III

II

IIIIIIIIII

I
I

‘ IIIII

SEQ III

 

I. THE ULTRAVIOLET ABSORPTION
OP VITAMIN ((1 AND THE
EFFECT OF LIGHT

I}. THE QUANTITATIVE

CHROMATOGRAPHIC DETER-
MINATION OF VITAMIN D IN

FISH LIVER OILS

The-sis for the Degree of Ph. D.
I‘I’IIr LIIGAN STATE I. OLLEGE

Frank Sargent Tom kins

I942

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
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_ DATE DUE DATE DUE DATE DUE

6/01 c10|F|CIDateDuap65pJS

 

I. THE ULTRAVIOLET ABSORPTION OF VITAMIN K1 AND THE
MECT OF LIGHT

II. THE QUANTITATIVE CHROMATOGRAPHIC DETERMINATION OF
VITAMIN D IN FISH LIVER OILS

by
Frank Sargent Tomkine

A THES IS

Presented to the Graduate School of Michigan State College of
IAgriculture and Applied Science in Partial Fulfillment
of Requirements for the Degree of Doctor

of Philosophy

Department of Chemistry
iMichigan State College

1942

“I

 

. acmonnncmrnm

The'writer wishes to express his
appreciation to Dr. D. T. Ewing for his help
and guidance throughout this investigation; and
to Parke-Davis and Company, without whose assis-

tance the work would have been impossible.

he
chl
CD
p...
33
CD

THE'ULTRAVIOLET ABSORPTION OF VITAMIN Ki.AND THE EFFECT OF
LIGHT ON THE VITAMIN

The relationship between chemical structure and
ultraviolet absorption spectra is nowhere illustrated bet-
ter than in the study of vitamin K and related compounds.
One generalization which we presented in a previous pub-
lication(1), namely that the vitamin K absorption curve
presents a summation of the benzenoid and the quinoid
components of the disubstituted naphthoquinone molecule,
recently has been extended by Morton and Earlan(2)to the
anthraquinone series. Because of the importance of this
aspect we have reinvestigated the structure of the ab-
sorption curve of the vitamin and have studied in detail
the influence of lightand other factors such as the
presence of acetic acid which sometimes is added as a
stabilizer. It was hoped also that the results of this
detailed study might clear up the controversy between
Karrer(3)(4)and Doisy(5)(6)regarding the absorption
coefficients of the pure vitamin. .Another object of our
'work was to check the identity of the natural with the

synthetic vitamin.

-2-
Apparatus and Materials

The samples of vitamin [1, both natural and
synthetic, used in this investigation were prepared by
Doisy and his associates at the Saint Louis University
School of Medicine. They were examined with a Bausch
and Lomb medium quartz spectrograph and ultraviolet
sector photometer, with a Hilger No. 3-698 hydrogen
discharge tube as the source of continuous ultraviolet -
light.

The hexane used as the solvent in this study
was the Eastman "Practical” grade, which was purified
and redistilled. The purification process consisted of
five to ten shakings with 10% fuming sulphuric ac id, two
washings with 10% Nazca3 solution, prolonged shaking
with 5% mnO‘-10%Na2003 solution, fifteen to twenty
washings with distilled water, drying for twenty-four
hours over calcium oxide, and distillation twice over
freshly fused calcium chloride. The purified hexane
boiled at 64.5-65.o°c, and its absorption spectrum between

A200 and A800 mp. did not show the presence of impurities.

The source of irradiation for the study of the
effect of ultraviolet light on the vitamin consisted of
a General Electric no. 3-4 mercury arc lamp, a condensing
lens, a Cenco ultraviolet transmitting filter, and a
Cenco infra-red absorbing filter. This combination

transmitted only the loose and 366.3 mp. lines of mercury.

-3-

Bausch and bomb 10mm. absorption cells with
detachable quartz ends and monel metal fittings were
selected for this investigation. The spectra were re-
corded on Eastman No. 40 plates, processed five minutes

in Eastman Developer Formula Ho. D-19.
Expo rimental Part

Specimens of the vitamin weighing between one
and two milligrams were dissolved in sufficient hexane to
give a 0.0025% concentration on the weight-volume basis.
Since in earlier work a trace of glacial acetic acid had
been added to all specimens of thevitamin for preserving
purposes and a question had arisen as to whether or not
the acetic acid was affecting the absorption and should
be removed, measurements were made both in the presence
and absence of acetic acid to disclose this effect if
present. It was found that the presence of acetic acid
in amount equal to the weight of the vitamin had no
noticeable influence on the absorption curve.

In studying the effect of ultraviolet light on
the vitamin, the hexane solution was placed directly in
the absorption cell and the spectrum.of the unirradiated
emmple determined. The cell containing the solution was
then exposed to the ultraviolet light generated as des-
cribed above, at a distance of 30 cm., for a definite
length of time, after which the cell was removed to the

spectrograph and the absorption spectrum again determined.

