Bridging the Polar and Hydrophobic Metabolome in Single-Run Untargeted Liquid Chromatography-Mass Spectrometry Dried Blood Spot Metabolomics for Clinical Purposes.

Hanne Bendiksen Skogvold,Katja Benedikte Prestø Elgstøen,Steven Ray Wilson,Anja Østeby,Per Ola Rønning,Helge Rootwelt,Camilla Løkken,Elise Mørk Sandås

doi:10.1021/acs.jproteome.1c00326

Hanne Bendiksen Skogvold, Katja Benedikte Prestø Elgstøen + Show 6 more

Open Access

https://doi.org/10.1021/acs.jproteome.1c00326

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Abstract

Dried blood spot (DBS) metabolite analysis is a central tool for the clinic, e.g., newborn screening. Instead of applying multiple analytical methods, a single liquid chromatography-mass spectrometry (LC–MS) method was developed for metabolites spanning from highly polar glucose to hydrophobic long-chain acylcarnitines. For liquid chromatography, a diphenyl column and a multi-linear solvent gradient operated at elevated flow rates allowed for an even-spread resolution of diverse metabolites. Injecting moderate volumes of DBS organic extracts directly, in contrast to evaporation and reconstitution, provided substantial increases in analyte recovery. Q Exactive MS settings were also tailored for sensitivity increases, and the method allowed for analyte retention time and peak area repeatabilities of 0.1–0.4 and 2–10%, respectively, for a wide polarity range of metabolites (log P −4.4 to 8.8). The method’s performance was suited for both untargeted analysis and targeted approaches evaluated in clinically relevant experiments.

Highlights

Reliable and accurate analyses of biological samples are essential for clinical purposes
We focus on factors including retention time/peak area stability, selectivity/peak capacity, Dried blood spot (DBS) extraction, and Mass spectrometry (MS) parameter settings
We find that a single liquid chromatography-mass spectrometry (LC−MS) method can be a compromise between multi-method deep profiling and fast “shotgun” approaches

Summary

Introduction

Reliable and accurate analyses of biological samples are essential for clinical purposes. Untargeted analyses of small biomolecules, i.e., metabolomics, provide the advantage of enabling the detection of large numbers of compounds at the same time. Inborn errors of metabolism (IEMs) represent a large and diverse group of diseases.[3−5] An IEM is typically caused by genetic mutations in a single gene, leading to changes in function or quantity of a vital enzyme. This in turn leads to deviations in the patient’s metabolism. For IEM diagnostics, targeted analyses are mostly used, i.e., monitoring a limited number of predefined analytes. Untargeted analyses can be of great importance for these diseases, as symptoms are often diverse and unspecific, making diagnostics based on targeted analyses difficult and potentially very time-consuming.[5]

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