Chemical Determination of Vitamin
A
in M i x e d Feeds
and Feedstuffs WILLIAM BREW AND MARY BETH SCOTT Research Laboratories, Ralston Purina Co.,St. Louis, Mo.
Skellysolve F, petroleum ether, boiling range 30 O to 60" C. EXTRACTION.Place a weighed 10-gram sample of feed in a 20 X 80 mm. paper extraction thimble and extract (all operations on vitamin A solutions should be carried out in subdued artificial light, preferably in amber glassware) for 4 hours in a Butt-type extraction tube with 35 ml. of Skellysolve F placed in a 100-ml. ' extraction flask. Other types of extraction equipment may require different volumes and extraction times. Evaporate off the solvent from the extract under a vacuum, add 30 ml. of 95% ethyl alcohol and 3 ml. of aqueous potassium hydroxide solution, and bring to a boil in a water bath. Cool and wash contents of flask into a 250-ml. separatory funnel with a minimum amount of Skellysolve F. Add 30 ml. of distilled water and sufficient Skellysolve F to make 25-ml. total volume of the latter. Shake and draw off the lower phase after settling, and transfer the Skellysolve phase to a clean 125-ml. flask. Repeat the extraction of the aqueous phase with 3 more 25-ml. portions of Skellysolve and combine the extracts in a clean 250-ml. separatory funnel. Wash free from alkali by repeated washings or with a continuous spray washing device. Filter alkali-free extract into a 300-ml. long-necked round-bottomed flask through about 15 grams of anhydrous sodium sulfate on a filter paper, and wash the filter paper with additional solvent. PURIFICATION. If little or no color is present in the final extract, this step may be omitted. If an appreciable arpount of colored pigments is present, evaporate the extract down to a 5ml. volume under a vacuum and wash into a chromatographic column with a minimum volume of Skellysolve. Prepare a 16 X 150 mm. chromatographic column with a 7.5cm. (3-inch) layer of dibasic calcium phosphate, topped by a 2.5-cm. (1-inch) layer of calcium carbonate. The dibasic calcium phosphate should be tested prior to use and shown to retain vitamin A without destruction and to pass &carotene using Skellysolve F as a solvent. The Skellysolve should be free from ether, alcohol, or other solvents and should be as dry IIS ossible. After the extract passes into the column, wa.8 at once with Skellysolve F and continue washing until all carotene passes through the column. This is indicated by a colorless filtrate. This solution may be retained and the carotene measured. The appearance of the column a t this point shows a sharp colored layer a t the extreme top, usually followed by a series of lines and a broad colored band, all in the carbonate layer. Remove all these with care, except for the lowest portion of the broad colored band, by digging out the adsorbent with a suitable instrumept. Change the receiving flask on the column and elute the vitamin A by passing 25 ml. of ether slowly through the column or until all the remaining yellow lines paw through the column. This eluate is to be used for the vitamin A reading. COLORREACTION.Divide the eluate from the column, or the original extract if the column purification was not r e y d , , into two equal portions of any convenient volume. At t is point it is desirable to estimate the approximate vitamin A content of the material being tested, in order that the final solution concentration of vitamin A will be approximately 15 units per ml., a concentration most suitable for measurement. A volume factor, 2, is approximated from the formula: (approximate units of vitamin A per gram in feed) X (weight of sample) x= 30
The antimony trichloride colorimetric reaction for determining vitamin A has been modified in order to correct for the color produced b y nonvitamin .A materials. The system of correction depends upon the action of light on the kinetics of the antimony trichloride reaction and the use of a preliminary chromatographic purification.
H E analysis of mixed feeds for their vitamin A content is complicated by the relatively low vitamin A level often present, the large number of different ingredients present in the mixture, and the wide variation in the physical and chemical characteristics of these ingredients. These complications have prevented the use of spectrophotometric methods for vitamin h measurement. The blue color reaction produced by vitamin A with antimony trichloride offers the best approach to the problem a t the present time.
