Review Article

Yonago Acta medica 2001;44:91–105

Internal Standard Compounds for Quantitative Determination of Bile
Acids by Gas Chromatography
Nobuo Yamaga, Yoshio Ogura, Kazuo Yamada, Hiromi Kohara* and Kiyohisa
Uchida†Department of Biochemistry and *Department of Pathological Biochemistry, Tottori University
Faculty of Medicine, Yonago 683-0826, and †The Cell Science Research Foundation, Osaka
541-0045 Japan
Gas chromatography is well recognized as a useful tool with several advantages for the
analysis of bile acids as well as various compounds. In gas chromatographic analysis,
bile acids in an analytical sample are subjected to a number of complicated procedures
involving many steps such as extraction, fractionation, solvolysis, hydrolysis, derivatization and injection to the gas chromatograph. These procedures result in the loss of bile
acids in the analytical sample. The addition of suitable internal standard compound(s)into the analytical sample prior to the extraction of bile acids is indispensable for an
accurate determination of bile acids. There are two methods for the quantitative determination of bile acids in a biological sample by gas chromatography: one is thedetermination of total bile acid amounts in the sample. The other is the determinationof bile acid amounts in each fraction after group separation of bile acids in the biological
β ,12αα -dihydroxy-5ββsample using an ion exchange gel column. The addition of 7ββ,12ββ-dihydroxy-5ββ-cholanoic acid as an internal standard compoundcholanoic acid or 7ββ,12ββ-dihydroxyis useful for the former method. On the other hand, the addition of 7ββ-cholanoic acid, glyco-7αα,12αα-dihydroxy-5ββ-cholanoic acid, tauro-7αα,12ββ-dihydroxy5ββ-cholanoic acid and glyco-7ββ,12αα-dihydroxy-5ββ-cholanoic acid 7-sulfate is a suitable
5β
combination as internal standard compounds for the latter method.
Key words: bile acids; biological sample; gas chromatographic analysis; internal standard compounds;
quantitative determination

class simultaneously. In 1960, Vanden Heuvel
and coworkers (1960) for the first time applied
gas chromatography to the analysis of bile acids
after converting them into methyl ester derivatives. Since then, gas chromatography has contributed greatly to the investigation of bile acidmetabolism, analyzing bile acids in several biological samples such as bile, feces, serum, urine,
gallstones or tissues.

The use of gas chromatography
in investigation of bile acids
Gas chromatography is well recognized as a
useful tool with several advantages for the analysis of bile acids as well as various compounds.
It especially has good sensitivity and is able todetermine a number of different compounds in a

Abbreviations: αα, 7α,12α-dihydroxy-5β-cholanoic acid; αβ, 7α,12β-dihydroxy-5β-cholanoic acid; βα,7β,12α-dihydroxy-5β-cholanoic acid; ββ, 7β,12β-dihydroxy-5β-cholanoic acid; Glyco-αα, glyco-7α,12αdihydroxy-5β-cholanoic acid; Glyco-αα 7-sulfate, glyco-7α,12α-dihydroxy-5β-cholanoic acid 7-sulfate;
Glyco-βα 7-sulfate, glyco-7β,12α-dihydroxy-5β-cholanoic acid 7-sulfate; Tauro-αα, tauro-7α,12αdihydroxy-5β-cholanoic acid; Tauro-αβ, tauro-7α,12β-dihydroxy-5β-cholanoic acid; Tauro-ββ, tauro7β,12β-dihydroxy-5β-cholanoic acid91

N. Yamaga et al.

(Garbutt et al., 1969; Neale et al., 1971; Takikawa
et al., 1983a, 1983b).

Metabolism and movementof bile acids

Derivatizations of bile acids forgas chromatography

Bile acids in the human body are mainly composed of cholic acid, chenodeoxycholic acid,deoxycholic acid, lithocholic acid and ursodeoxycholic acid (Beppu et al., 1982; Yamaga
et al., 1994a; Yamaga et al., 1996). Chenodeoxycholic acid and cholic acid are primary bile
acids synthesized from cholesterol in the liver,and they are major catabolic products of cholesterol (Danielsson, 1973). Deoxycholic acid,lithocholic acid and ursodeoxycholic acid aresecondary bile acids converted from primary
bile acids by the action of intestinal flora (Hilland Drasar, 1968; Shimada et al., 1969; Aries
and Hill, 1970). Most free bile acids are easily
conjugated with glycine or taurine in the liver.
Bile containing glycine- and taurine-conjugatedbile acids is stored and concentrated in the
gallbladder, and then released into the duodenum. Primary and secondary bile acids are absorbed almost exclusively from the ileum, return quantitatively to the liver by way of theportal circulation and are secreted into bile.
This is called enterohepatic circulation of bile
acids (Lack and Weiner, 1967; Dietschy, 1968;
Dowling, 1972; Heaton, 1972). During enterohepatic circulation, some parts of bile acids are
not absorbed to any significant extent from theintestine and excreted into feces (Eneroth etal., 1966; Dietschy, 1968; Tyor et al., 1971; Heaton,
1972). A part of bile acids leaks into the systemic circulation (van Berge-Henegouwen et al.,1974), and then excretes into the urine throughthe kidney.
Some parts of bile acids, especially secondary bile acids in the blood and urine are found as
their sulfates (Palmer, 1967; Makino et al.,
1973; Back, 1974), glucosides (Marschall et al.,
1988; Marschall et al., 1989), glucuronides(Palmer,1967; Stiehl, 1974; Alme and Sjövall,1980), and/or N-acetylglucosaminides (Marschall
et al., 1988; Marschall et al., 1989; Takikawa etal., 1982; Yamaga et al., 1994a). The formationof unusual bile acids and the ratios of various
conjugated bile acids offer useful information
on hepatobiliary and gastrointestinal diseases

