INDUSTRIAL AND ENGINEERING CHEMISTRY
416 Table
1.
Results of Titrations
Mscrotitration
Reagents
MF Relative mean w
Q./ml. NatCOrHCl 1.1204 CIH&OOH-NaOH0.4483 NaCI-AgNO: 0.5771
deviation Pnrfs per 1000
t0.4 t0.2 t0.3
"r
Microtitration Relative
Q./cm. 0,014542 0.005813 0.007500
mean
deviation Parla pcr 1000 -1.0 r1.0 -1.2
L
Ml./cm. 0,012979 0.012967 0.012998
normal sodium chloride with 0.1 N silver nitrate were performed. The indicator for the macrotitrations was 5 d r o p of 0.0025 M dichlorofluorescein; for the microtitration, one drop of O.OOO1 M solution of the same compound. FERROUS SULFATE WITH PERMANQANATE. Attem ta were made to use the microburet in a titration of ferrous sul?ate with permanganate. The mercury was oxidized so rapidly that even a fleeting end point was never attained. The results of the foregoing titrations are summariaed in Table I. The values in the second column are the mean ratios of W , the weight of the standard solution taken, over u, the volume of reagent used in the macrotitration. The values in the fourth column are the mean ratios of W over L, the quantity of reagent used in the microtitration measured in centimeters and corrected as indicated by the calibration. The values in the last column were obtained by dividing the values in the fourth column by the respective values in the second column.
the volume of dilute aqueous solutio11 delivered per centimeter is independent of the nature of the solute. The slight discrepancy for the silver nitrate is probably due to the fact that the end point of this titration was not so sharp as that of the others. The values of vlL obtained by titration are appreciably smaller than the value obtained by calibration with mercury. This is due to the fact that water wets glass, whereas mercury does not. The dimensions cited for this microburet need not be followed closely for other burets of this type. A capillary of smaller bore can be used if a smaller capacity is desired. A shorter buret would be more convenient and would require less time for a titration, but would be less accurate. The advantages of this buret in comparison with that of Hybbinette and Benedetti-Pichler are the following: It can be used in the titration of alcoholic solutions; the tip is coarser and hence less fragile; and the tip is longer and can accommodate taller titration vessels. On the other hand, this buret fails in titrations with permanganate and probably wjth any other strong oxidizing agent. In common with the buret of Hybbinette and Benedetti-Pichler, it requires a long time for a titration because of the small velocity of the meniscus. ACKNOWLE GMENTS
Suggestions from Robert Munch and Stephen H. Laning regarding the calibration are gratefully acknowledged. LITERATURE CITED
DISCUSSION
The values for the relative mean deviation of the microtitrations (Table I) indicate that the precision of this microburet is satisfactory. From the values of v / L , it may be concluded that
Vol. 16, No. 7
(1) (2)
Benedetti-Pichler, A. A,, "Microtechnique of Inorganic Analysis'', p. 256, New York, John Wiley & Sons, 1942. Hybbinette, A. G., and Benedetti-Pichler, A . A., Mikrochemie ow. Mikrochim. Acto, 30, 15 (1942).
Thiamine Content of Pharmaceuticals Comparative Study of Rat-Curative, Thiochrome, and Fermentation Methods DOUGLAS 1. HENNESSY AND SAMUEL WAPNER, Department of Chemistry, Fordham University, AND JOSEPH TRUHLAR, Laboratory of Industrial Hygiene, N e w York, N. Y. With 99 pharmaceutical products, no marked differencer were obtained b y the rat-curative, thiochrome, and fermentation methods. The average for the thiochrome values was 95.4% of the rat-curative values, 83% of the samples being less for the thiochrome than for the rat-curative. The average for the fermentation values was 98.3% of the rat-curative values, 69% of the samples being less for the fermentation than for the rat-curative. The ranges of values were moderate and indicate that, for these products, any one of the three methods was satisfactory.
