INTRODUCTION:

Chemically, Thiocolchicoside (THC) is 2-Demethoxy-2-glucosidoxythiocolchicine. Thiolchicoside is a semi synthetic derivative of naturally occurring compound colhcicoside from the seeds of various species of colchicum autumnale belonging to family liliaceae.(autumn crocus, meadow saffron, Gloriosa superba)¹. It is a muscle relaxant with anti-inflammatory and analgesic effects². Diclofenac (DCF) is monosodium salt of 2-[(2,6-dichlorophenyl) amino] benzeneacetic acid or 2-(2,6-dichloranilino) phenylacetic acid³. It is benzeneacetic acid derivative and belong to non-steroidal anti-inflammatory drug (NSAID) taken to reduce inflammation and as an analgesic reducing pain in conditions such as arthritis or acute injury⁴. Structures of both the drugs (THC and DCF) are shown in figure 1. Literature survey reveals that assay of DCF in bulk and dosage form is official in Indian Pharmacopoeia 2007⁵, British Pharmacopoeia 2008⁶ and United state Pharmacopoeia NF-27⁷.

Several analytical methods have been reported for estimation of THC are spectrophotometry⁸, RP-HPLC⁹, LC-MS¹⁰and capillary electrophoresis¹¹ and that of DCF are spectrophotometry^12-14 HPLC^15-18 colorimetric assay¹⁹, HPTLC^20-21supercritical fluid chromatography^22-24 The analytical methods reported for estimation of THC in combination with other drugs are spectrophotometry²⁵ and LC²⁶.For estimation of DCF with other drugs spectrophotometry²⁷, atomic absorption spectrophotometric^28-30, RP-HPLC^31-34 are reported. The present paper describes simple, accurate, specific and precise methods for simultaneous estimation of THC and DCF in their combined dosage form. The proposed method is optimized and validated as per the ICH guidelines^35-37. In the present work, a successful attempt has been made to estimate both these drugs simultaneously using two UV spectrophotometric methods (I) Absorbance Correction method and (II) Dual wavelength method.

MATERIALS AND METHODS:

Instrument used was an UV-Visible double beam spectrophotometer, make: Shimadzu (Model UV-1800) with a pair of 1 cm matched quartz cells. All weighing was done on Shimadzu analytical balance (Model AU-220). Pure drug samples of THC and DCF were obtained from reputed pharmaceutical company. Combined tablet formulation Thioact D8 (Brand A) and Mobiwok – plus (Brand B) was procured from local market. Distilled water was used as solvent.

Thiocolchicoside

Figure 1: Chemical structures of the Thiocolchicoside.

Preparation of standard stock solution:

Accurately weighed quantity of THC (100 mg) and DCF (100 mg) was transferred to two separate 100 ml volumetric flasks, dissolved in distilled water and diluted to the mark with same. (Stock solutions: 1000 μg/ml of THC and 1000 μg/ml of DCF).

Preparation of working standard solution:

100 mg/ml of THC solution was prepared by diluting 10 ml of stock solution with distilled water in 100 ml volumetric flask up to the mark.

200 mg/ml of DCF solution was prepared by diluting 20 ml of stock solution with distilled water in 100 ml volumetric flask up to the mark.

Absorbance correction method (Method I):

Absorbance correction method uses the absorbances at two selected wavelengths, one at λmax of one drug where other drug also shows considerable absorbance and other being the wavelength at which the first drug has practically nil absorbance. From the stock solutions, working standard solutions of THC (8μg/ml) and DCF (50μg/ml) were prepared by appropriate dilution and were scanned in the entire UV range to determine the suitable wavelengths (Fig. 3). THC and DCF have λmax at 371 nm and 276 nm respectively. Both the drugs were found to have considerable absorbance at 248.5 nm while at 371 nm only THC has absorbance. The wavelengths selected for analysis were 371 nm and 248.5 nm for THC and DCF respectively as shown in figure 2. A series of standard solutions ranging from 2-16 μg/ml of THC and 12.5-100 μg/ml of DCF were prepared separately and the absorbances of solutions were measured at 371 nm and 248.5 nm. A calibration curve was prepared by plotting absorbance versus corresponding concentration of drug and absorptivity of both drugs at both lambda was determined. The concentration of two drugs in sample solution was calculated by using following equations:

..………………………. (1)

…………….................. (2)

Where, A₁and A₂ are the absorbances of mixture at 371 nm and 248.5 nm respectively; a_x1and a_x2 are absorptivities of THC at 371 nm and 248.5 nm respectively; a_y2isabsorptivity of DCF at 248.5 nm; C_THCis concentration of THC; ^CDCFis concentration of DCF.

