Determination of Vitamin B, (Riboflavin) Comparison of Bioassay, Microbiological, and Fluorometric Methods A. D. EMMETT, 0. D. BIRD, R. A. BROWN, GAIL PEACOCK, AND J. M. VANDENBELT Research Laboratories, Parke, Davis and Company, Detroit, Mi&.
I n assaying an unknown, a t least three dilutions were made, preferably in the range of 0.1 to 0.2 microgram of riboflavin per cc. These were read against suitable standards and the potency was computed from the average values. ( d ) The photoelectric fluorescence measurements were made with the Pfaltz & Bauer fluorophotometer. In this instrument emission from a mercury capillary arc is filtered (Corning glmses 038 and 511) and directed through a solution of the riboflavin. The fluorescent light strikes a photocell a t the side of the cuvette and is transformed into electrical energy, the current of which is measured by a galvanometer. This photocell is protected from the incident intensity by Zeiss filter No. OG1. With a constant incident intensity, there is a linear relationship between the deflection of the galvanometer and the concentration of the riboflavin. In preparation of the sample, a 10-cc. portion of the riboflavin solution was diluted with twice the volume (20 cc.) of acetone as suggested by Hand ( 4 ) . The solution was agitated for a few seconds. If a precipitate formed the solution was filtered, the f i s t part of the filtrate being passed again through the filter. After suitable dilution, the fluorescence was then read directly in the cuvette, and the concentration of the riboflavin determined by reference to a standard curve prepared from dilutions of pure riboflavin. MICROBIOLOQICAL METHOD. This procedure w m carried out accordinz to the method of Snell and Strone (6). The erowth responseYof the bacteria was measured in t g i s of culture turbidity expressed as galvanometer extinction by means of the Pfaltz & Bauer fluorophotometer; and acidimetry, measuring the amount of acid produced in 72 hours' incubation. In this connection an innovation was introduced. The cultures were grown in carefully standardized 16 X 150 mm. test tubes which could be inserted directly into the light path of the fluorophotometer for extinction measurements at the end of 21 to 24 hours' incubation. Follom-ing this, the cultures were returned to the incubator to complete a 72-hour incubation, when the contents of each tube were removed and titrated against standard alkali. In this way both determinations were made on one and the same culture tube. Samples were prepared for assay by simple water dilution except in the case of dry yeasts. These were digested with the enzymes as outlined above. The standard riboflavin solution was run for each assay in duplicate a t five levels ranging from 0.05 to 0.15 microgram per 10-cc. culture tube. The unknowns were run in groups of 5 tubes covering this same range of dilutions. Response curves for the standard riboflavin were constructed from the turbidity values and from the acidimetric values. The unknowns were evaluated in terms of these and expressed as micrograms of flavin per gram.
T
HE increased interest in the possible sources as well as the biological properties and uses of vitamin Bz (riboflavin) has stimulated a desire for a more rapid method
of assay than the biological r a t growth procedure which is generally followed. At t h e present time, while there is no official biological method of assay, the one of choice is basically that of Bourquin and Sherman (1). This has been modified by different workers from time to time with the intent chiefly of improving upon the supplemental factors and thereby making i t more specific, One of the chief objections to the method is the time i t requires-at least 28 days-which not only entails the factor of cost but interferes markedly with the execution of many problems of practical interest. In contrast to the r a t growth method, two comparatively rapid procedures have been used to a limited degree for determining flavin. The first measures the degree of fluorescence given by flavin in violet light. This can be done with suitable filters using either a visual procedure with the Fluoray or similar type of instrument as described by Supplee and others ( 7 ) , or by photoelectric fluorescence measurement as developed by Cohen (2), and Euler (3) and adapted by Hand (4). The second method, the microbiological procedure of Snell and Strong (6), measures t h e influence of flavin on both the cell growth and the acid production of Lactobacillus casei E grown on a synthetic medium free of flavin. The present study is a report of progress with these methods as applied concurrently to several samples varying in type, composition, and potency.
