Development and Validation of Spectrophotometric Method for the Estimation of Chondroitin Sulfate in Bulk Drug and Pharmaceutical Formulations
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1 Chem Sci Trans., 2013, 2(4), Chemical Science Transactions DI: /cst ISSN/E-ISSN: / RESEARCH ARTICLE Development and Validation of Spectrophotometric Method for the Estimation of Chondroitin Sulfate in Bulk Drug and Pharmaceutical Formulations P. L. SMASHEKAR a*, K. P. SATHISH b, CHANDRASHEKAR JAVALI a, A. S. TRIPATHY c and R. B. KTANAL d a Govt College of Pharmacy, Bangalore,Karnataka , India b Micro Labs, Bangalore, Karnataka , India c Al-Ameen College of Pharmacy, Bangalore, Karnataka , India d BLDEA College of Pharmacy, Bijapur, Karnataka , India somashekarpl@gmail.com Received 2 February 2013 / Accepted 16 February 2013 Abstract: A simple and sensitive spectrophotometric method is described for the determination of chondroitin sulfate in bulk drug and pharmaceuticals. The proposed method involves strong acid hydrolysis of chondroitin sulfate into monosacchrides by concentrated hydrochloric acid and followed by formation of furfural. The condensation of furfural with resorcinol in the presence of ferric ions yields a yellow-orange coloured complex. This colour has absorption maxima at 435 nm against reagent blank. The absorbance was found to increase linearly with the concentration of the drug and formed the basis for quantification. The calibration graph was linear from 4-32 µg/ml with correlation coefficient was at The results presented are statistically validated in accordance with the guidelines provided by ICH. The recovery studies were carried out at three different levels. The precision was good with percentage relative standard deviation (RSD) lower than 2.0%. The Sandell s sensitivity was calculated and was found µg/cm 2. The detection limits and quantification limit are found to be µg/ml and µg/ml, respectively. Ruggedness of method was performed and the percentage relative standard deviation (RSD) was found below 2.0%. The proposed method was successfully applied to the determination of chondroitin sulfate in bulk drug and pharnarmaceutical tablet formulation. Keywords: Spectrophotometric method, Chondroitin sulfate, Resorcinol Introduction Chondroitin sulfate belongs to a family of heteropolysaccharides called glycosaminoglycans or GAGs. Glycosaminoglycans were formerly known as mucopolysaccharides 1,2. GAGs in the form of proteoglycans comprise the ground substance in the extracellular matrix of connective tissue. It is made up of linear repeating units containing D-galactosamine and D-glucuronic acid. It is found in humans in cartilage, bone, cornea, skin and the arterial wall.
2 Chem Sci Trans., 2013, 2(4), CHS is chemically poly- (1-3)-N-acetyl-2-amino-2-deoxy-3--ß-D-glucopyranurosyl (or 6-) sulfonyl-d-galactose. It is naturally found polymer of linear repeating units containing D-galactosamine and D-glucuronic acid. The amino group of galactosamines in the basic unit of CHS is acetylated, yielding N-acetyl-galactosamine. The sulfate group is esterified to the carbon 4 or 6 position in N-acetyl-galactosamine yield chondroitin sulfate A and chondroitin sulfate C respectively. The molecular weight of chondroitin sulfate A or C ranges from 5,000 to 50,000 Daltons and contains about 15 to 150 basic units of D-galactosamine and D-glucuronic acid 3. It is represented by the following structural formula: Figure 1. Chemical structure of chondroitin sulfate CHS mainly used in promotion and maintenance of the structure and function of cartilage, pain relief of osteoarthritic joints and anti-inflammatory activity. Chondroitin sulfate also useful in coronary artery disease, interstitial cystitis, iron absorption enhancement, ophthalmologic uses and muscle soreness. Literature review revealed that few analytical methods include Gel-exclusion chromatography 4,5, capillary electrophoresis 6 and titration with cetyl pyridinium chloride detecting end point with a phototrode 7,8. The other methods in the literature for quantitating chondroitin sulfate needs enzymatic digestion followed by disaccharide analyses, include reverse phase high performance liquid chromatography 9 (HPLC), anion-exchange chromatography 10, gel electrophoreses with fluorescence detection 11 and colorimetric metric method using orcinol reagent 12. Hence an attempt has been made to develop accurate, precise and reproducible colorimetric method for estimation of chondroitin sulfate in bulk drug and tablet formulation. The method was validated by using various parameters as per ICH guidelines 13. Experimental Perkin Elmer double beam UV-Visible spectrophotometer was used with 1 cm matched quartz cells. Chemicals and reagents Pure chondroitin sulfate was obtained as gift sample from Banner Pharmacaps (India) Pvt Ltd, Bangalore, India, Resorcinol Qualigens fine chemicals, AR Grade), Ferric chloride (Rasayan Laboratory), concentrated hydrochloric acid (Qualigens fine chemicals), ethanol and double distilled water were used. Reagents: 1. Resorcinol (0.2% w/v in concentrated hydrochloric acid and ethanol) 2. Ferric chloride (0.075% w/v in concentrated hydrochloric acid) Reagent preparation Dissolve 75 mg of ferric chloride in about 50 ml of concentrated hydrochloric acid. To this solution add 5 ml ethanol containing 200 mg of resorcinol and make up to the mark with concentrated hydrochloric acid.