THE
importance of laboratory control of the vitamin content of foods and pharmaceuticals needs no emphasis. While great progress haa been made in developing practical analytical methods, uncertainties of precision and of accuracy still beset the analyst. I n this paper, a comparative study of the assay of one class of materials is presented. Results are shown for the thiamine determination of a number of pharmaceutical products, for which three methods, involving quite different principles and procedures, were used. Considerable effort haa been spent on the estimation of thiamine by several methods and by various modifications thereof. However, many of the publications describing these efforts do not bear on the present problem. This study is deliberately
limited to the use of the rat-curative bioassay (U. S. P. XII), the thiochrome and the fermentation assays. These have been previously used in comparative studies and the results were sufficiently encouraging to warrant their use in this investigation. Hennessy and Cerecedo (6) reported fair agreement between the results of thiochrome and rat-growth assays on identical samples. Frey and Hennessy (4) summarized the results of comparative assays on cereal products by a group of collaborators, each of whom used one or more of the three m a y procedures. Cole, Jones, and Christiansen @) compared the results of thiochrome and biological assay on a limited group of pharmaceuticals without noticing serious disagreement. Conner and Straub (3)assayed a few cereal,- and one vegetablr by both biological and thiochrome assay, N ith satisfying resuits. Lane Johnson, and Williams (8) reported the thiochrome and biologid assay results concordant except for cooked ork. The fermentation method was shown to give somewhat gigher rcsults on beef than the Hennessy procedure by Hinman, Halliday, and Brooks ( 7 ) . Biological assays were reported by Brown, Hamm, and Harrison ( 1 ) to give significantly higher valucs than the thiochrome procedure. EXPERIMENTAL
The methods of assay were used without any attempt to modify the procedures aa described in the literature.
ANALYTICAL EDITION
Jdy, 1944 Table 1.
Thiamine Determinations
r2:& Ct$&e
Fermentation
No.
Contant
1 220 U.S.P.units/g. 2 1.5 mg./oape.ule 1.O mp./capsule 3 333 U.S.P. units/capsule 4 1.0 mg./capsule 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1.5 mg./tablet 29 72 U.S.P.unitdml. ‘I For riulfite correction.
chrome Thio217 1.60 1.05 351 1.02 1.50 502 528 569 1000 00 55 331 550 117 1.56 515 62 58 130 363 533 347 373 90 343 61 1.60 82
259 1.72 1.21 382 1.25 1.78 519 614 591 1028 74 65 377 083 136 1.75 600 80 70 124 384 550 366 386 111 373 73 1.80 96
212 1.63 1.13 343 1.08 1.63 500 563 662 974 64 59 326 662 113 1.40 649 70 63 117 351 494 360 381 92 371 68 1.73 84
268 1.66 1.05 333 1.17 1.84 527 585 680 1180 59 59 350 684 123 1.62 558 64 54 130 370 522 370 400 87 346 63 1.70 87
471
The order of the products given in Table I follows no set plan. If arranged by type of product, thiamine content. etc., no regularities were apparent. The products teated were sixteen capsules, seven tablets, none chocolate-coated, five liquids, and one powder. I n order to evaluate the significance of the experimental results, i t is convenient to recalculate to a common basis. This is done in Tables I1 and 111,in which the results of the rat-curative and thiochrome assays are placed a t loo%, respectively, and the other data recalculated to this new basis. These tables are summarired in Table IV to show the comparative ranges of the results obtained by the ditrerent methods of assay. A number of relations are evident in these groupinga. With the curative a t loo%, the thiochrome results showed 24 products below loo%, 5 of them below 90%. The fermentation results showed 19 products below loo%, also with 5 below 90%. When the thiochrome results were set at loo%, the fermentation results showed 18 products above 100%. I n general the two hiological methods tend to give higher thiamine content than
Table II.
