45.1.14 AOAC Official Method 967.21 Ascorbic Acid in Vitamin Preparations and Juices 2,6-Dichloroindophenol Titrimetric Method First Action 1967 Final Action 1968
(Applicable to determination of reduced ascorbic acid. Not applicable to highly colored juices or in presence of ferrous Fe, stannous Sn, cuprous Cu, SO2, sulfite, or thiosulfate. See Note.) A. Principle
Ascor bic acid re duces ox i da tion-reduction in di ca tor dye, 2,6-dichloroindophenol, to colorless solution. At end point, excess unreduced dye is rose pink in acid solution. Vitamin is extracted and ti tra tion per formed in pres ence of HPO 3 –CH 3 COOH or HPO3–CH3COOH–H2SO4 solution to maintain proper acidity for reaction and to avoid autoxidation of ascorbic acid at high pH. B. Reagents
(a) Extracting solutions.—(1) Metaphosphoric acid–acetic acid solution.—Dissolve, with shaking, 15 g HPO3 pellets or freshly pulverized stick HPO3 in 40 mL CH3COOH and 200 mL H2O; dilute to ca 500 mL, and fil ter rap idly through fluted pa per into glass-stoppered bottle. (HPO3 slowly changes to H3PO4, but if stored in refrigerator, solution remains satisfactory 7–10 days.) (2) Metaphosphoric acid–acetic acid–sulfuric acid solution.—Proceed as in (1), except use 0.15M H2SO4 in place of H2O. (b) Ascorbic acid standard solution.—1 mg/mL. Accurately weigh 50 mg USP Ascor bic Acid Ref er ence Stan dard (www.usp.org) that has been stored in desiccator away from direct sunlight. Transfer to 50 mL volumetric flask. Dilute to volume immediately before use with HPO3–CH3COOH solution, (a)(1). (c) Indophenol stan dard so lu tion.—Dis solve 50 mg 2,6-dichloroindophenol Na salt that has been stored in desiccator over soda lime, in 50 mL H2O to which has been added 42 mg NaHCO3; shake vigorously, and when dye dissolves, dilute to 200 mL with H2O. Filter through fluted paper into amber glass-stoppered bottle. Keep stoppered, out of di rect sun light, and store in refrigerator. (Decomposition products that make end point indistinct occur in some batches of dry indophenol and also develop with time in stock solution. Add 5.0 mL extracting solution containing excess ascorbic acid to 15 mL dye reagent. If reduced solution is not practically colorless, discard, and prepare new stock solution. If dry dye is at fault, obtain new supply.) Transfer three 2.0 mL aliquots ascorbic acid standard solution to each of thre e 50 mL Erlenmey ers con tain ing 5.0 mL HPO 3 –CH 3 COOH so lu tion, B(a)(1). Ti trate rap idly with indophenol solution from 50 mL buret until light but distinct rose pink persists ³5 s. (Each ti tration should require ca 15 mL indophenol solution, and titrations should check within 0.1 mL.) Similarly titrate 3 blanks composed of 7.0 mL HPO3–CH3COOH solution, B(a)(1), plus volume H2O ca equal to volume indophenol solution used in direct titrations. After subtracting average blanks (usually ca 0.1 mL) from standardization titrations, calculate and express concentration of indophenol solution as mg ascorbic acid equivalent to 1.0 mL reagent. Standardize indophenol solution daily with freshly prepared ascorbic acid standard solution.
