Quantification of serotonin O-sulphate by LC-MS method in plasma of healthy volunteers

Abstract

The objective of this study was to test the hypothesis that serotonin O-sulphate (5-HT-SO4) could be quantified in human plasma using modern liquid chromatography–mass spectrometry (LC-MS) method as well as develop and validate that method. First, a suitable LC-MS method for detection of 5-HT-SO4 in human plasma samples was developed and validated. Second, a Pilot phase involving four healthy volunteers was executed, where a basal plasma level of 5-HT-SO4 was measured for all subjects and for one after the intake of 100 mg of a 5-hydroxytryptophan (5-HTP) -containing food supplement used to promote serotonergic stimulation of the central nervous system. The basal level of 0.9–2.8 ng/mL of 5-HT-SO4 was observed. The changes of plasma 5HT-O-SO4 showed 1.2 ng/mL before and 22.6 ng/mL 1 h after stimulation. Finally, nine healthy volunteers were selected for the Study phase, where a basal plasma level of 5-HT-SO4 was measured before and after the intake of 5-HTP. One hour after stimulation, six study subjects showed a decrease in 5-HT-SO4 levels while three subjects showed an increase. The changes of plasma 5HT-O-SO4 from the Study phase showed an average 5-HT-SO4 level of 19.2 ng/mL before and 15.7 ng/mL 1 h after stimulation indicating ability of method to emphasize quantitative changes. This was the first study in which naturally occurring 5-HT-SO4 was detected in the samples of human plasma obtained from healthy volunteers. The method developed herein is specific to the measurement of 5-HT-SO4, sensitive enough to quantify intra-individual changes in the samples of plasma and opens up new possibilities to evaluate pathways of serotonin metabolism by minimally invasive methods. The discovery of novel biomarkers using such approaches is increasingly required to expedite development of mechanism-based therapeutics and patient stratification.

Application of 5-HT-SO4 in Biomarker Research

“…The intra-laboratory accuracy of the method over a 3-day period was characterised by a standard deviation of ± 11.95% [13]. Taking into account above mentioned results, the method was concluded to be a suitable technique for measuring 5-HT-SO 4 in human blood samples.…”

“…Speciicity of the method was assessed visually by comparing multiple reaction monitoring (MRM) chromatograms of plasma sample spiked with serotonin O-sulphate, samples of plasma and puriied water. As seen in Figure 2, in the plasma-based calibration standard (A) and plasma (B), some 1.79-1.80 min retention time peaks can be observed [13]. In the plasma-based calibration standard and "pure" plasma, some peaks with a retention time of 1.79-1.80 min were observed.…”

“…The puriied water samples treated similarly do not show such signals (C).This signal might be induced by native content of serotonin sulphate found in plasma samples. Conclusion was reached because in the analytical solution made of 5% serum albumin, such a signal was not seen [13]. For the MRM chromatograms, the test solution of 5% serum albumin (bufered to pH = 7 in a phosphate bufer) was prepared.…”

“…The linearity of detection was evaluated three times in diferent days by analysing calibration standard solutions of 5-HT-SO 4 [13]. This method resulted in a linear relationship between concentration of the analyte (from 10 to 225 ng/mL) and a mass spectral signal of 5-HT-SO 4 with a calibration curve correlation coeicient of >0.98.…”

“…Four diferent concentrations of 5-HT-SO 4 were used for a recovery testing and the method gave correct 5-HT-SO 4 detection results, which were justiied by the average level of recovery of the analyte at 116 ± 8%. The relatively high interval of recovery can be explained due to the matrix efect [13].…”