INTRODUCTION:

Thiocolchicoside (Fig. 1) is chemically N-[1,2-dimethoxy-10-methylsulfanyl-9-oxo-3-[3,4,5-trihydroxy-6-(hydroxymethyl) oxan-2-yl]oxy-6,7-dihydro-5H-benzo[a]heptalen-7-yl] acetamide.^1-2 It is used in the symptomatic treatment of painful muscle spasm. It is a muscle relaxant, which has been claimed to possess GABA mimetic and glycinergic actions.³ It is official in Indian Pharmacopeia, British Pharmacopeia, USP etc.^4-6

Fig. 1- Structure of thiocolchicoside

Etoricoxib (Fig. 2) is 5-chloro-2-(6-methylpyridin-3- yl)-3-(4-methylsulfonylphenyl) pyridine.¹It is used as a non-steroidal anti-inflammatory agent. It is selective inhibitor of COX-2 that decreases GI toxicity and is without effects on platelet function. It has powerful anticonvulsant activity.^7-9 It is official in Indian Pharmacopeia, British Pharmacopeia, USP etc.^4-6

Fig. 2- Structure of etoricoxib

There are number of analytical methods for the determination of various drugs from bulk and various formulations like tablets, capsules, injections, etc. These methods include Uv-spectrophotometry, HPLC, UPLC, Gas chromatography, etc.^10-40Literature survey revealed that there are various methods reported for determination of thiocolchicoside (THC) and etoricoxib (ETR) in alone and in combination with other drugs but no any method covers the complete validation of method as per ICH guideline.^41-48Validation studies which is essential to probe the chemical behaviour of the molecule. Validation shows the test performs according to specifications when executed for the first time using the personnel, equipment, and reagents available.^49-50 Hence, an attempts were made to develop new liquid chromatographic method for the analysis of same combination from tablet formulation.

MATERIAL AND METHODS:

Instrumentation:

Chromatography was performed with Agilent Technologies 1020 Compact LC (Germany) gradient pump, with variable wavelength detector and Nucleosil (4.6mm I.D × 250mm) C18 column was used for chromatographic separation.

Reagents and Chemicals:

All chemicals and reagents used in method were of HPLC grade. Before use, mobile phase and other solvents were filtered through 0.45µm Whatman filter paper. Standard drugs THC and ETR were provided as gift sample by Aden Healthcare, Chandigarh (India). Tablets (Edibox-T™) were purchased from the local market. Tablet contained containing THC 4mg and ETR 60mg.

Selection Detection Wavelength:

Appropriate dilution of each stock solution with mobile phase, various concentrations of THC and ETR were prepared separately. Each solution was scanned between the range 200 to 400nm in spectrum mode. The wavelength selected for the analysis was 220nm at which both drugs showed significant absorbance.

Preparation of Mixed Standard Stock Solution:

Standard stock solution containing THC and ETR in the concentration 50µg/mL and 750µg/mLwas prepared in mobile phase.

Preparation of Mixed Standard Solution:

Standard stock solution containing THC and ETR in the concentration 5µg/mL and 75µg/mL was prepared in mobile phase.

Linearity and Range:

Linearity and range of the method was studied by injecting series of dilution containing THC and ETR in the range of 2-10µg/mL and 30-150µg/mL, respectively. From graph, concentration 5µg/mL (THC) and 5µg/mL (ETR) were selected for further study.

Optimization of Chromatographic Conditions:

The chromatographic condition was optimized by using different mobile phases alone and in combination, at different flow rates, at ambient column temperature. Mobile phase consists of acetonitrile: water (0.05% w/v, Orthophosphoric acid (OPA)) in the ratio 25:75% v/v was proved to be more effective mixture. Flow rate 0.7 mL/min was selected as it gives better resolution and detection wavelength selected was 220nm.

System Suitability Parameters:

System suitability parameters were checked according to USP with mixed standard solution. About 20µL of the solution was injected into the chromatographic conditions and results were recorded.

Assay of Marketed Formulation:

For the assay of marketed formulation, 20 tablets were weighed, and their average weight was calculated and they were crushed to fine powders. Powder equivalent to 300mg of ETR (20mg THC) was weighed and transferred to 100ml volumetric flask. About 25mL of mobile phase was added and shaken for 30min and sonicated for 10 min. Final volume was made up to the mark with same solvent. The solution was further diluted to get 75µg/mL of ETR along with 5µg/mL of THC. It was then filtered through 0.45µm membrane filters. About 20µL volume was injected into the system and results were recorded. The drug content in the sample solution was calculated from the regression equation of the standard calibration graph.

Validation of Method:

Validation of proposed method was carried out in terms of recovery and precision, linearity and range, limit of detection (LOD), limit of quantitation (LOQ) and robustness.

Recovery study:

To study the accuracy of method, recovery study was performed by standard addition method at three level (i.e. 80, 100 and 120%). Accuracy was evaluated in triplicate by adding known concentration of standard solution to the preanalysed sample solution.

Precision:

The precision of method was checked by injecting three consecutive replicates of standard solution. Results were expressed in terms of %RSD calculated from the measurement of AUC (Table 1).

Linearity and range, LOD and LOQ:

To study the linearity and drugs, a series of dilutions were prepared from mixed standard stock solution. Calibration graph was plotted as concentration versus peak area response. The limit of detection and limit of quantitation were measured on the basis of signal-to-noise ratios.