~~
Methods BIOLOGICAL METHOD. The ration consisted of purified casein 18 per cent, sucrose 68 per cent, Crisco 9 per cent, McCollum salt mixture No. 185 4 per cent, and cod liver oil 1 per cent. This was supplemented with daily treatment of 16 micrograms of thiamine hydrochloride and 0.08 gram of Labco rice polish extract freed from flavin with fuller's earth. The rats a t the start were 22 to 25 days of age and weighed 40 to 50 grams. The depletion period ranged from 2 to 3 weeks. The test eriod was 4 weeks. Concurrently with the unknown samples, f e f a t different levels, 3 groups of rats were treated daily with 2.5 , 3.75, and 5.0 micrograms of riboflavin, respectively. These groups served as standard controls and permitted one to calculate the potency in terms of riboflavin. FLUORESCENCE METHODS. (a) In cases where the riboflavin might have been in partial combination, the sample w&sdigested as follows: five grams were suspended in 100 cc. of water, about 0.1 gram each of papain and of standard takadiastase were added, and the mixture was digested at 37"to 40" C. for 2 hours. Then 0.2 cc. of glacial acetic acid was added. The whole was heated to 80" C. to coagulate the protein and then filtered or centrifuged. The readings were made on the clear solution. (b) If the preparation was too dark, decolorizing was carried out by the method of Kemmerer (5)which is a modification of that of Van Eekelen and Emmerie (8). This consists of making a methanol extract, adding a few cubic centimeters of glacial acetic acid, filtering if necessary, adding a few drops of saturated solution potassium permanganate, and then a little fresh hydrogen peroxide. The fluorescence was measured on this decolorized final solution. I n some cases it was necessary to introduce adsorption and elution steps, making the determination of the riboflavin on the eluate. (c) For the visual reading the Fluoray Eveready carbon lamp was employed, fitted with a red-purple Corning filter No. 587 and a Corning a t e r No. 351 to screen out interfering blue fluorescence.
Discussion In Table I the results'are given as micrograms of riboflavin per gram of sample for the four procedures-biological rat growth, microbiological, fluorophotometric, and Fluoray . The type of samples begins at the top with relatively lowpotency extracts of grain which are rich in sugars, followed by samples of dried yeast and extracts of the same; by liver extracts, plain and fortified with other vitamins; by high concentrates; and finally by blends of the more potent types with the low ones, high in sugar. Observing these data for the various groups, the respective assay values for the four methods show a general tendency toward agreement. The largest differences occur, as one might expect, in the samples that are lowest in potency. This is brought out by comparing the values by the respective methods with the average of data by all the methods, given in the last column. If the average deviations from the mean are calculeted for the different methods, that for the biological 219
TABLEI. COMPARATIVE POTENCIES, DETERMINED BY DIFFERENT METHODS (Expressed in micrograms of riboflavin per gram) Sample Number 38,540 40,480 39,590 35,120 36,950 34,900 38,790 33,509 39,140 33,139 20,879 23,659 40,360 34,880 37,630 40,160
Average of Biohiicro- FluoroAll logibiologi- photoFluoMethDescription cal cal metric ods ray Wheat germ extract 2.7 3.7 4.5 3.2 3.5 Rice polish extract 9 7.1 10.9 6.4 8.4 Rice bran extract 9 10 8.5 6.5 8.5 Yeast, dried 62 46 51 65 56 Yeast, dried 57 50.5 54 48 52.4 Yeast concentrate 200 171 192 195 189.5 Liver extract, dried 208 195 246 230 220 Liver extract, dried 230 204 234 225 223 Liver extract, dried 285 207 226 300 254.5 500 501 507 537 511 Liver extract, dried Liver extract, fortified 250 277 244 302 268 241 216 244 Liver extract, fortified 250 269 Liver extract, fortified 682 670 685 727 691 Special concentrate 4690 4610 4800 4900 4750 Special concentrate 4685 5260 5470 4680 5024
40,170 40,420
40.430
Vol. 13, No. 4
INDUSTRIAL AND ENGINEERING CHEMISTRY
220
with wheat germ Yeast conccntrate with wheat germ
942
940
965
934
945
317
312
347
340
329
102
88
85
89
91
135
114
109
134
123