T
The antimony trichloride blue color reaction of Carr and Price ( 3 ) has high sensitivity to small amounts of vitamin A. This method as modified by Dann and Evelyn ( 5 ) has been widely used as the basis for a number of analytical methods adapted to a variety of materials. Oser, Melnick, and Pader ( 7 ) have used the blue color test in vitamin A measurements on food products in a procedure involving the use of an internal standard designed to correct for the presence of reaction inhibitors, temperature effects, reagent variations, turbidity, and extraneous color. Corbet, Geisinger, and Holmes (4)very early listed a large group of materials known to give a color reaction with antimony trichloride under conditions of the usual vitamin A test. The reaction has been utilized for the quantitative measurement of vitamin D and some of the related sterols and the blue color produced by carotene and the xanthophyll pigments is common knowledge. Dann and Evelyn (5) attempted to correct for the reaction of carotene with antimony trichloride and the resultant interference with the vitamin A test. Oser, Melnick, and Pader ( 7 ) attempted t o distinguish quantitatively between the blue color produced by the reaction of carotene and that produced by the reaction of preformed vitamin A by the difference in the intensity of the two color reactions with time. These authors found that the blue color of the reacted vitamin A faded almost completely in 2 hours, whereas the blue color of carotene increased in intensity over this period of time. The present authors in an attempt t o duplicate this work found that the period required for vitamin A blue color destruction was greatly influenced by both light and temperature conditions. The effect of temperature was noted by Norris and Church (6) and is a normal behavior, but the action of light has only recently been reported by Caldwell and Parrish (3) and appears to offer an explanation for some of the discrepancies in the vitamin A literature in regard t o the rate of fading of the blue color. Some types of mixed feeds and certain feed ingredients when reacted with antimony trichloride give strong color reactions of nonvitamin A origin that interfere greatly with the vitamin A tests. The action of light on the course of these nonvitamin A color reactions has been found to differ so greatly from the action on the true vitamin A blue color as to provide a means of differentiating between the color reaction of vitamin A and any interfering color reactions simultaneously produced.
by which x is found to the nearest whole number (but not less than 2). To one portion of the solution add 102 units of a vitamin A solution (obtained from a dilution of Distillation Products vitamin A concentrate capsules, Control No. PC-3, checked spectrophotometrically). Evaporate both fractions to dryness under a vacuum and dissolve each fraction in exactly x ml. of dry chloroform. By this system one portion of the sample has been fortified with an internal standard equipment t o 10 units per ml. Reaction readings are made on the Evelyn photoelectric colorimeter, using a 620 mp filter. Set a reagent blank containing 1 ml. of chloroform plus 10 ml. of antimony trichloride reagent to a reading of 1 0 0 ~ otransmission and find the corresponding reference no cell reading with blank solution tube removed. This
ANALYTICAL PROCEDURE
REAGENTS.Antimony trichloride reagent, 90 grams of reagent grade antimony trichloride in 240 ml. of reagent grade chloroform. Aqueous potassium hydroxide, 50 grams of reagent grade pellets in 50 ml. of distilled water. Calcium phosphate, dibasic, should be tested to retain vitamin .A and pass B-carotene (Mallinckrodt A. R. grade).
46
. A N A L Y T.1 C A L E D I T I O N
January, 1946
.3
E
0
0 min.
5
10
Reaction Time Figure I .
Derivation of Vitamin
A Calculation Formula
reading is used for subsequent settings for a series of readings on any one day with the same reagent. Place a 1-ml. sample of the unfortified unknown i n a reaction tube in the instrument, add 10 ml. of antimony trichloride reagent, and take a reading as soon as the galvanometer can be read (reading A ) . Place the reacted solution immediately in a glass-walled water bath (a rectangular battery jar is suitable) kept a t 30" C. midway between two 150-watt reflector flood lamps placed a t a distance of 30 cm. (12 inches) between the two bulb faces. After exactly 5 minutes read the reaction tube again (reading B ) and immediately return it to the water bath. A t the end of exactly 10 minutes obtain a third reading (reading C), React a 1-ml. sample of the vitamin A-fortified sample as above with 10 ml. of antimony trichloride and take only the initial reading (reading D). According to the data of Baxter and Robeson ( I ) , confirmed by the authors, this reading should not exceed a photometric density of 0.523, corresponding to a transmission of 30% in order to stay within the linear range for a vitamin A standard curve. Dilute a second 1-ml. aliquot of the unknown (if necessary a part of the fortified sample can be mixed with the unfortified for this reading) with 10 ml. of chloroform and read a t 440 mp (reading E ) against a blank of pure chloroform set to 100% transmission. Reading E is made to correct for the interference of certain carotenoid pigments which give color reactions with antimony trichloride. Calculate vitamin A concentration from the following formula in which all readings have been converted to photometric densities: 10(A - 2B C - 0.067 E ) ( ~ z ) Units of vitamin A per gram = (D - A) (wt. of sample) The factor 0.067 in this formula is explained below and should be determined by the user for the particular type of carotenoid pigments most commonly encountered (from alfalfa, corn gluten, etc.) and with the particulai. filters used in making the readings.
