Spectrophotometric and Biological Assay of Vitamin A in Oils N. H. COY, H. L. SASSAMAN, AND ARCHIE BLACK Vitamin Laboratory, E. R. Squibb and Sons, New Brunswick, N. J.
T
H E discovery in 1928 by Morton and Heilbron (16) that vitamin A has a characteristic absorption band in the region 320 to 330 pp has been followed by numerous publications on the use of this physical fact as a means of quantitative measurement of the amount of vitamin A in oils. Such literature covers the improvement in experimental technique, irrelevant absorption, proper solvents, forms of vitamin A, and, dependent on all these, the correlation of results obtained by physical tests with biological assay through the proper conversion factor. There is now available a wide variety of spectrophotometers and photoelectric photometers used in the physical assay of vitamin A. The Vitamin Assay Committee of the American Drug Manufacturers’ Association (1) in 1937 and again in 1939 made an attempt to correlate results obtained by such instruments. An examination of these reports reveals a divergence among the values of extinction coefficients obtained by different instruments on the same oils, attributed partially to errors in the instruments and partially to the technique of the operator and experimental conditions. The question of irrelevant absorption, or the presence in oils of substances which show ultraviolet absorption, not necessarily selective, in the region of 328 ,up is of great importance in the physical assay of vitamin A. Attempts to correct for such absorption have involved the application of correction factors (3, 12, 16) or the suggestion of Hume and Chick (9),who state that the irrelevant absorption bears no constant relationship to the amount of vitamin A present, that it is less for the unsaponifiable fraction than for the oil itself, but that in oils with large concentrations of vitamin A the error is insignificant. On the other hand, the problem is further complicated by the question as to whether the ultraviolet absorption a t 328 pp is a measurement of all the substances which cause vitamin A activity in rats. There are two aspects t o this problem. In the first place Gillam et al. ( 5 ) and Edisbury et al. (4) report the presence in oils of a new vitamin, vitamin A,, which also has biological activity, but with ultraviolet absorption maximum at 345 to 350 pp. One other vitamin in whale liver oil with absorption maximum at 290 pp has been reported by Willstaedt and Jensen ( I T ) , and it is possible that other forms of the vitamin exist. In the second place, Gray, Hickman, and Brown (6) report that vitamin A in fish liver oils is present mainly in the ester form. It is further implied by Hickman (7) that the esters of vitamin A have a greater biological activity than the alcohol. This may be due to the fact that the ester is more stable than the alcohol, as Mead, Underhill, and Coward ( I S ) have found.
All these factors influence the value of the conversion factor. The normal procedure of obtaining the conversion factor is by direct computation from the E and biological values of the pure substance. I n the case of vitamin A this procedure is not logical because of the instability of the vitamin and because some substances which have biological activity similar to vitamin A do not have absorption maxima a t 328 pp. The value of 1600 first reported (10) was based not on pure vitamin A but on the richest concentrate known a t the time. Since that time many values have been reported. Hume and Chick (9) state that the values vary from 1300 t o 2580. Morgan et al. (14) indicate that the factor may not be the same for cod liver oils as for oils of higher potency. Holmes and Corbett (8) report on tests made on crystalline vitamin A values from 1600 t o 1800; one test by Darby gave a value of
2100. The 1939 report of the A. D. M. A. committee ave conversion factors ranging from 1920 to 2780. Barthen anfleonard (2) on tests on U. S. P. standard oil report avalue of 2222; Mead, Underhill, and Coward (IS) report values of 1920 and 2150 for two esters and, having made the correction factor for the acid fraction of the ester molecules, compute 2000 as the conversion factor for vitamin A. Hickman (7),using data obtained mainly from molecular distillation studies, states that the conversion factor for the esters of vitamin A is higher than for the alcohol, that each preparation has a characteristic conversion factor, and that cod liver oils in general have higher factors than most other fish liver oils.
