Clinica Chimica Acta, 127 ( 1983) I - 17

Elsevier Biomedical Press

CCA 2359

Review

Serum bile acid analysis

K.D.R. Setchell * and A. Matsui **

(Received May 7th; revision August 31st, 1982)

Routine and research techniques are outlined for the analysis of bile acids in
serum. The basis of these techniques is the use of liquid-solid extraction and
liquid-gel chromatography coupled with the measurement of bile acids by gas
chromatography using high resolution glass capillary columns and mass spectrome-
try which provides increased sensitivity compared with conventional methods of
measurement. Problems associated with the isolation and purification of bile acids
from serum are discussed and rapid and flexible methods for their metabolic
profiling are described. The advantages and applicability of these procedures are
illustrated with examples of profiles of bile acids in the serum from normal subjects,
patients with liver disease and a patient with an ileal resection.

Despite the fact that serum bile acids are elevated in liver disease [l-5] and the
suggestion that their measurement may be a sensitive index of liver function, the
total concentration of bile acids in serum is extremely variable and provides little
diagnostic value in liver disease. The variability in the type of bile acids present in
serum together with their existence in different conjugated forms suggests that more
relevant information of liver dysfunction may be gained by examining patterns of
individual bile acids and their state of conjugation. Several groups have indicated the
potential of this type of approach; for example it has been suggested that the ratio of
the trihydroxy : dihydroxy bile acids in serum will differentiate patients with obstruc-
tive jaundice from those with hepatocellular injury [3,6] and that the diagnosis of
primary biliary cirrhosis and extrahepatic cholestasis can be made on the basis of the
ratio of the cholic : chenodeoxycholic acids [7-9).

* To whom correspondence should be addressed.

** Present address: Dept. of Pediatric Surgery, Faculty of Medicine, University of Tokyo. 7-3-1 Hongo
Bunkyo-ku, Tokyo, 113 Japan.

~09-8981/83/~-0~0/$03.~ Q 1983 EIsevier Biomedical Press

2

More recently, data have appeared indicating the value of measuring specific
types of conjugated bile acids in disorders such as Gilbert’s and Dubin-Johnson
syndromes [ 10.1 I]. Furthermore post-prandial increases in individual serum bile
acids are regarded as having greater sensitivity for detecting hepatic dysfunction
[9,12-14]. and. since intestinal input is a major determinant of the circulating
concentration of bile acids, they have also been proposed as a useful index of ileal
dysfunction in Crohn’s disease [15]. In addition, the fractionation of bile acids into
groups, on the basis of their polarity or mode of conjugation, is often essential to
studies of the elucidation of the metabolic pathways of administered bile acids, and
may also be of value in recognising pathological conditions involving the gastroin-
testinal tract.
With these considerations, procedures are described, based upon a recently
developed liquid-solid extraction procedure [ 161, liquid-gel chromatography, capillary
column gas chromatography and mass spectrometry, which are suitable for the
analysis of profiles of bile acids in serum samples, and which are sufficiently flexible
to allow modification depending upon the objective of the analysis.

Materials and reagents

Solvents and reagents were of analytical grade and were redistilled before use.
Diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20) was obtained from
Packard Instruments (Groningen, The Netherlands; marketed as Lipidex-DEAP),
and was washed with 72% ethanol and then converted to the acetate form by
washing with 0.2 mol/l acetic acid in 72% ethanol, followed by 72% ethanol [ 17,181.
Lipidex 1000 and Lipidex 5000 were obtained from Packard Instruments. Lipidex
1000 was washed with methanol and slurried into a glass column to give a bed size
of 5 X 1 cm. The gel was washed with 0.01 mol/l HCl (20 ml) before application of
the sample. SP-Sephadex (Pharmacia. Uppsala, Sweden) was converted to the [t-r”]
form by washing with 0.2 mol/l HCI in 70% methanol, followed by 70% methanol
prior to use. Reverse phase octadecylsilane bonded silica cartridges, Bond Elut
(Analytichem International, Harbor City, CA, USA) purchased from Jones Chro-
matography (Llanbradach, Mid Glam., UK) were washed with methanol (2 ml)
followed by water (5 ml) before use.
Cholylglycine hydrolase from Clostridium perfringens ( Welchii), 250 units/mg
protein (Biuret) was purchased from Sigma, St. Louis, MO, USA. The following
radiolabelled bile acids were obtained from the Radiochemical Centre, Amersham,
UK: [24-‘4C]lithocholic acid (specific activity 57 mCi/mmol), [24-‘4C]cholic acid
(specific activity 52 mCi/mm), [l-‘4C]glycocholic acid (specific activity 51 mCi/m-
mol), and ~24-‘4C]taurocholic acid (specific activity 60.9 mCi/mmol). A sampie of
[24-‘4C]lithocholic acid-3-sulphate was obtained from Dr. T.C. Bartholomew (Medi-
cal Unit, Royal Free Hospital, London, UK). Authentic bile acids were obtained
from Steraloids Inc. and the MRC Steroid Reference Collection (curator: Prof. D.N.
Kirk, Westfield College, London, UK). Diazald (~-methyl-~-nitroso-p-toluene
sulphonamide) was obtained from Aldrich Chemical Co., Gillingham, UK.
 3

