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RP-LC Gradient Elution Method for Simultaneous Determination of Thiocolchicoside, Aceclofenac and Related Impurities in Tablet Formulation

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RP-LC Gradient Elution Method for Simultaneous Determination of Thiocolchicoside, Aceclofenac and Related Impurities in Tablet Formulation Original Article RP-LC gradient elution method for simultaneous determination of thiocolchicoside, aceclofenac and related impurities in tablet formulation Pradnya A. Karbhari, Sneha J. Joshi, Suvarna I. Bhoir Department of Chemistry, ABSTRACT C.B. Patel Research Objective: The aim of the present study is to develop a simple and precise HPLC method for simultaneous Centre, 3rd floor, Bhaidas Hall, Vile Parle West, determination of thiocolchicoside, aceclofenac and related impurities in a tablet formulation and validate as Mumbai, Maharashtra, per ICH guidelines. The aim of study extends to perform forced degradation study to trace the degradation India pathways of potential degradant impurities. Materials and Methods: The separation was achieved on a 4.6 mm × 100 mm, 3 μm C18 column at 40ºC with the mobile phase containing 0.1 M ammonium acetate Address for correspondence: buffer and methanol in a gradient mode at a flow rate of 1.0 mL min−1. The UV detection was carried out Ms. Pradnya A. Karbhari, at 257 nm. Results: Acelofenac, thiocolchicoside and their related compounds were well separated from E-mail: pradnyapmhatre@ yahoo.co.in 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. Received : 20-12-13 Review completed : 02-02-14 KEY WORDS: Aceclofenac, colchicoside, diclofenac, gradient elution, related compounds, reverse phase Accepted : 17-03-14 high performance liquid chromatography, simultaneous determination, stability indicating, thiocolchicoside Introduction final pharmaceutical product reaching to patients should be safe, efficient and of desired quality. Therefore, it is necessary ablet formulation containing the NSAID aceclofenac to evaluate these before release in market. Bioavailability, T (AC) -[[[2-[(2,6-Dichlorophenyl)amino]phenyl] bioequivalence studies help to evaluate efficacy and safety acetyl]oxy]acetic acid and muscle relaxant thiocolchicoside whereas quality control testing evaluates the quality of (TC)- N-[(7S)-3-(beta-D-glucopyranosyloxy)-1,2-dimethoxy- product. Among these testing, potency test and impurity test 10-(methylsulfanyl)- 9-oxo-5,6,7,9-tetrahydrobenzo[a] are important. heptalen- 7-yl] acetamide is used in the treatment of severe pain. It is an important aspect of drug development that the The aim of present study is to develop and validate method for assaying the drug and related impurities of the combination Access this article online drug product. Quick Response Code: Website: www.jpbsonline.org Literature studies show various analytical methods reported for the estimation of AC in plasma by HPLC,[1,2] LC-MS,[3] [4] DOI: in other matrix by HPLC, stability-indicating method by [5,6] 10.4103/0975-7406.142955 spectrophotometry and densitometry, in combination with other NSAID like tramadol, paracetamol.[7,8] A UV-visible 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. 246 Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 Karbhari, et al.: Related compounds of aceclofenac and thiocolchicosides derivative spectroscopic method has been reported for AC in 100 mg AC working standards into two different 100 mL combination with paracetamol and tramadol.[9] 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 Analytical methods like densitometry, capillary electrophoresis capacity of the flasks and sonicated for few minutes to solubilize. and spectrometry have been reported for the determination of The solutions were individually diluted up to the mark with the TC either alone or in combination.[10-14] 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 In a regulated market drug product compliance to ICH a concentration of 100 μg mL−1 for AC and 4 μg mL−1 for TC. requirements for specification and impurities or degradation This solution was further diluted to prepare diluted standard products is a mandatory requirement.[15] for impurities test which contained 2.5 μg mL−1 for AC and 1 μg mL−1 for TC. In the present study, a simple, accurate and precise RP-HPLC method for simultaneous determination of AC and TC along Test solution preparation with related impurities from tablet formulation was developed and validated. Five tablets containing 4 mg TC and 100 mg AC each were transferred into a 200 mL volumetric flask. 