It would thus seem that, until some of these matters are further clarified, each laboratory should obtain its own conversion factors by direct comparison between the E and biological values of a great variety of oils. Such a procedure has been adopted in this laboratory. Experimental Procedure PHYSICAL. Two instruments, the Hilger vitameter and the ultraviolet spectrophotometer, were used in the physical assay of the oils. Experience with the vitameter has shown that variations in its performance lead to rather inaccurate results. The vitameter, however, is a simple and speedy instrument t o operate and has been found excellent for measurements preliminary to the biological and more accurate physical assays. The spectrophotometer used was a Judd Lewis ultraviolet photometer with vanes carefully refinished and reset and the various units rigidly mounted t o the frame of a 10 X 25 cm. (4 x 10 inch) Bausch and Lomb ultraviolet spectrograph. The original mounting of the Judd Lewis photometer has been reTABLEI. U. S. P. REFERENCE CODLIVEROIL 81% 1 om.
Oil No
Full Bottles 1.40 (unsap.) 1.36 (unsap.) 1.49 (whole) 1.39 (unsap.) 1.49 (whole) 1.36 (unsap.) 1.49 (whole)
1
2 3 4
Conversion Factorb
.. 0.10 0.13
Partially Filled Bottlesc 1.24 (unsap.) 1.12 (unsap.) *. 1.15 (unsap.) .. 1.17 (unsap.) 1.17 (unsap.) 0.09 1.26 (whole) 10 1.14 (unsap.) 0.12 1.26 (whole) 1.24 (unsap.) 11 0.08 1.32 (whole) Average conversion factor for unsaponified fraction Full bottles Partially filled bottles Average conversion factor for whole oil Full bottles Partially filled bottles
..
5 6 7 8 9
..
-
2140 2210 2010 2160 2010 2210 2010
0.13
2420 2680 2610 2560 2560 2390 2630 2390 2420 2270 2180 2550
* 1.4% * 3%
2010 2350
f
* 2% 07
whole E!?m. unsap. Biological value taken a8 3000 u. 8.P. Flushed with carbon dioxlde and allowed t o stand in refrigerator for several weeks. a
b
c
74
ANALYTICAL EDITION
February 15, 1941
TABLE11. CODLIVEROILS U.9. P, XI Conversion (Biological) Factor Units per gram 2730 0.75 0.25 2050 1872 (unsap.) 2050 (whole) 1.00 2820 0.26 3100 1.10 1891 (unsap.) 2280 (whole) 1.36 3030 2300 0.07 0.76 1870 (unsap.) 2770 (whole) 0.83 2760 2400 0.08 1885 (unsap.) 0 87 (whole) 2530 0 95 2400 3200 0.10 1847 (unsap.) 0.75 2820 (whole) 0.85 2860 0.25 1800 0.63 1838 (unsap.) (whole) 2050 0.88 2000 .. 2380 1859 (unsap.) 0.84 3490 0.18 2300 0.66 1857 (unsap.) 2740 (whole) 0.84 1650 0.06 2580 0.64 1928 (unsap.) 2360 (whole) 0.70 2730 0 . 6 6 1800 1667 (unsap.) 0.05 2500 (whole) 0.72 2570 6000 0.42 2.33 1850 (unsap.) (whole) 2.75 2190 2250 0.77 2920 1833 (unsap.) 0.08 (whole) 2650 0.85 2050 0.04 2360 1697 (unsap.) 0.87 (whole) 2260 0.91 2150 2620 1699 (unsap.) 0.09 0.82 2360 (whole) 0.91 1700 2430 0.70 1903 (unsap.) 0.09 2150 (whole) 0.79 2150 .. 2690 1879 (unsap.) 0.80 0.10 2150 2560 0.84 1978 (unsap.) 2290 (whole) 0.94 0.18 1600 2350 0.68 1987 (unsap.) (whole) 0.86 1860 0.09 1550 0.57 2013 (unsap.) 2720 (whole) 2350 0.66 1400 0.55 0.03 2004 (unsap.) 2550 (whole) 2410 0.58 0.02 2100 2021 (unsap.) 2700 0.78 (whole) 2620 0.80 2000 2022 (unsap.) 2440 0.11 0.82 (whole) 2150 0.93 Average conversion factor for unsaponifiable fraction 1700 * 8 % Average conversion factor for whole oil 2370 * 9 % Oil No.