Methods

A general outline of a variety of methods, based upon liquid-solid and liquid-gel

extraction and chromatography, for the analysis of different types of bile acids in
serum is shown in Fig. 1.

Liquid-s&d extraction of bile acids

Bile acids and their conjugates were rapidly and quantitatively extracted using
cartridges of reverse-phase octadecylsilane bonded silica (Bond Elut) as described
elsewhere [16]. Serum (1 ml) was diluted with 4 ~01s. of 0.1 mol/l sodium hydroxide
and heated in a water bath to 64X The diluted serum sample was passed rapidly
through a Bond Elut cartridge and discarded. The cartridge was washed with
distilled water (5 ml) and bile acids and their conjugates recovered by eluting the
cartridge with methanol (4 ml). The complete extraction procedure takes less than
5 min to complete.

(a) Analysis of the total non-su~hatgd bile acid concentration in serum

The methanolic extract was taken to dryness and re-dissolved in 2 ml sodium
phosphate buffer (0.1 mol/l, pH 5.8). Cholylglycine hydrolase (9 units) was added
and the sample incubated, either overnight at 37°C or for 4 h at 50’C. After
hydrolysis the pH of the solution was adjusted to pH 3-4 by the addition of 2 drops
of 1 mol/l HCl and the sample passed through a column of Lipidex 1000 (bed size
4 x 1 cm) pre-washed with 0.01 moi/l HCI. The sample tube was washed 4 times
with 3 ml of 0.01 mol/l HCl (pH 3.5) followed by 3 X 5 ml of distilled water. Bile
acids were then recovered from the Lipidex 1000 by elution with 68% methanol (20
ml). The sample was taken to dryness using a rotary evaporator and the bile acids
converted to methyl ester-trimethylsilyl ether derivatives for analysis by capillary
column gas chromatography.

(b) Analysis of unconjugated bile acids in serum

The methanolic extract from the Bond Elut cartridge was taken to dryness and
the extract resuspended in a solution of 0.01 mol/l HCl, (4 x 5 ml) and passed
through a small column of Lipidex 1000 (bed size 4 x 1 cm) prepared in this
solution. The column was then washed with distilled water (2 X 10 ml) and uncon-
jugated bile acids recovered by elution of the gel with 68% methanol (20 ml). The
sample was taken to dryness and methyl ester-trimethylsilyl ether derivatives pre-
pared for analysis by GC and GC-MS.

fc) Analysis of the individual bile acid conjugates in serum

The methanolic extract from the Bond Elut extraction was diluted with water to
give a final solution equivalent to 72% methanol. To remove any cations which may
interfere with the anion exchange chromatography [ 17,181, the extract was passed
through a small column of the cation exchange gel, SP-Sephadex (bed-size 5 x I cm)
prepared in the [H+] form in 72% methanol. The eluate was then applied directly to
4

TABLE I
SOLVENT SYSTEM FOR THE FRACTIONATION OF BILE ACIDS ON DEAP-LH-20

Fraction Eluting solution a ‘Apparent’ Applied

PH volume

Neutral compounds (SP-Sephadex eluate in 72% methanol) (-) (25 ml)

and sterols 72% ethanol 9 ml
&conjugated 0.1 moI/I acetic acid in 72% ethanol 4.0 6.5 mf
bile acids
Glycine and taurine 0.15 moI/l acetic acid in 72% ethanol 6.4 h 6.5 ml
conjugates
Sulphate conjugates 0.3 mol/l acetic acid in 72% ethanol 9.6 b 10 ml

