2254 12 archive 2 disk0/00/00/22/54 2008-07-01 14:04:56 2008-07-01 14:04:56 2008-07-01 14:04:56 article show Tham, Sock Ying Foo, C.H. School of Pharmaceutical Sciences School of Pharmaceutical Sciences pub Q R none Ascorbic acid, sequential injection analysis, peroxidase, inhibition A sequential injection analysis (SIA) method for ascorbic acid assay in pharmaceuticals utilizing crude zucchini (Cucurbita pepo) extract was compared with commercial horseradish peroxidase (HRP) assay. The peroxidase activity in the crude extract of zucchini was determined by the formation of a chromophore between guaiacol (0.05M) and hydrogen peroxide (10 mM) measured spectrophotometrically at 470 nm. The method adopted for assaying ascorbic acid is based on its inhibition of peroxidase catalyzing the formation of tetraguaiacol from guaiacol in the presence of hydrogen peroxide using a sequential injection analysis (SIA) manifold controlled by an in-house developed software. The percentage of inhibition of tetraguaiacol formation is proportional to the concentration of ascorbic acid. Km values for hydrogen peroxide using HRP and zucchini peroxidase were found to be 1.530mM and 0.868mM respectively. The conditions for the SIA system used to assay ascorbic acid in pharmaceutical and food samples were optimized at 0.9 ml/min flow rate, pH of 7 and hydrogen peroxide and guaiacol concentrations at 1.75 mM and 0.025M respectively as well as reaction time of 10 seconds. Linear range for the standards was found to be 0 to 120μg/ml (r = 0.9967 - 0.9998, n = 7). The recovery of ascorbic acid from samples ranged from 91.1 to 112.1% for the zucchini extract. Results were comparable with titrimetry using 2,6-dichlorophenolindophenol (USP method). The described SIA procedure presents a simple and rapid assay for ascorbic acid in pharmaceuticals where ingredients contained in the tablets do not interfere with the analytical method. 2005 published Malaysian Journal of Biochemistry and Molecular Biology 12 1 Malaysian Society for Biochemistry and Molecular Biology 8-13 TRUE ISSN 1511-2616 http://ejum.fsktm.um.edu.my/ArticleInformation.aspx?ArticleID=605 1. The United States Pharmacopeia 24, National Formulary 19, National Publishing, Philadelphia, 2000. 2. Leubolt R & Klein H. Determination of sulphite and ascorbic acid by high performance liquid chromatography with electrochemical detection. J. Chromatogr A, 1993;640:271-277. 3. Linquist J. Voltammetric determination of ascorbic acid by use of a carbon paste electrode. Analyst, 1975;100:339- 348. 4. Lau OW, Luk SF, Wong KS. Determination of ascorbic acid in pharmaceuticals using direct ultraviolet spectrophotometry. Analyst, 1987;112:1023-1025. 5. Zhu M Huang, M Li J Shen H, Peroxidase-based spectrophotometric methods for the determination of ascorbic acid, norepinephrine, epinephrine, dopamine and levodopa. Anal. Chim. Acta, 1997; 357:261-267. 6. Molkina-Diaz A, Ortega-Carmona I, Pascaual-Raguera MI. Indirect specrtrophotometric determination of ascorbic acid with ferrozine by flow injection analysis. Talanta, 1998;47:531-536. 7. Albero MI, Garcia MS, Sanchaz-Pedreno C, Rodriguez J. Determination of ascorbic acid in pharmaceuticals and urine by reverse flow injection. Analyst, 1992; 117:1635-1638. 8. Kleszczewski T, Kleszczewska E. Flow injection spectrophotometric determination of L-ascorbic acid in biological matters. J Pharm. Biomed. Anal, 2002; 29:755- 759. 9. Kleszczewski T, Kleszczewska E, Flow Injection Analysis of vitamin C in blood serum in humans at increasing ethanol concentration. J Pharm. Biomed. Anal 2001;25:477-481. 10. Iwado A, Mifune M, Akizawa H, Motohashi N Saito Y. Flow injection analysis of ascorbic acid using carriersmodified with metal-porphine as oxidative solid catalyses. J Pharm. Biomed. Anal 2003; 30:1923-1928. 11. Ma Y, Zhou M Jin X Zhang B, Chen, H Guo, N. Flow injection chemiluminescence determination of ascorbic acid by use of cerium-Rhodamine B system. Anal. Chim. Acta 2002; 464:289-293. 12. Casella L, Gullotti M, Marchesini A, Petrarulo M. Vitamin C assay in fruits and vegetables using peroxidase. J. Food Science 1989; 54:374-376 13. Uchiyama S, Umetsu Y. Concentration-step amperometric sensor of L-ascorbic acid using cucumber juice. Anal Chim. Acta 1991; 255:53-57. 14. Ruzicka J. Hansen, EH., Flow injection analysis: Principles, applications and trends. Anal Chim Acta 1980;114:19-44. 15. Ruzicka, J. Discovering flow injection: Journey from sample to a live cell and from solution to suspension. Analyst, 119; 1995:1925-1934. 16. Karlberg B, Pacey GE., Techniques and Instrumentation in Analytical Chemistry, Vol 10, Flow Injection Analysis : A Practical Guide, Chap. 2, Amsterdam, Elsevier, 1989. 17. Yebra-Biurrun, MC. Flow injection determination methods of ascorbic acid. Talanta 2000; 52: 367-383. 18. Ruzicka J, Gubeli T, Principles of stopped-flow sequential analysis and it’s applications to the kinetic determination of traces of a proteolytic enzyme. Anal. Chem. 1991;63:1680-1685. 19. Gubeli T, Christian GD, Ruzicka J. Fundamentals of sinusoidal flow sequential injection spectrophotometry. Anal Chem. 1991; 63:2407-2413. 20. Cruz Vieira I,Fatibello-Filho O. Flow injection spectrophotometric determination of hydrogen peroxide using a crude extract of zucchini (Cucurbita pepo) as a source of peroxidase. Analyst 1998; 123:1809-1812