A NEW RP HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ETODOLAC AND THIOCOLCHICOSIDE IN TABLET DOSAGE FORMS

A NEW RP-HPLC METHOD FOR THE SIMULTANEOUS

ESTIMATION OF ETODOLAC AND THIOCOLCHICOSIDE IN

TABLET DOSAGE FORMS

K. Sujatha*1, J.V.L.N. Seshagiri Rao2

1

Department of Pharmacy, Government Polytechnic, Visakhapatnam. 2

Yalamarty College of Pharmacy, Visakhapatnam.

ABSTRACT

An accurate, precise and reproducible high performance liquid chromatographic method was developed for quantitative estimation of etodolac and thiocolchicoside simultaneously in tablet dosage forms.

Separation of the drugs was achieved within 10 min on a Kromosil C18 (150 x 4.6 mm, 5) column by isocratic elution using a

mixture of acetate buffer (pH 3.7) and methanol (20:80 v/v) as the mobile phase at a flow rate of 0.8 mL/min. The analytes in the eluate were monitored at 242 nm. Under the optimized conditions, the retention times obtained for etodolac and thiocolchicoside were 3.176 min and 2.285 min respectively. The linearity of quantification was observed in the concentration range of 100 – 600 µg/mL for etodolac and 1 – 6 µg/mL for thiocolchicoside. The performance of the method was validated according to ICH guidelines. The method was found to be suitable for accurate determination of these drugs in tablet dosage forms without any interference from normal excipients.

Key words: Etodolac, Thiocolchicoside, Determination, HPLC, Isocratic.

INTRODUCTION

Etodolac [1-2], chemically (RS)-2-(1,8-Diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl)acetic acid, is a non - steroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic and antipyretic properties. Its therapeutic effects are due to its ability to inhibit cyclo-oxygenase enzyme and there by inhibit prostaglandin synthesis. It is indicated for relief of signs and symptoms of rheumatoid arthritis and osteoarthritis. Etodolac is official in the

Volume 3, Issue 2, 3994-4002. Research Article ISSN 2277 – 7105

Article Received on 10 January 2014,

Revised on 26 January2014, Accepted on 28 February 2014

*Correspondence for

Author

K. Sujatha

Department of Pharmacy,

Government Polytechnic,

Indian Pharmacopoeia[3], in British Pharmacopoeia[4] as well as in United States Pharmacopoeia[5].

Thiocolchicoside [6-8], chemically (N-[(7S )-3-(β-D-Glucopyranosyloxy)-1,2-dimethoxy-10-(methylsulfanyl)-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide), a semi-synthetic derivative of the naturally occurring compound colchicoside, is a muscle relaxant with anti inflammatory & analgesic properties. Thiocolchicoside binds to GABA-A and strychnine sensitive glycine receptors, acting as a GABA-A receptor antagonist [9]. It is primarily indicated in conditions like backache, neuralgia, pain, parkinsonism and sciatic pain.

(a) (b)

Figure 1. Chemical structures of Etodolac (a) and Thiocolchicoside (b)

Detailed literature survey was carried out and revealed that few HPLC techniques [10-15] have been reported for the simultaneous determination of etodolac and thiocolchicoside in pharmaceutical dosage forms. We have developed a new accurate and precise RP-HPLC method with short retention and run times for the determination of etodolac and thiocolchicoside in tablet dosage forms. The proposed method was duly validated as per ICH guideline [16].

MATERIALS AND METHODS

Drugs, Chemicals and Solvents

Rankem Fine Chemicals Ltd., Mumbai. HPLC grade water was prepared by using Millipore Milli-Q system.

Equipment and Chromatographic Conditions

A Waters Alliance liquid chromatograph (Model 2695) fitted with a diode array detector (Model 2996) and running on Empower2 data handling system was employed in the study. A Kromosil C18 (150 x 4.6 mm, 5) was used for resolving the drugs. All the chromatographic runs were carried out by using a mobile phase consisting of a mixture of acetate buffer (pH 3.7) and methanol (20:80 v/v) in isocratic mode at a flow rate of 0.8 mL/min. The injection volume of the samples was 10 µL. The detector wavelength was set at 242 nm for monitoring the drugs. The chromatographic run time was set as 10.0 min. The chromatography was carried out at 30°C. Under these optimized conditions, the retention time obtained for etodolac and thiocolchicoside were 3.176 min and 2.285 min respectively.

Preparation of the buffer

The acetate buffer was prepared by dissolving 0.77 gm of ammonium acetate in a beaker containing 1000 mL of water. The pH of the solution was then adjusted to 3.7 with dilute orthophosphoric acid solution. It was then sonicated for 15 min and then filtered through a 0.45µ membrane filter.

