ANALYTICAL CHEMISTRY
536 treated in a manner identical with that used for those containing only palladium. The results are shown in Table 11. The chloride complexes of bivalent. and quadrivalent platinum are sufficiently stable to prevent the reaction of these ions with ethylenediaminet,etraacetic acid under these conditions, hence, platinum does not interfere with the complesometric determination of palladium. However, if the titration is made at room temperature, and as rapidly as possible after the ethylenediaminetetraacetic acid is added, ruthenium or iridium does not interfere seriously. The rhodium chloride complex is sufficiently strong to prevent the formation of a rhodium-ethylenediaminetetraacetic acid complex in chloride solution. However, the presence of rhodium seriously interferes in another Ray Kith the complexometric titration of palladium. The effect of the rhodium is to increase the amount of zinc solution required for a back-titration, thereby decreasing the apparent concentration of palladium by as much as 50%. The nature of the reaction is not known. Chloride solutions of palladium containing even small amounts of osmium inactivate and decolorize several milliliters of Eriochrome Black T, making the end point almost impossible to detect. It was found that palladium solutions containing more t,han a trace of osmium could not be satisfactorily titrated complexometrically using Eriochrome Black T as an indicator. RECOMMENDED PROCEDURE
Add a slight excess of a standard solution of the disodium salt of ethylenediaminetetraacetic acid to a chloride solution of bivalent palladium. Regulate the p H to 10 =k 1 with 0.1N potassium hydroxide. Add 5 drops of the Eriochrome Black T indicator solution and titrate with a standard zinc solution until the equivalence point is reached, as shown by the color change from blue or green t o pink. REAGENTS
Standard Palladium Solution. A 2.5-gram sample of palladous chloride, from Coleman and Bell Co., was dissolved in 500 ml. of 0.2M hydrochloric acid. Spectrographic investigation showed only traces of platinum present. This solution was standardized by precipitation of the palladium with dimethylglyoxime followed by drying a t 110” C. ( 6 ) .
Standard Zinc Solution. A 1.8-gram sample of Mallinckrodt analytical reagent grade zinc oxide was dried for 2 hours a t 100,’ C., dissolved in a minimum amount of 1 to 1 nitric acid, and dlluted to 1 liter Lvith distilled water. This solution was standardized by precipitating with ammonium phosphate and weighing the zinc as the pyrophosphate according t o the procedure of Vance and Borup (14). Standard Ethylenediaminetetraacetic Acid Solution. A 5.5gram sample of the disodium salt of ethylenediaminetetraacetic acid, analytical reagent grade from Versenes, Inc., was dissolved in 1 liter of distilled water. The molarity of this solution was obtained by a complexometric titration using Eriochrome Black T and the standard zinc solution. Indicator Solution. A 0.1-gram sample of Eriochrome Black T from K , H. and L. D. Betz Co. was dissolved in 50 ml. of distilled water which had been made slightly basic with 3 drops of I N potassium hydroxide. Other Platinum Metals. Reagent grade perosmic acid, ruthenium chloride. olatinous chloride, olatinic chloride. rhodium chloride, and iridium chloride were used for the study of interferences. Spect,rographic analysis showed less than significant amounts of impurities. All solutions were standardized, using modifiratione of the Gilchrist-Wichers scheme (6). LITER4TURE CITED
(1) ,Itkinson, R. H., Analyst, 79, 368 (1954). (2) Burriel, F., and Pino PBrea, F., A n d e s real SOC. espaii. fis. 21 quim. ( M a d r i d ) , 47B, 261 (1951). (3) Flaschka, H., Mikrochim. A c t a , 1953, 226. (4) Gahide, Af., Bull. SOC. chim.Belg., 45, 9 (1936). (5) Harris, W. F.. and Sweet, T. R., iis.4~.CHEM.,26, 1649 (1954). (6) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A,, and Hoffman, J. I., “Applied Inorganic Analysis,” 2nd ed.. pp. 33883, Wiley, New York. 1953. (7) Kersting, R., Ann. Chem. Justus Liebigs, 87, 25 (1853). (8) RIacXevin, W. AI., and Kriege, 0. H., -bar.. CHEM.,26, 1768 (1954). (9) Schwaraenbach, G., and Ackermann, H., Helv. C h i m . Acta, 31, 1029 (1948). (10) Schwaraenbach, G., and Biedermann, W., Ibid., 31, 459 (1948). (11) Schwaraenbach, G., Biedermann, W., and Bangerter, F., Ibid., 29, 811 (1946). (12) Svrokomskii. V. S..and Gubel’bank. S. AI.. Zhur. A n a l . Khim.. 4, 146 (1949). (13) Ibzd., p. 203. (14) Vance, J. E., and Borup, R. E., XSAL.CHEM.,25, 610 (1953). RECEIVED for review August 9, 1954. Accepted December 7,
1954.