assay is lowest, about 8 per cent. The other three are very similar, 10 t o 12 per cent from the mean. I n Table I1 the microbiological, fluorophotometric, and Fluoray results in Table I are expressed as per cent difference (plus or minus) from the corresponding biological values. While the biological rat growth method is generally considered the standard criterion, such resiilts in themselves are not necessarily correct to within 10 to 15 per cent. Nevertheless it is of interest to make the comparison. It is seen that there are some rather wide variations which in general occur with the low-potency samples. The averages of the per cent differences (including sign) are, respectively, - 10.7, +5.3, and - 7.0 by the microbiological, fluorophotometric, and Fluoray methods. The question then arises as to what advantage, if any, one of these three methods has over the other, granting that they all require much less time than the biological method. I n general, with the fluorometric measurement, it is often difficult to know how and when to treat the samples so as to obtain 6he optimum value. As a result, there is entailed considerable preliminary experimentation by trial and error. I n addition, with the visual (Fluoray) procedure there is apt to be a large personal error unless the operator has had much experience. With the photoelectric procedure, there are also objections due t o possible solarizing ?r cell fatigue. This entails a frequent careful checkup and restandardization of the instrument before each series of runs. Since the microbiological method of Snell and Strong seemed to possess certain advantages, it was tested out further as to comparison of results obtained by turbidity extinction with those by acidimetry, reproducibility, and specificity in relation to the influence of other water-soluble vitamins. The data for the comparison of extinction and acidimetric determinations are given in Table I11 for the same samples that were reported on in Tables I and 11. As stated previously, the extinction values represent the culture turbidity formed in 24 hours' incubation and the acidimetric values represent acidity produced during 72 hours' incubation. The two sets of data show remarkable agreement throughout, irrespective of type or degree of potency of the samples. This fact is brought out clearly in the last column, giving the ratio of the two corresponding values, the average (19 samples) be-
ing 0.981. This shows that under the conditions as outlined, either procedure gives the same end results. The reproducibility of the method was tried on a liver extract (sample 39,140). I n all, fifteen separate runs were made. The data are given in Table I V and express extinction values. The micrograms of riboflavin per gram (column 2) ran very close, averaging 207, with an over-all range of 8.3 per cent and an average difference from the mean of only 2.5
TABLE 11. DEVIATION FROM BIOASSAY VALUES
Sample Number
Description
Difference from Bioassay Value Micro- Fluorobiologi- photom- Fluocal eter ray
Bioassay
%
%
%
-28.9 -21.2 -15.0 -25.8 -11.4 -14.5
-40.0
-17.8 -28.9 -35.0 4.8 -15.7 - 2.5
Iro./g.
38,540 Wheat germ extract 40,480 Rice polish extract 39,590 Rice bran extract 35.120 Yeast, dried 36,950 Yeast, dried 34,900 Yeast concentrate 38,790 Liver extract, dried 33,509 Liver extract, dried 39,140 Liver extract, dried 33,130 Liver extract, dried 20,879 Liver extract, fortified 23,659 Liver extract, fortified 40,360 Liver extract, fortified
4.5
9.0
10 62 57 200 208 230 285 500 250 250 682 4690 4685
- 6.3
-10.0
-17.8 -
5.3 4.0 $18.0 1.5 -20.7 1.4
-
+ + ++ +
-11.2 -29.8 0.2 +10.8 +20.8 3.6 6.5 1.8 0.5 1.9 2.4 f 1 2 . 3 +16.7
+
34,880 Special concentrate 37,630 Special concentrate 40,160 Special concentrate with wheat germ 942 40,170 Special concentrate with wheat germ 317 40,420 Yeast concentrate with wheat germ 102 40.430 Yeast concentrate with wheat germ 135 Average of 19, including sign
+21.1
-
-
0.3 1.6
-13.8 -15.6 10.7
-
+
+10.6 2.0 5.3
++- 7 . 5 -
2.4
+-13.6 6.6 +- 40 .. 25
+ 2.4 - 0.8 + 9.4 + 7 . 3 -16.7 -19.9
-12.8
-
0.7
+ 5.3 - 7.0
per cent. These values indicate that the method gives good reproducibility. In respect to specificity, Snell and Strong (6) reported that such substances as copper, manganese, iron, zinc, barium, pyridine, and alcohol did not inhibit the growth of the Lactobacillus casei. The question arose as to the specificity of the organism for riboflavin in the presence of the other water-soluble vitamins-that is, whether they might act as accelerators or inhibitors. The findings are given in Table
OF EXTINCTION AND ACIDIMETRIC TABLE111. COMPARISON RESULTS OF MICROBIOLOGICAL METHOD
Sample Number
38,540 40,480 39,590 35,120 36,950 34,900 38,790 33,509 39,140 33,139 20,879 23,669 40,360 34,880 37.630 40,160 40,170 40,420
40,430
Description Wheat germ extract Rice olish extract R'ice ran extract Yeast, dried Yeast, dried Yeast concentrate Liver extract dried Liver extract' dried Liver extract: dried Liver extract, dried Liver extract, fortified Liver extract fortified Liver extract: fortified Special concentrate Special concentrate Special concentrate with wheat germ p e c i a l concentrate with wheat germ east concentrate with wheat germ Yeast concentrate with wheat germ
g
Extinction,
Acidimetry,
Ratio
I I1 II/I Microgramr p e r gram 1.03 3.2 3.3 7.1 7 . 1 1.00 8 . 2 0.96 8.5 46 44 0.96 0.99 50.5 50 0.95 171 162 194 1.00 195 204 207 501 277 241 670 4610 5260
183 210 431 275 239 673 4520 6270
0.90 1.02 0.86 0.99 0.99 1.00
940 312 88 114
954 310 90 113
1.02
Av.
