Standard of Purity for Cholesterol
Nathan Radin and Adalbert L. Gramza
Three recrystallization technics were used to fractionate five commercial cholesterol
products and one artificial mixture. One technic involved the recrystallization of
cholesterol from absolute ethyl alcohol, the second technic involved the recrystallization of cholesterol from glacial acetic acid, and the third technic used the dibromide
derivative method. The molar absorptivities for the various crystal batches were in
the range of 1610 to 1750 L. mole-’ cm.-t at 620 m with a modified LiebermannBurchard procedure. The molar absorptivities were in the range of 9,800 to 11,500 L.
mole-’ cm.-1 at 560 m1Lwith a modified sulfuric acid-iron method. The original
products and the ethyl alcohol recrystallized products showed molar absorptivities
at the lower limits of the ranges, while the glacial acetic acid and dibromide derivative recrystallized cholesterol showed molar absorptivities at the higher limits of the
ranges. Absorption peaks at 235 m for methyl alcohol solutions of the cholesterol
preparations were used to estimate cholesterol impurities on the basis of 7-keto
cholesterol acetate absorption values. The decrease of absorption in the ultraviolet
spectral region established that impurities were removed from the cholesterol preparations studied.
THE
determination
presents
many problems
as
shown by discussion
and the many papers
covering
the subject.
The
subject of cholesterol
as a primary
standard
is usually not discussed
in cholesterol
methodology
articles.
A reliable criterion
for establishing the purity of cholesterol
has not been established.
The investigation
reported
in this paper is concerned
with the evaluation of the purity of a number
of commercial
cholesterol
preparations, preparation
of pure cholesterol,
and a study of the properties
of the purified
cholesterols.
SERUM
CHOLESTEROL
From the Division
of Biochemistry,
Rochester
General
Hospital,
Rochester
8. N. Y.
Presented
at the Fourteenth
Annual
Meeting
of the American
Association
of Clinical Chem.
ists, Aug. 28, 1962, Santa Monica, Calif.
The authors
wish to thank Galla Radiii for her aid in the statistical
analyses
of our data.
Received
for publication
Oct. 29, 1962.
121
122
RADIN & GRAMZA
Clinical
Chemistry
Experimental
Recrystallizationand Characterization of Commercial Cholesterol Preparations
Commercial
cholesterol
is usually prepared
by solvent extraction
of
spinal cords and brains
of cattle (1). Cholesterol
is then crystallized
and recrystallized
directly
or after the esterified
cholesterol
has been
saponified
with alkali,
information
from various
commercial
sources
for laboratory
quantities
of cholesterol
shows that the U.S.P. specifications
(2) are now the accepted
standard
for cholesterol
purity.
Cholesterol Recrystallization
The meaning
of pure leads to various
definitions,
starting
with: a
pure compound
is one in which all molecules
are identical.
In this investigation
the definition
of purity
used is in terms of operations
to
be performed.
A system
of molecules
is a pure compound
if an exhaustive
series of fractionations
fails to produce fractions
with different properties.
What one calls a pure compound
thus changes as new
methods
become available
for separating
material
into fractions,
or
for more accurately
measuring
the properties
of the fractions
(3).
The guiding
principle
in the present
study was to prepare
and evaluate cholesterol
as a primary
standard
using the equipment
available
within our laboratory.
As usually prepared,
cholesterol
is contaminated
by cholestanol
(dihydrocholesterol
or cholestan-3
$-ol), cholest-7-en-3
$-ol (lathosterol
or 7-cholestenol),
and traces of 7-dehydrocholesterol
(cholesta-5,
7dien-3$-oI)
(1).
Three recrystallization
methods
were chosen
for this study.
