STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF NIMESULIDE AND CHLORZOXAZONE IN PHARMACEUTICAL TABLET DOSAGE FORM

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Page5600 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876 STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF NIMESULIDE AND CHLORZOXAZONE IN PHARMACEUTICAL

1 Page5600 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: STABILITY-INDICATING RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF NIMESULIDE AND CHLORZOXAZONE IN PHARMACEUTICAL TABLET DOSAGE FORM Dr. Bhavsar Ankita *, Shah Dhwani Department of Quality assurance, Sat Kaival College of Pharmacy. Gujarat, Anand (Sarsa) India. ARTICLE INFO Article history Received 02/05/2016 Available online 31/05/2016 Keywords Stability Indicating, Hydrolysis, Oxidative, Nimesulide, Chlorzoxazone. Corresponding author Dr. Bhavsar Ankita Department of Quality assurance, Sat Kaival College of Pharmacy, Gujarat, Anand (Sarsa) India ABSTRACT A stability-indicating high performance liquid chromatographic method was developed and validated for quantitative determination of Nimesulide and Chlorzoxazone in tablet dosage form. Isocratic high performance liquid chromatographic method, using a ECO C 8 - (150mm*4.6mm*5μ) (particle size) as stationary phase with mobile phase Methanol: Acetonitrile: 1% Ammonium acetate (25:25:50%v/v/v) with flow rate of 1 ml/min retention time for Nimesulide and Chlorzoxazone were found to be min and 6.223min respectively. Nimesulide and Chlorzoxazone were exposed to acid/base hydrolytic, oxidative, thermal and photolytic stress conditions, and stressed sample were analysed by proposed method. There were no other co-eluting, interfering peaks from excipients, impurities or degradation products due to variable stress conditions. The linearity was obtained in the concentration range of µg/ml for Nimesulide and µg/ml for Chlorzoxazone. Limit of detection was found to be 4.71 μg/ml and μg/ml and data for limit of quantification were found to be μg/ml and μg/ml for Nimesulide and Chlorzoxazone respectively. The mean recoveries were % and % for Nimesulide and Chlorzoxazone, respectively. The accuracy of the method was in compliance with the proposed limits and the precision of the method was satisfactory. The system suitability of the method showed that the performance of the chromatographic system is not significantly influenced by variations of the operational parameters inside an accepted domain. The excellent separation was demonstrated in the chromatograms and no interfering peaks were observed. Hence, the present method was found to be sensitive, rapid, specific, accurate and robust. Please cite this article in press as Dr. Bhavsar Ankita et al. Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Nimesulide and Chlorzoxazone in Pharmaceutical Tablet Dosage Form. Indo American Journal of Pharmaceutical Research.2016:6(05). Copy right 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page5601 INTRODUCTION Nimesulide (NIM) chemically known as N-(4-nitro-2-phenoxyphenyl) methanesulfonamide (figure: 1). It is a nonsteroidal anti-inflammatory drug (NSAIDs) with broad effects on inflammation and other biochemical processes leading to a multifactorial mode of action. [1-5] NIM is a preferential cyclo-oxygenase-2 (COX-2) inhibitor but its anti-inflammatory and analgesic action also involves activity on a wide range of inflammatory and pain mediators and intracellular pathways including an effect on synovial concentration of substance P (SP) in patients with knee osteoarthritis. [6] NIM is official in British pharmacopeia (BP 2010). [7] Chlorzoxazone (CHZ) chemically known as a 5-chloro-3Hbenzooxazol-2-one (Figure: 2). [8] It is a colourless or white to creamy white crystalline powder, very slightly soluble in water. CHZ is a skeletal muscle relaxant. It acts by inhibiting multi synaptic reflexes involved in producing and maintain skeletal muscle spasm of varied aetiology. It acts on the spinal cord by depressing reflexes. CHZ is a synthetic compound, inhibits antigen-induced broncho spasms and hence, is used to treat asthma and allergic rhinitis. CHZ inhibits degranulation of mast cells, subsequently preventing the release of histamine and slow-reacting substance of anaphylaxis (SRS-A), mediators of type I allergic reactions. CHZ may reduce the release of inflammatory leukotrienes. CHZ is used to relieve pain and stiffness caused by muscle strains and sprains [9]. CHZ is official in United States Pharmacopeia (USP 2010) [10]. Literature survey reveals UV, HPLC and stability indicating HPLC methods have been reported for the estimation of NIM or CHZ alone and in combination with other drugs. Currently there is no stability indicating method has been reported for combination of NIM and CHZ till date. So the present study was carried out with the aim to develop simple, accurate, precise and sensitive stability indicating method for simultaneous estimation of NIM and CHZ in combined pharmaceutical dosage form. Figure: 1 Chemical structure of NIM. Figure: 2 Chemical structure of CHZ. MATERIALS AND METHODS Selection of analytical wavelength The standard solution of NIM (10 μg/ml) and CHZ (10 μg/ml) in methanol was scanned in the UV region of nm using methanol as a blank and the overlain spectra were recorded and 290 nm analytical wavelength was selected for estimation of NIM and CHZ. Chromatogram is shown in figure: 3. Selection of mobile phase The mobile phase should be sufficiently transparent at the wavelength of detection methanol: Acetonitrile (ACN), 1% ammonium acetate are the solvents which are tried for selecting solvent for mobile phase. In our studies various mobile phase with different ratio were used. The mobile phase consist of methanol: ACN: 1% ammonium acetate (25:25:50%v/v/v) provided optimum polarity for proper migration, separation and resolution of NIM and CHZ. Preparation of mobile phase A mixture of 25 ml of methanol and 25 ml of ACN in this solution add 50ml volume of 1% ammonium acetate (1gm in 100 ml water) and sonicated for 10 minutes. Preparation of mix standard solution of NIM and CHZ Accurately weighed NIM 50 mg and 125 mg CHZ in 50 ml volumetric flask and make up with methanol from this 2 ml was taken and diluted up to 10ml with methanol. This solution was containing 200 μg/ml of NIM and 500 μg/ml of CHZ. Solution was injected in HPLC chromatogram shown in figure: 4. Preparation of sample solution Twenty tablets were weighed and powdered equivalently to 50 mg of NIM and 125 mg of CHZ which were transferred in 50 ml volumetric flask. 25 ml of methanol was added, sonicated for 20 minute and diluted with methanol up to mark. The solution was filtered. First few ml of filter was discarded. 2 ml of filtrate was diluted to 10 ml with methanol to make 200 μg/ml NIM and 500 μg/ml of CHZ. Chromatogram is shown in figure: 5.

