Simultaneous determination of ergothioneine, selenoneine, and their methylated metabolites in human blood using ID-LC-MS/MS.

Abstract

Ergothioneine and selenoneine are structurally related dietary antioxidants and cytoprotectants that may help prevent several chronic diseases associated with inflammation and aging. Both compounds share pharmacokinetic characteristics such as cellular uptake through the ergothioneine transporter, accumulation in red blood cells, and biotransformation to methylated metabolites. A rapid, sensitive, specific, precise, and cost-effective analytical method is required to further investigate the potential health benefits of these compounds, individually or combined, in large epidemiological studies. We developed and validated an isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous specific quantification of these analytes in human blood following a simple sample preparation consisting of dilution in aqueous dithiothreitol followed by centrifugal filtration. Chromatographic separation of the analytes is achieved using a reversed-phase chromatography within an 8-min run. Analyte detection is performed using triple quadrupole mass spectrometry in multiple reaction monitoring mode. Each analyte is quantified against its corresponding isotopically labeled internal standard either commercially available or synthesized in-house (77Se-labeled selenoneine compounds). The validated method demonstrates excellent linearity and very good precision (all CV < 10%). Matrix effects are minimal, suggesting that this method could easily be adapted to other matrices. Freeze/thaw cycles have little effect on methylated metabolites but significantly reduced concentrations of the parent compounds. The method was successfully applied to a small set of volunteer blood samples containing low levels of the analytes. The developed ID-LC-MS/MS method opens new avenues for exploring the roles of these bioactive compounds and their metabolites in human health and disease.