Determination of Vitamin B, (Thiamine) in Extracts and Concentrates Comparison of Biological and Chemical Methods R . A. BROWN, EVA HARTZLER, GAIL PEACOCK, AND A. D. EMMETT Research Laboratories, Parke, Davis & Company, Detroit, M i c h .
I
FIELD METHODS. I n Table I the data obtained in assaying 11 different samples of thiamine, with potencies ranging from 1100 to 65,000 micrograms per ml., are given. The concordance of the results for the corresponding samples assayed by the three methods is very good. With but one exception the data agree within * 9 per cent of the values of the U. S. P. method-the mean differences being 4.4 for the rat-curative method and 5.3 per cent for the colorimetric method. Table 11gives a summary of these three methods as applied to 76 samples, which include pure thiamine, elixirs, synthetic vitamin mixtures, wheat germ extracts, fortified liver extracts, and miscellaneous samples. The thiamine solutions and the TABLEI. COMPARISON OF c.s.P., MODIFIED SMITH-CURATIVE, gave the best results. The vitamin were AXD MODIFIEDMELNICK-FIELD METHODS,USING THIAMIKE made up of additional components of the vitamin B complex, SOLUTIONS Modified Modified Difference0 supplied either in crystalline form or as natural concentrates. Sample SmithMelnickSmith Yji$$kIn addition, some of them contained the fat-soluble vitamins No. U. 5. P. Curative Field y/ml. y/mL y/ml. % % A and D. The variations between the three methods with 42,200 1,100 1,100 1,140 o , ~ +3,5 these samples are somewhat higher than for pure thiamine 41,680 6,138 6,600 6,600 +; : $::! and elixirs, but still within the range of accuracy of the 6,600 6,600 6,606 37,050 42,650 11,889 10,998 11,367 -7.5 -4.4 biological assays. The correlation between the data for the 24,000 27,240 0.0 +13.5 37,370 24,000 +o.g $0.6 three methods in the case of the wheat germ and liver ex41,340 25,944 26,178 26,100 42,440 25,998 28,164 28,236 +7.2 2::; tracts is satisfactory. The miscellaneous samples, which 42,640 28,100 25,998 26,700 -7.5 44,490 55,812 57,230 58,632 +2.4 +5.1 included rice-bran concentrates, dried yeast, thiamine hydro42,450 60,000 64,980 63,978 1-8.3 5::; chloride tablets, and alcoholic solutions of vitamin B complex 42,610 64,863 60,000 62,508 -7.5 Mean (11).. . , .... .... 4.4 5.3 mixtures, show the greatest discrepancies. Expressed as difference in per cent of value obtained by U. S. P. method' From t,hese data it is evident that any one of these three methods will give satisfactory results. The Smith-curative method has a distinct advantage, however, over the U. S. P. procedure in that it is more rapid. I n turn, the chemical Description of the Methods method has an advantage over the two biological procedures BIOLOGICAL METHODS.The U. S. P. method was carried out as as to time' described in the U. S. Pharmacopoeia XI (IO). The rat-curative method was that described by Smith (9) and modified by Birch COMPARISON OF THE MODIFIED SM1TH-CURATrvE, RATand Harris (b), so as to carry 0.5 per cent of yeast. This small GROWTH,AND PIGEON-WEIGHT-MAINTENANCE METHODS. I n Table 111 data are presented for 10 samples of wheat amount of yeast greatly increased the number of rats which developed typical polyneuritis and at the same time it did not ingerm extract and Of yeast concentrates, bioassayed by fluence the response to treatment, The rat-growth method was three methods. The mean values obtained agree within essentially that described by Chase and Sherman (8). The pi10 to 15 per cent, but the rat-growth and pigeon-weightgeon-weight-maintenance method was that of Cowgill (4). CHEMICAL METHODS.The colorimetric method of Melnick maintenance methods gave slightly higher results than the and Field (8) as modified by Emmett, Peacock, and Brown (6) curative methods. This may be due to a partial deficiency was used. This was further improved by introducing three in the rat-growth and pigeon-weight-maintenance basal changes: separation of the colored reaction product on SuperFiltrol by centrifuging, eluting the color with 10 cc. of 95 per cent diets, which was probably corrected by B vitamin factors ethyl alcohol, and measuring the depth of color with the Evelyn colorimeter, using filter No. 520. The thiochrome method of HenSMITH-CURATIVE, AND MODIFIED MELNICK-FIELD TABLE 11. COMPARISON O F u. s. P., MODIFIED nessy and Cerecedo (6) was used METHODS along with the adoption of Mason and Williams' (7) very Range of Values of Thiamine Content Mean Differencea No. of Modified Modified Melnioksimple and efficient procedure Type of Sample Samples U. S. P. Smith-curative Melnick-Field Smith Field for the oxidation and extraction y/ml. r/ml. y/ml. % % of the thiochrome with isobutaThiamine nol, and measuring the fluoressolutions 11 1,10@-64,863 1,100-60,000 1,140-62,508 4.4 5.3 cence with the Pfaltz and Bauer Elixirs, 7 150-31,410 156-31,410 159-33,750 1.4 6.8 fluorophotometer. Vitamin T IS the purpose of this paper to correlate the data obtained during the past several years, in determining thiamine by six different procedures. The following comparisons have been made: 1. The modified Smith-curative and modified Melnick-Field methods with the U. S. P. method. 2. The rat-growth and pigeon-weight-maintenance methods with the modified Smith-curative method. 3. The thiochrome method of Hennessy and Cerecedo with the modified Melnick-Field method.
