Abstract
An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.
Highlights
Dopamine (DA) and serotonin (5-HT) are monoamine neurotransmitters in the human brain that are involved in several physiological processes. 5-HT is involved in the regulation of several physiological functions, including the sleep-wake cycles, body temperature, blood pressure, perception of pain, hormonal functions of the hypothalamus and psychological functions, such as depression and anxiety [1,2]
We developed a sensitive and selective ultra-performance liquid chromatographic - mass spectrometric (UPLC-MS/MS) method, which provides direct and more reliable analysis of sulfate and glucuronide conjugates than the earlier methods employing hydrolysis of the conjugates
UPLC-MS/MS Method Development and Validation The UPLC-MS/MS method developed in this work for the analysis of DA and 5-HT and their phase I and phase II metabolites (Table S1) in human brain microdialysis and cerebrospinal fluid (CSF) samples was based on the HPLC-MS/MS method presented earlier by our group [40,41]
Summary
Dopamine (DA) and serotonin (5-HT) are monoamine neurotransmitters in the human brain that are involved in several physiological processes. 5-HT is involved in the regulation of several physiological functions, including the sleep-wake cycles, body temperature, blood pressure, perception of pain, hormonal functions of the hypothalamus and psychological functions, such as depression and anxiety [1,2]. The functions of DA have been linked to Parkinson’s disease, schizophrenia, depression and the regulation of motoric movements [3,4,5] The levels of both these neurotransmitters are regulated in the brain by reuptake and metabolism. DOPAC is further metabolized to homovanillic acid (HVA) by catechol-O-methyltrasferase (COMT) (Figure 1) Both DA and 5-HT, and their respective metabolites, can undergo conjugation with glucuronic acid or sulfonate mediated by catalysis with UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), respectively. In the human brain, which is the focus of this study, low mRNA levels of the UGTs 1A4, 1A5, 1A6, 2A1, 2A2, 2A3, 2B7, 2B11 and 2B17 have been reported. 5-HT has been shown to be a substrate for at least one human UGT, UGT1A6, and very low 5-HT glucuronidation activity was reported for UGT2B7 [6,11,12]. DA has been shown to be conjugated mainly by UGT1A10, and very low activities were reported for UGTs 1A6, 2A1, 2A3, 2B7, 2B11 and 2B17 [13]
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