Development and Validation of HPLC Method for Simultaneous Estimation of Etodolac andThiocolchicoside in Bulk and Pharmaceutical Dosage Form

INTRODUCTION

Etodolac’s anti-inflammatory and analgesic activity is related to its inhibitory action on prostaglandin and thromboxane synthesis throughthe inhibition of cyclooxygenase (both COX-1 and COX-2). As prostaglandins sensitize pain receptors, their inhibition accounts for the peripheral analgesic effects of Etodolac. Thiocolchicoside is muscle relaxant with anti-inflammatory and analgesic effects. It acts as a competitive GABA-A receptor antagonist and also inhibits glycine receptors with similar potency and nicotinic acetylcholine receptors to a much lesser extent. It has powerful convulsant activity and should not be used in seizure prone individuals.² Thus, a new fixed combination of Etodolac and Thiocolchicoside is proved to be effective in treatment of anti- inflammatory, analgesic, antipyretic. Analgesic effect is used in musculoskeletal and postoperative pain. Short- and long-term treatment of osteoarthritis, rheumatoid arthritis, acute pain, juvenile rheumatoid arthritis 1-3 Day to day numbers of newer drugs and their formulations, which are used in pain management in musculoskeletal condition, are marketed. Etodolac and Thiocolchicoside is one of them. In current scenario most of all the manufacturing units have sophisticated instruments like UV, HPLC, HPTLC GC; it would be beneficial if we can develop sensitive method to determine drug in combination usingone of these instruments. Literature survey revealed that Etodolac is official in IP’ 2010, USP’ 2008, and BP’ 2010. Thiocolchicoside is official in IP’ 2010. So, there are few official methods available for individual estimation of Etodolac and Thiocolchicoside. Literature review shows RP-HPLC method with buffered mobile phase. It would be advantageous if we develop a recent sensitive method todetermine both drugs in combination by using HPLC method.

The objectives of present work are

1) To develop HPLC method for combination drug product.

2) Validation of newly developed analytical method which should be; Simple, accurate, precise, selective, specific, reproducible, highly sensitivity.

3) To develop and validate High Performance Liquid Chromatography for simultaneous estimation of Etodolac and Thiocolchicoside.

4) To investigate ability of method to quantify two component mixture of Etodolac and Thiocolchicoside.

MATERIALS AND METHODS

Etodolac^4-10

Description : The sample of ETD was observed for its colour and texture. Solubility : The sample of ETD was taken in test tubes and observed for solubility invarious solvents like 0.1M NaOH, 0.1M HCl, water, methanol, acetonitrile, acetone andchloroform.

Identification test : Potassium Bromide IR disc was prepared using 1 mg of ETD on hydraulicpellet press. This disc was scanned in the region of 4000 – 400 cm^–1 in FTIR and obtained infrared spectrum was compared with the reference spectrum of ETD.

Water content : Water content can be determined using Karl Fischer Titrimeter. Karl Fischer reagent is standardized with sodium tartrate, then it is allowed to titrate with the known amount of sample i.e. ETD and then once the colour change is observed, it will indicate the end point of the titration and this in turn will give the amount of moisture present in the sample. Moisture/water content can be determine using following formula.

Thiocolchicoside

Description : The sample of THC was observed for its color and texture. Solubility : The sample of THC was taken in test tubes and observed for solubility in various solvents like water, 0.1M NaOH, 0.1M HCl, methanol, acetone and chloroform.

Identification test : Potassium Bromide IR disc was prepared using 1 mg of THC on hydraulic pellet press. This disc was scanned in the region of 4000 – 400 cm–1 in FTIR and obtained infrared spectrum was compared with the reference spectrum of THC.

Loss on drying : One gram of THC was transferred to previously dried shallow weighing bottle. The bottle was covered with its glass-stopper and weighed with the content. The content was distributed by gentle side wise shaking to a depth not exceeding 10 mm. THC was dried by placing the loaded bottle in an oven at 105 ºC, the glass-stopper was removed and bottle was left in the chamber. THC was dried to constant weight at 105 ºC for 3 hr. After drying was completed, the oven was opened; the bottle was closed promptly and allowed to cool to room temperature in a desiccator. Then, the bottle and content was weighed and loss on drying was calculated.

Reverse phase HPLC method

Chemicals and reagents used : Etodolac, thiocolchicoside, Proxym-MR forte tablet (Marketed Formulation, Label claim: Each Tabletcontains 300 mg of Etodolac and 8 mg of Thiocolchicoside), water, methanol and acetonitrile (HPLC Grade), triethylamine (HPLC Grade), acetic acid, ortho-phosphoric acid (HPLC Grade).

