A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS
2014; Elsevier BV; Volume: 55; Issue: 5 Linguagem: Inglês
10.1194/jlr.d047506
ISSN1539-7262
AutoresGenta Kakiyama, Akina Muto, Hajime Takei, Hiroshi Nittono, Tsuyoshi Murai, Takao Kurosawa, Alan F. Hofmann, William M. Pandak, Jasmohan S. Bajaj,
Tópico(s)Moringa oleifera research and applications
ResumoWe have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis. We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis. The composition of circulating bile acids in humans is a complex mixture of primary and secondary bile acids. In humans, primary bile acids are chenodeoxycholic acid (CDCA) and cholic acid (CA), each secreted into bile in mostly N-acyl amidated form, i.e., conjugated with glycine or taurine in an amide linkage (1Hofmann A.F. Hagey L.R. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.Cell. Mol. Life Sci. 2008; 65: 2461-2483Crossref PubMed Scopus (625) Google Scholar, 2Griffiths W.J. Sjövall J. Bile acids: analysis in biological fluids and tissues.J. Lipid Res. 2010; 51: 23-41Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 3Hylemon P.B. Zhou H. Pandak W.M. Ren S. Gil G. Dent P. Bile acids as regulatory molecules.J. Lipid Res. 2009; 50: 1509-1520Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). In the distal intestine, bile acids are modified by bacterial enzymes. Major modifications include deconjugation followed by dehydroxylation at C-7, by which CA is converted to deoxycholic acid (DCA) and CDCA is converted to lithocholic acid (LCA). Other bacterial modifications include epimerization at C-3 to form iso bile acids, oxidation of any of the hydroxyl groups, and desaturation of the side chain. The epimerization of the C-7 hydroxyl group of CDCA results in the formation of ursodeoxycholic acid (UDCA). Secondary bile acids are absorbed from the intestine to a varying degree, and are extracted by the hepatocyte where they may undergo additional modifications such as epimerization of iso bile acids to 3α-hydroxy bile acids, as well as reconjugation. LCA is also sulfated in part in addition to being N-acylamidated so that about half of the lithocholyl amidates are sulfated at C-3 (4Palmer R.H. Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat.J. Lipid Res. 1971; 12: 680-687Abstract Full Text PDF PubMed Google Scholar). In humans, UDCA is not epimerized during hepatocyte transport, and most people have a few percent of UDCA in their biliary bile acids that has been formed in the intestine (1Hofmann A.F. Hagey L.R. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.Cell. Mol. Life Sci. 2008; 65: 2461-2483Crossref PubMed Scopus (625) Google Scholar). Daily bile acid excretion to the feces is 300–600 mg/day in health (3Hylemon P.B. Zhou H. Pandak W.M. Ren S. Gil G. Dent P. Bile acids as regulatory molecules.J. Lipid Res. 2009; 50: 1509-1520Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar), and under steady state conditions is equal to bile acid synthesis from cholesterol. Bile acids play important roles in digestion, modulation of gut microbiota, and regulation of pathways necessary for cholesterol, lipid, and glucose homeostasis (3Hylemon P.B. Zhou H. Pandak W.M. Ren S. Gil G. Dent P. Bile acids as regulatory molecules.J. Lipid Res. 2009; 50: 1509-1520Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 5Trauner M. Claudel T. Fickert P. Moustafa T. Wagner M. Bile acids as regulators of hepatic lipid and glucose metabolism.Dig. Dis. 2010; 28: 220-224Crossref PubMed Scopus (231) Google Scholar). Therefore, evaluation of bile acid levels in healthy as well as diseased individuals is essential. Cirrhosis represents a particularly important clinical condition, in which the synthetic organ for bile acids, the liver, is damaged, and progression of disease is modulated by altered gut microbiota that can also impact bile acid metabolism (6Bajaj J.S. Hylemon P.B. Ridlon J.M. Heuman D.M. Daita K. White M.B. Monteith P. Noble N.A. Sikaroodi M. Gillevet P.M. Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation.Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 303: G675-G685Crossref PubMed Scopus (374) Google Scholar, 7Kakiyama G. Pandak W.M. Gillevet P.M. Hylemon P.B. Heuman D.M. Daita K. Takei H. Muto A. Nittono H. Ridlon J.M. et al.Modulation of the fecal bile acid profile by gut microbiota in cirrhosis.J. Hepatol. 2013; 58: 949-955Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar). A variety of methods have been reported for bile acid analysis in biological fluids, such as bile, plasma, urine, and stool (1Hofmann A.F. Hagey L.R. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.Cell. Mol. Life Sci. 2008; 65: 2461-2483Crossref PubMed Scopus (625) Google Scholar, 8Roda A. Francesco P. Mario B. Separation techniques for bile salts analysis.J. Chromatogr. B Biomed. Sci. Appl. 1998; 717: 263-278Crossref PubMed Scopus (54) Google Scholar). Analysis of fecal bile acids is by far the most difficult because of the multiplicity of fecal bile acids as well as the great range of polarity of fecal bile acids. For the quantification of individual bile acids, GC, HPLC, and their combination with MS are commonly utilized. Of these analytical methods, HPLC-MS is currently the most technically advanced. It allows screening of bile acid profiles without tedious prior sample purification. A number of reports based on this method have been published (1Hofmann A.F. Hagey L.R. Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.Cell. Mol. Life Sci. 2008; 65: 2461-2483Crossref PubMed Scopus (625) Google Scholar, 8Roda A. Francesco P. Mario B. Separation techniques for bile salts analysis.J. Chromatogr. B Biomed. Sci. Appl. 1998; 717: 263-278Crossref PubMed Scopus (54) Google Scholar). However, despite these advantages, the extraction of bile acids from feces has not been clearly described in prior studies. In addition, such methods have high overhead costs, limiting their routine use. Therefore, development of a simple and accurate HPLC method for fecal bile acids would be a useful advancement. In the present paper, we report the development of a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives, and its application to samples from healthy controls and patients with cirrhosis. Unconjugated bile acids, in contrast to conjugated bile acids, do not have appreciable absorbance at 205 nm (2Griffiths W.J. Sjövall J. Bile acids: analysis in biological fluids and tissues.J. Lipid Res. 2010; 51: 23-41Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 8Roda A. Francesco P. Mario B. Separation techniques for bile salts analysis.J. Chromatogr. B Biomed. Sci. Appl. 1998; 717: 263-278Crossref PubMed Scopus (54) Google Scholar, 9Roda A. Cerrè C. Simoni P. Polimeni C. Vaccari C. Pistillo A. Determination of free and amidated bile acids by high-performance liquid chromatography with evaporative light-scattering mass detection.J. Lipid Res. 1992; 33: 1393-1402Abstract Full Text PDF PubMed Google Scholar), explaining our use of phenacyl derivatives that have a strong absorbance at 254 nm. Our method provides nearly identical results to those obtained by LC-MS as well as those obtained by GC-MS, provided the bile acid content is sufficient. Abbreviations used for bile