Diclofenac

Figure 2: Chemical structures of the Diclofenac sodium.

Figure 3: Overlain zero order spectra of THC (8 µg/ml) and DCF (50 µg/ml) in distilled water.

Figure 4: Overlain zero order spectra of THC (8 µg/ml) and DCF (50 µg/ml) in distilled water.

Dual wavelength method (Method II):

In this method two wavelengths were selected (λ₁, λ₂) where the drug A showing equal absorbance (or difference between absorbance is zero) and drug B showing some response. Then different concentrations of drug A and drug B are prepared to confirm that at all different concentrations of drug A the difference between absorbance at two selected wavelengths (λ₁, λ₂) remain zero, and at all different concentration of drug B difference between absorbance at two selected wavelength (λ₁, λ₂ ) showing linear response. From the stock solutions, working standard solutions of THC (8μg/ml) and DCF (50μg/ml) were prepared by appropriate dilution and were scanned in the entire UV range to determine the suitable wavelengths. Then different concentrations of THC and DCF are prepared to confirm that at all different concentrations of THC the difference between absorbance at two selected wavelengths 248nm and 268nm remain zero, and at all different concentration of DCF difference between absorbance at two selected wavelength 248nm and 268nm shows linear response (Fig. 4). So the calibration curve is prepared for absorbance difference verses conc. of DCF (Absorbance difference is zero for TCH). A series of standard solutions ranging from 2-16 μg/ml of THC and 12.5-100 μg/ml of DCF were prepared separately and the absorbances of solutions were measured at 248 nm and 268 nm for estimation of DCF and THC was directly measured at 371nm where DCF shows no absorbance. A calibration curve was prepared by plotting difference in absorbances versus corresponding concentration of DCF. A calibration curve for THC was prepared by plotting absorbance at 371nm against respective concentration. The concentration of two drugs in sample solution was calculated by using linear equations of calibration data:

Assay of tablet formulation by method I and II:

Twenty tablets were weighed and crushed to obtain a fine powder. An accurately weighed tablet powder equivalent to about 8 mg of THC and 50 mg of DCF was transferred to 100 ml volumetric flask and the volume was made up to the mark using distilled water as solvent. The resulting solution was filtered through Whatman filter paper and 10 ml of this filtrate was appropriately diluted to get concentration of 8 μg/ml of THC and 50 μg/ml of DCF. Absorbance of sample solutions was measured at 248 nm and 268 nm and difference in concentration was measured for estimation of DCF .The concentration of THC in the sample was determined at 371nm.

Method validation:

Linearity and range:

Aliquots of standard stock solutions of THC and DCF were taken in volumetric flasks and diluted with distilled water to get final concentrations in range of 2-16 μg/ml for THC and 12.5-100μg/ml for DCF. This calibration range was prepared five times and absorbances were measured at respective wavelengths for each drug separately.

Precision:

Precision of the methods were determined by performing interlay variation, intraday variation and method repeatability studies. In interlay variation, the absorbance of standard solutions of THC (2-16 μg/ml) and DCF (12.5-100 μg/ml) were measured on five consecutive days. In intraday variation the absorbances were measured five times in a day. In repeatability study, three concentrations of both the drugs were analysed in triplicate.

Recovery studies:

To study the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels. A known amount of drug was added to preanalyzed tablet powder and percentage recoveries were calculated.

Ruggedness:

The data for ruggedness obtained from two different analysts for THC and DCF.

