Simultaneous Spectrophotometric Estimation of Lornoxicam and Thiocolchicoside in Combined Dosage Form

 

Madhusmita Sahoo, Pratima K. Syal, Snehal S. Ingale, Dipali M. Mehetre, Vishnu P. Choudhari* and Bhanudas S. Kuchekar

Pharmaceutical Analysis and Quality Assurance Department, MAEER’s Maharashtra Institute of Pharmacy, MIT Campus, Paud Road, Kothrud, Pune, 411038, MS, India.

*Corresponding Author E-mail: viraj1404@rediffmail.com

ABSTRACT:

Two simple, rapid, accurate and economic spectrophotometric methods are described for the determination of Lornoxicam (LOR) and Thiocolchicoside (THIO) in combined dosage form. The first method is Ratio Spectra Derivative and second is Absorption Corrected method. Methanol was used as solvent for both the methods. First method was measurement of amplitude of first order derivative of ratio spectra where LOR and THIO were determined at λ_max 445.21 nm and 371.79 nm, respectively. The second method was based on the absorption corrected method in which LOR and THIO exhibit λ_max at 435 nm and 333.74 nm, respectively in methanol. LOR has some interference due to THIO at 333.74 nm, while THIO do not show any absorption at 435 nm. Quantitative  estimation  of  THIO was  carried  out  by subtracting  the  absorption  due  to LOR at  333.74 nm  using  experimentally calculated absorption factor. Both the drugs obey Beer’s law in the concentration ranges employed for these methods. The results of analysis have been validated statistically and recovery studies confirmed the accuracy and reproducibility of the proposed methods which were carried out by following ICH guidelines.

 

 

INTRODUCTION:

Lornoxicam (LOR, 6-chloro-4-hydroxy-2-methyl- N-2-pyridyl-2H-thieno [2, 3-e]-1, 2-thiazine-3-carbox- amide-1, 1-dioxide) (Fig. 1) is a novel non-steroidal anti-inflammatory drug (NSAID) with marked analgesic properties. LOR, belongs to the chemical class oxicams, this includes piroxicam, tenoxicam, and meloxicam^{1- 3}.

 

Thiocolchicoside is (s)-N-[3-(B-D-glucopyranoxyloxy)-5,6,7,9-tetrahydro-1,2-dimethoxy-10-(methylthio) -9-oxobenzo[a]heptalen-7yl] acetamide (Fig.1). Thiocolchicoside is a synthetic sulphur derivative of colchicoside. Thiocolchicoside has a selective affinity for γ-amino- butyric acid (GABA) receptors and acts on the muscular contracture by activating the GABA-nergic inhibitory pathways thereby acting as a potent muscle relaxant⁴. Literature survey reveals that HPLC and UV Spectroscopic methods are reported for the estimation of LOR and THIO individually as bulk and in pharmaceutical formulations^5-16. The review of the literature revealed that there is no Spectrophotometric method available for determination of this combination.

 

Therefore the aim of the study was to develop simple, rapid, accurate, reproducible and economical spectroscopic methods for both the drugs in combined dosage forms. The proposed methods were optimized and validated as per the International Conference on Harmonization (ICH) analytical method validation guidelines².

 

MATERIALS AND METHODS:

Instrumentation:

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells was used. All weighing were done on electronic balance (Model Shimadzu AUW-220D).

 

Reagents and chemicals:

Pure drug sample of LOR, % purity 98.80 and THIO, % purity 99.92 was kindly supplied as a gift sample by Glenmark pharmaceuticals Ltd. Baddi and Medley Pharmaceuticals Ltd. Baddi, respectively. These samples were used without further purification. Two tablet formulations (Lot 302 and 304) were supplied by JPLC Pharma ltd., Jalgaon were used for analysis containing LOR 8mg and THIO 4mg per tablet. Spectroscopy grade methanol was used throughout the study. All the solvents and reagents used were purchased from LOBA Chemie Pvt. Ltd., Mumbai.

 

THIOCOLCHICOSIDE

 

LORNOXICAM

 

Preparation of Standard Stock Solutions and calibration Curve:

Standard stock solutions of pure drug containing 200 μg/ml of LOR and 400 μg/ml of THIO were prepared separately in methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in methanol. Ratio Derivative amplitudes of spectrum, by using the above mentioned procedure, were used to prepare calibration curves for both the drugs by Ratio derivative method. Absorbances were measured at selected wavelengths for absorbance corrected method and were used to construct calibration curves. Beer’s law obeyed in the concentration range of 12-36 μg/ml for LOR and 6-18 μg/ml for THIO for ratio derivative and 16-80 μg/ml for LOR and 8-40 μg/ml for THIO for absorption corrected method.