acids in this paper are based on the nomenclature recommendations reported by Hofmann et al. (10Hofmann A.F. Sjövall J. Kurz G. Radominska A. Schteingart C.D. Tint G.S. Vlahcevic Z.R. Setchell K.D.R. A proposed nomenclature for bile acids.J. Lipid Res. 1992; 33: 599-604Abstract Full Text PDF PubMed Google Scholar) with a few exceptions: We have used semi-trivial nomenclature for the Δ1-3-one, Δ4-3-one, and Δ4,6-3-one derivatives of the common bile acids by using the abbreviation for the saturated compound. The 3-oxo-5β-cholanoic acid is expressed as LCA-3-one (see Appendix). CA, glycocholic acid (GCA), taurocholic acid (TCA), CDCA, glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), UDCA, glycoursodeoxycholic acid (GUDCA), tauroursodeoxycholic acid (TUDCA), LCA, glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), DCA, glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), and hyocholic acid (HCA) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). isoDCA, isoLCA, allo-isoLCA, and norDCA were purchased from Steraloids Inc. (Newport, RI). The [2,2,4,4-d4]CA (d4-CA) was obtained from CDD Isotopes Inc. (Quebec, Canada). Unsaturated bile acids with the Δ4-3-one and Δ4,6-3-one configuration in the steroid nucleus for CA and CDCA, as well as sulfated bile acids were kindly donated by Professor Takashi Iida (Nihon University, Tokyo, Japan); methods for their synthesis have been reported (11Iida T. Momose T. Nambara T. Chang F.C. Potential bile acid metabolism X. Syntheses of stereoisomeric 3,7-dihydroxy-5alpha-cholanic acid.Chem. Pharm. Bull. (Tokyo). 1986; 34: 1929-1933Crossref Scopus (14) Google Scholar, 12Iida T. Momose T. Chang F.C. Nambara T. Potential bile acid metabolism XI. Syntheses of stereoisomeric 7,12-dihydroxy-5alpha-cholanic acids.Chem. Pharm. Bull. (Tokyo). 1986; 34: 1934-1938Crossref Scopus (10) Google Scholar, 13Björkhem I. Danielsson H. Issidorides C. Kallner A. On the synthesis and metabolism of cholest-4-en-7alpha-ol-3-one. Bile acids and steroids 156.Acta Chem. Scand. 1965; 19: 2151-2154Crossref PubMed Google Scholar, 14Goto J. Kato H. Hasegawa F. Nambara T. Synthesis of monosulfates of unconjugated and conjugated bile acids.Chem. Pharm. Bull. (Tokyo). 1979; 27: 1402-1411Crossref PubMed Scopus (61) Google Scholar, 15Suzuki M. Murai T. Yoshimura T. Kimura A. Kurosawa T. Tohma M. Determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related compounds in biological fluids of infants with cholestasis by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 1997; 693: 11-21Crossref PubMed Scopus (29) Google Scholar). All other bile acids such as 1β-, 2β-, 4β-, 6α-hydroxylated, Δ5 unsaturated, and C27 bile acids (DHChA and THChA) were from our collection (H.N. and T.K.'s laboratories). The reagents and enzymes, 2-bromoacetophenone, triethylamine (TEA), cholylglycine hydrolase (Clostridium welchii), and sulfatase (type H-1) were obtained from Sigma-Aldrich. All other chemicals used were of the highest grade obtainable except for water and methanol, which were of HPLC grade. After informed consent was obtained, fecal samples were obtained from three groups of subjects, early cirrhosis, advanced cirrhosis, and age-matched healthy controls. Cirrhotics were diagnosed using biopsy, radiological, or endoscopic evidence. Thirty-eight cirrhotic patients (age 54 ± 3 years, 30 men) and 10 age-matched healthy controls (age 54 ± 3 years, 8 men) provided fresh fecal samples. Early cirrhotics (n = 17) were Child class A without history of decompensation while advanced cirrhotics (n = 21) had experienced portal hypertensive complications (hepatic