V O L U M E 23, NO. 8, A U G U S T 1 9 5 1 Transfer this buffered solution to a 250-ml. separatory funnel, dilute to 50 ml., and add 75 ml. of ethyl acetate. Cool the mixture by placing in a refrigerator for 0.5 hour, or if this is impossible, in cold running water. When cool, add 1 ml. of 1 M ( 3.8Yc)hydrogen peroxide. After shaking the funnel vigorously for 0.5 minute, allow the layers to separate, and draw off the aqueous solution. Repeat the extraction of the aqueous layer a t least twice, using 15 ml. of ethyl acetate each time. Combine the ethyl acetate fractions. Add 1 ml. of lOY0 potassium hydroxide solution to the blue solution of perchromic acid, and shake until the blue color is replaced by yellow. Extract the \-ellow chromate with water, and boil the solution for 10 mink e s . Dilute to 50 ml. and determine the chromium with sdiphenylcarbazide. Analysis of Synthetic Samples. For the analysis of synthetic samples, 100 mg. of vanadic acid as ammonium metavanadate and known weights of sexivalent chromium from 0.1 to 3 nig. were used. The samples were prepared by mixing the proper amounts of standard solutions of vanadate and dichromate. Eleven such samples wercx analyzed (Table V). In the range from 0.1 to several per cent, the results are in good agreement n-ith the known values. Analysis of Standard Sample. Sational Bureau of Standards icarrovanadiuni, sample 61-1 con t,aining 0.6S70 chromiuni, was
1113 analyzed by this procedure. Parallel analyses were made using 0.100- and 0.250-gram samples and a value of 0.6.?17~chromium was obtained. LITERATURE CITED
Barreswil, L. C. A , , Ann. chini. p h y s . , (3) 20, 264 (1847). (2) Bobtelsky, RI.,Glasner, A , and Bobtelsky-Chaikin, L., J . Am. Chem. Soc., 67, 966-75 (1945). (3) Foster, 11.D., U. S. Geol. Survey, Bull. 950, 16-18 (1946). (4) JIellor, J. IT.,“Comprehensive Treatise on Inorganic and Theoretical Chemistry,” Vol. XI, pp. 353-61, London, Longmans, Green B. Co., 1931. (1)
( 5 ) Sandell, E. B., “Colorimetric Determination of Traces of Metals.” pp. 191-5, New Yo1 k, Interscience Publishers, 1936. (6) Snell, F. D., and Sriell, C. T., “Colorimetric Methods of Analysis,” DD. 671-5, New York. D. Van Nostrand Co.. 1936. (7) Spjtksky, E., 2. anorg. Ciiem., 56, 72-108 (1907). (8) Ihid., 69, 179-208 (1910). (9) JI-illaid and Diehl, “Advanced Quantitative Analysis,” p. 234, New Yolk, I).Van Nostrand Co., 1943. RECEIVEDDeoernber 20, 1950. I’iiblished by permission of the Director, Bureau of Mines, U. S. Deliartlnent of Interior.
Estimation of Ascorbic Acid in Pharmaceuticals With Particular Reference to Interfering Substances D. G. CHAPMAN, ODETTE ROCHON, AND J. A . C 4 l I P B E L L Food and Drug Divisions, Department of Sational Health and Welfare, Ottawa, Canada Several methods have been proposed for the estimation of ascorbic acid in the presence of interfering materials such as iron and copper salts, but few data exist on the comparative reliability and limitations of these procedures as applied to pharmaceuticals which may contain these and other interfering substances. Of the eight methods investigated, the procedure of Roe e t al. was found to be the most reliable under all conditions. The Brown and Adam method was the most satisfactory for routine analysis except in the presence of large amounts of copper. Iron does not interfere in the methods of Gawron and Berg and Frosst, but ascorbic acid may not be stable in the extracting agents used in these methods. The precision of all methods in the absence of interfering substances was found to be satisfactory. Great care must be exercised in the selection of a method for the estimation of ascorbic acid in certain complex pharmaceuticals containing minerals.
