549 JANGHEL et al: A NEW METHOD FOR DETERMINATION OF ASCORBIC ACID Journal of Scientific & Industrial Research Vol. 71, August 2012, pp. 549-555 *Authors for correspondence E-mail: **dr.ypervez@gmail.com; *eteshkumarjanghel@gmail.com A new method for determination of ascorbic acid in fruit juices, pharmaceuticals and biological samples Etesh K Janghel 1 * Santosh Sar 2 and Y Pervez 3** 1 Department of Applied Chemistry, Ashoka Institute of Technology & Management, Gram- Torankatta, Post-Somni, Rajnandgaon 491 441, India 2 Bhilai Institute of Technology, Bhilai House, Durg 491 001, India 3 Chhatrapati Shivaji Institute of Technology, Balod Road, Durg 491 001, India Received 23 March 2012; revised 22 June 2012; accepted 28 June 2012 This study presents a new, selective and accurate indirect spectrophotometric method for determination of L-ascorbic acid in fruit juices, pharmaceuticals and biological samples. Beer’s law is obeyed by ascorbic acid (conc. range, 0.8-6.3 μg) per 25 ml of final solution (0.032-0.252 ppm). The method showed: apparent molar absorptivity, 1.56 × 10 5 l mol -1 cm -1 ; Sandell’s sensitiv- ity, 0.0024 μg cm -2 ; limit of detection, 0.0086 μg ml -1 ; and limit of quantitation, 0.01 μg ml -1 . It was satisfactorily applied for determination of ascorbic acid in fruit juices, pharmaceuticals and biological samples. Reliability of method was established by parallel determination against Leuco crystal violet and Rhodamine – B method. Keywords : Ascorbic acid, Auramine O dyes, Biological Samples, Pharmaceuticals, Spectrophtotometry Introduction Ascorbic acid (AA; also known as L-ascorbic acid, antiscorbutic vitamin and vitamin C) is an important water soluble vitamin 1 . Humans and apes cannot synthesize ascorbic acid due to lack of gulonolactone oxidase enzyme, and hence ascorbic acid has to be supplemented from external sources, mainly through fruits, vegetables and tablets. Major metabolites of AA in human body are dehydroascorbic acid, 2,3- diketogluconic acid and oxalic acid 2 . Vitamin C is one of the most essential vitamins for both pharmaceutical and food processing industries in view of its nutritional significance, varied uses in food and high daily doses necessary for optimum health 3 . A large number of methods for determination of AA include titrimetry 4-5 , voltammetry 6 , amperometry 7 , conductometry 2 , potentiometry 8 , fluorometry 9-10 , chemiluminescence 1,11-13 , flow-injection analysis (FIA) 14-17 and chromatography 18,19 . Among spectrophotometric determination of AA, formation of an osazone [bis-(2,4-dinitrophenyl hydrazone)] derivative of AA 20 is a complex, time consuming method and subject to several interferences. Modifications have been suggested improving this method 21 . The method using 2,6-dichlorophenol indophenols sodium (DCPIP) 22 is subject to several limitations. Silver-gelatin complex has been used in reductive spectrophotometric method 23 . Oxidation of AA with Fe(III) and complexation of resulting Fe(II) with 1,10- phenonthroline 24 , besides a simple kinetic spectrophotometric measurement of AA based on the reduction of toluidine blue 25 are reported. An indirect spectrophotometric determination of AA based on extraction of iodine produced by reduction of potassium iodate 26 is also reported. Many other reagents [fast red 27 , leuco crystal violet 28 , methyl viologen 3 , Leuco malachite green 29 , rhodamine-B 30 , Cu(II) – ED + A-complex 31 , 2 – mercaptoethanol 32 and reverse flow injection analysis (rFIA) 33 etc.] have also been used in determination of AA. This study presents a simple method for determination of AA using auramine O as chromogenic reagent. Proposed method was successfully applied for determination of AA in fruit juices, pharmaceutical and biological samples. Experimental Section All chemicals used were of Anal R grade. Double distilled deionized water was used. For ascorbic acid (Loba Chemie), a stock solution (1 mg /ml) was prepared