 

Preparation of Sample Stock Solution and Formulation analysis:

Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 8  mg of LOR (4 mg of THIO) was weighed and dissolved in 40 mL of methanol with the aid of ultrasonicator for 15 min and solution was filtered through Whatman paper No. 41 into a 50 ml  volumetric  flask.  Filter  paper  was  washed  with  methanol,  adding  washings  to  the  volumetric  flask and volume was made up to mark. The solution was suitably diluted with methanol to get of 8μg/ml of LOR (4 μg/ml of THIO) and proposed methods were followed for analysis.

 

PROCEDURE:

Method A: Ratio Derivative Spectrophotometric method

Theoretical aspects:

The  method  involves  dividing  the  spectrum  of  mixture  by  the  standardized  spectra  of each  of  the  analyte  to get ratio spectra and first derivative of ratio spectrum was obtained which was independent of concentration of divisor (Fig. 2). The concentration of active compounds are then determined from calibration graph obtained by measuring amplitude at points corresponding to minima or maxima.

 

Fig 2: Spectrum of mixture of LOR and THIO for ratio derivative method

 

Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different LOR standards at increasing concentrations were obtained by dividing LOR+THIO scans with the stored spectrum of the standard solution of THIO (12 μg/ml) ( Fig 2A).  Wavelength 445.21 nm was selected for the quantification of LOR in LOR+THIO mixture (Fig 2B). The ratio and ratio derivative spectra of the solutions of THIO at different concentrations were obtained by dividing LOR+THIO scans  with the stored standard spectrum of the LOR (24 μg/ml) (Fig. 2C and 2D, respectively). Wavelength 371.79 nm was selected for the quantification of THIO in LOR+THIO mixture. Measured analytical signals at the selected wavelengths were proportional to the concentrations of the drugs. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The amount of LOR (C_LOR) and THIO (C_THIO) in tablets was calculated by using following equations-

At 445.21nm: CLOR = (Ratio derivative amplitude for LOR- 3.876)/ 6.845.... (1)

At 371.79nm: C_THIO = (Ratio derivative amplitude for THIO - 0.1732)/ 0.076.… (2)

 

Fig 2 (A): Ratio spectra of LOR using 12μg/mlsolution of THIO as divisor

 

Table 1: Optical characteristics of the proposed methods

Parameter		LORNOXICAM		THIOCOLCHICOSIDE
		Method A	Method B	Method A	Method B
λ (nm)		445.21	435	371.79	333.74
Beer’s law limit (μg mL-1)		12 – 36	16 - 80	6-18	8 - 40
Régression Equation (y = mx + c)	Slope (m)	6.845	0.016	0.076	0.001
	Intercept (c)	3.876	-0.051	0.173	-0.003
Correlation coefficient		0.999	0.999	0.999	0.998
Precision (%R.S.D.)	Repeatability (n=5)	0.86	0.76	1.12	0.82
	Intra-day(3×3 times)	0.98	0.78	1.28	1.40
	Inter-day(3×5 days)	1.24	0.95	1.36	0.83
Formulation Analysis (% Assay,  %RSD) n=6	Formulation I	99.01, 0.89	98.91, 1.1	100.89, 1.06	101.72, 1.04
	Formulation II	100.11, 0.63	99.24, 0.93	99.84,  0.55	101.5,  0.65

R.S.D. is relative standard deviation

 

 

Fig 2 (B): First derivative of the ratio spectra of LOR (12-36μg mL^-1).

 

Fig 2 (C): Ratio spectra of THIO using 24μg/mlsolution of LOR as divisor.

 

Fig 2 (D) First derivative of the ratio spectra of THIO (6-18μg    mL^-1)

 

METHOD B: ABSORPTION CORRECTED METHOD:

λ_max of  LOR and THIO was determined by scanning the drug solution in methanol was found to be at 435 nm and 333.74 nm respectively. LOR also showed absorbance at 333.74 nm, while THIO did not show any interference at 435nm. To construct Beer’s plot for LOR and THIO dilutions were made in the solvent using stock solution of 100 μg/ml. Also Beer’s plot was constructed for LOR and THIO in solution mixture at different concentration (16:8, 32:16, 48:24, 64:32, 80:40 μg/ml) levels. Both the drugs followed linearity individually and in mixture within the concentration range 16-80 μg/ml and 8-40 μg/ml for LOR and THIO respectively (Fig.3).

