Internal standards for GC of bile acids

Fig. 7. Internal standard compounds chemically synthesized for gas chromatographic analysis
after group separation.

Yamaga and coworkers (1983) adopted ββ as aninternal standard compound for the quantitative
determination of total bile acids in urine by gas
chromatography. Other workers used tauro7α,12α-dihydroxy-5β-cholanoic acid (Tauroαα) as an internal standard compound for thesame project (Ghoos et al., 1983). These synthesized compounds (αα, βα, ββ and Tauro-αα
except αβ, because the peak of αβ piles up onthat of lithocholic acid) are adequate internal
standard compounds for the quantitative determination of bile acids by gas chromatography.
However, as far as only one kind of internal
standard compound is used, the analysis after
group separation of bile acids can not attainaccurate data in all fractions, since ββ transfers
only into the nonamidate fraction after an ion
exchange gel column chromatography. On theother hand, Tauro-αα transfers only into thetaurine-conjugate fraction in a similar meaning
as above. Accordingly, adequate internal standard compounds must be added into other fractions except the nonamidate fraction in the caseof ββ and tauro-conjugate fraction in the case of
Tauro-αα on half way just after group separation in systematic analysis.

Internal standard compounds
for the determination ofgroup separated bile acids
Yamaga and coworkers (1987) have chemicallysynthesized four internal standard compounds
exactly transferred into each conjugate fraction
by group separation. They are four different compounds using 7,12-dihydroxy-5β-cholanoic acidisomers; 7β,12β-dihydroxy-5β-cholanoic acid(ββ) for the nonamidate fraction, glyco-7α,12αdihydroxy-5β-cholanoic acid (Glyco-αα) for theglycine-conjugate fraction, tauro-7β,12βdihydroxy-5β-cholanoic acid (Tauro-ββ) for
the taurine-conjugate fraction and glyco-7α,12α-dihydroxy-5β-cholanoic acid 7-sulfate(Glyco-αα 7-sulfate) for the sulfate fraction(Fig. 7). An artificial sample composed of thesefour compounds is fractionated into each fraction with PHP-LH-20 gel column, and theneach internal standard compound appears as apeak in each corresponding fraction coinciding
with the internal standard compound by gas
chromatographic analysis. However, when99

25.279

αα

30

26.7

23.3

20

Responsecholesterol25.102

N. Yamaga et al.

Fig. 8. Gas chromatogram of sulfate fraction fromhuman urine with ββ, Glyco-αα, Tauro-ββ and Glycoαα 7-sulfate. Cholesterol DMES ether and αα methyl
ester DMES ether. (See Table 1 for gas chromatographic condition.)

human urine with Glyco-αα 7-sulfate added isanalyzed in the same way, αα and cholesterol
appear as peaks on the same gas chromatogram
having almost the same retention time in sulfate
fraction in some biological samples. This evidence indicates that cholesterol sulfate in urine isfractionated into the sulfate fraction when theextracts from urine using Amberlite XAD-2 or
Sepak C18 are fractionated with PHP-LH-20 gel
column, and then cholesterol is detected in sulfate fraction (Fig. 8). In fact, cholesterol sulfate
is present in biological samples, especially inurine (Winter and Bongioanni, 1968: Muskiet etal., 1983). Therefore, it becomes necessary either to remove cholesterol from the solvolysate
by extraction with hexane after solvolysis of thesulfate fraction or to select another sulfated candidate from 7,12-dihydroxy-5β-cholanoic acidisomers except αα. Finally, the combination ofββ, Glyco-αα, Tauro-αβ and Glyco-βα 7-sulfate(Fig. 7) is most suitable as an internal standard
compound in gas chromatographic analysis
after group separation.
Figure 9 shows the result of an artificial
sample containing ββ, Glyco-αα, Tauro-αβ andGlyco-βα 7-sulfate as four internal standard
compounds being analyzed by the systematic
analysis shown in Fig. 1. Four internal standard
compounds are well fractionated into individual
corresponding fraction. Besides, Fig. 10 shows
the results that bile acids in human urine withand without the four internal standard compounds were analyzed by the same method described above. When both profiles from theanalysis of a urinary sample with and without
four internal standard compounds were compared, neither peaks of bile acid nor of non-bileacid compound piling up on the peak of the internal standard compound are detected on thegas chromatogram in any fraction. Moreover,the profile from the analysis of the urinary sample with the above combination of internal standard compounds lends a helpful suggestion. It
enables us to judge from the gas chromatogram
whether the fractionation with PHP-LH-20 gel
column is perfect or not, as the different fourinternal standard compounds were used. For
example, the peaks of different internal standard compounds more than two are detected in
one fraction when the fractionation is incomplete. Furthermore, the peak of an internal standard compound in each fraction acts as a peculiar indicator for the quantitative determinationof each bile acid in its fraction and for the identification of bile acid peaks by agreement withthe relative retention time of individual bile
acids to the internal standard compound appearing in its fraction (Table 1).
The amount of each internal standard compound, 1–5 µg in 1 mL of serum and in 5 mL ofurine, added in a biological sample should beenough in the analysis, though it depends on theconcentration of bile acids in the biologicalsample.

