Yonago Acta medica 2002;45:27–33

Utility of 19-Hydroxycholesterol as an Internal Standard Compound for
the Quantitative Determination of Sterols Using Capillary Gas ChromatographNobuo Yamaga, Yoshio Ogura, Md. Azharul Islam, Hiroko Matsuyama, Masahiro
Yamawaki*, Masachika Kai and Kazuo Yamada
Department of Biochemistry and *First Department of Internal Medicine, Tottori University
Faculty of Medicine, Yonago 683-0826 Japan
Gas chromatography is able to detect many kinds of sterols simultaneously. In order
to identify and estimate accurately many kinds of sterols in biological samples by gas
chromatography, it is indispensable to add an internal standard compound into theanalytical samples prior to the analysis. However, there is no report on a suitableinternal standard compound for the determination of sterols. In this study, 19-hydroxycholesterol was proved to be a suitable internal standard compound for simultaneousand quantitative determination of several sterols in various analytical samples by capillary gas chromatography, including the quantitative determination of β-cholestanol in
the human plasma. Moreover, the excellent condition of gas chromatographic analysis
for quantitative determination of several sterols in various analytical samples was found.
Key words: capillary gas chromatography; 19-hydroxycholesterol; internal standard compound;quantitative determination; sterols

in the serum of patients with CTX is usefulfrom the viewpoint of biochemical diagnosis(Seyama, 1999).
For the simultaneous determination of various sterols and bile acids, gas chromatography,gas chromatography mass spectrometry (GCMS) and/or high performance liquid chromatography (HPLC) are available means. In comparison with the above mentioned instruments,
gas chromatography is a better means for qualitative and quantitative determination of sterols
and bile acids, because a gas chromatograph isvery sensitive, is in widespread use in manylaboratories and is simple in its operation.
Determination of sterols by gas chromatography has been carried out by procedures suchas extraction, hydrolysis, derivatization and injection into the gas chromatograph. It is impossible to obtain 100% recovery of the sterols inthe sample. Moreover, recovery may differ from

There are many reports on the action of intestinal flora on several compounds such as
sterols and bile acids, in both in vivo and in vitro
experiments (Björkhem and Gustafsson, 1971;
Eyssen et al., 1973; Uchida et al., 1977; Macdonald
et al.,1983). However, the kinds of intestinal
flora concerned with the metabolism of sterols
in their host, and so on, have not yet been sufficiently solved. In order to investigate a numberof unsolved problems, the quantitative determination of various sterols in feeds, feces, bloodand intestinal contents of experimental animals
needs to be carried out precisely in various experiments using intestinal flora. Moreover, theconcentration of 5α-cholestan- 3β-ol (β-cholestanol), a kind of sterol, in the serum of patientswith cerebrotendinous xanthomatosis (CTX), a
genetic disorder of the cholesterol metabolism,
increases abnormally (Salen, 1971; Seyama etal., 1976). Therefore, analysis of β-cholestanol

Abbreviations: β-cholestanol, 5α-cholestan-3β-ol; CTX, cerebrotendinous xanthomatosis; GC-MS, gas
chromatography mass spectrometry; HPLC, high performance liquid chromatography; TMS, trimethylsilyl

27

N. Yamaga et al.

assay to assay. Therefore, it is indispensable to
add an internal standard compound into theanalytical samples for correcting the loss ofsterols during the analysis.
We have established a systematic method
for gas chromatographic analysis of bile acids
in biological samples using some internal standard compounds (Yamaga et al., 2001). However, a suitable internal standard compound forgas chromatographic analysis of sterols has not
yet been reported.
This paper describes the internal standard
compound and the excellent conditions of gas
chromatography for the qualitative and quantitative determination of several sterols simultaneously using a capillary gas chromatograph.

