Villaseñor Alma, Garcia-Perez Isabel, Garcia Antonia, Posma Joram M, Fernández-López Mariano, Nicholas Andreas J, Modi Neena, Holmes Elaine, Barbas Coral
Centre for Metabolomics and Bioanalysis (CEMBIO), Faculty of Pharmacy, Universidad San Pablo CEU, Campus Monteprincipe , Boadilla del Monte, 28668 Madrid, Spain.
Anal Chem. 2014 Aug 19;86(16):8245-52. doi: 10.1021/ac501853d. Epub 2014 Aug 6.
Breast milk (BM) is a biofluid that has a fundamental role in early life nutrition and has direct impact on growth, neurodevelopment, and health. Global metabolic profiling is increasingly being utilized to characterize complex metabolic changes in biological samples. However, in order to achieve broad metabolite coverage, it is necessary to employ more than one analytical platform, typically requiring multiple sample preparation protocols. In an effort to improve analytical efficiency and retain comprehensive coverage of the metabolome, a new extraction methodology was developed that successfully retains metabolites from BM in a single-phase using an optimized methyl-tert-butyl ether solvent system. We conducted this single-phase extraction procedure on a representative pool of BM, and characterized the metabolic composition using LC-QTOF-MS and GC-Q-MS for polar and lipidic metabolites. To ensure that the extraction method was reproducible and fit-for-purpose, the analytical procedure was evaluated on both platforms using 18 metabolites selected to cover a range of chromatographic retention times and biochemical classes. Having validated the method, the metabolic signature of BM composition was mapped as a metabolic reaction network highlighting interconnected biological pathways and showing that the LC-MS and GC-MS platforms targeted largely different domains of the network. Subsequently, the same protocol was applied to ascertain compositional differences between BM at week 1 (n = 10) and 4 weeks (n = 9) post-partum. This single-phase approach is more efficient in terms of time, simplicity, cost, and sample volume than the existing two-phase methods and will be suited to high-throughput metabolic profiling studies of BM.
母乳是一种生物流体，在早期生命营养中具有重要作用，对生长、神经发育和健康有直接影响。全球代谢谱分析越来越多地用于表征生物样品中的复杂代谢变化。然而，为了实现广泛的代谢物覆盖，有必要采用不止一种分析平台，通常需要多种样品制备方案。为了提高分析效率并保持对代谢组的全面覆盖，开发了一种新的提取方法，该方法使用优化的甲基叔丁基醚溶剂系统成功地在单相中保留了母乳中的代谢物。我们对一组具有代表性的母乳进行了这种单相提取程序，并使用液相色谱-四极杆飞行时间质谱（LC-QTOF-MS）和气相色谱-质谱（GC-Q-MS）对极性和脂质代谢物进行代谢成分表征。为确保提取方法具有可重复性且适用，在两个平台上使用18种选定的代谢物对分析程序进行了评估，这些代谢物涵盖了一系列色谱保留时间和生化类别。在验证了该方法后，将母乳成分的代谢特征绘制为一个代谢反应网络，突出了相互关联的生物途径，并表明液相色谱-质谱和气相色谱-质谱平台主要针对网络的不同领域。随后，应用相同的方案来确定产后第1周（n = 10）和第4周（n = 9）母乳之间的成分差异。这种单相方法在时间、简便性、成本和样品量方面比现有的两相方法更有效，将适用于母乳的高通量代谢谱研究。