RESULTS AND DISCUSSION:

The proposed methods were validated as per ICH guideline. The plot of absorbances versus respective concentrations of THC and DCF were found to be linear in the concentration range of 2-16 μg/ml and 12.5-100 μg/ml respectively with correlation coefficient 0.9998 at 371 nm and 0.9999 at 248.5 nm for absorbance correction method (Method I).For dual wavength method (method II) linearity range was same as for method I with correlation coefficient 0.9996 at difference in absorbance at 248nm and 268nm for estimation of DCF and 0.9998 at 371 nm estimation of THC. Precision was calculated as interday and intraday variations and % RSD was found to be less than 1 for method I and less than 2 for method II for both the drugs shown in Table 1 and Table 2. The accuracy of method was determined at 75, 100 and 125 % level. The % recovery ranges from 98.15 to 101.11 for both the methods as shown in Table 3. The % RSD of ruggedness for THC ranges from 2.11 to 2.88%, while for DCF it was found to be 2.30 to 2.57% for method I and for method II respectively. Results for all validation parameters are presented in table 1, 2 and 3. The two _methods can be successfully _used for simultaneous estimation of THC and _{DCF in
their combined tablet dosage form.}Marketed tablets were analyzed and results obtained were in the range of 98-102% (Table 4).

CONCLUSION:

The proposed methods give accurate and precise results for determination of THC and DCF in marketed formulation (tablet) without prior separation and are easily applied for routine analysis. The most striking feature of both the methods is its simplicity and rapidity. Method validation has been demonstrated by variety of tests for linearity, accuracy, precision and ruggedness. The developed methods have several advantages, as they are simple, accurate and precise. The proposed methods were successfully applied to determination of these drugs in commercial tablets.

Table 1: Validation Parameters for Absorbance Correction Method

PARAMETERS	THC	DCF
Linearity range	2-16 µg/ml	12.5-100 µg/ml
Correlation Coefficient	0.9998	0.9999
Precision	% RSD
Repeatability Intraday Interday	0.545% 0.234%-0.856% 0.121%-0.873%	0.528% 0.346%-0.876% 0.237% -0.994%
% Recovery	99.50%-101.83%	99.75%-100.56%
Ruggedness	2.119	2.307

*THC- Thiocochicoside; DCF-Diclofenac Sodium; RSD-Relative Standard Deviation.

Table 2: Validation Parameters for Dual wavelength Method

PARAMETERS	THC	DCF
Linearity range	2-16 µg/ml	12.5-100 µg/ml
Correlation Coefficient	0.9993	0.9999
Precision	% RSD
Repeatability Intraday Interday	0.207% 0.173%- 0.993% 0.182%- 1.654%	0.127% 0.069%- 0.919% 0.079%- 0.411%
% Recovery	99.08- 100.50	99.41-99.47
Ruggedness	2.885	2.577

*THC- Thiocochicoside; DCF-Diclofenac Sodium; RSD-Relative Standard Deviation.

_{Table 3:}_{Recovery studies}

_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_Method I (AC)		Method II (DW)
_{Name of Drug}	_{Amount of Drug Added (}µg/ml)	_%Recovery*	_SD	_%Recovery*	_SD
THC	2	101.83	0.05	100.17	0.01
DCF	37.5	100.56	0.04	99.41	0.01
THC	4	101.13	0.07	100.17	0.03
DCF	50	100.16	0.08	99.91	0.04
THC	6	101.10	0.03	99.15	0.03
DCF	62.5	99.75	0.02	99.47	0.01

*Mean of Three estimations; AC-Absorbance Correction; DW-Dual Wavelength; THC- Thiocolchicoside; DCF-Diclofenac Sodium; SD-Standard Deviation.

_{Table 4: Results of simultaneous
estimation of THC and DCF in marketed Formulation by Method I and II.}

Brand	Method	mg/tablet		%of label claim± S.D.*
		THC	DCF	THC	DCF
Brand A	Method-I(AC)	8	50	99.50+0.03	98.20+0.07
Brand A	Method-II(DW)	8	50	100.25+0.04	99.60+0.08
Brand B	Method-I(AC)	8	50	99.75+0.05	98.78+0.03
Brand B	Method-II(DW)	8	50	100.75+0.06	100.22+0.06

*Average of five determinations; AC-Absorbance Correction; DW-Dual Wavelength; THC- Thiocolchicoside; DCF-Diclofenac Sodium; SD-Standard Deviation.

ACKNOWLEDGEMENT:

The authors are also thankful to the Principal, Maliba Pharmacy College for providing necessary facilities.

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Received on 20.08.2010 Modified on 27.08.2010

Asian J. Research Chem. 4(1): January 2011; Page 123-127