V O L U M E 2 3 , NO. 3, M A R C H 1 9 5 1 recoveries were good where hoth nic.otinamide and iiicotinic acid were added. In Table I11 a comparison is made of values for aliquots of the same rat tissues, using the present procedure and that of Dann and Handler (3). For additional comparison the values originally reported by Dann and Handler (3)are given, as well as those obtained by Singal et al. ( 1 2 ) using a microbiological procedure (13). I t is the finding of this laboratory that many factors enter into the amounts of nicotinic acid and its derivatives which are found in tissues. 4ge, the amount of tryptophan in the diet, and the strain of rat must be considered when comparing values reported from different laboratories. When the Dann and Handler method and the present procedure were used on the same tissues, higher values were obtained by the latter. The microbiological procedure would be expected t o give higher values, as the values obtained by the cyanogen bromide procedures do not include any reduced diphosphopyridine nucleotide present in tissues. The cyanogen bromide reaction does not take place where the acarbon in nicotinic acid is substituted (14). In spite of this, the values of Singal et al. fall in the same range. Excellent recoveries of nicotinic acid in egg albumin digest were obtained when only 3 micrograms were present (Table I ) . In other recovery experiments values on the order of 1 microgram of nicotinic acid per h a 1 aliquot were determined quantitatively with an accuracy of 98%. Table I1 shon-s that 0.45 microgram of nicotinamide added per aliquot was quantitatively recovered. These recoveries show the applicability of the procedure for the accurate determination of small amounts of nicotinic acid and its derivatives. TIigonelline was not converted into nicotinic acid by this procedure (11). Xicotinuric acid was not tried. Kicotinic acid has been estimated in various ingredients of a rat's diet Dextrin gave no nicotinic acid, zein contained 791 microgram< per
487 100 grams, while celluflour contained 825 micrograms per 100 grams. Using a diphosphopyridine nucleotide preparation obtained from the Schwarz Laboratories, Xew York, which wae rated a t a 60% diphosphopyridine nucleotide content, the authors found values of 58.2 and 59.1%. The procedure as described appears applicable, therefore, to the accurate determination of nicotinic acid plus diphosphopyridine n icleotidr in a \vide variety of plant and animal products. ACKNOWLEDGMENT
The authors wish t o express their appreciation for the grants obtained from the University Research Committee of the Cnivcrsity of Utah, and the Sugar Research Foundation. LITER4TURE CITED
Bandiei, E., Btochenz. J . , 33, 1130 (1939). Bandier, E., and Hald, Jens, Ibid., 33, 264 (1939). Dann, W.J., and Handler, P., J . Biol. Chem., 140, 201 (1941). Friedemann, T. E., and Barborka, C. J., Ibid., 138, 786 (1941). Harris, L. J., and Raymond, IV. D., Beochem. J., 33, 2037 (1939).
Kodicek, E., Ibid., 34, i 1 2 (1940). McIlvaine, J. C., J . Biol. Chem., 49, 183 11921). Martinek, R. G., Kirch, E. R., and JTebster, G. L., Ibzd., 149, 245 (1943).
Melnick, D., and Field, I%.,Ibid., 134, 1 (1940). Roggen, J. C., .l'edrrland. Tijdschr. G e n e e s h n d e . 85, 4603 -8 (7941); Chein. Z e n t r . , 1942, I, 1917.
Sarett, H. P., Perlzweig, W. A., and Levy, E. D., J . Btol. Chem., 135,483 (1940).
Singal, S. A , , Sydenstricker, V. P., and Littlejohn, J. h l . , I b t d . , 176, 1069 (1948).
Snell, E., and Wright, L. D., Ibid., 139, 675 (1941). Waisnian, H. A,, and Elvehjem, C. -I., IND. ENG.CHEM.,ANAL. ED.,13, 221 (1941). RECEIVED March 20, 1950.