3 Page5602 Table 1: Optimized chromatographic condition. Parameters Conditions Mobile phase Methanol : ACN : 1%ammonium acetate (25:25:50%v/v/v) Stationary Phase ECO-C 8 (15mm*4.6mm*5μ) (particle size) suniest-eco- C 8 Flow rate 1ml/min Run time 20min Volume of injection 20μl Detection wave length 290nm Force degradation study Procedure for preparation of solutions for force degradation study It is a process in which the natural degradation rate of a pharmaceutical formulation is increased by applying the additional stress. The prepared standard solutions (20 mg of NIM and 50 mg of CHZ) and sample solutions (equivalent to 20 mg NIM and 50 mg CHZ) were treated with various degradation conditions, such as acidic (5 ml of 0.1, 1, 2, 5 N HCl, at room temperature for 8, 12, 24, 24 hours), basic (5 ml of 0.1, 1, 2 N NaOH, at room temperature for 8, 12, 24 hours), oxidization (5 ml of 3% H 2 O 2, at room temperature for 24 hours), thermal (20 mg NIM and 50 mg CHZ powder at 105ºC for 24 hours) and photo degradation (20 mg NIM and 50 mg CHZ powder for 24 hours). In above mentioned degradation conditions, each degradation samples were neutralized with respective degradants. Then diluted with 100 ml methanol to achieve the appropriate concentration of 200 μg/ml and 500 μg/ml for NIM and CHZ respectively. This resulting solutions were injected in HPLC. Degradation behaviour: Singh S and Bakshi M, in their article on stress testing suggested a target degradation of % of establishing the stability nature of assay method, as even intermediate degradation product should not interfere at any stage of drug analysis. 11 Although conditions used for force degradation were adjusted to achieve degradation in the range of %, this could not achieve in some cases even after exposures for a prolonged duration. But in basic degradation unknown impurity was found in 2 N NaOH. Figure 6 and 7 showed acidic degradation conditions (5 N HCl) for standard and sample respectively. Figure 8 and 9 showed basic degradation conditions (2N NaOH) for standard and sample respectively and figure 10, 11 showed oxidative degradation conditions for standard and sample respectively. Validation of developed method Validation of the optimized HPLC method was carried out with respect to the following parameters as per ICH Q2 (R1) guidelines. [12] Linearity Stock Solution 50 mg NIM and 125 mg CHZ was transferred in 50 ml volumetric flak add 25 ml methanol and sonicated for 20 min and make up the volume up to the mark. Dilution was done to achieve the appropriate concentration of 1000 μg/ml of NIM and 2500 μg/ml of CHZ. Linearity was prepared as follow. Aliquots from above stock solution (1, 1.5, 2, 2.5, 3) in 10 ml volumetric flask and diluted up to the mark with methanol to give solution having a strength of NIM 100 ppm, 150 ppm, 250 ppm 300 ppm, 350 ppm respectively and for CHZ 250 ppm, 375 ppm, 500 ppm, 650 ppm, 750 ppm respectively. The results are summarized in Table: 3 and calibration curve was shown in figure: 12 and 13 NIM and CHZ respectively. Limit of detection (LOD) and Limit of quantification (LOQ): The detection limit of an analytical method is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated. Limit of quantification is the lowest concentration of analyte in a sample which can be quantitatively determined with acceptable precision and accuracy. DL= 3.3 QL=10 σ =standard deviation of response, S= slope of the calibration curve. The slope S may be estimated from the calibration curve of the analyte. The results are summarized in Table: 4. ACCURACY The accuracy of the test method is demonstrated by % of recovery. The sample preparations are spiked with known amount of standard at three concentration levels and each concentration is injected three times (Like 50% 100% and 150%). Acceptance criteria: The % of recovery should be between 98 to 102%. The results are summarized in Table: 5.

