Biomolecular Analysis, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Anal Bioanal Chem. 2012 May;403(5):1353-60. doi: 10.1007/s00216-012-5928-4. Epub 2012 Mar 27.
A hydrophilic-interaction chromatography (HILIC) method coupled to electrospray ionization mass spectrometry (ESI-MS) was developed for the determination of trehalose-6-phophate (Tre6P) in Arabidopsis thaliana seedlings. The method was optimized for MS detection and separation of Tre6P from its isomers, such as sucrose-6-phosphate, by testing eluent pH, type of organic solvent and alkalinizer, and gradient conditions. Tre6P could be resolved from matrix components within 28 min by using a water-acetonitrile gradient (0.2 ml/min) at pH 12 with piperidine as alkalinizer. The method was validated for concentrations between 25 and 4,000 nM Tre6P in A. thaliana seedling extracts. Seedlings were extracted with consecutive liquid-liquid and solid-phase extractions, and analyzed with HILIC-MS. Obtained accuracy (80-120 %) and precision (<24 %) demonstrated the suitability of HILIC-MS for determining Tre6P level variations in plants. The limit of detection (LOD) was 3.5 nM Tre6P in extracts corresponding to 4.1 pmol.g(-1) fresh plant weight (FW). This is a considerable improvement with respect to anion-exchange chromatography (AEC)-MS (40 nM) and capillary electrophoresis-MS (80 nM). Furthermore, HILIC-MS analysis times were shorter than with AEC-MS (30 and 60 min, respectively). The applicability of the HILIC-MS method was demonstrated by the analysis of extracts from seedlings grown on medium containing 100 mM sorbitol or trehalose, resulting in mean Tre6P concentrations of 0.2 and 1.9 nmol.g(-1) FW, respectively. Similar concentrations were found with AEC-MS. HILIC-MS was also evaluated at a high flow rate (2.0 ml/min). This high-speed method resolved the Suc6P and Tre6P peaks within 3 min yielding a detection limit of 1.3 nM Tre6P.
建立了亲水作用色谱（HILIC）-电喷雾电离质谱（ESI-MS）法用于拟南芥幼苗中海藻糖-6-磷酸（Tre6P）的测定。通过测试洗脱液 pH 值、有机溶剂和碱化剂的类型以及梯度条件，对 MS 检测和 Tre6P 与其异构体（如蔗糖-6-磷酸）的分离进行了优化。采用 pH 12 的哌啶作为碱化剂，水-乙腈梯度（0.2 ml/min）在 28 min 内即可将 Tre6P 从基质成分中分离出来。该方法在 25 至 4000 nM Tre6P 浓度范围内对拟南芥幼苗提取物进行了验证。幼苗用连续液-液和固相萃取提取，并通过 HILIC-MS 进行分析。所获得的准确度（80-120%）和精密度（<24%）表明 HILIC-MS 适用于测定植物中 Tre6P 水平的变化。提取液中 Tre6P 的检测限（LOD）为 3.5 nM，相当于 4.1 pmol.g(-1)鲜重（FW）。与阴离子交换色谱（AEC）-MS（40 nM）和毛细管电泳-MS（80 nM）相比，这是一个相当大的改进。此外，HILIC-MS 的分析时间比 AEC-MS 短（分别为 30 和 60 min）。用含有 100 mM 山梨醇或海藻糖的培养基培养的幼苗提取物的分析证明了 HILIC-MS 方法的适用性，结果表明 Tre6P 的平均浓度分别为 0.2 和 1.9 nmol.g(-1) FW。AEC-MS 也得到了类似的浓度。还评估了 HILIC-MS 在高流速（2.0 ml/min）下的性能。该高速方法在 3 min 内分离出 Suc6P 和 Tre6P 峰，检测限为 1.3 nM Tre6P。