RAPCURATIVEBIOA~SAY. The method as described in the
U. S. Pharmacopoeia XI1 was used. N o difficulty was encountered in feeding a sufficient quantit of sample, since all products were of fairly high potency. $he assay was carried out a t a definite level in each individual case. To m i n u c e
errors due t o extrapolation, a scheme of interpolation was employed. This was made possible by running a curative series in which 5, 6, and 7 micrograms of thiamine chloride were fed to six groups of 10 polyneuritic rats each. It was found that the number of days in which 10 rats remained cured were: for 5 micrograms 82 days, for 6 micrograms 107 days, and for 7 micrograms 129 days. Since the unknown content did not differ from the assumed content by more than 21% in any case, the possible error due to interpolation could not be of significant ma nitude. THIOCHROM~ ASSAY. The method described by Hennessy and Cerecedo ( 5 )as modified by Hennessy (6) for routine analysis was followed. The maximum ratio of extracting solvent to sample and the minimum quantity of thiamine in the final aliquot, consistent with the necessary precision, were used. The sensitivity of the fluorophotometer permitted the measurement of 0.5 microgram of thiamine with a sensitivity of 0.005 microgram, so that this level of vitamin was approximated in the final aliquot in all assays. FERMENTATION A~SAY.The method as described by Schults, Atkin, and Frey (9) w a s followed, except that the larger apparatus described in their earlier papers was used. The sulfite correeCion was made in every determination. No unusual precautions were used in the assays; the customary care necessary in the analyses was observed. Duplicate determinations were made. If marked differences (greater than 10%) appeared in such duplicates, as happened occasionall the assay was repeated. f’wenty-nine pharmaceutical products, made up in various ways by different manufacturers and containing different ingredients, n ere tested. Included were capsule.; containing a number of the synthetic vitamins, fish liver oil concentrates, yeasts, liver and iron compounds, yeast and iron compounds, tablets containing synthetic vitamins, yeast concentrates, Solvamin and iron compounds, liquid reparations containing yeast extract, yeast concentrate, and mart extract. I n the following tables the products are listed by number and no statement is given as to their composition. Attention is focused entirely on the thiamine content. The values in the “Stated” column refer to the thiamine content of the products as stated by the manufacturer or on the label. These are generally 5 to 10% lower than the actual content because of the common practice of adding a slight excess to ensure adequate shelf life. The experimental values as given under the thiochrome, fermentation, and rat-curative headings were those found in the assays. Table I summarize- these values.
No 1
2 3
100 105 87 85 95 90 98 85 102 93 95 94 95 96 92 97 107 100 98 I02 94 93 103 99 97 94 94
4
5 6
7 8 9
10 11
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 2!4
Av.
95.4
Table 111. No. 1
2 3 4
a6 7
8 9 10 11
12 13 14 15 I6 17 18 19 20 21
22 23 24 25 26 27 28 20
-
Relative Thiamine Content
(Baaed on rat-curative Thiochrome 81 96
-
100 for each product) Fermentation Stated 79 82 90 98 95 108 100 103 92 86 81 89 95 9b 86 90 86 97 85 83 85 108 85 100 94 93 86 113 92 102 93 90 98 89 109 94 9a 117 85 90 90 95 96 95 90 97 88 9B 98 106 96 107 79 108 88 102 87 83 98 3 89.6
Relative Thiamine Content
(Rased on thiochrome Fermentation 98 102 108 98 106 105 100 107 99 97 107 107 99 120 97 94 107 113 109 90 95 93 104 102 102 108 112 109 102 Av. 102.9
100 for each produot) Rat-Curative Stated 124 104 100 95 115 118 105 111 102 118 98 107 106 106 105
104
108 103 93 100 102 98 107
107 97 101 103 106 106 105.1
101 94 95 95 98 96 100 95 88 100
83 91 100 91 107 96 97 97 86 86 90 94 97 94 94 97 82 94 88 94.0
INDUSTRIAL A N D ENGINEERING CHEMISTRY
478 Table IV. Percentage range8 of
No.
Comparison of Resulb by Three Methods of Assay
