T H E AMERICAN JOURNAL OP CLINICAL PATHOLOGY
Vol. 39, No. 5, pp. 450-455
May, 1963
Copyright © 1963 by The Williams & Wilkins Co.
Printed in U.S.A.
ANALYSIS OF TOTAL SERUM CHOLESTEROL BY MEANS OF GAS-LIQUID
CHROMATOGRAPHY
LEO P. CAWLEY, M.D., BILLY 0. MUSSER, B.A., C. (ASCP),
SALLY CAMPBELL, AND WENDELL FAUCETTE, B.S.
Division of Hormone Chemistry, Department of Laboratories, Wesley Medical Center, and the Wesley
Medical Research Foundation, Inc., Wichita, Kansas
Chromatographic technics for separation
and identification of various compounds in
biologic fluids is a recognized analytic tool
with widespread application in the clinical
laboratory.
Gas-liquid
chromatography
(GLC) has considerable advantage over
most technics of chromatography for certain
compounds, chiefly because of its excellent
resolving power. Separation of components
by means of GLC is relatively rapid, requires only micro quantities of sample, and
results in relatively clear separation of
closely related compounds with little carryover or trailing. GLC has been widely
applied to other areas of science but has only
recently become used in biology and medicine. It is a method of chromatographic
separation of compounds in their vapor state.
These compounds are first volatilized by
means of heat and carried by an inert gas
(mobile phase) through a column packed
with an inert support material (support
phase) that is coated with a liquid of low
volatility (stationary phase). Separation
takes place on the column by means of partition, and the individual components are
detected as they leave the column by means
of a sensing device, which then records its
impulses on a strip chart recorder. It is clear
that a number of biologic systems that contain chiefly steroids may be studied by
means of GLC without danger of disrupting
the molecular structure of the compounds.
It is the authors' purpose in this paper to
Received, July 30, 1962; revision received,
November 11; accepted for publication January
24, 1963.
Dr. Cawley is Clinical Pathologist and Associate Director of Laboratories; Mr. Musser is Head
of the Division of Hormone Chemistry; Miss
Campbell is Research Assistant in Pathology; and
Mr. Faucette is Chemist.
This work was supported by a grant from the
Kansas Division of the American Cancer Society.
describe a study of a technic based on the
use of GLC for the determination of total
serum cholesterol. Separation of steroids
including cholesterol or related steroids by
GLC has been demonstrated in several
laboratories.2' 6' u VandenHeuvel and associates13' u extended the use of GLC for
separation of steroids by using chromatographic columns containing a comparatively
thin coating of stationary liquid. Although
qualitative analysis of steroids separated
by GLC has been explored in some detail,
4, 5,7.9 methods for quantitative analysis are
still under development. Quantitative determinations by GLC of urinary estrogens in
urine of pregnant women,15 and aldosterone
in urine,16 have been reported. O'Neill and
Gershbein10 reported a method for quantitative analysis of cholesterol and squalene in
scalp sebum by GLC and applied it to
analysis of cholesterol and squalene in
ovarian dermoid cysts, shark liver oil, placental lipids, and tall oil. Sweeley and
Chang,12 in a study of the variations of
molecular response of various sterols with
the argon ionization detector, mentioned
that accurate measurement of the amount
of cholesterol in human serum was possible.
MATERIAL AND
METHODS
Apparatus—gas chromatograph. A BarberColman model 15 instrument* was used for
this study. The argon ionization detector in
the Barber-Colman instrument was 2.5 cm.
in diameter; it had a disk electrode and
56 ixc. of Ra226, plated on a stainless steel
strip, as a source of ionization radiation. A
U-shaped glass column with a total length
of 6 ft. and an internal diameter of J4 in.
was used. The column was packed with
0.75 per cent (weight for weight) silicone
gum (SE-30) liquid phase, on solid support
450
* Barber-Colman Company, Rockford, Illinois.
