768
ANALYTICAL CHEMISTRY
interfere much more in the argentometric titration than in that with cupric copper. Iodide strongly interferes in the argentometric method but not in the copper method. The accuracy and precision of the copper method are about *0.5% with cysteine concentrations between 4 X and 8 X 10-6 A’, and about *1.5% with cysteine concentrations between 6 X 10-6 and 10-6 -11. With these dilute solutions the copper solution should be air-free or be added from an ultra. microburet. ACKXOWLEDGM EYT
This inrestigation was supported by a research grant from the Sational Cancer Institute, U.S. Public Health Service.
accurate determination of traces of cysteine and cystine, which are more precise and accurate than the amperometric argentometric method. The accuracy and precision are *0.5% with cysteine conand 8 X 10-6 Jf and about * 1.5Oj, centrations between 4 X with cysteine concentrations between 6 X 10-6 and 10-5 M. With these dilute solutions the cupric copper solution should be ahfree or added from an ultramicroburet. Cadmium does not interfere. Zinc does not interfere when its concentration does not exwed that of cysteine more than 10 times, Cobalt interferes. Iodide doe. not interfere. LITERATURE CITED
SULMMARY
The reaction between cupric copper and cysteine in a suitable ammonia buffer and in the presence of sulfite has been made the basis of a simple and accurate amperometric titration of cysteine and cystine, using the rotating platinum wire microelectrode as indicator clcctrode. Procedures are given for the rapid and
(1) Benesch, R., and Benesch, R. E., Arch. Bmhem., 19,35-45 (1948) (2) Kolthoff, I. M., and Stricks, W., J . A m Chem. Soc., 72, 1952 11950). (3) Ibid., in press. (4) Lingane, J. ,J., and Kolthoff, I. M.,Ibid., 61, 825 (1939). ( 5 ) Michaelis, L., and Baron, E. S.G., J . B i d . Clrem., 81, 29 (192!)). RECEIYED October 20, 19.50
Determination of Small Quantities of Niacin in Presence of Niacinamide Separation by Paper Partition Chromatography E. G . FOLLISH, 3IORTON SCHMALL, AND E. G. E. SHAFER, Hoflrnann-La Roche IRC.,iVutZey, N . J .
A
S U l I B E R of investigators have developed methods for the determination of niacin and niacinamide in mixtures of these compounds. Lamb ( 8 ) and Melnick and Oser (10) determined both vitamins colorimetrically by means of the Koenig reaction (6). and by comparison of their respective reaction rates. First Scud1 ( 1 2 )and then Chaudhuri and Kodicek ( 2 , 5 )published fluorometric methods for the estimation of niacinamide in the presence of niacin. These authors determined total niacin colorimetrically after hydrolysis and subtracted the quantity of niacinamide found by their fluorometric procedure. Although adequate for the determination of niacinamide, these methods are apt to suffer from an appreciable inherent error if used for the determination of small quantities of niacin in presence of niacinamide. Recently another method has been described by Ciuza ( 3 ) . This author found that benzyl substitution on the pyridine nitrogen of nicotinamide prevented that compound from undergoing the Koenig reaction, whereas niacin is not easily benzylated under controlled conditions. By running total niacin and niacin after benzylation, both niacin and niacinamide may be determined by difference. The microbiological method of Johnson (4)and its modification by brehl ( 7 ) and associates, using B. leuconostoc mesaterozdes has been successfully applied for the determination of niacin in presence of niacinamide. I t occurred to the authors that it might be possible to determine more accurately small quantities of niacin in the presence of large amounts of niacinamide, if those substances could f i s t be separated. Paper partition chromatography appeared to present the most promising approach to making such separation.
in water. ( I t is stored in a refrigerator and used only if colorless.) p-Aminoacetophenone reagent. p-.$minoacetophenone is recrystallized from a warm saturated solution in 95% ethyl alcohol by addition of about 3% distilled water. After cooling in the refrigerator, the almost colorless crystals are filtered off and dried in vacuo at room temperature. A 5 % solution in 95% ethyl alcohol, when stored in the refrigerator, will keep for 1 week. Ethyl acetate, C.P. Ethyl acetate, C.P. Saturated with Kater. Hydrochloric acid, 3 IT. Niacin standard. iln aqueous solution containing 20 micrograms of United States Pharmacopoeia niacin per ml. Apparatus. The apparatus used is similar to that described by
PROCEDURE
Reagents. Cyanogen bromide reagent. A 4% aqueous solution, prepared by dissolving cyanogen bromide crystals, Eastman,
Figure 1. Apparatus for Chromatography
V O L U M E 23, NO. 5, M A Y 1 9 5 1
Niacinamide, a memher of the vitamin B complex family, is a component of almost every multivitamin preparation on the market. Recause niacin is apt to produoe side reactions such as flushing, while niacinamide does not produce such reactions, it is of concern t o the manufacturer to determine if a n d how much hydrolysis of niacinamide to niaoin has taken place in his products. No simple method for the accurate determination of small quantities of niacin in the presence of large quantities of niaeinamide has been available. In the present investigation niacin was separated from niacinamide by descending paper partition chromatography a n d both were assayed hy colorimetric procedure. The
769
stability of niacinamide in solutions of this vitamin alone and in multivitamin solutions at various pH levels and storage temperatures was investigated. Niacinamide was found to be stable in dry form as well as in solutions within the pH range 5.0 to 6.7. Less than 2% hydrolysis was observed at pH levels helow 5.0 and storage at 45' C. for 6 weeks. By the technique described i t was possible to separate niacin from niacinamide. The results indicate that niacinamide is a rather stable product within the pH levels indicated. A breakdown to niacin is likely to O E E I I ~only at alkaline or very acid pH levels, which would not he encountered under normal manufacturing or storage conditions.
