Application Investgation Of The Chromatographic Analysis Of Neurotransmitter Biogenic Amines Based On Fluorescent Probe

Biogenic amines?BAs?are a family of amino-containing,biologically active,and low molecular weight compounds that are ubiquitous in nature at low concentrations.BAs are a class of neurotransmitters with potential effects on multiple behaviors,and play an important role in various physiological functions.For example,proper intake can enhance immunity,promote growth and metabolism,but excessive amounts can cause some adverse physiological reactions or diseases,such as blood pressure changes,pheochromocytoma,headaches,abdominal cramps and so on.Moreover,BAs also exist in a variety of foods,especially fermented foods,aquatic products,and meat products.The content of BAs in these foods is closely related to their quality and is expected to be an important standard for evaluating the freshness of such foods.Therefore,it is of great significance to establish a rapid,accurate and sensitive analytical method for BAs.In this paper,high performance liquid chromatography with fluorescence detection?HPLC-FLD?and capillary electrophoresis with laser-induced fluorescence detection?CE-LIF?were combined with derivatization and solid-phase extraction?SPE?to analyze BAs in human blood and urine.The research mainly includes the following three parts:?1?Two analytical methods were established for the determination of norepinephrine,epinephrine,and dopamine with CE-LIF and SPE-CE-LIF.Using 5-?4,6-dichlorotriazinyl?amino fluorescein?DTAF?as the fluorescent derivatizing reagent,the derivatization can be achieved in borate buffer solution?pH 9.0?at 37? for 40 min.With?-aminobutyric acid?GABA?as the internal standard?I.S.?,it was found that the separation of I.S.and analytes could be completed within 8 min in 32mmol/L borate buffer solution?pH 9.5?containing 12 mmol/L Brij-35 at a wavelength of?_ex=473nm.With CE-LIF method,the detection limits of?S/N=3?norepinephrine,epinephrine,and dopamine were 1.2,0.1,and 0.4 nmol/L,respectively.With SPE-CE-LIF method,the detection limits of norepinephrine,epinephrine,and dopamine were 0.2,0.01,and 0.05 nmol/L,respectively.The spiked recovery of serum samples was 91.3%-104.6%.?2?A BODIPY fluorescent dye,1,3,5,7-tetramethyl-8-?N-hydroxysuccinimidyl propionic ester?-difluoroboradiaza-s-indacene?TMB-NHS?was used as pre-column derivatization reagent.A method using HPLC-FLD for the simultaneous determination of levodopa,dopamine,epinephrine and norepinephrine was developed.Derivatization and chromatographic separation conditions were discussed in detail.The derivatization reaction was completed at 35? for 30 min.At the wavelength of?_ex/?_em=493 nm/513 nm,norepinephrine,epinephrine,levodopa,and dopamine derivatives achieved baseline separation within 15 min.The detection limits?S/N=3?were 1.0,2.0,5.0,and 0.5 nmol/L,respectively.The new established method was applied to the determination of catecholamines and levodopa in human serum and urine samples with a recovery of 93.3%-106.7%.?3?A CE-LIF method was developed for the determination of serotonin,tyramine and dopamine using TMB-NHS as a fluorescent labeling reagent.When the separation voltage was set at 10.0kV,the three BAs reached baseline separation within 10 min at room temperature in 30 mmol/L sodium citrate solution?pH 9.1?at the wavelength of?_ex=473 nm.The limits of detection?S/N=3?for serotonin,tyramine,and dopamine were 0.3,0.02,and 0.2 nmol/L,respectively.The relative standard deviations?RSDs?of peak area and migration time were from 0.7%-4.4%and 0.2%-2.0%,respectively.This method was successfully applied to the determination of BAs in human serum and urine samples with a recovery of 91.0%-103.1%.