Spectrophotometric Determination of Vitamins D In Presence of Vitamin A D. T. EWING, T. D. SCHLABACHI,
and
M. 1. POWELLZ
Kedzie Chemical Laboratory, Michigan State College, East Lansing, M i c h .
J. W. VAITKUS and 0. D. BIRD Research Laboratories, Parke, Davis
& Co., Detroit 32, Mich.
The difficulties and limitations associated with the available physicocheniical methods for the determination of vitamin D in oil samples, including those containing vitamin A , led to the present investigation. The proposed method utilizes a two-step chromatographic process for the separation of vitamin D from the other nonsaponifiable oil components. In the first step an activated earth, Superfiltrol, is used to remove the vitamin A, related carotenoids, pigments, and some sterols. In the second step activated alumina is used to remove certain polyenes, as well as other residues from the oil and Superfiltrol itself. The vitamin D is ultimately determined by utilizing its absorption maximum at 265 mp as a measure of the amount present. The method has been successfully applied, within the limitations stated, to samples of irradiated ergosterol in corn oil, crystalline vitamin Dz and vitamin -4acetate in corn oil, irradiated ergosterol and vitamin A palmitate in corn oil, some fish liver oils, and some niiscellaneous samples.
several years work in these laboratories has been directed
F O toward R. the determination of vitamins D present in various oils using chromatographic techniques to separate t,he vitamin D from other nonsaponifiable oil constituents. Prior to this work two methods were proposed: the first by Ewing, Kingsley, Brown, and Emmett ( 5 ) , and the sevond by Ewing, Powell, Brown, and Emmett ( 6 ) . The latter method, developed to determine the amount of vitamin Dz present in samples of irradiated ergosterol in corn oil, employed a single chromatographic step using Superfiltrol as the adsorbent. The ultimate determination of vitamin D Z was carried out by measuring absorbancy a t 265 mp. Neither method was completely satisfact,ory with samples containing less than 50,000 U.S.P. units of vitamin Dz per gram. I n order to determine the vitamin D2 present in oil samples containing less than the above amount of vitamin D,, a modification of the method utilizing the absorbancy a t 265 mp was investigated. The method involves a two-step chromatographic process. In the first, step a Superfiltrol column is used to remove vitamin .i, carotenoids, pigments, some sterols, :tnd irradiation products of ergosterol other than vitamin DB. The use of this and similar adsorbents for like separations has been well estab6 , g-lf), and recently has been eniployed by lished ( 1 , i, Green ( 7 >8) for thc determination of vitamin D2 i n crude irradiation niistures, :tnd vitamin Ds in some fish liver oil?. The second chromatographic step uses an activated :iluinin:t to remove certain unsaturated (noinpounds of the equ:tlenc type, I tleconipo.*ition products, residual impurities cert:rin vitamin . from the Superfiltrol adsorherit, :tnd other niatr,rials present in the non.aponifiable portion of vegetable oils. ThiF: ad-orbent has been used by previous investigators for similar eeparations ( 2 , 3,8), but for the most part these studies have not dealt with the quantitative aspects of the separation. Present address, Bell Telephone Laboratories, N u r r a y Hill, N . .J. Present address, Attapulgas hlinerals and Chemicals Corp., Lakeland, Fla. 1
2
\-itamin D which is eluted from the :idyorbent column is finally determined by measuring the :xheorb:mce a t its 265 mp masininni rather than by the use of antimon>-chloride reagent. The shape of the absorption curve of vitaniin D i n the region of 265 nip is a valuable criterion of the purity of the compound. Marked distortion of the absorption curve is indicative of the probable presence of an impurity ait,h the vitamin D and may introduce an error. The method has been successfully applicd, within the stated limits, to samples of crystalline vitamin D, and vitamin A acetate ipalmitate in corn in corn oil, irradiated ergosterol and vitamin ; oil, some fish liver oils, and some miscellaneous samples. L
REAGENTS AND APPARATUS
