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 with reference standard and the mass balance (% assay + % impurities + % degradation products)  7KLRFROF 6 1^ > E' 'UXJ was calculated. KLFRVLGH JOXFRS\UDQQV\O R[\@± WHWUDK\GUR GLPHWKR[\  7& PHWK\OWKLR R[REHQ]R>i@ KHSWDOHQ\O`DFHWDPLGH Results and Discussion

 &ROFKLFL 1> 6 WHWUDPHWKR[\ 'HJUDGDQW QH && R[RWHWUDK\GUREHQ]R>D@ Development of chromatography KHSWDOHQ\O@ DFHWDPLGH Initially, a microsorb pursuit varian C18 column having  $FHFORIH >>  5 + 'HJUDGDQW dimensions 150 mm × 4.6 mm and particle size 5 µm was QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHWLF LPSXULW\ DFLG GLFORIHQDF  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  $FHFORIH >>>>  'UXJ TC almost in the void volume. When water in the mobile QDF $& 'LFKORURSKHQ\O DPLQR@SKHQ\O@DFHW\ O@R[\@DFHWLFDFLG phase was replaced with 0.5 M ammonium acetate, the retention of TC was increased but the polar component AC $FHFORIH  GLFKORURSKHQ\O GLK\GUR  'HJUDGDQW was highly retained on the column. For the faster elution of QDF +LQGRO RQH LPSXULW\ 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 $FHFORIH >>>>> >  3URFHVV QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHW\O UHODWHG other non‑polar, a gradient program was introduced. The final LPSXULW\ @R[\@DFHW\O@R[\@DFHWLFDFLG DFHWLF gradient program started with buffer: Methanol composition * $* DFHFORIHQDF  5 &+ 70:30 (v/v) and linearly changed to 70% methanol within &2 +  10 min and the isocratic gradient with buffer: Methanol  $FHFORIH >>>>>>> >  3URFHVV composition 30:70 (v/v) was run up to 25 min and then QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHW\O UHODWHG linearly changed to original composition 70:30 within LPSXULW\ @R[\@DFHW\O@R[\@DFHW\O@R[\@DFHWLF + $+ DFLG GLDFHWLFDFHFORIHQDF  next 3 min (28 min) and allowed to stabilize to original 5 &+&22&+&2+ composition till next 2 min so as to complete the run time

$FHFORIH PHWK\O>>> >  'HJUDGDQW of 30 min. QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHW\O LPSXULW\ @R[\@DFHWDWH PHWK\OHVWHURI ' $' DFHFORIHQDF  The gradient program was optimized in such a way that

5 &+ two impurities of TC and nine impurities of AC were well separated with a minimum resolution of 2.0 The USP  $FHFORIH PHWK\O>>  'HJUDGDQ resolution between peaks of TC and AC was 50 in the assay QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHWDW LPSXULW\ H PHWK\OHVWHURIGLFORIHQDF  method [Figure 2]. % $% 5 &+ The column length was reduced to 100 mm and particle size  $FHFORIH HWK\O>>> >  3URFHVV to 3 µm so as to achieve shorter run time and better resolution. QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHW\O UHODWHG LPSXULW\ @R[\@DFHWDWH HWK\OHVWHURI The combination of methanol and aqueous buffer increased the ( $( DFHFORIHQDF  viscosity of the mobile phase. The column temperature of 40°C 5 &+ was used to reduce the backpressure generated due to the high  $FHFORIH HWK\O> >  3URFHVV viscosity of the mobile phase. QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHWDW UHODWHG LPSXULW\ H HWK\OHVWHURIGLFORIHQDF  & $& The test sample concentration was finalized based on the 5 &+ requirement for limit of quantitation value being less than the [18]  $FHFORIH EHQ]\O>>> >  3URFHVV reporting threshold. Considering the maximum daily dose QDF GLFKORURSKHQ\O DPLQR@SKHQ\O@DFHW\O UHODWHG LPSXULW\ @R[\@DFHWDWH EHQ]\OHVWHURI for AC and TC, the reporting threshold is 0.1%. ) $) DFHFORIHQDF  Result from forced degradation studies 5 &+&+

Figure 1: IUPAC names, structure and category of the components Degradation was not observed when TC and AC were exposed to under study heat and photolytic degradation. TC was found to be degraded

 248 Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 Karbhari, et al.: Related compounds of aceclofenac and thiocolchicosides in oxidation and colchicoside was formed as a major degradant To test the linearity of the assay method, solutions at six which was confirmed by spiking colchicoside in degraded concentrations from 10% to 200% of the analyte concentration 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 a 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].

Method validation

Method validation was carried out as per ICH guidelines b 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 c 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].