+
BASIS OF COLOR-READING PROCEDURE
The vitamin A blue color reaction with antimony trichloride is usually read a t about 620 mp within 10 seconds after addition of the reagent to the A source. Unfortunately, if the vitamin A solution contains any considerable amounts of impurities of a variety of types, these impurities will produce colored reactions simultaneously with the formation of the vitamin A blue color. The best available means of purification do not completely remove these interfering materials from feed extracts. These interfering materials can be grouped into three broad reaction types: 1. Materials which produce a high initial color intensity which fades with time, similar to vitamin A itself. This is indicated by decreasing light absorption with time. 2. Materials which produce a low initial color intensity which increases with time. This is indicated by increasing light-absorption values. 3. Materials which produce a color reaction of an intensity relatively constant with respect to time. This is indicated by constant light-absorption values.
'
41
All classes of interference would be made up of one of these types or some combination of several types. If vitamin -4-antimony trichloride reaction solutions are subjected to controlled light of high intensity a t constant temperature, as outlined in the quantitative procedure, the blue color fades very rapidly, and a t the end of 5 minutes has completely disappeared. If reacted solutions of pure vitamin A are read a t 620 mp, the difference between the initial photometric density and that after 5 minutes is directly proportional to the A concentration over a wide range of concentration values. Extracts of feedstuffs known not to be sources of vitamin A have been tested under similar conditions with the following observed groupings: 1. Reacting materials which showed decreasing color intensities with time were mostly carotenoid in nature and had attained minimum values of photometric density a t 620 mp within 5 minutes. Moreover, the difference between the initial absorption a t 620 mp and the absorption after 5 minutes in each case was quantitatively related to the yellow color present in the unreacted solution. The latter could be measured by reading a t 440 mp. 2. Materials which showed increasing color intensities with time increased in photometric density a t approximately a uniform rate for short periods of time. The increase during the period 0 to 5 minutes was approximately the same as that for 5 to 10 minutes. In all cases this increase in photometric density a t 620 mp was relatively slow as compared with the rapid decreases observed for vitamin A and carotenoids and the difference between the changes in the two time intervals was negligible. 3. Materials which possessed a color of their own unrelated to the antimony trichloride reaction or which reacted to produce a color that did not change with short intervals of time. Utilizing the facts ascertained in these observations, it was decided that a procedure could be developed for reading vitamin -4 in the presence of all three types of interfering materials. This is best explained by means of Figure 1. Readings are made under controlled conditions of light exposure and temperature immediately after reaction (reading A), after 5 minutes' exposure (reading B ) , and after 10 minutes' exposure (reading C). I n addition, a second portion of the extract being tested is reacted in tkie presence of a known addition of pure vitamin A (reading D)and an independent reading is made a t 440 mp (reading E ) of the diluted, but unreacted, sample to be used as a correction for carotenoids present. Reading A is composed of the sum of the absorptions produced by an unknown amount of vitamin A, represented by amount L, and the three types of interfering colored reactions previously mentioned, and represented by quantities M , N , and 0, indicating decreasing color, increasing color, and stable color, respectively. Reading B, at the end of 5 minutes, shows that L and M have faded out completely, 0 has remained constant, and N has increased by increment X. At the end of 10 minutes, represented by reading C, an additional increment, X , has been added to t h e value of original quantity N , making its value N 2X. ReadingA = L M N 0 ( 1) Reading B = X N 0 (2) Reading C = 2 X N 0 (3) from which the formula L = A - 2 B C - ma be derived. M may be derived by a separate reading (reading I$of ) the carotenoid color a t 440 mp which will bear a relationship, K , to the blue color produced a t 620 mp for any particular group of pigments and pair of filters. Hence, A l = KE L = A - 2B + C - K E or To compensate for the fact that certain materials may inhibit the amount of absorption L produced by the antimony trichloride reaction with vitamin A, a known increment (10 units = S) of A is added to a second fraction of the original solution and after adjusting to the same volume for reaction, reading D is obtained. All color produced by preformed vitamin A has completely faded at the end of 5 minutes. Thus, after this eriod of time the sample with the added increment of vitamin will read the same as the sample without the vitamin A increment (Figure 1). This fact makes it possible to use the principle of the internal standard with its attendant advantages as stated by Oser, Melnick, and Pader ( 7 ) . D - A equals the amount of absorption produced by 10 units of vitamin A under the same reaction conditions as in the case of L absorption, produced by the unknown amount of vitamin A.