Action of NBromosuccinimide on Ascorbic Acid New Titrimetric Method for Estimation of Vitamin C MOHAMED ZAKl BARAKAT, MOHAMED FATHY ABD EL-WAHAB, and MOHAMED MAHMOUD EL-SADR Biochemistry Department, Faculty o f M e d i c i n e , Abbarria, Cairo, Egypt
Estimation of ascorbic acid with 2,6-dichlorophenolindophenol is limited by the presence of interfering substances such as reductones and reductic acid. Slow reducing substances that may be present in biological fluids render the end point less distinct. This defect induced the authors to investigate the estimation of ascorbic acid by N-bromosuccinimide, the oxidizing action of which might be selective. The fact that ascorbic acid is selectively oxidized by iVbromosuccinimide before other reducing substances that may be present provides a reliable titrimetric method of extensive applicability. The proposed method is simple, rapid, and sufficiently sensitive to determine a concentration as low as 7.04 y of ascorbic acid per milliliter. The experimental error does not exceed j=Z’%,
A
VARIETY of bioassay and chemical methods for the determination of vitamin C activity or ascorbic acid content has been developed ( 4 , 11, 12, 17). Although bioassays have the advantage of measuring the summation of chemical entities that possess vitamin C activity, they are time-consuming and expensive, and leave much to be desired in precision. Consequently, bioassays are now confined to use in comparative studies to establish the specificity of chemical methods for determining ascorbic acid in individual products. Chemical methods for the determination of vitamin C are based mainly upon the reducing properties of the vitamin. These methods include titration of an acid extract with iodine, methylene blue, ferricyanide, and 2,6-dichlorophenolindophenol (9, 19, 20). Oxidation of ascorbic acid with the dye 2,6-dichlorophenolindophenol is the most satisfactory and exteneively used method (5, 6 ) . However, the value of the 2,8dichlorophenol-
V O L U M E 2 7 , N O . 4, A P R I L 1 9 5 5 indophenol reagent for measuring ascorbic acid is limited by the presence of other reducing substances such as reductones, reductic acid, and dihydroxymaleic acid, which give rise to errors, particularly in food product,s. Inorganic and organic ferrous and ferric compounds also interfere n-ith the determination by 2,6dichlorophenolindophenol (3,13, 18). Ferrous ion reduces the dye in the presence of metaphosphoric acid, so that pharmaceutical preparations containing reduced iron should be titrated in 8% acetic acid solution free from metaphosphoric acid. On the other hand, ferric ion interferes with the end point in the absence of metaphosphoric acid, so that metaphosphoric-acetic acid mixture should be employed as the titration medium in testing pharmaceutical preparations containing oxidized iron (10). The end point with plant tissues is stable for some time, but many animal tissue extracts arid urines contain sufficient amounts of slow-reducing substances to make the end point less distinct, especially when small quantities of ascorbic acid are being titrated. I n such cases the accuracy of the titration depends on the skill of the observer in detecting the point a t which rapid reduction is replaced by a s l o fading ~ of the indicator. Colorimetric methods for estimating ascorbic acid, which are not based upon oxidation-reduction properties, include the 2,1dinitrophenylhydrazine met,hod ( 14-16), Tvhich is too tedious and time-consuming for routine analyses. The photoelectric colorimetric method for the determination of ascorbic acid, by means of uranium nitrate ( 1 j , has been applied only t o test solutions and pharmaceutical p1,oducts. X new easy, rapid, and accurate t,itrimetric method for the estimation of ascorbic acid in pure solutions, pharmaceutical preparations, biological fluids-e.g., blood and urine-and edible fruits, which overcomes the disadvantages previously mentioned, is based on the selective oxidation of ascorbic acid by S-bromosucciuimide. EXPERIMENTAL
