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 (α→β) and backward (β→α) 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 (α→β) and backward reaction (β→α) 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.

中文翻译：

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通过亲水作用色谱分离聚-N-（1H-四唑-5-基）-甲基丙烯酰胺键合的固定相上的碳水化合物异构体和异构体，并确定异构体互变能垒。

通过亲水相互作用色谱法（HILIC），系统地评估了新型的市售HPLC色谱柱，聚-N-（1H-四唑-5-基）-甲基丙烯酰胺键合的固定相（Daicel DCpak PTZ）的碳水化合物异构体分离能力。带电气溶胶检测（CAD）或（串联）质谱。还原糖倾向于分裂成两个端基异构体峰，这使得非衍生化形式的碳水化合物异构体分离更加复杂。因此，出于实用目的，理想地抑制了端基异构体的分离，这可以通过使用高温或高pH来实现，所述高温或高pH都与导致峰聚结的快速相互转化动力学相关，或者另一方面是通过具有低色谱端基异构体选择性的条件来实现的。四种主要己糖（葡萄糖，甘露糖，半乳糖，果糖），通过等度HILIC和0.1％（v / v）的甲酸和5种主要的戊糖（核糖，核糖，木糖，木酮糖，阿拉伯糖）和五个最重要的二糖（麦芽糖，纤维二糖，乳糖，蔗糖，海藻糖）作为单一碳水化合物标准品进行了分析。 2 mM乙酸铵在各种温度下以pH依赖的方式研究异构体互变平衡。计算了正向（α→β）和反向（β→α）异构化的速率常数以及相应的能垒。298 K时，异构化的能垒在83-91 kJ mol-1左右，正向反应（α→β）和反向反应（β→α）之间的差异通常在1-3 kJ mol-1之间。系统研究最终允许选择条件，以HILIC-ESI-MS（/ MS）和PTZ在梯度洗脱模式下同时分析所有14种碳水化合物。碳水化合物异构体选择性LC（带PTZ），串联MS（具有碳水化合物基团选择性MS1和某些物种特有的SRM过渡）与简单的反褶积策略相结合，可以测定复杂测试混合物中除二糖以外的所有碳水化合物乳糖和麦芽糖（可确定总和）对。因此，提出的方法代表了通过HILIC-ESI-MS / MS对单糖和双糖进行整体糖代谢组学分析的成功方法。