Linearity d Solution for testing linearity for the related substances were Figure 2: A-typical chromatogram of separation, As per order of prepared by diluting the impurity stock solution to different elusion, 1-Colchicoside, 2-Thiocolchicoside, 3-Colchicine, 4-Diclofenac, concentrations from the LOQ to 160% of the qualification 5-Aceclofenac, 6-Impurity I of Aceclofenac, 7-Impurity G of threshold of the impurity (i.e. LOQ to1.6% for an analyte Aceclofenac, 8-Impurity H of Aceclofenac, 9-Impurity D of Aceclofenac, −1 10-Impurity B of Aceclofenac, 11-Impurity E of Aceclofenac, 12-Impurity concentration of 100 μg mL TC) LOQ to 160% (i.e. LOQ C of Aceclofenac, 13-Impurity F of Aceclofenac, B- Chromatogram of −1 to 0.16% for an analyte concentration of 2500 μg mL for Oxidation of tablet Sample, C- Chromatogram of Basic hydrolysis of AC) [Tables 2 and 3]. tablet Sample, D- Chromatogram of Acidic hydrolysis of tablet Sample

Table 1: Forced degradation Stress Time (%) % Total Mass balance (% assay+ Remarks conditions Assay impurities % total impurities) TC AC TC AC TC AC Base/0.5 N NaOH/60ºC 5 min 99.9 40.65 0.16 59.3 100.06 99.95 Impurity I and ImpurityA of Aceclofenac formed Acid/0.5 N HCl/60ºC 60 min 98.31 87.83 1.69 12.17 100.00 100.00 Impurity A of Aceclofenac and Colchicine was formed

Oxidation/10% H2O2/60ºC 30 min 60.5 97.9 39.5 2.0 100.00 99.90 Colchicoside of Thiocolchicoside formed Thermal degradation 7 days 98.2 97.8 0.5 1.2 98.70 99.00 No major degradation observed Photolytic degradation 5 days 98.5 97.5 0.5 1.5 99.00 99.00 No major degradation observed TC: Thiocolchicoside, AC: Aceclofenac

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Table 2: Results from validation study of related substances method Parameters CS TC CC DC AC AI AG AH AD AB AE ACi AF Retention 1.59 3.24 5.66 9.22 9.8 10.03 10.3 10.58 13.3 14.5 14.9 17.1 20.24 time (min) Relative 0.16 0.33 0.58 0.94 1.00 1.02 1.05 1.08 1.36 1.48 1.52 1.74 2.07 retention time Tailing 1.55 1.11 0.97 0.95 0.98 1 1.02 1 0.98 0.99 0.94 1 1 factor Resolution - 8.6 18.3 32.4 5.4 2.4 2.2 2.1 4.8 6.0 2.3 8.1 5.1 (USP) Theoretical 772 6912 39925 169191 137042 140002 139401 166176 180013 178531 93748 100548 54497 plates Linearity 0.05-1.6 0.05-1.6 0.05-1.6 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 0.125-4.0 range (µgmL−1) LOD % 0.017 0.012 0.008 0.006 0.001 0.003 0.002 0.005 0.002 0.002 0.003 0.003 0.005 LOD 0.017 0.012 0.008 0.140 0.064 0.068 0.057 0124 0.061 0.059 0.067 0.067 0.114 (µgmL−1) LOQ % 0.057 0.041 0.026 0.019 0.003 0.009 0.008 0.016 0.008 0.008 0.009 0.009 0.015 LOQ 0.057 0.041 0.026 0.465 0.214 0.227 0.190 0.412 0.203 0.195 0.222 0.224 0.379 (µgmL−1) Slope (b) 46464 57484 61025 19616 21912 24673 25275 9548 22451 59448 20446 46720 16357 Intercept (a) −1118 738 1700 −812 721 50 42 972 −122 43 661 −254 −884 Correlation 0.999 0.9999 0.9986 0.9995 0.9999 0.9998 0.9999 0.9972 0.9999 0.9999 0.9998 0.9998 0.9992 coefficient Residual 0.9979 0.9999 0.9972 0.9989 0.9998 0.9996 0.9998 0.9944 0.9998 0.9998 0.9995 0.9997 0.9985 sum of squares (r2) Response 0.8 1 1.1 0.9 1 1.1 1.2 0.4 1.0 2.7 0.9 2.1 0.7 factor Correction 1.2 1 1.9 1.1 1 0.9 0.9 2.3 1.0 0.4 1.1 0.5 1.3 factor TC: Thiocolchicoside, AC: Aceclofenac, CS: Colchicoside, CC: Colchicine, DC: Diclofenac, AI: Aceclofenac impurity I, AG: Aceclofenac impurity G, AH: Aceclofenac impurity H, AD: Aceclofenac impurity D, AB: Aceclofenac impurity B, AE: Aceclofenac impurity E, ACi: Aceclofenac impurity C, AF: Aceclofenac impurity F, LOD: Limit of detection, LOQ: Limit of quantitation