A‘
IIOSG
the many methods for the determination of ascorbic acid, those using metaphosphoric acid as the extracting as the indicator appear agent and ~2,6-dichlorophenolindophenol to be the most widely used. .-\scorhic acid is stable in metaphosphoric acid ( 1 3 ) and a t the same time its oxidation by copper or enzymes is reduced (8). A disadvantage of metaphosphoric acid is that ferrous salts, which are present in many pharmacseutical products, are oxidized quantitatively by indophenol dye. To overcome this objection to nietaphosphoric acid, two possibilities are open to the investigat,or. A reagent may be added to the metaphosphoric acid which will remove the interference caused by iron, or an extracting agent may be sought in which ferrous iron does not react with the dye. Most of the common acids have been studied (13) as possible extracting agents, but ascorbic acid is relatively unstable in all except metaphosphoric and oxalic acids. However, ferrous iron
interferes with the estimat.ion of ascorbic acid in both of these, and both metaphosphoric acid ( 2 ) and oxalic acid (6) are themselves relatively unstable. T o eliminate the interference caused by ferrous iron in metaphosphoric acid extracts, Lugg ( 1 2 ) has suggested the use of formaldehyde, which at a pH of 3.5 condenses with ascorbic acid but not appreciably with ferrous salts. Because of the large number of titrations v hich are required for a single determination, this method did not appear to be adaptable to routine analysis and therefore was not studied by the authors. The addition of hydrogen peroxide \vas first used by Levy (9) to eliminate interference by sulfur dioxide. Huelin and Stephens ( 7 ) , however, have reported that for extrack containing ferrous iron, the use of hydrogen peroxide converts a positive error into a negative error of less magnitude. Hydrogen peroxide also promotes a slow reoxidation of the reduced dye and the titration must be completed as soon as possible after adding this reagent. Robinson and Stotz ( 1 4 ) have proposed a spectrophotometric method in which certain reducing materials are eliminated by the use of hydrogen peroxide or formaldehyde. Gawron and Berg ( 5 ) have suggest,ed using 8% acetic acid to extract ascorbic acid when ferrous iron is present. Brown and Adam (3)have reported that, a sodium acetate-hydrochloric acid mixture buffered to a pH of 0.65 overcomes the interference due to ferrous iron. Roe e l ai.( 1 6 ) have published a method for the determination of ascorbic acid based on the reaction between dehydroascorbic acid and 2,i-dinitrophenylhydrazine. Roe and Kuether ( 1 5 )liave stated that ferrous ion8 do not interfere in this procedure. It thus becomes apparent that a variety of methods has been proposed for eliminating or reducing the ferrous iron interference in ascorbic acid assays in foods. However, there is little published inforniation on the relative merits of these methods. Hence this study way undertaken for the purpose of comparing t,he reliability of eight methods in estimating ascorliic acid, in the presence of both ferrous iron and other substances commonly found in multivitamin pharmaceutical products.
ANALYTICAL CHEMISTRY
1114
