method for simultaneous determination of thiocolchicoside, aceclofenac and related impurities in tablet formulation

Department of Chemistry, C.B. Patel Research Centre, 3rdfloor, Bhaidas Hall, Vile Parle West, Mumbai, Maharashtra, India

Date of Submission20-Dec-2013
Date of Decision02-Feb-2014
Date of Acceptance17-Mar-2014
Date of Web Publication16-Oct-2014

Correspondence Address:
Pradnya A Karbhari
Department of Chemistry, C.B. Patel Research Centre, 3rdfloor, Bhaidas Hall, Vile Parle West, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.142955

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Objective: The aim of the present study is to develop a simple and precise HPLC method for simultaneous determination of thiocolchicoside, aceclofenac and related impurities in a tablet formulation and validate as per ICH guidelines. The aim of study extends to perform forced degradation study to trace the degradation pathways of potential degradant impurities. Materials and Methods: The separation was achieved on a 4.6 mm × 100 mm, 3 μm C 18 column at 40°C with the mobile phase containing 0.1 M ammonium acetate buffer and methanol in a gradient mode at a flow rate of 1.0 mL min−1 . The UV detection was carried out at 257 nm. Results: Acelofenac, thiocolchicoside and their related compounds were well separated from each other with good resolution and symmetry factor without interference of excipients. The method for assay was linear in the range of 10-200 μg mL−1 for aceclofenac and 0.4 to 8 μg mL−1 for thiocolchicoside. Conclusion: The method was validated according to ICH guidelines and the acceptance criteria for accuracy, precision, linearity, specificity, robustness, ruggedness and system suitability were met in all cases. The method was highly specific, as two related compounds of thiocolchicoside and nine related compounds of aceclofenac were well separated from each other. Stress study ensured the specificity of the method as the unknown degradation products formed during stress studies did not interfere with the determination of thiocolchicoside and aceclofenac, thus proving the stability indicating capacity of the method.

Keywords: Aceclofenac, colchicoside, diclofenac, gradient elution, related compounds, reverse phase high performance liquid chromatography, simultaneous determination, stability indicating, thiocolchicoside

How to cite this article:
Karbhari PA, Joshi SJ, Bhoir SI. RP-LC gradient elution method for simultaneous determination of thiocolchicoside, aceclofenac and related impurities in tablet formulation. J Pharm Bioall Sci 2014;6:246-52

How to cite this URL:
Karbhari PA, Joshi SJ, Bhoir SI. RP-LC gradient elution method for simultaneous determination of thiocolchicoside, aceclofenac and related impurities in tablet formulation. J Pharm Bioall Sci [serial online] 2014 [cited 2022 Aug 13];6:246-52. Available from:

   Introduction Top

Tablet formulation containing the NSAID aceclofenac (AC) -[2-[(2,6-Dichlorophenyl)amino]phenyl]acetyl]oxy]acetic acid and muscle relaxant thiocolchicoside (TC)- N-[(7S)-3-(beta-D-glucopyranosyloxy)-1,2-dimethoxy-10-(methylsulfanyl)- 9-oxo-5, 6, 7, 9-tetrahydrobenzo[a] heptalen- 7-yl] acetamide is used in the treatment of severe pain. It is an important aspect of drug development that the final pharmaceutical product reaching to patients should be safe, efficient and of desired quality. Therefore, it is necessary to evaluate these before release in market. Bioavailability, bioequivalence studies help to evaluate efficacy and safety whereas quality control testing evaluates the quality of product. Among these testing, potency test and impurity test are important.

The aim of present study is to develop and validate method for assaying the drug and related impurities of the combination drug product.

Literature studies show various analytical methods reported for the estimation of AC in plasma by HPLC, [1],[2] LC-MS, [3] in other matrix by HPLC, [4] stability-indicating method by spectrophotometry and densitometry, [5],[6] in combination with other NSAID like tramadol, paracetamol. [7],[8] A UV-visible derivative spectroscopic method has been reported for AC in combination with paracetamol and tramadol. [9]

Analytical methods like densitometry, capillary electrophoresis and spectrometry have been reported for the determination of TC either alone or in combination. [10],[11],[12],[13],[14]

In a regulated market drug product compliance to ICH requirements for specification and impurities or degradation products is a mandatory requirement. [15]

In the present study, a simple, accurate and precise RP-HPLC method for simultaneous determination of AC and TC along with related impurities from tablet formulation was developed and validated.