4 Page5603 System suitability parameters Sample solution of NIM and CHZ were injected in triplicates as per the procedure. From the standard chromatogram system suitability parameters like theoretical plates, tailing factor, retention time, area and resolution are evaluated. The results are given in Table: 6. Quantitative estimation of marketed pharmaceutical formulation. The proposed validation method was successfully applied to determine NIM and CHZ injection formulation. 50 mg of NIM and 125 mg of CHZ powdered mixture was transferred in to a 100ml volumetric flask and dissolved in methanol, it was shaken for 30 min and the volume was made up to the mark with methanol, the content was ultra-sonicated for 20 min it contains 1000 μg/ml NIM and 2500 μg/ml of CHZ. The solution was filtered through 0.45μm glass paper. Again from this solution 5ml taken and transferred in to 50 ml volumetric flask and make up with methanol up to the mark. It contain NIM 200 μg/ml and CHZ 500 μg/ml. The assay result obtained for NIM and CHZ was comparable with the corresponding labelled amount. Result are mentioned in Table: 7. RESULTS AND DISCUSSION Selection of analytical wavelength 290 Figure 3: Overlain spectra of NIM and CHZ. From overlain spectra of NIM and CHZ it is observed that Isoabsorptive point of NIM and CHZ is 290nm. Method development In order to develop an isocratic reverse phase HPLC method for the determination of NIM and CHZ in pharmaceutical dosage form the chromatographic conditions were optimized. For better separation and resolution the different solvents were tried. It has been found that methanol: Acetonitrile: and 1% ammonium acetate give better peak shape with ECO-C 8 (15mm*4.6mm*5μ) (particle size) suniest-eco- C 8. The analyte gave better response at 290 nm wavelength using UV detector. The flow rate was kept 1.0 ml/min. There was no peak tailing observed under these optimized chromatographic conditions. The retention times of NIM and CHZ were found to be min for NIM and min for CHZ respectively. Figure4: Chromatogram of NIM, CHZ and mix standard in methanol: ACN: 1%ammonium acetate (25:25:50% v/v/v).