One
involved
the recrystallization
of cholesterol
from ethanol,
one involved the recrystallization
of cholesterol
from acetic acid (4), and
one involved
the treatment
of cholesterol
in ether solution
with bromine, which precipitates
the insoluble
5, 6 ,8-dibromide
derivative,
which is then regenerated
with zinc and acetic acid (5). According
to
Fieser
(6) crystallization
of cholesterol
from ethanol does not remove
the cholest-7-en-3
$-ol and cholestanol
present,
but purification
through
the dibromide
eliminates
these companions
as well as two
products
of air oxidation,
7-ketocholesterol
and cholestane-3
$, 25-diol.
Cholesterol Recrystallizationfrom Ethanol
Cholesterol
lute ethanol
was added
(Publicker).
in the proportion
of 1 gm. per 5 ml. to absoA beaker containing
the mixture
was heated
Vol. 9, No. 2, 1963
STANDARD
CHOLESTEROL
123
on a hot plate, with mixing by swirling
until the cholesterol
was dissolved.
The beaker was then removed
from the hot plate, covered with
a watch glass, and was allowed
to cool at room temperature.
The
cholesterol
which precipitated
was separated
by filtration
through
Whatman
No. I filter paper iii a Buchner
funnel.
The damp crystals
were washed
with a small volume
of diethyl
ether, dried overnight,
then dried in an oven at 900 for 2 hr., and finally stored
over phosphorus pentoxide
in a vacuum desiccator.
Cholesterol Recrystallization from Acetic Acid
Glacial acetic acid (Baker’s,
ACS) was heated to the boiling point
in an Erlenmeyer
flask and then poured into a beaker which contained
cholesterol.
Eight milliliters
of acetic acid was used for each gram of
cholesterol.
The mixture
was stirred vigorously
with a wooden paddle
until tile solid material
was all dissolved.
The beaker was then plunged
into an ice bath, the contents
being stirred
vigorously.
Wheii room
temperature
was attamed
the complex was collected
on Whatman
No.
1 filter paper in a Buchner
funnel.
After a washing
with acetic acid
and methanol,
the crystals
were air dried overnight,
and then dried in
an oven at 900 for 2 hr. The cholesterol
was then stored in a vacuum
desiccator
over phosphorus
pentoxide.
Recrystallization of Cholesterol with Dibromide Method
Fifty grams of cholesterol
was dissolved
in 350 ml. of ethyl ether by
gentle warming
on a steam bath. After cooling the resultant
solution
to room temperature,
a solution containing
22.7 gm. bromine,
1.67 gm.
anhydrous
sodium acetate, and 200 ml. of glacial acetic acid was added.
A white paste resulted.
The mixture was stirred
about 10 mm., during
which time it was cooled to 20#{176}.
The material
was transferred
to a
Buchner
funnel
containing
Whatman
No. 1 filter paper
and was
washed with acetic acid until the filtrate was colorless.
The white material was then suspended
in 750 ml. of diethyl
ether and 10 ml. of
glacial acetic acid. Ten grams of zinc dust was slowly added and a reaction was evidenced
by bubbles
of hydrogen.
A white precipitate
formed which consisted
of zinc salts, and this was dissolved
by the addition of 10 ml. of water.
The ether solution was then decanted
from
the water layer containing
the excess solid zinc. By use of separatory
funnels
the ether solution
was washed
twice with 150-ml. portions
of
acid solution
(6 ml. of concentrated
hydrochloric
acid for each 100 ml.
of water) and then washed with 10% (w/v) sodium hydroxide
solution
124
RADIN & GRAMZA
Clinical
Chemistry
to remove the acetic acl(l. Five hundred
milliliters
of niethanol
was
theii added to the ether solution
and tile resulting
solution was evaporated slowly oii tile steam l)ath to tile poiiit
where most of the ether
was removed
aiid purified
cholesterol
began to crystallize.
Crystallization i)i’oceeded at room
temperature.
The product
was collected,
air
dried overnight,
dried ill an oven at 90#{176}
for 2 hr. and then stored in a
vacuum desiccator
over phosphorus
pentoxide.