Instruments used : Electronic weighing balance (Sartorius), Ultra sonicator (Toshniwal Pvt Ltd), UV–Visible spectrophotometer (ElicoModel: ELICO SL218 UV_Vis Spectrophotometer), digital pH Meter (Metrohm Model: 827 ph lab), vacuum pump (Rocker 300, HPLC pump), Supor 200 membrane filter, 0.2 μm (Pall India Pvt. Ltd.), cellulose acetate filter, 0.45 μm (Nylon 66), HPLC(Shimadzu), liquid chromatography: Shimadzu LC–20AT, UV–Visible detector: Shimadzu SPD-20A, guard column: Phenomenex (KJ0 – 4282), analytical column: Hypersil, B D S C 1 8 (250 mm×4.6 mm, 5 μm), data processor: LC solution software, Injector: Rheodyne – 7725i (Fixed Capacity Loop of 20 μL), Syringe:Hamilton, 25 μL

Selection of mobile phase and its strength : Mixed solution of ETD (300 μg/mL) and THC (8 μg/mL) was prepared and injected into the HPLC system. The solution was analysed first using different proportion of Acetonitrile: Water such as 50:50, 75:25, then using Acetonitrile:Phosphate buffer pH 4.75 (35:65), then using Acetonitrile: 0.1% Triethylamine Solution in water pH 4.5 adjusted with ortho phosphoric acid (60:40) then finally using Methanol: Water: Acetic acid (70:30:0.1% v/v/v) at flow rate of 1mL/min.

Selection of column (stationary phase) : To get well resolved, symmetric peak with highest number of theoretical plates, the solution of ETD and THC was analyzed using column as a stationaryphase like Hypersil BDS C18 column.

Selection of mobile phase pH : For the selection of a suitable pH for mobile phase, it was adjusted to different pH such as 4.5 using 1% Orthophosphoric acid and 4.75 phoshphate buffer. Mixed solution of ETD (300 μg/mL) and THC (8 μg/mL) was prepared at different pH and injected into the HPLC system.

Selection of analytical wavelength: The sensitivity of HPLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is the one that givesgood response for the drugs that are to be detected. In the present study, individual drug solution of ETD and THC (concentration: 10μg/ mL) was, therefore, prepared. Take 10 mg of ETD and THC in two separate 10 mL volumetric flask and addmethanol up to the mark. From that take 1 mL diluted with water upto 100 mL in individual 100 mL volumetric flask. This drug solution was than scanned in the UVregion of 200-400 nm and the spectrum was recorded.

Finalized chromatographic conditions: Analytical column: Hypersil B D S C 1 8 (250 mm×4.6 mm, 5 μm), mobile phase: methanol: water: acetic acid (70:30:0.1% v/v/v), injection volume: 20 μL, flow rate: 1 mL/min, detection wavelength: 260 nm.

Preparation of mobile phase : Mobile phase was Methanol: Water: Acetic acid in the ratio of 70:30:0.1%v/v/v. The mobile phase was filtered through 0.22 μm Millipore filter using vacuum filtration assembly and sonicated for 25min.

Standard stock solution of ETD : Accurately weighed 60 mg of ETD was transferred into a 50 mL volumetric flask. 25 mL mobile phase added to the flask. The drug was dissolved with sonication and the final volume was adjusted with mobile phase up to the mark to prepare 1200μg/mL stock solution of Etodolac.

Standard stock solution of THC : Accurately weighed and transferred 16 mg of THC working standard of known potency into a 50 mL volumetric flask. 25 mL mobile phase added to the flask. The drug was dissolved with sonication and the final volume was adjusted with mobile phase up to the mark to prepare 320 μg/mL stock solution of THC.

Combined standard stock solution of ETD and THC : Accurately weighed and transferred 300 mg of ETD and 8 mg of THC in 100 mL of volumetric flask and add 25 mL of mobile phase to the flask. The drug was dissolved with sonication and final volume was adjusted with mobile phase up to the mark to prepare 3000 μg/ mL solution of ETD and 80 μg/mL solution of THC.

Preparation of calibration curve for ETD and THC : From the combined standard solution (ETD3000 μg/mL and THC- 80μg/mL) 2.5, 5.0, 7.5, 10, 12.5 and 15.0 mL of aliquot were pipetted out in a series of100 mL volumetric flasks. The volume was made up to the mark with mobile phaseto obtain the concentration of 75, 150, 225, 300, 375, 450 μg/mL of ETD and 2,4, 6, 8, 10, and 12 μg/mL of THC.The solutions were filtered through 0.45 μm cellulose acetate filter usingsyringe and injected into the Rheodyne injector (20 μL) of HPLC system and theirchromatogram were recorded under the finalized chromatographic conditions asdescribed above after getting a stable baseline. Peak areas were recorded for all the peaks. Calibration curves of ETD and THC were constructed by plotting the peak area of ETD vs concentration of ETD and peak area of THC vs concentrationof THC, respectively.

Method optimization trials

Trial 1 : The first trial was taken on the basis of the literature survey. The method isdescribing theuse of ACN: Water (50:50) as mobile phase. But the method gave merged peak of ETD andTHC.