+ + + + + + + +
+
1
4a
.
INDU'STRIAL AND ENGINBERING CHEMISTRY
For low concentrations of vitamin A the readings given in terms a f photometric density are directly proportional to the amount of vitamin A present. Units of vitamin A -
Therefore .or
10
Unit,s of vitamin A
=
10(A
L D - A
- 2B
+ C - KE)
D-A
The value of K is derived by reacting with antimony trichloride and reading a t 620 mp portions of a series of solutions of varied carotenoid concentration and reading dilutions of the same solutions a t 440 mp. The reacted absorption values are plotted against the dilution absorption values and a straight-line relationship is found. The ratio of the photometric density a t 620 m p to that at 440 my is the value of K desired. This was found to be 0.067 for the authors' instrument for the carotenoids of alfalfa, the major carotenoid source in feeds. This value was obtained after a chromatographic column treatment and readings obtained according t o the procedure given. The chromatographic treatment removes pure carotene and only the related pigments are present. Table Ingredient Soybean oil meal C o r n meal Tankage Fish meal Ground oats Alfalfa Wheat germ Total
1.
Composition of Feed Mixtures
Sample 1 Sample 2 Samqle 3 Sample 4 Sample 5 Sample 6
%
%
30 50 20
..
...
30 50
20
%
%
30
...
...
30 20
...
50 ...
... ..,
20 100
100
30
... 20
,..
,..
100
100
%
%
30
... ,..
10 20
20 20 100
20 30 '
30
20 100
RECOVERY OF ADDED VITAMIN A
I n order to check on the accuracy of the reading procedure, samples of feedstuff mixtures (Table I) were prepared containing varying proportions of typical feed materials. Some of the mixtures were prepared with abnormally large amounts of certain types of ingredients known to interfere with vitamin A measurement. Portions of these mixtures were extracted and purified on chromatographic columns. Each extract was divided into two equal portions, each equivalent to 8 grams of original sample and one portion was fortified with 100 I.U. of vitamin A or 12.5 I.U. per gram of sample. The two portions of each sample were then treated as separate samples and their vitamin A content was read and calculated by the nrocedure given. The value of X for calculation is 2.5. According to the procedure later adopted a whole number, in this case 3, is recommended. Table I1 shows the results obtained on this experiment. The average recovery obtained, 12.61 I.U. per gram, checks well with a theoretical recovery of 12.5. The range 11.62 to 13.60 is within the range to be expected from a reading procedure depending upon five independent readings.
