Simultaneous Determination Of 5-hydroxytryptamine And Tryptophan By HPLC With Programmed Wavelength Fluorescence Detection

Objective:1. To establish a method for the determination of 5-hydroxytrypta-mine(5-HT) and tryptophan(TRP) by high performance liquid chromatog-raphy with programmed wavelength fluorescence detection, and the concentration of serum 5-HT and TRP in healthy persons were determined by this method.2. To explore the clinical significance of serum 5-hydroxytryptamine and tryptophan in patients with schizophrenia(Sch).Methods:1. Separation was achieved on the Hypersil C18 column (250mm×4.6mm i.d.,7μm particle size, Thermo Scientific Instrument Co., Ltd.) with a mobile phase consisting of 0.1mol/L phosphate solution and methanol (85:15). The flow rate was set at 1.0mL/min. The elute was monitored by the programmed wavelength detection, at the beginning of the run, fluorescence excitation and emission wavelengths were operated at 278 nm and 338nm, respectively, the excitation and emission wavelengths was changed to 280 nm and 340nm 6.0 min later. Serum samples were deproteinized with 5%(v/v) perchloric acid at equal volemn. The effects of various aspects on operation and determination were examined to establish optical assay conditions, such as detection wavelength, the content of organic solvent in the mobile phase and flow rate. The influences of common substances which produce natural fluorescence were investigated.2. The concentration of serum 5-HT and TRP in healthy persons (n=71) were analyzed by HPLC-FD, and the ratio of 5-HT to TRP were calculated.3. The concentration of serum 5-HT and TRP in patients with schizophrenia(n=70) were determined by HPLC-FD and the content ratios of 5-HT to TRP(5-HT/TRP) were calculated. The schizophrenia patients were divided into two groups with positive and negative symptom to explore the relation between 5-HT,TRP and schizophrenia.Results:1. The retention time of 5-HT was approximately 4.9 min, the linear equations was Y=4223.5X-35448.2, and the limit of detection was lnmol/L,and the recoveries of 5-HT were from 82.70%～95.40%, the intra-day and inter-day variations were 2.03% and 3.42%. The retention time of TRP was approximately 8.4 min, the linear equations was Y=421314X+38297.1, and the limit of detection was 0.007μmol/L,and the recoveries of TRP were from 83.67%～94.98%. the intra-day and inter-day variations were 1.32% and 3.31%. Phenyalanine, tyrosine, kynurenine and kynurenic acid didn't interfere in this assay.2. The serum 5-HT and TRP concentrations in 71 healthy persons were(544.8±156.8)nmol/L and (53.08±8.29)μmol/L respectively. The ratio of 5-HT to TRP were (10.45±3.25)X 10-3.3. The serum 5-HT and TRP concentrations in Sch patients were (609.5±238.3) nmol/L and (52.52±11.65)μmol/L, respectively, the ratio of 5-HT to TRP were (12.31±5.91)×10-3. Compared with the control group, the 5-HT concentrations were no statistically significant difference in schizophrenic positive-type patients, but the concentrations were significantly increased in schizophrenic negative-type patients.Conclusions:1. A new method was established for simultaneous determination of 5-HT and TRP in serum by high performance liquid chromatography with programmed wavelength fluorescence detection, and the concentration of serum 5-HT and TRP in healthy persons has been determined. The method is simple, rapid, sensitive and suitable for clinical and scientific study.2. Compared with the control group, the concentrations of TRP were no statistically significant difference, the concentration of 5-HT in patients with Sch were different in different subgroups, the ratio of 5-HT to TRP in Sch patients were significantly increased.