Center for Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain.
Anal Chem. 2012 Jul 17;84(14):5992-9. doi: 10.1021/ac300716u. Epub 2012 Jul 3.
Metabolic fingerprinting of biological tissues has become an important area of research, particularly in the biomarker discovery field. Methods have inherent analytical variation, and new approaches are necessary to ensure that the vast numbers of intact metabolites present in biofluids are detected. Here, we describe an in-vial dual extraction (IVDE) method and a direct injection method that shows the total number of features recovered to be over 4500 from a single 20 μL plasma aliquot. By applying a one-step extraction consisting of a lipophilic and hydrophilic layer within a single vial insert, we showed that analytical variation was decreased. This was achieved by reducing sample preparation stages including procedures of drying and transfers. The two phases in the vial, upper and lower, underwent HPLC-QTOF analysis on individually customized LC gradients in both positive and negative ionization modes. A 60 min lipid profiling HPLC-QTOF method for the lipophilic phase was specifically developed, enabling the separation and putative identification of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The aqueous phase of the extract underwent direct injection onto a 45 min gradient, enabling the detection of both polarities. The IVDE method was compared to two traditional extraction methods. The first method was a two-step ether evaporation and IPA resuspension, and the second method was a methanol precipitation typically used in fingerprinting studies. The IVDE provided a 378% increase in reproducible features when compared to evaporation and a 269% increase when compared to the precipitate and inject method. As a proof of concept, the method was applied to an animal model of diabetes. A 2-fold increase in discriminant metabolites was found when comparing diabetic and control rats with IVDE. These discriminant metabolites accounted for around 600 entities, out of which 388 were identified in available databases.
生物组织的代谢指纹分析已成为一个重要的研究领域，特别是在生物标志物发现领域。方法具有内在的分析变异性，因此需要新的方法来确保检测到生物流体中存在的大量完整代谢物。在这里，我们描述了一种管内双提取（IVDE）方法和一种直接进样方法，该方法从单个 20μL 血浆等分试样中回收的特征总数超过 4500 个。通过在单个管内插入物中使用由亲脂层和亲水层组成的一步提取，我们表明分析变异性降低了。这是通过减少包括干燥和转移步骤在内的样品制备阶段来实现的。管内的上下两层分别在正离子和负离子模式下，在单独定制的 LC 梯度上进行 HPLC-QTOF 分析。专门开发了一种用于亲脂相的 60 分钟脂质分析 HPLC-QTOF 方法，能够分离和推测鉴定脂肪酸、甘油脂、甘油磷脂、鞘脂和固醇。提取的亲水性相直接进样至 45 分钟梯度，可同时检测极性。将 IVDE 方法与两种传统提取方法进行了比较。第一种方法是两步乙醚蒸发和 IPA 重悬，第二种方法是甲醇沉淀，通常用于指纹图谱研究。与蒸发相比，IVDE 提供了 378%的重现性特征增加，与沉淀和进样方法相比增加了 269%。作为概念验证，该方法应用于糖尿病动物模型。与 IVDE 相比，糖尿病大鼠和对照组大鼠的判别代谢物增加了 2 倍。这些判别代谢物约占 600 个实体，其中 388 个在可用数据库中被鉴定。