Hexane. Skellysolve B (obtained from the Skelly Oil Co., :hatof pure vit,amin DPor D a i sindicative of a determination probably possessing a large positive error, particularly where small amounts of vitamin D are concerned. 8. Various approaches are possible for converting spectrophotometric readings t o units of vitamin I). I n the studies reported here this was done by multiplying the E;?;, 265 mp by 100,000 t o give U.S.1'. cquivnlcnt units of vitamin D per gram. This vias on the basis of data from a number of samples of irradiated ergosterol in corn oil which had previously been assayed spectrophotometrically by the method of Elving ef al. (6) and also biomsayed using the method and reference standard of r.S.1'. S I I I . The present U.S.P. 111- specifics t*q-staIlinevitamin D3 as wference standard and by definition establishes this as containing 40,000,000 U.S.P. units per gram. On the basis of the latest c7 reported E: &, for crystalline vitamin Da(1%')the factor for converting spcctropIiotomet,ric readings to U.S.P. equivalent units, assuming no loss of vitamin D in carrying out the various steps ofthe above described assay procedure, would be as follows: 40,000,000 (units of vitamin D per gram of vitamin D3) 485 ( E : Th,265 m p vitamin D3)
=
82,4i4
Theoretically, this would hold 0111~-if vitamin D, wa For assaying samples known to contain vitamin Dz, the E l1c%m , oi which is 460, the correct calrulatctl factor for converting spectrophotometric readingi to unit? i ~ o u l dI J8T~ 000. NOTES OS I'ROCEIJURE
The following general information \v:w acquired in the development of the method presented (11). T h r Superfiltrol used was selected froin various available lots; not all loti examined were satisfactory. I n general, the whiter grader bvere more effective than the darker grades for the desired separation. .ittempts to st~andardizcthe material by heating, or by ~vashingfolloived by drying nntl heating, yielded an adsorbent unsuitable for w r ; the material \ v : ~ used as received. Suitt be determined by treating ability of :i lot for use can m o ~ easily :t. krio1v11misture of vitamins A :incl D as described, and noting \ v h e t l i ~or i ~ not the desired sep:ir:ition is obtained. Moisture has pronounced adverse effect on the :\tl$orl)rnt; and must be (-0111pletely rscluded. The relative positions of the chromatographic zone8 for vitarnin D and the other nonsaponifiable oil component..9 were not found to vary appreciably under different eluent compositions. As the column length was increased, the threshold volume for vitamin D increased, as did the total volume required for complete elution. When pure, crystalline vitamin D4 and tho procedure outlined were used, the recovery was 987, from the - k i n . Superfiltrol column and 9670 from tho 6-cm. column. T h e vitamin A and related carotenoids are irreversibly adsorbed on this column.
1408
ANALYTICAL CHEMISTRY
Because the activity of different samples of alumina varies (often over a considerable range), i t was necessary to find some convenient method of standardizing the adsorbent with respect to the threshold volume of vitamin D, the banding of the vitamin D for a given solvent system, and rate of flow. As the commercially available alumina adsorbents usually adsorb vit,nmin D too strongly, the problem is one of controlled deactivation. The method used was to prewash the adsorbent with a definite volume (10 ml.) of a solvent composed of varying amounts of alcohol in ether, introduce pure, crystalline vitamin D2in hexane onto the column, and determine its threshold volume using io ether-30 hexane (v./v.) as the eluant. It is desirable to stand:irdize the alumina so that the threshold volume is 12 to 15 ml., using 70 ether-30 hexane (v./v.), and so that not more than 20 to 25 nil. of the ether-alcohol solvent (preferably having the same composition as the prewashing solvent) is required to elute the vitamin D completely. Csing the procedure outlined, the reof pure, crystalline vitamin D, from this column was found to be 96 to 987,. The interfering nonsaponifiable oil components present are much less strongly adsorbed than the vitamin D, and are eluted prior to it on the column. The rate of flow on this column is very important, as this rontrols the degree of equilibrium attained. I n the procedure outlined this factor n-as controlled hy utilizing alumina:: of two different mesh sizes, the finer mesh being a t the bottom. .is an alternative, a single alumina could be chosen with a pni.tivlr size such that the indicated rate of flow could be maintaiiicti using a sninll pressure difftm:ntial (10 to 20 mm. of mercni.y'i.
In Table I are listed the results obtained for various dilutions of a commercially prepared irrndiatrd ergosterol in corn oil. Procedure A for the Superfiltrol column step was used for these
Sample XO.