encephalopathy, ascites, variceal bleeding, hepatic hydrothorax) or were Child class B or C. The mean model for end-stage liver disease (MELD) score was 12.4 ± 6.5 and the etiology of cirrhosis was mostly hepatitis C (66%) or alcoholic liver disease (16%). Patients who had been abusing alcohol/illicit drugs over the past 3 months, those currently receiving absorbable antibiotics, subjects having coexisting inflammatory bowel disease or diagnosed irritable bowel syndrome, or subjects currently receiving UDCA therapy were excluded. After collection, stool was thoroughly mixed, and was snap-frozen at −80°C until analysis. The specimen was lyophilized before use. Steps in the extraction and derivatization procedures for the three different methods of fecal bile acid analysis are shown in Fig. 1. The lyophilized stool was thoroughly crushed to a powder before use. Powdered stool (10–20 mg, weighed exactly) was suspended in cold water (250 μl) and heated at 90°C in a screw-capped glass tube for 10 min. If any large particle was still left after heating, it was fragmented using the ultra-sonic bath. Sodium acetate buffer (100 mM, pH 5.6; 250 μl) containing 15 units of cholylglycine hydrolase and 150 units of sulfatase was added, and the solution was incubated at 37°C for 16 h. To stop the reaction, isopropanol (250 μl) was added and the mixture was heated at 90°C for 10 min. An internal standard (IS), 50 nmol of norDCA, and 0.1 N NaOH (3 ml) were added. The bile acids were extracted from the fecal matrix by ultra-sonication in a Branson type B-220 ultra-sonic bath (Danbury, CT) at room temperature for 1 h. After centrifugation, the supernatant was transferred to a glass test tube, and the pellet was washed with 0.1 N NaOH (2 ml). The combined extract was applied to a Waters Sep Pak tC18 cartridge (500 mg sorbent), which had been primed with methanol (10 ml) and water (10 ml). The cartridge was successively washed by water (5 ml), 15% acetone (4 ml), and water (5 ml). Retained bile acids were eluted with methanol (6 ml) and evaporated to dryness under an N2 stream below 40°C. After the step of cholylglycine hydrolase/sulfatase treatment (see method A), isopropanol (500 μl) and 1 N NaOH (100 μl) were added, and the solution was incubated at 60°C for 2.5 h. An IS, 50 nmol of norDCA, and 0.1 N NaOH (3 ml) were added, and the bile acids were extracted in the same manner as above. Extracted unconjugated bile acids (either by method A or B) were derivatized to their 24-phenacyl esters (16Stellaard F. Hachey D.L. Klein P.D. Separation of bile acids as their phenacyl esters by high-pressure liquid chromatography.Anal. Biochem. 1978; 87: 359-366Crossref PubMed Scopus (43) Google Scholar) as follows: to the dried extract, 10 mg/ml of TEA in acetone (150 μl) and 12 mg/ml of phenacyl bromide (2-acetobromophenone) in acetone (150 μl) were added, and the mixture was heated at 50°C with ultra-sonication in a screw-capped glass tube for 1.5 h. The reaction mixture was diluted with acetone (2 ml) and applied to a Waters Sep-Pak® silica cartridge (500 mg sorbent), which had been primed with acetone (5 ml). To elute the bile acid 24-phenacyl esters completely, the column was eluted with acetone (4 ml), and all the collected effluent was dried under an N2 stream. The obtained residue was resuspended in 82% methanol (200 μl), filtered through 0.45 μm filter, and an aliquot (20 μl) was injected to the HPLC instrument: The apparatus used was a Waters Alliance® series 2695 separation module equipped with 2487 dual λ absorbance detector, which was controlled by Empower Pro software. Waters Nova-Pak C18 column (300 mm × 3.9 mm inner diameter (id), particle size 4 μm) fitted with a guard column (20 mm × 3.9 mm id) was used for the separation, which was kept at 32°C during the analysis. Methanol (82%) was used as the mobile phase and its flow rate was kept constant at 0.65 ml/min. Individual bile acid 24-phenacyl esters were detected by monitoring their absorption at 254 nm. For the preparation of standard stock solutions, unconjugated bile acids were dissolved in 90% ethanol at a concentration of 200 μg/ml (500 nmol/ml). The sample was then diluted to the concentrations of 250, 50, 25, and 5 nmol/ml using 90% ethanol. IS stock solution containing norDCA (500 nmol/ml) was also prepared in 90% ethanol. In the calibration study, a 100 μl aliquot of each standard solution or stock solution was mixed with 100 μl of IS solution. A mixture of 500 μl stock solution and 100 μl of IS solution was also prepared. After evaporation under an N2 stream below 40°C, the bile acid mixture was subjected to the derivatization reaction as above. Each concentration of bile acid phenacyl ester mixture was dissolved in 200 μl of 90% methanol and a 20 μl aliquot was injected to the HPLC. Calibration curves were constructed by the peak-area ratio of each bile acid to the IS. For the recovery rate test, 100 μl aliquots of 50 or 500 nmol/ml stock solutions were spiked into the dried stool and the samples were subjected to the above entire clean-up and derivatization process (method A, without alkaline hydrolysis). The recovery (percent ± SD) was calculated as [observed concentration/(unspiked concentration + spiked concentration)] × 100 (n = 5). In order to check the deconjugation rate of the N-acylamidated bile acids, 500 nmol/ml of selected glycine and taurine amidated bile acid standard mixtures were also prepared, as above. A 100 μl aliquot of the respective stock solutions was spiked into the dried stool (10 mg). After incubation with cholylglycine hydrolase, the sample was processed and derivatizated (method A, without alkaline hydrolysis). The deconjugation rate was defined as the analytical recovery (percent) in the same manner as above (n = 4 for each sample). The LC-ESI-MS/MS analysis was conducted based on our previous method (17Muto A. Takei H. Unno A. Murai T. Kurosawa T. Ogawa S. Iida T. Ikegawa S. Mori J. Ohtake A. et al.Detection of Δ4-3-oxo-steroid 5β-reductase deficiency by LC–ESI-MS/MS measurement of urinary bile acids.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2012; 900: 24-31Crossref PubMed Scopus (32) Google Scholar) with modification: 5.0 mg of lyophilized stool (see HPLC method A) was suspended in 50 mM cold sodium acetate buffer (pH 5.6, 0.5 ml) and then refluxed with ethanol (1.5 ml) for 1 h. After centrifugation, the supernatant was diluted four times with water and applied to a Bond Elute C18 cartridge (500 mg/6 ml; Varian, Harbor City, CA). The cartridge was then washed with 25% ethanol (5 ml) and bile acids were eluted with ethanol (5 ml). After the solvent was evaporated, the residue was dissolved in 1 ml of 50% ethanol. To an aliquot (100 μl) of this solution, 0.9 ml of 50% ethanol and 1 ml of IS ([2,2,4,4-d4]CA, 200 pmol/ml in 50% ethanol) was added. Precipitated solids were removed by filtration through a 0.45 μm Millipore filter (Millex®-LG; Billerica, MA). A 10 μl aliquot of the filtrate was injected into the LC-ESI-MS/MS system. The LC-ESI-MS/MS system consisted of a TSQ Quantum Discovery Max mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an ESI probe and Surveyor HPLC system (Thermo Fisher Scientific). InertSustain C18 column (150 mm × 2.1 mm id, 3 μm particle