100

Internal standards for GC of bile acids

Taurine-conjugate fraction

Response

46.7

40

46.7

40

βα←Glyco-βα 7-sulfate

33.3

20

46.7

40

33.3

αα←Glyco-αα26.727.285

Response

Response20

33.3

Sulfate fraction

25.250Glycine-conjugate fraction

26.7

αβ←Tauro-αβ

20

46.7

40

33.3

20

26.7

ββ

26.7

Response

28.77224.211

Nonamidate fraction

Fig. 9. Gas chromatograms of nonamidate, glycine-conjugate, taurine-conjugate and sulfate fractions from
artificial sample added ββ, Glyco-αα, Tauro-αβ, and Glyco-βα 7-sulfate. (See Table 1 for gas chromatographic condition.)

Views on the future
It is inevitable that in analytical experiment a
number of complicated procedures result in theloss of the compound analyzed as mentioned
above. Therefore, the use of internal standard
compound(s) is indispensable for the correctionof the data obtained by quantitative analysis.
Very few investigators have described what compound(s) have been adopted as internal standard
compounds and many investigators are apt toomit the description of whether internal standard compound(s) were used or not in gas chromatographic analysis. The cause may be basically that there have been very few investigations
on internal standard compounds in the past; buton the other hand there seems to be an increasing trend that acquisition of experimental datais too far reaching for consideration in the experimental method. If so, the authors themselves imply no reliability in the data they have obtained.
It is important to obtain data by appropriate
experimental methods, and then, discussion is
to be established based on reliable analytical
data.
This article comprehensively described thenecessity of suitable internal standard compounds and their importance in gas chromatography at the present time as a general review.
At the present time, in the quantitative determination of bile acids using gas chromatography,
either ba or bb is the most suitable internal
standard compound for the determination of
total bile acid amounts in the biological sample.

101

102

46.7

40

33.3

46.97732.76634.84227.29228.24732.81734.86130.61931.11028.273

without
Glyco-αα30.783(βα)

20.77623.622Glycine-conjugate fraction

24.07723.593

46.7

40

33.3

26.7

20

46.7

40

33.3

47.04628.47830.62731.65733.96334.37937.19140.52630.48035.556(ββ)
28.74425.09127.72024.136(αβ)

40.62221.500

Taurine-conjugate fraction
30.61231.62033.92234.37035.57523.19524.30926.48127.68645.22247.052

Response

41.87235.63637.24030.514

without
ββ26.7

20.751

with
Glyco-ααResponse

42.61830.02430.58632.87334.34834.868

26.7

20

with
ββ

20

46.91325.359(αα)

Response

Nonamidate fraction

31.07040

46.7

39.69733.3

42.51329.93130.51532.77334.28234.77035.435

26.7

20

Response

N. Yamaga et al.

without
Tauro-αβwith
Tauro-αβSulfate fraction

without
Glyco-βα 7-sulfate

with
Glyco-βα 7-sulfate

Fig. 10. Gas chromatograms of nonamidate, glycine-conjugate, taurine-conjugate and sulfate
fractions from human urine with and without four internal standard compounds. (See Table 1 for gas
chromatographic condition.)

Internal standards for GC of bile acids
On the other hand, the combination of bb,
Glyco-aa, Tauro-ab and Glyco-ba 7-sulfate is
the the most suitable internal standard compound for the accurate determination of bile
acid amounts in each fraction after group separation of bile acids in the biological sample using the ion exchange gel column. However, it is
not because all problems in the accurate determination of bile acids using gas chromatography were completely solved. When unknown
bile acid(s) and new bile acid form(s) conjugating with other substance(s) are discovered, thedevelopment of new internal standard compound(s) and new systematic analyses may become necessary. It has already been requested
that new systematic analyses for glucoside,
glucuronide and N-acetylglucosaminide of bile
acids should be developed without delay including new internal standard compound(s).

Acknowledgments: We sincerely thank Ms. Yumiko
Uyama of the Department of Biochemistry, Tottori
University Faculty of Medicine for her help in clerical works.
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105

Received April 2, 2001; accepted May 10, 2001
Corresponding author: Nobuo Yamaga