Table 3: Validation study parameters of assay test method For the impurities, recovery was determined in triplicate at Parameters TC AC 0.8, 1.0 and 1.2 μg mL−1 for CS and CC and 2.0, 2.5 and −1 Retention time (min) 3.24 9.8 3.0 μg mL (0.08, 0.1 and 0.12%) of the analyte concentration Tailing factor 1.09 1.22 (2500 μg mL−1 for AC) and % recovery of the impurities was Resolution (USP) - 50 found within 85% to 115%[Table 2]. Theoretical plates 7468 104844 Linearity range (µgmL−1) 0.4-8.0 10-200 Intercept (a) −2927 −21727 Precision Correlation coefficient 0.9999 0.9999 Residual sum of squares (r2) 0.9999 0.9999 The precision of the related substance method was checked Precision (CV, %) (n=6) 0.5 0.8 by six‑fold analysis of tablet sample spiked with 0.5% of each (CV, %) (n=12) 0.9 0.9 impurity. The RSD (%) of peak area for each impurity which was Accuracy at 80% level 98.3 98.9 At 100% level 99.2 99.1 within 5% confirmed precision of the method. The precision of At 120% level 100.4 100.7 the assay was evaluated by performing six independent assays of a test sample and quantifying using an external standard AC: Aceclofenac, TC: Thiocolchicoside, USP: United States pharmacopoeia, CV: Coefficient of variance method using reference standard. The RSD (%) of the six results within 2% confirmed method to be precise for assaying (0.4 to 8, μg mL−1 for TC and 10 to 200 μg mL−1 for AC) were of AC and TC in the presence of its impurities. prepared from the stock solution. Least‑squares linear regression analysis was performed on peak area and concentration data To evaluate the intermediate precision (ruggedness) of the [Table 3]. method, the analysis was performed on a different day using a different instrument in the same laboratory [Tables 3 and 4]. Accuracy Robustness 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, To determine the robustness of the method the experimental 100, and 120 μg mL−1 for AC and recovery was calculated [Table 3]. conditions were deliberately altered and checked for the system

 250 Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 Karbhari, et al.: Related compounds of aceclofenac and thiocolchicosides

Table 4: Accuracy and precision study for impurities Parameters Related compounds CS CC DC AI AG AH AD AB AE ACi AF Precision (CV, %) (n=6) 0.90 0.42 1.20 0.28 0.20 1.70 0.43 0.71 0.65 0.77 1.49 (CV, %) (n=12) 0.80 0.35 1.51 0.32 0.42 2.01 0.51 0.94 0.57 1.03 1.25 Accuracy/recovery At 80% level 100.9 100.9 99.7 99.8 100.5 102.5 101.3 101.4 101.4 102.1 102.5 At 100% level 101.6 101.6 99.0 100.0 99.0 99.01 100.0 100.0 102.1 100.0 99.1 At 120% level 98.9 98.9 101.3 96.7 99.9 99.6 103.3 102.2 101.1 103.9 99.6 CV: Coefficient of variance, CS: Colchicoside, CC: Colchicine, DC: Diclofenac, AI: Aceclofenac impurity I, AG: Aceclofenac impurity G, AH: Aceclofenac impurity H, AD: Aceclofenac impurity D, AB: Aceclofenac impurity B, AE: Aceclofenac impurity E, ACi: Aceclofenac impurity C, AF: Aceclofenac impurity F