’ Between each change of solvent, 2 ml of 72% ethanol was added to wash out residuat buffer from the gel
bed.
b The pH of this solution was adjusted by the addition of cont. ammonia.

a small column of DEAP-LH-20 (0.6 g) prepared in the acetate form and bile acids
were fractionated on this anion exchange gel according to their mode of conjugation
[I: 81, by the stepwise elution of the solvents shown in Table I. To minilnise carry-over,
between the changes of buffers, the column was washed with 72% ethanol (2 ml). All
of these fractions were taken to dryness, ‘The conjugate fractions were treated as
follows:

(i) Unconjugated bile acids were converted directly to methyl ester-trimethylsilyi

ether derivatives and analysed by capillary column gas chromatography and mass
spectrometry.

(ii) G&he and taurine fractions were hydrolysed with cholylglycine hydrolase
and the deconjugated bile acids extracted using a column of Lipidex 1000 as
described above and converted to their methyl ester-t~methy~silyl ethers for analysis
by gas chromatography and mass spectrometry.

(iii) Suiphatefractions were solvolysed in methanol (9 ml) and acetone (1 ml) to

which 3 drops of 6 mol/l HCl were added [ 191. The sample was stoppered and
incubated at 39°C for 24 h. The sample was neutralised by addition of sodium
hydroxide and taken to dryness on a rotary evaporator. The residue was redissolved
in 0.01 mol/l HCl (2 x 5 ml), the solution was adjusted to pH 4.5 and passed
through a Bond-Elut cartridge. The cartridge was washed with water (2 ml) and bile
acids were recovered by elution with methanol (4 ml). The methanolic extract was
taken to dryness over nitrogen and bile acids hydrolysed with cholylglycine hydro-
Iase as previously described. ~nco~jugated bile acids were extracted using Lipidex
1000 as above and the extract finally purified, by passage through a small column of
DEAF-LH-20 prepared in the acetate form, and coltection of the u~~onjugated bile
acid fraction. The bile acids were converted to methyl ester-trimethylsilyl ether
derivatives and analysed by gas chromatography and mass spectrometry.

Derivatisation

After addition of the internal standard, coprostanol (O.l- 10 pg), the samples were
dried and dissolved in 0.3 ml of methanol. Methylation was carried out by reaction
for I.5 min with 2.7 ml of diazomethane in diethyl ether, freshly prepared from the
reaction between sodium hydroxide and N-methyl-N-nitroso-p-toluenesulfonamide
(Diazald). The reagent was removed by evaporating to dryness over nitrogen and the
trimethylsilyl ether derivative prepared by the addition of 50-100 ~1 of a solution of
pyridine : hexamethyldichlorosilane: trimethylchlorosilane (3 : 2 : 1, by vol.) and heat-
ing at 60°C for 15 min. The derivatised sample was purified and the excess reagents
removed by passage through a small column of Lipidex 5000 [20].

Gas chromatography

Gas chromatography was performed on a Pye 106 gas chromatograph, modified

to accept a 25-metre silicone OV-I wall-coated open tubular glass capillary column
(Jaeggi, Trogen, Switzerland) and equipped with an ail-glass solid injection device of
the type described by Van den Berg and Cox [21]. Helium was used as the carrier gas
and the flow rate through the column was approximately 2.5 ml/min. Temperature
programmed operation from 220-275°C with increments of 2”C/min was em-
ployed.

Gas chromatography-mass spectrometr+y (CC-MS)

GC-MS was carried out using a Varian 2700 gas chromatograph housing a 1%
Hi-eff &-BP conventional packed column (I metre) which was coupled to a Varian
MAT-731 double focussing mass spectrometer operated with electron impact ionisa-
tion. The temperature of the ion source was 250°C and the energy of bombarding
electrons 70 eV. Selected ion monitoring was carried out using accelerating voltage
switching to focus the ions of m/z 368, 369, 370 and 372 which are characteristic of
mono-, di- and tri-hydroxylated bile acid structures.