 

Table2 (A): Result of the recovery analysis for method A

Compound	Recovery Level (%)	aWt. spiked		Recovery (%)		R.S.D. (%) n = 3
		LOR	THIO	LOR	THIO	LOR	THIO
Tab I	50	6	3.0	99.96	99.33	0.52	0.38
	100	12.0	6.0	100.08	99.50	0.17	0.86
	150	18.0	9.0	100.22	100.22	0.39	0.18
Tab II	50	6.0	3.0	99.83	101.33	0.59	0.28
	100	12.0	6.0	99.83	99.33	0.58	0.78
	150	18.0	9.0	100.11	100.66	0.33	0.36

^a Wt. = Weight

 

 

Table2 (B): Result of the recovery analysis for method B

Compound	Recovery Level (%)	Wt. spiked		Recovery (%)		R.S.D. (%) n = 3
		LOR	THIO	LOR	THIO	LOR	THIO
Tab I	50	8.0	4.0	99.83	99.83	0.42	0.98
	100	16.0	8.0	100.16	99.83	0.47	0.26
	150	24.0	12.0	99.91	100.33	0.29	0.18
Tab II	50	8.0	4.0	100.75	100.83	0.75	0.29
	100	16.0	8.0	99.87	99.91	0.35	0.58
	150	24.0	12.0	100.13	100.33	0.13	0.32

 

 

Fig.3. Overlay spectrum of LOR and THIO in methanol. LOR (16-80µg mL^-1) and THIO (8-40 µg mL^-1)

 

Determination of Absorption Factor at Selected Wavelengths:

LOR and THIO solution in methanol of known concentrations were scanned against blank on spectrophotometer. The value of absorption factor was found to be 1.152. Quantitative estimation of LOR and THIO was carried out using following equation:

 

Corrected Absorbance of THIO at 333.74nm =

abs_333.74 (LOR+THIO) – [(abs_333.74 (LOR)/ abs₄₃₅ (LOR)] × abs₄₃₅ (LOR) or

 

Corrected Absorbance of THIO at 333.74nm = abs_333.74 (LOR+THIO) – 1.152 × abs₄₃₅ (LOR)

Where; abs: Absorption value at given wavelengths.

 

Recovery studies:

The accuracy of the proposed methods was checked by recovery study, by addition of standard drug solution to preanalysed sample solution at three different concentration levels (50 %, 100 % and 150 %) within the range of linearity for both the drugs. The basic concentration level of sample solution selected for spiking of the drugs standard solution was 12 μg/ml of  LOR and 6 μg/ml of THIO for method A and 16 μg/ml of LOR and 8 μg/ml of THIO for method B.

 

Precision of the Method:

To study intraday precision, method was repeated 5 times in a day and the average % RSD was found to be 0.86 and 0.78 for LOR by method A and B, respectively; 1.12 and 0.95 for THIO by method A and B, respectively.

Similarly the method was repeated on five different days and average % RSD was found to be 0.92 and 0.82 for LOR by method A and B, respectively; 1.09 and 0.83 for THIO by method A and B, respectively. These values confirm the intra and inter day precision.

 

RESULTS AND DISCUSSION:

Practically no interference from tablet excipients was observed in these methods. As  their  λmax  differ more  than 20 nm,  absorption corrected  method  was  tried  for  their  simultaneous  estimation  in formulation. Quantitative estimation of THIO was carried out by subtracting interference of LOR using experimentally calculated absorption factor. Both the methods are accurate, simple, rapid, precise, reliable, sensitive, reproducible and economical as per ICH guidelines. The values of % RSD and correlation of coefficient were satisfactory and results of the formulation analysis in Table 1 and result of the recovery study in Table 2(A) and 2(B) indicates that there is no interference due to excipients present in the formulation. It can be easily and conveniently adopted for routine quality control analysis.

 

CONCLUSION:

The proposed methods are simple, precise, accurate and rapid for the determination of LOR and THIO in combined tablet dosage forms. Analysis of authentic samples containing LOR and THIO showed no interference from the common additives and excipients. Hence, recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations. It can be easily and conveniently adopted for routine quality control analysis.

 

ACKNOWLEDGEMENT:

The authors are thankful to m/s Glenmark Pharmaceuticals Ltd. Baddi, India and Medley Pharmaceuticals Ltd., Baddi, India for providing gift samples of Lornoxicam and Thiocolchicoside, respectively. The authors are thankful to Management of MAEER’s Maharashtra Institute of Pharmacy, Pune for providing necessary facility for the work.