Stable Isotope Dilution Method for Nicotinic Acid Determination N. R. TRENNER, R. W. WALKER, BYRON ARISON, AND CAROL T R U M B i C ' E R .yerck & Co., Znc., Rahway, .V. J . The application of the isotope dilution technique to the determination of nicotinic acid was developed because it became necessary to assay accurately for the nicotinic acid content of crude products known to contain substantial amounts of other compounds containing pyridine rings, and because a review of the literature revealed the existence of no analytical method which would not be seriously affected by the presence of such impurities. A stable isotope dilu-
E
VER since the discovery ( 4 ) of the vitamin activity of nicotinic acid in 1937, there has been considerable interest in
methods for its determination, especially in complex mixtures. As a result there has appeared in the literature a relatively large number of papers describing such assay methods. K O attempt is made here to review these methods, as adequate bil liographies are available in a number of places ( 1 , s ) . In general, these methods of determination may be divided into two types, microbiological and chemical. Microbiological methods are characterized by relatively good specificity, but are most difficult to carry out with any high degree of precision. A great many papers have been written describing chemical methods of assay, but in every case the fundamental reaction involved is the
tion method is described in which deuteronicotinic acid is used as the tracer and its dilution is determined by infrared spectrophotometry. The method provides an absolute assay for nicotinic acid in complex mixtures, and has the general significance for analytical chemistry of further illustrating the importance of the isotope dilution principle in the development of specific (absolute) assay methods, a field which has been neglected in the past.
same-that is, the Konig reaction (?',8)in which the pyridine ring is opened by reaction with cyanogen bromide to give a product which is capable of further reaction with a primary or secondary amine to give a yellow colored product, the amount of which may be determined spectrophotometrically. Because many pyridine ring-containing substances will give this same reaction, such a method cannot be very specific and this fact is amply borne out by experience ( 2 ) . Under these circumstances and because the materials for which assays were required were known to contain closely related pyridine-ring compounds (Bmethyl nicotinic acid), the authors again (11)turned to the method of isotope dilution as one which would not be subject to any of the above disadvantages.
488
ANALYTICAL CHEMISTRY
A detailed discussion of the general nature of the authors' particular methods of application of the stable isotope dilution principle has been presented elsewhere (IO, 11). Briefly, the present method of assay is based upon the addition of a known amount of deuteronicotinic acid to a known amount of the sample to be assayed, followed by esterification of the whole mixture and qualitative isolation of a pure specimen of the mixed (isotopically) methyl nicotinates. The analysis is completed by determination of the ratio of methyl protionicotinate to methyl deuteronicotinate in the isolated specimen by direct infrared spectrophotometry, In the course of developing this assay procedure, the solubility properties of nicotinic acid were so unfavorable that no suitable solvent could be found in which the necessary infrared spectrophotometric measurements could be made. Attention was then turned to the methyl ester as possessing more favorable solubility properties. This proved to be the case and it was found that an additional advantage accrued through its use in that it could be more readily purified than the acid, a property vital to the ultimate reliability of any isotope dilution assay (IO). The low melting point of methyl nicotinate, moreover, proved a sensitive criterion of purity of the isolated specimen on which the spectrophotometric measurement of isotope dilution was made. The method of isotope dilution, when applied for the purpose of chemical analysis, suffers from the disadvantage that it is necessary to isolate qualitatively a pure specimen of the substance for which assay is required. Generally, this can seldom be achieved with le- than approximately 1 mg. of the sought substance. As a consequence, the method becomes progressively more difficult to apply to solid specimens which contain only small amounts of the compound for which assay is sought, unless some simple and selective process is known whereby the latter may be concentrated in relatively pure form. In many instances, therefore, the application of the isotope dilution method will not be practical as a general analytical method for everyday use. It will, nevertheless, be the method of choice when it is of paramount importance to obtain a completely reliable assay or when it is essential to check the reliability of some other more convenient but less certain assay method. In other words, the need for reliability must be sufficiently great to justify the additional expenditure in time and effort which may be required in order to achieve isolation a t the necessary level. Thus, modern micromanipulation techniques are of considerable value in rendering more feasible the achievement of the desired microscale isolations. PREPARATIOS OF DEUTERONICOTIYIC ACID AND ITS METHYL ESTER
Deuterization of nicotinic acid was effected by direct exchange using deuterosulfuric acid similar to that used with benzene (6). Deuterosulfuric acid (8OOjo) was prepared in an all-glass, watercooled apparatus equipped with a magnetic stirrer, by the addi-
20 10
0
tion of the required amount of sulfur trioxide (Baker and Adamson Sulfan B) to 99.8% deuterium oxide (Stuart Oxygen Co.). Four grams of pure nicotinic acid and 25 ml. of 80 weight % pure deuterosulfuric acid in pure deuterium oxide were heated a t 300" C. in a sealed Carius tube for 90 to 120 hours. After cooling to room temperature, the Carius tube was opened, the contents were transferred with water washing to a 250-ml. Erlenmeyer flask immersed in an ice bath, and 60 ml. of 30y0 sodium hydroxide solution were added slowly with stirring. Finally, the pH was adjusted to between 2 and 3 and the aqueous solution was extracted continuously for 24 hours with 200 ml. of ethyl acetate. After evaporation of the ethyl acetate extract to dryness, the residue was picked up in a minimum volume of boiling water, charcoaled lightly t o remove color, filtered hot, and allowed t o crystallize on cooling. Recrystallization was effected by solution in a minimum volume of hot absolute ethyl alcohol, hot filtration, and cooling. In this manner an 83% yield of deuteronicotinic acid was obtained. The deuteronicotinic acid v-as characterized by the following properties: observed melting point 234' to 235.5' C.; melting point of nicotinic acid 234" to 236" C; composition of CaH2D302N1 analytically calculated: carbon, 57.2, nitrogen, 11.1; composition found: carbon, 57.6, nitrogen, 11.3; ultraviolet absorption spectrum in 95% ethyl alcohol: pure nicotinic acid, molecdar extinctitin coc4icient, E M , 2900 a t 2630 A , ; deuteronicotinic acid, EM 2920 a t 2630 A. The atom per cent of deuterium u a s determined by combustion of the deuteronicotinic acid to water, the deuterium content of which was determined by the falling drop method ( 6 ) . The results indicated that the deuteronicotinic acid contained 54.7 * 3 atom yo of deuterium as the average of two determinations. For three atoms of deuterium in nicotinic acid, the atom per cent would be 60; thus, on the average, three quarters of the total number of nonexchangeable hydrogens have been exchanged in the tracer material, which proved sufficient to give the analytically required discriminatory properties in the infrared absorption spectra of the two isotopic analogs. The preparation of the methyl ester was carried out by treating each ram of deuteronicotinic acid with 10 ml. of methanol and 1.5 of fuming sulfuric acid (20% free sulfur trioxide) followed by refluxing on a steam bath for 1 hour. After transferring the reaction mixture to a separator? funnel and adding 20 ml of a standard neutralizing reagent (115 ml. of 6N ammonium hydroxide and 60 g r a m of ammonium sulfate in a 200-ml. total volume), the whole was twice extracted with two 100-ml. portions of petroleum ether. The petroleum ether extracts, after combination, were concentrated t o about 20 ml. or less, charcoaled with about 0.5 gram of Darco G60, and filtered and washed with a little petrolew ether; the total volume of the filtrate did not exceed 28 ml. On chilling the petroleum ether filtrate to 0" C. for 2 hours, white crystals formed which were isolated by cold filtration and dried in vacuo a t room tem erature for 0.5 hour. The melting point should be 41' to 42 E7 C. If the melting point is lower, the crystallization procedure should be repeated.
d.
In this manner a 70% yield of pure methyl ester was obtained.
-
-
2.0
I 3.0
k
4.0
I 5.0
1
6.0
7.0
8.0
9.0
I
I
I
10.0
11.0
12.0
WAVE LEUQTH I N MICRONS
Figure 1. Methyl Nicotinate in Carbon Disulfide Solution
1 13.0
I 14.0
I 15:o
16.0
V O L U M E 23, NO. 3, M A R C H 1 9 5 1
489
3 0
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In addition to the melting point, the pioduct mas characterized by its ultraviolet absorption and n ’ a found to give result8 identical t o those observed with an authentic sample of pure methyl nicotinate--E.v 3020 a t 2630 -4. SPECTROPHOTOMETRIC DETERMINATION OF lSOTOPIC DILUTION
h superficial comparison of the spectra in Figures 1 and 2 reveals a situation almost ideal for the spectrophotometric determination of the individual isotopic analogs in mixtures of the two. The introduction of deuterium into the pyridine ring of methyl nicotinate profoundly changes its infrared absorption spectrum. Particular attention is called to 8.05, 0.75, 12.10, 13.50, and 14.27 mu bands of the protio analog and to the 4.50, 7.37, 8.12, 9.22, and 11.21 mu bands of the deutero analog, any one of which could l)e used to determine the ratio of methyl protionicotinate to methyl deuteronicotinate in a mixture of the two. The authors found the 13.50 mu band of methyl protionicotinate, a region in which the deutero analog is transparent. the most favorable for :tnalytical use and consequently all subsequent n-ork was rarried out a t this wave length. The spectrophotometric techniques used in this work have lieeii improved over t,hose reported in the authors’ earlier publication (11). As in the earlier m-ork, a carefully calibrated (9) .\lode1 12A Perkin-Elmer infrared spectrometer was used. All measurements were carried out with the instrument set a t 13.50 mu; the ylobar was operated with a poa.er input of 125 Tvvatts, an amplifier gain corresponding to 1 microvolt full scale on the standard F3roR.n recorder chart, and slits of 0.900 mm. A sealed liquid cell wit.h rock salt windom of 0.10-mm. path length was used for the unknowns solution. For a reference state the authors found the use of a 0.20-mm. w l l containing pure liquid bcnzenc brst. Such a system had close to the same transmittance a t 13.50 mu as the 0.10-nim. cell when it is filled {Yith a carbon disulfide solution at a total concentration of 50 mg. per ml. of a 50 to 50 mixture of methyl protionicotinate and methyl deuteronicotinate. The chief advantage of such a reference state is its complete reproducibility over long periods of time in contrast with a standard solution (50 mg. per ml. of a 50 to 50 mixture of the two isotopic nicotinates in carbon disulfide), n-hich may change its concmtration on storage and handling. Any changes in the constants of the reference cell employed in this work were guarded against by daily check of its apparent transmittance, when filled with benzene, relative to a selected 16-mesh wire screen. In similar manner the benzene-filled unknowns cell was checked against the benzene-filled reference cell. This checking procedure, based on a stable standard, benzene, constitutes in effect a check of the reliability of the established calibration curve; it was found entirely satisfactory over the long period of time during which this technique was tested by repeated calibration-curve checks with
I
I
I
I
I
I
1
solutions of known composition. The transmittance values were always determined in terms of a 0% line set with the lithium fluoride filter in the incident beam to minimize scattered radiation effects. The actual calibration curve was established by successively filling the unknowns cell (0.10 mm. in length) with carbon disulfide solutions of fixed total concentration (50 mg. per ml.), but of various known ratios of methyl protionicotinate and methyl deuteronicotinate, and determining their apparent absorbance relative to the benzene-filled reference cell (0.20 mm. in length). The data are given in Table I. The carbon disulfide solutions were always made up by weighing on a microbalance a known amount of the sample to be examined in a glass-stoppered Erlenmeyer microflask (about 2- to 3-ml. volume) to eliminate sublimation of the relatively volatile methyl nicotinates; then the required volume of carbon disulfide was added from a 1-m]. microburet calibrated in 0.001 ml. in order to give a solution of standard concentration-that is, 50 mg. per ml. of solvent. Solution was effected in the stoppered flask and could be checked for correct amount of solvent by back-weighing if necessary. The solution was introduced into the unknown cell by means of a hypodermic syringe; the cell was placed in the spectrometer and its transmission was measured under the spectrometer s e t tings indicated earlier. Immediately the unknown cell was replaced by the benzene-filled reference cell and its transmittance \vas also determined. This was repeated alternately until a satisfactorily constant transmission ratio (tranqmittance), within 0.006 unit, is ohtainetl. Table I.
Calibration Data for Methyl ProtionicotinateMethyl Deuteronicotinate Mixtures
Deutero Component, % ’ 33.6 45.4 59.8 68.5
Absorbance, 13.50 mu (Av. of 2 Detns.) -0,074 +0.004
+o. 102 +0.162
Generally only two such cell interchanges are required to eatablish a satisfactory ratio from which the deutero-protio composition of an unknown can be evaluated by means of the calibration curve or the calibration equation. Beer’s law is followed closely; the data in Table I may be represented by the foilon-ing straight-line equation: log R = (1 = 0.680 PT - 0.305 (1) where R is the ratio of the transmission of the unknown solution to the transmission of the benzene reference standard, d is the absorbance, and PT is the per cent of the deutero component in the sample under analysis. I n order to preserve maximum precision in an isotope dilution assay, it is desirable to add an amount of tracer to the unknown as nearly equivalent as possible to the amount of substance whose assay is sought (10, 1 1 ) ; thus, it is not essential to extend the calibration much beyond the composition limits given in Table I.
490
ANALYTICAL CHEMISTRY Table 11.
Spectrophotometric Reproducibility
Date
PT
12-22-49 12-23-49 1-3-50 1-17-50 1-20-50 2-2-50
Deviation
44.9 44.9 45.5 45.0 45.3 45.2 Av. 4 5 . 1
-0.3 -0.3 f0.3 -0.2 f0.l
0.0 10.2
ISOTOPE DILUTION ASSAY PROCEDURE
A specimen of the sample t o be assayed is weighed out exactly ( W 8 ) ;the size of the sample is such that it will contain roughly 100 mg. of nicotinic acid, to which are added about 100 mg. (exact weight W T )of the deuteronicotinic acid tracer. The mixture is placed in a small (2- to 3-ml.) round-bottomed flask, 1.5 ml. of methanol and 0.3 ml. fuming (207, free sulfur trioxide) sulfuric acid are added, and the whole is refluxed for 1 hour on the steam bath. After transferring the crude methyl nicotinates to a 26-ml. glass-stoppered cylinder (graduated), containing 4 ml. of the standard neutralizing reagent (115 ml. of 6 S ammonium hydroxide and 60 grams of ammonium sulfate in a 200-ml. total volume), 15 to 20 ml. of petroleum ether are added and the whole is brought to equilibrium by vigorous shaking. After separation of the phmes, a second 15- to 25-mI. portion of petroleum ether is added and the extraction is repeated. The combined petroleum ether extracts are evaporated to about 5 ml. or less. Fifty milligrams of Darco G-60 are added, the solution is filtered, and the charcoal is washed with a little petroleum ether, keeping the total filtrate volume below 7 ml. (evaporate if neces; sary). The methyl nicotinates are crystallized by chilling t o 0 C. for 2 hours, filtering cold, and drying under vacuum a t room temperature for 0.5 hour. The melting point must be sharp a t 41’ to 42” C.; if it is lower, the crystallization procedure must be repeated. R h e n a satisfactorily pure specimen has been isolated, as judged by melting point, the per cent of the deutero component in the sample, PT,is determined spectrophotometrically. This procedure works well with the kinds of specimens the authors have assayed. In special cases a modified isolation procedure may be required becawe of the nature of the specimen to be assayed, but the petroleum ether extraction step seems very effective in separating methyl nicotinate from other substances. Operating with the quantities indicated, yields varying between 50 and 100 mg. were obtained. In preparing the carbon disulfide solutions for infrared measurement, a 25.0-mg. portion was generally weighed out and dissolved in 0.500 ml. of the solvent. Thus, all the quantities are sufficiently large to render all manipulations comfortable. If smaller samples are used, the problem can easily be met through the use of microabsorption cells which are now available from the Perkin-Elmer Corp. These cells may be obtained with a path length of 3 mm. li-hich makes possible the use of solutions of only 1 mg. per ml. concentration and, moreover, less than 0.1 ml. of solution is required to fill them. Thus, samples as small as 0.1 mg. may be analyzed for tracer dilution; this would reduce the required sample size by at least a factor of 100, relative to the amounts the authors used. COMPUTATION OF RESULTS