substances by each of the eight methods under investigation. Authors Extracting Agent, 70 WJV. Reagent Cod liver oil caused no appre1. Roe el al. (16) hletaphosphoric acid, 5 2.4-Dinitrophenylhydrazine gDichlorophenolindophenol ciable interference in any of the 2 . Huelin and Stephens using H20z ( 7 ) Metaphosphoric acid, 3 3. U.S. Pharmacopeia XIT' (12) Metaphosphoric, 3, plus 8% ( r / r i acetic Z:&Dichlorophenolindophenol m e t h o d s examined. Liver acid 4 . Gawron and Berg (6) Acetic acjd, S% ( v / v ) 2,6-Dichlorophenolindophenol fraction, on the other hand, 5 . Watanabe ( 1 7 ) Oxalic acid, o 2,6-Dichlorophenolindophenol gave a slightly positive error. (U.S.P. method) 6. Frosst ( 4 ) Sulfuric acid, 2.5 Iodine 0.005 A' In the titrimetric methods t.hia 7. Brown and Adam (3) Sodium acetate-hydrochloric acid buf- 2.6-Dichlorophenolindophenol v a s probably due to an obscurfered to p H 0.65 8. Robinson and Stotz using HzO? ( 2 4 ) Metaphosphoric acid, 6, buffered t o p H 2.&Dichlorophenolindophenol ing of the end point. ~ ~ ?.R I11 shows the mean and the standard deviation of five determinations of ascorbic acid EXPERIMENTAL alone and in the presence of ferrous iron by each of the eight methods. Methods Studied. Eight met.hods were chosen far study m representative of the available chemical procedures for the rouRoe et al. (16). The procedure for total ascorbic acid was tine analysis of ascorbic acid (Table I), and were follou-ed e.xactly used. This would give a measure of reduced ascorbic acid, dehydroascorbic acid, and diketo-L-guIonic acid. Blthough the as stated by the authors. In addition to the six metho& b-hich diketo-L-gulonic acid is biologically inactive, it is highly improbwere taken from the literature, two other estracting agents, 5% able that there would be any appreciable amounts of this subosalic acid and 2.5% sulfuric acid, were used. stance in pharmaceutical products. Roe and Kuether ( 1 5 )have Katanabe ( f 7 ) first recommended the use of oxalic acid as the stated that ferrous or stannous ions cause no interference. The authors' results indicate that none of the added materials cause best estractant for ascorbic acid, although he later used a mixture interference in the recovery of ascorbic acid. The precision of the of metaphosphoric and osalic acids (18). For the present method as s h o m by the small standard deviations is good. study the authors used a 5% osalic acid solution and titrated Huelin and Stephens (7). The hydrogen peroxide modificaagainst indophenol dye prepared according to the U.S. Pharmation of this procedure gave extremely low recoveries in the presence of ferrous sulfate. Unsatisfactory recoveries were also obcopeia method (12). tained when copper salts were present. Because stability of ascorbic acid in sulfuric acid is very poor United States Pharmacopeia ( 1 2 ) . This method gave an ex( 7 ) ,its use as an extractant for ascorbic acid is not recommemded. tremely high recovery of ascorbic acid when ferrous iron was Xevertheless, it has been found useful by Frosst ( 4 ) for the wtipresent. This was not unexpected, because the A.O.A.C. method ( f ) , vhich is essentially that of the U. S. Pharmacopeia, is not mation of ascorbic acid in the presence of iron. T h e titrarecommended for use in the presence of ferrous iron. Cuprous tions were carried out one minute after adding the ascorbic acid to chloride appeared to result in a low recovery, whereas cupric the sulfuric acid solution. sulfate caused no interference. As King (8)has found that for most investigations direct visual Gawron and Berg ( 5 ) . Ferrous iron caused no appreciable interference. However, the presence of cuprous chloride, cupric titrat,ion of ascorbic acid with indophenol dye gives reasonably acsulfate, and ferrous sulfate plus cupric sulfate resulted in low curate and satisfactory results, and this type,of method required a recoveries of the ascorbic acid. Gawron and Berg ( 6 ) did not minimum of equipment, sis of the methods chosen for study employed a titrimetric technique. Added Substances. The possible interference which might be caused by ferrous sulfate, cuprous chloride, cupric sulfate, ferTable 11. Effect of Certain Added Substances on Recovery of Ascorbic Acid as Determined by Eight Methods rous sulfate plus cupric sulfate, liver fraction (liver fraction L ob(Recoveries in per cent) tained from the Kutritional Biochemical Corp., Cleveland, Ohio), Substance Addeda and cod liver oil in the recovery of ascorbic acid was investigated. Ferrous To 100 mg. of ascorbic acid was added 1 gram of one of the interFerCusulfate rous prous plus Liver Cod fering materials (1 ml. in the case of cod liver oil) and both were SUIchlo- Cupric cupric frac- lirer quantitatively transferred to a 100-ml. volumetric 9ask and made Method of Analysis fate ride sulfate sulfate tion oil to volume with the respective estracting agents. This ratio of 1. Roe et al. ( 1 6 ) 1 0 0 . 3 1 0 1 . 2 1 0 1 . 2 101.2 100.0 100.0 2 . Huelin and Stephens 10 to 1 for ferrous sulfate to ascorbic acid'is similar to that which using H20s (7) 9.2 71.9 92.7 9.2 105.5 98.2 3. T.S.P. XIV (12) 378.8 89.5 9 8 . 5 248.2 106.9 99.2 is encountered in some pharmaceutical products. The same 4. G a w r o n a n d B e r g (6) 97.9 75,5 65.7 38.0 101.7 9i.R ratio was used for the copper salts, although smaller amounts are a. Watanabe (17) using 5% oxalic acid 146.4 9 7 . 1 100.6 234 7 108.2 99.4 usually present in pharmaceuticals. 6. Frosst using 2.5% HzSOI( 4 ) 9 9 . 7 9 9 . 5 100.6 8 8 . 8 112.4 98.5 The values reported for the recovery of the ascorbic acid are the 7. B r o w n a n d A d a m (3) 9 8 . 7 211.5 43.2 37.3 105.6 95.9 average of a t least two determinations when 1 nil. of the solution 8. Robinson and Stotz using H?Oz (14) 82 8 87.4 87 5 76 5 107.7 102 5 was taken for analysis. I n the case of pure ascorbic acid and asa Added a t rate of 1 gram t o 100 mg. of ascorbic acid. corbic acid plus ferrous iron, five det,erminations were made. Pharmaceuticals Examined. Ten multivitamin pharmaceuTable 111. Recoveries of Ascorbic Acid Alone and in tical products, five of which contained ferrous iron, were aaPresence of Ferrous Iron sayed for ascorbic acid by each of the eight methods. At least Recorerie-, 705 five tablets or capsules were taken for analysis and made to volMethod Ascorbic acid alone Ascorbic acid plus F e b ume with the various estracting agents, so that each millili1. Roe et 51. ( 1 6 ) 1 0 0 . 3 =t 0 5 100.3 f 0 . 9 2. Huelin and Stephens ter of final dilution contained approximately 1 mg. of ascorbic using H20s ( 7 ) 100.3 f 1 . 0 9.2 f 1.9 acid. The tablets were finely ground and quantitatively trans100.3 i 0.4 3 7 8 . 8 =t 1 3 . 2 3. E. S. P. XI\' (12) 4. Gawron and Berg ( 5 ) 100.2 i 0 . 6 97.9 f 0 . 5 ferred to a volumetric flask, using the respective estracting agents. 5. Watanabe ( 1 i )using 5T0 oxalic acid 101.0 f 0 . 3 146 4 f 24 3 The determinations were carried out in duplicate and the average 6. Frosst using 2.5% 9 9 7 f 1 3 99.9 i0 . 8 value was reported. HzSOa (4) Table I.
-
Methods Examined for Determination of Ascorbic Acid
,
RESULTS A N D DISCUSSION
Effect of Added Substances. Table I1 shows the per cent recoveries of ascorbic acid, in the presence of the various added
7. 8.
99.8 f 0 . 7 9 8 7 i 0 5 Brown and Adam ( 3 ) Robinson and Stotr 8 2 8 5 5 1 100.9 f 1 . 2 using Hz02 ( 1 4 ) a With standard deviations of fire determinations. b Ferrous iron added a t rate of 1 gram t o 100 mg. of ascorbic acid.