E1 % 1 om.
5 whole unsap. whole oil before saponification.
aEl%" 1 om.
One per cent refers t o Concentration of
placed by a more rigid and better aligned single unit mounting designed by the Squibb staff. In this mounting all the photometer units, including the source, are attached to the same unit. With such a mounting it has been the experience in this laboratory that in the 8 months of use, once the new photometer setup had been brought into adjustment (using approximately thirty plates), the only subsequent adjustments have been incidental to the removal and sharpening of the electrodes as they have worn away with use. On replacement of the electrodes a single plate taken varying the height of the arc was suf6cient to reset the source. The light source used was a tungsten-steel spark. On each assay plate one exposure with both apertures fully open and no absorbing cells in position was made as an adjustment check on the equality in intensity of the two beams. The density scale of the lower sector of the photometer was calibrated by comparing values found with solutions of potassium chromate and potassium nitrate with the values recorded in literature. Such concentrations were used as would include the entire scale. All absorption maxima of these absorbing salts were used. Such calibration was checked on the average after every ten plates had been exposed. Eastman spectroscopic plates No. I1 0 were used. Exposures with such plates, using Bausch & Lomb 1-cm. cells and slit width of 0.03 mm., varied from 2 to 10 seconds. Plates were developed for 5 minutes (at 20" C.) in Eastman D19 developer, washed and fixed for 15 minutes, then washed in running water for a t least 30 minutes. The actual procedure in the assay of any oil was to weigh out directly into a 100-ml. flask a quantity of the fresh oil accurate
75
to a 0.1 mg. This was then diluted with isopropanol and a vitameter reading was taken. Dilutions that would give a match a t a density reading of between 0.50 and 0.95 were used. A similar concentration was then placed in one of the photometer cells with isopropanol in the compensating cell, and a plate was taken varying the aperture of the density scale from 0.15 to 0.95 by steps of 0.05. On such a plate it was possible to read the match point a t 328 p p t o 0.05 density readings and also to plot the absorption curve of the oil. When such a density reading was obtained the value of was calculated from Beer's law and a second plate was taken. On this second plate two or three concentrations were used, each covering from six to nine exposures. The test solution was thus exposed to the ultraviolet light for not more than 1.5 minutes. Tests made for deterioration of the oils due to irradiation from the source when the cells were in their normal position with respect to the source and the aperture was fully open indicated that for exposures up to 3 minutes the decrease in the E value was well within the error of the instrument. A second weighing was also made and a third plate taken with two or three concentrations. If the extinction coefficients computed from the plates agreed to within 5 per cent for the different weighings no further data were taken; if not, a third weighing was made and plate exposed. Plates, when dry, were placed on a well-illuminated viewing stand and examined visually with a jeweler's loop for density match points a t 328 p p . If there %as doubt about any match point, the plate was projected on a screen using a Bausch & Lomb Balopticon projector and the projected image was examined for the match point. With the lower potency oils assays were made on both the whole oil and the unsaponifiable fraction. Two methods of saponification were used-one, that suggested by the Vitamin Assay Committee of the A. D. M. A. of 1937 (1)with the additional procedure that the ether was evaporated under carbon dioxide, and the other a modified form of this procedure by which in the process of saponification and extraction of 1 gram of oil larger amounts of ether, alcohol, and water were used than in the first procedure. Following this procedure, an accurate determination on a cod liver oil can be made in about 6 hours and on a n oil of higher potency in 4 hours. BIOLOGICAL. The U. S. P. XI procedure was followed in all cases. A master curve was used as a n aid in interpreting the results and to increase the accuracy. Precautions were taken in the biological assay to avoid the use of U. S. P. reference oil which had been exposed to air or had been stored in partially filled bottles any appreciable length of time. Fresh bottles of the oil, as distributed by the U. S. Pharmacopeia Vitamin Committee, were taken a t intervals of 2 to 3 months, and subdivided into three or five small vials which were thoroughly flushed, sealed, and stored in a refrigerator. These small vials were then consumed in periods not exceeding 2 to 3 weeks. These vials and various diluted solutions which were used in the tests were always thoroughly flushed with carbon dioxide after being opened and then returned to the refrigerator. Fresh dilutions were prepared weekly.
