RESUMO
A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100â¯×â¯2.1â¯mmâ¯×â¯1.8⯵m C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2â¯mL/min) from 2 to 20%. The eluent was initially composed of 10â¯mM KH2PO4 with 10â¯mM acetic acid and 25⯵M heptafluorobutyric acid. The total analysis time was 11â¯min. Validation of the method included detection of the limit of detection (2â¯nM), limit of quantification (5â¯nM), accuracy (97.2-101%), and intra- and interday precision (2.2-9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8â¯nM, respectively.