Results and discussion

1. Liquid-solid extraction of bile acids

The inclusion of an initial extraction step, which also affords a means of
removing proteins and inorganic ions, is essential in any scheme in which serum bile
acids are to be anaIysed by gas chromatography or gas chromatography-mass
spectrometry. Time-consuming liquid-liquid extraction or alcoholic protein precipi-
tation techniques, which were originally used to extract bile acids from serum, later
gave way to more convenient liquid-solid extraction techniques. These took the form
of columns of Amberlyst A-26 anion exchange resins [4] and later the neutral resin
6

Amberlite XAD-2 [22]. The need to increase sample throughput led to the descrip-
tion of a batch method for the extraction of bile acids from serum using the more
polar neutral resin Amberlite XAD-7, which afforded the extraction of up to 30
samples/day [23].
One disadvantage experienced with the Amberlite XAD resins is the poor
recovery of polar sulphated bile acids, and, although it has been suggested that the
recovery of bile acid sulphates can be improved by eluting the resin with ethanol
containing 8% of 12 mol/l HCl [24], this procedure may lead to their partial
solvolysis and the formation of artefacts. This supposedly neutral resin was recently
shown to exhibit weak anion exchange properties and this therefore accounts for the
strong irreversible adsorption of polar acidic steroids [25].
Recently the application of reverse-phase octadecylsilane bonded silica, in the
form of cartridges, increased dramatically the speed and efficiency of extracting
steroids from urine [26,27] and serum 125,271. As a result of these applications, a
method was developed for the quantitative extraction of bile acids and their
conjugates from serum [16]. Since this material behaves as a polar adsorbent and
does not possess any ion exchange properties, a quantitative recovery of extremely
polar sulphated bile acids is attained [ 161 while non-polar lipids, of which cholesterol
is quantitatively the most important, are largely excluded from adsorption [l&25].
The extraction procedure is rapid, taking only a few minutes to perform and, using a
specially designed vacuum apparatus, it may be semi-automated, thereby allowing
up to 60 samples/h to be extracted if required [ 161.
Once the bile acids and their conjugates are extracted, several procedures may be
adopted for their analysis, and these are outlined schematically in Fig. 1 and
discussed below.

2. Analysis of totalnon-sulphated bile acids in serum

This procedure, which is relatively rapid and convenient, is suitable as a routine
method for serum bile acid measurement.
Enzymatic hydrolysis of the bile acid extract with cholylglycine hydrolase will
result in the deconjugation of glycine and taurine conjugated bile acids [28,29]. The
extraction of unconjugated bile acids following hydrolytic procedures has almost
universally been carried out by either liquid-liquid partitioning after acidifying the
hydrolysate to pH 1, a procedure which may lead to partial solvolysis of sulphated
bile acids, or by liquid-solid extraction using Amberlite XAD-resins [22,23]. More
recently however, Lipidex 1000 was shown to be a very convenient and rapid
method for the extraction of steroids and bile acids from aqueous solutions [30,31];
however, the potential of this gel to bile acid analysis has yet to be widely realised.
The use of this gel for the quantitative extraction of unconjugated bile acids from
fecal extracts [32] and for their separation from conjugated bile acids in aqueous
solutions is described elsewhere [31]. Lipidex 1000 behaves as a non-polar adsorbent
and will quantitatively extract non-polar and medium polarity lipophilic molecules
from acidified aqueous solutions [30-341. It is, therefore, ideally suited to the
extraction of unconjugated bile acids from aqueous enzymatic hydrolysates. After
adjusting to pH 3-4, the hydrolysate is rapidly filtered through a small column of
 7

Fig. 1. A schematic representation of variety of approaches to the analysis of bile acids in serum samples.

Lipidex 1000, and after washing the gel with water, bile acids can be quantitatively
recovered by elution of the gel with methanol.
The extremely high capacity of this gel enables small column sizes and high flow
rates to be used, and affords a very convenient and rapid method for the quantita-
tive removal of unconjugated bile acids from enzymatic and alkaline hydrolysates
[32]. The recovery of [‘4C]cholic acid which was added to serum samples and
analysed by this procedure was 95.1 i 3.1% (n = 14).
Once compounds are sorbed to the gel, by increasing the polarity of the eluting
solvent it is possible perform reverse-phase partition chromatography and separate
unconjugated bile acids from neutral monohydroxy-sterols. Fig. 2 shows the elution
profiles of radiolabelled cholic acid, lithocholic methyl ester and cholesterol, when
the proportion of water in the eluting solvent is increased. By using 68% methanol,
cholesterol is retained by the gel and can be completely separated from bile acids.
This gel, therefore, has the additional advantage of providing a suitable method for
removing the small percentage (although relatively large amount) of cholesterol
remaining after the liquid-solid extraction, which would otherwise cause interference
with bile acid derivatives in the subsequent GC and GC-MS analysis.

