378
INDUSTRIAL AND ENGINEERING CHEMISTRY
were, respectively, 0.0263, 0.0138, 0.0007, and 0,0000 gram. Thus without undue haste on the part of the analyst, the copper and zinc content of a brass-plating bath (or the copper-zinc ratio of a brass plate) can be readily determined in 45 minutes. Perhaps the greatest disadvantage encountered is that some hydrocyanic acid is evolved during the electrolysis. However, experience has shown that if the electrolytic machines are placed in or close to a hood in a well-ventilated room, the method presents no hazard from cyanide fumes. The precision and accuracy of the method are satisfactory. The average weights of copper and zinc for twenty-five 10-ml. samples of standard brass solution were 0.2210 and 0.0169 gram, respectively, and the average deviation was *0.0003 gram for both copper and zinc. The solution was standardized by the methods of Miceli and Larson ( 1 ) and was found to contain 0.2211 gram of copper and 0.0167 gram of zinc per 10 ml.
Vol. 17, No. 6
While the method is hardly an umpire method, it has been found entirely satisfactory for production and experimental control work. The simplicity of the technique involved is also of benefit, especially when the analyses are turned over to a new operator. ACKNOWLEDGMENTS
The authors are grateful to V. F. Felicetta and C. A. Ihrcke for their contributions to the procedure during its many months of use. Thanks are also extended to the United States Rubber Company for permission to publish the work. LITERATURE CITED (1)
Miceli, A. S., and Larson, I. O., IXD.ESG.C m x . , ANAL.ED.,16, 165 (1944).
(2) Verdin, F. I., Zavodslcaya Lab., 10,648 (1941).
Standards in Vitamin A Assays U.S.P. Reference Cod Liver Oil vs. Beta-Carotene ELIZABETH CROFTS CALLISON AND ELSA ORENT-KEILES U. S. Department of Agriculture, Washington, D. C.
Bureau of Human Nutrition and H o m e Economics, Evidence from both biological and spectrophotometric data is presented showing that U.S.P. reference cod liver oil N o . 2 contains less than 1700 it,ternational units of vitamin A per gram and therefore that the U.S.P. vitamin A unit cannot b e considered identical with the international unit for that vitamin. As a result of this discrepancy, assays of vitamin A value performed b y bioassay using U.S.P. reference cod liver oil N o . 2 as the standard and expressed in terms of international units may b e from 30 to 44% higher than the actual value. Data are also presented showing the instability of vitamin A as it occurs in U.S.P. reference cod liver oil N o . 2. Pure p-carotene may b e used as a standard for the bioassay of vitamin A until a more stable and satisfactory standard i s developed.
IK
COXSECTIOK with a study of the utilization of carotene, in which the vitamin -1value of a number of samples of carrots was determined by both spectrophotometric and biological methods, evidence has been accumulated indicating that U.S.P. reference cod liver oil KO.2 is lower in vitamin .4 potency than 1700 international units per gram and thus that the U.S.P. and international vitamin A units are not "identical". -4s a result of this discrepancy, vitamin -4values determined by the bioassay method, using U.S.P. reference oil No. 2 as a standard and expressed in terms of international units, appear higher than they are in reality. BIOASSAY DATA
The hiological assays were conducted in accordance with the method outlined in the U. S. Pharmacopoeia XI, a total of 324 animals being used in the experiments. Both male and female rats from the authors' stock colony were used and the groups of animals to be compared were composed of equal numbers of sexlitter mates. The laboratory in which the animals were kept was maintained at 78" to 82" F. (25.56" to 27.78" (2.). One reference group of animals received U.S.P. reference oil KO. 2 in an amount providing 14 units of vitamin A per week, basing calculations on the value assigned to this oil of 1700 units per gram. I t was planned that a second reference group should receive @-carotene (pure &carotene, S l I A Corp.) equivalent to the cod liver oil in vitamin A value, considering 0.6 microgram of p-carotene as equivalent t o 1 international unit. As soon as it became apparent that there was a considerable difference between the growth rates of the animals receiving cod liver oil and those receiving 6-carotene, the 5-carotene content of
the solutions used for feeding was determined spectrophotometrically. An aliquot of 0.5 cc. of the B-carotene solution as diluted for feeding was weighed and diluted to 25 cc. with a 1to 1 mixture of purified Skellysolve, fraction C, and ethyl alcohol. The absorptions at wave lengths 430, 450, and 480 mp were read by means of a photoelectric spectrophotometer, using a Hilger double monochromator, and absorbencies of an equal amount of cottonseed oil measured a t the same wave lengths subtracted from these readings. The average ratios between the absorptions at 430 and 450 mp and at 480 and 450 mp were 73.6 and 88.170, respectively. These ratios indicate that some small amount of impurity was present in the supposedly pure p-carotene and point to the necessity for further purification of even the best commercial preparations. Calculation of the amount of p-carotene present in each solution was based on the average extinction coefficient for @-carotene of 0.2487. This coefficient has frequently been obtained with these same solvents in the laboratory of the Bureau of Dairy Industry, the highest extinction coefficient for pure @-carotenein petroleum ether-ethyl alcohol obtained in this laboratory being 0.2455. The average value thus obtained for the series of solutions was 25.6 micrograms of @-carotene per gram of solution, which is equivalent to 23.4 micrograms per cc., using 0.915 as t'he specific gravity of the cottonseed oil a t the temperature of the experiment room. This value represents the maximum amount of p-carotene present in the solutions and has been used in calculating the p-carotene intake of the experimental animals (Table I). The U.S.P. reference oil was diluted prior to feeding each week, using cottonseed oil containing 0.01 yo hydroquinone as the diluent. KO sample of reference oil was used beyond the expiration date marked on the bottle. The concentrated standard solutions of @-carotenein cottonseed oil were diluted at' weekly intervals for feeding. The solutions were stored in the dark a t a temperature below 0" C. a t all times when not actually in use. Supplements, fed thrice weekly from a calibrated syrin e, were prepared so that each dose was contained in 0.1 cc. of t f e cottonseed oil solution. The five assays reported viere performed over a period of 2 years; five different lots of U.S.P. reference oil and five different standard solutions of p-carotene mere used.