May 1963
SERUM CHOLESTEROL
451
FIG. 1. Series of peaks for serum cholesterol) revealing only slight trailing
gas chrome-P, 100 to 140 mesh. These
packings are commercially available.f The
column was packed by means of permitting
the powder to move through a funnel into
each arm of the U and gentle tapping of the
column. The column was conditioned initially at 250 C. for 24. hi\, in an atmosphere
of argon, and was subsequently operated at
235 C. The detector was operated at 270 C ,
and the flash heater at 270 C. The voltage
of the detector was set at 600. Argon pressure was 39 p.s.i. Samples were injected
with a 10-/il. No. 701-N Hamilton microliter
syringe.t
Standard. Cholesterol was prepared by
means of recrystallization of reagent grade
cholesterol from hot absolute ethanol and
dried to constant weight in a vacuum desiccator. The cholesterol, 160 mg., was dissolved in 50 ml. of chloroform and utilized
for the stock solution. The solution was kept
in the freezing compartment of the refrigerator when not in use. Dilute working
standards were made up in chloroform to
equal concentrations of 20, 40, 80, 120, 160,
and 320 mg. per 100 ml. Inasmuch as 5 /*1.
is the volume used for each injection, the
amount of each working standard placed
on the column is equal to 1, 2, 4, 6, 8, and
16 /ug., respectively, of cholesterol. These
amounts are equivalent to 50, 100, 200, 300,
400, and 800 mg., respectively, per 100 ml.
of serum cholesterol, inasmuch as the extract
from serum injected is equal to 2 /xl. of
original serum sample.
f Applied Science Laboratories, 140 N. Barnard
St., State College, Pennsylvania.
t Hamilton Co., Inc., P. 0. Box 307, Whittier,
California.
Afadtt of curves. Areas beneath curves
weHJ measured by computing the product of
peak height and width at half-height. Peaks
were symmetric (Figs. 1 and 2), and it was
possible to utilize peak height as a measure
of concentration, although more accuracy
was observed when area of curve was used.
Detector calibration. Samples, 5 jul. each,
of the 6 working standards with concentrations of 1, 2, 4, 6, 8, and 16 fig. per individual
injection were used for establishing tire
relation of the detector response. As may be
seen in Figure 3, the calibration curve
reveals a linear response of the detector with
slight deviation at the 1-jug. level. In Figure 3
a curve area of 2.2 cm.2 is equivalent to
1 Mg- of cholesterol. The area of curve equal
to 1 ^g. of standard (calibration factor) was
not always constant; it tended to vary
among different runs of standard from
1.95 cm.2 per jug. to 2.45 cm.2 per /ug. of
standard. The variation was somewhat
regular and was greatest when the column
was not used for several days. In a continuous series of analyses, the calibration factor
stabilized and became constant after the
instrument was in operation for a short
time. The factor was low at the onset of
operation, rose to a plateau in approximately an hour, and then remained constant.
Such a pattern suggests that there may be
active absorption sites on the inert packing
material that take up cholesterol until they
are completely saturated and that the
cholesterol bleeds off when the instrument is
not in operation. The value of tire calibration factor was determined at the beginning,
during, and at the end of a series of tests.
Occasionally the factors were different and
452
CAWLEY ET
(1
A
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'
Mi
FIG. 2. Three peaks from cholesterol standards.
A is 8 jug., B is 6 jug., and C is 4 ng.
an average value was used for calculation.
Concentration of cholesterol in an unknown
was computed by multiplying the area of
curve of the unknown by the calibration
factor. An internal standard, such as cholestane, could be added to each unknown and
used to control more closely the fluctuation
of the calibration factor; however, this practice would increase the length of time for the
analysis. Our experience with quantitation
of urinary 17-ketosteroids with internal
standard did not suggest that a similar
technic would increase the accuracy of analysis of cholesterol, and, therefore, we chose
to use a calibration factor.