Table
II. Recovery of A d d e d Vitamin A
Vitamin A RecovNo. Found ery Error I.U . / g . I.U./Q.I.C . i g . 1 0.027 0 . 0 2 2 0.082 0.214 0.034 0.43 IF^ 0.382 0.034 0.043 0 . 5 4 0 0.034 14.03 i3:60 + i : i o 0.10 .... 2 0.032 0.041 0.056 0.214 0.048 0.387 0.051 0.062 0.551 0.048 13.10 1i:OO + 0 . 5 0 2F' 0.00 3 0,149 0,122 0,122 0 . 3 2 8 0.403 3Fa 0.485 0.131 0.131 0.6Gl 0.403 11.62 li:62 -0.'&8 4 0.161 0.144 0.149 0.340 0.475 -0.35 4F" 0.512 0.158 0.161 0.671 0.475 12.77 13:12 + 0 : 6 2 0.17 .... 5 0.174 0.155 0.172 0.358 0,462 5Fa 0.509 0.164 0.181 0.677 0 . 4 6 2 12.32 l 2 : 1 5 -0.35 6 0.201 0.177 0.197 0.381 0.495 Q 38 6F0 0,530 0.184 0.204 0.696 0.495 12:54 l2:16 -0134 Av. 0.12 12.73 li:6l a Same as samples 1,2,etc., except 12.5 I.U. per gram added vitamin A.
Sample
Read- Read- Read- Read- Reading A ing B ing C ing D ing E
Vol. 18, No. 1
Kone qf the ingredients involved was believed to contain preformed vitamin A and an average value for the unfortified samples of 0.12 I.U. per gram with a range of -0.38 to f0.43 is acceptable. Previous experience in this laboratory has shown that the chromatographic procedure recommended completely separates the carotene from the vitamin A and that the amount of xanthophyll pigments present is reduced to a minimum. -4s a check on the recovery of vitamin A carried through the whole procedure, two formulas for commercial mixed feed concentrates containing no preformed vitamin A and known to give very bad reaction colors with antimony trichloride were fortified a t several vitamin A levels. These samples were carried through the complete analytical procedure and the recovery of vitamin A was measured (Table 111). The unfortified samples gave results of 0.07 and -0.21 I.U. per gram, which is in line with those expected, and assuming a zero value for the unfortified samples the amount of vitamin A lost in the complete procedure varied from 0.74 to 0.88 I.U. of vitamin -4per gram of sample. Since this is the combined loss of the extraction, saponification, and column procedure, plus losses in transferring and evaporation, it appears to be satisfactory for this type of procedure. However, this loss is apparently an absolute one and for very low potency feeds it may result in a high percentage error. No attempt is made in this paper to measure vitamin A activity other than that produced by preformed vitamin A. Carotene can be measured on the first eluate from the chromatographic column but the problems involved in the separation and measurement of different carotenoids having vitamin A activity and the subsequent conversion into units of preformed vitamin A are a t present only partially solved. Table 111. Sample
Recovery of A d d e d Vitamin Added Vitamin .4
I.U./Q.
A from M i x e d Feeds
Total Vitamin A
I.U./Q.
Loss of Vitamin A
I . UJQ.
SUMMARY
d modification of the antimony trichloride method for the determination of vitamin A in feeds and feedstuffs compensates for the presence of interfering reacting materials by means of the differential effect of light on the kinetics of the antimony trichloride reaction of vitamin A and the interfering materials, respectively. A chromatographic procedure suitable for the preparation of feed extracts intended for color reaction is given. The principles of the procedure appear favorable for use in a variety of applications in the determination of vitamin A in human foods. ACKNOWLEDGMENT
The authors wish to acknowledge the technical assistance of B. F. Beaver with regard to the chromatographic procedure. LITERATURE CITED
(1) Baxter, J. G., and Robeson, C. D., J. Am. Chem. SOC.,64, 2411 (1942). (2) Caldwell, M. J., and Parrish, D. B., J . Biol. Chem., 158, 181 (1945). ( 3 ) Carr, F. H., and Price, E. A., Biochem. J . , 20, 497 (1926). (4) Corbet, R. E., Geisinger, H. H., and Holmes, H. N., J. Biol. Chem., 100,657 (1933). (5) Dann, W. J., and Evelyn, K. A,, Biochem. J., 32, 1008 (1938). (6) Norris, E.R., and Church, A. E., J . Biol. Chem., 85,477 (1929). (7) Oser, B.L.,Melnick, D., and Pader, M., IND.ENQ.CHEM.,h A L . ED,,15, 724 (1943).