It was found on analysis of the results that in every case the animals receiving p-carotene made greater average gains ov& the 4-week period of assay than did their litter mates receiving a supposedly somewhat greater amount of vitamin A from the C.S.P. reference oil. .is a result of this discrepancy, the vitamin A value of t,he four samples of carrots under assay in this study appeared to be from 30 to 44% less when p-carotene was used as the standard than when U.S.P. reference oil was the standard on which the calculations were based (see Table I).
Table
I.
379
ANALYTICAL EDITION
June, 1945 Comparison of
U.S.P. Reference Cod Liver Oil No. 2 and P-Qrotene as Standards in the Bioassay of Vitamin A V a l u e
4 v . Weight Gain per Week with Standard Error of Animals Receiving: Vitamin A Value of Carrots Animals in U.S.P. reference On basis of Each Assay cod liver oil, p-carotene U.S.P. ref- On basis of Group5 14 I.U. per week 7.027 per we&b erence oil ?-carotene Difference Grams Grams ,--I.u./ioo Q.--. 1.1;./ioo Q. “o 6,365 4,180 2,185 34 10 12.8 * 1.37 15.8 * 2.07 1.48 17,500 9,750 7,750 44 14.0 15 9 . 2 f 1.77 4,775 2,055 30 8 11.0 f 0.91 14.6 * 1.88 6,830 15 12.4 f 2.34 14.4 * 2.02 13,335 9,360 3,975 30
rnnf -.
Assay
Carrots, No. 1 Carrots, N o . 2 Carrots, No. 3 Carrots, No. 4 6-Carotenec v.q. U.S.P.7;efZ ence oil N o . 2 28 10.0 * 0,683 1 3 . 3 =t 0.655 .... ... ... .. a Sex-litter mates were assigned to five groups which received U.S.P. reference cod liver oil, p-carotene. supplements of carrots a t two levels, and no supplement, respectively. b Determined by spectrophotometric analysis. Three groups of sex-litter mates were used in this experiment, receiving reference cod liver oil, ?-carotene, and n o supplement, respectively. f
In order to verify these data, 14 male and 14 female sex-litter mate pairs of animals were given the standard doses of reference cod liver oil and p-carotene used in the bioassays, respectively, resulting in similar findings. The rats receiving carotene gained approximately one third more weight during the assay period than did those receiving the U.S.P. reference cod liver oil (Table I). Baxter and Robeson (1) reported a biological potency for vitamin h 0-naphthoate of 3,440,000 U.S.P. XI units per gram and compared i t with a value of 2,225,000 international units per gram as found by Underhill and Coward (6) for the same vitamin A ester. This is a difference of more than SO%, which was attributed by Baxter and Robeson (1) t o a lack of uniformity in bioassay procedures among different laboratories. However, the discrepancy could well have been due, at least in part, to the fact that U.S.P. reference oil S o . 2 was used as a standard by Baxter and Robeson (1) while Underhill and Coward employed the international standard 0-carotene in their bioassays. SPECTROPHOTOMETRIC D A T A
The unsuitability of the U.S.P. reference cod liver oil KO.2 as a standard in spectrophotometric and colorimetric work is well known and the reasons therefor have been discussed (2, 6 ) , not the least of these being its chemical instability. This instability becomes of increased importance in biological assays, where the use of the sample for a single assay usually extends over a period of a t least 1 month. In a series of such assays one sample of the oil might be in use over the full 6-month period, during which it is guaranteed to be safe. Wiseman and Cary ( 7 ) of the Bureau of Dairy Industry, Agricultural Research Administration, found E: 325 mp values for the nonsaponifiable fraction varying from 0.764 to 0.88, in a series of U.S.P. reference oils No. 2 picked up from several laboratories in the Washington area and still considered as usable standards by the laboratories. I n an attempt to determine the sources of these variations, they analyzed immediately upon delivery two bottles of the U.S.P. reference oil S o . 2, which had been packed in dry ice and were in transit less than 24 hours. The absorption of the nonsaponifiable fraction a t both 325 mp and 620 mp, after treatment with antimony trichloride reagent, was measured by means of a photoelectric spectrophotometer using a Hilger double monochromator. The values for 325 mp were 0.870 and 0,873 and the values for E: 620 mp n-ere0.1242 and0.1244, respectively, using the rated potency of the oil in calculating the E per I.C. After aliquots had been removed for analysis, the bottles were resealed under nitrogen, stored a t reduced temperatures, and re325 mp value analyzed 11 months later. At this time the E:?,. bad dropped to 0.675 and the E: 620 miJ.value was lowered proportionately to 0.098. About a year after the first samples of reference oil had been received, two additional bottles were obtained, the same precautions being observed in shipping and handling; in this case the values for the freshly opened samples
Fm,
m’;1a:.