containing the solvent. The lower part of a 6inch desiccator, inverted, is used as a cover t o assure constant vapor saturation inside the spparatus. In order to obtain a good chromatogram, the sample should be applied to the filter paper in a very narrow band. The a p p a r e tus shown in Figure 2 permits the accurate addition of as much as 0.2 ml. of sample. It consists of a 0.2-ml. micropipet, connected to an all-metal needle valve of the Hershberg-Southworth type (id), which in turn is attached to a 30-ml. all-glass hypodermic svnnee. The Diston of the syringe is attached to the cylinder by ._j
....~.
~~
~~~~~
sample solution, which isyhhen dra&twnup t o a'l&el ab'ave the zero mark. The valve is quickly olosed and the solution is brought to the zero mark by opening the valve. One drop of the solution is placed on the paper; then the valve is closed. After the solution has evaporated on the paper, another drop is added until the required quantity of sample solution has been delivered. (When the solution was evaporated rapidly by application of hest to the paper, caking occurred and consequently a poor chromatogram was obtained.) METHOD
The sample is applied to the strip and ethyl acetate mturated with water is used as the mobile phase. I n approximately 3 hours a t 25' C . the solvent front moves 16 inches. At the end of that time, the paper strip is carefully removed, air dried, and cut into 1-inch sections. Each of these sections is assayed by a modification of the colorimetric method of Arnold, SchreBer, and Lipsius (1)as follows: Figure 2. Apparatus for Delivery of Sample
Shepherd (13) for the quantitative separation of sulfonamide8 (Figure 1). The setup described hy Winsten (16) might he used with equnllv good results. The apparatus consists of a %inch borosilicate glass tube, about 22 inches (55 em.) long, olosed a t one end. The other end is joined t o a 6inch borosilicate glass flange with ground surface. The solvent is contained in B 100-ml. hpnker, with its rim removed and with one Bide flattened. .4 miomscope slide is attached t o the flattened side of the beaker by means of a wire. Tx-o such beakers, each supporting R filter paper strip, can be used d h one tube, with the beakers standing on the flange a t opposite sides of the tube. About 50 ml. of et,hyl acetate, saturated with water, are placed in the bottom of the tube. The same solvent is used in the beakers. T h e ehromatoeram is develoDed on Whatman filter paper No. 1, cut into strips i f 1.25 X 22 hehes. Exactly 0.1 or 0.2 ml. of a m p l e solution is delivered along a horizontal line, marked with pencil on the paper a t a point about 3 inches from one end, and this end of the paper strip is immersed in the beaker
A I-inch section of the paper strip is placed in an amber 25-ml. glass-stoppered graduate. One milliliter of the phosphate huffer is added followed by 4 ml. of water. The graduate is plaeed in 8. water bath regulated a t 80"C. for 10minutes. Three milliliters of the cyanogen bromide reagent are added and the graduate is reheated in the 80' C. bath for exactly 5 minutes. The graduate is removed from the bath and cooled for 1 minute in a blast of air from a fan, then placed in an ice water bath for about 1 minute until the temperature of the solutipn is 20' to 22' C. pAminoacetophenone reagent IS added (0.5 ml.), the contents are well mixed, and 0.6 ml. of 3 N hydroohloric acid i s added. After thorough shaking, the graduate is allowed to stand in the dark for 15 minutes. Thirteen milliliters of ethyl acetate are added from a buret and the graduate is agitated in a mechanical shaker for 7 minutes. The contents of the graduate are transferred to a glass centrifuge tube equipped with a cork stopper. The tube is centrifuged a t 2500 r.p.m. for 2 minutes. The ethyl acetate layer is carefully decanted into a suitable colorimeter tube and the absorbance is determined a t 420 mw in a photoelectric colorimeter. Simultaneously a blank and a standard are run. T h e blank contains d l the reagents. The standard consists of 3 ml. of niacin standard solution, 1 ml. of phosphate buffer, and 1 ml. of water. tieltt,ed in the same manner as the sample.
ANALYTICAL CHEMISTRY
770
Experimental. In order to determine the rate of flow ( R j ) values of pure niacin and niacinamide, 60 micrograms of niacin were placed on the paper strip. All of the niacin was present on the first inch of the paper. The rate of flow was calculated as 0.10. When 450 micrograms of niacinamide were chroniatographed on another strip, all of the niacinamide was piesent within the fifth to the seventh inch (Figure 3). Its rate of flow value was calculated as G.37.
