Journal of Chromatographic Science 2015;53:1060– 1065

doi:10.1093/chromsci/bmu167 Advance Access publication December 17, 2014 Article

Rapid Determination of Bile Acids in Bile from Various Mammals by Reversed-Phase

Ultra-Fast Liquid Chromatography
Gu Leng Ri Si1,2, Peng Yao1 and Luwen Shi1*
1
Pharmacy Administration and Clinical Pharmacy of School of Pharmaceutical Science, Peking University, Xueyuan Road Haidian
District, Beijing 100191, China, and 2Inner Mongolia Autonomous Region People’s Hospital, Inner Mongolia Autonomous Region,
Hohhot 010010, China

*Author to whom correspondence should be addressed. Email: [email protected]

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Received 25 February 2014; revised 19 November 2014

A valid and efficient reversed-phase ultra-fast liquid chromatography reflect the status of liver disease, while the bile, which is directly
method was developed for the simultaneous determination of 13 bile formed in the liver, does (7, 8). So the alteration in bile can be
acids in the bile of three mammal species, including rat, pig and used as an indicator of various liver diseases, or as a marker of
human gallstone patients. Chromatographic separation was per- liver toxicity (7, 9). Moreover, it also can provide directly useful
formed with a Shim-pack XR-ODS column, and the mobile phase con- information about gallstone disease states (7, 10). Thus, screen-
sisted of acetonitrile and potassium phosphate buffer ( pH 2.6) at a ing and the determination of major bile acids may be crucial for
flow rate of 0.5 mL min21. The linear detection range of most bile the clinical practice.
acids ranged from 2 to 600 ng mL21 with a good correlation coeffi- However, it is difficult to develop a rapid analytical method to
cient (>0.9995). The precision of each bile acid was <1.8% for intra- determine bile acids in the bile, since they have similar chromato-
day and <4.8% for interday. All bile acids were separated in 15 min graphic retention behaviors in reverse-phase chromatography
with satisfactory resolution, and the total analysis time was 18 min, as well as complex composition of the bile. Until now, high-
including equilibration. The method was successfully applied in rapid performance liquid chromatography (HPLC) is the most univer-
screening of bile samples from the three mammals. Significant sal method for the bile acids determination in biological samples.
metabolic frameworks of bile acids among various species were Many LC-based methods have been reported to analyze major
observed, whereas considerable quantitative variations in both bile acids from mammal serum samples (11, 12), but it is difficult
inter- and intraspecies were also observed, especially for gallstone to obtain satisfactory resolution within short analytical time due
patients. Our results suggest that detecting the change of bile acid to the complex matrix in biological samples. Recently, more effi-
profiles could be applied for the diagnosis of gallstone disease. cient LC-based techniques, such as ultra-fast liquid chromatogra-
phy (UFLC), which employ fine particles (,2.2 mm) as the solid
phase to achieve extreme high-resolution and superior theoret-
ical plates with very short analytical time, have attracted wide
attention for the analysis of complex biological specimens (13).
Introduction Meanwhile, UFLC has showed higher sensitivity and peak capac-
Bile acids are synthesized from cholesterol through the action of ity than HPLC (14). These advantages ensure that they can be ap-
specific hepatic enzymes and excreted into the small intestine plied in rapid determination of bile acids from biological samples.
via the bile duct. They serve many important physiological func- In this study, a rapid and practical screening approach to com-
tions, such as cholesterol homeostasis, lipid absorption, the ex- plex bile samples was developed for determination of the com-
cretion and recirculation of drugs, vitamins and endogenous or mon bile acids of three different mammal species. Additionally,
exogenous toxin (1). They consist of free bile acids and their we also studied the qualitative and quantitative variations of
glycine- or taurine-conjugated forms at C24 site (Figure 1). Free bile acids among inter- and intraspecies.
bile acids can be divided into primary and secondary bile acids ac-
cording to their origins. Primary bile acids are synthesized within
hepatocytes and can be changed to secondary bile acids through
dehydroxylation by intestinal bacteria (2). The formation, secretion Experimental
and elimination of bile acids are closely related to the functions of Material and reagents
the digestive system, and the distribution and concentrations of Millipore water (Millipore, Bedford, MA, USA), LC grade acetoni-
bile acids may reflect the functions of the liver and intestines (3, 4). trile, methanol and o-phosphoric acid (Tedia, Fairfield, USA) were
Previous studies have mainly focused on the determination of used throughout the study. Thirteen bile acids standards ( purity
bile acids in biological samples, such as plasma, urine and feces .95%) used in this study were purchased from Sigma (St. Louis,
(5). Due to the low concentrations of bile acids in mammal plas- MO, USA), including glycodeoxycholic acid (G-DCA), taurocho-
ma and urine, it is difficult to quantify these bile acids in these lic acid (T-CA), cholic acid (CA), chenodeoxycholic acid (CDCA),
samples (6). Primary and secondary bile acids are always mixed deoxycholic acid (DCA), lithocholic acid (LCA), glycocholicacid
together in feces as a result of dehydroxylated and deconjugated (G-CA), glycochenodeoxycholicacid, taurochenodeoxycholic
effect by intestinal bacteria, making the determination of the bile acid, taurodeoxycholic acid (T-DCA), ursodeoxycholic acid
constituents in feces more complex (7). The distribution or con- (UDCA), tauroursodeoxycholic acid (T-UDCA) and glycourso-
centrations changes in above the biological samples do not truly deoxycholic acid (G-UDCA).
# Crown copyright 2014.
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Figure 1. Chemical structures of bile acids.

