Pharma Science Monitor 5(2), Sup-1, Apr-Jun 2014 PHARMA SCIENCE MONITOR
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1 Impact factor: /ICV: Pharma Science Monitor 5(2), Sup-1, Apr-Jun 2014 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: SIMULTANEOUS ESTIMATION OF TIZANIDINE HYDROCHLORIDE AND ROFECOXIB BY RATIO DERIVATIVE SPECTROSCOPIC METHOD IN TABLET Gajera Jatin B *, Hasumati A. Raj, Vineet C. Jain Shree Dhanvantary College of Pharmacy, Kim, Surat, Gujarat, India. ABSTRACT A simple, accurate and precise spectroscopic method was developed for simultaneous estimation of Tizanidine Hydrochloride and Rofecoxib using Ratio Derivative Method. Ratio was taken using divisor. Divisor for µg/ml Tizanidine Hydrochloride was 6µg/ml of Rofecoxib and for 6-30µg/ml Rofecoxib was 4µg/ml of Tizanidine Hydrochloride. After divisor spectra were converted to First derivative. Tizanidine Hydrochloride showed zero crossing point at nm while Rofecoxib showed zero crossing point at nm. The absorbance was measured at nm for Tizanidine Hydrochloride and nm for Rofecoxib and calibration curves were plotted as absorbance versus concentration, respectively. The method was found to be linear (r 2 >0.999) in the range of µg/ml for Tizanidine Hydrochloride at nm. The linear correlation was obtained (r 2 >0.998) in the range of 6-30µg/ml for Rofecoxib at nm. The limit of determination was 0.017µg/ml and 0.13µg/ml for Tizanidine Hydrochloride and Rofecoxib, respectively. The limit of quantification was 0.052µg/ml and 0.39µg/ml for Tizanidine Hydrochloride and Rofecoxib, respectively. The accuracy of these method were evaluated by recovery studies and good recovery result were obtained greater than 99% shows first order derivation zero crossing. The method was successfully applied for simultaneous determination of Tizanidine Hydrochloride and Rofecoxib in tablet. KEYWORDS: Tizanidine Hydrochloride, Rofecoxib, Simultaneous estimation, Ratio Derivative Method. INTRODUCTION Tizanidine Hydrochloride is It is a muscle relaxant and α 2 -adrenergic agonist (1). It is used to treat the spasms, cramping, and tightness of muscles caused by medical problems, such as multiple sclerosis, spastic diplegia, back pain, or certain other injuries to the spine or central nervous system.it is chemically 5-chloro-N-(4,5-dihydro- 1H-imidazol-2-yl)-2,1,3 benzothiadiazol-4- amine hydrochloride. Tizanidine HCl is a white or off- white amorphous powder having molecular weight 290.2g/mol. (2)
2 Impact factor: /ICV: FIG. 1 CHEMICAL STRUCTURE OF TIZANIDINE HCl Rofecoxib is a member of a subgroup of Non steroidal anti-inflammatory drugs(nasaids) known as Cycloxygenase-2(COX-2) selective inhibitors (3). The Anti-inflammatory, Analgesic and Antipyretic effects of NSAIDs appear to result from the inhibition of Prostaglandin synthesis so inhibition of inflammation responces. It is chemically 4-[4 (Methylsulfonyl) phenyl]-3- phenylfuran-2(5h)-one.rofecoxib is a light yellow powder having molecular weight g/mol. (3-4) FIG. 2 CHEMICAL STRUCTURE OF ROFECOXIB The review of literature regarding quantitative analysis of Tizanidine Hydrochloride and Rofecoxib revealed that no Ratio Derivative method attempt was made to develop analytical methods for Tizanidine Hydrochloride and Rofecoxib. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual and combination of drugs. (5-26) The focus of the present study was to develop and validate a rapid, stable, specific, and economic Spectroscopic method for the estimation of Tizanidine Hydrochloride and Rofecoxib in Tablet. MATERIALS AND METHODOLOGY Tizanidine Hydrochloride and Rofecoxib were obtained as gift samples from Purvi chemicals., Ahmedabad. A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software. An analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing the samples. Sonicator(D120/2H, TRANS-O-SONIC) Class A volumetric glassware were used (Borosillicte)