Equipment and Reagents. A microburet of 5-ml. capacity, graduated in hundredths of a milliliter. Graduated pipets of I-. 2-, and 5-ml. capacity. Erlenmeyer flasks of 25-, 50-, and 100-ml. capacity. Four per cent xeight per volume of potassium iodide in distilled water. Three per cent, volume per volume of acetic acid. One per cent starch solution prepared by dissolving 1 gram of soluble starch in 10 ml. of boiling water and adding to 90 ml. of saturated sodium chloride solution. A 0.1% ' weight. per volume S-bromosuccinimide aqueous solution which is freshly prepared and may be serially diluted 10 or 100 times as required. Concentration of S-bromosuccinimide solution used in titration. 0.1% weight per volume aqueous solution for pure ascorhic acid solutions and pharmaceuticals; 0.01 % 1Teight per volume aqueous solut,ion for urine and fruits; 0.001% weight per volume aqueous solution for blood. Action of AV-Bromosuccinimide on Ascorbic Acid. Ascorbic acid (1.76 grams, 0.01 mole) was dissolved in distilled water (30 ml.) and LV-bromosuccinimide (1.78 grams, 0.01 mole) was added t o the ascorbic acid aqueous solution. The mixt'ure was refluxed for 15 minutes. m-hen all S-bromosuccinimide went into solution. The colorless solution was distilled off in vacuo, and the solid residue n-as crystallized from benzene giving colorless crystals, with a melting point of 125-6' C., which were proved to he succinimide by comparison of the melting point and mixed melting point with an authentic sample. Yield was 0.7 grami.e., 71.42% of the theoretical yield. In another similar experiment, after the reaction v-as complete, a small portion of the colorless solution (5 ml.) was treated with dilute nitric acid and 10% silver nitrate solution, when a ve1lowi;zh white precipitate of silver bromide was deposited, indicating the presence of hydrobromic acid. To the rest of the colorless reaction mixture was added a mixture of phenylhydrazine hydrochloride (5.78 grams, 0.04 mole) and crystalline sodium acetate (5.44 grams, 0.04 mole); t h e mixture was heated in a boiling water bath for 30 minutes. Deep orange crystals were deposited from the solution while hot. The crystals were filtered off, dried, and recrystallized txvice from ethyl acetate, giving uniform orange crystals with a melting point of 218" C. which were proved to be dehydroascorbic acid osazone by
5 37 comparison with the melting point and mixed melting point of an authentic sample. Yield was 2.42 grams-Le., 81.92% of the theoretical yield. VALIDITY OF RE4CTIOY FOR QUANTITATIVE ESTIMATION
Before applying the reaction to the estimation of ascorbic acid in pure solutions, pharmaceuticals, biologicals, and fruits, the authors decided to verify the action of .V-bromosuccinimide on ascorbic acid from a quantitative point of view. It was assumed that 1 molecule of ascorbic acid was oyidized by 1 molecule of AV-bromosuccinimide and accordingly equimolecular and bimolecular solutions of ascorbic acid and S-hromosuccinimide were prepared. -
Table I.
Titration of Equimolecular Solutions of Ascorbic Acid and N-Broniosuccinimide .V-Bromosuccinimide S o h , hll. (1 hI1. = 1.78 M g . )
Ascorbic Acid S o h , 1\11, = 1 76 11g.)
(1 1\11.
1.00 1.99 3 01 4 00 5.00 10.00
1 2 3
4 10
Table 11. Comparative Analysis by Proposed AIethod and by 2,6-Dichlorophenolindophenol
Dilution 1OX. 176 y ascorbic acid per nil.
1OOX. 17.6 y as-
corbic acid per ml
.
Bscorbic Acid Soln., hI1. 5 4 3 2 1
2 1
Found Content,
NBSa,
y
1
880 704 528 352 170
882 704 529 352 176
33.2
17.6
5 8.80 corbic acid per nil. a S-bromosuccinimide solution.
lOOOX, 1 76 y as-
by
3.5.1
17.1; 8.80
Found Error,
%
0.24
0.'19
... ...
0.28
... ...
by
Dye, Y
877 704 530 352 176
33.1 17.5 8.80
Error,
%
0.31
...
0.38
... ...
0.28 0.X
...
Ascorbic Acid Solutions. Ascorbic acid (0.176 gram, 1 millimole), A, and ascorbic acid (0.352 gram, 2 millimole), B, were separately dissolved in distilled water and made up to 100 ml. with distilled mater in a standard flask. Solution A contained half the number of molecules of ascorbic acid present in solution €3.