Chromatography
Table I
Chromatographic separation was performed on a Shimadzu (Kyoto, Feed Formulation Rate of Rats and Pigs Diets (Weight %)
Japan) Prominence UFLC system equipped with a binary solvent
manager, a sample manager and a diode array detector. A Crude protein 18.8
Crude lipid 2.8
Shim-pack XR-ODS column (100  2.0 mm i.d., 2.2 mm) was used Crude fiber 5.2
as solid phase and the gradient system was used with a mobile Crude ash 5
Moisture 7
phase A [0.01 M potassium phosphate buffer (PPB), adjusted to pH Carbohydrate 61.2
2.6 with 80% o-phosphoric acid] and mobile phase B (acetonitrile). A
linear gradient was used for elution (0–8 min 30–55% B, 8–11 min
55–80% B, 11–14 min 80% B, and 14–18 min 30% B). The flow rate
was 0.5 mL min21 and the detection wavelength was set at 200 nm. Collection of samples
The column temperature was maintained at 608C. Samples were Male Wistar rats (n ¼ 10, 200 –250 g) and male pigs (n ¼ 10, 20 –
maintained at 48C and the UFLC injection volumes were 10 mL. 25 kg) were fed a commercial pellet diet (Department of
Laboratory Animal Science, Peking University Health Science
Center, China); the compositions of the diet were shown in
Standard solutions Table I. After 1-day fast with water ad libitum, rat bile samples
For all bile acid standards, the final solution was prepared in were collected by biliary drainage surgery. Pig bile samples
methanol with a serial dilution (10 and 1000 ng mL21). Calibration were purchased from Department of Laboratory Animal
standard samples for the mixture of 13 bile acids were prepared at Science of Peking University Health Science Center. Bile samples
eight different concentrations of 2, 4, 10, 50, 100, 200, 400 and of human were obtained from gallstone patients (n ¼ 10, male
600 ng mL21. Quality control samples for bile acids were prepared Chinese, from 60 to 81 years of age) during Endoscopic
at three different concentrations of 100, 200 and 400 ng mL21. Retrograde Cholangio-Pancreatography, at the Inner Mongolia
All biological samples were stored at 2208C and immersed in a People’s Hospital and kindly donated by the hospital. All bile sam-
constant temperature bath (378C) to thaw before use. ples were collected during the fasting state. Disease diagnosis

Rapid Determination of Bile Acids 1061

was based on conventional clinical chemistry parameters Results
(such as concentrations of alkaline phosphatase, gamma Development of the UFLC method
glutamyl-transferase, etc.). The study protocol for animal and In consideration of both conjugated and free bile acids maximal
human experiments was approved by the Ethics Committees of absorbance (200 nm) and low ultraviolet absorption, PPB and
Peking University and Inner Mongolia People’s Hospital. acetonitrile were used as the mobile phase. Moreover, the acidic
Research involving human subjects was performed under full mobile phase was crucial to the analysis of bile acids (16).
compliance with government policies and the Helsinki Figure 2A illustrates the separation efficiency of UFLC system
Declaration. for the mixture of bile acid standards. The UFLC method was de-
veloped and most of the bile acids were eluted with satisfactory
resolution within 15 min (Figure 2), and the re-equilibration time
Sample preparation was much shorter than that for the conventional LC. The UFLC
All bile samples were stored at 2208C in dark after collection. also produced a significant increase in peak capacity; the peak