Characterization
of Recrystallized Cholesterol
The con,niercial
cholesterol
preparations
chosen for this study were
from Armour
1.harnlaceutica1
Company,
I’fanstiehl
Laboratories,
inc., Steraloids,
Inc., Gemieral Biochemicals,
Iiic., and K & K Laboratories, Inc. The commercial
cholesterol
preparations
were all white,
powdery
substances
except for the K & K preparatiomi,
wilicil
was yelof cholesterol
was made by Combinilig
and mixing an
old yellow Eastman
preparation
with equivalent
amounts
of material
from the other companies
and heating
at 100#{176}
for 6 hr. After heating,
tile cholesterol
pooi was dark yellow.
Anhydrous
cholesterol
is obtained
by crystallization
from dry solvents an(i forms triclinic
needles.
Cholesterol
separates
as tile monohydrate,
rllombic-shaped,
triclinic
plates from moist solvents
(1). The
ethanol-recrystallized
cholesterol
formed
a white,
fluffy crystalline
material;
the acetic acid-recrystallized
cilolesterol
formed
a white,
low.
A mixture
packed material;
and the cholesterol
from the dibromide
recrystallizatioii method came out as a lustrous
white powder.
The yellow color of
the K & K product
and the mixture
was not completely
removed
with
recrystallization
from ethyl alcohol or acetic acid.
The cholesterol
monohydrate
loses water at 70-80#{176}
(1).
Thus, in
order to ensure anhydrous
cholesterol
after air drying,
tile recrystallized materials
were heated
in an oven at 90#{176}
for 2 hr. Tile cholesterol
preparations
treated
this way showed no cilange from the original
appeararice.
However,
it would he safer to use a vacuum
oven for this
operation.
The weight changes
due to the loss of moisture
after heating
and
drying
known amounts
of the commercial
cholesterol
preparations
were shown to be negligil)le.
The melting point of all the preparations
are shown in Table 1. The
method for obtaining
the melting
points in evacuated
capillaries
was
described
by Fieser
(7). Fieser
has observed
that melting
points
taken in open capillaries
are often several
degrees
lower than those
,-,
-
,-,
-
-
-
-
-
-
-
-
-
0
-
I
-
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I
1
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,
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-
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o
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00
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z
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#{149}1’‘‘
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t’-
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126
RADIN & GRAMZA
taken in evacuated
point for cholesterol,
14.93-150.0#{176}(4).
Clinical
Chemistry
capillaries.
Fieser
has shown that the melting
purified by recrystallization
from acetic acid is
Determination
of Commercial and
Recrystallized Cholesterol
of Absorptivities
The spectrophotometric
technic for characterizing
the cholesterol
products
under study was chosen as tile equipment
is generally
available in the clinical chemistry
laboratory.
The extinction
coefficient for
cholesterol
in ethanol
in the ultraviolet
region of the spectrum
Ilas
been reported
(8). As the maximum
of the absorption
peak for cholesterol is reported
at 203 m any measurements
made with the Beckman
Spectrophotometer,
Nodel DU, in the spectral
region
with the wavelength less than 210 m would be open to question.
The molar absorptivities were determined
for two commonly
used color reactions,
the
Liebermann-Burchard
reaction
and the sulfuric
acid-iron
reaction.
The Beckman
Spectrophotometer,
Model DU, was used for measuring
absorbancies,
and the Model DB was used for recording
wavelengthtransmittance
curves.
The cell compartments
were kept at 25#{176}
with
water
circulating
from a thermostat.
Ten-millimeter
light-pathmatched cells (Quaracell,
OS and QS) were used for the spectrophotometric studies.
Liebermann-Burchard Reaction
A modified Liebermann-Burchard
procedure
was adapted
from the
Carr and Drekter
(9) cholesterol
technic. The procedure
used to measure the absorbance
values and to calculate
the molar absorptivities
was as follows:
I. About
0.1000-gm.
portions
of the cholesterol
samples
were
weighed,
using weighing
boats and were then transferred
to 100-mi.
volumetric
flasks, using glacial acetic acid (Baker,
ACS) as the wash
solvent.
The flasks were kept at room temperature
overnight
to permit complete
solution
of the cholesterol
preparations.
The solutions
were then diluted to mark with glacial acetic acid and mixed well.
2. Five-milliliter
aliquots
of each stock solution were pipetted
into
50-mi. volumetric
flasks, and 5 ml. of acetic acid was pipetted
into another 50-mI. volumetric
flask for the reagent blank.
3. Twenty-milliliter
aliquots
of acetic anhydride
(Baker)
were
pipetted
into each volumetric
flask.
4. A 20% sulfuric
acid-glacial
acetic
acid solution
(V/V)
was
Vol. 9, No. 2, 1963
STANDARD
127
CHOLESTEROL
poured
up to about
the neck in the first flask as a stop watch was
started.
After swirling
to mix, the flask was placed in a water bath at
25#{176}.
The same procedure
was followed
with other flasks at exactly
1-mill,
intervals.
Each solution was diluted to mark with the acid mixture, mixed, and returned
to the water bath at exactly 10 mm. after the
illitial
operation.
5. The absorbance
of each solution was measured
at 620 m exactly
20 mm. after the initial operation.
6. The molar absorptivity
was calculated
for each solution
using
the Bouguer-Beer
Law, A = abc, where A is absorbaiice,
a, molar absorptivity,
b, light path in centimeters,
and c, concentration
of the final
solution in moles per liter.
For each commercial
or recrystallized
product
two stock cholesterol
solutions
were prepared
at different
times.
Duplicate
absorbance
measurements
of the color reaction
described
were made for each
stock solution
3 times.
Thus, for each cholesterol
preparation
there
was a total of 12 absorbance
measurements,
and 12 molar ahsorptivities were calculated.
The wavelength-transmittance
curve for this reaction
(Fig.
1)
shows a maximum
absorption
peak between
620 and 630 m.
The
shapes of the curves were essentially
the same whether
the cholesterol
was a commercial
preparation
or a recrystaliized
product.
Running
the curves sequentially
showed that there was about a 1% transmittance change for every 11 mm. By maintaining
exact intervals
during
color development
and absorbance
readings
equal periods
for color
development
was assured.
The relation
between absorbance
and choIesteroI was shown to be linear at 620 m.
Table 2 shows the mean
0
U
20
z
Fig. 1. Wavelength-transmittance
curve for the Liebermann-Burcharcl
cholesterol 1)rOcedure.
40
60
80
100
700
600
WAVELENGTh,
800
MILI.IMICRON$
400
128
Table
RADIN & RAMZA
2.
(ommerriol
MOLAR
ABsoJtp’rlvrrlEs
absorptivity:
CHOLESTEROL
PROCEDURE
Dthr
21
EtOli
IX
EtOll
21
IlAc
11
RAe
21
(0*)
(a*)
(0*)
(a*)
(0*)
1650
1670
1660
1670
1690
1700
1690
1660
1720
1700
1650
1610
1710
1730
1740
1720
1690
1710
1730
1690
1720
1700
1700
1750
1710
1740
6071 I
liters
LIEBERMANN-BURCHARD
Chemistry
Origna1
product
Armour
Lot V 31105
Pfanstiehl
Lot 4907
Steraloid
Batch 4907
K&.KLot555OL
General
Biochemienls
Mixture
*Molar
FOR THE
Clinical
cm.
moles
-
.
Each
value
1740
1750
1740
-
is the
mean
(0*)
(0*)
1740
1740
1740
1730
-
DBr
IX
-
of 12 single
incas-
uremeats.
molar absorptivity
terol I)I’Oducts.
values
foi’ commercial
and
recrystallized
choles-
Sulfuric Acid-Iron Reaction
The procedure
shown below, for the sulfuric
acid-iron
reaction,
was
adapted
from the Rosenthal,
Pfluke, and Buscaglia
(10) cholesterol
technic.
1. Three-milliliter
ahiquots of the cholesterol
stock solutions
prepared
for the Liebermann-Burchard
reaction
measurements
were
diluted to mark in 100-mi. volumetric
flasks with glacial acetic acid.
