Beppu T, Seyama Y, Kasama T, Yamakawa T
J Biochem. 1981 Jun;89(6):1963-73. doi: 10.1093/oxfordjournals.jbchem.a133398.
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, ad m/z 618, respectively. A serum sample of 200 microliters including 500 ng of internal standard was hydrolyzed with strong alkali, then acidified to pH 1 with 2N HCl 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 HCl) hydrolysate with ether and incubation at 40 degrees 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 micrograms/ml; deoxycholic acid, 0.462 micrograms/ml; chenodeoxycholic acid, 0.671 micrograms/ml; ursodeoxycholic acid, 0.070 icrograms/ml; and cholic acid, 0.217 micrograms/ml. Total lithocholic acid (non-sulfated and sulfated) in sera was 0.166 microgram/ml.
采用质量碎片分析法测定血清中单个非硫酸化胆汁酸和硫酸化石胆酸。胆汁酸的六氟异丙酯 - 三氟乙酰衍生物通过今井等人（《色谱杂志》120, 181, 1976）的方法制备。以氘标记的脱氧胆酸作为内标，在m/z 623处进行监测。通过分别监测m/z 622、m/z 620、m/z 620、m/z 620和m/z 618的强度来测定石胆酸、脱氧胆酸、鹅去氧胆酸、熊去氧胆酸和胆酸。将200微升含有500纳克内标的血清样品用强碱水解，然后在冰浴冷却下用2N盐酸酸化至pH 1，立即用乙醚萃取。乙醚萃取物不经进一步纯化直接进行衍生化。除了这种非硫酸化胆汁酸的测定方法外，血清中包括硫酸化形式的总石胆酸的测定方法如下：将酸化（用2N盐酸调至pH 1）的水解产物与乙醚混合，在40℃孵育2小时后进行萃取。这些血清提取物中的质谱图中的胆汁酸峰不受其他物质的影响。健康空腹受试者（n = 15）血清中单个非硫酸化胆汁酸的平均值如下：石胆酸，0.049微克/毫升；脱氧胆酸，0.462微克/毫升；鹅去氧胆酸，0.671微克/毫升；熊去氧胆酸，0.070微克/毫升；胆酸，0.217微克/毫升。血清中总石胆酸（非硫酸化和硫酸化）为0.166微克/毫升。
