Clin Biochem, Vol. 23, pp. 85-90, 1990 Printed in Canada. All rights reserved.

0009-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.

Simultaneous Determinatiun of Conjugated Bile Acids in Human Bile CHI-PUI PANG, 1 SHUK-DAI MOK, 2 PING-KUEN LAM, 2 JACK VARMA, 2 and ARTHUR K. C. LI2 1Department of Chemical Pathology, and 2Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong We describe an optimized liquid chromatographic method for simultaneous analysis of 10 conjugated bile acids in gall bladder and ductal bile. A quick and effective one-step purification with Sep-pak C-18 was adopted. We used a reverse phase C18 stainless steel column and an isocratic mobile phase in a flow programme and monitored the column effluent at 205 nm. The within-day CV ranged from 0.3 to 1.8%, and the between-day CV from 1.2 to 7.2%. Absolute analytical recovery ranged from 89 to 107%. Linearity ranged from 0.3 to 3 mg/mL for most bile acids. The chromatographic analysis was completed in 18 min.

KEY WORDS: HPLC; conjugated bile acids; human bile; cholestasis; cholelithiasis; gall bladder. Introduction

ile acids are 24-carbon steroids whose bipolar B nature is responsible for their detergent properties. In man, the primary bile acids cholic acid (3~, 7~, 12~-trihydroxycholanoic acid) and chenodeoxycholic acid (3~, 7~-dihydroxycholanoic acid) are derived from cholesterol in the liver by a series of hydroxylation and oxidation reactions. These primary bile acids are secreted in bile and enter the intestine where they undergo dehydroxylation at the 7~-position to form secondary bile acids, deoxycholic acid (3~, 12~-dihydroxycholanic acid) and lithocholic acid (3~-monohydroxycholanic acid) (1). Conjugation of bile acids normally occurs before they are secreted. The tauro- and glyco-conjugates are obtained via amidation of the carboxylic acid group at the C-24 position with a taurine and a glycine derivative respectively. There are also two types of minor conjugated bile acids: the glucuronidated and sulphated derivatives (1,2). Circulating bile acids in blood and in the enterohepatic system are mainly in conjugated forms. Impaired secretion and absorption of bile acids in the biliary system have been detected in cirrhosis

and acute hepatitis (3,4). Bile acid synthesis and metabolism are grossly affected in cholestasis. In various hepatobiliary disorders bile acid concentrations and composition in body fluids are altered (5,6). Simultaneous analysis of bile acids in biomedical samples requires chromatographic techniques (7), and HPLC provides a rapid and quantitative methodology (8,9). Different problems arise in analysis of various body fluids. Detectability and sensitivity are the challenges in assaying bile acids in blood and urine, while sample treatment for bile analysis is always time-consuming since solid extraction with Amberlite XAD-2 (8) or with Sephadex LH-20 are involved (10). Here we describe an optimized HPLC assay for quick and reliable simultaneous measurement of 10 conjugated bile acids in bile. Methods MATERIALS

Methanol (Chromatography grade) was from Merck (Darmstadt, FRG). Taurocholic acid (TC), glycocholic acid (GC), taurochenodeoxycholicacid (TCDC), taurodeoxycholic acid (TDC), glycodeoxycholic acid (GDC), taurolithocholic acid (TLC), and glycolithocholic acid (GLC) were obtained from Sigma (St Louis, MO, USA). Tauroursodeoxycholicacid (TUDC), glycoursodeoxycholic acid (GUDC) and glycochenodeoxycholic acid (GCDC) were from Hoechst (La Jolla, CA, USA), dexamethasone acetate from Fluka (Buchs, Switzerland) and Sep-pak C-18 cartridges from Waters (Milford, MA, USA). All other chemicals were of AnalaR grade from Merck or BDH (Poole, UK). APPARATUS

Correspondence: Dr. C. P. Pang, Department of Chemical Pathology, Prince of Wales Hospital, Shatin, N.T., Hong Kong. Manuscript received June 5, 1989; revised August 29, 1989; accepted September 1, 1989.

An automated liquid chromatographic system with integrated LC modules, HP1090 series M (Hewlett Packard, Palo Alto, CA, USA) was used. It consisted of a DR5 ternary solvent delivery system, a variable volume autoinjector, an autosampler, a Diode-array detector, a Thinkjet printer and an 79994A LC



PANG, MOK, LAM, VARMA, AND LI TABLE 1 Recovery Rates with the Present Analysis. The Average Rates Were Obtained from Analysis of 4 Bile Samples Before and After Being Spiked with Known Amounts of Bile Acid Standards





3o t9



Amount of Spiked Standard (~g)

Bile Acid





5 Time









IO 15 (min.)