5 Page5604 Figure 5: Chromatogram of marketed formulation containing 200μg/ml of NIM 500μg/ml of CHZ using mobile phase methanol: ACN: 1%ammonium acetate: (25:25:50%v/v/v). Above mentioned both the chromatogram showed that both the drugs had good resolution with good pick shape, sufficient retention time and tailing factor is also within the limit. Force degradation study Acid degradation study of 5 N HCl (with 5ml, 24hr) Figure 6: Chromatogram of mix std (NIM and CHZ). Figure 7: Chromatogram of marketed formulation. There was no impurity found in acid degradation so that it can concluded that drug is stable in acid degradation.

6 Page5605 Base degradation Base degradation with 2N NaOH (with 5ml, 24hr) Figure 8: Chromatogram of formulation (NIM and CHZ). Figure 9: Chromatogram of marketed formulation. Base degradation study showed unknown impurity in 2 N NaOH. Hence it can be said that drug is stable in base degradation up to 1 N NaOH but not stable in further base degradation conditions. Oxidative degradation (3% H 2 O 2, 24hr) Figure 10: Chromatogram of mix std (NIM and CHZ).

7 Page5606 Figure 11: Chromatogram of marketed formulation. It was observed from figure 10 and 11 that no interfering peaks were found with 3% H2O2. So drugs were stable in oxidative degradation. Table 2: Summary data of force degradation. Force degradation study Retention Time Result % Degradation Different condition NIM (min) CHZ (min) NIM (%) CHZ (%) 0.1N HCl(8hr) Negative 83.36% % 1N HCl(12hr) Negative 83.42% 68.43% 2N HCl(24hr) Negative 83.64% 68.44% 5N HCl(24hr) Negative 83.78% % 0.1N NaOH(8hr) Negative 77.90% 80.70% 1N NaOH(12hr) Negative 80.96% 80.69% 2N NaOH(24hr) Positive Unknown Impurity Unknown Impurity Oxidative degradation(3%h 2 O 2 24hr) Negative 81.17% 78.97% Thermal degradation Negative 81.17% 81.14% (105 o C 24hr) Photolytic degradation (longer wavelength 24hr) Negative 81.98% 79.98% Validation of the proposed method The proposed method has been validated for the simultaneous determination of NIM and CHZ formulation as per ICH guidelines. Linearity and Range Linear correlation obtained between peak area Vs concentration range of μg/ml for NIM and μg/ml for CHZ. Calibration curve of these two drugs at 290 nm are shown in figure: 13 and 14. Figure 12: Calibration curve of NIM.

8 Page5607 Figure 13: Calibration curve of CHZ. Table 3: Linearity values of NIM and CHZ. NIM(n=5) CHZ(n=5) Concentration (μg/ml) Mean peak area Concentration (μg/ml) Mean peak area Correlation coefficient Correlation coefficient Limit: Correlation coefficient NLT n=number of replicate injection. Correlation coefficient of NIM and CHZ was found to be and respectively which indicate that proposed method is linear. Table: 4 Limit of detection (LOD) and Limit of quantification (LOQ). Parameters NIM CHZ LOD μg/ml 4.71 μg/ml μg/ml LOQ μg/ml μg/ml μg/ml Accuracy (% Recovery) The recovery experiment was performed by the standard addition method. The % recoveries obtained were % and % for NIM and CHZ respectively. The low value of standard deviation indicates that the proposed method is accurate. Result are shown in table: 5 Table: 5 Accuracy data of NIM and CHZ. Drug % level Amount of sample Mean Area Amount of standard % recovery %RSD taken(mg) (n=3) recover(mg) NIM CHZ n= number of replicate injection. The percentage recovery for each level should be between % as per ICH guideline. The percentage recovery of NIM and CHZ were observed % and % respectively which was found within the limit.