0.98
1.00
0.99 1.02
0.99 0.981
April 15, 1941
ANALYTICAL EDITION
TABLEIv. REPRODUCIBILITY OF MICROBIOLOGICdL METHOD FOR RIBOPLAVIN Test 1
(Liver extract 39,140) Micrograms Difference per G r a m from Mean 207 0
Per Cent Difference 0.0
2.4 3.9 0.5 1.0 2.9 3.4 3.0 0.0
16
11 12 13 14 15
Av. (15)
198 199 207 206 211 202 207
-9
-5 0 -1
t;
4.4 3.9 0.0 0.5 1.9 2.4 2.5
221
by both culture turbidity and acidimetry. The four methods gave similar results; the greatest differences were with the low-potency samples. I n a further study the microbiological method showed excellent specificity and reproducibility. The results were almost identical whether measured as culture turbidity a t the end of 24 hours' or acidimetry a t the end of 72 hours' incubation. TABLE V. SPECIFICITYOF MICROBIOLOGICAL METHODFOR RIBOFLAVIN (Influence of other vitamins on galvanometer extinction) 0.1 fig. r Factor Added Added of RiboNice- PantoAsper 105itvin ThiPyritinic thenic corbio Cc.Tube Alone amine doxine acid acid acid
All
.
combined
Pg.
V as galvanometer extinctions for 0.1 microgram of riboflavin per 10-cc. tube, first alone as control, and successively when combined with varying concentrations of thiamine, pyridoxine, nicotinic acid, pantothenic acid, ascorbic acid, and finally a combination of all six vitamins. The values represent extinction readings for individual tubes. Since they do not vary in any case more than * 10 per cent from the readings for the tubes containing riboflavin alone, it is evident that the method measured only riboflavin and that the supplemental vitamins had no stimulating or retarding effect.
Summary A comparison has been made of four methods for determining vitamin B2 (riboflavin) : biological rat growth; visual fluorescence ; photoelectric fluorescence; and microbiological,
0.1 1 10 100 1000
0.52 0.52 0.53 0.52 0.53
0.55 0.55 0.54 0.53 0.53
0.53 0.51 0.53 0.53 0.52
0.52 0.53 0.51 0.53 0.53
0.54 0.51 0.53 0.53 0.52
0.54 0.52 0.49 0.54 0.57
0.53 0.50 0.53 0.50 0.56
Literature Cited (1) Bourquin, A., and Sherman, H. C., J . A m . Chem. SOC.,53, 3501 (1931). (2) Cohen, F. H., Rec. trav. chim., 54, 133 (1935). (3) Euler, H. von, and Adler, E., Z . physiol Chem., 223, 105 (1934). (4) Hand, D. B., IND.ENG.CHEM.,Anal. Ed., 1 1 , 306 (1939). ( 5 ) Kemmerer, A. R., J. Assoc. Oficial Agr. Chem., 23,346 (1940).
(6) Snell, E. E., and Strong, F. M., IND.ENQ.CHEM.,Anal. Ed., 11, 347 (1939) ; Strong, personal communication. (7) Supplee, G. C., et al., J. Dairy Sci., 19, 215 (1936); IND.ENQ. CHEM.,Anal. Ed., 11, 495 (1939). (8) Van Eekelen and Emmerie, Acta Brevia Neerland. Physwl. Pharmacol. Microbial., 6, No. 9-10, 136 (1936).
Chemical Estimation of Nicotinic Acid and Vitamin B6 HARRY A. WAISMAN AND C. A. ELVEHJEM University of Wisconsin, Rladison, Wis.
T
HE chemical methods now available for the determina-
tion of nicotinic acid depend upon the pyridine ring structure. It is well known that the pyridine ring is more reactive than the benzene ring and this greater reactivity is due to the nitrogen in the pyridine ring. The proposed methods depend on the breakdown of the pyridine nucleus with the liberation of nitrogen, thus opening the ring, followed by conjugation of the carbon chain with some aromatic amine. A number of compounds have been used to react with the tertiary nitrogen for the purpose of splitting the pyridine ring. Vongerichten (2%')and Reitzenstein (15) first proposed 2,4-dinitrochlorobenzene as the compound to add to the nitrogen. Konig (9) used cyanogen bromide, phosphorus trichloride, and other compounds. Once the nitrogen has its greatest valence satisfied by the addition of any one of these compounds, the ring is more susceptible to decomposition. The ring is easily broken with the liberation of the nitrogen in the form of a tertiary amine, thus yielding the carbon skeleton that combines with the amine to give a stable colored compound. The skeleton of the ring is a derivative of glu-
taconic aldehyde, which readily enolizes to give a reactive hydroxyl which can then combine with the organic base. The di-ani1 reaction illustrated
C NNH2