To the best of our knowledge, there is no stability indicating method available for simultaneous determination of AC with TC and related impurities.


Instrument and chromatographic conditions

Integrated HPLC system, Waters Alliance manufactured by Waters Corporation (Milford, USA) was used for method development and method validation. This system comprised of a quaternary gradient pump and auto sampler (Waters 2695 Separation module), column oven and a photo diode array detector (Waters 2998). PC installed Waters Empower software, Version 2.6, was used to record and to integrate the chromatograms.

A mobile phase consisted of 0.1 M ammonium acetate buffer and methanol filtered and degassed separately through membrane filter and delivered in a gradient mode. Thermo Hypersil C 18 (4.6 mm × 100 mm, 3 μm) analytical column from Thermo fisher scientific, (Mumbai, India) was used as a stationary phase. The flow rate was 1.0 mL min−1 and the detector was set at 257 nm.

The column oven temperature was maintained at 40°C and the volume of solution injected was 20 μL.

   Materials and Methods Top

AC and TC working standards were obtained from a reputed pharmaceutical company (Mumbai, India). Analytical reagent grade ammonium acetate and HPLC grade methanol were obtained from Merck (Mumbai, India). A Millipore Milli Q plus water purification system (Milford, USA), was used to prepare distilled water (>18 μ Ω). Test samples used were composed of AC and TC purchased from the local market, Zerodol-TH, Ipca Laboratories Ltd, India, of strength 100 mg and 4 mg per tablet, respectively.

Preparation of standard solution

AC and TC standard stock solutions were prepared by transferring accurately weighed quantities of 40 mg TC and 100 mg AC working standards into two different 100 mL volumetric flasks. Water: Methanol 50:50 (v/v) was used as a diluent. To both the flasks diluent was added up to the one-third capacity of the flasks and sonicated for few minutes to solubilize. The solutions were individually diluted up to the mark with the diluent and mixed. 1 mL of TC stock solution and 10 mL of AC stock solution were diluted to 100 mL with diluent to obtain a concentration of 100 μg mL−1 for AC and 4 μg mL−1 for TC. This solution was further diluted to prepare diluted standard for impurities test which contained 2.5 μg mL−1 for AC and 1 μg mL−1 for TC.

Test solution preparation

Five tablets containing 4 mg TC and 100 mg AC each were transferred into a 200 mL volumetric flask. 50 mL of diluent was added to disperse the tablets. The flask was shaken for 15 min so that the tablets disintegrate completely. The diluent was added up to the one third capacity of the flask and sonicated for 15 min. The flask was cool to room temperature. The volume was made up to the mark with diluent and mixed. The solution was filtered through a 0.45 μm PVDF membrane syringe filter from Merck Millipore and used for determination of related substances. 4 mL of the filtrate was diluted to 100 mL with diluent to give a test concentration containing 100 μg mL−1 for AC and 4 μg mL−1 for TC for an assay.

The amount of TC and AC per tablet was calculated from the peak area of AC and TC in the chromatograms of the test solution and standard solution, respectively.


The specificity of a method is its suitability for analysis of a substance in the presence of potential impurities. Stress testing of a drug substance can help identify likely degradation products, which can in turn help establish degradation pathways and the intrinsic stability of the molecule. It can also be used to validate the stability-indicating power of the analytical procedures used.

The specificity of the LC method for TC and AC was determined in the presence of 11 impurities and degradation products which included colchicoside, colchicine of TC and imp A, imp B, imp C, imp D, imp E, imp F, imp G, imp H, imp I of Aceclofenac [Figure 1].
Figure 1: IUPAC names, structure and category of the components under study

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Forced degradation of TC and AC was also performed to provide an indication of the stability-indicating properties and specificity of the method. [16],[17] The tablet formulation was also stressed along with active pharmaceutical ingredients. The stress conditions used for the degradation study included light (conducted as stipulated in ICH Q1B), heat (60°C), acid hydrolysis (0.5 M HCl), basic hydrolysis (0.5 M NaOH) and oxidation (10% H 2 O 2 ). The purity of peaks of TC and AC obtained from stressed samples was checked by use of the PDA detector. The purity angle is less than the purity threshold limit obtained in all stressed samples demonstrated the analyte peak homogeneity.