 

REFERENCES:

1.       The Merck Index (2001) Merck research Laboratories. 13^th edn, Maryndale J.O’Neil, Merck and Co., Inc., White house Station, NJ. USA.6281, 5602

2.       ICH-Q2B Validation of Analytical Procedures: Methodology International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1996.

3.       S. Radhofer-Weltea, P. Dittrichb, Determination of the novel non-steroidal anti-inflammatory drug lornoxicam and its main metabolite in plasma and synovial fluid, J. of Chrom. B, 1998,707, 151–159

4.       N.A.El-Ragehy*; Determination of thiocolchicoside in its binary mixtures (Thiocolchicoside_/glafenine and thiocolchicoside_/ floctafenine) by TLC_/densitometry, Il Farmaco.2003; 58: 463-/468.

5.       NEMUTLU E. ; DEMIRCAN S. ; KIR S. ; Determination of lornoxicam in pharmaceutical preparations by zero and first order derivative UV Spectrophotometric methods, Pharmazie   ISSN 0031-7144. 2005;60: 421-425

6.       Young Hoon Kim, Hye Young Ji, Eun-Seok Park 1, Soo-Wan Chae 2, and Hye Suk Lee; Liquid  Chromatography-Electrospray Ionization Tandem Mass Spectrometric Determination of Lornoxicam in Human Plasma ,Arch Pharm Res. 2007;30:905-910.

7.       Kiran R. Patil, Vipul P. Rane, Jaiprakash N. Sangshetti, Devanand B. Shinde , Stability-Indicating LC Method for Analysis of Lornoxicam in the Dosage Form, Chromatographia . 2009; 69: 1001-1005.

8.       Suwa, T., Urano, H., Shinohara, Y., and Kokatsu, J., Simultaneous high performance liquid chromatographic determination of lornoxicam and its 5’-hydroxy metabolite in human  plasma using electrochemical detection. J. Chromatogr.1993; 617:105-110.

9.       Emirhan Nemutlu, Şeyda Demircan, Sedef Kır Determination of lornoxicam in pharmaceutical preparations by using Spectrophotometric and chromatographic methods; Adnan Menderes University, 4th AACD Congress, Kuşadası-AYDIN/ TURKEY Proceedings Book 069.DOC. 2004 :135-136

10.     Rajesh S. Jadon*, Swadesh Nayak, Sabita Amlan Taste Masking of Lornoxicam by Polymer Carrier System and Formulation of Oral Disintegrating Tablets, Int .J. of Drug Delivery 1.2009: 27-31.

11.     Young Hoon Kim , Hye Young Ji , Eun -Seok Park , Soo -Wan Chae² and Hye Suk Lee  Liquid chromatography-electrospray ionization tandem mass spectrometric determination of lornoxicam in human plasma, A. Of Pharmacal Research.2007;30:905-910

12.     Mariella Artusi ¹, Sara Nicoli ¹, Paolo Colombo Effect of chemical enhancers and iontophoresis on thiocolchicoside permeation across rabbit and human skin in vitro, J.of pharmaceutical sciences , 93 : 2431 - 2438

13.     F.C.W. Sutherland*, M.J. Smit, Highly specific and sensitive liquid chromatography–tandem mass spectrometry method for the determination of 3- desmethylthiocolchicine in human plasma as analyte for the assessment of bioequivalence after oral administration of thiocolchicoside; J. of Chrom. A, 2002;949: 71–77

14.     M. Artusi a, P. Santi a, Buccal delivery of thiocolchicoside: in vitro and in vivo permeation studies, Int. J. of Pharmaceutics. 2003; 250: 203-/213.

15.     Priyabrata Pattanayak, Yogendra Prasad Chaudury; Simultaneous Spectrophotometric Estimation of Nitazoxanide and Ofloxacin in Combined Tablet Dosage Form; Research J. Pharm. and Tech.2 (2), ISSN 0974-3618. 2009,291-293.

16.     PS Tarte*, SP Wate; Absorption Correction Method for Estimation of Nebivolol and Hydrochlorothiazide in Combined Tablet Dosage Form; Asian J. Research Chem. 1(2): Oct.-Dec. 2008:74-76

 

 

Received on 25.01.2010       Modified on 28.02.2010

Accepted on 20.03.2010      © RJPT All right reserved