 

500

 

 

 

 

 

 

Ix
[Isles
\

 

 

 

 

/00

 

 

 

 

 

 

 

 

 

 

240 280 320
Wan- Length, m/x

FIG. 1 ABSORPTION CURVE OF NATURAL
VITAMIN K1 IN HEXANE

-4-

Proceeding in this manner, a single sample served for a
complete run, and errors involved in transferring volatile
hexane solutions from one container to another were
eliminated.

After samples of each solution were measured
they were placed in tightly stappered flasks, weighed, and
stored in the dark. These samples were re-run from thme
to thmo to determine whether or’not the vitamin decomposed
upon standing in the dark.

Figure 1 presents the detailed structure of the
absorption curve of the natural vitamin, and Figure 2 that
of the synthetic product. It will be noticed that the
curves are essentially identical. They differ from the
curves previously published(1)in that they show a new
maximum at X239 my and a minimum at X240 my. In both
cases the highest maximum is found at A249 my and has
an extinction coefficient of 438. The extinction co-
efficients of the many samples of vitamin Kl’ both na-
tural and synthetic, which we have evaluated during the
course of these investigations indicate that the 15%“, of
the pure vitamin is 43515. This value is in good agree-
ment with that of 425, which we previously reported for
the synthetic product (Reference [IZ'page 356). We feel
that this value more accurately represents the extinction
coefficient than that of 540 which we reported in the
same publication (Reference [27, page 350, Fig. 6).

 

I00

 

 

 

<:'**'
S

 

 

 

 

 

 

I

/\

 

 

 

 

 

 

 

 

 

 

\\// \
280 320 360
Wave—Length. 777/4
ABSORPTION CURVE OF SYNTHETIC

VITAMIN x1 IN HEXANE.

-5-

It is known that vitamin.xi is affected by
light with loss of physiological activity and modifica-
tion of the absorption spectrum. No systematic study
of the effect of light has been reported and little or
nothing is known concerning the chemical change that
occurs in the structure of the vitamin when exposed to
light. Accordingly, we have made a study of the pro-
gressive changes that occur when hexane solutions of
the vitamin in a quartz container are exposed to ultra-
violet light ovor definite time intervals.

Figure 3 shows nine absorption curves for'a
sample of natural vitamin [1 in hexane solution in the
absence of acetic acid which was exposed to A365.5 and
366.3 my.lines of mercury radiation, readings being
taken at o, 15, so, 45, so, 90, 155, 195, and 255
minutes, respectively. It will be noted that the ex-
posure produces a gradual lowering of the maxima at
A239, 245, 249, 260, and 269 my, and a less pronounced
decrease in the maximum.at X325 mum indicating a
gradual decomposition of the vitamin.

Figure 4 shows a similar set of curves for a
sample of synthetic vitamin ll containing a small amount
of acetic acid, determined under identical conditions as
those illustrated in Figure 3. The curves are essential-
ly the same except for a slight stabilizing effect of the

 

 

 

 

 

 

 

 

E

g;
/2}
%

 

 

IOO

 

 

 

 

 

 

 

 

 

 

 

w 250 320 new
Wave {claw/I, my

FIG. 3 ABSORPTION CURVES SHOWING: THE
EFFECT OF ULTRAVIOLET RADIATIONS 0N
NATURAL VITAMIN K1 IN HEXANE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.00 II
II/I/I
/"v “I
{175200 (i I
MU
\\._/ \\'\\\

 

 

 

 

 

 

 

 

 

2” 280 320 360
Wave -L snarl), my
FIG. h ABSORPTION CURVES SHOWING THE

EFFECT OF ULTRAVIOLET RADIATIONS ON
SYNTHETIC VITAMIN K1 IN HEXANE

-5- A

acetic acid during the first few exposures.

In both of the above cases, the initial effect
is most pronounced on the maxima at A260 and A269 my»
which we previously have shown to be associated with the
quinone structure (Reference [:7, page 350). It follows,
therefore that the point of attack is through the quinone
grouping. In connection with these figures it is interest-
ing to note that the nine curves intersect at approximately
A277 my, and that less definite iso-extinction coefficient
points occur at A2301mp.and A305 mp.

NO attempt was made to correct for the effect
of exposure of the sample to ultraviolet light from the
hydrogen discharge tube during the exposure of the plate,
because a series of runs made for the purpose showed that
the effect of this light was negligible.