IV-Bromosuccinimide Solution. S-Bromosuccinimide (0.178 gram, 1 millimole) x a s dissolved in hot distilled n-ater, and the aqueous solution was allowed to cool, and made up to 100 ml. Iyith distilled water in a standard flask. As the molecular weight of ascorbic acid is 176 and that of S-bromosuccinimide is 178, i t is clear that ascorbic acid solution A contains the same number of molecules as that present in the A'-bromosuccinimide solution; ascorbic acid solution B contains double that number of molecules. Each of the two above-mentioned equimolecular solutions of ascorbic acid and AT-bromosuccinimide (freshly prepared) was serially diluted 10, 100, and 1000 times n-ith distilled water in 100-ml. standard flasks; various concentrations such as 1 ml. of ascorbic acid solution containing 176, 17.6, and 1.76 y of ascorbic acid were obtained (Table 11). The above dilutions of ascorbic acid solution were estimated simultaneously, with 2,6-dichlorophenolindophenol. The dye was standardized so that 1 ml. was equivalent to 0.117 mg. of ascorbic acid, but in the case of urine 1 ml. of the dye was equivalent to 0.125 mg. of ascorbic acid (Tables I1 and IX). When 1 ml. of the ascorbic acid solution diluted 1000 timesi.e., containing 3.52 y per m1.-was estimated by N-bromosuccinimide oxidation, the end point was not clear. Practically it has been found that the reaction between ascorbic acid and L\r-bromosuccinimide proceeds quantitatively in equimolecular concentrations, and that the validity of the values reported (Tables I, 11, and 111)is also dependent upon that assumption.
ANALYTICAL CHEMISTRY
538 Table 111. Titration of Bimolecular Solutions of Ascorbic Acid and Molecular N-Bromosuccinimide Solution Ascorbic Acid NBS Soln. B, Used, MI. Mi. (1 M1. = Content, (1 M1. = 3 . 5 2 Mg.) Mg. 1 78 Mg.) 1 3.52 2.00 2 7.04 4.00 3 10.56 6.00 4 14 08 8 00 5 17 60 10 01 10 35 20 20 01
Dilution
l o x , 352 y per ml.
lOOX, 3 5 . 2
y
per ml.
IOOOX, 3 . 5 2 y perml.
Found, Mg.
3.52 7.04 10..56 14 08 17 62 35 22
Error,
70
0 05 0 06
5 4 3 2 1
1,760 1.408 1.056 0.704 0.352
9.99 8.00 6.00 4.00 2.00
1.758 1.408 1.056 0.704 0.352
0.11
2 1
0.0704 0.0352
4.00 2.00
0,0704 0.0352
... . ..
5 2
0.0176 0.00704
10.00 4.00
0.0176 0.00704
... . ..
... ...
...
._.
Table IV. Estimation of Pure Ascorbic Acid Solutions by 0.1% N-Bromosuccinimide Solution Ascorbic Acid Soln.,
%
0.5
Volume, hI1. 1
%8 M1.
Found,
hlg.
Mg.
%
5.05 10.10
4.99 9.98
0 2 0.2
Content,
2
5 10
0.4
1 2
4 8
4.06 8.10
4.00 8.00
0.3
1 2 3
3
3.02 6.05 9.10
2.98 5.98 8.99
0.1
6
9
9 8 7
10 9 8 7
6
6
5
5
10
10.10 9.06 8.05 7.02 6.02 5.05
9.98 8.96 7.96 6.94 5.95 4.99
Error,
0.66 0.33 0.11
0.2 0.4 0.5 0.85 0.83 0.2
method is recommended. A known volume of the solution is diluted with distilled water in a standard flask so that 5 ml. contain 0.5 to 10 mg. of ascorbic acid. Five milliliters of this dilution are accurately measured and introduced into an Erlenmeyer flask of 25- or 50-ml capacity. Then 5 ml. of 4% potassium iodide solution, 2 ml. of 3% acetic acid, and 2 drops of starch solution as an indicator are added. The 0.1% N-bromosuccinimide solution-i.e., 1 ml. contains 1 mg. of N-bromosuccinimide-is run into the ascorbic acid solution drop by drop with continuous shaking after each addition until the end point is reached. From the number of milliliters of N-bromosuccinimide solution used in the titration process, the amount of ascorbic acid present can be readily calculated. This method has been successfully applied to several kinds of injections obtainable in Egypt. One example is sufficient to illustrate the validity of the assay method. The injection consisted of 0.5 gram of ascorbic acid and 1 gram of calcium gluconate made up to 10 ml. with distilled water. One milliliter of this injection contained 50 mg. of ascorbic acid. One milliliter of this preparation was diluted with distilled water up t o 50 ml. in a standard flask. Therefore, 5 ml. of this diluted solution should contain 5 mg. of ascorbic acid (see Tables VI and VII). TABLETS. Ten tablets are weighed and powdered and an accurately weighed quantity of the powder, equivalent to 50 mg. of ascorbic acid, is introduced into a 50-ml. standard flask. Successive small quantities of distilled water (10 ml.) and glacial acetic acid (5 ml.) are added with continuous and vigorous shaking and the volume is made up to the mark with distilled water. The mixture is shaken well for 15 minutes and then filtered. The assay is carried out using 5 ml. of the filtrate, as described for injections. The result obtained, multiplied by 10, gives the amount of ascorbic acid in the original weight taken (see Tables VI and VII). The assay can be carried out even on one tablet with reproducible results (see Table VIII).
