May 15, 1943
ANALYTICAL EDITION
from which y may be determined, giving the micrograms of nicotinic acid, while y subtracted from T gives the micrograms of nicotinamide present in the sample aliquot. Small experimental errors in the determination of z or T or both become magnified in solving for z and y. As a result, individual determinations for nicotinic acid and nicotinamide in materials containing both forms may differ from one another by as much as 10 or 15 per cent. ~~
OF EQUIMOLECULAR QUANTITIES TABLE 111. EXTINCTIONS
Compound
Equimolecular Weights
I Log 2 I
PB.
Nicotinic acid Nicotinamide Pyridine 8-Picoline a
50.0 49.6 32.0 38.0
0.295 0.195 0.248 0.111
Time" Sec. 360-420 210 87 28
Length of time for development of maximum extinction.
Discussion I n Figure 1 time-reaction curves are given for /?-picoline and pyridine as well as for nicotinic acid and nicotinamide. A comparison of these curves shows that the more polar the group on the beta-carbon atom of the pyridine nucleus the longer the time required for maximum development of color and the more stable the color produced. These results indicate that the nature of the group on the beta-carbon atom of the pyridine nucleus exerts a decided influence on the mechanism of the dianil reaction and on the intensity of the color of the resulting compound. I n Figure 1 maximum extinction coefficients are plotted for equal quantities of compoundsnamely, 50 micrograms. Maximum extinction coefficients for equimolecular quantities of the four compounds have been calculated from Figure 1 and tabulated in Table 111. On an equimolecular basis the intensity of the color produced by these four compounds decreases in the following order:
355
nicotinic acid, nicotinamide, pyridine, and @-picoline. This is the same order as reported by Melnick, Robinson, and Field (6).
Summary
A method for the direct quantitative analysis of compounded vitamin mixtures for nicotinamide has been described, which has been used successfully in the analysis of several hundred samples during the past year. Time-reaction measurements of the color produced by the Konig reaction, using the reagents cyanogen bromide and aniline, form the basis of the method. The relationship between maximum extinction coefficients and time for their development has been found to be distinctly characteristic for nicotinic acid and nicotinamide as well as for pyridine and /?-picoline. A delay of a few minutes in the addition of aniline following the addition of cyanogen bromide introduces considerable error, while as much as a 15-minute delay in adding cyanogen bromide after first adding the aniline does not introduce an appreciable error. Results obtained by this method have been found to be reproducible to within *2 per cent for the direct quantitative determination of either nicotinic acid or nicotinamide in vitamin mixtures. Literature Cited (1) Bandier, E.,and Hald, J., Biochem. J., 33,264(1939). (2) Harris, L.J., and Raymond, W. D., Zbid., 33,2037 (1941). (3) Konig, W.,J . prakt. Chem., 69,105 (1904). (4) Melnick, D.,Cereal Chem., 19,553 (1942). (5) Melnick, D.,and Field, H., Jr., J . Bid. Chem., 134, 1 (1940);135, 53 (1940). (6) Melnick, D., Robinson, W. D.. and Field, H., Jr., Zbid., 136, 131 (1940). (7) Shaw, G. E.; and Macdonald, C. A., Quart. J . Pharm. P h a r m o l . . 11, No.3, 380 (1938). (8) Snell, E.E.,and Wright, L. D., J . Bid. Chem., 139,675 (1941). (91 Waisman, H., and Elvehjem, C. A., IND.ENCI. CHEM..ANAL.ED., 13, 221 (1941).
Chemical Differentiation between Niacinamide and Niacin in Pharmaceutical Products' DANIEL MELNICK AND BERNARD L. OSER Food Research Laboratories, Inc., Long Island City, N. Y.
HE microbiological method of Snell and Wright in its existing forms (1, 2, 6) does not distinguish between niacin and niacinamide, since both compounds exert the same microbiological stimulatory effects ( 5 ) . By taking advantage of the relative reaction rates, the chemical method offers a means of differentiating between the free acid and the amide. This distinction is of no little importance, since niacin produces in many individuals an uncomfortable sensation of flushing and erythema of the skin; moreover, the amide is more expensive. I n the chemical method (2, 3) the samples are first subjected to acid hydrolysis to convert biologically active niacin derivatives to free niacin. Niacinamide, however, reacts with the reagents prior to hydrolysis to yield the name color as niacin but of lesser intensity (3). Kiacinamide, like niacin, is quantitatively adsorbed on and eluted from Lloyd's reagent and neither is lost during the lead hydroxide 1
See Editor'B note on page 352.