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Samples were prepared as previously described (15). Briefly, widths generated by the UFLC system were between 7 and
100 mL of sample was mixed with 1 mL of 0.05% formic acid in 17 s, which resulted in a better chromatographic resolution
water; the mixture was loaded onto a reversed-phase extraction and increased signal-to-noise (S/N) ratio than the conventional
cartridge (C 18, 100 mg, Agela Technologies, Wilmington, LC. The limit of detection (LOD) was set at the concentration
Delaware, USA) which was previously conditioned with 1 mL with an S/N ratio of 3 : 1. Additionally, the LODs of analyzed
of 25% methanol and then 1 mL of 0.05% formic acid. The car- bile acids ranged from 0.2 to 0.6 ng mL21 and the corresponding
tridge was washed with 1 mL of 0.05% formic acid and then LODs were reported to be 2.0 –6.0 ng mL21 for the conventional
1 mL of 25% methanol for removal of water-soluble impurities. LC (16). The limits of quantification (LOQs) defined as the con-
Finally, bile acids were eluted with 1.5 mL of methanol and centration with the S/N ratio of 10 : 1. The LOQ of all analyzed
then dried at 608C in nitrogen. The residue was redissolved in bile acids was 0.7 – 600 ng mL21, except GUDC, TDCA and
100 mL of methanol before UFLC analysis. GLCA, whose LOQ ranged from 2 to 600 ng mL21. Especially,

Figure 2. Representative chromatograms of (A) standard of thirteen bile acids, (B) rat bile sample (2), (C) pig bile sample (4) and (D) gallstone patient bile sample (3). *Peak:1,
T-UDCA; 2, T-CA; 3, G-UDCA; 4, G-CA; 5, T-CDCA; 6, T-DCA; 7, CA; 8, G-CDCA; 9, G-DCA; 10, T-LCA; 11, CDCA; 12, DCA; 13, G-LCA.

1062 Si et al.
 99.83 + 39.80

568.6 + 186.3
both T-UDCA and TCA were separated using the UFLC, which has
not been achieved in the previous reports using LC. All results
indicate that the rapid UFLC-based method was suitable for large-

590.3

106.0
476.2

976.4

853.8
1169
3162

1368

1203

1240
Total

NQ
scale screening of complex bile samples.

5.72 + 4.37
44,.95
10.41

13.58

32.94

37.95
65.48
GLCA
Method validation

6.85
8.28

9.87
11.5

NQ

NQ
There was a good linearity in the concentration ranging from 2 to
600 ng mL21 for all analyzed bile acids, except GUDC, TDCA and

6.37 + 3.99
GLCA, whose linearity in the concentration ranged from 5 to

TLCA
600 ng mL21. The calibration curves of 13 bile acids were con-

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ

NQ
structed by calculating the peak area of each bile acid against

41.49 + 33.30

280.9 + 89.98

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the known concentrations. The intra- and interday precision
and accuracy were evaluated by assaying the low, medium and

GDCA

35.86
16.19
47.61

10.89

65.93
53.57
100.8
high concentrations of quality control samples containing bile

2.11

5.45
76.5

NQ
acids in methanol solution. The intraday variation was deter-
mined by assaying three replicates on the same day and interday

15.31 + 9.37
variation was assayed for three consecutive days. The intraday
CVs were ,1.8% and the interday CVs were ,4.8% for all studied

TDCA

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ

NQ
bile acids. The correlation coefficients were over 0.9995 for all
studied bile acids. Solid-phase extraction (SPE; Waters

88.44 + 40.40
Sep-Pakw, USA) was used for sample preparation, and the SPE re-

5.38 + 2.53
covery of 13 bile acids ranged from 85.6 to 109.8% with satisfac-

TCDCA
tory reproducibility. These results indicated that the proposed

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
SPE-UFLC method was valid for determination of bile acids in

GCDCA
complex biological samples.