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''
Results and Discussion COMPARISON OF THE U. 8. P., MODIFIED SMITH - CURATIVE, AND
a
mixtures Wheat germ extracts Liver extracts fortified hliscellaneous Expressed as difference
11
9-13,140
24
90-201
14 9
8-11,499
87-216
6-11,328
87-231
17-26,667 1526,253 13-25,500 4243,778 51-37,562 48-39,072 in per cent of value obtained by U. S. P. method.
M O D I F I E DMELNICK494
7.3
10.5
8.8
12.6
4.1 13.5
7.9 14.6
August 15, 1943
ANALYTICAL EDITION
present in the wheat germ extracts and the yeast concentrates. With certain types of materials of low potency the curative methods are not suitable because of the inherent difficulty of giving large amounts of samples orally to polyneuritic animals. Obviously then, for this type of sample, it is necessary to use the rat-growth or the pigeon-weight-maintenance methods. COMPARISON OF THE MODIFIEDMELNICK-FIELDAND THIOCHROME METHODS.The values obtained with these two methods are given in Table IV. An inspection of the results shows a very good agreement. I n general, however, the values obtained by the thiochrome method were slightly lower than those found with the colorimetric method except in the case of the samples of thiamine (at the top of the table) which contained little or no interfering material. In the course of many assays, carried out in this laboratory by the colorimetric method, the authors have found the reproducibility of values obtained on any given sample to be very good, while with the thiochrome method this has not always been the case. With the thiochrome method a repeat assay will occasionally yield a value 15 to 20 per cent different from the original value for no apparent cause. Whether these variations are primarily due to faults in the chemical procedure or to errors incurred in the measurement of the fluorescence has not as yet been definitely determined. I n this laboratory, the colorimetric method has definitely proved to be the more reliable of the two. It is, however, less sensitive in that it requires a concentration of 2 to 3 micrograms per milliliter while the thiochrome method can be carried out on a concentration of as little as 0.05 microgram of thiamine per milliliter. Further, in the case of very potent samples, the base exchange step is unnecessary because the samples can be diluted to the point where interference by foreign material is negligible. However, in cases where the base exchange procedure must be employed, the thiochrome method has no advantage over the colorimetric procedure with respect to time. I n general the fluorometric method has been used much more extensively than the Melnick-Field method. The possibility of adapting the latter for the determination of
TABLE 111. COMPARISON OF MODIFIED SMITH-CURATIVE, GROWTH, AND PIGEON-WEIGHT-MAINTENASCE METHODS Sample N 0.
Thiamine Content PigeonweightRatmaingrowth tenance
Modified Smithcurative
-!/a.
Y/B.
Y/B.