For gas chromatographic analysis the compounds
analyzed are required to be volatile. Therefore,
nonvolatile compounds should be converted
into volatile derivatives quantitatively. It is truein the case of bile acids since they are polar
compounds having a carboxyl group and somehydroxyl groups in their molecules. Usually,
the carboxyl group of bile acids is methylatedwith diazomethane or hexafluoroisopropylatedwith hexafluoroisopropanol (Imai et al., 1976).
The hydroxyl group is acetylated with acetic
anhydride (Roovers et al., 1968) or trifluoroacetylated with trifluoroacetic anhydride (Endoet al., 1979). In addition, the hydroxyl group issilylated with both hexamethyldisilazane andtrimethylchlorosilane (Makita and Wells, 1963),
N-trimethylsilylimidazole (Karlangnis andPaumgartner, 1979; Amuro et al., 1983) or dimethylethylsilylimidazole (Miyazaki et al., 1977;
Arimoto et al., 1982). In these situations, directconversion of original conjugated bile acids
into volatile derivatives has not been succeeded
up to the present.

Fractionation and gas chromatographic analysis by difference inconjugated bile acid forms
As mentioned above, bile acids in human andanimal bodies are present as a nonamidated
form (free form) and forms conjugated withglycine, taurine, sulfuric acid, glucuronic acidand/or others. Therefore, in gas chromatographic analysis, bile acids must be converted
into volatile derivatives after carrying out anumber of complicated procedures. Bile acids
extracted from an analytical sample are converted into free bile acids by solvolysis withhydrochloric acid (Back, 1937; van BergeHenegowen et al., 1976; Alme et al., 1977;
Yamaga et al., 1994a), followed by alkaline
92Internal standards for GC of bile acids

Fig. 1. Outline of procedures for systematic analysis of bile acids in a sample.

hydrolysis with 2 M sodium hydroxide solution(Nair and Garcia, 1969; Campbell et al., 1975;
Yamaga et al., 1997) or enzymatic hydrolysis(Nair and Gordon, 1967; Cantafora et al., 1979),
and free bile acids are converted into volatile
derivatives. Bile acid derivatives are analyzed
by gas chromatography.
However no information on the original
conjugated forms of bile acids is obtained fromthe gas chromatogram. In order to solve thisproblem, crude bile acid extract from theanalytical sample is first subjected to column
chromatography with an ion exchange gel suchas diethylaminohydroxypropyl-Sephadex-LH20 (DEAP-LH-20) (Alme et al., 1977) or piperidinohydroxypropyl-Sephadex- LH-20 (PHP-LH20) (Goto et al., 1978), fractionating into five
fractions; each containing neutral compounds,
nonamidated bile acids, glycine-conjugated bile
acids, taurine-conjugated bile acids and sulfatedbile acids. Each of all the fractions is subjected
to solvolysis with hydrochloric acid, followedby hydrolysis with enzyme or 2 M sodium hydroxide solution, and then free bile acids thus
obtained are converted into volatile derivatives

for gas chromatographic analysis. Outline ofcommon procedures for systematic analysis ofbile acids in sample is shown in Fig. 1. By theseprocedures, the information on original conjugated bile acids is available.
Glucosides, glucuronides and N-acetylglucosaminides of bile acids (particular bile
acid conjugates) are present in only their forms
or double conjugated forms with glycine or
taurine. When bile acid extract from biologicalsample was fractionated with PHP-LH-20 gel
or DEAP-LH-20 gel column, these particularbile acid conjugates are not fractionated independently. No fractions are restricted where theparticular bile acid conjugates are fractionated.
For example, bile acid N-acetylglucosaminides
are fractionated into both nonamidate fraction
and glycine-conjugate fraction (Yamaga andKohara, 1994). There are no reports describing
the quantitative determination of bile acid glucosides, bile acid glucuronides and/or bile acid Nacetylglucosaminides in systematic analysis. The
method for their analysis is referred to the individual reports (Takikawa et al., 1982; Marschall etal., 1988, 1989; Yamaga and Kohara, 1994).

93

N. Yamaga et al.

iii) Internal standard compound(s) should bestable. That is, during the analysis, theymust not decompose to other compounds.

Indispensability ofinternal standard compoundsiv) After the fractionation with PHP-LH-20
gel column or DEAP-LH-20 gel column,
the characteristic internal standard compound(s) should be transferred into each of
all fractions. For this reason, four internal
standard compounds consisting of nonamidated, glycine-conjugated, taurineconjugated and sulfated forms, are necessary for the quantitative and qualitative
determination of bile acids in each fraction
after group separation.