An assay of stressed samples was performed by a comparison with reference standard and the mass balance (% assay + % impurities + % degradation products) was calculated.

   Results and Discussion Top

Development of chromatography

Initially, a microsorb pursuit varian C18 column having dimensions 150 mm × 4.6 mm and particle size 5 μm was used with water: Methanol as a mobile phase in a ratio of 60:40 (v/v) at a flow rate of 1.0 mL min−1 using 257 nm as a detection wavelength. This resulted in too early elution of TC almost in the void volume. When water in the mobile phase was replaced with 0.5 M ammonium acetate, the retention of TC was increased but the polar component AC was highly retained on the column. For the faster elution of AC peak, it was necessary to increase the eluotropic strength of the mobile phase. Therefore, to obtain desirable retention for two components having opposite nature, one polar and other non-polar, a gradient program was introduced. The final gradient program started with buffer: Methanol composition 70:30 (v/v) and linearly changed to 70% methanol within 10 min and the isocratic gradient with buffer: Methanol composition 30:70 (v/v) was run up to 25 min and then linearly changed to original composition 70:30 within next 3 min (28 min) and allowed to stabilize to original composition till next 2 min so as to complete the run time of 30 min.

The gradient program was optimized in such a way that two impurities of TC and nine impurities of AC were well separated with a minimum resolution of 2.0 The USP resolution between peaks of TC and AC was 50 in the assay method [Figure 2].
Figure 2: A-typical chromatogram of separation, As per order of elusion, 1-Colchicoside, 2-Thiocolchicoside, 3-Colchicine, 4-Diclofenac, 5-Aceclofenac, 6-Impurity I of Aceclofenac, 7-Impurity G of Aceclofenac, 8-Impurity H of Aceclofenac, 9-Impurity D of Aceclofenac, 10-Impurity B of Aceclofenac, 11-Impurity E of Aceclofenac, 12-Impurity C of Aceclofenac, 13-Impurity F of Aceclofenac, B- Chromatogram of Oxidation of tablet Sample, C- Chromatogram of Basic hydrolysis of tablet Sample, D- Chromatogram of Acidic hydrolysis of tablet Sample

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The column length was reduced to 100 mm and particle size to 3 μm so as to achieve shorter run time and better resolution. The combination of methanol and aqueous buffer increased the viscosity of the mobile phase. The column temperature of 40°C was used to reduce the backpressure generated due to the high viscosity of the mobile phase.

The test sample concentration was finalized based on the requirement for limit of quantitation value being less than the reporting threshold. [18] Considering the maximum daily dose for AC and TC, the reporting threshold is 0.1%.

Result from forced degradation studies

Degradation was not observed when TC and AC were exposed to heat and photolytic degradation. TC was found to be degraded in oxidation and colchicoside was formed as a major degradant which was confirmed by spiking colchicoside in degraded samples. TC was also degraded in acidic hydrolysis to form unknown impurity at RRT 0.59 (RRT wrt AC).

AC was found to be degraded in basic hydrolysis, acidic hydrolysis and very slight degradation was observed in oxidation.

Diclofenac and impurity I were found to be the major degradants of AC in basic hydrolysis. Other degradants of AC found were impurity D and impurity B [Figure 2].

Peak purity results from the PDA detector for the peaks of TC and AC produced after degradation were homogeneous and pure for all the stress samples analyzed.

The mass balance for the stressed samples was close to 100 for TC and AC [Table 1].
Table 1: Forced degradation

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Method validation

Method validation was carried out as per ICH guidelines for parameters such as precision, linearity, accuracy, limit of detection and quantitation, robustness, response factor and stability in solution. [19]

Limit of detection (LOD) and quantification (LOQ)

LOD and LOQ for the impurities and analytes were estimated as the amounts for which the signal-to-noise ratios were 3:1 and 10:1 respectively. The study was performed by injecting a series of dilute solutions of known concentration within the developed chromatographic conditions. The limit of detection for all studied impurities was below 0.01% and limit of quantitation was below 0.05% of the test concentration which is well below the reporting threshold of 0.1% [Table 2].
Table 2: Results from validation study of related substances method

