Enantiomeric separation of 1-methyl-tryptophan on a mixed-mode stationary phase via pre-column derivatization and in vivo assessment of chiral inversion in rats

Abstract

1-Methyl-d-tryptophan (d-MT) is an inhibitor of the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). d-MT has been used in studies on Trp metabolism inhibition and as a concomitant drug with antitumor agents. In this study, we investigated the enantiomeric separation of d-MT and its enantiomer, l-MT, via LC-MS/MS following pre-column derivatization with succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl-5-oxoimidazolidin-4-yl) acetate ((R)-CIMa-OSu). Consequently, the use of a mixed-mode stationary phase, Scherzo^Ⓡ SM-C18, provided a separation factor (α) of 1.44 and a resolution (R_s) of 6.14, with both values being higher than those obtained with a reversed-phase column (α: 1.29, R_s: 2.69). Using the proposed LC-MS/MS method, the time-course profiles of plasma d-MT concentrations in rats administered d-MT were investigated. Consequently, a chiral inversion of d-MT to l-MT was observed in rats administered d-MT, whereas no inversion of l-MT to d-MT was observed in rats administered l-MT. The involvement of d-amino acid oxidase (DAO) in the chiral inversion of d-MT to l-MT was suggested, as pre-administration of DAO inhibitors significantly increased the d-MT concentrations in rat plasma.