(6)

(8)
(9)

(2)
(3)

(7)
(5)

Response

(1)

43.1

35.4

27.7

(10)

20

Experimental Procedures

(4)

min

Materials
Cholesterol, 19-hydroxycholesterol (an internal
standard compound), lanosterol, stigmastanol
and 5β-cholestan-3α-ol were purchased from
Sigma, St. Louis, MO. Coprostanol was purchased from Gasukuro Kogyo Inc., Tokyo, Japan.
β-Cholestanol and N-trimethylsilylimidazole
were purchased from Tokyo Kasei Kogyo Co.,Tokyo, Japan. Campesterol, β-sitosterol andstigmasterol were purchased from Tama Biochemical Co., LTD, Tokyo, Japan. Other chemicals were of analytical reagent grade, and allorganic solvents were distilled before use.

Fig. 1. Capillary gas chromatogram of several authentic sterols. The peaks of sterols are as follows:( 1) Coprostanol,( 2) 5β-Cholestan-3α-ol,( 3) Cholesterol,( 4) β-Cholestanol,( 5) 19-Hydroxycholesterol (internal standard
compound),( 6) Campesterol,( 7) Stigmasterol,( 8) Lanosterol,( 9) β-Sitosterol,(10) Stigmastanol.
See the text on derivatization and gas chromatography of sterols.

Extraction of sterols from analytical samples

Derivatization

Biological samples (20–50 mg for feces and feed,and 10–100 µL for human plasma) with andwithout 5–10 µg of 19-hydroxycholesterol as aninternal standard compound were hydrolyzed
with 1 mL of 1 M ethanolic sodium hydroxide for
30 min at 80˚C. After addition of 0.5 mL of distilled water into the hydrolysate, nonsaponifiable materials (sterols) were extracted with nhexane, and then the solvent was evaporated todryness.

Extracted sterols were converted into trimethylsilyl (TMS) derivatives with N-trimethylsilylimidazole at room temperature overnight or at
80˚C for 30 min. The sterol-TMS derivatives
were extracted with n-hexane and evaporated todryness after washing with distilled water. An
aliquot of the sterol-TMS derivatives dissolved
in n-hexane was injected into the gas chromatograph.

28

Internal standard for GC of sterols

pound (the retention time of 19-hydroxycholesterol = 1.00). The relative factors for quantitative analysis in the gas chromatography between
the peak area ratio and the weight ratio of eachsterol to 19-hydroxycholesterol were estimatedautomatically by injecting a known amount ofauthentic sterols and the internal standard compound into the gas chromatograph.

Gas chromatography
The gas chromatograph (Model GC-14A,
Shimadzu, Kyoto, Japan) was equipped with
HiCap CBP-1 capillary column (25 m × 0.25
mm I.D., Shimadzu) and with a solventless injector (Shimadzu). The capillary column was
programmed at 240˚C for 20 min, from 240˚C
to 260˚C at a rate of 2˚C/min, and then at 260˚C
for 20 min. Helium was used as the carrier gas.
Capillary gas chromatograms and analytical
data of the sterols were obtained using a computerized data system (Model C-R4A Chromatopac, Shimadzu) connected with the gas chromatograph.

Results and Discussion
In Fig.1, many kinds of authentic sterols, which
are found in specimens of animals and plants,
have been detected separately as peaks of sterolTMS derivatives under the excellent conditionsof capillary gas chromatography described inexperimental procedures. Figure 2 shows capillary gas chromatograms of nonsaponifiable
sterol-TMS derivatives extracted from feed andfeces of mice and rats as well as plasma of humans under the same conditions of capillary gas
chromatography for analyzing the authentic
sterols. These results suggest that it is possible
to detect separately many kinds of sterols from

(c)

I.S.

Response

Response

I.S.

(d)

Response(b)

Response

(a)

min

min

20

min

27.7

I.S.

43.1

35.4

27.7

20

43.1

35.4

27.7

20

43.1

35.4

27.7

20

I.S.