1
2 3
Potency o f Samples o f Irradiated Ergosterol in Corn Oil U.S.P. Units of Vitamin
DdG. 44,000 44,000 44,000
4 5
25,620 25,620 25,620 23,360
8 9 10 11 12 13 15 16 17 18 19 20 21 22 23 24
12,420 12,420 12,420 12,150 12,150 12,150 12,150 10,630 10,630 9,200 8,700 8,040 8,040 8,040 8,040 8,040 8,040
23 26 27 28 29 30 31 32 33 34 35 36 37 38
6,160 6,100 5,980 5,980 5,980 5,980 5,980 5,075 5,075 5,075 5,070 5,040 4,450 4,000
$
14
Observed T.S.P. Equivalpnt Units of Vitamin D d G . 38,800 39 , (io0 38,401) 21,350 21,600 28.200 21 ,3no 11 ,0;i(l 1O..i00 10,830 10,400 10,300 1 0 , ,500 11,300 io.ooo r(,!?Jil
8 , 3.50 8.100 8,580 7,100 7,100 7 , 070 7,200 7,920
Deviation
Alean Deviation,
-16.2 -10.0 -12 ti
-12.9
70
%
-16.1 -15.8 +10.0
-
8.ti
-11.1 -15 1 -12 cj -14.6 -15.1 -13 8 - 7.1 - 0.3 - 6.4 - 9.1 - 7.0 G.2 -11 -11 7 -12.0 -10.1 - 1 4 -10.8
- 7.6
-10.7
+ ,
-
7.1
-
5.0
-
1.4
+-- 530.t;.. f3i +- 1 16. .43 -16.3 4,800 420 ,,,200 5,370 0,500 4,300 4,170
t,
Observed U:S.P. Equivalent Mean Sample Units of Deviation, Deviation, .uo. B/G. D/G. Vitamin D/G. % 7% Irradiated Ergosterol and Vitamin A Palmitate in Corn Oil 1 27,600 5,525 5,900 6.9 2 43,400 8,676 6 9 8,280 3 (0.05 9 . ) 810,000 162,000 162,000 0 0 U.S.P. Units of Vitamin
- 2 4
- 3 0 f " 3 -13.8 + 9 2 - 3.3 4.3
+
U.S.P.
Units of Vitamin
++
4 5
2 , 8 9
10 11 12 13 14
1.5 l!i 17 18 19 20
Crystalline Vitamin D2 and Vitamin A Acetate in Corn Oil 18,500 23.600 20,400 - 9.3 25,600 20,400 17,000 -13.3 28,800 19,300 17,130 -10.3 17,540 28,800 19,300 - 9.3 -10.5 9,650 27,600 11,200 -13.8 10,100 27,600 11,200 - 9.8 27,600 11,200 10,100 - 9.8 25,600 12,000 10,650 -11.3 2 5 , GOO 13,700 13,200 - 3.6 - 9.7 46,000 12,200 11,000 - 9.9 10,700 47,800 11,150 - 4.0 - 6.9 (0.1 9 . ) 135,600 30,880 29,800 - 3,s 30,880 29,600 - 4.2 (0.1 a . ) 135,600 30,880 28,900 - 6.5 - 4 7 (0.1 e , ) 135,600 22,100 4,490 5,280 +17.5 4.7 22,100 4,490 4,700 22,100 4,490 4,600 2.5 8 2
--
21 22 23 24
28.800 28,800 28,800 28,800
?i ?!i
59,400 59,400 59,400 ~9,400
2; 28
"9 b 30 c
R E s t ' LTS
Table I.
Table 11. Potency of Samples Containing Both Vitamins A and Da
b c
6.400 8,100 6,100 6,100
6,100 5 , ,500 6,300 6,500
10,850 10,830 10,850 10,850 20,000 250,000
11,800 12,300 9,820 10.890 20,700 201,500
+
-
5.0 - 9 8 3.3 7.4.
+ +
+ 8.7 +13.3 - 9.5
0.0
-
1.0
+
3.1
30,000 - 3 5 33,800 -19.3 Procedure A for Superfiltrol column step used for these samples. Tuna liver oil. Irradiatpd ergosterol in fish liver oil.
