Chemical Determination of Nicotinic Acid AARON ARNOLD, C. B. SCHREFFLER, AND S. T. LIPSIUS Nopco Vitamin Laboratories, National Oil Products Company, Harrison, N. J.
T
HE role of nicotinic acid in the prevention or cure of
Some nicotinic acid-rich materials do not require this preliminary extraction treatment, but yield reliable values when treated directly in the same way as the extracts, A few such cases are shown in Table I.
black tongue in dogs (5) and pellagra in humans (14) has stimulated studies on methods for the quantitative determination of this compound. The chemical methods are based on its reaction with 2,4-dinitrochlorobenzene (7, 17') or with cyanogen bromide and aniline (9, 10, 11, 16) or p-methylaminophenol sulfate (2, 3, 12) as color reagents. Recently, Harris and Raymond (6) have reported considerable success for the evaluation of nicotinic acid in urine with p-aminoacetophenone as the color reagent. Since the authors have been engaged in utilizing rapid laboratory methods for the determination of the factors of the vitamin B complex, they have adapted this latter method for the evaluation of nicotinic acid in natural and concentrated source materials in a manner somewhat different from the procedure developed b y Kodicek (8). They believe the general interest in the antipellagra factor warrants recording their method and results a t this time.
The combined extracts of the nicotinic acid-rich materials, made up to a volume of about 80 cc. with water, are made alkaline with 5 cc. of 20 per cent sodium hydroxide solution and are heated for half an hour on a steam bath or hot plate to liberate nicotinic acid from its amide. Then 2 cc. of 4 per cent sodium bicarbonate solution are added to the cooled solution to aid in adjusting the pH, 1.5 cc. of concentrated hydrochloric acid are added, and the solution is brought to a pH of 6.0 to 6.2 with hydrochloric acid (10 per cent). The solution is transferred to a 100-cc. volumetric flask and diluted to the mark with water. TREATMENT OF EXTRACT.Aliquots (5 cc.) of the solution containing the unknown are measured into each of four 15-cc. amber glass graduated cylinders (Otto R. Greiner Co., Newark, N. J., 1.0-cc. graduations). These are diluted with 5 cc. of pH 6.0 potassium dihydrogen phosphate-sodium hydroxide buffer solution (4) to help ensure comparable pH conditions between separate determinations. With nicotinic acid-low materials 10cc. aliquots are sometimes necessary, in which event the buffer Experimental solution is omitted. PREPARATION OF EXTRACT.An amount of finely ground or Twenty and 40 micrograms of nicotinic acid, as a solution conminced sample, designed to supply about 0.4 mg. of nicotinic acid, taining 100 micrograms of nicotinic acid per cc., are added to two is sus ended in 75 cc. of water in a centrifuge bottle and autoof the graduates, all the cylinders being then placed in a water clavecffor 15 minutes at 10,500 kg. per sq. meter (15 pounds per bath a t 80" C. After 10 minutes, 2.0 cc. of freshly prepared sq. inch). This step is similar to the one used in the preparation cyanogen bromide (saturated bromine water just decolorized with of riboflavin extracts, which has been found fully satisfactory 10 per cent potassium cyanide) are added to three of the gradufor adequate extraction (1, I S ) . After cooling, the bottles are ates, including those containing the added nicotinic acid, and all centrifuged and the supernatant liquid is decanted into a 125-cc. four cylinders are held a t 80' C. for an additional 4 minutes. Erlenmeyer flask. The residue is taken up with enough water The solutions are then rapidly cooled to room temperature and to bring the total amount of extract up to about 80 cc., centriafter 4 minutes, 0.2 cc. of p-aminoacetophenone solution (10 fuged, and decanted for addition to the first extract. grams dissolved in 28 cc. of 10 per cent hydrochloric acid and diluted to 100 cc.) is added to each graduate. After mixing the contents, the cylinders are allowed to stand in a darkened TABLEI. NICOTINIC ACID CONTENT OF SOURCE M.4TERIALS place for 15 minutes. After that time, 0.4 cc. of 10 per cent p-Aminoacetophenone Procedure Other Methods Direct Chemically hydrochloric acid is measured -4mount Extraction alkali Bioassay obtained with a microburet into each Material assayeda procedure treatment values ( 1 8 ) values cylinder and the solutions are Gram 1Mg. % 1Mg. 5% M P . 70 AMQ. % allowed to stand for an addi0.125 9.2 Fresh beef liver 9 . 3 (7) 9.2 25-27.5 tional 15 minutes in the dark. 12.2 (2) The volume in each cylinder is 1 8 . 0 (oj Fresh beef muscle 0.4 2.4,2.5 ... 3.8-10.9 3 . 8 (7) then adjusted to 13 cc. with 4 . 1 (2) water. The reaction mixtures are 0.1 Ether-extracted peanut meal A 22 ... transferred to 25-cc. centrifuge 0.1 Peanut meal B 6% fat 20, 23 ... 13 (raw meal) separatory funnels (Pfaltz & Defatted wheai germ A 0.4 3.4 .. . .... Bauer, Inc New York, N. Y.) Defatted wheat germ B 0.4 2 . 9 - ' 3 : 9 (8) 2.9,3.0 . . . Less than 6 0.05 Brewer's yeast 41 38 34-93 33.9-36.7 (9) containing y5 cc. of ethyl acetate. Baker's yeast 0.06 4 4 . 6 , 4 5 . 5 (3) 29, 32 ... 50 The funnels are shaken for 5 0.07 Feed yeast 34, 37 .... minutes, though a somewhat Rice bran extract 0.0137 .... 17i;iso 165 0.075 Rice bran 28, 31 .... .. shorter period may be equally 2l a5, 29 0.1 Rice germ .. IO ... .... satisfactory, and the mixtures are 0 Expressed in terms of weight of original sample supplied by 5-00. aliquot used, allowed to separate, centrifuging for half a minute to clear the
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ANALYTICAL EDITION
January 15, 1941
emulsion if necessary. The aqueous lower layer is drawn off and discarded. The ethyl acetate layer is clarified by adding about 2 grams of anhydrous sodium sulfate, whereupon it is ready for the colorimetric reading. INsTnmimT. A recently developed fluorometer (Pfaltz I% Bauer) has proved entirely satisfactory for estimating the concentration of the color complex. The characteristics of the instrument rendered it unsuitable for use in direct readings of the unextracted color complex, which was therefore extracted with ethyl acetate as described above. The authors determined the extinction curve of the nicotinic acid-p-aminoacetophenone color complex in this solvent, and noted a marked inflection at 420 mw which led them to use Corning filters 038 and 511. The filters are placed in the metal tube of the instrument between the capillary mercury arc tube and the cuvette containing the color complex in ethyl acetate. The following instrument settings have been found satisfactory: rheostat setting at 2.0, resistance box setting a t 2.0 (coarse), and an iris diaphragm setting at approximately 20. The light intensity will vary xith the age of the tube and is adjusted to bring the galvanometer indicator to 0.0 on the extinction scale when a standardizing cuvette with ethyl acetate is in position. The low-sensitivity range of the galvanometer is used for the readings. READINGS AXD DETERMISATION. Twelve cubic centimeters Of the clarified ethyl acetate solution are placed in a cuvette and the concentration of the color complex in each of the aliquots is estimated on the basis of the observed extinction values. The extinction value for the blank (no cyanide added) is subtracted from the values obtained for the aliquots containing 0, 20, and 40 micrograms of added nicotinic acid. The corrected extinction coefficients are plotted against the amounts of added nicotinic acid. The nicotinic acid content of the unknown can then be read from the graph, by extending the line to its intersection with the abscissa, as shown in Figure 1 for two determinations which are discussed below. In general, the authors prefer to obtain a reading of about 20 micrograms for the aliquot of the unknown. The amount of extract prepared permits four determinations, so that little difficulty is experienced in obtaining an aliquot which will supply approximately the required amount of nicotinic acid.
RECOVERY OF ADDEDNICOTINICACID. Figure 1, which illustrates the determination of the nicotinic acid content defatted wheat germ, also shows the excellent recovery which may be expected in the determination of the added nicotinic acid. An 8.0-gram sample gave a value of 13.7 micrograms of nicotinic acid per 5-cc. aliquot (curve B), representing (13.7 X 20) 274 micrograms for the 8.0-gram sample, or 3.4 mg. per cent; 4.0 grams of the same wheat germ to which had been added 200 micrograms of nicotinic acid gave a reading of 17.0 micrograms per 5-cc. aliquot (curve A), representing a total of (17.0 X 20) 340 micrograms of nicotinic acid for the sample. This represents a recovery of 102 per cent for the added nicotinic acid. The extinction coefficient for the blank aliquot (no cyanide added) had a value of 0.044 in the case of curve B and half this figure in the case of curve A. The amount of interfering color mas therefore relatively low.
