Establishment Of A Quantitative Analysis Method For Degradation Of Neurotransmitters Based On MPST

With the development of neurobiology,a large number of neural active substance neurotransmitters are found in the nervous system depression,Parkinson’s,alzheimer’s disease and other disease are related to the neurotransmitter.At present,various etiologies can cause neurological diseases,and the effects of neurotransmitters in human body on nervous system diseases are getting more and more attention.This research in the early stage of the development of MPST reagent for pre-column derivatization reagent,due to high sensitivity and high selectivity,the HPLC pre-column derivation method has become the frist choice of analysis of neurotransmitters in drug and clinical sample.Preliminary research in the development of MPST reagent containing carboxyl groups which can derivated with amino functional groups of neurotransmitter,and MPST reagent containing positively charged triphenyl phosphine structure,which can improve the UV detection sensitivity.In this study,a quantitative analysis method based on MPST derivative neurotransmitter was developed to analyze and identify the contents of neurotransmitters in the brain of mice.In this study,5-hydroxyltryptamine(5-HT),norepinephrine(NE),glycine(Gly)and gamma-aminobutyric acid(GABA)were used to derivate with MPST reagents.Using HPLC method chromatographic column for SHIMADZU C18(250 mm x 4.6 mm,5.0 μm),column temperature at 40℃,with 0.5%of formic acid in aqueous solution and 0.1%formic acid in methanol solution as mobile phase,detection wavelength of 254 nm were used to detect detecting derivative products.The optimum reaction conditions and the separation efficiency of the four kinds of neurotransmitters,eventually the best condition about MPST reagent reacted with four kinds of neurotransmitters was at 60℃ maintaining 60 min were investigated.Separation efficiency of 4 kinds of neurotransmitters,Gly and NE of separating degree was 2.6,NE and GABA separating degree was 1.7,GABA and 5-HT separating degree was 5.4,All neurotransmitters were achieved complete separation in 30 min.The four neurotransmitters(NE,Gly,GABA,5-HT)had a good relationship between 1-100 nM(R2=0.9990).The lowest detection limit of NE and Gly(S/N=3)was 5 pmol,and the quantitative limit(S/N=10)was 10 pmol;The lowest detection limit of NE and Gly(S/N=3)was 17 pmol,and the quantitative limit(S/N=10)was 50 pmol;The inter-day precision range of the four neurotransmitters(NE,Gly,GABA,5-HT)were 0.14%-5.67%,0.12%-8.35%,0.14%-2.76%,0.29%-2.18%.Inter-day precision range were 0.26%-6.43%,0.18%-6.53%,0.16%-2.56%,1.12%-2.71%.The average recovery rate of four neurotransmitters(NE,Gly,GABA,5-HT)were 95.55%-110.36%,which met the requirements of biological sample determination.In addition,about 30 healthy mice(15)male and female brains in four kinds of neurotransmitters(NE,Gly,GABA,5-HT)content of quantitative analysis,the results show that the content of Gly in mice brains was 4.416-12.884(pmol/mg),the content of NE was 0.546-4.773(pmol/mg);The content of GABA was 0.616-42.247(pmol/mg);The content of 5-HT was 1.054-6.838(pmol/mg).Based on MPST derivate with neurotransmitters of HPLC-UV detection technology,established a rapid analysis method,four kinds of neurotransmitters(NE,Gly,GABA,5-HT)in the brains of mice was developed.This method is simple,rapid and accurate,and a novel analytical method for the diagnosis and drug safety evaluation of neurotransmitter diseases will be provided.