43.1

Identification of peaks on the gas chromatogram from the specimens was carried out inagreement with the found and calculated relative retention time between them and the authentic sterols to the internal standard com-

35.4

Qualitative and quantitative determination of sterols by gas chromatographyminFig. 2. Capillary gas chromatograms of nonsaponifiable fractions with 19-hydroxycholesterol. (a) mouse
feces, (b) rat feces, (c) mouse and rat feed and (d) human plasma. I.S. indicates the peak of 19-hydroxycholesterol as internal standard compound. See the text on gas chromatography.

29

N. Yamaga et al.

Table 1. Retention times and relative retention times of internal
standard compounds and several authentic sterols
Sterols
( 1)
( 2)
( 3)
( 4)
( 5)
( 6)
( 7)
( 8)
( 9)
(10)

Retention time (min)Relative retention time

28.54729.08332.36732.79536.50537.76839.51742.19943.19743.834

0.782
0.797
0.887
0.898
1.000
1.035
1.083
1.156
1.183
1.201

Coprostanol
5β-Cholestan-3α-olCholesterol
β-Cholestanol
19-Hydroxycholesterol
Campesterol
Stigmasterol
Lanosterol
β-Sitosterol
Stigmastanol

Gas chromatographic condition is described in experimental procedures.
Capillary column is HiCap CBP-1 capillary column (25 m × 0.25 mm I.D.).
Sterols are derivatized to TMS-ether.
Relative retention times are expressed relative to 19-hydroxycholesterol.

analytical samples as peaks on a capillary gas
chromatogram, as shown in Fig. 2, under thesame experimental conditions as gas chromatography.
However, in order to accurately identify andestimate these detectable peaks of sterols fromsamples on a gas chromatogram, it is indispensable to add an internal standard compound into
the analytical samples before the extraction of
the sterols.
The fundamental requirements for selecting
a suitable internal standard compound for thedetermination of sterols by gas chromatography
are as follows —iv) the retention time of the internal standard
compound must not be largely distant fromthose of sterols which are being assayed; andv) a linear relationship should be maintained
between the measured peak height or area
and the weight of the sterol to the internal
standard compound.
Previously, among 5α-cholestane (Setoguchi
et al., 1974; Ishikawa et al., 1976), 5β-cholestan3β-ol (Kasama et al., 1987), 5β-cholestan-3αol (Byun et al.,1988) and 5α-cholestan-3α-ol(Serizawa et al., 1981), each one has been used
as an internal standard compound for only
quantitative determination of β-cholestanol inhuman serum by gas chromatography, GC-MS
and/or HPLC for the biochemical diagnosis of
CTX patients. But these internal standard compounds are homologous in structure and appear
earlier than many other kinds of sterols such as
campesterol, stigmasterol and lanosterol, etc.,
in gas chromatographic analysis which strongly
differs from the above mentioned 1st and 4th
criteria for internal standard compounds.
During metabolic oxidation of cholesterol,
19-hydroxycholesterol (a particular oxysterol)
is formed (Verhagen et al., 1996). So far as weknow, there is no report on the detection of 19hydroxycholesterol in biological samples. Wei) an internal standard compound must be
added to the analytical samples before theextraction of sterols, and then the peak of
an internal standard compound must appear
together with peaks of sterol derivatives in
the analytical samples on the same gas
chromatogram;ii) the internal standard compound must be
stable. During the analysis, it must notdecompose into other compounds;iii) the peak of the internal standard compound
must not pile up on the peaks of sterol
derivatives and other mixed compounds inthe biological sample;

30

Internal standard for GC of sterols

4

Coprostanol
5β-Cholestan-3α-ol

Weight ratio

3

Cholesterol
β-Cholestanol
Campesterol2

Stigmasterol
Lanosterol1β-Sitosterol
Stigmastanol

0
0

1

2

3

4

5

Peak area ratio
Fig. 3. Calibration curves of several sterols to 19-hydroxycholesterol for quantitative determination.
Weight ratio =Weight of each sterol
Weight of 19-hydroxycholesterol