PROCEDURE
For the determination, 0.5 ml. of serum
was placed in a large, round-bottomed,
glass-stoppered, 40-ml. test tube and saponified by means of adding 5 ml. of freshly
prepared alcoholic KOH and incubating for
60 min. in a 37 C. water bath, according to
Abell and associates.1 Extraction was performed with 10 ml. of petroleum ether. To
this mixture, 5 ml. of water were added, and
Vol. 39
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the mixture was shaken for 1 min. and centrifuged for 5 min. at 2000 r.p.m., or until
2 clear layers were obtained.
Two 4-ml. aliquots of the layer of petroleum ether were removed, and each was
placed in a test tube, 13 by 100 mm., and
evaporated to dryness in a 50-C. water
bath under a nitrogen stream. The dried
contents were dissolved in 0.5 ml. of chloroform. Reconstituted specimen, 5 jul., was
then injected into the column. Cholesterol
peaks appeared on the recorder within
5 min., and it was possible to inject as many
samples at 6-min. intervals as desired. A
chromatogram of a series of cholesterol
peaks is illustrated in Figure 1. It was not
necessary to adjust the instrument between
specimens, and standards were inserted in
the test series at desired points. Colorimetric
analysis of total serum cholesterol of each
serum was made by means of the method
of Bloor.3
RESULTS AND DISCUSSION
The response of the argon detector in our
instrument for cholesterol was linear (Fig. 3),
through a range of 50,100, 200, 300, 400, and
800 mg. of cholesterol per 100 ml. of serum,
with slight deviation from a straight line
at the 50-mg. level. The actual amount
placed on the column was equivalent to 1,
2, 4, 6, 8, and 16 ;ug. of cholesterol.
It is important to know which portion of
an analytic technic is most likely to contribute to the greatest variation. In order to
obtain this data, we analyzed 10 serums
selected at random in 2 sets of studies. In
the first, the over-all procedure was evaluated
by means of pairing each specimen and
carrying these paired samples through the
complete procedure. In the second, the same
10 samples were taken through the extraction procedure as single specimens and were
then paired before being placed on the gas
chromatograph. The difference between
these 2 sets of figures should, then, give
some information as to how much variability
was introduced by the extraction procedure
in the same set of 10 specimens. The over-all
procedure yielded a standard deviation of
±9.6 mg. per 100 ml. In the study of paired
tests on the chromatographic portion of the
May 1963
SERUM CHOLESTEROL
453
Ow
CHOLESTEROL C O N C E N T R A T I O N
(microqra ms)
FIG. 3. Plot of concentration of cholesterol and peak area
procedure, a standard deviation (S.D.) of
± 6 . 2 mg. per 100 ml. was obtained. The
mean was 205 mg. per 100 ml. in both sets of
analyses. Use of the Bloor method on these
same specimens resulted in an S.D. of 3.5 mg.
per 100 ml. In a further study pertaining to
the precision of GLC, a single specimen was
analyzed 20 times and yielded an S.D. of
±7.5 mg., with a mean of 155 mg., per
100 ml. These figures tend to suggest that
the extraction portion of the analytic technic
does not contribute greatly to the over-all
variation. It seems that the major part of
the variability is in the chromatographic
portion of the technic. Such factors as injection technic, consistency of packing material, and variability in instrumentation are
to be considered. Other factors as yet unex-
plored concern nonspecific absorption of
cholesterol by the inert packing material.
The question of whether or not the single
peak, as demonstrated in the chromatogram, is represented by a single substance,
namely, cholesterol, was not fully investigated during this study. It is interesting to
note that the difference between values obtained by the Bloor method and those
obtained by the GLC method indicates that
the 2 technics were not necessarily measuring the same substances. In a comparative
study between the cholesterol values obtained by the Bloor method and those
obtained by the GLC method, 24 separate
serum specimens were analyzed by means of
both teclinics (Table 1). The mean by tlie
Bloor method was 230 mg. per 100 ml.,
454
CAWLEY ET
TABLE 1
COMPARISON
OF V A L U E S FOR C H O L E S T E R O L
BY M E A N S OF B L O O R ' S M E T H O D AND
G A S - L I Q U I D CHROMATOGRAPHY
No.
