Separation of carbohydrate isomers and anomers on poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase by hydrophilic interaction chromatography as well as determination of anomer interconversion energy barriers
Separation of carbohydrate isomers and anomers on poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase by hydrophilic interaction chromatography as well as determination of anomer interconversion energy barriers
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DOI：
10.1016/j.chroma.2020.460981
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发表时间：
2020-06-07
影响因子：
4.1
通讯作者：
Laemmerhofer, Michael
中科院分区：
文献类型：
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作者：
Fu, Xiaoqing;Cebo, Malgorzata;Laemmerhofer, Michael
采用亲水相互作用液相色谱（HILIC）、电荷气溶胶检测（CAD）或（串联）质谱法对新型市售高效液相色谱柱聚n -（1h -四唑-5-酰基）-甲基丙烯酰胺键合固定相（Daicel DCpak PTZ）的碳水化合物异构体分离能力进行了系统评价。还原糖倾向于分裂成两个异头峰，这使得非衍生形式的碳水化合物异构体分离更加复杂。因此，对于实际用途来说，理想地抑制了异象分离，这可以通过使用高温或高pH来实现，这两者都与导致峰聚结的快速相互转化动力学相关，或者另一方面，通过低色谱异象选择性的条件来实现。以0.1% （v/v）甲酸和2 mM乙酸铵为溶剂，在不同温度下，用等温HILIC法分析了四种主要己糖（葡萄糖、甘露糖、半乳糖、果糖）、五种主要戊糖（核糖、核酮糖、木糖、木酮糖、阿拉伯糖）和五种最重要的二糖（麦芽糖、纤维素糖、乳糖、蔗糖、海藻糖）作为单一碳水化合物标准，研究了异头物在ph依赖性下的相互转化平衡。计算了正向（α -> β）和反向（β -> α）异构化的速率常数和相应的能垒。298k时，异构化的能垒在83-91 kJ mol（-1）之间，正向反应（α -> β）和反向反应（β -> α）之间的差值通常在1-3 kJ mol（-1）之间。系统研究最终确定了梯度洗脱模式下HILIC-ESI-MS（/MS）同时分析所有14种碳水化合物的条件。碳水化合物异构体选择性LC（带PTZ），串联质谱（带碳水化合物基团选择性MS1和一些物种特异性SRM过渡）和简单的反卷积策略的组合可以确定复杂测试混合物中的所有碳水化合物，除了双糖对乳糖和麦芽糖（可以确定为sum）。因此，所提出的方法代表了通过HILIC-ESI-MS/MS建立单糖和双糖全球糖代谢组学分析方法的成功一步。(C) 2020 Elsevier B.V.版权所有
A new commercially available HPLC column, poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase (Daicel DCpak PTZ), was systematically evaluated for its carbohydrate isomer separation capability by hydrophilic interaction liquid chromatography (HILIC) with charged aerosol detection (CAD) or (tandem) mass spectrometry. Reducing sugars tend to split into two anomer peaks which makes carbohydrate isomer separations in non-derivatized form even more complicated. For practical purposes anomer separations are therefore ideally suppressed which can be accomplished by using high temperature or high pH that are both associated with fast interconversion kinetics leading to peak coalescence, or on the other hand by conditions with low chromatographic anomer selectivity. Four major hexoses (glucose, mannose, galactose, fructose), five main pentoses (ribose, ribulose, xylose, xylulose, arabinose) and five most important disaccharides (maltose, cellobiose, lactose, sucrose, trehalose) were analyzed as single carbohydrate standards by isocratic HILIC with 0.1% (v/v) formic acid and 2 mM ammonium acetate at various temperatures to study anomer interconversion equilibria in a pH-dependent manner. Rate constants of forward (alpha -> beta) and backward (beta -> alpha) anomerization and corresponding energy barriers were calculated. The energy barriers of anomerisation were in the range of around 83-91 kJ mol(-1) at 298 K and the difference between forward (alpha -> beta) and backward reaction (beta -> alpha) was typically between 1-3 kJ mol(-1). The systematic studies finally allowed to pick conditions for the simultaneous analysis of all 14 carbohydrates by HILIC-ESI-MS(/MS) with PTZ in gradient elution mode. A combination of carbohydrate isomer-selective LC (with PTZ), tandem MS (with carbohydrate group-selective MS1 and some species-specific SRM transitions) and a simple deconvolution strategy allowed the determination of all carbohydrates of the complex test mixture except for the disaccharide pair lactose and maltose (which can be determined as sum). Consequently, the proposed method represents a successful step towards a global glycometabolomics profiling method of mono- and disaccharides by HILIC-ESI-MS/MS. (C) 2020 Elsevier B.V. All rights reserved.