Biological Fluids. DETERMISATION OF ASCORBIC ACID IN WHOLEBLOODAND PLASJIA.Five milliliters of oxalated whole blood or plasma are added, dropwise with shaking, to 5 ml. of 20YG trichloroacetic acid in a 25-ml. centrifuge tube, and stirred to obtain a fine suspension. The suspension is allowed to stand 5 minutes, then centrifuged. The clear supernatant solution is filtered and 5 ml. of the filtrate are titrated immediately against 0.001 7,solution of N-bromosucrinimide; 1 ml. contain8 0.01 mg. of N-hromosuccinimide (Tablt. IX).
Table V.
Comparison of Results by N-Bromosuccinimide and 2,6-Dichlorophenolindophenol
ESTIMATION OF PURE ASCORBIC ACID SOLUTIONS
Procedure. Into a 50-ml. conical Erlenmeyer flask, a known volume of the unknown ascorbic acid solution is introducedLe., 1 ml. contains 1 mg. of ascorbic acid. Then 5 ml. of 4% potassium iodide solution, 2 ml. of 3% acetic acid, and 2 drops of starch solution as an indicator are added. The aqueous A'bromosuccinimide solution (0.1% ' weight per volume) is introduced into the microburet and is allowed to run drop by drop, into the ascorbic acid solution, with continuous shaking. The end point is reached when the last drop of the A'-broniosuccinimide solution added produces a permanent blue color in the ascorbic acid solution (Table IF'). Each of the above two solutions of 0.1% ascorbic acid and 0.1% N-bromosuccinimide v a s diluted 10 and 100 times, 80 that 1 ml. contained 100 and 10 y of ascorbic acid, respectively. The estimation was done a t the same time by means of 2,6dichlorophenolindophenol (Table ir).
Dilution l o x , 100 y per mi. 100 X , 10 y per ml.
Asrorbic .4cid ConS o h , tent, MI. y 5 500 2.5 250 1 100 5 50 2.5 25 1 10
Found by XBS, Error, r % 497 0.6 248 0.8 100 ... . 60 . 25 ... 10 ...
..
Found by Dye,
Error,
Y
%
496 248 99 50 24.6 10.5
0.8 0.8 1.0
...
1.6 5.0
Table VI. Estimation of Ascorbic Acid in Injections and Tablets by 0.1% AV-Bromosuccinimide Solution
Sample Injection Tablet
Diluted Ascorbic Acid Solution, bll. 5 5
Ascorbic Acid Content,
0 . 1%
JIg.
MI.
5 5
5.05 5.1
NBS
Used,
Ascorbic Acid Found, Mg. 4.99 5.04
Error,
% 0.2 0.8
CALCULATION
176 Ascorbic acid content = V X C X - (mg. or y) 178 where V = volume of N-bromosuccinimide solution C = concentration of N-bromosuccinimide solution either in milligrams or micrograms METHOD OF ASSAY
Pharmaceutical Products. INJECTIONS. For the estimation of ascorbic acid in solutions prepared for injection the following
Table VII. Comparison of Results by N-Bromosuccinimide and 2,6-Dichlorophenolindophenol
Sample Injection, 10 ml. Tablet
Asrorbic Acid per Ampoule or Tablet, hlg. 500
Found by NBS. M g. 494
Error.