Method of
Bloor
Gas-Liquid
Chromatography
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
225
155
280
205
230
145
270
270
280
170
225
210
205
530
130
145
105
205
225
235
195
255
485
150
235
125
240
195
225
125
265
265
285
165
260
205
205
480
90
125
80
195
210
195
180
215
545
125
Average
230
218
with a range of values from 105 to 530. The
mean by the GLC method was 218 mg. per
100 ml., with a range of 80 to 545. The GLC
method yielded values that were 5.2 per
cent lower than those obtained by the Bloor
method. The Bloor method yields higher
values with reference to the actual amount
of cholesterol present in serums, inasmuch
as substances other than cholesterol reveal
color with a Liebermann-Burchard color
reagent. 1 The peaks seen in the chromatogram of Figure 1 are relatively symmetric
and seem to represent a single compound.
Preparations of steroids related to cholesterol, B-sitosterol, 7-dehydrocholesterol, and
desmosterol, singly and as a mixture, were
clearly separated from cholesterol, with
retention times referable to cholesterol of
1.31, 1.10, and 0.94, respectively.
Vol. 39
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From these preliminary studies it is
apparent that GLC is applicable for the
determination of total serum cholesterol, but
the technic is slower than the available
colorimetric methods, and at the moment
not as precise. Analysis of single components, such as cholesterol, in biologic
fluids is helpful in understanding the problems related to quantitation by GLC applied
to biologic systems. Once the difficulties of
analysis of single components are overcome,
more information will be available for
application to measurement of mixtures of
steroids such as 17-ketosteroids in urine.
Continued investigation of the use of GLC
for the analysis of lipids, including cholesterol, other steroids, and fatty acids, in
blood, other body fluids, and tissue extracts
can be contemplated. A very important
consideration with respect to GLC is its
resolving capabilities in separating mixtures
of compounds into individual components,
which make it ideally suited to the analysis
of lipids removed from various tissues.
Quantitation of extracts containing many
components is, as already mentioned, somewhat more complex than the procedure
described here for a single component.
SUMMARY
Determination of total serum cholesterol
by means of a gas chromatographic technic
was evaluated and compared with the Bloor
colorimetric method for total serum cholesterol. The argon detector responds linearly
with concentration of cholesterol through a
range of 2 to 16 Mg-, which is equivalent to
a serum range of 100 to 800 mg. per 100 ml.
Total serum cholesterol can be determined
within 90 min. Improvement in technics of
injection, column packings, and instrumentation can be expected to reduce the
total time for analysis and to increase accuracy. Further increases in the use of gasliquid chromatography for analysis of
steroids, fatty acids, and other lipids in
body tissues and fluids may be contemplated
and designed.
STJMMARIO IN INTERLINGUA
Le determination del total cholesterol del
sero per medio de un technica de chroma-
May 1963
SERUM CI
tographia a gas esseva evalutate e comparate con le methodo colorimetric de Bloor
pro total cholesterol serai. Le detector de
argon responde in linearitate con le concentration de cholesterol intra le region ab
2 ad 16 ng, lo que equivale a un region in le
sero ab 100 ad 800 mg per 100 ml. Le total
cholesterol del sero pote esser determinate
intra 90 minutas. II pare justificate predicer
que meliorationes technic in le injection, le
paccage del columna, e le instrumentation va
reducer le requirimentos in tempore e
augmentar le grado de accuratia. Plus
extense usos additional del chromatographia
a gas-liquido in le analyse de steroides,
acidos grasse, e altere lipidos in tissus e
liquidos del corpore pote esser considerate
e elaborate.
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