2. A stock reagent
was prepared
by dissolving
and diluting
5 gm.
of ferric
chloride
(FeCla
6 H20, Baker,
ACS)
with concentrated
ortilo-phosphoric
acid (85%, Baker, ACS) to mark in a 30-ml. volumetric flask. Five milliliters
of the stock reagent and 15 ml. of concentrated
phosphoric
acid were diluted
to mark with concentrated
sulfllric acid in a 250-mi. volumetric
flask for the coloring
reagent.
This
dilute coloring
reagent
was freshly
prepared
every day that measure.
ments
were
made.
3. Five-milliliter
ahiquots of each diluted cholesterol
solution
(Step
1) were transferred
into 50-ml. Erlenmeyer
flasks. Five milliliters
of
glacial acetic acid was transferred
into a flask for the blank.
The
flasks were then placed in a water bath at 25#{176}.
Four milliliter
ahiquots
of the dilute coloring reagent
was transferred
into each flask at minute
intervals
and mixed
by swirling.
4. The absorbancies
for each solution were measured
at 560 m 30
mm. from the initial addition
of the dilute coloring
reagent.
5. The molar ahsorptivities
were calculated
using the BouguerBeer Law as described
for the Liebermann-Burchard
reaction.
However, in this case the concentration
value was based on a 9-mi. final
Vol. 9. No. 2. 1963
STANDARD
129
CHOLESTEROL
volume which was not corrected
for any volume change that took place
when the glacial acetic acid and sulfuric
acid solutions
were mixed.
A typical
wavelength-transmittance
curve for the cholesterol
sulfuric acid-iron
reaction
is shown in Fig. 2. There is a maximum
absorption
peak between 540 and 560 m.
The spectral
curves recorded
for tIns study reproduce
the spectral
curves shown by Rosenthal
and
Jud (ii).
The shapes of the curves were essentially
the same whether
the cholesterol
examined
was a commercial
or a recrystallized
product.
Running
the curves sequentially
showed that there was a 1% transmittance change every 11 mm. Thus, for this reaction,
as with the Liebermann-Burchard
reaction,
equal intervals
of color development
and absorbance
measurements
were maintained.
The relation
between
absorbance
and cholesterol
concentration
at 560 m was shown to be
linear.
Table 3 shows the mean molar absorptivity
values for the commercial and recrystallized
cholesterol
products.
Each mean molar absorptivity
value
represents
12 values obtained
as described
for the
Liebermann-Burchard
procedure.
0
20
I
U
z
t
Fig. 2. Wavelength-transmittance
furic
curve
acid-iron
for
40
I
the
siil-
cholesterol
4
60
5..
procedure.
so
100
700
600
500
400
WAVELENGTh, MILLIMICRONS
Table
3.
MOLAR
ABSORPTIVITIES
FOR THE
SULFURIC-IRON
ItOH
Cowmrciot
Original
(a*)
product
ArmourLotV3llO5
Pfanstiehl Lot 4907
SterahoidBatch49O7
K&KLotS55OL
General Biochemicals
6071 I
Mixture
*Molar
is the
meun
absorptivity:
of 12 single
liters
11100
10800
11300
11000
10700
9800
cm.1
measurements.
ItOH
11
21
(a*)
(a*)
11100
11200
11400
11200
11100
11000
11200
11300
11400
moles1,
not
-
corrected
CHOLESTEROL
RAe
IX
DiBr
HAc
21
(0*)
IX
11300
11300
11500
volume
-
change.
DiBr
21
((1*)
(a*)
11300
11200
11500
11400
11400
11200
for
PROCEDURE
11300
11300
11400
11500
11400
11300
(l*)
11300
11300
11500
-
Each value
130
RADIN & RAMZA
Clinical
Chemistry
Ultraviolet Spectral RegionStudies
Solutions
of all the commercial
amid recrystallized
nets in methanol
(Eastman,
Spectro.