A part of bile acids in an analytical sample,
especially in a small volume sample, is certainly lost during procedures such as extraction,
fractionation and other treatments. The usual
method for correcting the loss of bile acids
during analytical procedures is based on either
adding an internal standard compound to thesample in the course of the systematic analysis(Fig. 1) or performing a recovery test usinglabeled and non-labeled bile acids under a condition similar to the analytical condition. For
gas chromatographic analysis, the latter method
does not ensure that the recovery from abiological sample will be the same as that froman artificial sample that has been prepared withlabeled or non-labeled bile acids. Moreover,recovery may differ from assay to assay. Therefore, the use of internal standard compounds is
entirely indispensable for correcting the loss ofbile acids.

v) The peak(s) of internal standard compound(s) must not pile up on the peaks ofbile acids and the peaks of other mixed
compounds in the biological sample.
vi) The retention time of the internal standard
compound peak in each fraction must not
be largely distant from those of bile acidpeaks. Preferably, the peak of the internal
standard compound should appear in therange between the first and the last bile
acid peaks detectable on the gas chromatogram.

The minimum requirements forselecting internal standard compoundfor gas chromatography of bile acidsvii) Some linear relationship should be kept
between the peak high or area and theweight of each bile acid to the internal
standard compound.

The minimum requirements for selecting a suitable internal standard compound for the determination of bile acids by gas chromatography
are as follows.

When the selected internal standard compound(s) satisfy the requirements above mentioned, the area of an internal standard compound peak on the gas chromatogram is regarded as the amount (weight) of the internal standard compound added to the analytical sample,
irrespective of the recovery rate during all theprocedures. There is no occasion for considering the recovery ratio. It is only required to obtain linear calibration curves between the peakarea ratios and the weight ratios of each bile
acid to the internal standard compound.
Up to now, some kinds of unique bile acids
such as 5β-cholanoic acid (Campbell et al., 1975),i) Internal standard compound(s) must be
added to the analytical sample before theextraction of bile acids. Then, the peak ofcharacteristic internal standard compound
must appear together with peaks of bile
acids in the analytical sample on the samegas chromatogram.
ii) It is not necessary for internal standard compound(s) to be bile acid(s). However, it isbetter for them to be non-natural 5βcholanoic acid homologues with somehydroxyl groups.
94Internal standards for GC of bile acids

Fig. 2. Artificial bile acids chemically synthesized from cholic acid.

Internal standard compounds
for the determinationof total bile acid amounts

nordeoxycholic acid (van Berge-Henegouwen
et al., 1977; Roda et al., 1978), hyodeoxycholicacid (Ali and Javitt, 1970; Karlangnis andPaumgartner, 1978; Cantafora et al., 1979), 7ketolithocholic acid (Klaassen, 1971), 7-ketodeoxycholic acid (van Berge-Henegouwen etal., 1976) and hyocholic acid (Subbiah, 1973;
Yamaga et al., 1983) have been used as an internal standard compound for gas chromatographic analysis. Certainly, these compounds areable to be used as an internal standard compound. But they have some drawbacks. They
are sometimes found in samples from healthy
subjects, neonates and some patients (Alme etal., 1977, 1978, 1980; Back and Walter, 1980;
Sawada, 1981). Furthermore, oxo-5β-cholanoic
acids such as 7-ketolithocholic acid and 7-ketodeoxycholic acid are easily vulnerable to decomposition during alkaline hydrolysis withsodium hydroxide solution (Lepase et al., 1978).
For these reasons, it is necessary to develop
other internal standard compounds.

Natural bile acids originally have a hydroxyl
group at the C-3α position in the steroid nucleus. First, we chemically synthesized four isomers of 7,12-dihydroxy-5β-cholanoic acidlacking a hydroxyl group at C-3α position ofcholic acid; 7α,12α-dihydroxy-5β-cholanoic
acid (αα), 7β,12α-dihydroxy-5β-cholanoic
acid (βα), 7α,12β-dihydroxy-5β-cholanoic
acid (αβ) and 7β,12β-dihydroxy-5β-cholanoic
acid (ββ) (Fig. 2) (Arimoto et al., 1982). An
outline of the chemical synthesis of these artificial bile acids from cholic acid is shown in
Fig. 3. These compounds in the derivatives ofmethyl ester dimethylethylsilyl (DMES) ether
have different retention times compared to eachother on the gas chromatogram (Fig. 4). In addition, the retention times of these four compounds
are different from those of many authentic bile
acids. But, only the retention time of αβ is very
close to that of lithocholic acid (Table 1) and95

N. Yamaga et al.

Fig. 3. Outline of chemical synthesis of four 7,12-dihydroxy-5β-cholanoic acid isomers
from cholic acid.

27.292βαββ24.13628.744

αβ

46.7

40

33.3

26.7

20

Response

25.359ααFig. 4. Gas chromatogram of αα, αβ, βα and ββ (methyl ester DMES ether).
(See Table 1 for gas chromatographic condition.)96