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Solution for testing linearity for the related substances were prepared by diluting the impurity stock solution to different concentrations from the LOQ to 160% of the qualification threshold of the impurity (i.e. LOQ to1.6% for an analyte concentration of 100 μg mL−1 TC) LOQ to 160% (i.e. LOQ to 0.16% for an analyte concentration of 2500 μg mL−1 for AC) [Table 2] and [Table 3].
Table 3: Validation study parameters of assay test method

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To test the linearity of the assay method, solutions at six concentrations from 10% to 200% of the analyte concentration (0.4 to 8, μg mL−1 for TC and 10 to 200 μg mL−1 for AC) were prepared from the stock solution. Least-squares linear regression analysis was performed on peak area and concentration data [Table 3].


The accuracy of the assay method for TC and AC was evaluated in triplicate at three concentrations 3.2, 4, 4.8 μg mL−1 for TC and 80, 100, and 120 μg mL−1 for AC and recovery was calculated [Table 3].

For the impurities, recovery was determined in triplicate at 0.8, 1.0 and 1.2 μg mL−1 for CS and CC and 2.0, 2.5 and 3.0 μg mL−1 (0.08, 0.1 and 0.12%) of the analyte concentration (2500 μg mL−1 for AC) and % recovery of the impurities was found within 85% to 115%[Table 2].


The precision of the related substance method was checked by six-fold analysis of tablet sample spiked with 0.5% of each impurity. The RSD (%) of peak area for each impurity which was within 5% confirmed precision of the method. The precision of the assay was evaluated by performing six independent assays of a test sample and quantifying using an external standard method using reference standard. The RSD (%) of the six results within 2% confirmed method to be precise for assaying of AC and TC in the presence of its impurities.

To evaluate the intermediate precision (ruggedness) of the method, the analysis was performed on a different day using a different instrument in the same laboratory [Table 3] and [Table 4].
Table 4: Accuracy and precision study for impurities

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To determine the robustness of the method the experimental conditions were deliberately altered and checked for the system suitability criteria, i.e. resolution between TC and AC and tailing factor. The chromatographic conditions which were altered are flow rate (±0.2 mL min−1 ; column oven temperature (±5°C), molarity of buffer solution (±0.002 units), different column make. The tabular results show that the method is robust for the studied changes [Table 5].
Table 5: Robustness study

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Relative response factor (RRF) and correction factor (CF) study

Response factor is a relative term, being the response of equal weights of one substance relative to that of another in the conditions described in the test. The RRF value for given impurity as the ratio of response of peak at a particular concentration to that of analyte peak at that concentration. The RRF values less than 0.2 and more than 5.0 are not acceptable as per European pharmacopoeia. In such cases there is a need for change in chromatographic parameters like wavelength or different method of visualization is used. The extrapolation of linearity study was done to determine RRF and CF [Table 2].

Stability in Solution

The stability of TC and AC and their impurities in solution was determined by keeping test solutions of the sample and reference standard and spiked sample solution volumetric flasks at room temperature for 24 h at bench top and withdrawn at regular time intervals to assay the analytes. The absolute difference of assay values within 2% and impurities from initial value concluded solution stability for 24 h for assay however related substances were found stable up to 12 hours.

   Conclusions Top

The proposed method is simple, specific, accurate and precise for determination of AC and TC in the presence of degradants and process related impurities. The method is also applicable to study content uniformity and assay of dissolution test. Thus, the method can to use for quality control of all types of pharmaceutical preparations like modified release tablets containing AC and TC.

   References Top

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Musmade P, Subramanian G, Srinivasan KK. High-Performance liquid chromatography and Pharmacokinetics of aceclofenac in rats. Anal Chim Acta 2007;585:103-9.  Back to cited text no. 2
Kang W, Kim EY. Simultaneous determination of aceclofenac and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 2008;46:587-91.  Back to cited text no. 3
Bhinge JR, Kumar RV, Sinha VR. A simple and sensitive stability indicating RP-HPLC assay method for the determination of Aceclofenac. J Chromatogr Sci 2008;46:440-4.  Back to cited text no. 4
el-Saharty YS, Refaat M, el-Khateeb SZ. Stability indicating spectrophotometric and densitometric methods for determination of aceclofenac. Drug Dev Ind Pharm 2002;28:571-82.  Back to cited text no. 5
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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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