50 500
50.9 499.9
Error,
1.2
Found by Dye, Mg. 495
1.8 0.02
51 503
2.0 0.6
%
% 1.0
V O L U M E 27, NO. 4, A P R I L 1 9 5 5 Table VIII.
Assay on Ten Tablets and One'Tablet
Original Weight, Grams 2.0 0.198 5.00 0.803
No. of
Tablets 10
1 10 1
539
Weight of Powder Assayed, Gram 0.20 0.198 0.50 0.503
Ascorbic Acid Content, >Ig.
Ascorbic Acid Found, Mg.
%
50 50 100 100
50.03 49.95 100.1 100.2
0.06 0.1 0.1
Error,
0.2
Table IX. Estimation of Ascorbic Acid in Blood and Urine hy N-Bromosuccinimide and 2,6-Dichlorophenolindophenol 0 001%
Blood Filtrate, 1\11.
NBS Used, hI1. 2.25 2.50 5.00 3.00
5
Acidulated Urine, 1\11, 2 2 2 2
2 5 5
0 01% NBS Lsed, 1\11. 3.80 3.70 1.80 2.60 2.28 1.35 2.80
round hlg. % 0 89 n- qq _. 1 98 1 18 Found Xlg. %, 23.48 22 86 1 1 12
16.0li 14.08 3.33 6.92
Dye Used, MI. 0 117 Mg.)
( 1 111. =
Found. h1g. %
0 45 0 25
2.11 1.17
Dye Used, ZII.
...
Found,
( 1 Ml. = 0 125 Mg.)
Rig. %
3.00 2.90 1.40 2.10 1.80 1.08 2.20
23,43 22.65 10.94 16.40 14.06 3.37 6.88
DETER~IINATION OF ASCORBIC ACIDI N URINE. T o 40 ml. of the urine sample (better freshly passed) 10 ml. of 20% metaphosphoric acid are added to retard the oxidation of ascorbic acid, and mixed well. Two or 5 ml. of the acidulated sample are measured accurately according to the ascorbic acid content; then 5 ml. of 4% potassium iodide solution and 2 drops of starch solution are added and titrated against 0.01% ' N-bromosuccinimide solution; 1 ml. contain- 0.1 nig. of S-bromosuccinimide (Table I S ) . Table X.
Recovery of Added Ascorbic Acid to Whole Blood and Urine
1 .O6
1.00
0.99
1.04
Blood 1.06 0.93
2.00 10.00
2.00 9.98
2.11
10.00
9.98
I O . 89
20.00 35 00 2.00 14.00
19.97 35.04 1.96 14.04
20.74 35.06 2.02 14 05
14.06 Urine 14.06 22.75 3.35 14.07
~~~~~~
XII).
RESULTS
Interfering Substances. The following substances which might interfere have no influence on the titration process: carbohydrates such as glucose, lactose, sucrose, and starch; diketogulonic acid; reductones and reductic acid; urea; uric acid and creatinine; alcohols; formaldehyde; acetone; ethyl acetate and acetoacetic ester; thiamine hydrochloride and riboflavine; oxalates, tartrates, and citrates; amino acids such as glycine, alanine, valine, and isoleucine; and ferrous and ferric salts. The only interfering substances that are also oxidized by N-bromosuccinimide before iodine is liberated from potassium iodide in acetic acid medium include sodium sulfite, sulfide, thiosulfate, and thiourea. Experimental Error. From the results given in Tables IV to VII, it has been deduced that the error of the proposed method does not exceed &2%. Every result is the average of a t least duplicate or triplicate titration processes. DISCUS SIOY
.Y-Bromosuccinimide acts as an ovidizing agent-e.g., it converts primary and secondary alcohols into the corresponding aldehydes and ketones, respectively (a). I t s oxidizing action is peculiar and highly selective in many cases, as is shown in the oxidation of the ia-hydroxyl group of cholic acid ( 7 )and the 6phydroxyl group of cholestane-3@,5a,6p-triolby use of N-bromosuccinimide. Selective oxidation of a 3-acyl derivative of methyl cholate (8) to the i-ketone can be accomplished in high yield iTith S-bromosuccinimide. There seem to be no references in the literature to the reaction between S-bromosuccinimide and ascorbic acid. N-Bromosuccinimide in aqueous medium readily oxidizes an aqueous solution of ascorbic acid to dehydroascorbic acid, while N-bromosuccinimide is irreversibly reduced to succinimide with the formation of hydrogen bromide. Dehydroascorbic acid has been isolated in the form of its osazone. Succinimide has been separated from the reaction mixture and the formation of hydrogen bromide has
...