Area ratio =Peak area of each sterol
Peak area of 19-hydroxycholesterol

mens of animals and plants, excellent condition
for gas chromatography was established as described in experimental procedures.
The amount of β-cholestanol in humanserum is usually determined for the biochemical
diagnosis of CTX. The level of β-cholestanol isless than 0.2 mg/100 mL on an average inhealthy subjects, and in the serum of CTX patients the level of β-cholestanol increases about
20–30 times compared with that of healthy
humans (Seyama, 1999). The cholesterol levelin the serum of CTX patients is usually within
the normal range (130–260 mg/mL) (Salen,1971). As we have had no chance to collect blood
samples from CTX patients, different amounts(1.1, 2.2, 3.1, 5.2, 6.1 and 8.6 µg) of authentic βcholestanol were added into 100 µL of healthy
subject plasma, (cholesterol concentration, 219
mg/100 mL in plasma) to prepare artificial plasma samples of CTX patients, and then thesesamples were analyzed. Although the retention
times of β-cholestanol and cholesterol from artificial plasma samples were adjacent in the gas
chromatogram, the larger cholesterol peakwere interested in investigating this particular
sterol, 19-hydroxycholesterol, as an internal
standard compound for the quantitative determination of sterols using a capillary gas chromatograph. The peak of 19-hydroxycholesterol
was in the middle domain between those of the
1st and the last sterols among many sterol peakson the gas chromatogram (Fig. 1), and the retention time of 19-hydroxycholesterol was also
different from those of many authentic sterols(Table 1). Besides, there is no peak which overlaps at the position of the 19-hydroxycholesterol peak in all analytical samples. Calibration
curves for the quantitative determination ofseveral sterols maintained a linear relationshipgoing through the origin between the weightratio and the peak area ratio of each sterol to 19hydroxycholesterol in the range of the measurement (Fig. 3). From our present experimental
findings we can conclude that 19-hydroxycholesterol fullfills all the requirements of an internal standard compound. Using both HiCap
CBP-1 capillary column made with methyl silicone and major authentic sterols found in speci-

31

N. Yamaga et al.

Response(c)

Response

(b)

min

min

32.6

28.8

25

36.3

32.6

28.8

25

36.3

32.6

28.8

25

I.S.

I.S.

I.S.

36.3

Response(a)minFig. 4. Gas chromatograms of nonsaponifiable fractions from healthy subject plasma and artificial plasma
samples. (a) Healthy subject plasma (100 µL), (b) Artificial plasma sample (100 µL) with 3.1 µg of authentic
β-cholestanol and (c) Artificial plasma sample (100 µL) with 6.1 µg of authentic β-cholestanol. The arrow
point indicates the peak of β-cholestanol. I.S. means the peak of 19-hydroxycholesterol. See the text on gas
chromatography.

hardly ever overlapped onto the smaller βcholestanol one in the same gas chromatogram(Fig. 4). Besides, the amount of β-cholestanol ineach artificial plasma sample of CTX patientswas as expected (Fig. 5). Cholesterol amount in
the serum of healthy subjects is 130–260 mg/
100 mL on the average. Cholesterol level in theserum of CTX patients is usually within thisnormal range (Salen, 1971). But in these analytical conditions it is uncertain whether or not
the cholesterol peak may pile up on that of βcholestanol if the cholesterol level increases
above the normal level. For this reason, the relation between the amount of cholesterol and βcholestanol was investigated. A sharp peak of
β-cholestanol appeared as a discernible shoulder at the end side of the larger cholesterol peak.

When the level of cholesterol was above 300
mg/100 mL, the level of β-cholestanol was determined differently. In these cases, the level of
β-cholestanol could not be determined accurately, but it could be judged that the level ofmeasured β-cholestanol in artificial samples
was more than that of β-cholestanol in the plasma of normal subjects.
Due to the large difference between the level of cholesterol and β-cholestanol in the humanplasma of both normal individuals and patientswith CTX and CTX-like disease, 19-hydroxycholesterol should not be considered as an internal standard compound only in the case of thesimultaneous determination of cholesterol and
β-cholestanol. So, it is better to be determinedthe cholesterol level by the enzymatic method.
32