MacCorquodale, Binkley, et al.(7)report that
vitamin K2 is unstable when exposed to light, and curves
'were presented in our previous article showing the de-
terioration of the absorption curves of samples of
vitamins K1 and [2 when exposed to diffuse daylight. In
order to determine, if possible, the wave lengths or
'wave length of light causing this decomposition, samples
of vitamin Ki'wero exposed to various wave length regions

from.infra-red to ultra-violet. The results are shown

in the following table.

 

 

 

Source of
Radiation Filters Used Time Effect
Nornst Glower None 3 hrs. Slight decomp
position
' " Zeiss R-30 " ' lo effect
Tungsten Filament Wratten 45 min. u w
Lamp ELF
' ' " Wratton a . u ,
IBI
" ” " Wratten s w w u
"OI
‘Hg.Arc Cenoo ultra- 15 min. Decomposition
violet transmit-
ting plus Cenco
infra-red absorb-
ins
Diffuse daylight lens 2 hrs. Decomposition

 

These data show that light radiations between
A400 and 800 mp.have no appreciable effect on the vitamin.
The slight decomposition shown in the case of the Hornet
glower with no filter was probably due to the ultraviolet
light present in the radiation from the incandescent
filament of the glower. Thus, the decomposition report-
ed earlier as being due to the effect of visible light
probably was due to the small amount of ultraviolet pres-
ent in diffuse daylight.

We reported previously that vitamin Ii was un-
stable in hexane solution in the dark. However, using

specially purified hexane, we can now report that vitamin K1

-3-

in dilute hexane solution is stable for periods up to

five months when stored in the dark at room.temperature.

Discussion

Since narrer and Doisy first began publication
on vitamin Ki there has been a discrepancy between the
two laboratories concerning the correct value for EIém at
A249 my. In their first publication Dam et al.(8)gave
a value of 250 and McKee et al.(5)a value of 385. In
subsequent publications Karrer(3)(‘)has clahmed that
his vitamin preparation was pure and that 280 was the
correct value for the extinction coefficient. In an
effort to discern the cause of the discrepancy, we have
made numerous measurements on both natural and synthetic
vitamin K1 samples which were prepared and supplied to
us by Doisy. We find that the égm at A249 mp, is 435:5
(105 3m 3 4.29). This value is in good agreement with
values reported by D. M. Bowen(9)(log Tm.= 4.24-4.27)
(in alcoholic solution) and T. J. Webb‘9)(1og Tm : 4.26)
(alcoholic solution). We believe that this is the
correct value for either pure natural or synthetic vit-
amin K1 in hexane solution.

Of particular interest in connection with

the controversy between the two laboratories is the

fact that(both)groups agree on the values forififignfor

1)(4
vitamin K2 and for the diacetate of dihydrovitamin

(1H4)
Ki (log Em.= 4.93) .

In view of our close agreement with other
laboratories it is Iarrer's responsibility to explain
his low values for lTém for vitamin Ki. From a
structural point of view the chief difference between
vitamin Xi and vitamin I2 is the size of the side-chain
in the 3-position. Since the side-chain in each case is
aliphatic and contains no conjugated double bonds, the
absorption spectra for the two compounds would be ex-
pected to be quite similar. If the absorption is due to
the naphthoquinone portion of the molecule and is not
influenced by the size of the aliphatic side-chain in
the 3-position, the $2,, values for the two vitamins
should be inversely preportioned to their molecular
'weights and the log !m values should be equal. The
same reasoning holds true for the diacetates of vitamins
Xi and K2. IActually this is the case. The log Em values
for vitamins Xi and 13 are 4.27 and 4.29 respectively,
and the values for the corresponding diacetates are 4.93
and 4.93. It is significant that the molar extinction
coefficients obtained for vitamins I; and Kg agree well
‘with values obtained by Tishlor et al.(9)for three
crystalline 2,3-dialky1-l,4-naphthoquinones. These
quantitative relationships are good evidence in support
of the correctness of our values.

A comparifon of the absorption curves previous-

ly published by us , as well as those now presented,

-10-

(8)
‘with that illustrated in the article by Dam, ot al.

suggests that there might be a proportionate dis-
crepancy between the heights of the respective extinction
coefficients at A249 my. and A325 mu. As a matter of
fact, the discrepancy is not serious. Karrer's curve

‘was plotted as log E§§m versus wave-length, which tends

to enhance the 325 m. maximum, and was compared in

1%
lcm

wave-length. This latter method shows better the fine

this form with our curve which was plotted as E versus

structure in the region A239 mp.to A270 my, but gives
a less pronounced maximum.at A325 my.

An examination of the absorption curves shows
ing the effect of ultraviolet light on solutions of
vitamin K1 gives a certain amount of information as to
the actual chemical change involved. We have stated
in the experimental part of this paper that the point of
attack probably is through the quinone grouping, and
may add that the A325 mu maximum which we previously
associated with the ring structure changes more slowly
than the rest of the curve.