Mean Differencea Weight mainGrowth tenance
70
%
$36.4 f8.7 $4.2 0.0 +20.0 +26.9 $15.4 $15.4 -16.7 14.4
$22.7 +13.0 0.0 +4.0 +8.0 -23.1 $3.9 4-15.4 +13.3 -16.7 12.0
0.0 $20.0 $10.0 0.0 0.0 $20.2 $16.8 0.0 fll.0 -19.8 9.8
0.0 $20.0 -10.8 +lO.l -25.0 +20.2 $5.3 0.0 $9.0 -25.0 12.5
Wheat Germ Extracts 95 647 93:337 94,287 93,567 95,507 93 967 95:197 95,797 92,716 94,907 Mean (10)
94,537 95,807 96,897 95,417 97,097 92,977 93,797 94,547 95,917 97.087 Mean
... 750 750 750 77 1 800 831 s55 900 900 999
...
...
...
Yeast Concentrate 750 750 900 900 825 669 771 849 800 600 999 999 999 900 900 900 999 981 800 750
...
...
0.0
Expressed as difference in per cent of value obtained by modified Smithcurative method.
TABLEIV.
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3,422 7,962 9,542 92,982 7,072 5,562 59,471 4,092 94,472-A 94,472-B 99,642 1,592
65,881 70,652 74,002 88.432 99,582
495
COMPARISON OF MODIFIEDMELNICK-FIELD AND THIOCHROME METHODS Modified MehickField
Sample Thiamine solutions
Elixir Wheat germ extract
Yeast
Liver extract fortified Vitamin mixture (ABCD)
Vitamin
99,592 5,572 98,482 Ration Mean (20)
complex
Thiamine Content Thiochrome
Differencea
y/mL
y/ml.
70
5,350 23,600 55,400 57,300 111,700 23 1 ’ 99 191 306 348 14.4
5,600 24,800 56,100 62,000 109,000 237 105 181 282 336 14.0
$4.7 f0.5 $1.3 +8.2 -2.4 +2.6 +6.1 -5.2 -7.8 -3.4 -2.8
2,450
2,290
-6.5
Y/B.
Y/Q.
8,200 8,200 8,100 8,900 4,010 3,900 3,900 4,000
7,150 7,750 7,400 9,000 3.880 3,810 3,630 3,640
.......
. . . .. . .
-12.8 -5.8 -8.6 fl.1 -3.4 -2.3 -6.9 -9.0 5.1
Expressed as difference in per cent of values obtained by modified Melnick-Field method. Q
thiamine in samples of low potency has not been thoroughly studied. The method has, however, recently been adapted to urine by Alexander and Levi (I), and should therefore be of particular interest to laboratories which are not equipped for measuring fluorescence.
Summary The choice of a method for the determination of thiamine will be governed, in the main, by the potency of the samples to be assayed. From the standpoint of pharmaceutical preparations, the modified Smith-curative method is preferable to the U. s. P., rat-growth, or pigeon-weight-maintenance methods because of the rapidity of obtaining results. However, if the samples are low in potency, it is necessary to employ the rat-growth or the pigeon-weight-maintenance method, because of the difficulty of administering large doses to a polyneuritic rat. Good agreement was found between the values obtained with the modified Melnick-Field and the thiochrome methods. The modified Melnick-Field method has proved to be the more reliable of the two, but the thiochrome method is more sensitive and is, therefore, the method of choice for samples of very low potency.
Literature Cited (1) Alexander, B., and Levi, J. E., J . Bid. Chem., 146,399 (1942). (2) Birch, T.W., and Harris, L. T., Biochem. J.,28, 602 (1934). (3) Chase, E.F.,and Sherman, H. C., J. Am. Chem. Soc., 53, 3506 (1931). (4) Cowgill, G. R., “Vitamin B Requirements of Man”, p. 33, New Haven, Yale University Press, 1934. (5) Emmett, A. D.,Peacock, Gail, and Brown, R. A,, J . Bid. Chem.. 135. 131 (1940). (6) Hennessy, D: J., and Cerecedo, L. R., J. Am. Chem. Soc., 61, 179 (1939). (7) Mison, H.L.,and Williams, R. D., J. Bwl. Chem., 146, 589 (1942). (8) Melnick, D.,and Field, H., Ibid., 127, 505,515. 531 (1939). (9) Smith, M. I., U. S. Pub. Health Service, Pub. Health Rept. 45, 116 (1930). (10) U. S. Pharmacopoeia, 11th decennial revision, 2nd supplement, p. 129,1939. \ - - - - ,
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P R E ~ N Tbefore E D the Division of Agricultural and Food Chemistry at the 105th Meeting of the AMERICANCHEMICAL SOCIETY, Detroit, Mich.
