Department of Clinical Pharmacology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jan 1;878(1):51-60. doi: 10.1016/j.jchromb.2009.11.019.
We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography-tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005-5micromol/L. The extraction recoveries for all the analytes fell in the range of 88-101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6h at room temperature, at least three freeze-thaw cycles, in the -70 degrees C or -20 degrees C freezer for 2 months, and also in the reconstitution solution at 8 degrees C for 48h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.
我们报道了一种灵敏而稳健的方法，可使用液相色谱-串联质谱法测定人血浆中的胆酸（CA）、鹅去氧胆酸（CDCA）、脱氧胆酸（DCA）、石胆酸（LCA）、熊去氧胆酸（UDCA）及其牛磺酸和甘氨酸缀合物浓度。我们使用活性炭制备无胆酸血浆，作为制备标准品和质控样品的生物基质。血浆样品制备涉及固相萃取。总共 16 种胆汁酸和 5 种内标在反相柱上通过梯度洗脱分离，并在负离子模式下通过串联质谱检测。所有胆汁酸的校准曲线在 0.005-5μmol/L 范围内呈线性。所有分析物的提取回收率均在 88-101%范围内。日内和日间变异系数均低于 10%。稳定性测试表明，所有胆汁酸在室温下至少 6 小时、至少三个冻融循环、-70°C 或-20°C 冰箱中放置 2 个月以及在 8°C 的复溶液中放置 48 小时内均稳定。无胆酸血浆与真实血浆的基质效应比较表明，活性炭净化过程不会影响作为校准基质的活性炭净化血浆的性质。该方法已用于测定 300 多个健康个体的血浆中胆汁酸浓度。总之，该方法适用于人血浆中单个胆汁酸的同时定量。