Discussion Data on the nicotinic acid content of various source materials are given in Table I together with those of other investigators. The results of Waisman, Mickelsen, McKibbin, and Elvehjem (18) are of particular interest, since they are reported on the basis of bioassays with dogs. The variation in the values for the nicotinic acid content of fresh beef liver may be due to the variation in the age and nutritive condition of the animal, as indicated b y Waisman et al. (18). The bioassays of fresh beef muscle also indicate a similar variation in nicotinic acid content. It is important to note, however, that the authors’ results, the bioassay values, and the figures obtained by other chemical methods are essentially of the same order on comparable materials. The nicotinic acid values for samples of low-fat peanut meal, 20 to 23 mg. per cent, and the biologically assayed raw peanut meal, 13 mg. per cent, agree when compared on the
2 w 9 k
63
0.4 ;x
A /->:
0.3
tIN
SAMPLE+
-ADDED
P
Ug. NICOTINIC
ACID
FIGURE 1. RECOVERY O F ADDED1\;ICOTINIC ACID A.
B.
4.0 gram8 of defatted wheat germ 8.0 grams of defatted wheat germ
+ 200 micrograms of nicotinic acid
same basis, since raw peanuts contain approximately 50 per cent fat. The authors’ results with defatted wheat germ, 2.9 to 3.4 mg. per cent nicotinic acid, are in accord with the observation of Kaisman et al. (18) t h a t this dietary supplement cannot be regarded as a good source of the antipellagra factor. It is interesting to observe that the rice kernel furnishes by-products which are a good source of the antipellagra factor. The germ layer, which contains approximately 15 mg. per cent nicotinic acid, is a much better source of this factor than wheat germ. Rice bran ranks close to yeast as a source of the antipellagra factor and was, in fact, early (16) recognized as a source of both nicotinic acid and the antiberiberi vitamin. The results demonstrate that the method is applicable t o a wide variety of materials. From the standpoint of laboratory procedure, i t appears simpler than some of the methods submitted by other investigators, while the color reagent proposed by Harris and Raymond (6) also appears to be more stable than those previously suggested. The color complex is readily extracted with ethyl acetate and yields, in general, blanks with very little color due to interfering substances.
Literature Cited Arnold, A., Lipsius, 5 . T., and Greene, D. J., Food Research (in press). Bandier, E., Biochem. J., 33, 1130 (1939). Bandier. E., and Hald. J., Ibid.. 33, 264 (1939). Clark, W. M . , “Determination of Hydrogen Ions”, p. 200, Baltimore, Williams and Wilkins Co., 1928. Elvehjem, C. A., Madden, R. J., Strong, F. M . , and Woolley, D. W.,J . Biol. Chem., 123, 137 (1938). Harris, L. J., and Raymond, ’w. D., Biochem. J., 33, 2037 (1939).
Karrer, P., and Keller, H., Helv. Chim. Acta, 22, 1292 (1939). Kodicek, E., Biochem. J . , 34, 712, 724 (1940). Kringstad, H., and Naess, T., 2. physiol. Chem., 260, 108 (1939). hlelnick, D., and Field, H., Jr., J . Bid. Chem., 134, 1 (1940). Pearson, P. B., I b i d , 129, 491 (1939). Rosenblum, L. A., and Jolliffe, N., Ibid., 134, 137 (1940). Snell, E. E., and Strong, F. bl., IND.EXG.CHEM.,Anal. Ed., 11, 346 (1939). Spies, T. D., Bean, W.B., and Ashe, W. F., Ann. Intern. Med., 12, 1830 (1939). Suzuki, U., Shinamura, T., and Odake, S., Biochem. Z., 43, 89 ( 19 12). Swaminathan, M., Indian J . M e d . Research, 26, 427 (1938). Vilter, S. P., Spies, T. D., and Mathews, A. P., J . Biol. Chem., 125, 85 (1938). Waisman, H. A., Mickelsen, O., MoKibbin, J. hl., and Elvehjem, C. A., J . Nutrition, 19, 483 (1940).