3 1429 Chem Sci Trans., 2013, 2(4), Preparation of standard stock solution About 100 mg of chondroitin sulfate working reference standard of known purity (92.42% pure) was accurately weighed into a 100 ml volumetric flask, dissolved & volume was made up to 100 ml with water (1 mg/ml). 10 ml of above solution was diluted to 50 ml with water (200 µg/ml). Determination of wavelength of maximum absorbance 2 ml of working reference standard solution was pipetted into a 25 ml volumetric flask, 10 ml of resorcinol reagent was added and solution was cooled to room. The solution mixture was heated in a boiling water bath for 1 hour and cooled to room temperature by placing in ice-cooled water. The volume was made up to 25 ml distilled water. The yellow-orange coloured solution was scanned in the nm range against reagent blank. The wavelength of maximum absorbance of yellow-orange colour chromogen was found at 435 nm as shown in Figure 2. Figure 2. Absorbance maxima of chondroitin sulfate Reaction scheme for chondroitin sulfate with resorcinol: CH CH 2 R R 1 H H NHCCH nm ABS Conc. HCL Fecl 3, Heat CH H H H H Glucuronic acid Chondroitin sulfate C H 2 + CH 2 R 1 R H H NHCCH3 N- Acetyl galactosamine CH Furfural H H Resorcinol H Scheme 1 C Yellow-orange colour
4 Chem Sci Trans., 2013, 2(4), Assay of marketed formulation Twenty tablets were accurately weighed and crushed to obtain fine powder. An accurately weighed tablet powder equivalent to about 100 mg of chondroitin sulfate was transferred to 100 ml volumetric flask and sonicated for 15 minutes in about 50 ml of double distilled water and made up the volume with double distilled water. The resulting solution was filtered through Whatman filter paper. 10 ml of filtrate solution was diluted to 50 ml with distilled water. 2 ml of above solution was transferred into 25 ml volumetric flask, 10 ml of resorcinol reagent solution was added and solution was cooled to room temperature. The solution mixture was heated in a boiling water bath for 1 hour and cooled to room temperature by placing in ice-cooled water. The volume was made up to 25 ml distilled water. The absorbance of yellow-orange colour chromogen was measured at 435 nm against reagent blank. Method validation Validation is the process of established documented evidence which provides high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. The method was validated by various parameters as per ICH guidelines (Table 2). Linearity Varying concentrations of chondroitin sulfate were treated with carbazole within the range 4 µg/ml to 32 µg/ml corresponding to 50%, 75%, 100%, 125% and 150% of chondroitin sulfate. The linearity of chondroitin sulfate was found to be 4-32 µg/ml and linear regression was found to be r 2 = Accuracy Accuracy of the method was determined in terms of % recovery of standard chondroitin sulfate at three different concentrations (50%, 100% and 150%). Result of the recovery study were found to be within the acceptable criteria 100±10%, indicates sensitivity of the method towards detection of chondroitin sulfate and non interference of excipients in the method (Table 1). Spike level Table 1. Recovery calculation for chondroitin sulfate Theoretical Value, mg/ml Practical Value, mg/ml Recovery, % Level I 50% % % Level II 100% % % Level III 150% % % Average RSD, %
5 1431 Chem Sci Trans., 2013, 2(4), System precision The precision of the system was determined by 6 repetitive absorbance of the same standard solutions by using 2 ml of stock solution. As the value of % RSD of system precision study were in within the acceptable limit (less than 2%). Hence the method provides good precision. Method precision The precision of the method for the assay of chondroitin sulfate was determined by the assay of six aliquots of the homogeneous sample. As the value of %RSD of system precision study were in within the acceptable limit (less than 2%). Hence the method provides good precision and reproducibility. Specificity The study was conducted to prove that the absorbance obtained in the samples is only due to chondroitin sulfate without any interference from other excipients. Placebo solution at varying concentration does not show any absorbance at 435 nm. Hence the method is specific for the determination of chondroitin sulfate. Detection limit The detection limit was determined by the analysis of chondroitin sulfate with known concentrations and by establishing the minimum level at which the chondroitin sulfate was reliably detected. The detection limit (DL) may be expressed as; DL =3.3 σ/ S where σ = the standard deviation of the response S = the slope of the calibration curve Quantitation limit The quantitation limit was determined by the analysis of chondroitin sulfate with known concentrations and by establishing the minimum level at which the chondroitin sulfate was quantified with acceptable accuracy and precision. The quantitation limit (QL) may be expressed as: QL=10 σ/ S where σ = the standard deviation of the response S = the slope of the calibration curve Solution stability The stability of the analytical solution for assay of chondroitin sulfate was determined by the assay of sample preparation at fixed intervals of time. The % RSD for the assay values for chondroitin sulfate up to 24 hours was found to be This indicates that the analytical solution is stable up to 24 hours. Ruggedness The ruggedness of the method for assay of chondroitin sulfate was determined by the assay of six aliquots of the homogeneous sample on different instrument and by different analyst. The % RSD for the assay values for chondroitin sulfate in ruggedness study was found to be This indicates that the method has good reproducibility and very less random error. The results of the method developed and validated for chondroitin sulfate in formulation are depicted in the Table 2. The results developed method showed good agreement with the labeled claim in the formulation analyzed.