24Methyl-l,4-naphthoquinone upon exposure to
light for long periods of time is decolorized and
forms a polymer of known structure(10). It is possible

that a similar reaction occurs when vitamin K1 is ex-

posed to light.

-11-

Summary

1. A.more careful examination of the absorp-
tion curve of vitamin Ii shows the presence of a new
maximum at A259 mu.

2. The 1&5 of pure vitamin 11 at A249 mr-is
455:5. '

3. Vitamin K1 in hexane solution is stable
upon standing in the dark at room.temperature for as
long as five months.

4. Vitamin K1 in hexane solution is decomp
posed rapidly by the action of ultraviolet light, while
visible and infra-red radiation have no effect. The
point of attack probably is through the quinone group.

5. The presence of acetic acid has no
noticeable effect on the absorption curve.

6. 0n the basis of the absorption values re-
ported by Dam, Geiger, Glavind, Karrer, Rothschild, and
Solomon, their product appears to have been 60 to 80

per cent pure.

*********

4-12-

Rofer cases

(1) Ring, D. T., Vandenbelt, J. M., and Kamm, Oliver,
J. Biol. Chem. 33;, 545 (1959).

(2) Morton, R. A. and Earlan, W. T., J. Chem. Soc.

_1_5_9_ (1941).

(3) Karrer, P. and Geiger, A., Helv. chim. Acta
3.3, 945 (1959).

(4) Karrer, P., Geiger, A., Legler, R., Ruegger, A.,
and Solomon, R., Helv. chim. Acts 22, 1464 (1939).

(5) McKee, R. 91., Binkley, S. B., MacCorquodale, D. W.,
Thayer, S. A., and Doiey, E. A., J. Amer. Chem. Soc.
g, 1295 (1959).

(6) Binkley, S. B., MacCorquodale, D. W., Thayer, S. A.,
and Doiey, a. 11., J. Biol. Chem. $.52» 219 (1959).
('7) MacCorquodalo, D. W., Binkley, S. B., McKee, R. 91.,
Thayer, S. A... and Doiey, I. A., Proc. Soc. Expor.

Biol. 4 Med. 59, 482 (1959).

(8) Dam, B., Geiger, A... Glavind, J., Karrer, P.,
Rothschild, B., and Solomon, R., Helv. chim. Acta
23, 510 (1959).

(9) Tishler, M., Fieser, L. P., and Wendler, N. In,

J. Amer. Chem. Soc. _6_2_, 1982 (1940).

(10) Rev. Acad. Cienc. 33;, 617 (1934).

 

-1-
II

THE QUANTITATIVE CHROMATOGRAPHIC DETERMINATION OF
VITAMIN D IN FISH LIVER OILS

The quantitative determination of vitamin D
by chemical means has been the object of several inves-
tigations in recent years. Brochnann and Chen(1)report-
ed that vitamins D2 and D3 when treated with a solution
of antimony trichloride in chloroform give an orange-
yellow color which soon reaches a maximum at A500 my,
and that the measurement of the absorption at this wave-
length gives a measure of the amount of vitamin present.

This procedure has been reinvestigated and
amplified by Marcussen(2), Nield, Russell, and Zimerliwz
and Miles, Hoggio, and Reynolds“). In each of these
reports it has been shown that the Brockmann and Chen
color reaction is applicable only where the vitamin is
present in the pure form or where interfering biological
materials have been reduced to very low concentrations.
The most troublesome of these interfering materials are
vitamin A, sterols, irradiation products of the sterols
other than vitamin D, and the tocopherols. Among the
methods suggested for separation of such materials were
chromatographic adsorption, freezing out of sterols, and
the treatment of the sample with maleic anhydride for

the decomposition of vitamin A.

-2-

The method to be described here involves an
improved chromatographic treatment to remove vitamin.A,
the removal of interfering sterols by freezing and pre-
cipitation with digitonin, and the measurement of the
absorption at A500 muprodueed when the purified sample
is added to a solution of anthmony trichloride in

chloroform.containing a small amount of acetyl chloride.

Apparatus and‘Materials

The extinction coefficient measurements were
made on a Bausch and Lomb polarization type visual
spectrophotometer. The absorption cells were Bausch and
Lamb one centimeter cells with glass spacers, quartz and
plates, and monel metal fittings. Adsorption tubes for
the chromatographic procedure were made by sealing a
10 cm. length of 7 mm. pyrex tubing to the bottom of a
5/8"x 6" pyrax test tube.

The SbClBCHClchzCOCI reagent was prepared
according to Nield et al‘z), by dissolving 22g. of C.P.
antimony trichloride in purified chloroform, diluting
to 100 m1., and adding 2 ml. of redistilled acetyl chlor-
ide to the resulting solution. The chloroform for this
reagent must be carefully purified and dried as follows:
Shake repeatedly with distilled water to remove the
alcohol, distill and discard the first quarter of the
distillate, reflux for two hours over P205 and filter.