Like any isotope dilution assay, and as described in detail in an earlier paper (11))the desired numerical result is computed from the following equation:
where F = weight per cent of nicotinic acid in sample PT = per cent of deutero component in sample W , = weight in milligrams of sample taken W r = weight in milligrams of tracer added t o W , Table I1 presents data illustrating the reproducibility of infrared spectrophotometric measurements as a function of time. All measurements were made with a standard mixture containing
45.2y0 methyl deuteronicotinate and 54.8y0 methyl protionicotinate. Table I11 presents data illustrating the reliability of the overall assay procedure including isolation. Known mixtures were assayed as though theywere unknownswith results as in Table 111. The data in Table I11 clearly reveal the absence in the isolation procedure of any significant degree of fractionation of the isotopic analogs. Table IV presents a complete summary of the results obtained using the techniques described herein with actual unknowns. 1111 these results led the authors to evaluate the average precision as a t least * 1%; inasmuch as precision and reliability are equal (11),the authors believe that the assays are within 1% of the absolute amount of nicotinic acid in the unknowns studied. DISCUSSIO\
The general factors which play a role in the spectrophotometric-isotope dilution assay technique described herein have been discussed in earlier publications (10, 11). Some of the generalities, only envisaged then, have now been successfully reduced t o practice, a justification of the broad scope claimed for this a p proach to the analysis of specific organic entities in complex media. ACKNOWLEDGMENT
The authors wish to express their sincere thanks to R. S . Boos and his colleagues for the ultimate analyses and to L. J. Wissow for his help in preparing some of the pyridine carboxylic acids used in this investigation.
Table 111.
Over-all Reproducibility P T , Found 44.8 48.7 33.7
PT, As Made U p 44.4 49.6 33.7 60.2
60.6
Deviation +0.4 -0.9
0.0 f0.4 -
______~
Table
IY.
Sample 1 2 3 4 5 6 7 8 9
Anal>sisof Crude Nicotinic Acids Sicotinio Acid, R7t.% ( F )
LITERATURE CITED Association of Vitamin Chemists, “Methods of Vitamin bssay.” p. 125, New York, Interscience Publishers, 1947. Brown, E. B . , Thomas, J. M., and Bina, ;i. F., J . Biol. Chem., 162, 221 (1946). Dann, W. J.,and Satterfield, G. H . . Biological Symposia, Vol. XII. “Estimation of the Vitamins,” Lancaster, Pa., Jaquea Cattell Press, 1947. Elvehjem, C. A,, Madden, R . J., Strong. F. B.I., and Wooley. D . IT., J . Am. Chem. SOC.,59, 1767 (1937). Innold. C . K . . Raisen. C. G.. and Wilson. C. L., J. Chem. Soc., 7936,915. Keston, A. S., Rittenberg, D . , and Schoenheimer, R . , J . Biol. Chem., 122,227 (1937). Konig, J . prakt. Chem., 69, 105 (1904). Ibid., 70, 19 (1904). Oetjen, R. A , , Kao, C. L . , and Randall, H. SI.,Rev. Sci. Inrlrumenta, 13, 515 (1942). Trenner, N. R., and Walker, R. IT., ANAL. CHEM.,21, 314 (1949) (Abstract). Trenner, N. R . , Walker, R . W.,A4rison,B., and B u b , R. P., Ibid., 21,285 (1949). ~
RECEIVED August 10, 1950.