Robustness:

Robustness of the method was determined by making changes in the chromatographic conditions, such as slight change in mobile phase composition, change in the flow rate was varied by ± 0.1ml min, and change in wavelength.

RESULTS AND DISCUSSION:

We developed liquid chromatographic method (RP-HPLC) simultaneous estimation of TCH and ETR from tablet formulation. The combination of the drugs and method were selected on basis of literature survey. For the analysis, mobile phase and other chromatographic conditions were selected on the basis of trial and error. The mobile phase selected was Acetonitrile: water (0.05% Orthophosphoric acid V/V) in the ratio 25:75 V/V and flow rate was kept at 0.7 mL/min at ambient temperature. At this chromatographic conditions both the drugs were separated with minimum tailing and maximum resolution. Both the peaks were of Gaussian shape. The retention time observed for THC and ETR was 3.75 and 6.13, respectively. The chromatogram of sample solution is shown in Fig. 3. As THC is more polar than ETR, THC eluted first. Depending on the sensitivity and concentration of drugs, the peak height and peak area for both drugs were variable.^41-52

Fig. 3- Chromatogram of sample solution of THC and ETR

To ascertain the resolution and reproducibility of the proposed chromatographic system for estimation of THC and ETR, suitability parameters were studied. The results are shown in Table 1. At the selected chromatographic conditions, assay of marketed tablet formulation (Edibox-T) was performed by injecting sample in triplicate. Method was validated as per ICH guideline. The parameter studied are discussed below.

Accuracy and precision:

Accuracy of method was determined by performing recovery study. Recovery study was performed by standard addition method. It was performed at 3 levels i.e. 80, 100, 120%. Average recovery of both drugs from tablet formulation were found to be 99.98% and 99.69% for THC and ETR, respectively. Precision was determined by injecting standard solution in triplicate. The mean of inter-day precision was found to be 0.78 % and 0.43% for THC and ETR, respectively. The results of System Suitability Parameters are shown in Table.1.

Table 1: Result of System Suitability Parameters.

Sr. No.	System Suitability Parameters	Result
Sr. No.	System Suitability Parameters	THC	ETR
1	Retention Time	3.75	6.13
2	Area	379.23	7577.10
3	Theoretical Plate Number	2373.1	5246.4
4	Tailing Factor	1.71	1.25

Linearity study:

Linearity of THC and ETR was observed in the range of 2-10μg/ml and 30-150μg/ml. Detection wavelength used was 220nm. The calibration curve yielded correlation coefficient (r²) 0.998 and 0.998 for THC and ETR, respectively. The Calibration curves are shown in Fig.4.

Fig. 4- Calibration curve for THC (left) and ETR (right)

Limit of detection and limit of quantitation (LOD and LOQ):

The LOD and LOQ of THC and ETR were determined from standard deviation of the response and the slope. LOD value for THC and ETR were found to be 0.03 µg/ml and 0.35µg/ml, respectively. Whereas LOQ value for THC and ETR were found to be 0.09µg/ml and 1.07 µg/ml, respectively. For results, See Table 2.

Table 2- Results of Linearity and range, LOD and LOQ

Parameters	Result
	THC	ETR
Linearity range	2-10µg/ml	30-150µg/ml
% RSD	1.19	0.97
Slope	80.77	96.2
LOD	0.0132	0.0115
LOQ	0.04	0.0348

Robustness study:

It is the capacity of a method to remain unaffected by small, deliberate variations in method parameters. It was indicated by changing the mobile phase composition, flow rate and change in detection wavelength. It was observed that method remain unaffected with small but deliberate changes in chromatographic conditions. The results shown in Table 3.

CONCLUSION:

The proposed A new simple, reproducible and efficient liquid chromatographic method (RP-HPLC) was developed for simultaneous estimation of thiocolchicoside and etoricoxib for tablet formulation is simple, precise and accurate. It be used for routine quality control analysis.

ACKNOWLEDGMENTS:

Authors are thankful to Shri. Yogendra Gode, President, IBSS's Dr. Rajendra Gode Institute of Pharmacy, Amravati and Dr. Rajendra Gode College of Pharmacy, Amravati for providing excellent research facility to undertake this work. Authors are also thankful to Aden Healthcare, Chandigarh (India) for providing gift samples of drugs.

Disclosure of conflict of interest

The authors declare no conflicts of interest.

Table 3- Results of robustness study

Drug	Factor	Level	Mean+-SD	% RSD
THC	Flow rate	0.6 ml	439.59±4.34	1.23
	Flow rate	0.8 ml	590.68±7.26	0.99
	Wavelength	257 nm	488.61±7.16	1.47
	Wavelength	259 nm	510.52± 2.60	0.51
	Mobile phase composition (A: B)*	26:74	484.11±5.64	1.17
	Mobile phase composition (A: B)*	24:76	481.31±6.30	1.30
ETR	Flow rate	0.6 ml	7646.14±5.88	0.08
	Flow rate	0.8 ml	8321.67 ± 9.08	0.09
	Wavelength	257 nm	8719.00±6.97	0.11
	Wavelength	259 nm	8814.56± 9.90	1.01
	Mobile phase composition	26+74	8779.39±7.12	0.31
		24+76	8819.27±9.23	1.22

* A- Acetonitrile, B- Water (0.05% Orthophosphoric acid, V/V)

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Received on 26.11.2020 Revised on 15.01.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):118-122.

DOI: 10.52711/2231-5675.2021.00020