Yang Y, Griffiths W J, Nazer H, Sjövall J
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Biomed Chromatogr. 1997 Jul-Aug;11(4):240-55. doi: 10.1002/(SICI)1099-0801(199707)11:4<240::AID-BMC686>3.0.CO;2-6.
Solid-phase extraction and group separation by anion exchange chromatography were combined with capillary column liquid chromatography-mass spectrometry (LC/MS) to permit a thorough characterization of bile acids and intact conjugates of bile alcohols in human urine. Groups of compounds were separated according to acid strength and were analysed on a capillary column, 0.25 x 500 mm, packed with 5 microns particles of Chromasil C18, and connected via a fused silica capillary to the continuous-flow fast atom bombardment (CF-FAB) or electrospray (ES) sources of an AutoSpec-TOFFPD hybrid mass spectrometer. Acetonitrile:water mixtures containing 30 mM ammonium acetate pH 7.2 were used as mobile phases, with 5% glycerol added for FAB Ionisation. Bile acids were analysed directly or after derivatization of carboxyl groups with 4-aminobenzenesulphonic acid. Negative-ion spectra (m/z 1000 or 800 to 300 or 100) were recorded using the point detector or, in the case of ES ionization, the focal plane array detector (FPD). Deprotonated molecules of bile acids containing a sulphonic acid group were detected with a spectral signal to noise ratio of 5:1 when about 90 fmol were injected onto the column of the LC/CF-FAB system. The corresponding peak in the reconstructed ion chromatogram gave a signal-to-noise ratio of about 25:1. The sensitivity could be increased 20-50 times by using ES ionization and the FPD. Bile acids without a sulphonic acid group gave about 70% of the signal of sulphonic acids using ES ionization. The capillary column LC/MS systems were evaluated by analyses of urine from an infant with cholestatic liver disease. More than 150 different bile acids and bile alcohol conjugates were detected, some of which were partially characterized using collision induced dissociation (CID) of the deprotonated molecules and B/E linked scans. A number of compounds were detected for the first time, e.g. di-, tri-, and tetra-hydroxycholestanoic acids conjugated with N-acetylhexosamine and cholestenediol, cholestenetriol and cholestanetriol doubly conjugated with sulphuric acid and glucuronic acid. The relative merits of ES and FAB ionization are discussed.
将固相萃取和阴离子交换色谱分离法与毛细管柱液相色谱 - 质谱联用（LC/MS）相结合，用于全面表征人尿中的胆汁酸和胆汁醇的完整共轭物。根据酸强度对化合物组进行分离，并在一根0.25×500 mm、填充有5微米Chromasil C18颗粒的毛细管柱上进行分析，该毛细管柱通过熔融石英毛细管连接到AutoSpec - TOFFPD混合型质谱仪的连续流快原子轰击（CF - FAB）或电喷雾（ES）源。含有30 mM乙酸铵（pH 7.2）的乙腈 - 水混合物用作流动相，添加5%甘油用于FAB电离。胆汁酸可直接分析，也可在其羧基用4 - 氨基苯磺酸衍生化后进行分析。使用点探测器记录负离子谱（m/z 1000或800至300或100），对于ES电离情况，则使用焦平面阵列探测器（FPD）。当向LC/CF - FAB系统的柱中注入约90 fmol含磺酸基团的胆汁酸去质子化分子时，检测到的光谱信噪比为5:1。重建离子色谱图中的相应峰给出的信噪比约为25:1。使用ES电离和FPD可将灵敏度提高20 - 50倍。使用ES电离时，不含磺酸基团的胆汁酸给出的信号约为磺酸的70%。通过对一名患有胆汁淤积性肝病婴儿的尿液分析对毛细管柱LC/MS系统进行了评估。检测到150多种不同的胆汁酸和胆汁醇共轭物，其中一些通过去质子化分子的碰撞诱导解离（CID）和B/E关联扫描进行了部分表征。首次检测到许多化合物，例如与N - 乙酰己糖胺共轭的二、三、四羟基胆甾酸，以及与硫酸和葡萄糖醛酸双重共轭的胆甾烯二醇、胆甾三醇和胆甾烷三醇。讨论了ES和FAB电离的相对优点。