Grade)
were
cholesterol
l)rod-
preiared.
Traiismittance-wave
length curves are shown for Armour
Cholesterol
and
7-keto cholesterol
acetate
(Steraloids)
methanol
solutions
in Fig. 3.
The shape of the curves for the cholesterol
preparations
were all imilar.
As the absorbance
values for equimolar
solutions
decreased
at 235
m with recrystallizations
the change was taken as a measurement
for
impurities.
in order to estimate
the amount of impurity
in each cholesterol
preparation
7-ketocholesterol
acetate
was choseii as a standard. As tile free sterol, 7-ketocholesterol,
is an oxidation
product
of
cholesterol,
the use of the acetate
which was available
commercially
was felt to he justifiable.
The 7-ketocholesterol
acetate
in methanol
solution
(lid show a linear
relationship
between concentration
and absorhance.
Table 4 shows the estimate
of the cholesterol
preparations
purity in terms of 7-ketocholesterol
acetate.
u
w2o#{149}
I,
____
Fig.
/2’\
_
40
S
#{149}
\
/
\
/
60
/
I
250
I
.1”
WAVELENGTH,
Table 4.
(ournerrial
PURITY
I
I
Lot V 31105
Lot 4907
Sterahoid Batch 4907
K & K Lot 5550 L
99.5
99.0
99.5
99.4
General Biocheniicals6071 I
Mixture
98.0
94.2
Pfanstiehl
*Grams
acetate).
of cholesterol
per
I
220
MILLIMICRONS
Original
(%*)
Armour
I
240
OF CHOLESTEROL
product
mg. per 100 nil,methanol.
.
260
Transmittance-
spectral curves:
cholesterol,
450
fig, per 100 ml. methanol. B.
7-ketocholesterol
acetate,
1
I-
100
300
3.
wavelength
A. Armour
IN TERMS
OF 7-KEro
ElOR
#{163}1011
liAr
JIAc
1)iIlr
1)iBr
IX
(%*)
21
(%*)
IX
21
IX
(f*)
21
(%*)
99.8
99.5
99.7
99.7
98.7
98.4
99.9
99.8
99.9
-
CHOLESTEROL
(*)
(9*)
99.9
99.7
99.7
99.9
99.7
98.4
100 gIn, of cholesterolplus impurities (grams
99.9
99.9
99.9
ACETATE
-
99.9
99.9
99.8
99.9
99.7
-
99.5
-
99.9
99.9
99.9
-
of 7-ketocholesterol
Vol. 9 No. 2, 1963
STANDARD
131
CHOLESTEROL
Discussion
The data shown in Table 2 indicate that the melting points generally
increase
with recrystallization.
The original
Armour,
Pfanstiehl,
and
Steraloid
cholesterol
products
show similar
melting
point ranges,
while the K & K product
shows a wider melting
point range than the
former products.
The General Biochemical
cholesterol
melting range
not only is greater
than that of the other products,
but the temperature
values are lower than those of the other commem’cial products.
The
melting point range for the General Biochemicals
cholesterol
was miarrowed with recrystallization.
The melting
point ranges
do point to
differences
between
the commercial
products.
Differences
between
recrystallized
products
become less apparent,
however,
the greater
the melting
point range
for the original
commercial
products
the
greater
the range for the recrystallized
products.
The molar absorptivity
values determined
with both procedures,
as
shown HI Tables 2 an(l 3, show an increase
with recrystallization.
The
higher
tile molar ahsorptivity
the purer
the product
should
be. In
order to determine
whether
the mean molar absorptivities,
each Of
which was calculated
from 12 replicate
determinations,
differ significantly an analysis
of variance
computation
was made for each commercial
cilolesterol
and its recrystallized
preparations.
An example
of an analysis
of variance
computation
for Armour
cholesterol
is
shown in Table 3. As the F ratio of 3.3954 exceeds the F ratio for the
99% significance
level it can be said that there is a significant
difference between the molar absorptivities,
as determined
with the Liebermann-Burchard
procedure,
of the Armour
cholesterol
and its recrystallized
preparations.
