A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort. A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 μL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8-250 ng mL-1 for free hydroxycholesterols, 0.2-10 ng mL-1 for dihydroxycholesterols, 0.2-500 ng mL-1 for bile acids and 16-2000 μg mL-1 for cholesterol with acceptable accuracy and precision. In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained. In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.

中文翻译：

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游离氧固醇和胆汁酸，包括结合物 - 通过液相色谱-串联质谱法同时定量人血浆和脑脊液

开发了一种液相色谱-电喷雾电离-串联质谱 (LC-ESI(+)-MS/MS) 测定法，可用于分析胆固醇代谢的 35 种分析物，包括游离胆固醇、17 种游离、非酯化氧甾醇和 17 种游离胆固醇。以及血浆和脑脊液中的结合胆汁酸。作为内标，使用了分析物的 25 种市售稳定的氘标记类似物。分析前研究包括对受不同抗凝添加剂影响的分析物浓度的稳定性测试：肝素锂、柠檬酸盐、EDTA-K3 稳定的血浆和血清，以及 EDTA 全血在 RT 中的稳定性。该 LC-ESI(+)-MS/MS 方法成功应用于分析有和无血脑屏障障碍患者的配对血清/脑脊液样本，以及 LIFE-Adult 研究子队列的 100 个血浆样本。开发了一种快速简单的样品制备方法，包括蛋白质沉淀和在线固相萃取。分析只需要 55 μL 的人血浆/血清或脑脊液。使用 C18 核壳柱可以在 23 分钟内分离异构氧甾醇和胆汁酸。该测定能够在 0.8-250 ng mL-1 的线性范围内定量游离羟基胆固醇、0.2-10 ng mL-1 的二羟基胆固醇、0.2-500 ng mL-1 的胆汁酸和 16-2000 μg mL-1以可接受的准确度和精密度检测胆固醇。在脑脊液中，可以量化一种游离氧甾醇、五种游离胆汁酸和五种结合胆汁酸。在有和没有血脑屏障障碍的患者之间没有获得显着差异。在 LIFE-Adult 亚组中，可以在 EDTA 血浆中量化两种游离氧固醇、四种游离胆汁酸和七种结合胆汁酸。男性的 26-OHC 浓度明显高于女性（p = 0.035）。此外，在女性中，较低水平的胆酸、甘氨胆酸、甘氨脱氧胆酸、鹅脱氧胆酸、甘氨鹅脱氧胆酸、甘氨熊去氧胆酸、乙醇石胆酸和较高水平的牛磺胆酸、牛磺鹅脱氧胆酸、熊去氧胆酸/猪脱氧胆酸被量化。