size; G and L Science, Tokyo, Japan) was employed at 40°C. A mixture of 5 mM ammonium acetate, ethanol, and methanol was used as the eluent, and the separation was carried out by linear gradient elution at a flow rate of 0.2 ml/min. The mobile phase composition of ethanol and methanol was gradually changed as follows: ammonium acetate-ethanol (8:2, v/v) for 0–3 min, ammonium acetate-methanol (8:2, v/v) at 3.1 min, ammonium acetate-methanol (2:98, v/v) for 3.1–42 min, ammonium acetate-methanol (2:98, v/v) for 42–46 min; the column was reequilibrated for 4 min. The total run time was 50 min. To operate the LC-ESI-MS/MS, the spray voltage and vaporizer temperature were set at 3,500 V and 330°C, respectively. The sheath and auxiliary gas (nitrogen) pressure were set at 50 and 10 arbitrary units, respectively, and the ion transfer capillary temperature was carried out at 330°C. The collision gas (argon) pressure and the collision energy were kept at 1.3 mm Torr and 27–55 eV, respectively, all in the negative ion mode. Lyophilized stool powder (5.0 mg, weighed exactly) was heated with water, and then incubated with cholylglycine hydrolase/sulfatase for 16 h as described in the HPLC section. Ethanol (1.5 ml) was added and the solution was refluxed for 1 h. After centrifugation, the supernatant was evaporated under an N2 stream. The extracted bile acids were resuspended in 1 ml of 90% aqueous ethanol and applied to a piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20) (18Goto J. Hasegawa H. Kato H. Nambara T. A new method for simultaneous determination of bile acids in human bile without hydrolysis.Clin. Chim. Acta. 1978; 87: 141-147Crossref PubMed Scopus (173) Google Scholar) column (30 × 6 mm id) which had been primed with 90% aqueous ethanol. The column was washed with 90% ethanol (4 ml) and the unconjugated bile acids were eluted with 0.1 N acetic acid in 90% ethanol (5 ml). After evaporation, the purified bile acids were derivatizated to their corresponding methyl ester-dimethylethylsilyl ether derivatives as previously described (15Suzuki M. Murai T. Yoshimura T. Kimura A. Kurosawa T. Tohma M. Determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related compounds in biological fluids of infants with cholestasis by gas chromatography-mass spectrometry.J. Chromatogr. B Biomed. Sci. Appl. 1997; 693: 11-21Crossref PubMed Scopus (29) Google Scholar). GC-MS was performed with a Hewlett Packard 5890 gas chromatograph and Hewlett Packard 5973 mass selective detector instrument (Agilent, Santa Clara, CA) using a DB-5MS gas chromatographic column (30 m × 0.25 mm id, and a 0.25 μm-film-fused silica capillary column (Agilent). The column temperature was programmed to rise from 170°C to 230°C/min at 10°C/min, from 230°C to 300°C at 5°C/min, and to remain at 300°C at 20 min. Helium was used as the carrier gas with the flow rate of 1.4 ml/min. The mass spectra were recorded at an ionization energy of 70 eV with an ion source temperature of 250°C. A chromatogram using bile acid standards is shown in Fig. 2A. The retention times, calibration curves, and quantification and detection limits of each bile acid are listed in Tables 1 and 2. The calibration curves showed excellent linearity for a 500-fold dynamic range for all bile acids that were examined (correlation coefficient >0.999 for most bile acids). The detection [signal-to-noise ratio (S/N) > 3] and quantification (S/N > 10) limits were 1.2–1.5 pmol and 2.4–7.3 pmol applied to the column, respectively, depending on the bile acid species. The assay validation in Table 3 shows excellent recovery of seven dominant fecal bile