Table 6 shows the results
for all the commercial products
and for both procedures.
The data indicate
that with
the Liebermann-Burchard
procedure
the mean molar absorptivities
are shown to he significantly
different
for 3 out of 5 commercial
prodTable
Source
5.
ANALYSIS
OF VARIANCE FOR ARMOUR
CHOLESTEROL
DETERMINED
WITH THE LIEBERMANN-BVRCHARD
Sum of
equarco
of e,timate
Within
materials
Between
materials
Total
69,934
18,499
88,433
-
3083.167
-
908.234
=
1).
MOLAR ABSOEPTIVITY,
REACTION
5.
Variance
77
6
83
908.234
3083.167
-
< 3.12
AS
132
RADIN & GRAMZA
Table 6.
RESULTS
TALLIZED
OF ANALYSIS
CHOLESTEROL
OF VARIANCE
PRODUCTS
BETWEEN
AND THE
Commercial
product
Armour
Lot V 31105
Pfanstiehl
Lot 4907
Steraloid
Batch 4907
K & K Lot 5550 L
General
Mixture
Biochelnicals
Chemistry
AND RECRYS-
OF EACH
MOLAR
Sulfuric
acid-iron
MEAN
n-B urcho
rd
S.D.
Significant
Significant
30
30
30
30
Not
CHOLESTEROL
DEVIATION
Significance
significant
Significant
6071 I
ORIGINAL
STANDARD
ABSORPPIVITY
L jehe rman
Clinical
at 95%
Significant
Significant
level
20
20
Significance
Not significant
Significant
Significant
Significant
Significant
Significant
SD.
200
100
100
200
200
200
ucts. The data calculated
for the sulfuric
acid-iron
procedure
indicate
that the mean molar absorptivity
values were significantly
different
for 4 out of 5 commercial
products.
Tile estimates
of cholesterol
purity measured
in terms of 7-ketocholesterol acetate are shown in Table 4. When the estimate
of the original commercial
cholesterol
purity was less than 99.4% the analysis
of
variance
results
show that both the Liebermann-Burchard
and the
sulfuric
acid-iron
procedures
can detect
significant
differences
between molar absorptivity
values for the original
and recrystallized
cholesterol
preparations.
With purity
estimates
above 99.4% the
sulfuric
acid-iron
procedure
was sensitive
enough to detect differences
due to recrystallization
for the Steraloid
and the K & K cholesterol
preparations.
The ratio of the molar absorptivity
values for tile sulfuric acid-iron
procedure
to the Liebermann-Burchard
procedure
is
about 6.5, indicating
the former method to be a more sensitive
method
than the Liebermann-Burchard
method for the analysis
of cholesterol.
The standard
deviations
which are pooled estimates
for the precision of tile measurements
for each commercial
cholesterol
and its
recrystallized
preparations
are shown in Table 6. The coefficient
of
variation
(100 X S.D./mean
molar absorptivity),
for both methods,
shows that for 68% of the measurements
for one material
the variation in precision
is within 2%. It can also be seen that the standard
deviations
may be affected by the materials
being measured.
Comparison
of the mean molar ahsorptivity
values
for 1- and 2times-recrystallized
cholesterol
preparations
by means of tile statistical “t-test”
did not show any significant
differences
between
the
preparations.
Here, again, the standard
molar ahsorptivity
value was significantly
between the 1- and 2-times-recrystallized
deviation
greater
cholesterol
around
the mean
than the difference
molar absorptivi-
Vol. 9, No. 2, 1963
Table
7.