Figure 1-Chromatographic separation of conjugated bile acid standards. Column: Hibar prepacked LiChrosorb RP18 (250 × 4 mm ID, 5 ~m). Flow programme as described in text. Mobile phase: 15 mmol/L potassium dihydrogen phosphate (pH 6.25) - methanol 25:75. Detector: Hewlett Packard Diode-array detector, at 205 nm. w o r k s t a t i o n complete w i t h an 10MHz 68010 processor, a W i n c h e s t e r disc drive a n d 79995A software. W a t e r purification was p e r f o r m e d w i t h a Milli-Q W a t e r P o l i s h i n g S y s t e m (Millipore, Bedford, MA,



91.5 92.8 93.8 97.3 99.0 96.0 95.8 97.3 95.8 103.3

(1.8) (5.6) (4.1) (3.1) (8.0) (7.7) (7.2) (15.2) (1.9) (7.5)

TABLE 2 Analytical Precision of the Present Analysis

Concentration mmol/L

Within-day "CV (%) (n = 10)

Between-day aCV (%) (n = 4)


1.88 0.38

1.3 0.3

3.5 2.0


2.08 0.42

0.6 0.6

3.0 4.9


1.92 0.39

0.4 1.6

1.4 8.9


2.46 0.49

0.8 0.4

1.7 2.8


2.01 0.40

1.4 0.6

5.3 3.4


2.07 0.42

3.1 0.8

7.3 8.2


2.07 0.41

1.0 0.5

1.3 7.2


2.14 0.42

1.8 0.4

2.0 2.1


0.83 0.18

0.5 2.1

1.2 2.5


0.67 0.14

0.75 0.4

1.7 2.8

Bile Acid


An aliquot (0.5 mL) of a bile sample after thawing and centrifugation (3000 x g, 10 min) was added to 25 mL of 30 mM potassium dihydrogen phosphate

1.12 0.93 1.02 1.33 0.84 1.18 1.14 1.10 0.65 0.47

buffer, pH 6.25. To a 5 mL portion, 10 mL water and 100 ~L of 1.33 mmol/L dexamethasone acetate in phosphate buffer as internal standard were added. The diluted bile was then applied to a Sep-pak C-18 cartridge which had been treated with 5 mL meth-


Bile was collected from the gall-bladder during cholecystectomy or bile duct exploration from patients suffering from different biliary disorders such as cholecystitis, cholecystolithiasis and cholangitis. They were all cholelithic. Bile from nonbiliary patients, mostly with colon carcinoma, was also obtained during endoscopic examination. Both groups of patients were all adults aged 25 or above. The specimens were collected in polystyrene bottles and stored at - 7 0 °C for not more than 3 months before analysis.

Average Percentage Recovery (SD)

"Coefficient of variation.







280 8~

11] IE E

D cr






40 ¸



5 Time

18 [5 (m~n.)


5 Time

II~ 15 (m~n.)


Figure 2--Conjugated bile acid profile of gall bile in (A) a 67-years-old male patient with cholesterol gall stones; (B) a 60-years-old male patient with chronic cholecystitis. Chromatographic conditions as described in Figure 1. anol and 30 mL water in a Sep-pak cartridge rack (Waters Associates, Milford, MA, USA). The cartridge was washed in turn with 10 mL water, 3 mL 10% acetone (v/v) and 10 mL water. The bile acids were then eluted from the resin with 3 mL methanol. The eluate was evaporated to dryness in a stream of air at 37 °C and reconstituted in 0.6 mL mobile phase prior to injection (10 ~LL) into the chromatographic system. CHROMATOGRAPHY

We used an isocratic mobile phase of 75% (v/v) methanol in 15 mM potassium dihydrogen phosphate, pH 6.25. Its aqueous and organic components had been filtered through a 0.45 ~Lm nitrocellulose membrane and a 0.22 ~tm fluorcarbon membrane (Millipore, Bedford, MA, USA) respectively. Degassing was carried out by sparging with helium before and during chromatography. A reversed-phase Hibar C18 column (Particle size 5 ~Lm, 250 X 4 mm ID) was used at ambient temperature. The eluate was monitored at 205 nm. A flow programme was adopted and the change in flow-rate was in linear mode. An initial flow rate of 0.5 mL/min was maintained for 12.5 min and was increased to 0.65 mL/min in 1 min. It was then maintained for 6 min before readjustment to 0.5 mL/min in 1 min. The total run time was 23 min and the system was then ready for another injection.