Analytical values of β-cholestanol (µg)
in artificial plasma samples (100 µL)

Internal standard for GC of sterols

10

Biochem 1973;36:411–421.
4 Ishikawa TT, Brazier JB, Stewart RW, Fallat
RW, Glueck CJ. Direct quantitation of cholestanol in Plasma by gas-liquid chromatography. J
Lab Clin Med 1976;87:345–353.
5 Kasama T, Byun D, Seyama Y. Quantitative analysis of sterols in serum by high-performanceliquid chromatography. J Chromatogr 1987;400:
241–246.
6 Macdonald IA, Bokkenheuser VD, Winter J,
McLernon AM, Mosback EH. Degradation ofsteroids in the human gut. J Lipid Res 1983;24:
675–700.
7 Salen G. Cholestanol deposition in cerebrotendinous xanthomatosis. A possible mechanism. Ann Intern Med 1971;75:843–851.
8 Serizawa S, Seyama Y, Otsuka H, Kasama T,
Yamakawa T. Simplified determination of cholesterol in serum by gas-liquid chromatography.
Biochemical diagnosis of cerebrotendinous
xanthomatosis. J Biochem 1981;90: 17–21.
9 Setoguchi T, Salen G, Tint GS, Mosbach EH. A
biochemical abnormality in cerebrotendinous
xantomatosis. Impairment of bile acid biosynthesis associated with incomplete degradation of thecholesterol side chain. J Clin Invest 1974;53:
1393–1401.
10 Seyama Y, Ichikawa K, Yamakawa T. Quantitative determination of cholestanol in plasma withmass fragmentography. Biochemical diagnosis
of cerebrotendinous xanthomatosis. J Biochem
1976;80:223–228.
11 Seyama Y. Cholestanol metabolism: nutrition
and its molecular pathology. Nihon Eiyo Shokuryogaku Zasshi 1999;52:321–328.
12 Uchida K, Nomura H, Kadowaki M, Takeuchi N,
Yamamura Y. Effect of dietary cholesterol oncholesterol and bile acid metabolism in rats. Jpn
J Pharmacol 1977;27:193–204.
13 Verhagen JCD, ter Braake P, Teunissen J, vanGinkel G, Sevanian A. Physical effects of biologically formed cholesterol oxidation products
on lipid membranes investigated with fluorescence depolarization spectroscopy and electron
spin resonance. J Lipid Res 1996;37:1488–1502.
14 Yamaga Y, Ogura Y, Yamada K, Kohara H,
Uchida K. Internal standard compounds forquantitative determination of bile acids by gas
chromatography. Yonago Acta Med 2001;44:
91–105.

7.5

5

2.5

0
0

2.5

5

7.5

10

Amount of β-cholestanol (µg) added into
artificial plasma samples (100 µL)

Fig. 5. Recovery experiments of β-cholestanol (µg)
in artificial plasma samples (100 µL) prepared fromhealthy subject plasma by adding different amountof authentic β-cholestanol.

As a result, we conclude from our experimental findings that 19-hydroxycholesterol becomes an appropriate internal standard compound for the simultaneous determination ofseveral sterols, including β-cholestanol in theserum of CTX patients.

Acknowledgments: We sincerely thank Ms. Yumiko
Uyama of the Department of Biochemistry, Tottori
University Faculty of Medicine for her help in clerical work.

References
1 Björkhem I, Gustafsson J-A. Mechanism of
microbial transformation of cholesterol into
coprostanol. Eur J Biochem 1971;21:428–432.
2 Byun DS, Kasama T, Shimizu T, Yorifuji T,
Seyama Y. Effect of cholestanol feeding on sterol concentrations in the serum, liver, and cerebellum of mice. J Biochem 1988;103:375–379.
3 Eyssen HJ, Parmentier CG, Compernolle FC,
DePauw G, Piessens-Denef M. Biohydrogenation of sterols by Eubacterium ATCC. Eur J

Received December 19, 2001; accepted January 15, 2002
Corresponding author: Nobuo Yamaga

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