-3-

The filtrate is shaken with activated carbon to remove
the small amount of phosgene formed during the refluxing
with P205, filtered and the filtrate redistilled. The
purified chloroform should be stored in the dark. This
product is relatively unstable and the purification
should be carried out on small lots.

The mixed solvent for the chromatographic pro-
cedure was prepared by adding 10 parts of C.P. anhydrous
ethyl ether to 50 parts (by volume) of commercial hexane
(Eastman Practical grade).

The other was purified by washing repeatedly
with distilled water, distilling, and redistilling over
sodium.

The hexane was used as received.

The adsorbent was a fine grade of activated
Bentonite clay or "Superfiltrol".

Samples of fish liver oils and vitamin D

concentrate were supplied by Parke-Davis and Company.

Experimental

A.sample of fish liver oil or concentrate to
contain 20,000 to 80,000 vitamin D units was weighed,
10 ml. of N/2 alcoholic KOH added, and the sample heated
for one hour on the steam bath. The resulting solution
was cooled, 20 ml. of distilled water added, and the
solution extracted three times with 25 ml. portions of

other. The combined ether extracts were washed with

Dark blue____ 7/ \
Yellowish green ----- 5.7: \

90": \
eye. )— -Activated
Colorless—— - — 3:3; ,’ bentonite

CSTIction Re- — — Cotton pad

 

 

Fig. 1. SCHIMATIC DIAGRAM SHOWING CHROMATOGRAPH
SET-UP AND APPEARANCE 0F BANDS AT END OF RUN.

-‘-

distilled water in a separatory funnel until the wash-
ings gave no reaotion with phenolphthaloin, dried over
anhydrous sodium sulfate, and filtered.

The filtrate was evaporated to dryness under
reduced pressure, the residue taken up in 10 ml. of
absolute methanol, and 5 ml. of the resulting solution
pipetted into a 15 ml. centrifuge tube. The centrifuge
tube and metal holder were cooled to -15° C. in an
acetone bath kept at this temperature by additions of
dry ice. The cooled solution was centrifuged, the super-
natant liquid poured off, the precipitate washed twice
‘with 2 ml. portions of methanol--cooling and centrifuging
after each addition--and the liquid and washings trans-
ferred to a second 15 ml. centrifuge tube.

To the resulting 9 ml. of solution was added
1 ml. of distilled water and 2 m1. of 29!, digitonin in
90% methanol solution and the mixture allowed to stand
at least two hours. at the end of this time the mixture
‘was centrifuged and the precipitate washed twice with
2 ml. portions of 90%:methanol; the washings were added
to the bulk of the separated liquid. The solution was
then evaporated to dryness under reduced pressure and
the residue taken up in 5 ml. of the ether-hexane mixture.
The resulting solution was chromatographed as follows:

The adsorption tube was prepared by placing
a mmall cotton wad in the bottom of the tube and adding
the adsorbent to a depth of about 50mm., tapping the tube

-5-

aftcr each small addition to pack the adsorbent. Slight
suction (4 cm. Hg) was applied to the tube and 5 ml. of
the mixed solvent added tO‘IOt the adsorbent. The 5 ml.
sample from.the sterol separation was then added, followed
by 5 ml. of the solvent used to rinse the flask, and
finally 20 ml. of the solvent to develOp the colored bands
formed in the adsorption tube. Each addition of solvent
was made before the liquid from.the previous addition had
quite disappeared, in order that drying out of the ad-
sorbent be prevented. if the column becomes dry, further
addition of the solvent destroys the bands and the sample
is lost. During the washing of the column with the last
20 ml. of solvent a very narrow and sometimes rather in-
distinct yellowish-green band appears at the lower edge
of the blue band formed by the vitamin A. This band
provides a convenient reference point for the separation
of the column later and should be marked Just before the
last of the wash sdlvent disappears. When the last of

the liquid had gone through, the column was dried by
pulling air through it for five to ten.minutee and the
upper portion of the column--to the mark previously made--
removed with a bent spatula and discarded. This portion
of the column contains all of the vitamin A. The remain-
ing portion of the column was removed to a small erlen-
meyor flask and the adsorption tube rinsed with 25 ml.

of anhydrous ether which.was added to the same flask.