samples The original oil had been carefully bioassayed previously and found to contain 500,000 U.S.P. XI11 units of vitamin D1 per gram. This potency was also checked several times wing the spectrophotometric method of Ewing et al. ( 5 ) , which gave an average value of 474,000 units. Using the former value, dilutions of the original oil were made with corn oil to give the units. This resulted in calculated potencies indicated as U.S.P. a series of samples ranging in potency from 44,000 to 2980 vitamin D units per gram. One or more replicate samples of each group having similar potencies were assayed. I n Tables I1 and I11 are listed the results obtained for the following sample types: (1)crystalline vitamin D, and vitamin A acetate in corn oil, (2) irradiated ergosterol and vitamin A palmitate in corn oil, (3) some fish oils, and (4) some miscellaneous samples. For type 1, samples of the indicated potency were made up in corn oil using pure crystalline vitamin D, and crystalline vitamin -4acetate (potency given as 2,906,000 U.S.P. units per gram). For type 2, an oil containing 810,000 U.S.P. units of vitamin A palmitate and 162,000 U.S.P. units of vitamin D.: per gram (present as a bioassayed irradiated ergosterol) was diluted as required with corn oil. Where it was desired to alter the r:itio of vitamin 9 to D in this oil, either irradiated ergosterol or vitsmin A palmitate of a known potency was added as required. I n the columns headed U.S.P. units of vitamin A or D per gram of oil are given the assay values for the original oil, or those cxlculated from the known weight dilutions of these original oils. Knlese otherwise indicated after the sample number, all the determinations listed were made on 1-gram samples. DISCUSSION
The t:thles list determinations made on samples of irradiated ergosterol in corn oil, crystalline vitamin Dz and vitamin -4in corn oil, irradiated ergosterol and vitamin A palmitate in corn oil, two fish liver oils, and some miscellaneous samples. Of the 110 determinations listed, only five differ from the bioassay value (or
1409
V O L U M E 2 6 , NO. 9, S E P T E M B E R 1 9 5 4 the bioassay value calculated from the dilution factor) by niore than 20%. I n Figure 1 are given some typical absorbancy curves for thc indicated samples in Table 111. The maximum absorbancy is a t 265 mp, identical with that, for pure vitamin D, and Ds.The distortion of the absorption spectra, although very slight in the critical region of 260 to 2 i O mp, is more marked in the region of 230 to 240 m,u, especially for samples containing smaller amounts of vitamin D. Three principal factors must be considered in stating the range of application for the proposed method. For samples containing less t,han about 4000 U.S.P. units of vitamin D per gram of oil the error begins to rise rapidly, and this value may be taken at; a lower limit for the satisfactory determination of vitamin D in oil samples. Furthermore, t h r ratio of vitamin -4t o D is important, and for the procedure outlined the error begins to rise rapidly when this ratio exceeds 10 to 1. Some limited work using an S-cm. Superfiltrol column indicates that oils with a soniewh:rt higher ratio of -1to D may be satisfactorily determined (in the neighborhood of a 15 t o 1 ratio). Lastly, the amount of vitamin -4 introduced onto the Superfiltrol column is important, and sliould not exi,eed the saturation levels given in the procedure. Kithin the above stated limitations, very satisfactory deterniinations may be made, and for the results given the mean deviation varied from - 12.9'% to +9.3%. The interferences which determine the range of application of the method stem from two sources. The first is due to a enixll amount of residue in the oil itself which cannot be completely removed. Ite cont#ribution t,o the total light absorption i n the final step becomes more pronounced as the amount of vitamin 1) decreases and consequently the error increases. Where thc oil content per gram is low in relation to the vitamin D present, smaller amounts of vitamin D can be satisfactorily determined (around 3000 U.S.P. units). The second source of interference i p the decomposition product(s) of vitamin A formed on the Superfiltrol column. Wlien present in large amounts, a portion of it tends to be eluted with the vitamin D from the alumina column. Its absorption curve is similar to t h a t obtained by Enibrec and Shantz ( 4 ) for subvitamin 4 and kitol. Green also ohaervrd similar absorption curves in his work with vitamin A on floridin earths ( 7 ) . A larger Superfiltrol column, however, will allow B greater amount of vitamin -4 t o be introduced onto the column,
'Table 111. Potency of Samples C o n t a i n i n g Both \'itarnins .\ and lYL U.S.P.
Sauiple No.
Units of Vitamin A/G.
I..S.P. Units of Vitainin DIG.
U.S.P.
l; Dr) 12.200 io,m 10,600 - 0.1 - 3 4 12,200 1 0 ,G30 9,930 - 6.7
32 (2.0 g . ) 33 (2.0 9.)
34 (2.0 g . ) 35 (2.0 9 . )
.i,m
Abdcc llropsb 1,760 2, 1,7IiO 2,160 1 ,i m 1,620 1,760 2,170
nnn
Abdol Capsulese
f13.8 f22.8 - 7.9 f23.3
+ I 3 I1
+
1.200 9.1 + i i -I S,650 1.230 i13.7 Procedure B for Superfiltrol colunin s t e p used for these samples. Parke, Dayis 6- Co. water-inisrihle nliiltivitaniin preparation. Parke, Davis 6- Co. niultivitnriiin crapsille.
36 (3.0 g . ) 37 (3.0 6 . ) b
8,800 8,800 8,800 8,800
1.100 1.100
arid will yield satisfar~tory i,cwiltr a t higher vitamin .I to D ratios. ;ittempts to apply tlicl iiir~tliocl t o lialihut liver oils met \\-it11 failure due to the prc'seiirc oi' t i t i interfering substancc whose chromatographic hehavior 011 110th rolunins, under widely differing conditions, was identiwl {vith that of vitamin D.
0 26
0 24
LITER.4TUKE CITED
> V
20.22 a m
.\Inore, L. P.. Coutrib. Can. B i d . F i ' a h Fries, 7 , 413 (1933). ( 2 ) Drunimond, J. C., et al., I t i a l y s t . 71, 261 (1946). (:
