Profiling of polar metabolites in biological extracts using diamond hydride‐based aqueous normal phase chromatography

Abstract

Highly polar metabolites, such as sugars and most amino acids are not retained by conventional RP LC columns. Without sufficient retention low concentration compounds are not detected due ion suppression and structural isomers are not resolved. In contrast, hydrophilic interaction chromatography (HILIC) and aqueous normal phase chromatography (ANP) retain compounds based on their hydrophilicity and therefore provides a means of separating highly polar compounds. Here, an ANP method based on the diamond hydride stationary phase is presented for profiling biological small molecules by LC. A rapid separation system based upon a fast gradient that delivers reproducible chromatography is presented. Approximately 1000 compounds were reproducibly detected in human urine samples and clear differences between these samples were identified. This chromatography was also applied to xylem fluid from soyabean (Glycine max) plants to which 400 compounds were detected. This method greatly increases the metabolite coverage over RP-only metabolite profiling in biological samples. We show that both forms of chromatography are necessary for untargeted comprehensive metabolite profiling and that the diamond hydride stationary phase provides a good option for polar metabolite analysis.

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

“…It is important to note that the gradients and mobile phase composition can be modified to favor the separation of particular compound classes on each column. A number of methods using the diamond hydride column have been reported which include chromatographic conditions optimized for the separation of specific metabolite classes such organic acids or amino acids (Callahan et al 2009;Pesek et al 2008Pesek et al , 2009Young et al 2011). The tandem LC configuration is flexible and could incorporate these different gradients.…”

“…The ANP column is used in preference to the HILIC column as it allows shorter re-equilibrations times, and is less sensitive to retention time shifts in response to changes in pH or salts (Callahan et al 2009). This procedure utilizes a trapping step, in which hydrophobic compounds are retained to a RP column, followed by a mobile phase conversion from high aqueous concentration to high organic concentration to allow hydrophilic molecules to be fractionated by the ANP column.…”

Liquid chromatography/mass spectrometry compatible approaches for the quantitation of folic acid in fortified juices and cereals using aqueous normal phase conditions

“…In this manner, the columns can be used for retention of both hydrophilic and hydrophobic analytes depending on the mobile phase composition. Under ANP conditions, they have been found to be especially useful in metabolomics applications [28][29][30], since a given metabolome often contains a variety of polar species which are difficult to retain and separate by conventional RP techniques.…”

Aqueous normal phase chromatography improves quantification and qualification of homocysteine, cysteine and methionine by liquid chromatography–tandem mass spectrometry

“…Retention mechanism of ANP chromatography on silica hydride stationary phases is currently not completely understood [1,5]. It is presumed that, in addition to partitioning effects similar to HILIC chromatography, analytes may also interact directly with the stationary phase surface [5,32]. The water layer on the stationary phase seems to be weaker and less dense in comparison to HILIC, permitting rapid equilibration [4,5].…”