This ether-adsorbent mixture was shaken vigorously,

-6-

allowed to stand until the adsorbent had settled, and the
supernatant liquid poured off through a coarse filter
into a second erlenmeyer; This extraction was repeated
with three additional 25 ml. portions of other and the
combined filtrate added to that from.the adsorption pro-
cess. This solution was evaporated to dryness under
reduced pressure, taken up in 10 ml. purified chloroform,
and 1 m1. of the resulting solution added to 10 ml. of
the antimony trichloride reagent. The log 10/]: value of
the resulting Mixture at A500 “P" was determined, using
the Bausch and Lamb spectrophotometer, and the ngm
value calculated. I

The procedure may be interrupted after the
saponification and extraction, or after the extraction
of the lower portion of the adsorption column, when the
vitamin D active materials are in other solution. Such
a solution can stand overnight without measurable loss

in potency.

Data and Results

Table I shows the values obtained on a series
of seventeen fish liver oil and vitamin.D concentrate
samples by the procedure described above. The calculated
potency values are on the basis of sample #44090 taken
as a standard. In each case the l§§m value given in the
table is the average value obtained from a set of two

duplicate samples.

-7-

 

 

 

 

 

 

 

 

Sample Type 1%& Bio. lpotency Ca%378£ctcncy
44090 Vit. D Distillate 1.30 22,000 22,000
44080 Vit. D Distillate 1.74 31,000 29,500
47761 High vit. D Oil 1.01 15,000 17,100
41860 High vit. D Oil .807 16,000 13,600
56021 High vit. D Oil .670 13,000 11,300
55691 High vit. D 011 .709 12,000 12,000
40090 High vit. D Oil .912 20,000 15,400 //

18119 Bonita liver oil 1.09 36,000 18,400 /
55031 Vit. D Distillate .859 18,500 14,600
44470 Tuna liver conc. 14.2 240,000 240,000
56961 Vit. D Distillate 1.05 20,000 17,800
57481 High vit. D Oil .495 15,000 8,400 v/”
57951 Blue fin tuna oil 1.43 28,000 24,200
57971 Albacore liver 011 2.80 55,000 47,400
57991 Yellow fin tuna oil .946 3,000 16,000 /'
58011 Bonita liver oil 5.20 65,000 54,000 x
57381 Tuna liver conc. 15.1 260,000 255,000

Table I

Sample #18119 was in very poor physical condition
--discolored and full of sediment and suspended particles--
‘which.may account for the very low result obtained in this

case. There is no obvious explanation for the low value

obtained for sample #57481 or the high value for #57991;

assuming that the biological value is correct as given.

-3-

The values obtained by the chromatographic
procedure show good agreement with the biological values
--with the exception of the instances cited above--and
represent an appreciable saving in time and expense over

the biological assay method.

Discussion

Vitamin D containing biological materials such
as milk, butter, yeast, fish liver oils, etc., are ex-
ceedingly complex mixtures in which the vitamin D, although
it may be very active biologically, represents but a
minute fraction of the total amount of material present.
At the same time the vitamin "D" in the given material
may be any one of many vitamin D active substances, or a
combination of two or more such substances.

Fish liver oil, for example, is a mixture of
fatty acids, sterols, carotenoids, vitamin A, vitamin "D",
and in some instances tocopherols; in which, as it has
been shown by Brockmann and Busse(5), the characteristic
antirachitic principle is vitamin D3, the vitamin D3
being accompanied by varying quantities of vitamin D2.

The problem of chemically determining the antirachitic
potency of such a sample then, offers two possibilities.
Either we must separate the vitamin D active principle
from the bulk of the material sufficiently well that the
interferences from these materials will be small, or we

must find a reaction specific to the vitamin D active

-9-

substanco which will work in the presence of other bio-
logical materials and be sensitive to the small amount

of vitamin D present. Since the first alternative seemed
to offer the most possibilities, it was along this line
that the work was directed.

The antimony trichloride color reactions of
vitamins D2 and D3(TT° identical, as first stated‘gy
Brockmann and Chen and confirmed by Nield et a1 ,
and the improved reagent described by the latter in the
same paper gives agroatly increased sensitivity to the
test. The difficulty in the use of this reagent lies in
the fact that vitamin A, certain sterols, and fatty acids,
carotenoids, and tocopherols; all give color reactions
which interfere with the vitamin D color. The vitamin A
color is particularly intense and troublesome.

Miles, Reggie, and Reynolds(4)describe a
method in which the non-saponifiable fraction of a fish
liver oil is heated with maleic anhydride in 1,4 dioxane
in order to destroy the vitamin A, carotenoids, and
possibly 7-dehydrocholesterol; after which the treated
non-saponifiable fraction is added to antimony trichloride
solution and the rig, at A500 n,» taken as an indication
of the amount of vitamin D present. Several samples were
run using this method but consistent results could not
be obtained. Upon further investigation it was found

that, contrary to the statement of the authors, maleic

anhydride in dioxane reacts with vitamin D under the

-10-

conditions recommended and results in the destruction of
part of the vitamin D as well as the vitamin A. This
‘was tested by running samples of irradiated ergosterol
in corn oil, containing no vitamin A,'with and without
the maleic anhydride treatment. The treated samples
gave lower ngm values at A500 mfrwith antimony tri-
chloride than did the untreated samples, as shown in the

accompanying Table II.