INCWES FROY STARTINO POINT O f OHROYATOORAY
Figure 3.
Chromatograms of Niacin (left) iand Niacinamide ( r i g h t )
levels 4.9, 5.8, and 6.6, the R/ of the niacinamide appeared to be constant a t 0.37. -4s added proof that the &st inch of the strip contained all of the niacin added, a niicrobiological assay, using R. leuconostoc rtiesenteroides, was run on duplicate chromatograms. It was found that all of the niacin was present Tyithin the first inch of the paper. Solutions were prepared containing 5 mg. of thiamin hydrochloride, 3 mg. of riboflavin, 3 mg. of pyridoxine hyd.rochloride, 3 mg. of panthenol, 50 mg. of ascorbic acid, 50 mg. of niacinamide and 5 nig. of niacin per in]. These solutions mere buffered a t various pH levels. When chromatographed in the manner described previously, only fair separation was obtained at a p€I below 4, while at pH 5 separation xws almost complete and a t pII levels above 5 niaciii could be completely separated froni niacinamide (Figure 7 ) . Therefore, in sulxcquent determinations a sodium acetate-acetic acid buffer of pH 6.0 was added to unknown multivitamin solutions beforr application to the chroniatographic paper. JI-heii determilling t,he niacin content in niultivitaniiri prepnutions, aliquots of the same suniple solution should be applied to t KO separate strips and allowed to develop simultaneously. The same section used for the assay from one of the strips should be used as a blank for the second st'rip. This blank is run in the same manner as described for the sample except that the cynnogen bromide solution is omitt,ed, and inst'ead, 3 ml. of aatcr are substituted. STABILITY OF NIACINAMIDE
JIikkelsen (II), and later Meier (9), investigated the stability of solutions of pure niacinamide under various pH conditions. Using a direct titration with the addition of formaldehyde to bind any liberated ammonia, these authors found no hydrolysis in ampoules at pH 5 to 7 and only 0.3% a t pH 8 after storage for 2 years.
?a
INCUES FROY STARTIN0 POINT OF CHROYATOORAY
Figure 4. Separation of Niacin from Niacinamide from Solution of Equal Quantities of Each Vitamin
Experiments were conducted with solutions containing equal quantities of each vitamin (50 micrograms of each, Figure 4), and also 1 part of niacin in 100 parts of niacinamide (10 micrograms of niacin and 1 mg. of niacinamide, Figure 5 ) . When these solutions were chromatographed, good separation and quantitative recovery of niacin were obtained. One division of the galvanometer reading shown in Figure 4 was equivalent to 1 microgram of niacin. When U.S.P. reference standard niacinamide was used as a standard, one division of the galvanometer reading was equivalent to approximately 2 micrograms of niacinamide. pH EFFECT ON SEPARATION
Solutions containing 1part of niacin to 10 parts of niacinamide were buffered a t p H levels of 3.1, 4.1, 4.9,5.8, and 6.6 with 0.1 M acetic acid-sodium acetate. Aliquots of these solutions equivalent to 50 micrograms of niacin were chromatographed. It can be seen from Figure 6 that good separation and recovery of niacin were obtained at all pH levels. However, the Rj of niacinamide was 0.43 at pH 3.1, and 0.40 a t pH 4.1. At p€I
INCHES FROY STARTIN6 POINT OF GHROYATOQRAY
Figure 5. Separation of 1 Part of Niacin in 100 Parts of Niacinamide at pH 6.0
Using the previously described method of separations, the stability of niacinamide solutions at various conditions of pH and temperature was investigated. Five solutions, each containing 10 mg. of niacinamide per ml., were buffered to pH 3.1, 4.1, 5.0, 5.8, and 6.7, respectively, and sealed in ampoules. Four additional solutions containing, per millilter of solution, 10 mg of niacinamide, 5 mg. of thiamin hydrochloride, 3 mg. of riboflavin, 3 mg. of pyridoxine hydrochloride, 3 nig. of panthenol, and 50 mg. of ascorbic acid, and buffered to pH 3.0,4.0, 4.9,and 5.6, respectively, were also sealed in ampoules. The ampoules were sterilized at 122" C. for 30 minutes. One half of these sterilized ampoules were placed in an oven at 45' C., m-herem the remainder were kept a t room temperature. Initially and after 6 weeks of aging, each of the solutions \\-as chromatographed and the quan-
V O L U M E 23, NO. 5, M A Y 1 9 5 1
1 VI
it.
I
77 1 Table I.
Stability of Niacinamide Solutions
250
4
150
L
50
PH f3.1
:100
904
Solution containing 10 niacinamide per ml.
YA
M I
m g . of
Solution containing 10 mg. of niacinamide per ml. plus ascorbic acid and B-complex vitamins
YA
14.0 5.0 15.9
[ 6.8 3.0
Niacinamide Hydrolyzed to Niacin, % 6 weeks at 6 weeks at room temp. 4 5 O C.