3. The measurement of unconjugated bile acids in Serum

It has recently been shown that unconjugated bile acids present in serum are
8

80

fi 100 % methanol
60

40

20

0 :
r
0 4 8 12 16 20
100
80 % methanol
80 T

68 % methanol
40 i

0 10 20 30 40 50
Volume (ml)
Fig. 2. The elution profiles of radiolabelled lithocholic acid, lithocholic-methyl ester and cholesterol which
were extracted from an acidic aqueous solution by a small column of Lipidex 1000 and &ted using
solvents of increasing polarity.

quantitatively of greater importance than had previously been believed [35,36]. These
studies also revealed that meal-induced changes in the concentration of uncon-
jugated bile acids in the serum of normal subjects occurred, indicative of a pool of
bile acids sequestering in the intestine [35,36].
Unconjugated bile acids can be measured by either of two approaches outlined in
Fig. 1.
(i) As already discussed, Lipidex 1000 will effectively remove unconjugated bile
acids from acidified aqueous solutions. Therefore, by reconstitution of the bile acid
extract in 0.01 mol/l HCl and passage of the acidic solution through this gel,
unconjugated bile acids are retained and can be quantitatively recovered [3 1,321 and
separated from monohydroxy sterols using 68% methanol (Fig. 2).
The recovery of [r4C]cholic acid added to serum samples and isolated by this
procedure was 93.8% & 3.0% (n = 11). Adopting this approach, the sample is suffi-
ciently purified to enable unconjugated bile acids to be measured directly by
capillary column gas chromatography.
(ii) Fractionation of the serum bile acid extract on the lipophilic gel DEAP-LH-20
[ 181 offers an alternative method for the isolation and purification of unconjugated
 9

lieal resection patient V.R age 23 P

Meal Meal
2.5 CDca l

CA n

p 2.0 UCCA .

s I5
‘8

Fig. 3. Changes in concentration (pmol/l) of unconjugated bile acids in the serum from a patient with an
ileaf resection measured over a 24 h period by CC-MS after their isolation by chromatography on the
anion exchange gel DEAP-U-I-20.

bile acids and this is discussed below. When CC-MS is employed to measure the bile
acids isolated in the unconjugat~d bile acid fraction following their derivatisation,
diumai variations are seen to occur in normal subjects and the significance of these
physiological changes have been reported and discussed elsewhere [35,36], Fig. 3
shows that, as wet1 as having sufficient sensitivity to detect and measure serum
unconjugated bile acids, post-prandiat changes, atthough reduced in amplitude
compared to normals [36], may also be detected in a subject with an ifeat resection
when these techniques were empioyed.

4. The analysis of individual bile acid coqjugates in serum

The separation of bile acids into groups based upon the use of the lipophilic
anion exchange gel DEAP-LH-20 was first described in detail by AlmC et al [ 181 and
applied to the analysis of bile acids extracted from the urine of patients with
cholestatic liver disease. More recently, the technique was shown to be applicable to
the analysis of bile acids in serum [37,38]. Most of the technical details relating to
the use of this gel have been discussed in detail previously [ 1st.
Differences from the method originally described include the use here of smaller
elution volumes of buffers (Table 1) and the colfection of the glycine and taurine
conjugates as a single fraction. although, if required, the separation of these two
conjugate groups can be achieved using the original solvent system. It should be
stressed that the eluting volumes of the buffers and their ‘apparent’ pH are critical to
prevent overlap between the different conjugate groups, and this may explain some
of the difficulties which have been experienced with this chromatographic step [39].
Furthermore, we have chasen to substitute the cation exchange gel, SP-Sephadex, for
the Amberlyst A-15 resin originally described [IS] because unlike the resin, the inert
nature of the gel is less likely to lead to irreversible adsorption of compounds, and
gives a higher degree of sample purification. The inclusion of this cation exchange
gel, which should be prepared and run in methano~ic solution, is essential to remove
cations which may interfere with the anion exchange chromatography. The re-
coveries of several radiolabelled bile acids which were added to serum samples,
IO