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ

NQ

NQ
60.15 + 27.80

22.89 + 10.31
Discussion
With a multitude of new roles for bile acids in several diseases
TCA

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
coming to light, the need to investigate and understand the spe-
cific role of each member of the bile acid family has gained sig-

45.12 + 25.57

97.18 + 57.45
nificance (1, 17, 18). In the study, elution profiles of the three

4.61 + 2.59
mammal bile samples obtained by the UFLC demonstrated excel-
35.19
20.57
27.91

47.82

65.42
55.43
76.75
84.03
3.44

34.6
GCA

lent resolution among the analyzed bile acids, even when a large
amount of sample was placed on the column (Figure 2B–D). All

35.36 + 16.10
the samples were analyzed by the UFLC and the representative
chromatograms of three species were shown in Figure 2.
Among these bile acids, ten of which were detected from three
GUDC
Bile Components Determined from Bile of 10 Human Gallstone Patients, Rats and Pigs

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ

NQ
species and their distribution profiles and corresponding con-
centrations were presented in Table II. 53.17 + 23.33
14.52 + 8.31

The method developed in the present study had the shortest

chromatographic retention time compared with other bile acid
TUDCA

detection methods (over 60 min) (17, 19, 20). Meanwhile, injec-

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ

tion volume (10 mL) and sensitivity (2 ng mL21) were much su-
DCA

perior to most of previous methods (16, 21 –23).

NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ
NQ

NQ

NQ

Due to the ethical reason, it was inaccessible to obtain bile

446.5 + 268.9

30.62 + 14.62

samples of healthy volunteers, so we failed to compare the bile

acids profile between gallstone patients and healthy individuals.
CDCA

308.2
660.7
205.0
34.93
205.6
872.3
485.8
378.3
548.6
765.8

So we chose pig and rat as control species, considering pig having

NQ

a close relation to human in terms of biological consanguinity,

NQ, less than limit of quantification.
Bile acid (ng mL21)

and rat taken as the first choice for medical experiments (15).
490.3 + 248.4

In rat bile samples, six conjugated bile acids and one free bile
acid were detected. Notably, .95% of the total bile acids were
200.6
464.6
727.6
551.9
100.7
367.8
875.0
684.7
633.7
296.4

comprised of taurine conjugates, while glycine conjugates and

NQ

NQ
CA

free bile acids also existed at very low levels in 10 rat bile samples.
Mean + SD

Mean + SD

Mean + SD

A large abundance of TCA (60.25%) and a low level of GCA were

Samples
Table II

Patients

found in the rat bile while CA and DCA not existed. These results
Rats

Pigs
10

were consistent with a previous report that the content of

1
2
3
4
5
6
7
8
9

Rapid Determination of Bile Acids 1063

glycine-conjugated bile acids was much lower than that of Especially, UFLC method founds the bile acids metabolic charac-
taurine-conjugated bile acids in rat (19). It may be for the high teristics which reflect the formation of gallstone.
concentration of taurine and potent N-acyltransferase activity
for taurine in rodent livers (20). In pig bile samples, five conjugat-
ed bile acids and one free bile acid were detected. More than 66%
Conclusion
of the total bile acids were comprised of glycine conjugates. The
major components were GDCA (49.40%) and GCA (17.08%), A rapid, valid, sensitive, specific and accurate method (UFLC)
while taurine-conjugated bile acids were relatively minor compo- which can detect large amounts of biological samples is devel-
nents. Similar results were also observed in a previous study using oped for simultaneous determination of 13 bile acids from bile
LC – MS (17). To obtain some clinical results, the UFLC-based of three mammal species. Significant interspecies differences
method was also utilized to analyze bile samples from 10 gall- both in distribution and in concentrations of bile acids are ob-
stone patients in the present study. In comparison with rat and served among the species using the UFLC method. This method
pig bile samples, a much higher concentration of total bile may be suitable for routine clinical practice for the diagnosis of

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acids from gallstone patients was displayed, which may be ex- gallstone-related diseases, although further studies are required.
plained by the position of bile collection (21). Notably, 84.06%
of total bile acids were the free bile acids, and glycine- and
taurine-conjugated bile acids were present at very low levels in Acknowledgment
all bile samples of gallstone patients. The major components The authors thank the support of Fund of Peking University and
were CDCA (43.99%) and CA (40.06%), while GLCA and GCA the Inner Mongolia People’s Hospital.
were also detected. These results agreed with a previous report
that the major bile acids in bile samples of liver disease patients
were CDCA (57.63%) and CA (40.79%) (22). Previous studies also
showed that both CDCA and CA can be detected in healthy References
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Rapid Determination of Bile Acids 1065