b
*
]
V O L U M E 2 3 , NO. 8, A U G U S T 1 9 5 1
1115
of the method of Huelin and Stephens ( 7 ) gave consistently Product A B C D E F G H I J low recoveries. G e n e r a l l y Ferrous iron label claim, mg. S o n e Pione S o n e S o n e None 130 162 110 65 300 speaking, the more iron that Ascorbic acid, mg. was present, the lower was the Label claim 17.5 50 30 50 10 50 15 25 40 25 Found by method of Roe et a l . recovery of the ascorbic acid. (16) 17.5 51.0 29.4 50.0 7.0 59.0 22.0 28.0 55.0 23.3 A s was to be expected, the Ascorbic acid, as 70of Roe's method, found by methods of U.S.P. method ( 1 2 )gave values Huelin and Stephens using HzOz ( 7 ) 98.3 101.2 99.3 1 0 2 . 0 98.6 61.0 27.8 47.5 65.5 22.7 far in excess of that found by T.S.P. XIV ( 1 2 ) 100.6 101.6 100.7 9 9 . 8 102.9 190.2 440.5 211.1 298.2 300.4 Roe's method. The procedure Gawron and Berg (6) 97.7 1 0 1 . 8 99.3 1 0 2 . 0 100.0 103.7 97.3 93.6 98.2 97.9 Watanabe ( 1 7 ) using 5 % oxalic of Gawron and Berg (5)gave acid 100.0 99 4 100.3 100.0 100.0 118.5 124.5 103.6 104.7 135.2 r r o s s t using 2.5% IIzSOa (4) 97.7 103.3 101.4 102.8 101.4 1 0 4 . 1 104.1 110.0 98.5 106.4 results which with but one e\Brown and Adam (3) 98.3 98.2 100.7 98.0 9 7 . 1 101.2 100.9 100.4 99.6 100.9 ception were in good agreeRobinson and Stotz using HzOz (24) 98.9 100.0 9 8 . 0 102.0 94.3 102.5 88.6 96.4 99.1 93.1 ment with those given by a Results exuressed in mg. for Roe's method and as percentage of t h a t found by Roe's method for each of other Roe's method. When usingpropedures. 5% oxalic acid the recoveries of ascorbic acid were high. This is consistent with the study the possible interference due to copper salts, nor did they values reported in Table 11, which show that iron introduces a report the stability of ascorbic acid in acetic acid. However, positive error in the presence of osalic acid. Ponting ( I S ) has shown that ascorbic acid is not stable in the The 2.5% sulfuric acid solution gave slightly high recoveries in presence of acetic acid. the presence of ferrous iron. This may be explained by the fact Watanabe ( 1 7 ) . Ferrous sulfate and ferrous sulfate plus cupric sulfate resulted in high and variable recoveries of ascorbic that many pharmaceuticals impart considerable color to the exxcid in oxalic acid. This is consistent with the findings of Loren2 tracting solutions, making the detection of the iodine-starch end and Arnold ( I O ) , who reported that ferrous salts titrate with indopoint very difficult to observe. This difficulty had the effect phenol dye in solutions of oxalic acid. The chief advantage of of lowering the precision of the method. Furthermore, ascorbic osalic acid is its stabilizing action on ascorbic acid even in the presence of copper (13). Under the conditions of the authors' acid is unstable in sulfuric acid (7'). The method of Brown and esperimept the copper salts caused no interference in the recovery hdam ( 3 )gave values that were in excellent agreement with thoie of ascorbic acid. found by Roe's method. Khile copper may interfere in the Frosst ( 4 ) . When this method was used, ferrous sulfate, cuBrown and Adam method (Table 11),product J was the only one prous chloride, and cupric sulfate caused no interference. Ferrous sulfate plus cupric sulfate, however, resulted in a low recovery containing copper (1.6 mg. per tablet), and this amount caused no of ascorbic acid. It is evident from Table I11 that this procedure discrepancy. The hydrogen peroxide modification of the method can give reproducible results if sufficient care is taken to allow of Robinson and Stotz ( 1 4 ) gave slightly low recoveries for three for the instability of ascorbic acid in sulfuric acid. of the five products that contained iron. Brown and Adam ( 3 ) . Unfortunately, these authors do not indicate how the sodium acet,ate-hydrochloric acid solution buffered to pH 0.65 was prepared. The present authors prepared CONCLUSIOY a 1 S hydrochloric acid solution and added 1 sodium acetate to this until a pH of 0.65, as indicated on the Beckman pH meter, I