9 Page5608 System suitability parameters The column efficiency, resolution and peak asymmetry were calculated for the standard solution. From the chromatogram of standard solution containg 200 μg/ml NIM and 500 μg/ml CHZ. The value obtained demonstrated the suitability of the system for analysis of this drug combination. Chromatogram given in table 6. Table 6: System suitability data of NIM and CHZ. Parameters NIM±RSD CHZ±RSD Acceptance criteria Theoretical plates 5247± ±0.17 >2000 Tailing factor 1.29± ±0.14 <2 Resolution 11.19±0.40 >2 Retention time 6.251± ±1.69 <20 Area ± ± Flow rate 1ml/min 1ml/min - This method showed all the system suitability parameters were within the limits only. Table no: 7 Analysis of marketed formulation. Sr. no. Actual conc. Conc. Found %Assay NIM CHZ NIM CHZ NIM CHZ (%) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (%) Mean SD %RSD (Limit: NMT 2%) The percentage assay should be between % as per ICH guideline. The % assay of NIM and CHZ was found % and % respectively which is within the limit. CONCLUSION Rapid separation of NIM and CHZ was successfully attained with a relatively short retention time, provides outstanding resolution, good peak shape gave reliable and highly reproducible results on C8 HPLC column. Separation of NIM and CHZ mixture was achieved with a total run time less than 20 minutes. Based on the results obtained from the analysis of forced degraded samples using the described method, it can be concluded that there was no other co-eluting peak with the main peaks and the method was specific for the estimation of NIM and CHZ in the presence of degradation product(s) / impurities. Excellent values for linearity and recovery were achieved together with low LOD and LOQ. The ease in preparation of mobile phase and economy of the components of mobile phase showed explicitly the applicability of this method the best choice in routine analysis of NIM and CHZ in pharmaceutical quality control departments for routine analysis. Conflict of interest The authors declared that they have no conflict of interest. ACKNOWLEDGEMENT The authors are indebted to Indica laboratories for providing the sample of CHZ and NIM. We are highly thankful to Aum research laboratories for providing the necessary guidance to carry out this research work.

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10 Page5609 REFERENCES 1. Rainsford KD. Nimesulide a multifactorial approach to inflammation and pain: scientific and clinical consensus. Curr Med. Res. Opin. 2006; 22(3): Warner TD, Giuliano F, Vojnovic I. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc. Natl. Acad. Sci. U S A. 1999; 96(2): Bennett A, Villa G. Nimesulide: an NSAID that preferentially inhibits COX-2, and has various unique pharmacological activities. Expert Opin Pharmacother 2000; 1: Suleyman H, Cadirci E, Albayrak A. Nimesulide is a selective COX-2 inhibitory, atypical non-steroidal anti-inflammatory drug. Curr Med Chem. 2008; 15: Dogan MD, Ataoglu H, Akarsu ES. Nimesulide and Diclofenac inhibit lipopolysaccharide-induced hypothermia and tumour necrosis factor-alpha elevation in rats. Fundam. Clin. Pharmacol. 2002; 16(5): Bianchi M, Broggini M, Balzarini P. Effects of Nimesulide on pain and on synovial fluid concentrations of substance P, interleukin-6 and interleukin-8 in patients with knee osteoarthritis: comparison with celecoxib. Int. J. Clin. Pract. 2007; 61: British pharnacopoiea;6th Edn; the stationary office, London medicines and health care product Regulatory Agency,2009,vil II, pp Neil M J. drugs and biological.14th ed. USA: Merck Research Laboratories: pp Rang H P. Dale M M. Ritter J M. Pharmacology. 6th Ed. Churchill Livingston: New York: pp United state oharmacopoiea-34 and national formulary-29; Asia; United States Pharmacopoeia convention, Rockville MD, USA, 2011 vol II, pp Singh S, Bakshi M. Development of validated stability-indicating assay methods-critical review. J. Pharm. Biomed. Anal. 2002; 24: Validation of analytical procedure Methodology ICH Q2 (R1) ICH Harmonized Tripartite Guidelines,

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