 

 

 

Sample no. Treatment sigh
55209“ As received 7.71
' Non-saponifiable fraction 8.12
" ‘Milas procedure using fresh maleic 2.79
anhyd. and commercial dioxane
" Miles procedure with Eastman Kodak
Co. maleic anhyd. and commercial 3.76
dioxane
' fMilas procedure with fresh.maleic 3.59
anhyd. and purified dioxane**
" Heated 1 hr. with dioxane alone 7.52
" Heated 1 hr. with dioxane and .23 4.93
maleic anhyd.
l'(I.) Miles procedure , 4.02
"(IIa) Miles procedure 3.50
"(Ib) iMilas procedure plus 15 min.
heating after second addition 4.87
of XOR ‘
"(IIb) iMilas procedure plus 15 min.
heating after second addition 3.63
of XOR
Table II

* Sample #33209 is irradiated ergosterol in corn oil.

** The dioxane used in these tests was purified accord-
ing to: g; Amer. Chem. Soc. §§, 2264 (1936)

-11..

These results show that in any case in which
the sample is heated with maleic anhydride, a breakdown -
of the vitamin D2 occurs; and that the breakdown is not
due to impurities in the dioxane. The last four samples
‘were heated after the second addition of alcoholic KOH
to determine whether the breakdown product could be con-
verted back to the free vitamin. Hewever, if the effect
takes place it is very sma11--as shown by the very slight
increase in 3%gl value of the heated samples. This
indicates that either the loss in vitamin D2 upon heat-
ing with maleic anhydride is not due to esterification
or that the vitamin D2 esters are not easily hydrolyzed.

Attention was next directed toward chromato-
graphic adsorption as a means of separating vitamin D
from interfering materials. Marcussen 2 reported that
an effective separation of vitamin A from vitamin D
could be made by adsorbing the vitamin A from.a heptano
[solution of the non-saponifiable fraction of a fish liver
oil. Hydraffin 14, an activated carbon, was used as the
adsorbent. Since neither Hydraffin Kg nor heptano was
available, a series of tests were made using Norite A.as
the adsorbent and hexane as the solvent, following the
Marcussen procedure in other respects. It was found
that only a slight separation of the two vitamins was
possible by this method--both vitamins being very strong-
ly held by the Noritc A. Slightly better separation was

obtained when a small amount of other was added to the

VD

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/.6

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/ I
/ I

/I I '
\

 

 

 

 

 

 

 

\
‘\~

 

 

 

 

 

 

 

 

 

 

4&9 JR? 650 XX?
Wave-Length, rn/u

FIG. 2 ABSORPTION CURVE 0F IRRADIATED
ERGOSTEROL IN CHLOROFORM TREATED WITH
813013 REAGENT

O

5‘)

 

 

 

[.10

int}; fl

 

 

 

 

 

 

I
I

\

450 .550 650 7.50
Wave-Length, m/u

 

 

 

 

 

 

 

 

 

 

 

FIG. 3 ABSORPTION CURVE OF CHOLESTEROL

IN CHLOROFORAI TREATED WITH SbCll.3 REAGENT

-13-

hexane, but there was still too slight a difference in
the adsorptive power of the Norite A, with respect to
the two vitamins, to make the separation complete.
Several other adsorbent-mixed solvent can»
binatims were investigated before the activated benton-
ite-ether-hexane combination was found. These included
activated aluminum oxide, calcium.hydroxide, dicalcium. '
phosphate, zinc carbonate, and.bentonite; chloroform,
benzene, carbon tetrachloride, and hexane. 0f the ad-
sorbents, zinc carbonate, bentonite clay, and activated
bontonitc were the only ones which gave positive results.
The action of the zinc carbonate was particularly
interesting because of the large number of sharply de-
fined bands which were visible in ultraviolet light after
the non-saponifiable fraction of a fish liver oil had
been run through a column of this material. No sharp
separation of vitamins A and D was obtained with this
adsorbent, but a.moro complete investigation of the bands
produced would make an interesting problem. In each of
the above trials the adsorption column was out into
sections, each section extracted with other, the result-
ing solution evaporated to dryness and the residue
taken up in chloroform, and antimony trichloride solution
added. A qualitative examination of the absorption
spectrum of the resulting solution, particularly in the
regions A620 and 500 my, gave an indication of the degree

of separation of vitamins A and D.