3 Impact factor: /ICV: Standard stock solution for Tizanidine HCl An accurately weighed quantity equivalent to 10mg of Tizanidine HCl was transferred to 100 ml volumetric flask made up to the mark with the methanol. Sonicate for 15 mins. Filter Stock solution. Pipetting 5 ml was transferred into a 50 ml volumetric flask from above stock solution and volume was adjusted up to mark with methanol so it give 10 μg/ml. Standard stock solution for Rofecoxib An accurately weighed quantity of RFC (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with methanol to obtain standard solution having concentration of RFC (100 μg/ml). Preparation of Standard Mixture Solution ( + RFC): 2 ml of working standard solution of (50 μg/ml) and 2.4 ml of standard Stock solution of RFC (100 μg/ml) were pipetted out into two separate 10 ml volumetric flasks and volume was adjusted to the mark with Methanol to get 2 μg/ml of and 24 μg/ml of RFC. Preparation of test solution: Twenty ROFINOL-TZ Tab Tablets (containing 2mg of Tizanidine HCl and 25 mg of Rofecoxib) were accurately weighed and ground to fine powder. An accurately weighed quantity equivalent to 2mg of Tizanidine HCl and 25mg of Rofecoxib from the formulation fine powder was transferred to 100 ml volumetric flask made up to the mark with the methanol. Sonicate for 15 mins. Filter Stock solution. Take 1 ml dilute up to 10 ml (Tizanidine HCl)(2 μg/ml). From above solution take 1 ml dilute up to 10 ml (Rofecoxib) (25 μg/ml). Calibration curve for Tizanidine HCl This series consisted of five concentrations of standard solution ranging from 0.5 to 2.5 μg/ml. The solutions were prepared by pipetting out Standard stock solution (100 μg/ml).then pipetting 5 ml was transferred into a 50 ml volumetric flask and volume was adjusted up to mark with methanol. Then pipetting out (0.5ml, 1ml, 1.5ml, 2ml, 2.5ml ) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with methanol. A Ratio Derivative spectrum of the resulting solution was recorded, measured the absorbance at nm against a reagent blank solution (methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of. Calibration curve for Rofecoxib This series consisted of five concentrations of standard RFC solution ranging from 6 to 30 μg/ml. The solutions were prepared by pipetting out Standard RFC stock solution (0.6ml, 1.2ml,
4 Impact factor: /ICV: ml, 2.4ml, 3.0ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with methanol. A Ratio Derivative spectrum of the resulting solution was recorded, measured the absorbance at nm against a reagent blank solution (methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of RFC. Ratio Derivative method: Thus obtained spectra were then processed to ratio by dividing the spectra of all series of µg/ml of by the spectra of concentration 6µg/ml of RFC and dividing the spectra of all series of 6-30µg/ml of RFC by the spectra of concentration 4µg/ml of. (Figure ) The divided spectra were further processed to obtain first order derivative. The overlain second order spectra (fig ) of and RFC reveal that showed zero crossing at nm, while RFC showed zero crossing at nm. At zero crossing point (ZCP) of (253.00nm), RFC showed a First-derivative absorbance, whereas at ZCP of RFC (316.00nm), showed a Firstderivative absorbance. Hence nm and nm were selected as analytical wavelengths for determination of Tizanidine HCl and Rofecoxib, respectively. FIG.3 OVERLAIN LINEAR SPECTRA OF DIVISION BY 6 μg/ml OF RFC (BLUE) AND RFC BY 4 μg/ml OF (RED) IN 1:12 RATIOS. FIG.4 OVERLAIN LINEAR FIRST ORDER SPECTRA (BLUE) AND RFC (RED) IN 1:12 RATIOS.
5 Impact factor: /ICV: RESULT AND DISCUSSION Validation Parameters (I) Linearity Six point calibration curve was obtained in the concentration range of µg/ml for Tizanidine HCl and 6-30µg/ml for Rofecoxib. The response of drug was found to be linear in investigation range and the regression equations was found to be y = x for (n=6) and y = x for RFC (n=6), with the correlation coefficient and (n=6) respectively, is listed in Table. TABLE. 1 CALIBRATION DATA FOR AND RFC AT NM AND NM, RESPECTIVELY. *(N=6) Sr no. Concentration Absorbance Absorbance (μg/ml) (316.00nm)±SD* (253.00nm)±SD* RFC RFC ± ± ± ± ± ± ± ± ± ±0.041 FIG. 5 CALIBRATION CURVE FOR AT NM
6 Impact factor: /ICV: FIG. 6 CALIBRATION CURVE FOR RFC AT NM (II) Precision The precision of the method was evaluated in terms of inter-day and intra-day by carrying out independent assays of three concentrations chosen from range of the standard curves (1, 1.5, and 2µg/ml of and 12, 18, 24µg/ml of RFC) and the %RSD of assay (inter-day and intra-day) was calculated. The results of study are shown in Table 2 and 3. TABLE 2. INTRADAY PRECISION DATA FOR ESTIMATION OF AND RFC *(N=3) Conc. (μg/ml) RFC RFC Abs. ±% RSD* Abs. ±%RSD* ± ± ± ± ± ±0.24 TABLE 3. INTERDAY PRECISION DATA FOR ESTIMATION OF AND RFC *(N=3) Conc. (μg/ml) RFC RFC Abs. ±% RSD* Abs. ±%RSD* ± ± ± ± ± ±0.12 (III) Accuracy The accuracy of the method was determined by spiking of EDA and CIT to prequantified sample solutions of (1µg/ml) and RFC (12.5µg/ml) in triplicate at three concentration level of 80, 100, 120% of the specified limit. The percentage recoveries of and RFC were calculated and the result is nearer to 100% shown in Table 4 and 5.
7 Impact factor: /ICV: TABLE 4. RECOVERY DATA OF *(N=3) Conc. Amount of Total Total amount % % of from Std. amount of of found Recovery RSD formulation added (µg/ml)* (n=3) (µg/ml) (µg/ml) (µg/ml) Mean ± SD ± % ± % ± % TABLE 5. RECOVERY DATA OF RFC *(N=3) Conc. Amount of Total Total amount % % of RFC from Std.RFC amount of of RFC found Recovery RSD formulation added RFC (µg/ml)* (n=3) RFC (µg/ml) (µg/ml) (µg/ml) Mean ± SD ± % 0.08% ± % 0.59% ± % 0.42% (IV) Limit of Detection and Limit of Quantification The limit of detection (LOD) and limit of quantitation (LOQ) of the method were evaluated by standard deviation of response and slope method. LOQ and LOD were calculated by the equation LOD = 3.3 N/B and LOQ = 10 N/B, where N is standard deviation of the absorbance, and B is the slope of the corresponding calibration curve. The limit of detection (LOD) were found to be 0.017µg/ml for and 0.128µg/ml for RFC and respectively and limit of quantitation (LOQ) were found to be 0.052µg/ml for and 0.390µg/ml for RFC presented in Table 6. TABLE 6. LOD AND LOQ DATA OF AND RFC *(N=10) Conc. (μg/ml) Avg. ± SD(316.00nm)* % Avg.±SD(253.00nm)* % RFC RSD RFC RSD ± % 0.193± % LOD (μg/ml) LOQ (μg/ml)
8 Impact factor: /ICV: (V) Robustness and Ruggedness Robustness was done by different instrument and difference in preparation of stock solution. The result was decided by %RSD which is in the limit which is mentioned in table no 7. TABLE 7. ROBUSTNESS AND RUGGEDNESS DATA OF AND RFC *(N=3) Condition Conc. (μg/ml) Different Instrument Different Stock Solution Preparation UV-2450 UV-1800 Stock-1* Stock-2* Tizanidine HCl Mean (n=3) ± % RSD Rofecoxib Mean(n=3) ± %RSD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.33 (VI) Assay As per the test solution preparation, the solutions were prepared and further proceed for UV Spectrum. A zero order derivative spectrum of the resulting solution was recorded and processed division and after that proceed to first derivative spectra. A first order derivative spectrum of the sample solution was recorded and the absorbance at nm and nm were noted for estimation of and RFC, respectively. The concentrations of and RFC in formulation were determined using the corresponding calibration graph. The result was as per the Table 8. TABLE 8. ANALYSIS DATA OF COMMERCIAL FORMULATION *(N=3) Sr. No. Formulation (ROFINOL TZ) Absorbance (316.00nm) % Assay ±SD* Absorbance (253.00nm) % Assay RFC±SD* RFC RFC ± ±
9 Impact factor: /ICV: Summary Table SR. NO. TABLE. 9 SUMMARY OF VALIDATION PARAMETERS PARAMETER Tizanidine HCl Rofecoxib 1 Wave length Max nm nm 2 Linearity (µg/ml) (n=6) 0.5 to 2.5 µg/ml 6 to 30 µg/ml 3 Regression equation y = 0.119x y = x Correlation coefficient (r 2 ) Accuracy(%Recovery) (n=3) LOD (µg/ml) (n=10) LOQ (µg/ml) (n=10) Precision Intra-day (%RSD)(n=3) Inter-day (%RSD)(n=3) Robustness and Ruggedness (%RSD) Assay 99.77% 98.25% CONCLUSION A new, Ratio Derivative method has been developed for estimation of Tizanidine HCl and Rofecoxib. The method was validated by employment of ICH (27) guidelines. The validation data is indicative of good precision and accuracy, and prove the reliability of the method. ACKNOWLEDGEMENT We are sincerely thankful to Shree Dhanvantary Pharmacy College, Kim, Surat, for providing us Infrastructure facilities and moral support to carry out this research work. We are also thankful to SDPARC for giving us their special time and guidance for this research work. We also thank our colleagues for their helping hand.
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