title: A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS.
abstract: 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.
authors.affiliation: Division of Gastroenterology, Hepatology, and Nutrition, Virginia Commonwealth University and McGuire VA Medical Center, Richmond, VA 23249.
authors.forename: Genta
authors.initials: G
authors.lastname: Kakiyama
authors.forename: Akina
authors.initials: A
authors.lastname: Muto
authors.forename: Hajime
authors.initials: H
authors.lastname: Takei
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nittono
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Murai
authors.forename: Takao
authors.initials: T
authors.lastname: Kurosawa
authors.forename: Alan F
authors.initials: AF
authors.lastname: Hofmann
authors.forename: William M
authors.initials: WM
authors.lastname: Pandak
authors.forename: Jasmohan S
authors.initials: JS
authors.lastname: Bajaj
mesh_terms: D001647:Bile Acids and Salts / Q000032:analysis* / Q000737:chemistry; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005243:Feces / Q000737:chemistry*; D005260:Female; D005355:Fibrosis; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008297:Male; D008875:Middle Aged
chemical_list: D001647:Bile Acids and Salts
keywords: bile acid 24-phenacyl ester; derivatization; esterified bile acids; extraction; gas chromatography-mass spectrometry; high-performance liquid chromatography; liquid chromatography-tandem mass spectrometry; liver cirrhosis; routine analysis