(d) Thymol blue pH indicator.—0.04%. Dissolve 0.1 g indicator by triturating in agate mortar with 10.75 mL 0.02M NaOH and dilute to 250 mL with H2O. Transition range: 1.2 (red)–2.8 (yellow). C. Preliminary Test for Appreciable Amount of Basic Substances
Grind representative test sample or express contents from capsule and add ca 25 mL HPO3–CH3COOH solution, B(a)(1). Test pH by placing drop thymol blue pH indicator on pestle or by using spot plate. (pH >1.2 indicates appreciable amounts of basic substances.) For liquid preparations, dilute representative test sample ca 2-fold with HPO3–CH3COOH solution, (a)(1), before testing with indicator. D. Preparation of Test Solution
(a) For dry products containing no appreciable amount of basic substances.—Pulver ize test portion by gen tle grind ing, add HPO3–CH3COOH solution, B(a)(1), and triturate until test portion is in suspension. Dilute with HPO3–CH3COOH solution, B(a)(1), to measured volume. Designate this volume as V mL. (Use ca 10 mL extracting solution/g dry test portion. Final test solution should contain 10–100 mg ascorbic acid/100 mL.) (b) For dry products containing appreciable amounts of basic substances.—Pulver ize test portion by gen tle grind ing, add HPO3–CH3COOH–H2SO4 solution, B(a)(2), to adjust pH to ca 1.2, and triturate until test portion is in suspension. Dilute with HPO3–CH3COOH solution, B(a)(1), to measured volume. Designate this volume as V mL. (Use ca 10 mL extracting solution/g dry test portion. Final solution should contain 10–100 mg ascorbic acid/100 mL.) (c) For liquid products.—Take amount of test portion containing ca 100 mg ascorbic acid. If appreciable amounts of basic substances are present, adjust pH to ca 1.2 with HPO3–CH3COOH–H2SO4 solution, B(a)(2). Dilute with HPO3–CH3COOH solution, B(a)(1), to measured volume containing 10–100 mg ascorbic acid/100 mL. Designate this volume as V mL. (d) For fruit and vegetable juices.—Mix thoroughly by shaking to ensure uniform test portion, and filter through absorbent cotton or rapid paper. Prepare fresh juices by pressing well-pulped fruit and filtering. Express juice of citrus fruits by commercial device and filter. Add aliquots of ³100 mL prepared juice to equal volumes of HPO3–CH3COOH solution, B(a)(1). Designate total volume as V mL. Mix, and filter through rapid folded paper (Eaton-Dikeman No. 195, 18.5 cm, or equivalent). E. Determination
Titrate 3 test solution aliquots each containing ca 2 mg ascorbic acid and make blank determinations for correction of titrations as in B(c), using proper volumes of HPO3–CH3COOH solution, B(a)(1), and H2O. If ca 2 mg ascorbic acid is contained in test solution aliquot <7 mL, add HPO3–CH3COOH solution to give 7 mL for titration. mg Ascorbic acid/g, tablet, mL, etc. = (X - B) ´ (F/E) ´ (V/Y) where X = average mL for test solution titration, B = average mL for test blank titration, F = mg ascorbic acid equivalent to 1.0 mL indophenol standard solution, E = number of g, tablets, mL, etc. assayed, V = volume initial test solution, and Y = volume test solution titrated. [Note: Products containing ferrous Fe, stannous Sn, and cuprous Cu give values in excess of their actual ascorbic acid content by this 2006 AOAC INTERNATIONAL
ã 2005 AOAC INTERNATIONAL
method. Following are simple tests to determine whether these reducing ions are present in such amounts as to invalidate test: Add 2 drops 0.05% aqueous solution of methylene blue to 10 mL freshly prepared mixture (1 + 1) of test solution and HPO3–CH3COOH reagent and mix. Disappearance of methylene blue color in 5–10 s indicates presence of interfering substances. Stannous Sn does not give this test and may be tested for as follows: To another 10 mL test solution to which 10 mL HCl (1 + 3) has been added, add 5 drops 0.05% aqueous solution of indigo carmine and mix. Disappearance of color in 5–10 s indicates presence of stannous Sn or other interfering substance.]
References: J. Biol. Chem. 103, 687(1933); 112, 625(1936); 116, 409, 563(1936); 126, 771(1938). Biochem. J. 27, 580(1933); 30, 2273(1936); 36, 115(1942). Physiol. Rev. 16, 238(1936). J. Am. Med. Assoc. 111, 1290(1938). Biochemical Z. 301, 229(1939). JAOAC 27, 537(1944); 28, 559(1945); 29, 69(1946); 30, 673(1947); 32, 479(1949); 50, 798(1967). CAS-50-81-7 (ascorbic acid)
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