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. 2017 Mar 30;7(2):13.
doi: 10.3390/metabo7020013.

Sample Preparation Strategies for the Effective Quantitation of Hydrophilic Metabolites in Serum by Multi-Targeted HILIC-MS/MS

Elisavet Tsakelidou  1 , Christina Virgiliou  2 , Lemonia Valianou  3 , Helen G Gika  4 , Nikolaos Raikos  5 , Georgios Theodoridis  6
Affiliations

Sample Preparation Strategies for the Effective Quantitation of Hydrophilic Metabolites in Serum by Multi-Targeted HILIC-MS/MS

Elisavet Tsakelidou et al. Metabolites. .

Abstract

The effect of endogenous interferences of serum in multi-targeted metabolite profiling HILIC-MS/MS analysis was investigated by studying different sample preparation procedures. A modified QuEChERS dispersive SPE protocol, a HybridSPE protocol, and a combination of liquid extraction with protein precipitation were compared to a simple protein precipitation. Evaluation of extraction efficiency and sample clean-up was performed for all methods. SPE sorbent materials tested were found to retain hydrophilic analytes together with endogenous interferences, thus additional elution steps were needed. Liquid extraction was not shown to minimise matrix effects. In general, it was observed that a balance should be reached in terms of recovery, efficient clean-up, and sample treatment time when a wide range of metabolites are analysed. A quick step for removing phospholipids prior to the determination of hydrophilic endogenous metabolites is required, however, based on the results from the applied methods, further studies are needed to achieve high recoveries for all metabolites.

Keywords: endogenous metabolites; sample preparation; serum; targeted metabolomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the sample treatment protocols applied in the study indicating the steps needed prior to LC-MS/MS analysis, (a) standard protein precipitation procedure; (b) Dispersive-Solid Phase Extraction (d-SPE) (QuEChERS); (c) Liquid–Liquid Extraction (LLE); (d) HybridSPE.
Figure 2
Figure 2
Plot showing the number of detected compounds (bars) and the sum of their areas (line) in the extract of pooled serum sample at different Hybrid SPE conditions.
Figure 3
Figure 3
Pearson correlation heat-map indicating the response of the detected metabolites under PPT treatment and different HybridSPE conditions. Colour coding: signal response (peak area) increases from light to dark blue. Hierarchical clustering shows metabolites with similar trends with the applied protocols.
Figure 4
Figure 4
Distribution of recovery values for the 53 analytes under optimum HybridSPE conditions at (a) high and (b) low concentration. The respective recovery values for simple PPT are shown in (c) and (d) for high and low concentration in spiked pooled serum.
Figure 5
Figure 5
Overlay extracted ion chromatograms (m/z 184) from full scan analysis of samples after PPT and HybridSPE procedures.
See this image and copyright information in PMC

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