Standard mixtures of the 10 major conjugated bile acids were well separated (Figure 1). The use of our isocratic mobile phase with flow programming was satisfactory for the analysis of bile samples from patients with or without biliary diseases (Figure 2). The buffer concentration of 15 mM and pH of 6.25 were found to be critical for the resolution of the bile acids. A relatively high content of organic solvent, 75% methanol, was used to avoid prolonged retention of nonpolar substances in the column. Exact overlap of corresponding bile acid peaks was obtained when a bile sample was spiked with authentic bile acid standards prior to sample treatment. The chromatographic analysis required about 18 min. No guard column was used to avoid peak broadening. We found our extraction method to be adequate for sample clean-up and for satisfactory recovery rates (Table 1). With proper handling a Sep-pak C-18 cartridge could be reused 10 times for sample preparation in our analysis with no change in recovery rate, and more than 400 injections could be carried out with one HPLC column. Analytical data were obtained from the analysis of bile samples spiked with low and high concentrations of a mixture of the 10 conjugated bile acid standards. The intraassay CVs were less than 3.1% and the interassay CVs less than 8.9% (Table 2). We also assessed the analysis of bile specimens by


PANG, MOK, LAM, VARMA, AND LI TABLE 3 Precision of Absolute Retention Time and Retention Time Relative to Internal Standard (I.S.) in 26 analyses of the Same Sample Mixture During a Period of 5 Months Absolute Retention Time

Relative Retention Time

Bile Acid

Mean (min)


CV (%)

Mean (min)


CV (%)


5.76 6.30 6.74 7.56 9.18 9.96 10.69 11.65 14.58 17.06 8.45

0.093 0.09 0.12 0.17 0.26 0.30 0.33 0.37 0.45 0.58 0.15

1.6 1.5 1.8 2.2 2.9 2.9 3.0 3.1 3.1 3.4 1.7

0.680 0.745 0.798 0.897 1.087 1.180 1.265 1.379 1.728 2.021 1.000

0.005 0.005 0.003 0.007 0.014 0.017 0.022 0.024 0.029 0.043 --

0.68 0.66 0.42 0.72 1.28 1.44 1.70 1.70 1.71 2.10 --

determining the precision of the retention time of individual conjugated bile acid relative to dexamethasone in the chromatogram. During a period of 5 months in 26 chromatographic assays the CVs of absolute retention times and relative retention times were less than 3.4% and 2.1% respectively (Table 3). The use of either peak height measurement or peak area integration was satisfactory in providing wide l i n e a r i t y ranges. The d a t a p r e s e n t e d in this r e p o r t were o b t a i n e d by p e a k a r e a i n t e g r a t i o n (Figure 3). The l i n e a r i t y r a n g e d from 0.3 to 1.2 m g / m L for both GLC a n d TLC (not s h o w n in F i g u r e 3).

We also examined the possible effect of interference in our analysis by drugs given to the patients in our study. These included cefuroxime, cefotaxime, ceftazidine, amphicillin, metronidazole, pethidine, fentanyl and phytomenadione. We analysed pure drug standards and bile samples spiked with drug s t a n d a r d s at 10 t i m e s t h e i r t h e r a p e u t i c c o n c e n t r a tions. N o n e of these m a t e r i a l s , w h e t h e r as p u r e s t a n d a r d s or b e i n g spiked in bile, was detected in our analysis. Cefuroxime, cefotaxime, ceftazidine a n d a m p h i c i l l i n were completely r e m o v e d by t h e solid-phase s a m p l e t r e a t m e n t procedure, w h i l s t phy-

TABLE 4 Concentrations (mmol/L) of Bile Acid Components in Gall Bladder Bile of Nonbiliary Patients as Controls (A) and Cholelithic Patients (B) A. Control (n = 20)


B. Cholelithiasis (n = 25)







36.7 13.6 66.1 22.6 14.1 4.6 9.6 1.8 0.99 0.43 a171.3 a127.5 b43.0 4.4

20.8 10.1 29.7 13.8 18.3 9.2 7.6 1.8 1.7 0.61 60.3 46.4 26.6 3.2

7.7-87.5 2.4-34.7 12.9-104.3 2.6-51.1 0-76.0 0-41.6 1.2-29.2 0-6.9 0-7.8 0-2.2 48.2-241.7 41.8-196.6 6.4-99.2 1.1-14.0

24.4 9.6 27.1 11.4 14.6 6.4 3.7 1.3 0.49 0.07 103.7 70.3 28.8 3.2

13.3 7.3 14.3 6.3 10.0 13.4 4.6 1.9 0.64 0.18 55.3 34.3 20.5 2.0

4.4--49.4 0.90-25.2 1.7-53.8 1.7-24.7 0-33.4 0-66.7 0-22.8 0-9.3 0-2.4 0-0.75 11.4-199.3 7.5-136.7 3.9-90.5 0.54-8.6

aControls significantly higher than cholelithiasis, p

Simultaneous determination of conjugated bile acids in human bile.

We describe an optimized liquid chromatographic method for simultaneous analysis of 10 conjugated bile acids in gall bladder and ductal bile. A quick ...
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