extracted and fractionated on DEAP-LH-20 were essentially quantitative and com-

parable to values reported previously [ 18,321.
Enzymatic hydrolysis of the combined glycine and taurine fraction was carried
out and the deconjugated bile acids extracted using the Lipidex 1000 system
described. When radiolabelled [24-‘4C]taurocholate was added to sera, the overall
mean recovery of radioactivity by this method following deconjugation was 98.7%
(n = 5, cv = 5.7%).
The sulphate fraction was first solvolysed and then enzymatically hydrolysed.
Solvolysis in methanol/acetone/HCl 1191 was recently shown to give higher re-
coveries of bile acids which were sulphated in positions C-3 and C-7 compared with
other methods [40]; however, it should be realised that acetonides of cis-glycol
structures will result. In cases where there may occur bile acids with this type of
structure it is preferable to solvolyse the sample in ethyl acetate/ethanol/H,SO,
[41] followed by an aqueous alkaline hydrolysis. Final purification of this fraction
can be achieved using the anion exchange gel DEAP-LH-20 and collection of the
unconjugated bile acid fraction.

5. Gas chrornaaographic analysis

Capillary column gas chromatography is a widely employed technique for the
qualitative and quantitative analysis of steroids [42], yet in spite of the advent of
open-tubuIar glass capillary columns with greatly increased chromatographic resolu-
tion, their application to bile acid analysis has been limited [43-451. The higher
chromatographic resolution of glass capillary columns compared with conventional
packed columns affords greatly increased sensitivity, and is potentially a powerful
tool for the metabolic profiling of complex mixtures of bile acids [32,34]. With the
use of an all-glass dropping needle solid injection device [Zl] it is possible to
introduce onto the capillary column a much greater proportion of the sample than is
possible by liquid injection and this means, in effect, that the sensitivity of the
analysis is improved. The limit of detection for serum bile acids by this method is
approximately 0.01 umol/l, and this sensitivity compares favourably with that of
most radioimmunoassay methods for serum bile acids.
An example of this high sensitivity is seen from the chromatogram shown in
Fig. 4, which represents the total non-sulphated bile acids in the serum (1 ml) from a
healthy adult (fasting) man. Using conventional packed column gas chromatogra-
phy, chromatograms suitable for the quantitative determination of bile acids in the
serum of healthy subjects are difficult or even impossible to attain, except where
large volumes of serum are taken for analysis.
Concentrations of the principal serum bile acids determined by this procedure for
a number of normal subjects and a group of patients with liver disease are shown in
Table II. The improved extraction, isolation and purification procedures described
here, coupled with the increased sensitivity and specificity possible using capillary
column GC, most probably accounts for the slightly lower range in the total serum
bile acid concentration of normals compared with the ranges reported by techniques
in which packed column gas chromatography is employed [4,5,46-481.
 11

td

4
;,w
40mins. 20 0
Fig. 4. Capillary column gas chromatographic profiles of the fasting total non-sulphated bile acids in the
serum of a healthy adult man. Bile acids were analysed as their methyl ester-trimethylsilyl ether
derivatives using a 25-m glass capillary column coated with silicone OV-1 using conditions described in
the text.

The potential of gas chromatography-mass spectrometry to the qualitative de-

termination of bile acids is well recognised [49,50]. When the technique is employed
in the selected ion monitoring mode, the quantitative determination of analytes can
be carried out with greatly increased sensitivity and specificity compared to capillary
column gas chromatography. This added sensitivity may be particularly useful where
extremely low concentrations or small sample volumes are encountered, as was the
case in a recent study of bile acids in the interstitial fluid of patients with cholestatic
liver disease and pruritus [38]. Selected ion monitoring of the ions m/z 368, 369, 370
and 372 (Fig. 5) affords a means of detecting with high sensitivity, all saturated and
unsaturated bile acids which are substituted with one, two and three hydroxy’
functions, irrespective of their positions in the bile acid nucleus [36,38]. Identifi.
cation of a bile acid can be made on the basis of the GC retention time and the ion
response relative to authentic compounds, One of the problems in selecting these
particular ions for recording, is the fact that the trimethylsilyl ether derivative 01
cholesterol gives rise to a relatively intense ion at m/z 36X and is also difficult tc
separate from, either lithocholic acid on non-selective GC phases such as SE-30 01
OV-1, or cholic acid when a selective GC phase such as Hi-eff-8BP or Carbowar
12