Results I n Table I values obtained for the U. S. P. reference cod are tabulated. Each E value recorded represents an average value obtained by following the procedure outlined above for the assay of cod liver oils. I n some cases more than two saponifications mere made. In Tables I1 t o IV the results for a series of oils are recorded. The conversion factors were obtained by dividing the biological value by the E value. The average conversion factors for the various types of oils and the average per cent errors were calculated. Many absorption curves of the oils have been plotted. For the oils of higher potency the curves are fairly symmetrical about a pronounced peak around 328 pp, I n some of these there is evidence of a "flat" in the curve at 310 and 340 t o 350 pp. A comparison of the curves of the whole oils and unsaponifiable fractions of cods is interesting. I n both cases there is a pronounced peak at approximately 328 pp, also evidence of a flat at 310 and 340 to 350 pp, but the curves for the whole oils are more irregular, showing evidence of selec-
INDUSTRIAL AND ENGINEERING CHEMISTRY
76
TABLE 111. TUNA AND HALIBUT LIVEROILS E1 % NO.
1 cm.
u. s. P. XI (Biological) Units p e r gram Tuna Liver Oil 120,000 26,000
1959 58 1902 12 1897 34 1909 49 1623 56 1741 12 1916 23 1917 26 2016 55 2023 44 Average conversion factor
1617 1619 1863 1860 1936 1937 1914 1915 1692 1988 1989 1990
Conversion Factor
2070 2170 2350 1580 2690 2500 2420 2130 2000 2090
ao,ooo
75,000 145,000 30,000 55,600 55,300 110,000 92,000 2180 * 10% 2260 * 9% (omitting 1909)
Halibut Liver Oil 67 150,000 70,000 33 140,000 56 110,000 41 100,000 46 105,000 50 61,700 26 42,400 18 62,000 31 26 56,000 37 so,ooo 93 202,000 Average conversion factor 2250
2240 2120 2500 2680 2180 2100 2380 2360 2000 2150 2160 2170 6%
tive absorption in the region 260 to 280 pp, and the curves as a whole are higher than those for the unsaponifiable fractions.
for the whole oils themselves are greater than those for the other oils. There is no explanation a t the present time for the higher conversion factor, but the inference might be made that there are present in cod liver oils, in proportions greater than in oils of higher potency, substances which have biological activity similar t o that of vitamin A but do not have maximum ultraviolet absorption a t 328 pp. Further work on this point is in progress.
Summary The details of a spectrophotometric method of assay of vitamin A in fish liver oils and the results of the biological and physical assay of 53 such oils are recorded. I n all cases assays were made on fresh oils and all biological assays were made on the whole oils. The average conversion factors computed from the measurements on 22 cod liver oils yield values of 2700 and 2370 for the unsaponifiable fractions and whole oils, respectively. The average conversion factors for oils of higher potency are 2260 for tuna liver oils, 2250 for halibut liver oils, and 2270 for miscellaneous oils as listed, giving an average of 2260 for these oils of higher potency. Studies on the U. S. P. reference standard have shown that the E value gradually decreases when the oil remains in partially filled bottles, even though they have been flushed with carbon dioxide and stored in a refrigerator.
TABLE IV.