acids from the fecal matrix. The assay accuracy, which was defined as analytical recovery of calibration standards at nominal bile acid levels (5 and 50 nmol), was satisfactory, ranging from 85 to 102% (except for UDCA which was 72%). The recoveries of the taurine and glycine conjugated bile acids by the cholylglycine hydrolase treatment are also shown in Table 3. The deconjugation reaction was almost quantitative (90–104%) for most conjugated bile acids except GUDCA, which was 82%.TABLE 1Retention data of bile acid 24-phenacyl estersCA (1)CDCA (2)DCA (3)LCA (4)UDCA (5)isoDCA (8)isoLCA (9)isoLCA-Δ5 (24)7Keto-DCA (33)7Keto-LCA (34)LCA-3-one (35)norDCA (IS) (36)Retention time (min)12.70 ± 0.0322.05 ± 0.0623.98 ± 0.0646.08 ± 0.129.68 ± 0.0313.76 ± 0.0442.39 ± 0.1239.07 ± 0.117.27 ± 0.0211.78 ± 0.0350.14 ± 0.1418.21 ± 0.05Relative retention time to IS0.70 ± 0.001.21 ± 0.001.32 ± 0.002.53 ± 0.000.53 ± 0.000.76 ± 0.002.33 ± 0.002.15 ± 0.000.40 ± 0.000.65 ± 0.002.75 ± 0.00—Values are expressed as mean ± SD (n = 7). Abbreviations of bile acids and their compound numbers in parentheses are given in the Appendix. Open table in a new tab TABLE 2Calibration curve, limit of detection, and limit of quantification for selected bile acidsEquationCorrelation Coefficient (r2)LOD (pmol)LOQ (pmol)CA (1)y = 0.3380x + 0.00450.99991.222.44CDCA (2)y = 0.3471x − 0.00270.99991.342.68DCA (3)y = 0.3122x − 0.00380.99991.275.08LCA (4)y = 0.3646x − 0.00980.99961.335.31UDCA (5)y = 0.3842x − 0.00280.99991.282.56isoDCA (8)y = 0.3704x − 0.00340.99991.342.68isoLCA (9)y = 0.3756x − 0.01210.99951.467.30Limit of detection (LOD), S/N > 3; limit of quantification (LOQ), S/N > 10 (on column). Abbreviations of bile acids and their compound numbers in parentheses are given in the Appendix. Open table in a new tab TABLE 3Recovery of selected bile acids5 nmol50 nmolAdded (nmol)Found (nmol)Recovery (%)Added (nmol)Found (nmol)Recovery (%)Unconjugated bile acidsCA (1)4.94.9 ± 0.499.9 ± 10.149.049.9 ± 3.2102.0 ± 6.4CDCA (2)5.35.2 ± 0.2100.0 ± 8.253.551.8 ± 1.196.8 ± 1.9DCA (3)5.14.7 ± 0.8191.9 ± 17.150.951.8 ± 4.0102.1 ± 7.7LCA (4)5.34.8 ± 0.489.7 ± 9.353.152.3 ± 4.098.5 ± 7.6UDCA (5)5.13.7 ± 0.772.1 ± 18.350.936.9 ± 2.372.3 ± 5.0isoDCA (8)5.44.9 ± 0.193.3 ± 4.953.445.6 ± 1.185.3 ± 2.1isoLCA (9)5.95.4 ± 0.592.2 ± 9.858.456.6 ± 2.196.8 ± 3.6N-acylamidated bile acidsGCA (39)———51.349.2 ± 1.196.0 ± 2.2GCDCA (40)———55.153.0 ± 2.696.1 ± 4.7GUDCA (41)———54.144.4 ± 2.582.2 ± 4.6GDCA (42)———54.155.3 ± 1.5102.2 ± 2.7TCA (44)———47.442.7 ± 2.090.1 ± 4.3TCDCA (45)———49.848.2 ± 2.896.6 ± 5.6TUDCA (46)———49.445.0 ± 2.5104.2 ± 5.2TDCA (47)———47.946.8 ± 2.497.6 ± 5.1Results are expressed as percent ± SD; n = 5 for unconjugated bile acids, n = 4 for conjugated bile acids. Abbreviations of bile acids and their compound numbers in parentheses are given in the Appendix. Open table in a new tab Values are expressed as mean ± SD (n = 7). Abbreviations of bile acids and their compound numbers in parentheses are given in the Appendix. Limit of detection (LOD), S/N > 3; limit of quantification (LOQ), S/N > 10 (on column). Abbreviations of bile acids and their compound numbers in parentheses are given in the Appendix. Results are expressed as percent ± SD; n = 5 for unconjugated bile acids, n = 4 for conjugated bile acids. Abbreviations of bile acids and their compound numbers in parentheses are given in the Appendix. The method was then applied to the determination of fecal bile acid concentration in samples from patients with cirrhosis (17 early and 21 advanced) and from 10 healthy controls. 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