TABLE II

CONCENTRATIONS (pmol/l) OF THE PRINCIPAL NON-SULPHATED BiLE ACIDS IN THE

SERUM OF NORMAL SUBJECTS AND PATIENTS WITH LIVER DISEASE

Sex Age Cholic Cheno- Deoxy- Litho- Total

deoxy- cholic cholic
cholic

Normal (fasting) F 28 0.01 0.10 0.10 n.d. 0.2 I

Normal (fasting) F 25 0.12 0.17 0.33 0.07 0.69
Normal (fasting) F 23 0.12 0.29 0.08 nd. 0.49
Normal (fasting) F 24 0.16 0.37 0.21 nd. 0.74
Normal (fasting) F 21 0.12 0.08 0.03 n.d. 0.23
Normal (fasting) F 21 0.03 0.06 0.01 n.d. 0.10
Normal (fasting) M 33 0.32 0.33 0.24 n.d. 0.89
Normal (fasting) M 50 0.10 0.15 0.03 nd. 0.28
Normal (fasting) M 40 0.08 0.17 0.08 n.d. 0.33
Normal (fasting) M 41 0.31 0.23 0.04 nd. 0.58
Normal (fasting) M 31 0.12 0.18 0.01 0.05 0.35
Normal (fasting) M 28 0.13 0.10 0.05 0.0 1 0.28
Normal {fasting) M 27 0.14 0.44 0.52 0.02 1.12

Primary biliary cirrhosis F 52 81.79 51.75 n.d. 0.01 133.54

Primary biliary cirrhosis F 40 1.30 4.23 nd. 0.0 1 5.53
Primary biliary cirrhosis F 59 10.78 15.61 n.d. 0.01 25.39
Primary biliary cirrhosis F 53 39.14 26.14 3.47 n.d. 68.75
Primary biliary cirrhosis M 67 4.65 0.76 0.26 n.d. 5.67

Biliary atresia F 4/12 19.06 13.97 1.12 0.74 34.89

Biliary atresia F 7/12 9.17 49.64 6.63 0.11 65.55
Biliary atresia F 2/12 8.69 16.56 3.27 0.85 29.37
Biliary atresia F 6/12 24.61 2.21 2.60 0.43 29.85
Biliary atresia F 24 21.74 34.80 nd. n.d. 56.54

Benign recurrent intra-

hepatic cholestasis F ? 54.53 7.64 13.47 2.23 77.97
-
n.d.. not detected.

20000 is used. Different derivatives have been suggested as a means of overcoming

this problem. However, the only satisfactory solution, is to remove cholesterol from
the sample prior to derivatisation, and in this respect the combined use of the
Iiquid-soIid and liquid gel techniques described here are particularly advantageous.
Fig. 5 shows ion current chromatograms obtained by this procedure for the
individual conjugate fractions isolated by chromatography on DEAP-LH-20 typical
for the serum of a patient with primary biliary cirrhosis. A detailed account of the
quantitative levels and the qualitative profiles of indi~dua~ bile acids in each
conjugate class for a group of patients with cholestatic liver disease is reported
elsewhere [38], and a striking feature is the relatively high proportion of sulphated
bile acids present in the serum [38].
Quantification is carried out by comparing, against calibration standards, the
 13

389

--I
18 Timelminrl 018 ,,metmtnri 018 TlrnPl”l”5i 020 Timelrn,“il 0

Fig. 5. GC-MS selected ion current recordings of the ions m/r 368, 369, 370 and 372 (gain settings
shown) obtained following the analysis of methyl ester-trimethylsilyl ether derivatives of bile acids on a
Hi-eff 8BP packed column at 23O’C. Shown is a chromatogram obtained for a mixture of the authentic
bile acids and chromatograms typical of the glycine, taurine and sulphate conjugates which were isolated
by chromatography on DEAP-LH-20 from the serum of a patient with primary biliary cirrhosis.

