V O L U M E 2 3 , NO. 7, J U L Y 1 9 5 1 LITERATURE CITED (1)
.Iheledo, C. A. and Kolthoff. I. XI., J . A m . Chem. SOC., 53, 2893 (1931).
Bennett, H.,and Hartwood, H. F.,A n a l y s t , 60, 677 (1935). (3) Burriel, F., and Suarez. R., Aiirrlcs real soc. espafi. jfs. y q u i m . , @)
44, 449 (1948). (4) Cool, R. D., and Yoe, .J. H., IND.EXG.CHEM.,ANAL.E D . , 5, 112 (1933). (5) Cummings, R’. bI., and Alexander, W. A , , A n a l y s t , 68, 273 (1943). ( 6 ) Endres, G.. and Kaufmaiin, L., Ann., 530,184 (1937). (7) Feigl, F., A r i o i s assoc. q u i m . Brasil, 1, 234 (1942). (8) Feigl, F., J . Ch.em. Education, 21, 347 (1944); 20, 139 (1943). (9) Fischbach, IT., 2. anal. Chem., 96, 443 (1934). (10) Keilin, R.. and Otvos, J. W., J . Am. C h e m . Soc., 68, 2665 (1946). i l l ) Kinnicut, I,. P., and Sef, S. U., Am. Cheni. J . , 5, 388 (1883). (12) Klemec, .I., 2. anal. C‘hem., 61, 448 (1922). (13) Klenienc, A., and Klima, L., 2. anorg. n l l g e v i . C h o n . , 179, 379 (1929). (14) Kolthoff, I. hl., and Furinan, S . H., ”Volumetric .kiialysis,” Vol. 2, p. 603, New York, John Wley & Sons, 1929. (15) Kolthoff, I . M., and Saiidell, E. B., “Textbook of Quantitative Analysis,” p. 603, K‘ew York, hlacmillan Co., 1943. (16) Korenman, I. XI., et al.. %niodskoyn h h . , 16, 3 (1950).
983 (17) Kricheviskii, I., and Kantorovich, L., J. Chem. I d . (U.S.S.R.). 12, 139 (1935). (18) Lang, F. M.,Compt. rend., 227, 849 (1948); 226, 1381 (1948). (19) Lang, F. M., and Aunis, G.,Chem. anal., 32, 139 (1950); Compt. rend., 230,208 (1950). (20) Lunge, G., Ber., 10, 1074 (1877); 2. angew. Chem.,4, 629 (1891); Chem.-Ztg., 28, 501 (1904). (21) Raschig, F., Ber., 38, 3911 (1905). (22) Rubel, W.M.,2. Untersuch. Lebensm., 60, 588 (1930). (23) Rylioh, -4.,Collection Czechoslov, Chem. Communs., 7, 288 (1935). (24) Schulek, E., and Floderer, I., 2. anal. Chem., 123, 198 (1942). (25) Scott, IT. W., ”Standard Methods of Chemical Analysis,” p. 521, KeF York, D. Van Nostrand Co., 1917. (26) Juzuki, K., J . SOC.Chem. I d . , J a p a n , 43,404 (1940). (27) Tokuoka, M., Collection Crcchoslov. Chem. Communs., 4, 444 (1932). (28) Treadwell. W. D., and 1-ontobt.1, H., H&. (’him. Acta, 20, 573 (1937). (29) Ubalkini, I., and Guerrieri, F., A 7 u i . (,him.rcpplicata, 38, 702 (1948). (30) \Villard, H. H., and Young, P., -1..lm. C’hem. SOC.,50, 1379 (1 928). (31 1 \Vinograd, -4., Chernist-d,iirly.~f,20 (3) 15 (1931). R E C E ~ V E D L>rrrrnher 5.
1950.
Chemical Differentiation between Nicotinic Acid and Nicotinamide JAMES P. SWEENEY A ~ I )U’ILL4CE L. HALL Dic*ision qf \iilrition. Food a n d Drug I d m i n i s t r a t i o n . Federul Seciirity . - l p e n q . . IInshinpton 15, D . C . It niaj be of importance to the clinician and to the control chemist to be able to differentiate between nicotinic acid and nicotinamide. In the chemical estimation of these compounds their pyridine rings are split with cyanogen bromide by the Koenig reaction and the reaction products are coupled with sulfanilic acid. At 430 mp, where the color complexes are measured, the absorbancy of the nicotinic acid color is approxiniately twice that of the color produced writh nicotinamide. I s nicotinaniide is converted quantitatively to nicotinic acid by hydrolysis, the percentage composition of a mixture may he determined by absorbancy measurements before and after h?drolj sis. Tobias acid (2-naphthylamine-1-sulfonic acid) may be used in place of sulfanilic acid for the development of the color complexes; colors produced with nicotinic acid and nicotinamide are differentiated visuallJ. This permits its use in a qualitative test, Data and graphs demonstrate the applicahili t y nf these methods.
I
K THE examination of vitamin products available conimer-
cially it is a t times important t o differentiate between nicotinic acid and nicotinamide. Because of an unpleasant flushing reaction in some patients to whom nicotinic acid is administered, there is a clinical preference for the amide, nhich is without such effect (6). The microbiological procedure foi the determination of nicotinic acid included in the U. S. Pharmacopeia 9IV does not distinguish betweeu the two forms, for the organism used can utilize the acid and amide equallr. Chemical methods described by Lamb (4)and Melnick arid Ober ( 6 ) ,in which differences in color intensitv are obtained \Then the acid and amide react n ith cyanogen hromide and aniline, have beeii criticized by Ciusa ( 2 )from the standpoint of lack of piecision. Ciusa demonstrated that the amide, hut not the acid, will react with henzyl chloiide to form a complex that is not converted to a colored rompound in the presence of cyanogen hromide and a n aromatic amine, and proposed this as a method for differentiation. Such a method is too cumbersome for routine control purposes. C‘haudhuri and Kodicek ( I ) describe a procedure in iyhich a rnwsuw I S madcl of thtx f l u o i r ~ c e ~pioduced ir~ nhcn the ainidt.
react.< ivith cyanogen hroniide and the solution is subsequently made alkalinc~. d number of compounds, among them thiamine arid pyiidosine, are known to give a similar fluorescence. Therefore, it is necessary t’o pretrwt the vitamin solution prior t o the cyanogen bromide reaction in order to remove the interfering fluorescence. This niet,hod also is too elaborate for routine use, X niethod for the determination of nicotinic acid, developrd in this laboratory, is bawd OII the color reaction of Konig ( 8 ) but makes use of sulfanilic acid as tht. aromatic amine. The :idvantages of the use of sulfanilic acid have bcrn covered (7-9). This method has been applied t o a variPtJ- of natural materials and commercial products containing t h r vitamin and, follo\~ingcollaboriitivr study, has been adoptrd for control purposes hl- the -1sfiociation of Official ,igriculturaI Chrniists. Provision is made in this method for conversion of thc, amide to the acid form by nd measurement by nieans of a spectrophotomet,erof the color formed a t wavc length 450 nip, the point of maximum ahsorptioii. -4comparison of the absorption spertra of the nicotinic acid and nicotinamitir rolors as tieveloprd with cyanogen bromide and PUI-
ANALYTICAL CHEMISTRY
984
aliquot of the unhydrolyeed solution is also made to a volume that contains approximately 10 micrograms of total nicotinic acid or nicotinamide per ml. COLORDEVELOPMENT.The color development of these solutions is carried out in selected 18-mm. test tubes according to Table I. The sample solution or standard solution, water, and ammonium hydroxide are added to the tubes as indicated. Sulfanilic acid and one drop of concentrated hydrochloric acid are added to the blank tube. The tube is placed in the instrument, which is adjusted to read 100% transmittance a t 430 mp. The cyanogen bromide is added to a sample or standard tube from a pipet with swirling to ensure mixing, and immediately after this addition the sulfanilic acid is measured into the tube in a similar manner. The tube is placed in the instrument and the transmittance reading is taken a t the time of maximum absorption (1.5to 2 minutes).
fanilic acid revealed that between wave lengths 400 and 430 mp the nicotinic acid color has approximately double the absorbancy of the nicotinamide color. Figure 1shows the absorption spectra of nicotinic acid and nicotinamide colors. The concentration of nicotinic acid and nicotinamide in each case was 10 micrograms in a volume of 10 ml. Because the molecular weights of nicotinic acid and nicotinamide are 123 and 122, respectively, the weights used can be considered molecular equivalents. It is possible to measure accurately the difference in absorbancy between nicotinic acid and nicotinamide a t any wave length between 400 and 475 mp. However, the difference in absorbancy is greatest a t 430 mp, and all results reported in this paper have been determined on the basis of the absorbancy a t 430 mp. As the amide is readily converted to the acid by hydrolysis, the percentage composition of nicotinic acid and nicotinamide can be determined by measuring the absorbancy a t 430 mp of the color produced with cyanogen bromide and sulfanilic acid before and after hydrolysis. Presented here are a procedure for the quantitative differentiation of nicotinic acid and nicotinamide, and the details of a simplified method for qualitative differentiation of these compounds employing the aromatic amine 2-naphthylamine-1-sulfonic acid (Tobias acid), in place of sulfanilic acid. The method described is applicable to pharmaceutical preparations containing amounts of nicotinic acid or nicotinamide usually dispensed.
0.4
0.3 F
QUANTITATIVE DIFFERENTIATION O F NICOTINIC ACID AND NICOTINAMIDE
z 4
m K
0 Reagents. Ammonium hydroxide, 5 ml. of concentrated anim monium hydroxide diluted to 250 ml. 4 Hydrochloric acid, 1 volume of Concentrated hydrochloric 0.2 acid added to 4 volumes of water. Cyanogen bromide, 10% aqueous solution. This should be prepared under a hood. Sulfanilic acid, 10% solution. Place 20 grams of sulfanilic acid in 170 ml. of water, and add concentrated ammonium hydroxide, 1 ml. a t a time, until solution is obtained. Adjust p H to 4.5, using bromocresol green indicator. Use a spot plate for testing 0.I pH. Make up to 200 ml. with water. Nicotinic acid stock solution, 50 mg. of U.S.P. nicotinic acid reference standard made up to 500 ml. with 95% ethyl alcohol. (Keep in refrigerator.) Nicotinic acid standard solution, 5 ml. of stock solution diluted to 50 ml. with water; 1 m]. of this solution contains 10 micrograms of nicotinic acid. Xicotinamide standard solutions are prepared in the same C manner aa the nicotinic acid standard solutions. 400 450 500 550 WAVE LENGTH, m p Instrument. A spectrophotometer or filter photometer should be used for absorbancy measurements. If a simple nonamplifyFigure 1. Absorption Spectra of Nicotinic Acid ing filter photometer is used, it must be equipped with a filter or and Nicotinamide Colors Developed with Cyanogen filters which have a high percentage transmittance a t a narrow Bromide and Sulfanilic Acid band range. Interference filters are useful for this purpose. Procedure. PREPARATION OF SAMPLE. For many samples it is convenient to prepare a dilution or extract that contains approximately 100 micrograms of total nicotinic acid or nicotinCALCULATION. The color is first developed on an aliquot of the amide per milliliter. For tablets, the sample is finely ground in a test solution which contains both nicotinic acid and nicotinamide. mortar and extracted with hot water for 15 minutes. Then the The nicotinamide on another aliquot is then converted to nicoextract and residue are transferred to a volumetric flask and made to the proper volume. For capsules, the sample is dispersed in hot water and made t o a similar volume. d 10Table I. Development of Color ml. aliquot is added to 10 ml. of concentrated hydrochloric Unhydrolyzed Hydrolyzed acid, the resulting solution is Sample Unhydrolyzed Sample Hydrolyzed Nicotinic NicotinBlank Sample Blank amide Pample Acid Blank evaporated on a hot plate to a volume of about 2 ml., and 50 Nicotinic acid 1 1 .. standard, ml. t o 75 ml. of water are added. Xicotinamide The solution is made alkaline 1 standard ml. . . t o bromothymol blue indicator Cnhydrolyked 1 1 ... sample ml ... b addition of sodium hydroxHydro1yzkd iL (use of pellets is con1 ... ... 1 ... ... sample ml. ... 6.5 1.5 6.5 1.5 venient). A spot plate is used 1.5 Water &I. 6.5 1.5 0.5 0.5 0.5 0.5 0.5 0.5 “,OB, ml. 0.5 for the p H adjustment. The 5.0 5.0 ... 5.0 5.0 ,.. $. CNBr,, solution is then made to a 2.0 2.0 i:o 2.0 2.0 Sulfanilic acid, ml. 2.0 2.0 1 ... 1 ... volume that contains approxi1 ... Concd. HC1, drop ... 1 1 ... 1 ... ... 1 Water. drop mately 10 micrograms of total nicotinic acid per ml. A 10-ml. ~
V O L U M E 2 3 , NO. 7, J U L Y 1 9 5 1 0.400
-
20.350
-
9ss 10 ml. The readings were taken a t 430 mp with a Beckman Model B s p e c t r o p h o t o m e t e r , using selected 18-mm. test tubes. To demonstrate the applicability of the quantitative method t o pharmaceutical products, a series of determinations was made on various types of vitamin preparations. Results are presented in Table 11, in which amounts of nicotinic acid and nicotinamide,
z >-
yO.300 -a
-
m
-
K n
ul
obtained by differentiation, are compared with the total amount 2 found by the method of the 6 4 3 10 9 8 7 :Issociation of Official Agricultural Figure 2. .4hsorhancy Values at 430 m p Chemists. Colore produced b? reaction of cyanogen bromide and sulfanilir acid with mixture. of nicotinic acid Samples 1 to 3 and 11 t o 15, and nicotinamide. Solutionc contained a total of 10 micrograms af t w o compoundfi in indicated ratios inclusive. contained mixtures of nicotinic acid and nicotinamide. The percentage composition, as tirtermined by the authors, $vas in close agreement with that tinic acid by hydrolysis. Any increase in color developed in this stated by the label claim. Samples 4 to 10, inclusive, were laaliquot as compared to that produced in the previous aliquot is the heled nicotinamide and XTere proved to be nicotinamide by analyresult of the conversion of nicotinamide to nicot,inic acid, and the sis. Sone of the camplee teqtetl gave any evidence of a breakamount of increase is proportional t o the amount of nicotinamide down of nicotinamide to nicotinic acid. originally present. .4 wmplc cnlrulation follons: PRECISIOS. The results obtained in the determination of total nicotinic acid by the sulfanilic acid method (1, 9) are reproducible 1. .-ihsorbancy o f 1 0 m i ~ ~ i ) ~ i ~ofa nnirotinii, is :rr*itl t o within *2%. However, such precision is not possible in the standard = 0.400 differentiation method proposed here. Because of the number of 2. Absorbancy of I0 niicwgrams of nicwtin:Lmid(. measurements required, and the possibility that errors may be standard = 0 200 additive, individual drterminations may differ from rach other 3. Absorbancy of 1 nil. of unhydrolj.zcd sample = 0.320 by as murh as *5y0 4. Absorbancy of 1 nil. of hy(Irolyz(4sample = 0,400 5. hicrease in atw)rl)an(~,v tlnc' t o h ~ ~ t l r o l ~ ~ s i s 0 080 0
2
I
3
I
I
4
5 5
I 6
I 7
I
I
8
9 I
I I O - Y N I C O T I N I C ACID 0-YNICOTINAMIDE
Tahle 11.
-
~1
of nicotinic acicl
0.400
Determination of Sicotinic icid and Nicotinamide
:in(I
1 mirrograrn of nicotinamide)
=
0.020
0.320 = 0.080 = 1 niicrogJ,ams of nicotinaniide in 0.020 0.02 unhydrolyzed .samplv -
~
The use of a standard r e t ( ~ r t ~ i i(i ~w(v* e has proved to be a morr practical means of determining the perrcntage composition of a mixture of nicotinic ncid and nirotinnniide. For this purpose a standard curve similar to t h a t illurtrated in Figure 2 may used, in which the absorbancy v:ilues of colors produced by thr reaction of cyanogen bromidc rind sulfanilic. acid with standard nic.otinir acid and nicotinamide are plotted. The tot,al nicotinic; itrid content of an aliquot of thc Iiyclrolyzed teat solution is'first determinecl. An aliquot of the uiili!-drolyzed test solution is then dilutrd so that it contains the s:tInc' total nicotinic acid plus nirot,inamide concentration as do th(, ytandards used in construction of the reference curve. The nbsorlxincy of the color produred by the unhydrolyzed test Polution, when developed with cyanogen bromide and sulfanilic acid, is then read a t 430 mp and the percentage composition i q rlrtermined by interpolation from thr standard curve. Results. Standard solutions containing known amounts of nicotinic acid and nicotinamide were tested. Figure 2 shows the effect of increased ratios of nicotinic acid to nicotinamide on the absorbancy of the color produced by the reaction with cyanogen bromide and sulfanilic acid. The total amount of nicotinic acid plus nicotinamide in each rase was 10 micrograms in a volume of 1
~
('oinlnercial Samples 1 2 3 4 5 6 7 8 9 10 11 12
13 14 15
Amounts Found by Differentiation Analyses _ _ Nicotinic acid Sirotinarnide 25 2 27 0 9 6 70 4 18 0 77 0 Sone 52 0 Xone 100 0 None 102 0 None 13.0 None 2750 0 None 103.0 Pione 103.0 11 9 25 13 12
8 5 0 5 4
8.8 9 8 25.0 15.9 9.8
Equivalent Piicotinic Acid, Columns 2 + 3 j2.2 80.0 96.0
52.0
100.0 102,o 13.0 2750.0 103.0 103.0 20.6 19.3 50.0 29.4 22.2
Total Nicotinic Acid by AOhC Method 51.0 82.0 95.0 52.0 100,o 102.0 13.0 2750.0 103.0 103.0 20.0 19.7 50.0 30 0 21.0
1
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