Analysis of intact glucuronides and sulfates of serotonin, dopamine, and their phase I metabolites in rat brain microdialysates by liquid chromatography-tandem mass spectrometry.
A method for the analysis of intact glucuronides and sulfates of common neurotransmitters serotonin (5-HT) and dopamine (DA) as well as of 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in rat brain microdialysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Enzyme-assisted synthesis using rat liver microsomes as a biocatalyst was employed for the production of 5-HT-, 5-HIAA-, DOPAC-, and HVA-glucuronides for reference compounds. The sulfate conjugates were synthesized either chemically or enzymatically using a rat liver S9 fraction. The LC-MS/MS method was validated by determining the limits of detection and quantitation, linearity, and repeatability for the quantitative analysis of 5-HT and DA and their glucuronides, as well as of 5-HIAA, DOPAC, and HVA and their sulfate-conjugates. In this study, 5-HT-glucuronide was for the first time detected in rat brain. The concentration of 5-HT-glucuronide (1.0-1.7 nM) was up to 2.5 times higher than that of free 5-HT (0.4-2.1 nM) in rat brain microdialysates, whereas the concentration of DA-glucuronide (1.0-1.4 nM) was at the same level or lower than the free DA (1.2-2.4 nM). The acidic metabolites of neurotransmitters, 5-HIAA, HVA, and DOPAC, were found in free and sulfated form, whereas their glucuronidation was not observed.
Uutela
Päivi
P
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland.
Reinilä
Ruut
R
Harju
Kirsi
K
Piepponen
Petteri
P
Ketola
Raimo A
RA
Kostiainen
Risto
R
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002853:Chromatography, Liquid; D004298:Dopamine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D020719:Glucuronides / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008297:Male; D017551:Microdialysis; D008862:Microsomes, Liver; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D012701:Serotonin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013431:Sulfates / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D020719:Glucuronides; D013431:Sulfates; D012701:Serotonin; D004298:Dopamine