RESULTS
STANDARD
OF ANALYSIS
CHOLESTEROL
Choleaterol
Original
prepa
ration
OF VARIANCE
PREPARATIONS
133
CHOLESTEROL
BETWEEN
FROM DIFFERENT
L iebe rio,,,, n-B,,
rcha rd
THE MOLAR
ABSORI’TIVITIES
COMMERCIAL
S ulph uric
Significant
Significant
Significant
Not
HAc LX
Not
Significant
DiBr 1X
Significant
EtOli
lx
significant
OF THE
SOURCES
acidirun
sigiiificamit
Significant
ties, indicating
again that the precision
of the methods
is not good
enough to detect small differences.
It is for this reason that 2-timesrecrystallized
cholesterol
was not prepared
for all commercial
products.
Analysis
of variance
computations
were made to detei’mine
whether
the commercial
cholesterol
products
differed
significantly
between
companies
or the similarly
recrystallized
preparations
differed
significantly
because of their origins.
The results
are showmi ill Table 7.
Both methods
show significant
differences
between
the molar ahsorptivities
of the original
cholesterol
preparations.
This is to be expected, as the purity of the original
preparations
were shown to differ
widely by the melting
point determinations
and the ultraviolet
spectral studies.
However,
tile results
showmi for the ethyl alcohol and
acetic acid recrystallized
cholesterol
preparations
show no conclusive
results,
as the molar absorptivities
are significantly
different
for one
method or the other.
According
to the results,
the dihi’omide
derivative recrystallization
method
cholesterol
preparations
show significant differences
of the molar absorptivity
values for both methods.
While specific impurities
in each cholesterol
could be influencing
the
results
it is more probable
that the variability
of tile methods
could
explain inconclusive
results.
Conclusions
The dibroniide
method is recommended
for cholesterol
recrystallization as the results
show that the materials
obtained
have greater
molar absorptivities
than the materials
obtained
with the classic ethyl
alcohol or the acetic acid recrystallization
methods.
An example of the
effect of no recrystallization
may 1)e shown by the molar absorptivity
values for General Biochemicals
cholesterol.
There is an approximately 6% difference
between
the molar ahsorptivities,
for 1)0th methods,
between
the commercial
and the dibromide
recrystallized
preparatiolls. This would lead to a 6% error in concentration
if the commer-
134
RADIN & GRAMZA
Clinical
Chemistry
cial product was used as a standard.
Another
example of an error due
to the use of impure cholesterol
for a standard
is shown by our molar
absorptivity
measurements
of a 1 ml. = 2 mg. cholesterol
in glacial
acetic acid Harleco
standard
solution.
There would be an approximately 4% concentration
error with the Liebermann-Burchard
procedure
and a 14% concentration
error with the sulfuric
acid-iron
method.
In order to check cholesterol
preparatiolls
for use as standards
it is
recommended
that the molar absorptivity
values of 1750 ± 30 (1 S.D.)
for the Liebermann-Burchard
procedure,
as described,
and 11,500 ±
100 (1 S.D.) for the sulfuric
acid-iron
procedure
should be used as a
guide to ascertaill
purity.
References
1. Cook, B. P., Cholesterol.
Academic Press Inc.,New York, 1958.
2. The Pharnmacopei
of the United States of Anterica
(ed. 16.
The United
States Pharmaeopeial Convention, Inc.,Washington, D. 0., 1960.
3. Eyring, H., Aaal. Chem. 20, 98 (1948).
4. Fieser, L. F., J. Ant. Chein. Soc. 75, 4395 (1953).
5. Fieser,L. F., J. Am. Giesn. Soc. 75, 5421 (1953).
6. Fieser,
L. F., Private
communication.
7. Fieser, L. F., Experiments
in Organic
Chenmistry.
D. C. Heath and Company, Boston,
Mass.,
1955, p. 23.
8. Dorfman,
L., Chein. Rev. 53, 47 (1953).
9. Carr, J. J.,and Drekter,
I. J., Clin. Chem.. 2, 353 (1956).
10. Rosenthal,
H. L., Pfiuke,
M. L., and Buseaglia,
S.,J. Lab. Clin. Med. 50, 318 (1957).
1]. Rosenthal,
H. L., and Jud, L., J. Lab. Clin. Med. 51, 143 (1958).