ratio of the peak height response of each bile acid to the peak height response
obtained for the ion of m/z 370 arising from the internal standard, coprostanol,
which is added to the sample prior to derivatisation. The precision of the method
determined from replicate analysis (n = 5) of the serum from a patient with liver
disease and a normal subject, gave coefficients of variation of 6% for cholic acid and
5 % for chenodeoxycholic acid.
The concentrations of the principal bile acids in each of the conjugate classes
measured by GC-MS is shown in Table III for five normal subjects. The range for
the total bile acids of 0.27-1.09 pmol/l is comparable to the values found when
capillary column GC was employed (Table II), and similar to those reported
previously using this lipophilic gel and repetitive magnetic scanning GC-MS analysis
[371.
The use of bile acids labelled with stable isotopes as internal standards [51,60],
when they become less expensive and more widely available, would be expected to
improve the accuracy and the precision of the method.
One drawback, however, of the selected ion monitoring technique is the limited
number of ions which can be monitored simultaneously, which therefore gives the
analysis a biased or selective nature. Repetitive magnetic scanning over the entire or
partial mass range has been used effectively [ 18,373, and, while this approach offers
greater flexibility in terms of the identification of potentially new bile acids, it has
14

TABLE III
THE CONCENTRATION (rmol/l) OF THE PRINCIPLE BILE ACIDS IN THE SERUM OF 5
NORMAL FASTING ADULTS, MEASURED BY CC-MS WITH SELECTED ION MONITORING
AFTER FRACTIONATION INTO CONJUGATE GROUPS USING THE ANION EXCHANGE
GEL DEAP-LH-20

6 31 yrs 6 28 yrs 6 27 yrs P 25 yrs P 24 yrs

Glycine conjugates
cholic acid 0.053 0.063 0.049 0.072 0.1 I5
chenodeoxychoiic acid 0.117 0.074 0.218 0.136 0.335
deoxycholic acid n.d. 0.05 1 0.249 0. I67 0.165
lithocholic acid 0.005 n.d. n.d. n.d. n.d.

Taurine conjugates
cholic acid 0.029 0.029 0.026 0.022 0.023
chenodeoxycholic acid 0.025 0.010 0.029 0.010 0.014
deoxycholic acid n.d. n.d. 0.016 0.019 0.014
lithocholic acid n.d. n.d. n.d. n.d. n.d.

Sulphate conjugates
cholic acid 0.040 0.033 0.064 0.023 0.017
chenodeoxycholic acid 0.042 0.013 0.194 0.024 0.017
deoxycholic acid n.d. n.d. 0.253 0.044 0.029
lithocholic acid 0.051 0.001 0.192 0.065 0.022

Total bile acids 0.362 0.274 1.090 0.482 0.75 1

n.d., not detected.

less sensitivity than selected ion monitoring and furthermore requires the use of a
computer to store and interpret the vast amount of data which is recorded.

Conclusions

The potential of the liquid-solid and liquid-gel extraction procedures outlined has
yet to be fully realised. However an expansion in their use, not only in connection
with CC and GC-MS techniques, is anticipated. As well as for serum bile acids
[ l&31,34-38] these general techniques have been effectively applied to the multicom-
ponent analysis of bile acids in urine [l&52-54], feces ]32,34], and meconium [55J, in
addition to bile alcohols [56], bile acid glucuronides [57] and neutral steroids [ 171.
As is evident from the literature over the last year [16,58-601, an exponential
increase in the application of reverse-phase octadecylsilane bonded silica cartridges
can be expected and, in our opinion, this liquid-solid extraction procedure will
eventually replace more conventional methods of extracting bile acids from biologi-
cal samples, particularly since the procedure can be semi-automated [ 161.
The inert nature, high capacity and versatility of the liquid-gel extraction and
chromatographic procedures described here and elsewhere [ 17,18,3 1,32,34] provides
a high degree of sample purification and compound enrichment, factors which are
important when GC and GC-MS are to be used for the identification and quantifica-
 15

tion of trace amounts of compounds. Compared to conventional packed column GC,

capillary column GC is far superior in terms of chromatographic resolution, specific-
ity and sensitivity and together with the allied techniques of sample pre-treatment
described, should be considered as a powerful complementary procedure rather than
an alternative one to TLC, radioimmunoassay and enzyme immunoassay methods
for bile acid measurement.

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