Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry

• Published in: Analytica chimica acta 2018-12, Vol.1037, p.245-255
• Main Authors: Reinicke, Madlen, Schröter, Jenny, Müller-Klieser, Daniel, Helmschrodt, Christin, Ceglarek, Uta
• Format: Article
• Language: English
• Subjects: Acids; Additives; Anticoagulants; Bile acids; Bile Acids and Salts - blood; Bile Acids and Salts - cerebrospinal fluid; Bile Acids and Salts - chemistry; Bile Acids and Salts - metabolism; Blood plasma; Blood-brain barrier; Cerebrospinal fluid; Chemical precipitation; Chenodeoxycholic acid; Cholesterol; Cholic acid; Chromatography, Liquid; Citric acid; Deuterium; Disturbance; Edetic acid; Esterification; Ethylenediaminetetraacetic acids; Footnote; Free bile acids; Free oxysterols; Heparin; Humans; Ionization; LIFE-Adult; Lipid metabolism; Liquid chromatography; Lithium; Mass spectrometry; Mass spectroscopy; Metabolism; Oxysterols - blood; Oxysterols - cerebrospinal fluid; Oxysterols - chemistry; Oxysterols - metabolism; Patients; Plasma; Pre-analytics; Proteins; Sample preparation; Solid Phase Extraction; Solid phases; Spectroscopy; Stability analysis; Stability tests; Tandem Mass Spectrometry; Targeted metabolomics; Taurocholic acid; Ursodeoxycholic acid; Validation
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2018.02.049

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. [Display omitted] •A LC-MS/MS method is developed for the simultaneous quantification of oxysterols and bile acids in one method.•For oxysterols, enhanced signal intensities and robustness is achieved by using the electrospray ionization in positive mode.•7α-HOCA is included a