a$’”’*
were lower-Le., the 325 mp values were 0.772 and 0.785 and the E: :.Im1. 620 mM values were 0.1117 and 0.1135, respectively, again calculating the E per I.U. as above. This clearly demonstrates that the variability observed in the vitamin A content of the samples of the reference cod liver oil No. 2 is not entirely due to destruction of vitamin A following the opening of the bottle in the laboratory. Three of the samples of the reference oils used in the bioassays described in this report gave 325 mp values of 0.728, 0.790, and 0.756, respectively.
DISCUSSION
Oser, Melnick, and Pader ( 6 ) state that “when parallel tests on the reference oil are used as the basis for arriving a t factors for converting E values to biological unitage in unknown oils, serious complications result”, and that “errors in estimating potency in relation to the U.S.P. reference oil may fall within the limits of error of the bioassay and are difficult to prove”. Hume (3) has questioned the assumption that the international and U.S.P. vitamin A units are equivalent, basing this conclusion upon the results of a series of collaborative tests using the C.S.P. reference cod liver oil No. 1; an exhaustive statistical analysis of these results has recently appeared. I n the case of vitamin A bioassays, t,he limits of error are rathcr wider than in other types of assay-for instance, as reported by Irwin (4) in his statistical examination of the accuracy of vitamin A assays, the limits of error of vitamin h value ( P = 0.99) obtained from testing several thousand animals lay between 65 and 154%. It is when attempting more precise measurements, such as the establishment of a conversion factor for spectrophotometric values, that the inaccuracies of the bioassay become troublesome and for this reason are considered significant. However, even from a practical viewpoint, a systematic error of 30 to 44% invariably in the same direction-Le., increasing the apparent vitamin A value-becomes important, especially when one considers that the only direct way a t present of arriving at the vitamin -4values of food is by bioassay. Figures of this kind are in widespread use as the basis of calculations for dietary surveys. To the rather wide range of error inherent in the vitamin A bioassay there should not be added a constant error of this magnitude through the use of a st,andard as unstable as the U.S.P. reference cod liver oil No. 2. .it present, pure p-carotene appear.3 to be a more reliable standard for use in vitamin A4bioassay than the U.S.P. reference cod liver oil No. 2 and one which could easily be replaced by a vitamin h standard, when a satisfactory preparation is available. ACKNOWLEDGMENT
The authors are indebted for the spectrophotometric measurements to H. G. Wiseman, Bureau of Dairy Industry, Agricultural Research Administration. LITERATURE CITED
(1) B a x t e r , J. G., a n d Robeson, C. D., J . A m . Chem. Soc., 64, 24071 0 (1942). ENG.CHEZI.. (2) Coy, N. H., Sassaman, H. L., a n d Black, A.. IXD. AXAL.ED., 15,441-3 (1943). (3) Hume, E. M.,S u t u r e , 151,535-6 ( 1 9 4 3 ) . (4) I r w i n , J. O., J. H y g . , 43,291-314 (1944). ( 5 ) Oser, B. L., Melnick, D., a n d P a d e r , M . , IXD.ENG.C H E W . -LNAL.ED., 15,717-24 (1943). (6) Underhill, S. TV. F., a n d C o w a r d , K . H., Biochem. J., 33, 594-600 (1939). (7) W i s e m a n , H . G.. a n d Cary, C. A , , unpublished d a t a .