Preparation of the mobile phase

The optimized mobile phase consisted of a mixture of the above-mentioned phosphate buffer (pH 3.7) and methanol in a ratio of 20:80 v/v. This solution was also used as a diluent for preparing the drug solutions.

Preparation of the mixed working standard solution of etodolac and thiocolchicoside

Calibration Curve

Various dilutions of the mixed standard stock solution of etodolac and thiocolchicoside were prepared at different concentration levels including the working concentration. Ten microlitres of each concentration were injected into the HPLC system. The response was read at 242 nm and the corresponding chromatograms were recorded. From these chromatograms, the mean peak areas were calculated and linearity plots of concentration over the mean peak area were constructed for the individual drugs.

Estimation of the drug from the tablet dosage forms

Ten tablets of ‘Etova – MR’ (etodolac (400 mg) and thiocolchicoside(4 mg)) were crushed and ground to a fine powder. A quantity of powder equivalent to 400 mg of etodolac was transferred into a 100 mL volumetric flask. 80 mL of the diluent was added and sonicated for 30 min. The volume was made up with the diluent and the contents were mixed well. This mixture was filtered through a 0.45µ membrane filter (discarding the first few mL of the filtrate). 1.0 mL of the filtrate was transferred into a 10 mL volumetric flask and made up to volume with the diluent. This solution was then chromatographed six times. From the chromatograms obtained, the average drug content in the formulation was calculated.

RESULTS AND DISCUSSION

Table 1. Optimized chromatographic conditions

Stationary Phase Kromosil C18, (150 x 4.6 mm, 5)

Mobile Phase Acetate buffer : Methanol = 20:80 v/v

Flow Rate 0.8 mL/min

Column Temperature 30°C

Injection Volume 10 µL

Detection Wavelength 242 nm

Run time 10.0 min

Specificity

The chromatogram shown in Figure 2 reveals good baseline separation of etodolac and thiocolchicoside from working standard solution. A good analytical method should be able to measure the drug accurately in the presence of probable interferences from the excipients of its formulation. Fig. 3 thus demonstrates that no interfering peaks arise due to the excipients of its tablet at the retention times of etodolac and thiocolchicoside.

Figure 2. Representative chromatogram showing the separation of etodolac and

thiocolchicoside from the working standard solution.

Figure 3. Representative chromatogram obtained from analysis of etodolac and

Linearity

The regressions of the plots were computed by least square regression method and were shown in the Fig. 4 and 5. The calibration curves (n=3) constructed for each drug were linear over the concentration range of 100 – 600 μg/mL for etodolac and 1 – 6 μg/mL for thiocolchicoside. The correlation coefficients were greater than 0.999 and the % RSD for each concentration studied was less than 2%.

Figure 4. Linearity plot for etodolac

Figure 5. Linearity plot for thiocolchicoside

Accuracy and precision

3 and Table 4. From the data obtained the developed RP-HPLC method was found to be precise.

Table 2. Accuracy data of the proposed method

Analyte

Amount of the analyte added (µg/mL) Mean recovery (n=3) (µg/mL) SD of recovery % Mean recovery Etodolac

200 201.4 0.29 100.7

400 397.8 0.31 99.5

600 600.1 1.47 100.0

Thiocolchicoside

2 2.0 0.02 100.3

4 4.0 0.01 101.0

6 6.0 0.05 100.6

Table 3. Precision data for etodolac

S.No. Repeatability Intermediate

precision

1 969579 970071

2 968112 971654

3 969414 967825

4 970135 968474

5 969958 969554

6 970066 969974

Average 969544.0 969592.0

SD 756.56 1341.71

%RSD 0.078 0.138

Table 4. Precision data for thiocolchicoside

S.No. Repeatability Intermediate

precision

1 77513 77712

2 77553 77045

3 76996 76995

4 77374 77034

5 77294 77001

6 77579 77451

Average 77384.8 77206.3

SD 219.98 302.69

%RSD 0.284 0.392

System suitability parameters

Table 5. System suitability parameters of the proposed method

Parameter Etodolac Thiocolchicoside

Retention time (min) 3.176 2.285

Tailing factor 1.52 1.05

Theoretical plates 5105 4226

HETP 0.0294 0.0355

Method suitability

The commercial tablet formulation, ‘Etova – MR’ [etodolac (400 mg) and thiocolchicoside (4 mg)] was analyzed by the proposed method. The recovery obtained (100.0 %) by the proposed method was found to be in good agreement with the labeled amount of the drug, which confirms the suitability of the method for the analysis of etodolac and thiocolchicoside in tablet dosage forms.

ACKNOWLEDGEMENT

The authors are thankful to the authorities of the College of Pharmaceutical Sciences, Andhra University, for providing laboratory facilities for carrying out this study.

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