Table 5: Robustness study Conclusions Robustness parameters Tailing factor Resolution TC AC (USP) between The proposed method is simple, specific, accurate and precise TC and AC for determination of AC and TC in the presence of degradants Change in molarity of buffer and process related impurities. The method is also applicable to 0.048M 1.11 0.98 50 study content uniformity and assay of dissolution test. Thus, the 0.052M 1.10 0.98 50 method can to use for quality control of all types of pharmaceutical Change in column brand (unisphere C18) 1.0 1.0 52 Change in flow rate preparations like modified release tablets containing AC and TC. 0.8 mL min−1 1.1 0.98 48 1.2 mL min−1 1.1 1.0 51 References Change in column oven temperature 0 35 C 1.2 1.1 50 1. Hinz B, Auge D, Rau T, Rietbrock S, Brune K, Werner U. Simultaneous 0 45 C 1.1 1.1 50 determination of aceclofenac and three of its metabolites in human TC: Thiocolchicoside, AC: Aceclofenac, USP: United states pharmacopoeia plasma by high‑performance liquid chromatography. Biomed Chromatogr 2003;17:268‑75. 2. Musmade P, Subramanian G, Srinivasan KK. High‑Performance liquid suitability criteria, i.e. resolution between TC and AC and tailing chromatography and Pharmacokinetics of aceclofenac in rats. Anal factor. The chromatographic conditions which were altered are Chim Acta 2007;585:103‑9. 3. Kang W, Kim EY. Simultaneous determination of aceclofenac and its −1 flow rate (±0.2 mL min ; column oven temperature (±5°C), three metabolites in plasma using liquid chromatography–tandem molarity of buffer solution (±0.002 units), different column mass spectrometry. J Pharm Biomed Anal 2008;46:587‑91. make. The tabular results show that the method is robust for 4. Bhinge JR, Kumar RV, Sinha VR. A simple and sensitive stability indicating RP‑HPLC assay method for the determination of the studied changes [Table 5]. Aceclofenac. J Chromatogr Sci 2008;46:440‑4. 5. el‑Saharty YS, Refaat M, el‑Khateeb SZ. Stability indicating Relative response factor (RRF) and correction factor (CF) study spectrophotometric and densitometric methods for determination of aceclofenac. Drug Dev Ind Pharm 2002;28:571‑82. 6. Hasan NY, Abdel‑Elkawy M, Elzeany BE, Wagieh NE. Stability indicating Response factor is a relative term, being the response of methods for the determination of aceclofenac. Farmaco 2003;58:91‑9. equal weights of one substance relative to that of another 7. Jamil S, Talegaonkar S, Kheu RK, Kohli K. Development and validation in the conditions described in the test. The RRF value for of a stability indicating LC method for simultaneous Analysis given impurity as the ratio of response of peak at a particular of Aceclofnac and paracetamol in Conventional Tablets and in Microsphere formulations. Chromatographia 2008;68:557‑65. concentration to that of analyte peak at that concentration. The 8. Kachhadia PK, Doshi AS, Ram VR, Joshi HS. Validated method for RRF values less than 0.2 and more than 5.0 are not acceptable simultaneous analysis of tramadol hydrochloride and aceclofenac in as per European pharmacopoeia. In such cases there is a need a commercial tablets. Chromatographia 2008;68:997‑101. for change in chromatographic parameters like wavelength or 9. Srinivasan KK, Alex J, Shiwalkar AA, Jacob S, Sunil Kumar MR, Prabu SL. Simultaneous derivative spectrophotometric estimation different method of visualization is used. The extrapolation of of aceclofenac and tramadol with paracetamol. Indian J Pharm Sci linearity study was done to determine RRF and CF [Table 2]. 2007;69:540‑5. 10. El‑Ragehy NA, Eilaithy MM, El‑Ghobashy MA. Determination of Stability in Solution thiocolchicoside in its binary mixtures (thiocolchicoside‑glafenine and thiocolchicoside‑floctafenine) by TLC – densitometry. Farmaco 2003;58:463‑8. The stability of TC and AC and their impurities in solution 11. Walash MI, El Tarras FM, Ef sheriff ZA, Mohamed AO. was determined by keeping test solutions of the sample and Spectrophotmetric determination of two N‑(4‑quinolyl) anthranilic acid derivative (glafenine and floctafenine). J Pharma Biomed Anal reference standard and spiked sample solution volumetric flasks 2000;23:483‑91. at room temperature for 24 h at bench top and withdrawn 12. Ghielmetti G, Bocchia A. Spectrophotometric characterstics of at regular time intervals to assay the analytes. The absolute colchicines, colchicoside and thiocolchicoside a significant derivation difference of assay values within 2% and impurities from initial of recent use in neurology. Farmaco Prat 1958;13:256‑61. 13. Lu Q, Copper CL, Collins GE. Ultraviolet absorbance detection value concluded solution stability for 24 h for assay however of colchicine and related alkaloid on a capillary electrophoresis related substances were found stable up to 12 hours. microchip. Anal Chim Acta 2006;572:205‑11.

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14. Sutherland FC, Smit MJ, Elst Hundt HK, Swait KJ, Hundt AF. methods‑critical review. J Pharm Biomed Anal 2002;28:1011‑40. Highly specific and sensitive liquid chromatography tandem 17. Cartnsen JT, Rhodes CT. Drug stability principles and practices. 3rd ed. mass spectrometry method for the determination of 3 New York: Marcel Dekker; 2000. desmethylthiocolchicine in human plasma as analyte for the 18. ICH guidelines Q3 B (R2) Impurities in new drug product: International assessment of bioequivalence after oral administration of Conference on Harmonisation 1995. thiocolchicoside. J Chromatogr A 2002;949:71‑7. 19. ICH guidelines Q2(R1) Validation of Analytical Procedures: Text and 15. ICH guidelines Q6A Specifications: Test Procedures and acceptance methodology: International Conference on Harmonization, 2005. criteria for new drug substances and new drug products: Chemical substances: International Conference on Harmonisation; 1999. Source of Support: Nil, Conflict of Interest: None declared. 16. Bakshi M, Sing S. Development of validated stability‑indicating assay

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