Quantitative Determination of Individual Non-Sulfated Bile Acids and Sulfated Lithocholic Acid in Serum by Mass Fragmentography

Abstract

Individual non-sulfated bile acids and sulfated lithocholic acid in serum were determined by mass fragmentography. A hexafluoroisopropyl ester-trifluoroacetyl derivative of bile acid was prepared by the method of Imai et al. (J. Chromatogr. 120, 181, 1976). Deuterium labeled deoxycholic acid was used as an internal standard monitoring at m/z 623. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were determined by monitoring the intensities of m/z 622, m/z 620, m/z 620, m/z 620, and m/z 618, respectively. A serum sample of 200 μl including 500 ng of internal standard was hydrolyzed with strong alkali, then acidified to pH 1 with 2 N HC1 under cooling on ice, and extracted with diethyl ether immediately. Ether extracts were derivatized without further purification. Besides this assay of non-sulfated bile acids, total serum lithocholic acid including the sulfated form was determined as follows: extraction was performed after mixing the acidified (pH 1 with 2 n HC1) hydrolysate with ether and incubating at 40°C for 2 h. Bile acid peaks in the mass fragmentogram were not affected by other materials in these serum extracts. The average values of individual non-sulfated bile acids in sera from healthy fasting subjects (n=15) were as follows: lithocholic acid, 0.049 μg/ml; deoxycholic acid, 0.462 μg/ml; chenodeoxycholic acid, 0.671 μg/ml; ursodeoxycholic acid, 0.070 μg/ml; and cholic acid, 0.217 μg/ml. Total lithocholic acid (non-sulfated and sulfated) in sera was 0.166 μg/ml.