Gregus Michal, Roberg-Larsen Hanne, Lundanes Elsa, Foret Frantisek, Kuban Petr, Wilson Steven Ray
Department of Chemistry, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic.
Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway.
Chem Phys Lipids. 2017 Oct;207(Pt B):87-91. doi: 10.1016/j.chemphyslip.2017.05.014. Epub 2017 Jun 3.
Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.
毛细管电泳（CE）能够通过非常简单的仪器实现高分离效率，但在氧甾醇/胆固醇方面尚未得到充分探索。胆固醇和25-羟基胆固醇（两者均为4-烯-3-酮类固醇）在被胆固醇氧化酶酶促氧化后，使用吉拉德P试剂定量转化为腙。采用非水毛细管电泳并在280nm处进行紫外检测实现分离；“电荷标记”的吉拉德P试剂确保了电荷和发色团（这是CE-UV的要求）。过量的试剂也与两种分析物分离，无需在分析前进行去除。使用与质谱完全兼容的分离电解质，化合物在不到5分钟内分离，分离效率极佳。CE-UV方法用于优化电荷标记步骤，结果表明该过程受分析物/试剂比例、反应时间以及分析物结构的影响。