RECOVERY OF ASCORBIC ACIDADDEDTO WHOLE BLOODA K D URINE. Some recovery experiments were carried out in which the ascorbic acid added t o whole blood and urine was in the same range, less than the amount originally present and also several times as great in amount as the amount originally present. T h e original sample content was estimated by means of N-bromosuccinimide; and the recovered ascorbic acid determined by N-bromosnccinimide . ...-... . and dve methods was exoressed in terms of the added ascorbic acid (Table X). Edible Fruits. The new method has been applied to the estimation of ascorbic acid in certain edible Egyptian fruits known to contain ascorbic acid-e.g., oranges, lemons, tomatoes, grapes, watermelons, and water cress. The fruits used in the estimation of ascorbic acid were almost ripe. The sampling and extraction of the material under examination must be carried out with the minimum delay, so t h a t no significant change in ascorbic arid content takes place prior t o analysis. Each sample is squeezed or bruised and the juice is directly received in a known volume of 20% trichloroacetic acid, so that the juice is diluted with the stabilizing acid a t least twice, t o avoid the oxidation of ascorbic acid and also to precipitate inter~~
fering substances such as protein and thereby facilitate subsequent clarification of the extract. The acidulated juice is filtered and the determination is carried out on a known volume of the clear filtrate as described previously. The N-bromosuccinimide solution used in t h e titration is 0.01%-Le., 1 ml. contains 0.1 mg. of N-bromosuccinimide (see Tables XI and
Table XI.
Estimation of Ascorbic Acid in Fruits by N-Bromosuccinimide Ascorbic Acid Determined by NBS, M g . ?& 72.78 56.15 52.57 35.59 37.57 32.00 24.80 29,84 27.90 13.02 33.59
Fruit Sample
Tomato Grapefruit Watermelon Water cress Pomegranate
__
~
Table XII.
Comparative Determination of Ascorbic Acid in Fruits
Fruit Sample Orange (Seifi) Orange (Seifi) Water cress Watermelon Lemon Banaaheir Tomato Guava ._ _ .
Grapefruit Mango
Found by NBS, .%i % 39.55 35.63 17.79 27.68 45.44 27.66 43.96 24.90 55.72
Found by Dye, Mg. 9i 39.60 36.00 18.40 28.23 46.56 29.10 44.62 25.29 57,04
540
ANALYTICAL CHEMISTRY
been confirmed. The reaction proceeds quantitatively in equimolecular concentrations according to the equation:
o=c--, I 1 HO-6
1
CHzCO
A ?I
+
HOH-A-J
'
i
I
\
S.Br
/
CH&O
H0-L-H
i
the titration process. The end point is thus easily observed by the appearance of a blue color in the ascorbic acid solution or extract. The presence of other reducing substances does not interfere with the titration with ,V-bromosuccinimide, since iodine is selectively liberated from potassium iodide before reducing substances present, other than ascorbic acid, are oxidized by X-bromosuccinimide, and consequently t,he end point is easily determined in the presence of starch. The end point is definitely blue in the case of pure solutions and pharmaceuticals, but violet in the case of biologicals and fruits.
CHzOH
LITERATURE CITED
H - L I
HO-~--H
I
CHiOH The fact that ascorbic acid reacts very rapidly with N-bromosuccinimide, whereas many of the interfering substances react more slowly or even do not react a t all, provides a reliable titrimetric method for the determination of ascorbic acid. N-Bromosuccinimide is an oxidizing agent and thus can liberate iodine from potassium iodide in aqueous acetic acid medium, but it oxidizes ascorbic acid to dehydroascorbic acid preferentially. Until all the ascorbic acid present in the solution is oxidized, no iodine is liberated from potassium iodide. The slightest excess of N-bromosuccinimide added, after all the ascorbic acid content has been oxidized, Fill liberate iodine from potassium iodide, n-hich is easily detected by the blue color developed with a few drops of starch solution added a t the beginning of
(1) Barakat, h l . Z., Badran, S . ,and Shehab, S. K., J. Pharm. and Pharmacol., 4 , 46 (1952). (2) Barakat, R l . Z., and Mousa, G. XI,,Ibid., 4, 115 (1952). (3) Basu, K. P., and Nath, XI. C., J . I n d i a n Chem. Soc., 15, 133 (1938). (4) Bessey, 0. A., J . Am. M e d . Assoc., 111, 1290 (1938). ( 5 ) Bessey, 0. A , J . Bid. Chem.. 126, 771 (1938). (6) Bessey, 0. A., and King, C. G., Ibid., 103, 687 (1933). (7) Fieser, L. F., and Rajagopalan, S., J . Am. Chem. Soc., 71, 3935, 3938 (1949). (8) Ibid., 72, 5530 (1950). (9) G I , Imre, AVafure,138, 799 (November 1936). (10) Gawron, O., and Berg, R., IND. EX. CHEY.,A h - a ~ED., . 16, 757 (1 944). (11) Hochberg, A I . , XIelnick, D., and Oser, B. L., Ibid., 15, 182 (1943). (12) King, C. G., Ibid., 13, 225 (1941). (13) Penney, J. R., and Zilva, S. S.,Biochem. J . , 39, 392 (1945). (14) Roe, J. H., and Kuether, C., J . B i d . Chem., 147, 399 (1943). (15) Roe, J. H.. Mills, R l . B., Oesterling, XI. J., and Damron, C. M., Ibid.. 174, 201 (1948). (16) Roe, J. H., and Oesterling, RI. J., Ibid., 152, 511 (1944). (17) Sherman, H. C.. La Mer. V. K., and Campbell, H. L., J . A m . Chem. SOC., 44, 165 (1922). (18) Snow, G. A . , and Zilva, S.S.,Biochem. J . , 38, 458 (1944). (19) Tauber, H., and Kleiner, I. S., J . Bid. Chem., 108, 563 (1935). (20) Tillmans, J. Z., 2. L'ntersuch. Lebensm., 54, 33 (1927).
RECEIVED for reriew February 3,
1954.
Accepted November 22, 1954.
Method -of Assay for Ethylenimine Derivatives .L
EUGENE ALLEN and WILLIAM SEAMAN Research Division, American Cyanamid Co., Bound Brook,
A method was devised for the determination of ethylenimine derivatives, which are being studied experimentally for use in the treatment of cancer. It is based on a rapid reaction between the ethylenimino group and thiosulfate ion at pH 4. This reaction consumes 1 mole of acid for each ethylenimino group. A large excess of thiosulfate is needed to suppress competing reactions. -4s this excess is difficult to measure with precision, the acid consumption is measured instead, by adding standard acid and thiosulfate and titrating the excess acid with standard alkali. The method has been studied for the assay of the following ethylenimine derivatives and homologs: 2,4,6-tris(laziridy1)-s-triazine (triethylenemelamine), 2-amino4,6- bis(1- aziridyl) s - triazine (diethylenemelamine), N , N ',N " triethylenephosphoramide, iV,N ', N " - triethylenetbiophosphoramide, N ( 3 oxapentamethylene"N',N''-diethylenephosphoramide, N,N',N"-tris(1methylethylene) phosphoramide, iV,N',N"-tris(1,l-dimethylethylene) phosphoramide, p-toluenesulfonN-ethyleneamide, and iV,N'-diethylenebenzene-1,3disulfonamide. The standard deviation of these determinations, where applicable, is in the neighborhood of *O.l%.
-
N. 1,
S
EVERAL derivatives of ethylenimine have recently been
studied as possible agents for the treatment of certain types of cancer. These compounds have physiological effects similar to those of the nitrogen mustards, which have been used for the palliative treatment of cancer ( 3 ) . However, the ethylenimines are reported to be easier to administer and to bring about unpleasant side effects less often ( 2 ) . When a nitrogen mustard is dissolved in water, it is transformed to an ethylenimonium ion ( 1 ) . Equation I shows such a reaction for methylbis (6-chloroethyl)amine, a typical nitrogen mustard:
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- -
The ethylenimonium ion is thought to be the active chemotherapeutic agent. The ethylenimino group has a structure similar to that of the ethylenimonium ion, except that the nitrogen atom does not have a positive charge. 2,4,6-Tris( 1-arizidy1)-s-triazine (triethylenemelamine) (I), also known as TEM, is the ethylenimine derivative which has been most thoroughly studied clinically. Another typical ethylenimine derivative is X,X', N"-triethylene phosphoramide (11).