Trial 1 method parameter

Mobile phase Acetonitrile: Water [50:50]; Column Hypersil BDS C18 (250mmx4.6mmx5μ); Flow Rate 1.0mL/min; Injection Volume 20 μL; Detection λ 260 nm; Column Temp. Ambient.

Trial 2: The second trial was taken on the basis of resolution. But, the resolution of ETD and THC isnot good and peaks were not resolved. The method is describing the use of ACN: Water(75:25) as mobile phase.

Trial 2 method parameter

Mobile phase Acetonitrile: Water [75:25]; Column Hypersil BDS C18 (250mmx4.6mmx5μ); Flow Rate 1.0mL/min; Injection Volume 20 μL; Detection λ 260 nm; Column Temp. Ambient

Trial 3: The third trial was taken on the basis of the literature survey. The method is describing the useof Acetonitrile: Phosphate Buffer of pH4.75 (35:65) as mobile phase. But the method did notgive sharp peaks of ETD and THC.

Trial 3 method parameter

Mobile phase Acetonitrile: Phosphate Bufferofp H4.75 [35:65]; Column Hypersil BDS C18 (250mmx4.6mmx5μ); Flow Rate 1.0 mL/min; Injection Volume 20 μL; Detection λ 260 nm; Column Temp. Ambient

Trial 4: The third trial was taken on the basis of the literature survey. The method is describing the useof Acetonitrile: Phosphate Buffer of pH 4.75 (35:65) as mobile phase. But the method did not give sharp peaks of ETD and THC.

Trial 4 method parameter

Mobile phase: Acetonitrile: 0.1%Triethylamine solutionin Water, pH4.5 adjusted with orthophosphoric acid [60:40]; Column Hypersil BDS C18 (250mmx4.6mmx5μ); Flow Rate 1.0mL/ min; Injection Volume 20 μL; Detection λ 260 nm; Column Temp. Ambient.

Trial 5 (Optimized method)

The method is describing the use of Methanol: Water: Acetic acid (70:30:0.1% v/v/v). Themethod gave sharp and resolved peaks of ETD and THC.

Trial 5 Optimized method parameter

Mobile phase Methanol: water: Acetic acid(70:30:0.1% v/v/v); Column Hypersil BDS C18 (250mmx4.6mmx5μ); Flow Rate 1.0mL/min; Injection Volume 20 μL; Detection λ 260 nm; Column Temp. Ambient; Retention time in min, THC: 3.76 , ETD:13.18

Validation of RP-HPLC Method

Accuracy : The accuracy of the method was determined by adding known amount of individual working standard solutions of ETD and THC viz.0%, 80%, 100%, 120% of the sample solution concentration, to a pre-quantified sample solution of ETD (300μg/mL) and THC (8μg/ mL). Each solution was injected in triplicate and the percentage recovery and mean recovery was calculated at each level and recorded.

Precision : The precision of an analytical method was studied by performing inter-day precision and intra-day precision.

Intra-day Precision : Intra-day precision was determined by analysing the combined standard solution of ETD (300 μg/mL) and THC (8 μg/mL) at five different times intervals on same day.

Inter-day Precision : Inter-day precision was determined by analysing the combined standard solution of ETD (300 μg/mL) and THC (8 μg/mL) at five times on different daysover a period of 1 week.

Linearity and range : Linearity and range was determined at six levels over the range of 25, 50, 75, 100, 125, and 150% of sample concentration. The area at each level was calculated and a graph was plotted by taking area on Y-axis & concentration on X-axis.

Limit of detection and limit of quantitation: The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated using the standard deviation of intercept (N) and slope (S) of the calibration curve using signal-to-noise ratio.

Robustness : The robustness was studied by analyzing the samples of ETD and THC by deliberate variation in the method parameters. The changes in the response of ETD and THC were noted and compared with the original one. The robustness of themethod was established by making deliberate minor variations in the following method parameters:

Š Organic phase ratio: ± 2%

Š Flow rate: ± 0.2 mL/min

Blank, standard preparation and sample preparation were prepared and injected. The effect of changes observed on system suitability parameters, theoretical plates and asymmetry were recorded.

System suitability: Sample solutions of ETD (300 μg/mL) and THC (8 μg/mL) were prepared andanalyzed. Chromatograms were studied for different parameters such as tailing factor, resolution and theoretical plates to see that whether they comply with their commended limit or not.

Assay of Marketed Formulation

Amount of drugs present in the marketed formulation (Proxym-MR Forte Tablet) were calculated using equation mentioned in the section no 4.2.11. Amount of ETD and THC were found in the range from 98.99 and 99.37%, respectively.

CONCLUSION

In the present investigation, the developed and validated RP-HPLC method was found to be simple, rapid, accurate, sensitive, precise and robust for determination of Etodolac and Thiocolchicoside in combined solid oral dosage (Tablet) formulation. The excipients usually present in the pharmaceutical formulation did not interfere with determination of Etodolac and Thiocolchicoside.This developed method can be successfully used for routine quality control of Etodolac and Thiocolchicoside in their combined dosage form.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.