Development And Application Of LC-MS/MS Method For Determination Of Amino Acid Neurotransmitters And Melatonin Concentrations In Plasma Of Patients With Schizophrenia

ObjectiveSchizophrenia is a chronic mental disorder characterized by a range of delusions,hallucinations,thinking disorders,and negative symptoms such as apathy and depression.Changes in amino acid neurotransmitters and melatonin levels have been shown to be associated with the development of schizophrenia.However,to date,quantitative analyses of plasma amino acids and melatonin related to this disease have not been developed.Therefore,it is necessary to establish an accurate and mature method for quantifying the amino acids(glutamic acid,aspartic acid,gamma-aminobutyric acid,glycine,taurine)and melatonin in human plasma,which can provide reference for the diagnosis and prognosis of clinical schizophrenia.MethodsIn this study,a triple quadrupole liquid chromatography-tandem mass spectrometer was used to quantify five amino acids and melatonin in human plasma,and stable isotope internal standard was used for all of them.For the determination of amino acids,the protein in plasma was removed by organic solvent,and then the amino acids were pretreated with derivatization.The amino acids were separated by Waters XSelect (?) HSS T3 column,and mass spectrometry was performed by multiple reaction monitoring(MRM)mode in positive ion mode.For the determination of melatonin,the plasma was pretreated by liquid-liquid extraction method and separated by Waters BEH C18 column.Multiple reaction monitoring(MRM)mode was used for mass spectrometry in negative ion mode.Both methods were validated according to CLSI-62 A.Plasma levels of five amino acids and melatonin in 150 patients with schizophrenia were analyzed.ResultsThe two LC-MS/MS methods developed in this study have good results after method verification.The six indexes have good linearity within the set linear range,and the correlation coefficient r is greater than 0.99.The matrix effect deviations were all lower than 20%,the recoveries were 97.06%-103.36%,the precision was 0.54%-14.43%,and the accuracy was 96.39%-104.42%.The stability of plasma during storage,transport and pretreatment was well investigated.The relative standard deviation was-7.82% to 14.42%.The plasma samples of schizophrenia patients showed that the content of excitatory amino acid neurotransmitters glutamic acid(P<0.01)and gamma-aminobutyric acid(P<0.01)in plasma was higher than that of healthy control group,and the content of melatonin was lower than that of healthy control group(P=0.000),with significant differences.ConclusionsIn this study,two rapid,sensitive and robust liquid chromatography-tandem mass spectrometry methods were established for the determination of amino acid neurotransmitters and melatonin in human plasma,which met the requirements of international methodology validation standards and had good sensitivity and accuracy.The preliminary study on schizophrenic patients shows that the method may be used to assist the diagnosis of this disease and provide a strong basis for the prognosis.