Identification and characterization of reaction products of 5-hydroxytryptamine with methylglyoxal and glyoxal by liquid chromatography/tandem mass spectrometry.

RATIONALE

Methylglyoxal (MGO) and glyoxal (GO) are known to be at high levels in humans with diabetes. They react with amine-containing proteins and amino acids to form advanced glycation end products, however, their reactivity with other amine-containing metabolites, such as neurotransmitters, has not been explored. In this study, we aimed at studying the reactivity of 5-hydroxytryptamine (5-HT) with MGO or GO, which may alter the metabolic function of 5-HT.

METHODS

Stock solutions of 5-HT, MGO and GO were made in PBS buffer at pH 7.4 and 5-HT was incubated with MGO or GO at different concentrations. The reactions were also performed at physiological concentrations. The reaction mixtures collected at different incubation times were analyzed by direct ESI-HRMS, LC/MS and LC/MS/MS to detect/characterize the products. Agilent 6545 Q-TOF and Agilent 6420 triple quadrupole mass spectrometers were used for the study, and LC separations were performed on a C18 column.

RESULTS

The direct ESI-HRMS data of the reaction mixtures showed formation of three and four reaction products when 5-HT was reacted with MGO and GO, respectively. All the products showed dominant [M + H] ions. The products were characterized by HRMS, LC/MS/MS and literature reports on similar compounds. The products can easily be identified by LC/MS based on the accurate mass values together with retention time information. The MS/MS of the reaction products showed structure-indicative fragment ions.

CONCLUSIONS

5-HT reacts with one or two MGO/GO to form a set of reaction products. The reaction between 5-HT and MGO or GO was faster at higher concentrations of MGO/GO (<10 min), and the same products were found even at physiological concentrations (<48 h). The LC/MS/MS (SRM) method can be used to screen the reaction products when present at low levels.