 

[.5

 

 

/20 \\I {‘A

 

. I / I
(“1%-

 

 

 

 

 

I

 

 

 

 

 

 

 

 

 

 

 

450 550 650
Wave-Length m/u
'FIG. I. ABSORPTION CURVE OF ADSORBATE

FROM CHROMATOGRAPH COLUMN IN CHLOROFORM
TREATED WITH 811013 REAGENT

7.50

 

M N

 

 

 

E“ \

lc'm. \
4.0

 

 

 

\

 

\

240 280 320 350
Wave-Length, m/L

 

 

 

 

 

 

 

 

 

 

FIG. 5 ABSORPTION CURVE OF IRRADIATED
ERGOSTEROL IN CORN OIL IN HEXANE '

 

 

 

 

 

 

 

.60
£13...
N
\
\

 

 

X,

 

 

 

 

 

 

 

 

Z40

 

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280
Wan-L enjth, Tn/u-

»\‘L—52o

'0

FIG. 6 ABSORPTION CURVE OP ADSORBATE
FROM CHROMATOGRAPR COLUMN IN HEXANE

-13-

The first positive separation was obtained
‘using bentonite as the adsorbent and a hexane solution
of the saponified sample. With this combination a blue
band was formed at the top of the adsorption column
'which when extracted and tested with antimony trichloride
solution, showed very strong absorption at A620 my.
This was due to the presence of a large concentration of
vitamin A. The lower portion of the column and the
filtrate, when tested.in the same way, showed strong
absorption at X500 my.and very slight absorption at
)680 mp. A better separation was obtained when a small
amount of ether was added to the hexane used as the sol-
vent. This was to be expected since the adsorption pro-
cess had already shown the vitamin D to be less tenacious-
ly held by the bentonite, and consequently the desorbing
action of the ether would be more effective toward this
vitamin. The final modification of the adsorption
procedure was the substitution of activated bentonite
for bentonite as the adsorbent. This is a finely divided
product which results in more uniform.adsorption columns.

Figure 2 shows the absorption spectrum of
irradiated ergosterol (vitamin D2), Figure 3 that of
commercial cholesterol, and Figure 4 that of the adsor-
bate from.the ehromatograph column--all after treatment
'with antimony trichloride solution. It will be noted
that Figure 4 is the type of curve which would be expect-

ed to result from a mixture of the pure vitamin D2 and

-14-

cholesterol. This is consistent with the statement by
earlier workers that sterols are among the interfering
substances present in fish oils. riguge 5 and Figure 6
show the similarity between the ultraviolet absorption
curves of the adsorbate from a chromatograph column and
a solution of irradiated ergosterol in corn oil, in hex-
ane.

Assuming that free sterols constitute the
greater part of the interfering materials left in such
an adsorbate, the next step was to find a means of re-
moving these sterols-~either before or after the
chromatographic adsorption. Freezing out of the sterols
from a methyl alcohol solution by cooltng to -15° C. was
tried, but it was found that the removal by this means
'was not complete. The method finally adopted combined
the freezing process with a precipitation of the remain-
ing sterols with digitonin--before the adsorption.

Figure 7 is the absorption curve obtained from.a sample

 

of the same oil as was used for Figure 4, after the
sterols had been removed by the above treatment. It
shows a marked decrease in absorption in the region below
460 m , and more nearly matches the curve for the pure
vitamin. It was this treatment that was incorporated

into the final procedure.

 

 

 

/.ZO

A
a 7

VI

 

 

 

 

\\
”AP/M

.40

 

 

 

 

 

 

 

 

 

 

\
4.50 5.50 650 7.50
Wave-Lenjtlo, m/u

 

FIG. 7 ABSORPTION CURVE OF ADSORBATE

IROM CRROMATOGRAPH COLUMN IN CHLOROFORM

TREATED WITH 81:01 REAGENT. SAMPLE
TREATED V TH DIGITONIN

Sumagy

l. A method for the. quantitative determination
of vitamin D in fish liver oils has been presented; in
which the oil is saponified, cooled and treated with
digitonin to remove sterols, chromatographed to separate
the vitamin A and D, and the vitamin D determined by
measuring the absorption at X500 my. produced when the
sample is added to a solution of antimony trichloride
in chloroform.

2. A'table is presented showing the results
obtained on a series of seventeen samples, using the

procedure outlined above.

*********

-16..

References

(l) Brockmann and Chen, Zeit. physiol. Chem.
241. 129-33 (1936)

(2) Marcussen, Dansk. Tids. Farm. ;2. 141-159 (1939)

(3) Hield, Russell, and Zimmerli, J. Biol. Chem.
136, 73-79 (1940)

(4) Milas, Heggie, and Reynolds, J. Ind. Eng. Chem.
Anal. Edillg, 227-231 (1941)

(5) Brockmann and Busse, Zeit. puysiol. Cham.
856, 252 (1938)

 

 

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