clarification procedure ( 2 ) . The color reactions due to these two compounds in one solution are additive. When the color reaction is carried out according to the collaborative chemical procedure (2), the maximal color developed (the photometric density 3 to 8 minutes after the addition of the aniline reagent) is 35 to 40 per cent greater with niacin than with an equivalent amount of niacinamide. With the latter, the maximal photometric density develops in approximately 2.5 minutes but is stable for only 30 seconds. If the cyanogen bromide and aniline reagents are added in rapid sequence as in the method of Melnick and Field (3)i. e., omitting the 10-minute period during which the cyanogen bromide alone is allowed to react-the sensitivity of the method for niacin is reduced by 30 to 35 per cent, but the maximal color due to reacted niacin is now practically twice that of reacted niacinamide. Figure 1 shows these differences in reaction rates. Readings were taken a t 15-second intervals
INDUSTRIAL AND ENGINEERING CHEMISTRY
356
in a n Evelyn photoelectric colorimeter (Rubicon Company, Philadelphia, Penna.) with filter 420. I n conducting a test for differentiating niacin from niacinamide, the material is tested before and after acid hydrolysis. The molecular weights of niacin and niacinamide are almost the same, 123 and 122, respectively. The details for the
Then 0..438
niacin
-
0.016 = 0.422 photometric density due to reacted
0.699 - 0.438 = 0.261 photometric density due to 10 pg. of
reacted niacin
0.422/0.261 X 10
solution I
0600
1
1
I
n
Vol. 15. No. 5
=
16.2pg. of niacin in 3-ml. aliquot of test
16.2 X 10/3 X l 5 / 8 X 1000/1 X 1/10 X 1/1000 = 1 0 . 1 mg. of total niacin per capsule
Test for niacinamide: repeat above, omitting acid hydrolysis 0.000 = photometric density of the blank 0.216 = photometric density of test solution 0.475 = photometric density of test solution containing 10 pg. of added niacin 0.216
- 0.000
0.475
- 0.216
niacin compounds added niacin
6u a100 $
k Nior/namide
0 2 4 6 8 / 0 0 2 4 6 8 / 0
7/ME /N MIIUTES FIGURE 1. TIME-REACTION CURVES 10-microgram quantities of niacin and niacinamide with cyanogen bromide and aniline reagents. I. Reagents added in rapid sequence (3) 11. Cyanogen bromide allowed to react with vitamin for 10 minutes before addition of aniline reagent (2)
Typical Calculation Test substance: vitamin capsule subjected to acid hydrolysis. Preparation of test solution: 20 capsules dissolved in 200 ml. of water; 1-ml. aliquot carried through the collaborative chemical method (2) but the maximal yellow color due to reacted niacin measured after the cyanogen bromide and aniline reagents were added in rapid sequence (3). Eight milliliters of the final neutralized supernatant solution are diluted to 10 ml. and 3-ml. aliquots taken for testing. 0.016 = photometric density of the blank
photometric density of the test solution 0.699 = photometric density of the test solution containing 10 pg. of added niacin 0.438
=
= 0.259
photometric density due to reacted photometric density due to 10 pg. of
0.216/0.259 X 10 = 8 . 3 pg. of apparent niacin in sample 8.3
x
10/3,X 15/8 X 1000/1 X 1/10 X 1/1000 = 5 . 2 rng. of
apparent niacm per capsule
Since this is one half of the total niacin, found after hydrolysis, the material is present in the capsule entirely as niacinamide. Had the above value for apparent niacin been 7.0 mg. per capsule, then 10.1 - 7.0
3.1 n I
6.2mg. of niacinamide per capsule and 10.1 - 6 . 2
preparation of the reagents, the hydrolysis, subsequent clarification, and the setting up of the solutions for the color development have been described in detail elsewhere ( 2 ) . The final color development, however, follows the earlier method of Melnick and Field ( 3 ) . [For the first test of total niacin content the collaborative chemical procedure (2) may be used. However, for testing the unhydrolysed sample the addition of the reagents in rapid sequence (3) is preferred, in order that the photometric densities of reacted niacin and niacinamide may show a bigger difference. The readings should be taken periodically during the color development, since maximal constant values are required for the calculation and the reaction, particularly with niacinamide, fades rapidly once the maximum is attained.]
= 0.216
= 3.9 mg. of
niacin per capsule
Some latitude ( * 15 per cent) should be allowed in interpreting the results, since the accuracy of the partition of the niacin ingredient is dependent upon the precision of two assays and errors may be additive. I n testing samples such as pure niacin, niacinamide, or mixtures of the two, clear colorless solutions are obtained; the adsorption-elution and decolorization procedures are then omitted. Greater precision is also obtained. For determinations conducted on pure niacin or niacinamide preparations, direct reference to the reaction curves in Figure 1 may suffice to identify and allow rapid quantitative estimation of the compound with a good degree of precision ( * 2 per cent). The nicotinamide-containing coenzymes fail to yield a yellow reaction product with the reagents unless first hydrolyzed, Nicotinuric acid reacts with the reagents to yield some color prior to hydrolysis. Strong acid hydrolysis is required to convert it quantitatively to free niacin (4). However, these compounds are not readily available. The test procedure described is intended for use only in assaying the niacin ingredient in vitamin capsules and tablets containing the synthetic vitamin.
Literature Cited (1) Isbell, H., J. Biol. Chem., 144, 567 (1942). (2) Melnick, D., Cereal Chem., 19, 553 (1942). (3) Melnick, D., and Field, H., Jr., J. Biol. Chem., 134, 1 (1941). (4) Melnick, D., Robinson, W. D., and Field, H., Jr., Ibid., 136, 131 (1940). (5) Snell, E. E., and Wright, L.D., Ibid.. 139, 675 (1941).
