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2293892 
Journal Article 
Development of a simple and accurate HPLC method for measurement of fecal bile acids: validation by GC-MS and LC-MS and application in healthy and cirrhotic patients 
Kakiyama, G; Muto, A; Takei, H; Nittono, H; Murai, T; Kurosawa, T; Hofmann, AF; Pandak, WM; Bajaj, JS 
2014 
Yes 
Journal of Lipid Research
ISSN: 0022-2275
EISSN: 1539-7262 
55 
5 
978-990 
English 
24627129 
WOS:000335335100021 
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 stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60oC to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of de-conjugated 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. 
extraction; esterified bile acids; bile acid 24-phenacyl ester; derivatization; liver cirrhosis; high-performance liquid chromatography; gas chromatography-mass spectrometry; liquid chromatography-tandem mass spectrometry; routine analysis