Comparative evaluation of phenyl isothiocyanate derivatization and "dilute-and-shoot" methods for HPLC-MS/MS-based targeted metabolomics analysis of amine-containing metabolites in plasma samples.
Kangkang Xu, Markus Aigensberger, Franz Berthiller, Heidi E Schwartz-Zimmermann
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引用次数: 0
Abstract
Metabolomics, the study of small molecule metabolites in biological systems, is essential for disease diagnosis and biomarker discovery. A key consideration in developing targeted metabolomics methods using HPLC-MS/MS for human or animal plasma is whether to employ derivatization of amino acids, amino acid-related compounds, and biogenic amines. Derivatization with phenyl isothiocyanate (PITC) enhances ionization and LC-separation, but complicates sample preparation and introduces potential errors. This study focuses on (1) the validation of a PITC derivatization method employing reversed-phase (RP) HPLC-MS/MS analysis and (2) a comparison of two analytical approaches for targeted metabolomics analysis of animal and human plasma: the PITC derivatization-based RP-LC-MS/MS method and a "dilute-and-shoot" approach using hydrophilic interaction chromatography (HILIC)-MS/MS and RP-LC-MS/MS analysis. The derivatization method was validated for porcine plasma, assessing limits of detection, lower limits of quantification (LLOQs), linearity, repeatability, recovery, and trueness. Derivatization reduced LLOQs for derivatized compounds in pure solvent solutions but, due to higher dilution factors, resulted in similar LLOQs for derivatized compounds and higher LLOQs for non-derivatized compounds in plasma compared to the "dilute-and-shoot" method. Derivatization improved chromatographic separation of isomers and reduced carryover but introduced challenges such as matrix effects, coelution with impurities, and calibration issues. The "dilute-and-shoot" method performed better for non-derivatized compounds and was less error-prone. Both methods were applied to plasma from various species, demonstrating comparable concentrations for most metabolites. The results also emphasize the importance of using different approaches for cross-validation. Above all, this study highlights the strengths and limitations of both the derivatization method and the "dilute-and-shoot" approach, providing guidance for their application in targeted metabolomics.
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