50 mL of diluent To the best of our knowledge, there is no stability indicating was added to disperse the tablets. The flask was shaken for method available for simultaneous determination of AC with 15 min so that the tablets disintegrate completely. The TC and related impurities. diluent was added up to the one third capacity of the flask and sonicated for 15 min. The flask was cool to room temperature. Experimental The volume was made up to the mark with diluent and mixed. The solution was filtered through a 0.45 μm PVDF Instrument and chromatographic conditions membrane syringe filter from Merck Millipore and used for determination of related substances. 4 mL of the filtrate was Integrated HPLC system, Waters Alliance manufactured by Waters diluted to 100 mL with diluent to give a test concentration Corporation (Milford, USA) was used for method development containing 100 μg mL−1 for AC and 4 μg mL−1 for TC for and method validation. This system comprised of a quaternary an assay. gradient pump and auto sampler (Waters 2695 Separation module), column oven and a photo diode array detector (Waters The amount of TC and AC per tablet was calculated from 2998). PC installed Waters Empower software, Version 2.6, was the peak area of AC and TC in the chromatograms of the test used to record and to integrate the chromatograms. solution and standard solution, respectively. A mobile phase consisted of 0.1 M ammonium acetate buffer and Specificity methanol filtered and degassed separately through membrane filter and delivered in a gradient mode. Thermo Hypersil C18 (4.6 The specificity of a method is its suitability for analysis of mm × 100 mm, 3 μm) analytical column from Thermo fisher a substance in the presence of potential impurities. Stress scientific, (Mumbai, India) was used as a stationary phase. The testing of a drug substance can help identify likely degradation flow rate was 1.0 mL min−1 and the detector was set at 257 nm. products, which can in turn help establish degradation pathways and the intrinsic stability of the molecule. It can also be used The column oven temperature was maintained at 40ºC and the to validate the stability-indicating power of the analytical volume of solution injected was 20 μL. procedures used. Materials and Methods The specificity of the LC method for TC and AC was determined in the presence of 11 impurities and degradation AC and TC working standards were obtained from a reputed products which included colchicoside, colchicine of TC and pharmaceutical company (Mumbai, India). Analytical reagent imp A, imp B, imp C, imp D, imp E, imp F, imp G, imp H, imp I grade ammonium acetate and HPLC grade methanol were of Aceclofenac [Figure 1]. obtained from Merck (Mumbai, India). A Millipore Milli Q plus water purification system (Milford, USA), was used to prepare Forced degradation of TC and AC was also performed to distilled water (>18 μ Ω). Test samples used were composed provide an indication of the stability-indicating properties of AC and TC purchased from the local market, Zerodol-TH, and specificity of the method.[16,17] The tablet formulation was Ipca Laboratories Ltd, India, of strength 100 mg and 4 mg per also stressed along with active pharmaceutical ingredients. tablet, respectively. The stress conditions used for the degradation study included light (conducted as stipulated in ICH Q1B), heat (60°C), Preparation of standard solution acid hydrolysis (0.5 M HCl), basic hydrolysis (0.5 M NaOH) and oxidation (10% H2O2). The purity of peaks of TC and AC AC and TC standard stock solutions were prepared by obtained from stressed samples was checked by use of the PDA transferring accurately weighed quantities of 40 mg TC and detector. The purity angle is less than the purity threshold limit Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 247 Karbhari, et al.: Related compounds of aceclofenac and thiocolchicosides 6U &RPSRQ ,83$&QDPH 6WUXFWXUH&DWHJRU\ obtained in all stressed samples demonstrated the analyte peak HQW homogeneity. &ROFKLFR 1>WULPHWKR[\ R[R 'HJUDGDQW VLGH >WULK\GUR[\ K\GUR[\PHWK\O R[DQ \O@R[\ An assay of stressed samples was performed by a &6 GLK\GUR+EHQ]R>D@KHSWDOHQ \O@DFHWDPLGH comparison
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