A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatography-nanoelectrospray ionisation mass spectrometry

• Methods were developed to profile the plasma (xeno)metabolome.
• Samples were concentrated by mixed-mode solid-phase extraction.
• Plasma extracts were profiled using nanoflowUHPLC-nanoESI-TOFMS.
• The new method resulted in greater coverage of the (xeno)metabolome.

Current metabolite profiling methods based on liquid chromatography-mass spectrometry (LC-MS) platforms do not detect many of the components present at trace concentrations in extracts of plasma due to their low ionisation efficiency or to interference from highly abundant compounds. Nanoflow LC-nanospray MS platforms, which are commonly used in proteomics, could overcome these limitations and significantly increase analytical sensitivity and coverage of the plasma (xeno)metabolome (i.e., metabolites and xenobiotics), but require small injection volumes (<0.5 μL). In this study, we developed sample preparation methods to remove ion suppressive phospholipids and concentrate remaining components of the plasma (xeno)metabolome in order to analyse sub-microliter volumes of plasma extracts for nanoflow ultra-high-performance liquid chromatography-nanoelectrospray ionisation-time-of-flight mass spectrometry (nUHPLC-nESI-TOFMS). These methods use phospholipid filtration plates in combination with polymeric or mixed mode exchange solid-phase extraction (SPE). The phospholipid filtration plates removed >94% of the predominant phospholipid/lysophospholipid species from plasma, whilst absolute recoveries of 63 selected (xeno)metabolites from spiked plasma were generally between 60 and 104%. After a further SPE step, recoveries of test compounds were between 50 and 81%. Studies revealed that both the sample preparation methodology and nUHPLC-nESI-TOFMS analyses gave acceptable repeatability. A qualitative comparison of SPE methods revealed that sample concentration by either polymer or mixed mode ion-exchange SPE gave comprehensive metabolite coverage of plasma extracts, but the use of cation exchange SPE significantly increased detection of many cationic compounds in the sample extracts. Method detection limits for steroid, eicosanoid and bile metabolites were <1.0 ng/mL plasma and for pharmaceutical contaminants were between 0.01 and 30 ng/mL plasma. Comparison of the phospholipid removal/cation exchange SPE and the classical protein precipitation (PPT) sample preparation methodologies revealed that both methods detected the same range of (xeno)metabolites. However, unlike PPT extracts, the SPE preparations allowed direct injection of more concentrated plasma extracts onto the nUHPLC-nESI-TOFMS platform without blockage of the nanocolumn or nanospray, thus resulting in a wider coverage of the (xeno)metabolome. This is the first work to demonstrate the significantly enhanced sensitivity arising from the use of concentrated SPE sample preparations and direct nUHPLC-nESI-TOFMS analysis for untargeted profiling of plasma samples and constitutes a step forward for identifying mixtures of chemical stressors accumulated in blood as well as the disruption of key metabolite pathways in the same sample.