n the absence of ferrous iron, all methods investigated gavL' was obtained. Brown and .Idam have shown that ferrous iron satisfactory results for the determination of ascorbic acid in does not cause interference with the titration of ascorbic acid when using their extracting medium. The data reported here pharmaceuticals. I n the presence of ferrous iron the, method of would confirm this. When cuprous chloride was added to asBrown and Adam proved to be most suitable for routine analycorbic acid, it int,roduced a large positive error. Cupric sulfate, ses. However, if both ferrous iron and copper were present, the on the other hand, resulted in a very low recovery of the ascorbic method of Roe et a2. was most reliable. arid. although this is contrary to the findings of Brown and hdam, who report that copper sulfate does not interfere, those authors were using a solution containing 2 mg. of ascorbic acid LITERATURE CITED and 5 mg. of copper sulfate in 100 ml., whereas the solution used by the present authors contained 100 mg. of ascorbic acid and (1) hssoc. Offic. Agr. Chemists, "Official and Tentative Method3 1000 mg. of copper sulfate in 100 ml. Using the amounts sugof Analysis," 1945. gested by Brown and Adam, no interference in the recovery of ( 2 ) Bessey, 0.A., J . Bid. Chem., 126, 771 (1938). ascorbic acid was encountered. Both ferrous sulfate and cupric (3) Brown, F., and hdam, W.B., J . Sci. Food Agr., 1, 5 1 (1950). sulfate together gave a low recovery. The precision of this (4) Frosst, Charles E., Br Co., Montreal, personal communication. method 1%-asgood, as can be seen in Table 111. ( 5 ) Gawron, O., and Berg, R., IND.ESG. CHEM.,ANAL.ED.,16, 757 Robinson and Stotz using hydrogen peroxide ( 1 4 ) . Under the (1944). conditions of this experiment, ferrous sulfate, cuprous chloride, (6) Huelin, F. E., Analyst, 75, 391 (1950). cupric sulfate, and cupric sulfate plus ferrous sulfate all interfered (7) Huelin, F. E., and Stephens, I. AI., Australian J . Exptl. Bid. with the recovery of ascorbic acid. If, however, an amount of M e d . Sci., 25, 17 (1947). ferrous iron similar to that suggested by Robinson and Stotz (8) King, C. G., IXD.ESG. CHEY.,-1x.iL. ED..13, 225 (1941). (molar ratio of ferrous ion to ascorbic acid of 1 to 1) was used, (9) Levy, L. F., Biochem. J . , 37, 713 (1943). 100% recovery of ascorbic acid was obtained. The standard devi(10) Lorens, A. J., and Arnold, L. J., ISD. E x ; . CHEM.,ANAL.ED.. ation for pure ascorbic acid, when using this method, was 1.2. 10,687 (1938). (11) Lugg. J. Vi. H., Australiax J . Erpt. Biol. M e d . Sci., 20, 273 Assay of Ascorbic Acid in Pharmaceuticals. The results of Table IV.
Determination of Ascorbic Acid in Pharmaceutical Products"
tlie ascorbic acid determinations on the pharmaceutical products :ire given in Table IT'. As the procedure of Roe et al. ( 1 6 ) was found to be the most reliable in the presence of possible interfering materials, i t was used as a standard and the values obtained by the ot'her methods are expressed as a percentage of it. -411 the methods gave very similar and satisfactory results for products that cont,ained no ferrous sulfate. Product E appeared to be below the labeled potency of 10 my. per tablet, as no method gave a value higher than 7 . 2 mg. If ferrous iron was present, the niethoda gave varying values for tlie ascorbic acid content. The hyd:ogen peroside modification
(1942). (12) Pharmacopeia of the United States of America, 14th revision. Easton, Pa., Mack Printing Co., 1950. (13) Ponting, J. D., IND.EX. CHEM.,.ISIL. ED.,15, 389 (1943). (14) Robinson, IT.B., and Stotz, E., J . Biol. Chem., 160, 217 (1945). , I h i d . , 147, 399 (1943). (15) Roe, J. H., and Kuether, C, (16) Roe, J. H., Mills, 51.B.. Oesterling, 52.J., and Damron, C. >I., Ihid., 174, 201 (1943). (17) Watanahe, K.. J . SOC.T i o p . A g r , T n i h o k u I m p . L - ~ L ~ E8,. , 381 (1937). (18) I h i d . , 9, 162 (1937).
RECEIVED February 12. 1931. Presented t o the Analytical Division of the Chemical Institute of Canada. Montreal. February 16, 1931.