6 Chem Sci Trans., 2013, 2(4), Table 2. Results for the validation of the analytical method for chondroitin sulfate Parameter Acceptance criteria Results btained 1. Linearity Regression coefficient (r 2 ) not less than Beer s Range µg/ml y = x Regression Equation 2. Accuracy Recovery between % % 2. System precision % RSD should be less than 2.0% 0.46% 4. Method precision % RSD should be less than 2.0% 1.08% 5. Specificity Non interference of placebo and Complies blank in analysis 6. Detection Limit µg/ml 7. Quantitation Limit µg/ml 8. Ruggedness % RSD should be less than 2.0% % 9. Sandell s sensitivity (mg/ml/0.001 abs units) µg/cm 2 Results and Discussion The proposed method for the estimation of chondroitin sulfate is based on the reaction between resorcinol and hydrolyzed component of chondroitin sulfate in presence of ferric ions yields a yellow-orange coloured complex. This colour has absorption maxima at 435 nm against reagent blank. This method obeyed Beer s concentration range 4-32 µg/ml. The accuracy of the method was established by recovery study, as per ICH guidelines, at three different levels viz. 50%, 100% and 150% and the % recovery ranged from Statistical analysis of the results shows that all the proposed procedures have good precision and accuracy. The method was found to be sensitive with respect to Sandell s sensitivity. The detection limits and quantification limit are found to be µg/ml and µg/ ml, respectively. Ruggedness of method was performed and the percentage relative standard deviation (RSD) was found %. Results of analysis of pharmaceutical formulations reveal that the proposed methods are suitable for their analysis with virtually no interference of the usual additives present in pharmaceutical formulations. Conclusion The proposed method is simple, sensitive and selective with reasonable precision and accuracy for the quantification of chondroitin sulfate in bulk drug and pharmaceutical dosage forms. Hence developed method was conveniently adopted for routine quality control analysis. Acknowledgement The authors are thankful to Banner Pharmacaps (India) Pvt Ltd, Bangalore, India for providing the drug sample and facilities to carry out the present work. The authors also thankful to Principal, Govt College of Pharmacy, Bangalore for providing the facilities to carry out the present research work. References 1. Pamela C C and Richard A H, Lippincott s Illustrated Reviews: Biochemistry. 2 nd Ed., Lippincott Williams & Wilkins Publishers, Philadephia (USA), 1994,
7 1433 Chem Sci Trans., 2013, 2(4), Satyanarayana U, Biochemistry 2 nd Ed., Books and Allied (P) Ltd Publishers, Vijayawada (India), 2002, The Merck Index, 13 th Ed., Merck & Co, INC Whitehouse station (USA), 1997, Way W, Gibson K and Breite A, J Liquid Chromatogr Relat Technol., 2000, 23(18), Choi D W, Kim M J, Kim H S, Chang S H, Jung G S, Shin K Y and Chang S Y, J Pharma Biomed Anal, 2003, 31(6), Al-Hakim A and Linhardt R J, Anal Biochem., 1991, 195(1), Liang Z, Bonneville C, Senez T and Henderson T, J Pharma Biomed Anal., 2002, 28(2), USP, Pharmacopeial Preview, Pharmacopoeial Forum Sept-ct, 2000, 26(5), kamoto H, Nakajima T, Ito Y, Shimada K and Yamato S, J Chroma A, 2004, 1035(1), Murata K and Yokoyama Y, Anal Biochem., 1985, 146(2), Karousou E G, Porta G, De Luca G and Passi, J Pharma Biomed Anal., 2004, 34(4), Kinoshita T, Kanada K and Tsuji A, J Biochem., 1975, 77(2), International Conference of Harmonization (ICH) of Technical Requirement for the Registration of Pharmaceuticals for Human Use. Validation of Analytical procedures; Methodology. ICH-Q2B, Geneva (1996); CPMP/ICH/281/95).
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