Discussion of Results An examination of Table I reveals the fact that the E values listed for the U. S. P. reference cod liver oil vary over a considerable range from the higher consistent values for the assays on full bottles to the lower varied values on bottles which were only partially filled and were handled as indicated above. A similar falling off of the E values for this reference oil was reported by McFarlan (11). There has been no evidence that any loss of vitamin A has occurred in the original bottles of U. S. P. reference samples as determined by repeated physical tests and biological assays. Whether or not the decrease in the E values which occur in the U. S. P. reference oil after standing for some time in partially filled bottles is also accompanied by a decrease in vitamin A activity has not been determined but is not believed to affect the interpretation of the biological assays, since the use of such oil has been avoided. In Table I1 are listed the conversion factors for 22 cod liver oils as obtained by direct computation of the biological and physical values for the individual oils. The average of the unsaponifiable fraction for these oils is 2700 and that for the whole oils 2370. Conversion factors for oils of higher potencytuna, halibut, and other fish liver oils-are listed in Tables I11 and IV, giving average values of 2260, 2250, and 2270, respectively. It would appear that the conversion factors of the cod liver oils or a t least a part of them were higher than that of the U. S. P. reference cod liver oil or samples of the more highly active tuna, halibut, and other fish oils which were studied. These differences cannot be explained by variations in the instrument, since the various oils have been run as received in the laboratories. Different oils were run concurrently and results should be comparable. Furthermore, the instrument was calibrated a t frequent intervals with standard inorganic solutions. ' Neither is it considered that these differences are due to the loss of the vitamin in the process of saponification, since two methods of saponification were used and results agree to within 5 per cent. Moreover, the values
Vol. 13, No. 2
Type of Oil
NO.
1839 1878 1899 1948 1977 1934 1944 1979 2019
MISCELI,ANEOUS OILS El% 1 cm.
Pollack 3.8 Pollack 4.5 Pollack 3.7 Pollack 3.5 Pollack 4.2 Shark 56 Shark 57 Sword 28 Shark 109 Average conversion factor
u. s. P. XI
(Biological) Units p e r gram 10,500 9,500 8,500 7,800 10,200 135,000 109,000 67,000 206,000 2270 9%
Conversion Factor 2760 2110 2300 2230 2430 24 10 1910 2390 1890
f
Literature Cited Am. Drug. Mfrs. Assoc., Vitamin Assay Committee, J . A m . Phurm. Assoc., 26, 525 (1937). Barthen, C. L., and Leonard, C. S., Zbid., 26, 515 (1937). Drummond, J. C., and Morton, R. A,, Biochem. J . , 23, 785 (1 929).
EckLYy, T. R., Morton, R. A,, Simpkins, G. W., and Lovern, J. A,, Zbid., 32, 118 (1938). Gillam, A. E.,Heilbron, I. M., Lederer, E., and Rosanova, V.. Nature, 140, 233 (1937). Gray, E.LeB., Hickman, K. C. D., and Brown, E. F., J . Nutrition, 19, 39 (1940). Hickman, K. C . D., J . B i d . Chem., 128, XLIII (1939). Holmes, H. N., and Corbett, R. E., J . A m . Chem. Soc., 59, 2042 (1937). Hume, E. M., and Chick, H., Med. Research Council (Brit.), Spec. Rept. Ser. No. 202, 61 (1935). Lovern, J. A., Edisbury, J. R., and Morton, R. A., Biochem. J . , 7, 1461 (1933). McFarlan, R. L., Bates, P. K., Merrill, E. C., IND. ENQ.CHEM., Anal. Ed., 12, 645 (1940). McFarlane, W. D., and Sutherland, A. J., Can. J . Research, 16B, 421 (1938). Mead, T. H., Underhill, S. W., and Coward, K. H., Biochem. J., 33, 589 (1939). Morgan, R. S., Edisbury, J. R., and Morton, R. A., Ibid., 29, 1645 (1935). Morton, R. A , , and Heilbron, I. M.,Zbid., 22, 987 (1928). Morton, R. A., Heilbron, I. M., and Thompson, A., Ibid., 25, 20 (1931). Willstaedt, H., and Jensen, H. B., Nature, 143, 474 (1939).
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