Abstract
In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks.In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%).
Highlights
Due to the incredible heterogeneity of all the metabolites present in the human body, it has been quite difficult so far to develop generic analytical techniques for their determination [1,2,3,4]
Ion suppression was the main source of matrix effects (ME) for urine, while in plasma the presence of a more complex profile of endogenous compounds translates into the presence of both ion suppression (10% of metabolites) and, in a major form, ion enhancement (20%)
The extremely low values of average RSDs calculated in all conditions (0.3 - 0.5%) represent another demonstration of how modern UHPSFC has evolved into a stable and robust technique, with performance very similar to the wellestablished ultra-high-performance liquid chromatography (UHPLC)
Summary
Due to the incredible heterogeneity of all the metabolites present in the human body, it has been quite difficult so far to develop generic analytical techniques for their determination [1,2,3,4]. The Human Metabolome Database (HMDB) has registered around 110,000 metabolites in its database, and around 30,000 human metabolic and disease pathways are present in the Small Molecule Pathway Database (SMPDB) [12,13,14,15]. Considering this impressive number of potential compounds and targets, there is a strong need to increase the number of metabolites that must be tested under SFC conditions to check their detectability with this technique
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